Chapters Archive - Endotext (2023)

Table of Contents
ABSTRACT INTRODUCTION NON-EXERCISE ACTIVITY THERMOGENESIS: A SIGNIFICANT COMPONENT OF DAILY ENERGY EXPENDITURE Basal Metabolic Rate Thermic Effect of Food Physical Activity: EAT and NEAT Measurement of NEAT INDIVIDUAL AND ENVIRONMENTAL DETERMINANTS OF NEAT Effects of Occupation, Gender, and Age Industrialization and Societal Status Seasonal Effects Alteration of NEAT with Varying Energy Availability from Foods PHYSIOLOGICAL AND MOLECULAR DETERMINANTS OF NEAT Principles of NEAT Regulation Tissues, Organs, and Central Mechanisms Involved in NEAT Regulation The Role of Peripheral Tissues for NEAT Regulation: Adipose Tissue and Adipokines Skeletal Muscle as Peripheral NEAT Modulator Associations of NEAT with Type 2 Diabetes Mellitus and NAFLD/MAFLD as Common Sequelae of Obesity Personalized Approaches versus Environmental Re-engineering for Promoting NEAT Increasing NEAT: Realistic Goals, Pitfalls and Appropriate Activities Limitations to Increasing NEAT CONCLUSION REFERENCES ABSTRACT INTRODUCTION IS EATING BEHAVIOUR REGULATED? FOOD INTAKE AND APPETITE CONTROL The Motivation to Eat A CONCEPTUAL THEORETICAL MODEL LINKING PHYSIOLOGY, MOTIVATION, AND BEHAVIOR TO FOOD INTAKE EPISODIC AND TONIC SIGNALS OF APPETITE CONTROL Episodic Appetite Signals Cholecystokinin Glucagon-Like-Peptide-1 Peptide YY 3-36 Amylin Ghrelin Satiety Cascade Peptides TONIC SIGNALS OF APPETITE CONTROL Ghrelin and the Hunger Drive The Role of Leptin Fat-Free Mass and Resting Metabolic Rate and Associations with Appetite Studies Examining the Associations between Fat-free Mass, Resting Metabolic Rate and Energy Intake Factors Affecting the Strength of Association Between Fat-Free Mass and Energy Expenditure with Energy Intake Associations Between Body Composition and Energy Intake During Prolonged Negative or Positive Energy Balances HOMEOSTATIC AND HEDONIC PROCESSES OF APPETITE CONTROL Homeostasis and Hedonics: Cross-Talk and Interaction Liking vs. Wanting Food MODULATION OF APPETITE THROUGH PHYSICAL ACTIVITY Physical Activity and Control of Food Intake Does Habitual Physical Activity Level Affect Appetite Sensitivity? CONCLUSION REFERENCES ABSTRACT INTRODUCTION DIAGNOSING DIABETES INSIPIDUS Confirmation Of Hypotonic Polyuria Conformation Of Polyuria Excluding Other Causes of Polyuria Initial Serum/Plasma and Urine Investigations DIAGNOSIS OF THE TYPE OF DIABETES INSIPIDUS Water Deprivation Test Measurement Of Plasma AVP Measurement Of Plasma Copeptin Hypertonic Saline Infusion Test IDENTIFICATION OF THE UNDERLYING CAUSE OF THE DI FUTURE DIRECTIONS CONCLUSIONS REFERENCES ABSTRACT INTRODUCTION ETIOLOGY OF HYPOPITUITARISM Congenital Tumors Vascular Causes Immunological/Inflammatory Disease Infections Radiation Therapy Traumatic Brain Injury Empty Sella Syndrome CLINICAL MANIFESTATION OF HYPOPITUITARISM Growth Hormone Deficiency Adrenocorticotrophic Hormone Deficiency Gonadotrophin Deficiency Thyroid Stimulating Hormone Deficiency Prolactin Deficiency Antidiuretic Hormone Deficiency INVESTIGATION OF SUSPECTED HYPOPITUITARISM Baseline Investigations Dynamic Testing Imaging the Pituitary MANAGEMENT OF HYPOPITUITARISM Hypocortisolism Secondary Hypothyroidism Gonadotrophin Deficiency Growth Hormone Deficiency Diabetes Insipidus GUIDELINES REFERENCES ABSTRACT INTRODUCTION ANATOMY, CELL BIOLOGY AND PHYSIOLOGY OF THE OF THE HYPOTHALAMO-POSTERIOR PITUITARY AXIS Anatomy Of the Neurohypophysis Molecular-Cell Biology of Vasopressin and Oxytocin Synthesis And Release of Vasopressin and Oxytocin The Physiology of The Secretion of Vasopressin and Thirst The Physiology of Oxytocin CLINICAL PROBLEMS SECONDARY TO DEFECTS IN THE HYPOTHALAMO-POSTERIOR PITUITARY AXIS Diabetes Insipidus Syndrome Of Inappropriate Antidiuresis Adipsic And Hypodipsic Syndromes REFERENCES ABSTRACT CLINICAL RECOGNITION PATHOPHYSIOLOGY DIAGNOSIS and DIFFERENTIAL DIAGNOSIS THERAPY Acute Adrenal Insufficiency (Adrenal Crisis) Management of Chronic or Insidious Onset of Adrenal Insufficiency FOLLOW-UP Patient Education PROGNOSIS GUIDELINES REFERENCES ABSTRACT INTRODUCTION EPIDEMIOLOGY PATHOPHYSIOLOGY CLINICAL FEATURES External Genitalia Internal Genitalia Postnatal Effects and Growth Puberty Gender Role Behavior and Cognition Fertility Salt-Wasting 21-Hydroxylase Deficiency Simple-Virilizing 21-Hydroxylase Deficiency Non-Classical 21-Hydroxylase Deficiency OTHER FORMS OF CONGENITAL ADRENAL HYPERPLASIA 11-β Hydroxylase Deficiency 3-β Hydroxysteroid Dehydrogenase Deficiency 17 α -Hydroxylase/17,20 Lyase Deficiency Congenital Lipoid Adrenal Hyperplasia Cytochrome P450 OxidoReductase Deficiency GENETICS 21-Hydroxylase Deficiency DIAGNOSIS Hormonal Diagnosis Prenatal Diagnosis of 21OHD Non-Invasive Prenatal Diagnosis of CAH Preimplantation Diagnosis Prenatal Treatment Outcome of Prenatal Treatment of 21OHD Prenatal Diagnosis and Treatment of 11β-OHD CAH TREATMENT Hormone Replacement Bone Mineral Density Surgery Other Treatment Strategies and Novel Therapies CONCLUSION REFERENCES ABSTRACT INTRODUCTION EMBRYOLOGY SEPTO-OPTIC DYSPLASIA HESX1 SOX2 SOX3 OTX2 PAX6 RAX TCF7L1 Other Genes SYNDROMIC CAUSES OF HYPOPITUITARISM LHX3 LHX4 GLI2 GLI3 Hypopituitarism Associated with Holoprosencephaly Pituitary Stalk Interruption Syndrome FOXA2 EIF2S3 BMP4 ARNT2 TBC1D32 NON-SYNDROMIC CAUSES OF HYPOPITUITARISM POU1F1 PROP1 ISOLATED HORMONE DEFICIENCIES Isolated Growth Hormone Deficiency Central Hypothyroidism Isolated ACTH Deficiency Central Hypogonadism Central Diabetes Insipidus SYNDROMES ASSOCIATED WITH HYPOPITUITARISM Prader-Willi Syndrome Axenfield-Rieger Syndrome (PITX2) Johanson-Blizzard Syndrome (UBR1) Oliver-McFarlane Syndrome (PNPLA6) Wiedemann-Steiner Syndrome (KMT2A) Kabuki Syndrome (KMT2D) Williams Syndrome PHACES INDICATIONS FOR GENETIC TESTING Septo-Optic Dysplasia Syndromic Causes Non-Syndromic Isolated Hormone Deficiencies Syndromes Associated with Hypopituitarism ABBREVIATION LIST REFERENCES ABSTRACT PINEAL PHYSIOLOGY Pineal Anatomy and Structure Main Function of the Pineal Gland Melatonin Synthesis Control of Melatonin Synthesis: A Darkness Hormone Melatonin Metabolism Melatonin Production During Development and Across Life MELATONIN’S MECHANISMS OF ACTION Melatonin Target Sites and Receptors Chronobiotic Effects of Melatonin MELATONIN PHYSIOLOGY AND PATHOPHYSIOLOGY Melatonin During Puberty, Menstrual Cycle and Reproductive Function Melatonin and Core Body Temperature Melatonin and Energy Metabolism and Glucose Homeostasis Melatonin, Antioxidant Properties and Cancer Miscellaneous MELATONIN, CLINICAL APPLICATION AND THERAPEUTIC USE Melatonin and Sleep Melatonin and the CNS Melatonin: Therapeutic Use REFERENCES ABSTRACT INTRODUCTION THE OVERWEIGHT AND OBESITY-FOCUSED ENCOUNTER History, Physical, and Laboratory Testing of the Patient who is Overweight or Has Obesity CLINICAL IMPORTANCE OF A WEIGHT TRAJECTORY Early Growth, Childhood, and Puberty Pregnancy, Breast Feeding, and Menopausal Transition CASE LESSONS IN PATIENT-GENERATED WEIGHT GRAPHS Impact of Medications Effects of Situational Life Changes That Impact Weight Identification of Response to Weight Loss Interventions Identification of Weight Regain after Successful Weight Loss: Importance of Prompt Intervention and Identification of Lifetime Maximum Weight Communicating with Patients About the Disease of Obesity CONCLUSIONS REFERENCES Post navigation


Low levels of physical activity combined with food intake in excess of daily energy expenditure over extended time periods precede weight gain and promote increases in body fat. Obesity and related insulin resistance are common sequelae of a chronically positive energy balance, potentially resulting in type 2 diabetes (T2D) and nonalcoholic/metabolic dysfunction associated fatty liver disease (NAFLD/MAFLD). The percentage of individuals considered as obese and morbidly obese is continuously rising and developing countries are catching up quickly as compared to industrialized nations. If the observed trend continues, global obesity prevalence will prospectively reach more than 21% in women and 18% in men by 2025. In addition to poor dietary habits, physical activity levels have decreased in recent decades in parallel with an increase in sedentary behavior. Given the technological advances in domestic, community, and working spaces in the last century it is not uncommon for people in industrialized countries to spend one half of their day sitting. As well, for a majority of people, voluntary physical exercise remains of minor importance. Non-Exercise Activity Thermogenesis (NEAT) refers to that portion of daily energy expenditure resulting from spontaneous physical activity that is not specially the result of voluntary exercise. Levels of NEAT ranges widely, with variance of up to 2000 kilocalories per day between two individuals of similar size. These differences are related to complex interactions of environmental and biological factors, including people’s differing occupations, leisure-time activities, individual molecular and genetic factors, and evidence that food intake has independent effects on spontaneous physical activity. Available data support the hypothesis that targeting NEAT could be an essential tool for body weight control. This comprehensive review systematically describes the definition, evaluation methods, and environmental and biological factors involved in the regulation of NEAT. It further emphasizes the association with obesity and related disorders and suggests practical relevant implications, but also potential limitations for the integration of NEAT in daily life.


The prevalence of obesity and resultant adverse health implications continues to rise in both sexes in the United States and worldwide (1). In 2011 Finucane et al. estimated international trends of mean, age-standardized, body mass index (BMI) and suggested that an estimated 1.46 billion adults worldwide had BMI of 25 kg/m² or greater, of these 205 million men and 297 million women were considered obese (2). Indeed, between 1980 and 2008 the prevalence of adult obesity (BMI ≥ 30 kg/m2) has risen in every sub-region of the world except for Central Africa and South Asia (2). The mean BMI increase was reported to be 0.4 kg/m2 per decade for men and 0.5 kg/m2 for women (2). Comparable data have been presented by the NCD Risk Factor Collaboration Trial (3). With a focus on industrialized countries, the most prominent increases of mean BMI have been observed in the United States, followed by the United Kingdom and Australia. The United States had the highest BMI of all studied high-income countries, recent data from the U.S. National Health and Nutrition Examination Survey indicate that obesity affects approximately 35% of the male and 40% of the female population (1, 4, 5). Similarly, populations of European and Eastern Mediterranean countries show an overweight prevalence of up to 50% (6). Further data indicate that compared to 1975, childhood obesity is much more common in 2016, with one obesogenic key driver in this group representing reduced physical activity (3, 7). This continuous rise in the incidence and prevalence of overweight and obesity in children, adolescents, and adults in the past century has created major burdens for national health care systems worldwide.

Obesity-related diseases are typically of chronic nature and include type 2 diabetes mellitus (T2D), nonalcoholic/metabolic dysfunction associated fatty liver disease (NAFLD/MAFLD), peripheral artery and cardiovascular disease, sleep apnea, hypertension, as well as various cancers. In the United States, seven out of the leading ten causes of premature death and disability are represented by chronic diseases related to obesity (8). In fact, obesity is one of the most important avoidable risk factors for significant morbidity and premature mortality, leads to impaired quality of life, can elevate disability rates, and statistically reduces expected life span by seven years (9, 8, 10, 11).

The etiology of obesity is multifactorial, resulting from genetic and epigenetic, physiological, behavioral, sociocultural, and environmental factors leading to an imbalance between energy intake and expenditure (12). To this end excess body fat consistently results from a period of sustained net-positive energy balance, followed by homeostatically regulated weight maintenance. The roles of diet and caloric intake in these processes have been the principal focus of most mechanistic studies. In countries with high obesity prevalence rates, nutritional quality is mostly poor, but there is a controversy as to whether increased energy intake alone can explain the obesity epidemic. Trends for improved dietary quality and reduced energy intake have been observed in recent decades in the United Kingdom and among parts of the U.S.-population (13–15). For instance, since the 1980s obesity prevalence has substantially risen in Great Britain, while caloric intake appeared to decline on a population level (12). In order to explain such findings it was hypothesized that if physical activity decreases, body weight may increase if energy intake is not altered (16). Therefore, under conditions of unchanged caloric intake, the amount of daily physical activity emerged as major predictor (1, 17). Accordingly, the National Weight Control Registry of the United States has identified strategies for maintaining weight loss, which include engagement in higher levels of physical activity (1). Therefore, greater attention has been paid to addressing the energy expenditure side of the caloric balance equation, in particular with regards to prevention of body weight regain after successful weight loss.


Daily total energy expenditure (TEE) is the net amount of energy utilized by animals and humans to maintain core physiological functions and locomotion. Three main components of energy balance determine TEE: Basal metabolic rate (BMR), the thermic effect of food (TEF), also called diet-induced thermogenesis, and the energy expended for physical activity (18). Additional components may exist (e.g. energy costs of emotion), but apparently play a minor role with respect to energy balance (19–22). Multiple factors significantly affect our daily energy needs, including age, body composition, thyroid hormone status, catecholamine levels/sympathoadrenergic activity, ambient and body temperature, disease states, and certain medications (19, 18).

Basal Metabolic Rate

Basal metabolic rate (BMR) represents the minimal amount of energy expended to maintain all the body’s vital processes at rest (the basal state). BMR is essentially a function of lean body mass (LBM), which is estimated to account for 80% of BMR within and across species (23–29) . BMR is sometimes misinterpreted to imply the lowest level of energy expenditure during the day. This is, however, not true, since during sleep or undernutrition metabolic functions may be lower than that observed under basal conditions (27). In free-living individuals, BMR accounts for the main percentage of TEE (Figure 1), in healthy subjects with mainly sedentary occupations it predicts around 60% of the variance (22). Otherwise it is proportionally larger in individuals with minimal physical activity (e.g., in sedated and ventilated patients on an intensive care unit) (30).

Of note, it is not uncommon that the term resting energy expenditure (REE) is synonymously interchanged with BMR. However, BMR is measured in completely rested subjects, both before and during the measurements. Therefore, measurements are classically taken in the morning after 8 hours of sleep. Subjects should be lying supine, fully awake, and be fasted for at least 10-12 hours. The environment in which the measurements are taken should be thermo-neutral (22–26 °C) so that no thermoregulatory effect on heat production biases the results (19); and subjects should be free from emotional stress and familiar with the measurement apparatus (27). By contrast, REE is equivalent to postabsorptive energy expenditure at complete rest at any time of the day and can vary as much as 10% from BMR (21).

Some studies suggest BMR is reduced in those prone to become obese (31–33). It is, however, a matter of controversy as to whether a reduction of energy expenditure due to a decrease in BMR sufficiently explains the positive net energy balance that contributes to excess weight gain in the majority of subjects who become obese (34–36). On the other hand, LBM can be enlarged or even preserved by means of regular physical activity, which in turn can favorably modulate BMR (1, 37–39).

Thermic Effect of Food

Thermic effect of food (TEF) is the increment of energy expenditure above REE following meal ingestion that reflects the energy cost (burned) during food digestion, absorption, and storage. It is a relatively stable component of total energy expenditure (Figure 1). Thermic effect of food typically ranges between 8-15% of TEE and the variance of TEF has been associated with nutrient composition and energy content of consumed foods (40).

Physical Activity: EAT and NEAT

Physical activity is the second most significant contributor to TEE in most people (Figure 1). It is defined as the additional energy expenditure above REE and TEF that is required for performing daily activities and therefore also called physical activity-related or simply activity-related energy expenditure (PEE/AEE). PEE/AEE can be categorized into exercise-related activity thermogenesis (EAT) and non-exercise activity thermogenesis (NEAT). Both vary widely within and between individuals.

For the majority of subjects in industrialized countries, exercise is believed to be negligible (20). According to NHANES data, 36.1% of the studied US population was categorized as sedentary, while a further 47.6% were physically active at low levels (40, 41). Remarkably, only around 16% of subjects in NHANES met recommended guidelines for physical activity or were highly active. Even so, the latter subjects did not necessarily exercise (42, 43, 41). Thus, it is reasonable to conclude that on a population level the percentage of subjects engaging in regular, intense physical exercise is low. In those who habitually participate in purposeful physical training, EAT is believed to maximally account for 15-30% of TEE (18, 44). Other authors suggest that the majority of subjects undergoing regular physical training, defined as “bodily exertion for the sake of developing and maintaining physical fitness,” do not exercise more than two hours a week, accounting for an average energy expenditure of 100 kilocalories (kcal) per day(45). Such expenditure would contribute to only 1-2% to the variance of TEE. Taken together, for most human subjects EAT seems to not be a major contributor to TEE variance.

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Figure 1. Model of human energy expenditure components (adapted from (46)). Exercise-related physical activity is comparable to exercise-related activity thermogenesis (EAT), while spontaneous physical activity is comparable to non-exercise activity thermogenesis (NEAT); for further explanations see text. Note that parts of spontaneous physical activity are beyond voluntary control, also called “fidgeting.” Arrows symbolize the multiple and varying impacts of individual factors, genetic background, and environment.

In contrast, NEAT comprises the largest share of daily activity-related thermogenesis, including for most subjects engaging in regular physical training. It is important to note that both NEAT and spontaneous physical activity are not interchangeable but represent complementary concepts (47): NEAT refers to energy expenditure, while spontaneous physical activity describes the types of bodily activity that are not defined as purposeful movements but still contribute to NEAT. Some authors categorize NEAT into three main subcomponents comprising body posture, ambulation, and all other spontaneous movements including “fidgeting” (48). Accordingly, a certain percentage of spontaneous physical activity (and therefore NEAT) is beyond voluntary control (e.g., “fidgeting”).

NEAT corresponds to all the energy expended with occupation, leisure time activity, sitting, standing, stair climbing, ambulation, toe-tapping, shoveling snow, playing the guitar, dancing, singing, cleaning, and more (20, 22). These activities do not characteristically involve moderate- to vigorous-intensity activities in comparable manner as voluntary exercise, but occur at a low energy workload for minutes and up to hours (12).

The importance of NEAT becomes apparent when considering the following points: The variability in BMR between individuals of similar age, BMI and of equal gender ranges around 7-9% (49), while the contribution of TEF is maximally 15%. Thus, BMR and TEF are relatively fixed in amount and account for approximately three quarters of daily TEE variance. As EAT is believed to be negligible on a population level, NEAT consequently represents the most variable component of TEE within and across subjects. It is responsible for 6-10% of TEE in individuals with a mainly sedentary lifestyle and for 50% or more in highly active subjects (47, 22, 29).

Taken together, with respect to body mass regulation and modification of energy balance, NEAT must be considered as a factor with potentially major impact. This is of particular interest in terms not only of developing therapeutic strategies for the patient with obesity, but also for designing obesity-prevention strategies for populations. The following sections will focus on these issues, including the environmental and biological modification of NEAT.

Measurement of NEAT

For a better understanding of the potential role of NEAT in the context of obesity it is essential to recognize the strengths and limitations of available techniques used for the quantification of NEAT.

NEAT can be principally measured by two approaches: 1) by assessing total NEAT, and 2) by using the factorial approach (for extensive reviews of available techniques see (50, 51)).


Assuming that EAT is negligible, total NEAT can be calculated by subtracting BMR and TEF from TEE (16):

Equation 1: NEAT = TEE – (BMR + TEF)

To complete equation 1, TEE, BMR and TEF need to be measured. Under free-living conditions TEE can be reliably measured by using the doubly-labeled water method (16). This approach requires the application of stable isotopes (deuterium/2H2 and O18) containing water (2H2O18). The difference in the clearance rate of the two isotopes equals carbon dioxide (CO2) production generated during energy production and thus represents quantification of TEE (52)(Figure 2).

One necessary precondition of the doubly-labeled water method is that the O2 of expired CO2 is in equilibrium with the O2 in body water. Once ingested, O18 will be readily distributed in the systemic H2O, H2CO3 and CO2 pools. 2H2 will be dispensed in body water and the H2CO3 pool (equation 2).

Equation 2: CO2 + H2O ↔ H2CO3

The concentration of labeled O2 in body water will then decrease over time due to a loss of CO2 with expiration, perspiration, and in excreted body water (urine). 2H2 is mainly lost through excreted body fluids, yet a small percentage can be incorporated into body fat or protein. Since body water becomes tagged with known amounts of tracers at the same time at the beginning of the measurement period, the difference of elimination rates of both tracers equals CO2 excretion.

Doubly labeled water is usually administered at baseline after samples of blood, urine and saliva have been collected (21). Subsequent sample collections occur within 7-21 days after administration, when the isotopes are completely dispersed within body compartments. Isotope-ratio mass spectroscopy is used to measure 2H2 and O18 enrichments in collected samples, which are needed to finally calculate CO2 production. Thereby, TEE can be ascertained under free-living conditions with an error of about 6-8%. This error can be minimized by repeatedly gathering samples over the measurement period rather than relying on only two time points.

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Figure 2. Schematic picture of the doubly labeled water method. CO2, carbon dioxide; 2H2, deuterium; O18, labeled oxygen.

It should be mentioned that the study of TEE classically relied on direct calorimetry (49, 18). Direct calorimetry, or direct measurement of heat loss from a subject, is achieved by using well-controlled environmental chambers. However, this requires participants to be confined to small rooms, which fails to capture the enormous variety of NEAT components in free-living individuals (21, 22).

By contrast, indirect calorimetry is an easily accessible and portable method commonly used to assess BMR, RMR and TEF. Indirect calorimetry is based on the principle that by measuring oxygen consumption (VO2), CO2 excretion (VCO2), or both over a defined time span, the metabolic conversion of fats, carbohydrates, and proteins into energy can be calculated using established formulae (18). The net energy released is typically expressed as kcal or Kilojoule (kJ). The small percentage of physiological protein oxidation can be estimated by nitrogen excretion in urine, or can be neglected without adding a substantial error in subjects who are in stable nitrogen balance (18).

Indirect calorimetry is accordingly one of the most commonly used approaches in clinical investigations, either in controlled metabolic-ward studies or in field settings. For instance, Levine et al. in their study on human NEAT applied indirect calorimetry to measure BMR and TEF (53). To measure TEF they provided participants with a meal containing a third of the subject’s daily weight maintenance energy needs. To estimate TEF over 24 hours, the measured energy expenditure area-under-the-curve obtained when they ate the standardize meal was multiplied by three (16). Alternatively, study protocols may ignore TEF as a non-substantial variable, or simply multiply TEE by 0.1 to yield a crude estimate.

When sensitive techniques are not available, BMR can be alternatively estimated by using validated age-, gender- and population-specific equations. For instance, the Harris-Benedict equation is widely used in clinical settings (18). However, this equation was derived from about 300 healthy, normal-weight Caucasian adults between 1907 and 1917 and its application, therefore, has limitations (18). Substantial error could be introduced by applying it to an inappropriate collective, including subjects who are with obesity, which is also true for other validated equations estimating BMR (18).

The physical activity level (PAL) is frequently used in studies to provide an index of activity when measurement of total NEAT is not possible. It is calculated by expressing TEE relative to BMR (15). Sedentary subjects in industrialized countries typically have a PAL of approximately 1.5 (see Table 1). The PAL rises to values of 2.0-2.4 with strenuous work and under certain conditions it can increase to values up to 3.5-4.5 (22). Using PAL, however, can also introduce significant bias in the assessment of NEAT, as under free-living conditions the cumulative error of PAL measurements can be as high as 7% (21).

Table 1. Physical Activity Levels (PAL) Predicted From Lifestyle (from 16).

Chair or bed bound


Seated work with no option of moving around and little or no strenuous leisure activity

1.4 – 1.5

Seated work with discretion and requirement to move around but little or no strenuous leisure activity

1.6 – 1.7

Standing work (e.g., homemaker, shop assistant)

1.8 – 1.9

Strenuous work or highly active leisure

2.0 – 2.4


Measuring total NEAT or using PAL provides no information regarding the individual components contributing to PEE/AEE. Therefore, the factorial approach is widely used to estimate NEAT constituents. Accordingly, all physical activities of a subject of interest are recorded over a defined time span, typically seven days. The energy equivalent of each activity is determined, and these equivalents are then apportioned according to the time spent by the individual with the respective activities. Finally, the data is totaled to get an estimate of the energy expenditure attributable to NEAT (21, 22). The first step when using the factorial approach is the quantification of a subject’s physical activities. Several methods are available to obtain such information, including questionnaires, interviews, and activity diaries. These approaches might be useful for estimates of particular activities, such as those related to occupational duties. Otherwise, they have substantial limitations, including inadequate or incomplete data recording, alteration of habits during assessment periods, and others (21, 22).

Measuring NEAT is a complex task when intensities of transition movements and fidgeting-like activities are the matter of research interest. By applying a combination of direct calorimetry and motion detectors, Ravussins’s group have shown that there is a considerable inter-person variability of daily energy expenditure, even after adjustment for differences in LBM (29). A large percentage of this variability among individuals was due to variability in the degree of e.g., "fidgeting." Thus, estimation of fidgeting-like activities represents a substantial subcomponent under conditions of NEAT measurement. However, subcomponents such as “fidgeting” might not be captured by many methods. Levine et al. proposed an accelerometer method that could predict 86% of activity related energy expenditure for the posture and locomotion component of NEAT compared with a room calorimeter, but predicted only 50% of the variance in fidgeting and did not discriminate various types of fidgeting (50). Therefore, more technically advanced approaches have been developed. Among these are motion detectors, floor-pressure-pad displacement, cine photography, pedometers, accelerometers, and global positioning systems (GPS) (21, 22). Recently, portable intelligent devices for energy expenditure and activity assessment were used to study subtypes of NEAT by the method of mechanical modeling (54).

However, depending on the degree of sophistication, all these approaches have limitations. While data obtained from triaxial accelerometers typically found in wearable activity monitors is often used as an estimate of activity-related energy expenditure, they only measure actual movements (not energy expended), and a combination of methods probably yields the most appropriate estimates (21, 22). For example, energy costs of single NEAT components are typically measured by means of indirect calorimetry, for which highly sophisticated portable systems are now available. Alternatively, tables with listed energy costs of NEAT activities may be used. This latter method is convenient and inexpensive, but for similar reasons as pointed out for PAL, substantial systematic errors can be introduced (21). Similar problems will arise when NEAT is assessed by multiple linear regression approaches at a population level (55).

Furthermore, principal problems ascend with the NEAT measurement per se, as little validated information is available concerning the time necessary to representatively assess spontaneous physical activity. Many studies report extrapolation of measured results of an individual’s energy expenditure obtained during a limited timeframe to longer intervals. In this regard, experienced investigators believe that measurements of approximately seven days will likely provide a representative assessment regarding a 2-4 month time span (21).

Taken together, the difficulty of getting true estimates under free-living conditions is a major reason why available information on human NEAT physiology is limited. Representative studies are needed that are conducted over appropriate time spans (months) and utilize a combination of the factorial approach and total NEAT measurement to overcome major limitations in current estimates.


There is a close interplay between environmental factors and individual characteristics with respect to NEAT as discussed below.

Effects of Occupation, Gender, and Age

Highly physically-active individuals expend up to three times more energy in 24-hours than subjects with negligible bodily activity (56). As previously discussed, NEAT accounts for a significant proportion of the observed differences at the population level TEE. Thereby, occupation clearly represents the key determinant of NEAT (45). Indeed, NEAT can vary by as much as 2000 Kcal per day when comparing two adults of similar body size, lean body mass, age and gender (Figure 3).

For an average worker spending most of her/his time in a seated position, occupational NEAT is relatively low and associated energy costs range at a maximum of 700 kcal per day (Figure 3). A comparable person working mainly in a standing position can increase their occupational NEAT to up to 1400 kcal per day, while an agricultural occupation would theoretically result in NEAT categories ranging around 2000 kcal per day or more (Figure 3) (45). Thus, occupations relying on intense physical activity can expend 1500 kcal per day more than a sedentary job.

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Figure 3. The effect of occupational intensity on NEAT (adapted from (45)). The data assume a BMR of 1600 kcal per day.

This variation in daily NEAT between individuals and populations becomes even more accentuated when considering research from non-industrialized countries (57, 58). For instance, a study by Levine and colleagues included more than 5000 inhabitants of agricultural regions of the Ivory Coast of Africa (Figure 4).

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Figure 4. Gender- and age-dependent physical activity levels (time engaged in work activities) of people living in agricultural communities of the Ivory Coast in Africa (adapted from reference (58)).

Each subject was studied over seven days and all of their daily tasks were recorded by a trained evaluator (58). Several lines of available evidence regarding NEAT were established from this study. First, a substantial effect of gender was observed as women apparently worked more than men in these societal constructs. Women were responsible for more than 95% of domestic and for an additional 30% of agricultural tasks. Otherwise, men exclusively worked in agricultural occupations and had more leisure time, resulting in substantially lower NEAT than women (45, 58). In contrast, men in Canada, Australia or the United Kingdom are reported to be up to three times more physically active than females, while data from the United States indicate comparable activity levels of genders (59, 60). Even less data is available on NEAT in children, but in general boys seem to be reproducibly more active than girls (61, 62).

A second important observation from the study of Levine et al. is the fact that decreased occupational NEAT was observed with aging (Figure 4) (58). An age-related decline of NEAT had been previously reported across species (63). The underpinnings for this age-effect on NEAT in free-living humans has been reported by Harris et al. (64), who showed that the substantial decrease in elderly as compared to younger subjects was mainly attributable to the ambulation subcomponent of NEAT. Elderly subjects walked less distance despite having a comparable number of daily walking periods compared to their younger counterparts. Indeed, elderly subjects performed 29% less non-exercise activity, corresponding to three miles less ambulation per day (64).

A further factor that should be considered to potentially impact NEAT could be sleep restriction. It was shown that subjects with 5.5 compared to 8.5 hours nighttime sleep opportunity had 31% fewer daily activity counts, spent 24% less time engaged in moderate plus vigorous physical activity and became more sedentary (65). However, the significance of these findings is currently limited to time spans over few weeks and it remains therefore unclear as to whether the results are also valid in terms of common problems, such as chronic sleep insufficiency or shift working. Although, some short-term experiments support the latter findings, conflicting data have also been reported on that matter (66–69). Future research will need to validate these associations and, if true, better understand mechanisms underlying restricted sleep time and alterations in PEE/AEE. Sleep restriction should be nevertheless regarded as potential biasing factor in future research projects on NEAT.

Industrialization and Societal Status

Industrialization and societal status are distinctive factors affecting NEAT. Urban environment and mechanization are associated with a decrease in daily physical activity. For example, it was shown that sales of labor-saving domestic machines (e.g. washing machines) and obesity rates are closely correlated in the U.S. population, while this association was not present with respect to energy intake (20, 22). Furthermore, it is known that poverty and societal aspects comprising neighborhood and educational status are associated with increased consumption of energy-dense foods, which is thought to contribute to higher obesity rates reported in lower socioeconomic groups (70, 71). Highly educated individuals report more leisure-time physical activity and are three times more likely to be physically active as compared to less educated individuals (20, 22).

Seasonal Effects

Seasonal variations also play a role for NEAT. Time spent in activity is twice as likely during the summer as compared to winter months and contributions to activity from agricultural or construction work plays a greater role in the summer season (20).

Together, available data indicate close relationships between environmental conditions and biological aspects of NEAT. Evidence suggests that the contribution of occupational activity to NEAT can vary by 2000 kcal per day. Since the accumulation of excess body fat is a result of positive energy balance, subtle perturbations of energy balance over sustained time periods are theoretically capable of contributing to unwanted weight gain. But once weight stability is again established at a higher weight, energy expenditure and energy intake need to be precisely matched to achieve a long-term persistency of body mass, as an error of 1% would lead to a gain or loss of 1 kg per year or some 40 kg between the ages of 20-60 years. Therefore, small alterations of energy balance by means of NEAT could theoretically result in significant effects with respect to severity of obesity in an individual and the prevalence of obesity in a population. Consequently, when considering how to influence NEAT as a strategy for the treatment or prevention of obesity, a central question arises: In what fashion does NEAT interact with another environmentally-influenced factor involved in energy balance, that of energy intake?

Alteration of NEAT with Varying Energy Availability from Foods

Alteration of energy balance is followed by multiple adaptations, but relatively few studies have addressed modulation of spontaneous physical activity and NEAT by variations in nutritional energy intake. The latter is part of the concept of adaptive thermogenesis (72).

Adaptive thermogenesis refers to changes in REE, EAT and NEAT, which are independent of changes in LBM and are, instead, modulated by other factors, including increased or decreased caloric intake (72). With caloric restriction, both REE and non-resting energy expenditure substantially contribute to changes in adaptive thermogenesis (72). Interestingly, in most studies, alterations in adaptive thermogenesis in response to overfeeding are in large part explained by raised non-resting energy expenditure (e.g., NEAT). However, the individual ability to adapt NEAT to manipulations of energy balance is highly variable between subjects and situations, particularly with respect to overfeeding (39). But if an individual is able to generate a significant upregulation of NEAT in the face of positive energy balance brought about by an increase in food intake, they may be more able to prevent obesity (34).


As discussed above, by using a combination of direct calorimetry and motion detectors, Ravussin et al. have shown that the inter-person variability of TEE is considerable and remains significant even after adjustment for LBM (29). A substantial percentage of this variability is explained by differences in spontaneous physical activity including “fidgeting” (29). Fidgeting-like activities accounted for a range in energy expenditure of 100-800 kcal per day in the subjects. Interestingly, the researchers observed elevated spontaneous physical activity levels in subjects with obesity as compared to those who were lean, indicating a positive relationship with elevated body mass. However, the latter study was performed under isocaloric conditions and over a short observation period. Therefore, the importance of this report was to establish the significance of spontaneous physical activity for energy balance, but whether acute variability in energy intake affects NEAT was not addressed. In their “weight clamping” study, Leibel et al. (73) overfed adults who were lean and overweight/obese by 10%, or approximately with 5000-8000 kcal per day over 4-10 weeks, followed by a weight maintenance period. Body composition, fecal caloric loss, TEE, REE, TEF and spontaneous physical activity were measured using multiple technical approaches including indirect calorimetry, the doubly labeled water method, and activity trackers in a subgroup (73). They showed that maintaining a 10% elevated body mass induced a significant increase of TEE by 9±7 kcal per kg LBM per day in subjects who had not been previously obese and by 8±4 kcal per kg LBM per day in those who had formerly been obese (73). The majority of this rise in TEE was attributable to non-resting energy expenditure. The magnitude of this effect was comparable in those who had previously not been obese and those who had been overweight or obese, indicating similar adaptations to overfeeding conditions (73). However, a related review pointed out the large inter-individual variability regarding the capability to adjust energy expenditure with overfeeding in the “weight clamping” experiments (46) and emphasized that in other human over-feeding studies there is a wide range of individual weight gain per unit of excessively consumed energy. Therefore, it can be proposed that some individuals show a remarkable capacity to increase energy expenditure in response to overfeeding, while others do not (46). It was further suggested that spontaneous physical activity, and accordingly NEAT, likely play an important role regarding the impact on body weight under such conditions.

Levine’s group was the first to systematically investigate the effect of overfeeding on the individual ability to adapt NEAT in free-living subjects (53). Using sophisticated methods and measuring NEAT over a representative time span, the authors overfed 16 volunteers (12 males, 4 females; age ranging from 25-36 years) by 1000 kcal per day over their weight maintenance requirements (20% of the calories came from protein, 40% from fat, 40% from carbohydrates). The energy surplus was paralleled by a mean TEE increment of 554 kcal per day. In this study 14% of this TEE increase was attributable to a rise in REE, approximating by 79 kcal per day. A further 25% of the TEE increment (136 kcal per day) corresponded to an increase in TEF, probably due to the relatively high percentage of protein intake. The most prominent effect was, however, attributable to enhanced physical activity thermogenesis, corresponding to around 336 kcal per day. As volitional exercise of the study subjects remained at a constant low level and since the authors did not detect changes in exercise efficiency, they concluded that about 60% of the increase in TEE due to overfeeding was attributable to NEAT (53). Again, however, the change in NEAT varied remarkably between subjects, ranging from -98 to +692 kcal per day. The maximal individual increase of NEAT constituted 69% of the excessively consumed 1000 kcal per day in this study (74). Moreover, the change in NEAT was directly predictive of the individual vulnerability or resistance to body fat accumulation (53).

A recent randomized study enrolled young aged female and male participants with variable BMI (19-30 kg/m2) and used gold standard methods to investigate not just the effect of caloric overfeeding, but also of protein intake on PEE/AEE (75). 140% of caloric needs including 5, 15, or 25% of energy from protein was fed for 56 days. Caloric overfeeding resulted in increased physical activity and correspondingly PEE/AEE including NEAT, even after adjustment for changes in body composition. By contrast, changes in physical activity were not related to protein intake. The authors concluded that increased PEE/AEE in response to weight gain might be one mechanism of modulating adaptive thermogenesis (75). But the results indicate also that the observed increase in activity was not likely effective at attenuating weight gain.

Other recent reports have failed to detect compensatory increases in spontaneous physical activity during short-term overfeeding in humans (76–80). While in several cases, methodological differences make them less directly comparable to Levine’s study, (76, 56, 77, 79), a study by Siervo et al. used state-of-the-art methods and applied a highly standardized 17-week protocol with progressive overfeeding from 20-60% energy in excess of maintenance needs (81) in lean, healthy men. Three sequential intervals of stepwise overfeeding were each separated by one week of ad libitum energy intake. In agreement with previous findings, between-subject variability concerning weight change during ad libitum phases was high (63). However, in contrast to Levine’s study (53), Siervo et al. failed to detect a significant systematic change in spontaneous physical activity (81). The authors therefore concluded that systematic elevations in energy intake induce very limited counter-regulatory responses in energy expenditure (63).

In summary, these data underscore the highly individual compensatory response regarding adaptive thermogenesis to overfeeding. From Levine and Apolzan et al., supportive evidence is provided for the susceptibility to gain body mass under standardized overfeeding conditions and identifies an individual’s ability to adapt NEAT as a potential modulator (75, 53, 20, 34). In accordance with findings of other overfeeding studies (reviewed in (74)), adaptations in thermogenesis by changes in NEAT observed between individuals may explain why some individuals are particularly susceptible or resistant to weight gain. The potential impact of age on these adaptational mechanisms remains unclear and should be addressed by future research.


While evidence with respect to systematic NEAT upregulation under overfeeding conditions remains somewhat controversial, TEE and spontaneous physical activity are consistently influenced with energy deprivation in the majority of available studies (72, 80). This is of importance as most subjects who are overweight or have obesity try to lose body mass through various calorie-restricted dietary strategies.

The first study to quantitatively investigate adaptive thermogenesis under conditions of food restriction was the Minnesota Starvation Experiment (reviewed in (46, 49, 72)). This evaluation showed a marked reduction in TEE that was later reproduced by numerous studies, independent of pre-starvation BMI or method used to induce weight loss (reviewed in (72)). For instance, Leibel et al. in a second arm of their “weight clamping” experiment evaluated the effect of a 10% body mass reduction, followed by weight maintenance at this level (73). While overfeeding was accompanied by a 16% increase of adjusted TEE, weight reduction from underfeeding induced a 15% decrement (57). As compared to overfeeding, it is well accepted that both REE and non-resting energy expenditure are affected by energy deprivation in humans and animal models (82, 72, 80).

These findings are supported by results from the so-called Biosphere 2 experiment, where the authors demonstrated a major reduction of TEE and spontaneous physical activity with body mass reduction over a two-year time period, which persisted after six months of body weight regain (46, 83). Demonstrated TEE reductions by adaptive thermogenesis in weight-regainers has been reproduced in other human studies (72). Of note, when subjects recover from starvation they spontaneously overeat and, moreover, if weight loss-induced adaptive thermogenesis persists (e.g. reduced NEAT), there is a significant risk of regaining weight and “overshooting” pre-starvation body mass (72). Thus, changes in non-resting energy expenditure with underfeeding (or overfeeding) appear to occur in a direction tending to return subjects to their initial body mass and to conserve body energy as recently described in humans as so-called “thrifty phenotype” compared to a “spendthrift phenotype” (84).

Regarding this, findings from a recent study in sedentary overweight females are of potential practical relevance (85). In this investigation, 140 pre-menopausal females reduced their body mass by an average of ~11 kg while undergoing a total of 800 kcal per day diet. Subjects were randomly assigned to one of three groups: no exercise, a structured aerobic training schedule, or an exercise program incorporating resistance training. Body composition, strength, TEE, REE and NEAT were measured using various approaches. One main finding was that TEE, REE and NEAT all declined with weight loss in the no-exercise group. Non-exercise activity thermogenesis was reduced by 150 kcal per day, which was equivalent to 27% compared to baseline levels. However, in subjects undergoing the exercise regimens only REE was reduced, while NEAT remained unaffected. Thus, since caloric deprivation appears to consistently decrease spontaneous physical activity, untrained subjects with obesity who plan to lose body mass by means of dietary intervention alone would be advised to engage in a concomitant exercise program so as to avoid negative effects on NEAT and mitigate weight cycling after reaching a lower body weight. Of note, the authors of this study emphasized that, consistent with the concept that “more is not always better,” exercising two days a week was capable of increasing NEAT but, paradoxically, an intense three days weekly schedule was followed by a substantial decrement in NEAT (85).

At this end, one further study of Levine’s group should be considered (78). NEAT as measured by gold standard methods and posture allocation was evaluated in mildly obese subjects and normal weight controls over a period of ten days. This baseline evaluation showed a 164 minute per day longer seated time in obese compared to lean subjects. A subset of eight obese subjects then underwent an intentional weight loss program over a time span of eight weeks and thereby lost significant body weight. Posture allocation was measured for another ten days after intervention. Interestingly, the weight losing obese subjects did not further reduce their NEAT in this experiment (78). The latter study has several limitations, e.g., the small number of enrolled subjects and the relatively short intervention period when compared to some of the above-mentioned trials. Levine and colleagues themselves emphasized that it was a pilot experiment (78). The data otherwise suggest that interindividual differences in posture allocation could at least in part be biologically determined and are therefore not necessarily influenced in every individual by e.g., reduced energy intake over short time periods. Over- and underfeeding studies in identical twins indeed suggest an important role of genetics regarding the variability underlying body mass regulation (86, 87). Specifically regarding NEAT, it is likely that due to the polygenetic nature of obesity, numerous pathways are involved in the regulation of spontaneous physical activity, as the latter has multiple environmental cues and affects a multitude of behaviors (8, 88). Zurlo et al. have shown that spontaneous physical activity levels cluster in families and could prospectively help to explain the propensity for weight gain (89). Moreover, according to a recent review up to 57% of the variability of spontaneous activity has been attributed to inheritance (90).

Otherwise, biological and genetic determinants can only partially explain NEAT variance. Models of human energy budgets related to exercise can provide a conceptual framework to better understand NEAT regulation in humans (reviewed in (39)). For instance, the so-called independent model predicts that changes in basal TEE have no impact on the energy budgeted for behavior, e.g., spontaneous physical activity. This model is analogous to the factorial model of exercise in humans that assumes that exercise has an additive effect on TEE (39). In contrast, the allocation model suggests that the total energy budget is constrained, and therefore, an increase in the energy cost related to maintaining basal functions will reduce the amount of available energy to support other functions, finally altering an individual’s behavior (39). The independent model would for example predict that exercise additively increases TEE, while the allocation model predicts that exercise leads to a reduction in some components, e.g., NEAT. Indeed there is evidence that each of these models is evident in different human populations, although studies suggest that e.g. allocation may be affected by confounders such as age (older more likely to reallocate), sex (males more likely to reallocate), and exercise volume (allocation more likely with higher volumes) (39). Such aspects have to be considered and could help to integratively explain the contradictory findings of Levine et al. and from other researchers.

In summary, an integrative view of existing human overfeeding studies proposes that the magnitude of adaptations regarding energy expenditure varies largely between individuals. Subjects capable of responding with increased spontaneous physical activity to overfeeding can be categorized as “compensators” and are therefore less susceptible to obesity (“spendthrift phenotype”). Subjects unable to respond to a continued energy surplus with increased NEAT (e.g., “non-compensators” or “thrifty phenotype”) seemingly represent the majority of subjects on a population level, of which a major percentage will attempt dietary weight-loss strategies at some time in their lives. However, REE and non-resting energy expenditure including NEAT are reduced with underfeeding in the majority of subjects. They therefore represent a risk factor for weight regain after dietary intervention. Mild voluntary exercise during and maybe shortly after periods of dietary weight loss could be a promising strategy to avoid decreases in NEAT, although this alone will probably not fully compensate for the obligate decline in REE. Whether the majority of dieting subjects with obesity will continuously adhere to a structured exercise program during extended periods of weight maintenance as suggested by current recommendations is also uncertain (and unlikely) (42, 8, 43, 41).

Therefore, to advance anti-obesity strategies that systematically increase or even conserve NEAT it is essential to understand the mechanisms contributing to regulation of spontaneous physical activity.


Principles of NEAT Regulation

The ability to adapt thermogenesis, and specifically NEAT, presupposes existing mechanisms in the body that sense, accumulate, and integrate internal and external directionality signals with respect to energy balance (20). Figure 5shows a proposed model of the principal regulation of spontaneous physical activity and correspondingly NEAT. In this model, physiological data regarding caloric intake, energy depots and energy expenditure are compiled centrally by the brain. These signals converge at the data acquisition center, are rectified to a common signal, and directed to a NEAT accumulator, which is constantly summing the net amount of NEAT per unit of time. The latter continuously refers to the energy balance integrator, which, in turn, modulates spontaneous physical activity in response to changes in energy intake. Thereby, energy balance resulting from these three components is in constant flux.

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Figure 5. The NEAT hypothesis of energy balance (adapted from (20)).

Continuous arrows represent energy flow through the system, broken arrows stand for putative signaling pathways. BMR, basal metabolic rate; NEAT, non-exercise activity thermogenesis; TEF, thermic effect of food.

For example, under circumstances of positive energy balance during increased food intake, the energy balance integrator may mediate deposition of the caloric surplus in energy stores (adipose tissue) or alternatively dissipate the excess energy in the form of NEAT. More likely, since BMR is essentially fixed, some energy will be spent on food digestion and absorption (increased TEF), some will be deposited as body fat or in other depots, and the rest will be dissipated as NEAT (20). This model also supports the observation that although BMI and energy balance are continuously oscillating around a weight set point (or “set range”) under daily life conditions, body mass is remarkably stable in the long term. This is exemplified by data from the Framingham Study showing a mean body mass increase of only 10% over a 20 year time span in average adults (cited in (91, 92)).

Thus, it appears that the regulation of energy balance and body mass is realized by a complex network of precisely working auto-regulatory structures controlling effector systems, one of which being spontaneous physical activity. However, while ample available data detail the regulation of food intake, there is lack of information concerning the biological mechanisms driving NEAT (34). Most animal evidence supports the concept that NEAT can help to protect against obesity (93). Therefore, spontaneous physical activity has certainly potential for impacting body mass and energy balance. It is very likely that many of the biological systems involved in the regulation of energy intake are also involved in gathering and integrating information regarding NEAT and determining NEAT adaptation in humans (34).

Tissues, Organs, and Central Mechanisms Involved in NEAT Regulation

Several well-described central neuroendocrine systems are thought to regulate NEAT through interactions with peripheral tissues/organs known to affect energy balance (Figure 6).

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Figure 6. Model for the neuroendocrine regulation of NEAT in the service of energy balance (adapted from (34)). Multiple external and internal signals are sensed and integrated, whereby defined brain structures (e.g., arcuate nucleus of the hypothalamus, area postrema and nucleus of the solitary tract of the hindbrain, dopamine pathway of the mesolimbic system) interpret a multitude of sensory cues of energy availability. The involved brain systems have multiple ascending and descending projections affecting the amount of physical activity through arousal and limbic pathways, and descending neural projections and endocrine signals to modulate the energy efficiency of physical activity. Thereby, the central nervous system could adapt NEAT to adjust energy balance under conditions of caloric excess or starvation. Ach, acetylcholine; AgRP, agouti-related peptide; CART, cocaine- and amphetamine-regulated transcript; CCK, cholecystokinin; LC, locus ceruleus; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; (P)SNS, (para-)sympathetic nervous system; SNS, sympathetic nervous system; TMN, tuberomammilary nucleus; VP, ventral pallidum

These associated neural mechanisms of brain regions involve a multitude of neurotransmitters and neuropeptides (for extensive review see (34) and (94)). While all are important, this section will focus on the biological role of central orexin peptides as promising and potentially targeted regulators of spontaneous physical activity (for extensive review see (47)).

Experimental data indicate that injections of orexin A into defined brain regions reproducibly induce a dose dependent increase in spontaneous physical activity along with significant increments in NEAT. Moreover, current knowledge suggests a role for the orexin system in the control of arousal and sleep, and for reward and stress reactions. This has led to conduction of extensive research showing that the orexins A and B are produced by cleavage from a single pro-peptide (44). The majority of orexin A is synthesized in the lateral hypothalamus and perifornical area. Orexin A binds to two G protein-coupled receptors, namely orexin receptor type 1 (OXR1) and 2 (OXR2). OXR1 has high affinity for orexin A, while OXR2 has equal affinity for the A and B peptide. Receptor binding of orexin A is associated with increased intracellular calcium levels and followed by enhanced neuronal signaling (44).

Physiologically, orexin A is capable of modulating spontaneous physical activity, food intake, and sleep as evidenced by an obesity-resistant rat model with high orexin A activity (34). These animals show more ambulatory and vertical movement, independent of age or food availability (34). Intriguingly, this rat model exhibits lower body weight gain when fed a high-fat diet, despite consuming significantly more kcal per gram body mass. From this study, it appears that orexin A simultaneously enhances feeding behavior and induces physical activity, but the consumed calories are outweighed by those expended with spontaneous physical activity (47, 34). These data were confirmed by a mouse model with postnatal loss of orexin neurons (47). These mice exhibit hypophagia, decreased spontaneous physical activity, and develop spontaneous-onset obesity while consuming a regular chow-diet. Furthermore, in recent years so called DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) have been developed and were applied to manipulate orexin neuron activity in specific animal models. Compared with previous work of targeted infusions of orexin and/or orexin agonists into the brain, the use of DREADDs allows for the sustained activation of the neurons of interest without needing to inject directly into the brain (95).

A high-fat diet model was used to evaluate the effect of DREADDs on NEAT, since spontaneous physical activity can be decreased under such circumstances. It was shown that NEAT was decreased in animals on the high-fat diet, and was increased to the NEAT level of control animals following activation of orexin neurons with DREADDs (95). Thus, these preliminary results clearly support the hypothesis that the orexin signaling system could be a promising target of modulation by drugs.

However, from the perspective of humans, data on orexin are currently scarce. Otherwise it was recently shown in a pilot study that plasma levels of orexin A correlated with physical activity levels including many NEAT features (96). Therefore, plasma orexin A could be considered as a biomarker of NEAT in future studies. Furthermore, the orexin system has been shown to be defective in the sleep disorder narcolepsy. Since obesity is a recognized comorbidity of narcolepsy in both animal models and humans (47), regulation of this signaling system has the potential to impact clinical endpoints, probably including the development of overweight and obesity.

The Role of Peripheral Tissues for NEAT Regulation: Adipose Tissue and Adipokines

Peripheral tissues are also believed to contribute to NEAT regulation (Figure 6). That NEAT can be influenced by over- and underfeeding has been extensively discussed in earlier sections; but it is essential to also reconsider that longitudinal studies of fasting humans show significant decreases in adaptive thermogenesis with starvation, while refeeding may induce a variable elevation (72). These adaptations are likely the result of feedback mechanisms between thermogenesis regulation and energy depots (Figures 5 and 6). It can be further hypothesized that adaptations of thermogenesis in response to fasting or overfeeding result, at least in part, from an adipose tissue-specific impact on thermogenesis (reviewed in (46)). Indeed, studies of white adipose tissue (WAT) have led to the recognition that this energy store represents an important endocrine organ communicating with the brain through secretion of so-called “adipokines.” Accordingly, adipokines could play a role for NEAT regulation (Figure 7) (97).

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Figure 7. Essential elements in the regulation of thermogenesis including effects of WAT (adapted from (34, 98)). BAT, brown adipose tissue; cAMP, cyclic 5” adenosine monophosphate; COX, cyclooxygenase; IL, interleukin; LPS, lipopolysaccharide; PAMPs, pathogen associated molecular pattern; PGE, prostaglandin E; PKA, protein kinase A; SNS, sympathetic nervous system; UCP, uncoupling protein; WAT, white adipose tissue.

Leptin is such an adipokine signal that is the product of the rodent obese (ob) gene and human homolog (LEP) first characterized in 1995 (99). Due to its role regarding the sensing of energy stores and regulation of food intake, it was also suggested as an important signal with respect to adaptive thermogenesis (reviewed in (34, 98)). For instance, Dauncey and Brown studied ob⁄ob mice lacking leptin production and their lean littermates at comparable body weights (100). Leptin-deficient mice expended less energy and showed less motor activity as compared to control animals. The energy expended relative to metabolic size was greater in lean littermates, who were also more active than the ob⁄ob mice. It was calculated that activity-related energy expenditure accounted for at least part of the body mass difference observed between ob⁄ob and wild-type mice (100, 101). A central mediator of leptin action on NEAT partly includes the orexin signaling system, since the activity of orexin neurons has been shown to be modulated by leptin among other metabolic indicators (reviewed in (93)).

With respect to humans, Franks et al. have shown that fasting leptin levels were significantly associated with physical activity-related energy expenditure (101). Moreover, it was pointed out in a recent review that replacement of leptin in weight-reduced subjects was capable of reversing neuroendocrine adaptations that accompany caloric reduction as evidenced by an increase in SNS activity, suggesting that this component of the non-resting energy expenditure and adaptive thermogenesis is at least in part under endocrine control (72). In response to caloric restriction, the authors therefore suggest an adaptation model where in an initial fast “first set,” reduced insulin secretion and glycogen stores are accompanied by a reduction of both REE and non-resting energy expenditure within days, while in the long term, with maintenance of reduced body weight, a “second set” mediated by a drop in leptin levels becomes active that keeps energy expenditure low so as to preserve fat depots and maintain reproductive function (72). This endocrine action resulting from the drop in leptin levels with related activation of multiple pathways then enhances the risk of weight regain (72).

Overall, available data suggest that leptin’s central actions include not only reducing food intake but potentially also increasing energy expenditure mediated, in part, through increasing physical activity levels (reviewed in (34)). The potential role of leptin on NEAT in human obesity is of particular interest given the phenomenon of central leptin insensitivity under conditions of overfeeding and related obesity (102, 103). However, it must be also kept in mind that the leptin system represents only one component of peripheral tissue signaling that impacts NEAT. Other endocrine systems, such as thyroid hormone status, also have major influences on energy expenditure. For example, hyperthyroid rats have been shown to have significantly increased NEAT (104).

Energy availability from food and internal energy stores, ambient and body temperature, and systemic inflammation are further determinants of thermogenesis regulation. In WAT, two relevant factors converge: WAT represents the principal energy storage depot and, under conditions of significant obesity, WAT is a site of low-grade inflammatory activity that contributes to systemic inflammation. Recent epidemiological data from humans support this hypothesis (105). It was shown that levels of systemic low-grade inflammation (e.g., circulating interleukin-6 and interleukin-1β) were related to non-exercise physical activity. Although no causal relationship can be drawn from these findings, inflammation is theoretically one signaling system involved in NEAT regulation.

Signals from WAT can induce enhanced sympathetic nervous system (SNS) activity and affect downstream regulators of adaptive thermogenesis (Figure 7). Indeed, experimental data indicate that NEAT is one component affected by WAT signaling (93). Related to the hypothesis of “adaptive thermogenesis”, Dulloo et al. in their review regarding human starvation and overfeeding suggest SNS as key regulatory effector mechanism (46). Of note, activation of brown adipose tissue, which has recently gained a lot of interest with respect to human obesity (106–108), is suggested to be an additional site of adaptive thermogenesis.

Skeletal Muscle as Peripheral NEAT Modulator

Skeletal muscle is a major contributor not only to REE, but also to spontaneous physical activity and has been newly recognized as important NEAT effector. Human subjects maintaining a 10% weight loss in response to caloric restriction have increased skeletal muscle work efficiency (92, 109). This mechanism is believed to be responsible for the phenomenon that, even after adjustment for the reduced body mass, weight-loss maintaining subjects need proportionally fewer calories for performing comparable physical-activity tasks compared to before weight loss (92, 109). Therefore, adaptations of skeletal muscle work efficiency can contribute to reduced NEAT after low-calorie induced weight loss. Several recent animal models are in line with this hypothesis (82, 110). For example, a recent study in rats investigated the contribution of REE and non-resting energy expenditure to reduced TEE observed after three weeks of 50% energy deprivation (82). They reported a 42% reduction in TEE, even after correction for the loss in body weight and LBM (82). Moreover, 48% of the detected TEE reduction was explained by a significant decline in non-resting energy expenditure, e.g. spontaneous physical activity (82). Reduced baseline and activity-related muscle thermogenesis was also found in this study. Reduced skeletal muscle norepinephrine turnover as a surrogate of SNS drive and increased expression of the more energy “economic” myosin heavy chain (MHC) 1 isoform were identified potential mechanisms for the observed changes (82, 92, 109). Thus, according to these findings, restriction of caloric intake apparently not only suppresses spontaneous physical activity, but in addition reduces related caloric demand, which is mediated by reduced SNS activity to skeletal muscle and altered expression of MHC isoforms. These findings are supported by data from another recent animal study investigating rodents with “high intrinsic physical activity” and counterparts with “low intrinsic running capacity” under eucaloric conditions (87). It was found that animals with high intrinsic physical activity were lean, had lower skeletal muscle economy along with increased skeletal muscle heat dissipation during activity. This resulted in higher TEE and NEAT and was related to increased activation of skeletal muscle by SNS (110).

Taken together, skeletal muscle is an intrinsic site for NEAT and not only of great importance quantitatively, but also represents an important effector system for NEAT mediated through modifications on the level of SNS activity and gene expression, particularly under conditions of altered caloric intake. Therefore, evidence indicates that NEAT is modified by a complex network regulation of central and peripheral systems. The latter involve a variety of redundantly organized immediate and delayed mechanisms, including central neuropeptides and signaling systems to and from peripheral tissues. Involvement of such biochemical signaling systems identifies spontaneous physical activity and NEAT as a potential site for pharmaceutical targeting. Pharmaceutical manipulation of such a system could be envisioned to either increase the amounts of activities people undertake, or elevate activity-related energy expenditure.

As stated in the introduction, obesity is associated with a many adverse health outcomes (1, 111). Energy expenditure in occupational activities has declined by a mean of 140 kcal/d since 1960 in the United States and this reduction is thought to account for a significant portion of the observed increase in mean BMI (reviewed in (1)).The latter hypothesis is supported by observational data showing that reduced energy expenditure of 100 kcal/d below expected values corresponds to a 0.2 kg/year weight gain, of which 0.1 kg/year are fat mass (112). In addition there is ample evidence from epidemiological, cross-sectional, and longitudinal studies showing that individuals with higher physical activity levels have lower mean body weight, gain less weight and fat mass over time, and show reduced weight regain after intended loss in body mass (113–117). Moreover, regular physical activity can moderate or even eliminate weight gain among those carrying a risk at the FTO (fat mass and obesity associated) gene variants ((118), reviewed in (93)). It has further been evidenced that levels of habitual or spontaneous physical activity are positively related to reduced risk of major cardiovascular disease and premature mortality, while an increase of sitting time correlates with a raise in complications (119–123).

Associations of NEAT with Type 2 Diabetes Mellitus and NAFLD/MAFLD as Common Sequelae of Obesity

In industrialized countries the daily time spent sitting is estimated to be 6-7 hours per day, which is closely correlated not only with obesity rates but also with the incidence of T2D (120, 124). A plethora of data suggest, on the other hand, beneficial effects of lifestyle interventions on insulin resistance, metabolic control, pain and clinical endpoints in these patients (e.g. (125, 126), reviewed in (127)). In subjects with impaired glucose tolerance or prediabetes, lifestyle interventions have proven to decrease the incidence of T2D, of which prominent examples comprise the DaQuing Study, the Finish Diabetes Prevention Study, the Diabetes Prevention Program and others (128–131). This was newly confirmed by the lifestyle intervention and impaired glucose tolerance Maastricht (SLIM) trial, which has shown to impact clinical endpoints even four years after stopping the intervention (132, 133).

The prevalence of NAFLD/MAFLD parallels the pandemic rise in T2D. A recent meta-analysis indicates that on a global perspective more than 55% of T2D patients suffer from bland liver steatosis, while a further 37% show signs of non-alcoholic steatohepatitis (NASH) (reviewed in (127)). “NAFLD” was defined in the 1980s to describe excess hepatocellular lipid accumulation in absence of significant alcohol intake, autoimmune, or viral liver disease, and the course of this pathology was long believed to follow the so-called “two-hit hypothesis” (134). Manifestation of bland steatosis was defined as first hit, while histological signs of liver inflammation, fibrosis and hepatocyte injury were proposed as succeeding second hit (NASH). Insulin resistance has now been recognized as the most valid predictive parameter of NAFLD/MAFLD progression to NASH. Moreover, presence of insulin resistance puts these patients to an elevated risk of morbidity and mortality, while sedentary behavior constitutes a complementary risk factor and correlates with clinical outcomes (reviewed in (135, 127)).

Since physical exercise is capable of improving and maybe reversing insulin resistance, while short term decreases in physical activity reduce multiorgan insulin-sensitivity and in parallel increase liver fat (reviewed in (127)), it is reasonable to assume that physical activity represents a potent treatment modality for NAFLD/MAFLD. There is indeed some evidence from prospective controlled randomized intervention studies that liver fat can be reduced independently from changes in body weight by structured exercise regimens in a limited, yet significant manner, while effects on liver inflammation and fibrosis remain to be investigated (extensively reviewed in (127)). Although no direct research of NEAT effects on NAFLD/MAFLD is currently available, one well-designed study using brisk walking as an intervention has shown reduced liver fat after 6 and 12 months (136). This suggests that low to moderate intensity activities potentially impact hepatic steatosis, which should be further evaluated.

For weight maintenance purposes, current recommendations encourage subjects with obesity to engage in 200-300 minutes per week in activities as e.g. walking (8). Yet, patients with T2D show markedly reduced TEE (< 300 kcal per day), number of steps taken (<1500 per day), physical activity duration (<130 min per day), and activity related energy expenditure (<300 kcal per day) as compared to subjects without diabetes (137). Accordingly, only 28% of patients with T2D in the U.S. achieve the recommended physical activity levels and less than 40% engage in regular voluntary exercise (120, 138, 139). In line with this, recent observational studies have shown that reduced NEAT is related to measures of insulin resistance, glycated hemoglobin A1c, and other features of the metabolic syndrome, consistent with the hypothesis that increasing spontaneous physical activity could be beneficial for those with T2D and NAFLD/MAFLD (140–142). This is supported by data coming from the Nurses’ Health Study showing that regular walking at normal pace (e.g. 3.2-4.8 km h-1) was associated with a 20-30% relative risk reduction of T2D, while in another study, frequent walking was correlated with an equally remarkable risk reduction of mortality in T2D (120, 143, 144).

On the other hand, it must be acknowledged that data on NEAT, T2D and NAFLD/MAFLD are limited. First, evidence suggests interindividual differences in the response to a standardized exercise schedule at a given dose and there is, moreover, strong indication of genetic components to the variation in human trainability (reviwed in (145)). Second, available evidence almost exclusively comes from observational and cross-sectional studies. Furthermore, the data on lifestyle interventions on sequalae of insulin resistance and manifest T2D also have limitations (146). For instance the 10-year prospective randomized LookAHEAD study in T2D patients was stopped early based on a futility analysis, since the trial was unable to detect substantial effects on cardiovascular events, although the intervention group experienced significant weight loss (147). Otherwise, secondary analyses of this hallmark study were able to show that the magnitude of weight loss may be predictive of outcome measures (148). In more detail, obese and overweight subjects suffering from T2D were examined regarding the association of the magnitude of fitness change (n=4406 and weight loss (n=4834) over a median of 10 years of follow-up for the primary outcome. The primary outcome was a composite of death, cardiovascular disease, myocardial infarction, hospitalization for angina, and stroke. Subjects losing more than 10% of body mass in the first year of the intervention had a 21% lower risk of the primary outcome relative to participants with stable body mass or weight gain. Interestingly, achieving a > 2 metabolic equivalents (MET) fitness change was not significantly associated with the primary, but with the secondary endpoint (composite of coronary–artery bypass grafting, carotid endartectomy, percutaneous coronary intervention, hospitalization for congestive heart failure, peripheral vascular disease, or total mortality (148). Therefore, it appears that life style modifications are principally capable of beneficially modulating clinical endpoints in, while the optimal exercise (and lifestyle intervention) therapy for individuals with T2D, particularly with co-existing complications, remains unknown (120). These findings suggest that priority should be made for subjects who are obese to engage in any type of regular physical activity before T2D and NAFLD/MAFLD become manifest ((144), reviewed in (120, 8)). Accordingly, the “Step It Up! (The Surgeon General’s Call to Action to Promote Walking and Walkable Communities)” program was recently released, focusing on promoting optimal health before disease occurs (149).

In summary, since EAT is negligible in the vast majority of subjects and NEAT represents the main contributor to daily PEE/AEE and therefore TEE in Western populations NEAT can be recognized as a major potential target for lifestyle modification in subjects suffering from T2D and probably also under conditions of NAFLD/MAFLD.

Personalized Approaches versus Environmental Re-engineering for Promoting NEAT

In free-living individuals, counterbalancing effects of an obesity-promoting environment involves addressing several central questions:

  • How can the amount of NEAT be increased or preserved under conditions of caloric restriction?
  • What are realistic goals for daily spontaneous physical activity and what are appropriate activities for increasing NEAT?

When aiming to prevent or treat obesity, it can be argued that in an obesogenic environment each person needs to increase physical activity levels (45). Alternatively, it could be argued that the epidemic of obesity needs to be addressed at the population level, since obesity has emerged as a result of environmental pressures that have led to decreased population-wide activity levels (e.g., more sedentary jobs, more time at work and less in leisure) (45). In reality, these contributors are not mutually exclusive. However, in order to substantially increase NEAT it is useful to categorize these perspectives as the “individualized approach” versus the “environmental re-engineering approach”(45). Accordingly, Levine and Kotz have developed the “egocentric” and the “geocentric” models, which provide a theoretical framework to understand important environmental determinants of NEAT from these two perspectives (20).The egocentric model focuses on a single person. Accordingly, environmental factors that impact a particular person’s spontaneous physical activity levels are considered, such as “my occupation”, “my transportation to work”, or “my leisure time activities” (Figure 8) (20). By contrast, the geocentric model is focused on how the environment impacts NEAT of multiple subjects, such as city planning to ensure walk-friendly or bike-accessible environments (Figure 8) (20). These models may help to elucidate how NEAT can be effectively modulated.

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Figure 8. Environmental determinants of NEAT – the egocentric versus the geocentric model (adapted from (20)).

In the geocentric model a variety of environmental factors impact spontaneous physical activity. The most prominent negative influences on NEAT are represented by urbanization and mechanization, which are largely a phenomenon of high- and middle-income countries. Examples include televisions, drive-through restaurants, clothes washing machines, motorized walkways, and others (20).

Accordingly, when comparing daily energetic costs of mechanized tasks with the same tasks performed manually a century ago, the difference in daily energy costs approximates 111 kcal, or more than 40,000 kcal per year (150). Evidence such as this has been used in support of the proposal that urbanization and mechanization have likely had a dramatic impact on energy balance (20). Hence, proposals to increase NEAT on a population level have included environmental “re-engineering,” including provisions for adequate walkways, to build schools within walking distance, and to adapt employment laws for promoting office fitness. The problem with this argument is that it includes imposing high economic costs on business and localities without agreed upon standards for success (45).

It is self-evident that to intentionally increase NEAT over a protracted time period presupposes a significant amount of self-discipline. To facilitate behavior modification Levine has published approaches for promoting NEAT that focus on behavioral economic theory (for extensive review see (45)). Behavioral economic theory is a framework for conceptualizing how people make behavioral choices based upon their perceived relative value. When applied to NEAT, behavioral economic theory is concerned with how people choose between various activity/inactivity options. Consequently, Levine proposes four key elements (45): 1) It is critical to provide individuals with free-choice. By contrast, forcing a subject to choose a specific NEAT-promoting activity is likely to have the opposite to the intended effect. Otherwise, if an activity is self-selected, it is likely to be more reinforcing and consequently self-selected more often; 2) The delay between performing a NEAT-promoting behavior and the outcome needs to be minimized. While sedentary behaviors that people enjoy have immediately reinforcing consequences, health benefits of standing or ambulation may take longer to manifest. Therefore, it is important to choose NEAT-promoting physical activities; that are pleasing. For example, walking while listening to music or walk-and-talk with a friend. 3) Behavioral “costs” determine which sorts of activity/inactivity become selected. If a person has to work strenuously to participate in a given activity, they will be less likely to do it. For example, it is unlikely that many people will drive 40 minutes to the gym long-term just to engage in fitness training. People are more likely to choose physical activities that are easily accessible, such as home- or even office-based activities that do not require changing location or clothes; and 4) For an individual to choose a NEAT-promoting activity, it has to be more attractive than available alternatives. For example, behavior will change when providing a competing behavior that is more valued: a given person may prefer to surf the internet while seated rather than visiting the gym for a fitness workout. If, however, walk-and-talk with a friend was an option, the individual could choose that instead of internet-surfing. These behavioral components have been synthesized into a simplified approach termed, STRIPE (45): STRIPE is an acronym that represents S = Select a NEAT-activity that is enjoyed and start it; T = targeted, specific individual goals must be defined; R = rewards need to be identified for reaching the defined goals; I = identify barriers and remove them; P = plan NEAT-activity sessions; E = evaluate adherence and efficacy.

Overall, to increase NEAT in the prevention and treatment of obesity, egocentric and geocentric approaches should be considered. Occupation and leisure time are the two principal time frames that have to be targeted for promoting individual NEAT. To increase NEAT by means of changing behavior, the STRIPE approach is considered as safe and well-grounded in conceptual evidence. Unfortunately, there is no evidence as to whether an individualized or the population-based approach is more effective in terms of increasing physical activity levels and/or to affect body mass. Despite this, however, it is our opinion that both approaches should be still considered when counseling patients regarding ways they can improve their daily life conditions (45).

Increasing NEAT: Realistic Goals, Pitfalls and Appropriate Activities

Obesity is not a problem in only one component of energy balance and food restriction alone is surely not the complete answer on a long-term perspective. Hill et al. hypothesized that a person who is physically highly active could maintain energy balance and a healthy body weight by eating and expending at, say, an estimated 3000 kcal per day. This person, if adopting a sedentary lifestyle, could alternatively maintain energy balance and consequently body weight by eating and expending 2000 kcal per day. If, however, this sedentary person fails to strictly keep energy intake at 2000 kcal per day to match reduced energy expenditure over time, experiencing weight gain would be the unavoidable consequence (16). In other words, for a majority of subjects in a given population, maintaining energy balance at substantial higher levels by increasing NEAT could be in the long term much more realistic under free-living conditions (e.g. in an environment with high access to energy dense foods), than maintaining caloric intake at lower levels (16). Therefore, when attempting to increase NEAT, it is important to establish what goals have to be addressed for prevention or treatment of obesity. According to a review, the amount of physical activity necessary for weight loss approximates 2000-2500 kcal per week or about 2 ½ hours of additional daily ambulation, which is considered realistic for a majority of subjects with obesity (45). This is in line with current recommendations, suggesting ≥ 150 minutes per week of aerobic exercise in combination with a hypocaloric diet (8).

However, evidence is lacking from randomized, controlled studies as to whether strategies to promote NEAT in absence of dietary manipulation are effective for obesity prevention or treatment. Moreover, some lines of evidence suggest a compensatory upregulation of energy intake with rising physical activity energy expenditure, especially at higher workloads and energy expenditure levels (reviewed in (90, 112, 151, 80). Therefore, it remains unclear whether substantially increasing NEAT for body mass control under free-living conditions will be capable of inducing weight loss without caloric restriction. Probably this shows large inter-individual variability, since it was recently shown that subjects losing weight over a two year time span increased their daily activity by about 35 minutes, while subjects gaining weight showed a reciprocal decrease (113). Energy intake was comparable in both of these groups. Contrasting with that, subjects either losing body weight or weight maintainers did not show alterations in daily activity as compared to weight gainers in another study (152). Thus, even under real life conditions there might be “responders” and “non-responders” in terms of daily activity/NEAT and body weight control. These groups remain, however, to be better defined.

Recent work could help to better categorize “responders” and “non-responders” by applying the above mentioned constrained energy expenditure model (153). The authors hypothesized that total energy expenditure would increase with physical activity at low levels, while plateauing at higher activity levels. It was found that after adjusting for body size and composition, TEE is indeed positively associated with PEE/AEE, but the relationship was markedly stronger over the lower range of physical activity. For subjects in the upper range TEE plateaued (Figure 9). According to the author’s hypothesis, body fat and activity intensity appeared to modulate the metabolic response to physical activity (153). In other words, subjects who maintain high levels of physical activity in their daily lives could show a smaller response to attempts of further increasing NEAT as it contributes to TEE increment, while subjects with habitually low activity levels could increase NEAT contribution and can therefore possibly be categorized as “responders”.

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Figure 9. Effects of physical activity on PEE/AEE and its components (adapted from (153)).

Since hypocaloric diets can significantly reduce NEAT (see previous sections), a major goal has to include prevention of this reduction in NEAT. There is strong evidence that physical activity is essential for the prevention of weight regain after weight loss, and for weight maintenance (16, 80). For instance, it was shown that low physical activity levels are related to significant weight regain at follow up after hypocaloric diets (154). Furthermore, when subjects gaining weight under free-living conditions are compared to weight stable counterparts, the “weight gainers” demonstrated markedly lower physical activity related energy expenditure and less muscle strength (155). Of note, after a one year period, lower physical activity energy expenditure explained approximately 77% of body mass increase in the “weight gainers” as compared to “weight maintainers” (155). A recent meta-analysis on weight loss maintenance shows that physical activity has a significant treatment effect of approximately 1.6 kg in the short term (156). However, long term this effect was lost, probably due to reduced compliance since effects of voluntary physical exercise were examined (156).

As discussed above, for prevention of a decrease in NEAT resulting from hypocaloric dieting, an effective strategy could therefore be to recommend moderate voluntary exercising during weight reduction, and then to engage in NEAT-enhancing activities at the initiation of weight maintenance. This strategy could mitigate poor long-term compliance and substantially contribute to lower body mass preservation. However, when purposeful training is initiated in combination with a hypocaloric diet, the exercise schedule must be moderate as too intense of an exercise regimen during caloric restriction can disproportionally reduce NEAT rather than stabilize it (157, 85). Otherwise, even purposeful exercise training cannot completely overcome hypocaloric diet-induced reductions of TEE, as the REE-component remains largely unaffected by this kind of intervention even when LBM is preserved (85, 72, 36, 80).

From a practical point of view this is a central issue, as after weight loss reduced TEE will persist over years. This was recently shown by a follow up study on participants of the “The Biggest Loser” televised weight loss competition in the U.S.(158). Fourteen subjects who were severely obese lost approximately 59 kg during a 30 week period of strenous physical exercise in combination with dieting. Fat mass was primarily lost, while LBM was largely preserved (158). Compared to baseline, by the end of the 30-week competition TEE decreased significantly by 800 kcal per day, which was mainly explained by the decline in RMR, approximating 600 kcal per day (158). Unfortunately, NEAT was not directly measured. However, keeping in mind that subjects underwent an intense physical exercise schedule it can be hypothesized that the observed difference of TEE and RMR (e.g. 200 kcal per day) was contributed to by a decline in spontaneous physical activity, which is comparable to earlier studies (157, 39, 72). After six years, the participants had regained 41 kg, TEE was reduced by roughly 400 kcal day-, and in spite of the significant weight regain, RMR remained reduced by 600 kcal per day (158). Therefore, after six years, “The Biggest Loser” televised weight loss competition resulted in a total weight loss of approximately 12 kg in subjects who were previously severely obese. The data indicate that these individuals will have to live with remarkably reduced dietary energy needs, even after adjustment for body weight and age (158) that contributes to their ongoing weight regain. Remarkably, the fact that after six years the reductions in RMR were lower than TEE indicates that some form of compensation is occuring, but it is unclear which component of daily energy needs is accounting for it (Figure 1). From the previously reported findings it can surmised that decreased NEAT was one of the factors contributing to the large weight regain (158, 39). Indeed, it was shown that participants of the “The Biggest Loser” television show with high levels of physical activity over years had significantly lower body mass regain compared to subjects with low physical activity levels. Weight loss maintainers had physical activity thermogenesis of roughly 12 kcal/kg/d as compared to weight regainers with approximately 8 kcal/kg/d, which was reflectetd by a significant negative correlation of body mass regain and physical activity levels (114).

Overall, moderate voluntary exercise results in significantly preserved weight loss during hypocaloric dieting (159, 160), which can be a helpful tool for preventing significant decreases in NEAT that will later help to stabilize body weight loss. With initiation of weight maintenance, subjects should be encouraged to engange in actvities increasing NEAT, to compensate at least in part for the obligatory weight loss-induced reduction in RMR. As stated previously, additional voluntary exercise is a useful tool for weight maintenance, but engagement in NEAT-related activities can be more easiliy integrated in daily life and will therefore likely result in higher adherence and compliance rates.

In terms of appropriate activities, it can be relatively easy to increase NEAT (Figure 10). Standing instead of sitting burns three times more kcal per hour, gum chewing increases energy expenditure four times, and stair climbing more than 40 times above resting levels (45). Ambulation (e.g., walking) in particular can raise NEAT and is easily performed at almost any place and at any time. But how can a goal of 2.5 hours of additional daily ambulation/standing time be realistically be integrated into daily routine? A key problem is that people’s occupations and personal lives regularly contribute to prohibit this degree of adaptation (45). As one’s occupation is the principal determinant of NEAT in adulthood and can represent an efficient means of promoting physical activity (45), how NEAT can be primarily integrated at the site of occupation needs examining. One important issue in this context addresses transportation to work. Similar to the NHANES data for the United States (42, 43, 41), recent results from the English and Welsh 2011 Census show that among the 23.7 million adult commuters, approximately 67% used private motorized transport as their usual main commute mode, while about 18% used public transport (161). In contrast, only 10.9% walked and 3.1% cycled (161). Thus, the majority of subjects rely on motorized transport. Promoting cycling or walking as a daily routine, not only with regard to transportation to work but also with respect to other daily needs (such as going to the grocery store), could represent a promising and realistic way for a majority of subjects to increase individual NEAT levels.

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Figure 10. Effects of physical activity on PEE/AEE and its components (adapted from (153)).

Together, 2.5 hours of additional ambulation and standing time per day are proposed as goals for those considering weight loss. However, potential beneficial effects on weight loss could be at least partially compensated for by increasing energy intake from food. Increasing NEAT as an adjunct for weight maintenance could overcome potential compensatory effects to the low-calorie state and hypothetically represent an alternative strategy for body mass control.

Limitations to Increasing NEAT

A number of factors potentially affect time spent in NEAT, of which some (sociological and environmental factors) can be influenced, while others cannot (genetic and endocrine factors) (Figure 11). Two points are important to make. First, as was shown previously in this chapter, skeletal muscle can increase in fuel economy and work efficiency not only in response to the loss of body mass, but also so that benefits of NEAT may be diminished. Moreover, it has also been reported that under weight-loss conditions, subjects with obesity oxidize proportionally more carbohydrates and less fat as compared to lean counterparts, and that differences in skeletal muscle metabolism and SNS activity underlie some of the observed differences between subjects who are and are not obese (reviewed in (21)). And second, even though regular physical activity can increase the capacity of skeletal muscle to oxidize lipids and to store glycogen (85), regular physical activity can also increase work efficiency and thereby possibly reduce the energetic costs of NEAT (85). Even so, given the known contribution of physical activity to body weight maintenance, the recommendation to patients who are overweight and with obesity to increase NEAT-related activities such as ambulation or bicycling is logical, even before definitive evidence is reported.

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Figure 11. Factors interfering with spontaneous physical activity and NEAT (adapted from (120)).


The epidemic of worldwide obesity in past decades is contributing to serious health concerns. Apart from poor diet, reduced physical activity and increased sedentary behavior contribute to the pathogenesis of obesity. Non-exercise activity thermogenesis is a highly variable component of daily total energy expenditure and essentially a function of environmental and individual factors. Whether caloric overfeeding systematically affects non-exercise activity thermogenesis is a matter of debate, while a negative energy balance due to voluntary caloric restriction and/or exercise can decrease it. As physical activity contributes to weight maintenance and prevention of body mass regain after hypocaloric dietary interventions, overcoming current obesogenic environmental pressures and increasing non-exercise activity thermogenesis could holds tremendous promise as a tool for body weight control.


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Knowledge of the factors influencing food intake is crucial to form an understanding of energy balance and obesity. Classical physiological feedback models propose that eating behavior is stimulated and inhibited by internal signaling systems (for the drive and suppression of eating, respectively) to maintain stability of the internal environment (usually energy or nutrient stores). However day-to-day food intake involves complex interactions of both internal and external inputs coordinated through homeostatic, hedonic, and cognitive processes in the brain. Twenty-five years ago, the term ‘obesogenic environment’ entered into scientific discourse and implies that prompts, cues, and triggers from the external environment are largely responsible for the increases in food intake that underlie the epidemic of obesity. This approach revitalized interest in the sensory and external stimulation of food intake and has drawn attention to the hedonic dimension of appetite. There is now a very strong current of thought that energy balance regulation is asymmetric and excess food intake is due to poor homoeostatic defense against positive energy balances in an environment rich in available, accessible, and readily assimilated food energy. This does not mean that regulatory signals concerned with energy balance regulation are unimportant. Indeed, they appear to be of incremental importance in prolonged negative energy balances and help explain control and loss of control of food intake as a dynamic continuum.


Traditionally food intake has been researched within the homeostatic approach to physiological systems pioneered by Claude Bernard (1), Walter Cannon (2) and others, and because eating is a form of behavior, it forms part of what Curt Richter referred to as the behavioral regulation of body weight (or behavioral homeostasis) (3). This approach views food intake as the vehicle for energy supply whose expression is modulated by the metabolic requirement to replenish energy stores. The idea was that eating behavior is stimulated and inhibited by internal signaling systems (for the drive and suppression of eating, respectively) in order to maintain stability in the internal environment (energy stores, tissue needs). Since this time, it has become clear that energy intake is not as tightly regulated under modern environmental conditions as symmetrical negative feedback models initially suggested. Compensatory changes in physiology and behavior are more pronounced in response to negative than positive energy balances. Furthermore, energy intake is determined by eating behavior, which itself, may be determined by a number of complex physiological, environmental, social, and cultural factors. One should not perhaps expect to see strict regulation of food or energy intake on a day-to-day basis in modern environments.


The control of appetite is often viewed to operate within an energy balance model of body weight regulation, but this should not lead to the view that appetite is controlled simply as an outcome of energy balance. Eating (food, energy, and nutrient intake) is a form of behavior, and like many aspects of volitional behavior, the factors leading to changes in that behavior are complex and are the outcome of factors which can be described by behavior change models. Using a COM-B model of behavior (Capability, Opportunity, Motivation, Behavior) (4), these factors include capability (psychological or physical ability to enact the behavior), motivation (reflective and automatic mechanisms activate or inhibit behavior), opportunity (physical and social environments that enables the behavior). Given the interacting and complex nature of these influences it is to be expected that unless homeostatic or other physiological feedback signals are particularly powerful, their effects on eating behavior would be difficult to detect. It is often implied (but not explicitly stated) that eating behavior is part of a feedback loop that is subsumed to the regulation of energy balance, but under the conditions of modern environments, there is little rationale for why eating behavior per se should be a regulated phenomenon as described by classic homeostatic models. Eating behavior may show repeated patterns which are stable over time, but these patterns may be quite different from the concept of regulation described in typical homeostatic models of blood glucose regulation or thermoregulation for example.

While eating behavior is influenced by a number of factors, it is reasonable to argue that some of those factors may become particularly salient under specific physiological or environmental circumstances. This is important as energy balance regulation appears asymmetric, with compensatory changes in physiology and behavior more pronounced in response to negative than positive energy balances. The exact mechanisms that oppose energy deficits are complex, inter-related, and individually subtle (5, 6). While energy expenditure and its components change in response to energy deficits in a quantitatively important manner, it is likely that changes in energy intake (EI) have a greater capacity to produce relatively large alterations in energy balance and body composition (7). The physiological and psychological impacts of weight loss likely occur on a continuum, with the point on this continuum influenced by (i) the degree of energy deficit, (ii) its duration, (iii) body composition at the onset of the energy deficit, and (iv) the psychosocial environment in which it occurs (8). In contrast, as weight is progressively gained there is very little evidence of physiological or behavioral systems exerting negative feedback to actively limit further weight gain. However, there is considerable heterogeneity in the rate of weight gain during overfeeding (9), which may in part, reflect inter-individual variations in physical activity and/or partitioning the excess energy between fat mass (FM) and fat-free mass (FFM) (10, 11).

The implication of asymmetric energy balance regulation would be that appetite is under stronger physiological control in relation to negative energy balances, whereas in a state of energy balance or positive energy balances weak linkages (negative feedback) exist between physiological functioning, food intake, and the motivation to eat. If energy balance regulation is asymmetric and modern environments are spatially and temporally rich in energy dense foods, it is logical to assume that many of the factors that shape eating behavior and energy intake are due to environmental influences such as sensory and environmental cues for food intake (which are presumably mediated through hedonic and other affective mechanisms). There is now a strong current of thought that a major cause of an increase in food intake associated with the rise of obesity resides in the hedonic rather than the homeostatic system. Some authors argue that in the resource limiting environments in which we evolved, hedonic and homeostatic systems functioned in a synchronized manner to facilitate over consumption during relatively brief periods of food abundance. In an environment where food resources are unpredictable and finite, overconsumption would have been an adaptive behavior limited (capability, opportunity) by environmental uncertainty. Natural selection would favor such behaviors. There would have been little need to evolve systems that protect against weight gain as it would be an improbable outcome in resource limited environments. This does not mean that the so-called ‘energy homeostasis system’ is no longer important in modern environments. Modern day environments have changed very rapidly and radically relative to the environment shaping energy balance regulation. Therefore, to understand how homeostasis and hedonics may influence food intake in modern environments requires an appreciation of the asymmetry of energy balance regulation, the time course over which such regulation may operate, and the very rapid time-course over which the modern food environment has changed.


The Motivation to Eat

Within the COM-B model of behavior, motivation is an important factor influencing behavior if capability and opportunity do not constrain behavior (as is the case in today’s so called ‘obesogenic’ environment). In the field of ingestive behavior, motivation to eat usually refers to reflective processes that are subjectively experienced or expressed. They are often believed to relate to underlying physiological or external environmental influences, but have the status of a self-reported, subjectively expressed psychological construct. Appetite has been defined as the subjective expression of willingness or motivation associated with qualitative selection and quantitative consumption of specific foods during an ingestive event (12). Appetite is not necessarily solely related to situations of nutritional depletion and can be influenced by a number of physiological and non-physiological factors. Appetites are specific to certain foods, often learned and frequently sensory specific (13). Unfortunately, the term appetite control is often used without specifying what is being controlled. Does eating behavior change to maintain some constancy and motivation to eat? Does appetite change in order to control food intake around some central tendency? Does appetite change with the aim of changing food intake to regulate energy balance? Often it is the latter view that is implied but not clearly articulated.

Table 1. Key Psychobiological Components of Appetite (14, 15)




The subjective sensation described as the primary motivation to eat. An increase in subjective hunger usually predicts meal initiation underad libitumfeeding situation. It does not necessarily predict type or amount of food eaten.


The process during a meal that generates the negative feedback leading to its termination (within-meal inhibition).


The degree of satisfaction and/or fullness following food consumption. This bears some reciprocal relationship to hunger and inhibits further motivation to eating.


The sensory pleasure elicited by contact with food contributing to the hedonic motivation to consume (wanting).


The motivation to consume a specific food, manifesting explicitly (desire to eat) or implicitly.

Because a great deal of human behavior is both reactive and learned, it is possible that the environment can produce prompts, cues, and stimuli that influence learned patterns of motivation to eat. Because eating behavior is a significant determinant of energy balance it is often argued that manipulation or control of motivation to eat (commonly termed appetite control) can be used as a means to prevent excess energy intake and obesity, usually via putative mechanisms of satiety. This is a logical proposition particularly if appetite is not actually very tightly controlled with reference to overconsumption and the development of obesity. Some characteristics of the expression of appetite do appear to render individuals vulnerable to over-consumption of food - these characteristics can be regarded as risk factors that vary between individuals (16). Other significant and salient environmental, acquired, and inherited influences on eating behavior aside, there has been considerable work dedicated to trying to conceptualize and understand putative mechanisms that may link motivation to eat to food and energy intake.


One of the most commonly accepted theoretical models for the control of appetite is the satiety cascade, a putative network of interactions between physiological, psychological, and behavioral factors which form a psychobiological system. The term psychobiological assumes that physiological functions provide internal cues that may impact motivation to eat, and can be conceptualized on three levels (Figure 1). These are the levels of psychological events (hunger perception, cravings, and hedonic sensations) and behavioral operations (meals, snacks, energy, and macronutrient intakes); the level of peripheral physiology and metabolic events; and the level of neurotransmitter and metabolic interactions in the brain (17) (see for Andermann & Lowell (18) for a review of the central control of food intake). Appetite reflects the synchronous operation of events and processes in the three levels. Implicit in this theoretical model is the notion that the physiological signaling systems influence motivation to eat and that motivation to eat shapes eating behavior. The model suggests that neural events trigger and guide behavior, but each act of behavior involves a response in the peripheral physiological system. In turn, these physiological events are translated into brain neurochemical activity that is related to the strength of motivation to eat and the willingness to refrain from eating. In this model it is assumed that motivations to eat change with the aim of changing food intake to regulate energy balance. The lower part of the psychobiological system (Figure 1) illustrates the satiety cascade links motivation and behavior to peripheral and central signals related to eating. It also includes those behavioral actions which actually form the structure of eating, and those processes which follow the termination of eating and which are referred to as post-ingestive or post-prandial events.

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Figure 1. The satiety cascade, as originally presented (17), showing the expression of appetite as the relationship between three levels of operations: the behavioral pattern, peripheral physiology and metabolism, and brain activity. See for Andermann & Lowell (18) for a more recent review of the central control of food intake. PVN, paraventricular nucleus; NST, nucleus of the tractus solitarius; CCK, cholecystokinin; FFA, free fatty acids; T: LNAA, tryptophan: large neutral amino acids.


Traditionally a distinction has been drawn between episodic and tonic signals in the control of appetite (19). Episodic signals are mainly inhibitory (but can be excitatory) and are usually generated by episodes of eating. These signals oscillate in accordance with the pattern of eating, and most are closely associated with the signaling of satiety. Tonic signals arise from tissue energy stores such as adipose tissue and metabolically active tissues to exert some degree of feedback on the expression of appetite to match day-to-day food intake with longer-term energy needs. These two sets of signals, one set responding sharply to nutrient flux and the other providing a slow modulation of appetite and food intake, are integrated within complex brain networks that control the overall expression of appetite. Examination of these putative mechanisms tend to be more common in acute or short-term studies of ingestive behavior in which the primary change is motivation to eat or eating behavior rather than longer term studies. Short-term experiments are valuable for mechanistic understanding, but such studies often make the assumption that changes in the motivation to eat or eating behavior will translate in the long term into aspect of energy balance regulation. However, concentrating only on short-term effects without considering the longer-term time scale may fail to reveal the way that food intake is affected or energy balance is regulated as the experimental time window is much narrower than is relevant for such regulation to occur (e.g., weeks and months rather than minutes, hours or days). Put simply, many investigators may be looking for evidence of regulation over a period where no such regulation is likely to occur. It is therefore important to distinguish between longer term (tonic) mechanisms of putative energy balance regulation and shorter term (acute, episodic) mechanisms that may affect motivation to eat or EI.

Episodic Appetite Signals

Episodic signals are those physiological events that are triggered as responses to the ingestion of food. These form the inhibitory processes which first of all stop eating and then prevent its re-occurrence and are therefore termed satiety signals. The types of signals involved in terminating a meal (satiation) and preventing further consumption (post meal satiety) can be represented by the satiety cascade. Initially the brain is informed about the amount of food ingested and its nutrient content via sensory input. The gastrointestinal tract is equipped with specialized chemo- and mechano-receptors that monitor physiological activity and pass information to the brain mainly via the vagus nerve (20). This afferent information constitutes one class of ‘satiety signals and forms part of the pre-absorptive control of appetite. It is usual to identify a postabsorptive phase that arises when nutrients have undergone digestion and have crossed the intestinal wall to enter the circulation. These products, constitute the flux of energy and nutrients into the circulation, may be metabolized in the peripheral tissues or organs, or may enter the brain directly via the circulation. In either case, these products constitute a further class of metabolic satiety signals. Additionally, products of digestion and agents responsible for their metabolism may reach the brain and bind to specific chemoreceptors, influence neurotransmitter synthesis or alter some aspect of neuronal metabolism. In each case the brain is informed about some aspects of the metabolic state resulting from food consumption. It seems likely that chemicals released by gastric stimuli or by food processing in the gastro-intestinal tract are involved in the control of appetite (21). Many of these chemicals are peptide neurotransmitters, and many peripherally administered peptides cause changes in food consumption (22). (Please refer to ENDOTEXT chapter ‘Endocrinology of The Gut and the Regulation of Body Weight and Metabolism’ by Andrea Pucci and Rachel L. Batterham for additional information on gut hormones physiology).


Cholecystokinin (CCK) is a hormone released in the proximal small intestine mediating meal termination (satiation) and possibly early phase satiety. CCK reduces meal size and also suppresses hunger before the meal; these effects do not depend on the nausea that sometimes accompanies an IV infusion (23). Food consumption (mainly protein and fat) stimulates the release of CCK (from duodenal mucosal cells), which in turn activates CCK-A type receptors in the pyloric region of the stomach. Fat in the form of free fatty acids (FFA) of carbon chain lengths C12 and above produce pronounced CCK releases (24, 25). This signal is transmitted via afferent fibers of the vagus nerve to the nucleus tractus solitarius (NTS) in the brain stem. From here the signal is relayed to the hypothalamic region where integration with other signals occurs.

Animal data suggest that endogenous CCK release mediates the pre-absorptive satiating effect of intestinal fat infusions, and may in turn be critical in regulating the intake of fat (26). As in rats, intestinal infusions of fat produce a reduction in food intake and promote satiety in humans (27). In humans the satiety effect of fat infused directly into the duodenum can be blocked by the CCKA receptor antagonist loxiglumide (28). High-fat breakfasts have been shown to produce both greater feelings of satiety (signified by reduced levels of hunger, desire to eat and prospective consumption) and elevated endogenous plasma CCK levels. Collectively, these studies support the theory that CCK plasma levels are a potent fat (or fatty acid) -stimulated endogenous satiety factor, whose effects on food intake and eating behavior are mediated by CCKA receptors.

It has also been shown that synthetic CCK-A type agonists suppress food intake in humans. A drug, known by the number ARL1718, caused a significant reduction in meal size and had a longer duration of action than observed after infusions of CCK itself. A number of other CCK analogues / CCK 1 receptor agonist treatments have been developed including most recently GW181771 (GlaxoSmithKline) and SR146131 (Sanofi-Aventis). Studies with such drugs, together with those on the peptide hormone itself, do suggest that CCK has the properties of a true satiation signal which contributes, under normal circumstances, to the termination of a meal. However, CCK is not uniquely involved in the expression of satiety and is also involved in a spectrum of physiological responses generated following nutrient consumption. The action of CCK certainly acts in concert with other meal-related events, such as gastric distention for example.


Glucagon-like peptide (GLP)-1 is an incretin hormone, released from the gut into the blood stream in response to intestinal nutrients. Endogenous GLP-1 levels increase following food intake, particular of carbohydrate (29, 30). These studies suggest a role for GLP-1 in mediating the effects of carbohydrate (specifically glucose) on appetite. In healthy men of normal weight, infusions of synthetic human GLP-1 (7-36) during the consumption of a fixed breakfast test meal, enhanced ratings of fullness and satiety when compared to the placebo infusion (31). During a later ad libitum lunch, food intake is also significantly reduced by the earlier GLP-1 infusion. Intravenous GLP-1 also dose-dependently reduces spontaneous food intake and adjusts appetite in healthy weight male volunteers. This marked reduction in food intake and enhancement in satiety is also observed in male patients living with overweight or obesity and type 2 diabetes. In men living with obesity, intravenous GLP-1 potently reduces food intake either during or post-infusion (32) and, at lower sub-anorectic doses, slows gastric emptying. Reductions in intake and slowed gastric emptying are accompanied by decreased feelings of hunger, desire to eat and prospective consumption, and a prolonged period of post-meal satiety. These data demonstrate that exogenous GLP-1 reduces food intake and enhances in satiety in humans, both those healthy weight and living with obesity. However, it should be kept in mind that the doses of GLP-1 often administered are usually higher than the normal values seen in blood after a meal. Consequently, although GLP-1 receptors could be a possible target for anti-obesity drugs, the physiological role of GLP-1 itself in the normal mediation of satiety is still not confirmed. Nonetheless, GLP-1 through its action as an incretin which prompts the release of insulin, will certainly have some indirect role on the pattern of eating behavior. Interestingly, two of the most promising drug options for people living with obesity are liraglutide and semaglutide; both GLP-1 agonists. These drugs have been shown to decrease hunger (33) and/or increase satiety (34). The action of the pharmacological agents may mean that people living with obesity gain some control over their eating behaviors, however longer-term studies are required to investigate their true potential. In addition, it should be noted that the GLP-1 receptors responsible for the anti-obesity action of semaglutide and liraglutide are located in the brain rather than the periphery.

Peptide YY 3-36

Peptide YY 3-36 (PYY 3-36) is one of the two main endogenous forms of PYY. It is produced from the cleavage of PYY 1-36 (the other major form of PYY) by dipeptidyl peptidase IV (DPP IV). PYY is a 36 amino acid ‘hind gut’ peptide released from endocrine cells in the distal small intestine and large intestine. This hormone is similar in structure to the orexigenic neuropeptide NPY (70% amino acid sequence identity), and in the past, PYY has been regarded, like NPY, as a potent stimulator of food intake. However, in a series of studies in rats, mice and in one human study (all included in one paper), Batterham et al. (35) have demonstrated that peripheral PYY 3-36 administration reduces food intake and inhibits weight gain in rodents. These effects on intake and body weight are not observed in transgenic animals lacking NPY Y2 receptors (the NPY Y2 receptor knock-out), thereby implicating these receptors in mediating the anorectic effects of PYY. PYY release in the distal intestine is triggered by a variety of nutrients, including fats (particularly FFA), some forms of fiber and bile acid (24, 25). In humans, endogenous PYY is released predominantly after, rather than during a meal (35, 36) and causes a decrease in gastric emptying (the so-called ‘ileal brake’). Thus, it is more associated with post-meal satiety. PYY (including PYY 3-36) can cross the blood brain barrier via a non-saturable mechanism. Moreover, some of the effects of peripheral PYY 3-36 on food intake are either independent of or dependent on vagal afferents running from the periphery to the brain (37, 38).

With regard to the effect of PYY on human appetite, Batterham et al. (35) demonstrated that in healthy humans a 90-minute PYY 3-36 infusion reduced hunger and subsequent food intake two hours later. In a further report, PYY infusions in people who were either a healthy weight or living with obesity caused a 30% reduction in lunch intake post infusion and decreased the 24 h energy intake by 23% in those with a healthy weight and by 16% in those living with obesity (36). The natural plasma levels of PYY were lower in those with living with obesity than in the healthy weight participants, and were inversely correlated with the body mass index. The lower levels of PYY in those with living with obesity could mean a weaker satiety signaling through this hormone and therefore a greater possibility of over-consumption. However, as the authors noted these effects required doses greater than the normal physiological range of endogenous PYY and marked nausea was observed in one experiment (39-41).


Research has also focused on amylin, a pancreatic rather than a gastrointestinal hormone, which also has a potent effect on both food intake and body weight (42). Peripheral administration of amylin reduces food intake in mice and rats, and meal size in rats. Chronic or peripheral administration of amylin over a period of 5 to 10 days produces significant reductions in cumulative food intake and body mass of rats (43). Thus, amylin appears to be a component part of the appetite regulation system. The effects of amylin on human food intake, food choice or appetite expression has yet to be fully assessed. However, pramlintide (a human amylin analogue), given to replace deficits in endogenous amylin in people with diabetes, has been shown to alter body weight in people living with obesity and diabetes treated with insulin (44-46) and people with obesity without diabetes (47). In healthy weight participants pramlintide induces reductions in meal intake and duration, and reduces pre meal appetite (48). Similar effects of pramlintide on intake and eating behavior are reported in people living with obesity (with and without type 2 diabetes) (49, 50).


In contrast to the peptides mentioned above, ghrelin is the only known excitatory peptide released in the gastrointestinal system. Ghrelin is a 28-amino acid peptide that stimulates the release of growth hormone from the pituitary (51). Secreted primarily from the stomach, it is also found in a number of other tissues (52, 53). Ghrelin was the first excitatory peptide discovered and acts upon the hypothalamic arcuate nucleus (51, 54, 55). The composition and action of ghrelin is uniquely modified by the addition of an octanoyl group to the serine residue at position three. Some studies suggest this acylation is crucial for ghrelin to bind to the growth hormone-secretagogue receptor (GHS-R) and cross the blood-brain barrier (56). Ghrelin’s effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein in the central nervous system (57).

The majority (80-90%) of circulating ghrelin is in the deacylated form (51). Two theories have been proposed, firstly that deacylated ghrelin could result from incomplete acylation of the peptide, with both forms utilising differently regulated pathways, or secondly, that DG could result from the deacylation of ghrelin (58). More recently, deacylated ghrelin has been termed the inactive form. Ghrelin is thought to be involved in meal initiation as it is high during periods of fasting and decreases in response to food intake, thus suggesting a physiological role for ghrelin in meal initiation (59). Intravenous infusion or subcutaneous injection of ghrelin in humans increases both feelings of hunger and food intake (56, 60) and to promote increased food intake, weight gain and adiposity in rodents (60).

Satiety Cascade Peptides

In the overall control of the eating pattern, the sequential release and then de-activation of the peptides described above, can account for the evolving biological profile of influence over the sense of hunger and the feeling of fullness (61). The actions of these hormones therefore contribute to the termination of an eating episode (thereby controlling meal size) and subsequently influence the strength and duration of the suppression of eating after a meal. Evidence of this is shown by Gibbons et al. (62) whereby the post meal period was separated into early and late phases of satiety. It should be noted that whilst the profiles of peptide response shown in papers often show the expected increase and decrease (in the case of satiety peptides) and decrease and increase (in the case of ghrelin), there is a wide degree of individual variability in peptide responses. This is not often commented on, or shown. Furthermore, the majority of papers do not measure a range of peptides but rather focus on one or two. Individual variability in the release and maintenance of the levels of hormones (or the sensitivity of receptors) may determine whether some individuals are prone to snacking between meals or to other forms of opportunistic eating. The overall strength or weakness of the action of these peptides will help to determine whether individuals are resistant or susceptible to weight gain. Individual variability in the response of gut peptides to different food types has been shown more recently (63) and can be seen in Figure 2. At present, it does not appear that a poor response in one peptide means a poor response in all peptides, and it is likely that the cumulative response of the peptides (of which there are many) is key for the modulation of appetite and EI. Since different foods may produce the same effect on hunger and fullness but display quite distinctive profiles of post-prandial peptides, this suggests that the satiety signaling system is complex and there is no single unique pattern of peptides that defines satiety. This questions to what extent short term satiety can be accounted for by individual changes in putative satiety peptides, and what other factors may contribute to subjective satiety or cessation of eating behavior in humans.

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Figure 2. Panel A shows the average ghrelin suppression after high fat and low-fat meals and Panel B and C shows the individual profiles of ghrelin for each participant after both high and low-fat meals. Adapted from (63).


Ghrelin and the Hunger Drive

As noted above, ghrelin is an episodic peptide increasing during periods of fasting and decreasing in response to food intake. However, ghrelin is also unique since it has been proposed that in addition to being linked to the initiation of eating, ghrelin also acts as a compensatory hormone. Circulating ghrelin decreases in response to overfeeding and increases in response to chronic negative energy balance such as occurs with exercise or anorexia nervosa (64). This means that in people living with obesity and in animals experimentally made fat, circulating ghrelin levels would be reduced in an apparent attempt to restore a normal body weight status. Therefore, ghrelin illustrates the characteristics of both an episodic and tonic signal in appetite control. From meal to meal the oscillations in the ghrelin profile act to initiate and to suppress hunger; over longer periods of time, some factor associated with fat mass applies a general modulation over the profile of ghrelin and therefore, in principle, over the experienced intensity of hunger. At some point, it seems likely that people living with obesity are insensitive to lower ghrelin levels and/or other factors outweigh the relative importance of circulating ghrelin. When weight is lost, for example following a period of food restriction and weight loss, ghrelin levels would rise (or normalize), and therefore promote the feeling of hunger. This is likely to be one of the signals that makes the loss of body weight difficult to maintain. Ghrelin blockade therefore may prove a useful anti-obesity treatment. Whilst people living with obesity have lower fasting ghrelin levels, they have been shown to show a similar response to infused ghrelin as normal-weight participants, that is, increased food intake (65). Ghrelin levels in people living with obesity do fall after food, but not to the same degree as healthy weight participants in whom different calorie loads were shown to decrease ghrelin levels in a dose-response manner, but in people living with obesity this clarity was not shown as clearly (66). This points towards a potential mechanism for weight gain to be a consequence of a down regulation of gut peptide signaling and that the sensitivity to ghrelin is being overridden by other factors, for example, hedonic control of appetite.

The Role of Leptin

One of the classical theories of appetite control has involved the notion of a long-term signal, leptin, which informs the brain about the state of energy stored in adipose tissue (67). In 1994 a mouse gene that controls the expression of a protein by adipose tissue which could be measured in the peripheral circulation (leptin) was discovery. It is now well accepted that there is a good correlation between the plasma levels of leptin and adipose tissue (68), and leptin interacts with NPY, one of the brain’s most potent neurochemicals involved in appetite, and with the melanocortin system, in the central control of appetite. Alongside other neuromodulators involved in a peripheral-central circuit, leptin links adipose tissue with central appetite mechanisms and metabolic activity. A number of mutations in the genes controlling molecules in the leptin-insulin pathways are associated with the loss of appetite control and obesity. For example, the MC4-R mutation (melanocortin concentrating hormone receptor 4) leads to an excessive appetite and massive obesity in children, similar to leptin deficiency. Studies have shown for individuals with a genetic form of leptin deficiency that leptin treatment produces dramatic weight loss, an effect associated with marked decreases in hunger (69-71). Further, the administration of exogenous leptin to humans, with either an insufficiency in or a specific deficit of endogenous leptin appears to strengthen within meal satiation and post meal satiety (71, 72). However, leptin-based obesity treatments for most individuals with obesity seem inappropriate given they appear leptin insensitive rather than leptin deficient. Indeed, despite extensive literature on leptin and other putative feedback signals arising from adipose tissue (73, 74), there appears to be limited evidence in humans of the extent to which changes in adipose tissue exert strong negative feedback on motivation to eat or EI in individuals in approximate daily energy balance or modest positive imbalances (as characterize the majority of individuals in the modern environment). As a number of questions still exist regarding the applicability of a 'lipostatic' control system to the regulation of appetite in humans free from congenital leptin deficiency (75). Consequently, recent models of human appetite have attempted to integrate the role of both FM and FFM into the control of appetite and energy balance to better account for the peripheral signals of appetite.

Fat-Free Mass and Resting Metabolic Rate and Associations with Appetite

A conceptual model of human appetite that incorporates the energetic demands of metabolically active tissues has been proposed, with a tonic drive to eat arising from components of energy expenditure (e.g., resting metabolic rate; RMR) and its main determinants (e.g., FFM). This model is based on a series of studies demonstating that FFM, but not FM, is associated with hunger and EI under conditons of energy balance (76-81) (see Figure 3). For example, Blundell et al. (78) reported that FFM was associated with self-selected (ad libitum) meal size and total daily EI in 93 individuals living with overweight or obesity. In contrast, no associations were found between FM and EI. Resting metabolic rate, of which FFM is the main determinant (82), has also been found to be associated within-day hunger sensations and EI (80, 81, 83). These findings have been replicated in studies employing a wide range of participants and under a variety of experimental conditions, with the associations between FFM and EI observed under laboratory (79-81, 84) and free-living settings (85, 86), in new born babies (87), adolescents (88, 89), normal weight women (90),people living with moderate (91) or extreme obesity (92), and people of varying ethnic origin (93, 94). These studies provide evidence that the relationship between FFM and EI exists across the entire age spectrum from birth (87), through childhood and adolescence (88) and into adulthood (78, 81, 84, 93, 95) and older age (96). There is also limited evidence that losses of both FFM and FM may be associated with changes in appetite (97) and weight regain (98) following weight loss, suggesting that integrated models of weight loss that account for FFM and FM losses may better explain changes in appetite during prolonged energy deficit (see below sections). However, there are few prospective longitudinal studies relating changes in functional body composition to appetite or EI, and where there is evidence, it often from studies with extreme weight loss induced via semi-starvation or military training.

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Figure 3. Association between fat-free mass, resting metabolic rate and daily energy intake (top panels), and a path diagram illustrating a mediation model for the direct effects of FM and FFM on RMR and RMR on EI, the indirect effect of FM and FFM on EI mediated by RMR, and the squared multiple correlations (R2) for RMR and EI. Data originally reported in Hopkins et al. (80, 85).

While often not explicitly discussed in relation to models of human appetite, the concept that energy needs exert influence on food intake is not new. In 1962, Kenneth Blaxter (99) noted that the basal metabolism of mature animals of different species increases to a fractional power of weight, with small animals have a higher basal metabolism per kilogram of weight than the large ones. This implies that to maintain body weight, small animals must obtain each day from food a larger number of calories per unit of the weight the large ones. If the total habitual energy expenditures of different species are all about the same multiples of their basal energy expenditure, then both the calorie intake and the basal metabolism are likely to be proportional to the same fractional power of bodyweight. Furthermore, the energetic demands of individual tissue-organs such as the liver (100) or the growth and maintenance of lean tissues (101, 102) have previously been suggested as sources of appetitive feedback. For example, Millward’s protein-stat theory suggests that lean mass, and in particular skeletal muscle mass, is tightly regulated such that food intake (specifically, dietary protein) is directed to meet the needs of lean tissue growth and maintenance (101). This theory is based on the existence of an ‘aminostatic’ feedback mechanism in which food intake is adjusted in response to amino acid availability to meet the protein demands of lean tissue growth and maintenance.

Studies Examining the Associations between Fat-free Mass, Resting Metabolic Rate and Energy Intake

Using statistical mediation models, a number of cross-sectional studies conducted under conditions of energy balance have demonstrated that the effect of FFM on EI is mediated by RMR (80, 103) and total daily energy expenditure (TDEE) (104), suggesting that energy expenditure per se may exert influence over EI. For example, Hopkins et al. (80) reported that the effect of FFM on EI was fully mediated by RMR i.e. FFM had no ‘direct’ effect on EI but rather ‘indirectly‘ influenced EI via its effect on RMR. In agreement with these findings, Piaggi et al. (104) reported that TDEE accounting for 80% of the observed effect that FFM exerted on EI in 107 healthy individuals. Such findings suggest that the associations between FFM and RMR with EI may reflect a ‘mass-dependent’ effect arising from the energetic demands of FFM and its constituent tissue-organs rather than a specific endocrine signal secreted by these tissue-organs. However, it should be acknowledged that such findings represent statistical rather than biological pathways. Furthermore, skeletal muscle, a major component of FFM by weight, secretes a large number of myokines (105)which provide a molecular signal for bi-directional communication with other organs (106). While myokines such as interleukin 6 (107) and irisin (108) have been linked to food intake and energy expenditure, the specific role that these (and other myokines) play in the control of appetite is unclear.

An important consideration in the proposed relationship between FFM and EI is that FFM is a heterogeneous tissue compartment that is comprised of numerous individual tissue-organs with wide ranging metabolic functions and mass-specific metabolic rates (109-111). Tissue-organ structure and function are tightly coupled and determine their tissue-specific metabolic rate (112). In turn, the tissue-specific metabolic rates of individual organs summate to determine whole-body metabolic rate (e.g., RMR). The maintenance of tissue-organ structural integrity and function is therefore a metabolic priority (112), but to date there has been little attempt to integrate individual tissue-organs and their mass-specific energy expenditures into homeostatic models of human appetite. Recently however, Casanova et al., (113)used whole-body magnetic resonance imaging to examine whether the masses of high-metabolic rate organs (brain, liver, heart and kidneys) were associated with fasting hunger in 21 healthy males (age= 25 ± 3 years; BMI = 23.4 ± 2.1 kg/m2) (114). As expected, fasting hunger was associated with FFM (r = 0.39; p = 0.09) but not FM (r = -0.01; p = 0.99). Interestingley, the association between the combined masses of the high-metabolic rate organs and fasting hunger (r = 0.58; p = 0.01) was stronger than with FFM as a single uniform body compartment. In particular, liver (r = 0.51; p = 0.02) and skeletal muscle mass (rs = 0.57; p = 0.04) were strongly associated with fasting hunger. As the masses of the liver and skeletal muscle explained ~17% and ~21% of the variance in RMR, respectively, these findings again suggest that energy expenditure per se may exert influence over food intake.

Another important consideration is how behavioral components of total daily energy expenditure (e.g., physical activity or activity energy expenditure) influence energy intake. The effects of physical activity and/or exercise on appetite is discussed below. As noted, physical activity may influence the control of appetite via a number of physiological and psychological pathways (e.g., alterations in gastric emptying (115), appetite-related hormones (116), food reward (117), and eating behavior traits (118)). In addition, physical activity or exercise may also exert influence, albeit modestly, on appetite and EI via its contribution to TDEE. Hopkins et al. reported in 242 individuals in which physical activity and EI were measured under free-living conditions that activity energy expenditure was independently associated with daily EI alongside FFM and RMR (86). As activity energy expenditure only explained 3% of the variance in total daily EI, its effect on daily EI was much more modest than that seen for FFM or RMR. This is perhaps not surprising given the smaller and more variable contribution of physical activity energy expenditure to TDEE as compared to FFM and RMR (119). It could be argued that while FFM and RMR are well placed to exert stable influence over day-to-day food intake, the contribution of physical activity energy expenditure to daily EI is likely to be weaker and more variable (and therefore, also harder to quantify).

Factors Affecting the Strength of Association Between Fat-Free Mass and Energy Expenditure with Energy Intake

It has been suggested that excess FM may disrupt the coupling between FFM and EI, with associations between FFM and EI weaker in those living with obesity than in healthy weight individuals (90, 95, 104, 120). Early work by Cugini et al. reported that a positive association existed between FFM and hunger, while FM and hunger were negatively associated, in healthy weight individuals (121). However, no such associations were seen between FFM or FM and hunger in those living with obesity (120). Based on these data, the authors suggested that FM accumulation may disrupt the feedback mechanisms linking these tissues to hunger. More recently, Grannell et al. reported a positive association between FFM and EI during an ad libitum test meal in 43 individuals living with severe obesity, but the strength of this association was weaker in individuals with a higher BMI (92). To further explore the moderating effect of FM, Casanova et al., (90) examined the linear and non-linear associations between body composition (FFM and FM), energy expenditure (RMR and TDEE) and EI (ad libitum test meal intake and free-living 24-hour EI) in 45 healthy weight and 48 individuals living with obesity. Percentage body fat moderated the associations between RMR (β=-1.88; p=0.02) and TDEE (β=-1.91; p=0.03) with free-living 24-hour EI. Furthermore, FM was negatively associated with test meal EI only in the leaner group (r=-0.43; p=0.004), with a weak non-linear association observed between FM and EI in the whole sample (r2=0.092; p=0.04).

Such findings point to a non-linear relationships between FM and EI, and this may help account for why negative associations between FM and EI have been observed in healthy weight individuals (121, 122), but studies in those living with overweight or obesity often report no association between FM and EI (79, 88, 91, 123). A weaker negative association between FM and EI at higher body fatness is in line with the notion of leptin and insulin resistance (124, 125), which may alter central and peripheral sensitivity to appetite-related feedback signals (126-128). Furthermore, while the contribution of FM to RMR is smaller than FFM (129), its contribution to RMR becomes proportionally larger as FM increases with excessive weight gain. Therefore, differences in the strength and direction of association between FM and EI at higher body fatness may reflect the increased contribution of FM to body weight and RMR alongside a blunting of its inhibitory influence on EI. It should also be acknowledged that the associations between FM and EI likely reflects both biological and psychological factors. Indeed, Hopkins et al., (85) have demonstrated that psychological factors such as cognitive restraint are robust predictors of EI when considered alongside physiological determinants of EI (e.g. FFM and RMR), and have the potential to play a mediating role in the overall expression of EI (85). A recent paper also suggests that the associations between FFM and TDEE with EI may become weaker with age (96). Based on a secondary analysis of the Interactive Diet and Activity Tracking in AARP Study, a biomarker validation study of self-reported diet and PA measures in older adults, Hopkins et al., reported that FFM and TDEE (derived from doubly labelled water) predicted self-reported EI in 590 older adults (mean age 63.1 ± 5.9 years). Interestingly, while the associations between FFM or TDEE and EI existed across age quintiles, age moderated the associations between FFM and TDEE with EI such that these associations weakened with increasing age (96). Please refer to ENDOTEXT chapter ‘Control of Energy Expenditure in Humans’ by Klaas R Westerterp for additional information).

Associations Between Body Composition and Energy Intake During Prolonged Negative or Positive Energy Balances

Another important point to note is that the aforementioned associations between body composition, energy expenditure, and EI are from cross-sectional analysis performed in weight stable individuals at or close to energy balance. However, the effect of FFM on appetite appears to be dependent on energy balance status, with evidence suggesting that losses of FFM may also act as an orexigenic signal during energy deficit. During the Minnesota semi-starvation study (130), 32 healthy men undertook 24 weeks of semi-starvation (25% of weight loss), 12 weeks of controlled refeeding and 8 weeks of ad libitum refeeding. During the last phase (n = 12), hyperphagia remained until baseline levels of FFM were restored. This led to FM accumulation that surpassed baseline levels (i.e., “fat-overshoot”), a phenomenon that has been reported elsewhere following underfeeding or military training (131, 132). As hyperphagia persisted until FFM had been restored to pre-weight loss levels, it was suggested that independent appetitive feedback signals from both adipose tissue and FFM (e.g., a ‘proteinostatic’ mechanism) contributed to the changes in hunger and food intake seen and restoration of body weight (133, 134).

While the demands imposed by semi-starvation or military training on energy balance clearly exceed those experienced during common diet and/or weight loss interventions, evidence also exists to suggest that FFM loss during clinically relevant weight loss may also act as an orexigenic signal. Following weight loss, it is commonly suggested that subjective hunger and orexigenic hormone concentrations increase (135, 136), but studies have also reported no change or reduced hunger following weight loss (137-139). The composition of the weight lost, which is a function of initial body fat, the rate and extent of weight loss, diet composition and exercise (140, 141), may also influence any accompanying changes in appetite. It has been suggested that greater FFM loss during weight loss is associated with increased hunger (97) and weight regain (98) following weight loss. To assess whether changes in body composition occurring during weight loss were associated with subsequent energy balance behaviors under conditions of therapeutic weight loss, Turicchi et al. conducted a systematic review and meta-regression examining weight loss studies in weight clinical weight loss was achieved (mean = 10.9%) and weight regain occurred in the follow-up period (mean = 5.4%) (98). They found that while both greater rate and amount of WL predicted weight regain, the composition of weight loss i.e. the (amount of FM and FFM) explained greater variance in weight loss alone (40% vs 29%) (98). Furthermore, Turicchi et al., reported that greater FFM loss following a 12% reduction in body weight via a low-calorie diet was associated with greater increases in hunger in men (r = 0.69, p = 0.002) but not women (r = 0.25, p = 0.24) (97). In line with these findings, after 5 weeks of very-low calorie diet (500kcal/d) or 12 weeks of low-calorie diet (1250kcal/d) Vink (142) reported that greater FFM loss during energy restriction was associated with greater weight regain during a subsequent 9-month follow-up period. Data examining FFM loss during extensive periods of energy deficit are therefore suggestive that FFM loss may be part of an integrated response driving post-weight loss increases in EI and weight regain, potentially as a means to restore the structural integrity of FFM compartments (although the influence of FFM loss appears more modest than FM loss). These data also emphasize the importance of developing integrative models of energy balance that consider the dynamic relationships between body structure, physiological function, and the way these mechanistic interactions influence key psychological and behavioral determinants of energy balance such as appetite. However, there are few prospective longitudinal studies relating changes in functional body composition to appetite or EI, and further research using advanced imaging methods for tissue-organ composition and multi-compartmental body composition models across a range of initial body compositions and weight losses would provide additional mechanistic insight (97).

Taken together, cross-sectional research in weight stable individuals indicates that greater FFM is associated with increased EI, but research also indicate that FFM loss is associated with increased appetite and EI. If greater FFM is associated with increased appetite, how is it that FFM loss during weight loss is also associated with increased appetite? One explanation is that FFM exerts ‘passive’ and ‘active’ effects on appetite under situations of differing energy balance (6, 143). At or near to energy balance, Dulloo et al. (143) has suggested that the energy demand of FFM and its constituent components create a ‘passive’ background pull on EI that ensures the energetic demands of metabolically active tissues are met through day-to-day food intake. In contrast, during weight loss, FFM loss may act as an ‘active’ orexigenic signal that stimulates increased hunger and EI in an attempt to ensure the preservation of FFM and the functional integrity of its constituent tissue-organs (143). However, it should be noted that long-term studies with longitudinal tracking of appetite, body composition and energy expenditure are rare, particularly under-conditions in which body composition is systematically manipulated.

Body weight gain leads to an expansion of FFM which increases RMR, but how such changes causally influence appetite or food intake has not been examined. As weight is gained, both FM and FFM expand but at different rates. While such changes may not drive weight gain per se, a higher FFM and associated RMR may increase the background tonic drive to eat, favoring maintenance of a higher body weight. Expansion of FM over the long term induces insulin and leptin resistance, expansion of FFM and, in extremis, some slight elevation of RMR, could account for the apparent diminishing negative feedback from FM as adipose tissue expands (90, 95, 104, 120). Therefore, it may be argued that any putative effect of FFM or RMR on the drive to eat may decrease with increasing BMI, since FFM and RMR increase at a decelerating rate with increase in weight, while the energy content of the body expands disproportionately as FM expands. While factors associated with energostatic models of appetite may be unlikely to drive body weight up in the first place, it is not inconsistent with maintenance of a higher body weight once this is achieved by other means. Thus, it may be that RMR is associated with EI at or close to energy balance, but that RMR (and its primary determinant FFM) become dissociated from the process of overconsumption during significant weight gain as the signal(s) becomes ‘overwhelmed’ by other stimuli important in driving weight gain e.g., food availability, sensory variety, dietary energy density and food reward.


Food intake is clearly influenced by homeostatic processes of hunger and satiety, modulated according to short term and long-term signals of nutritional and energy status and moderated by lifestyle factors such as physical activity. However, human eating behavior is a complex phenomenon and people eat for many other reasons too: for friendship and celebration, in response to sadness or stress. All these cognitive and emotional motives converge on the fact that eating is a potent source of reward. It provides the eater with an instant but temporary hit of gratification. The greater the reward, the harder it is to resist the behaviors that produce it even when the consequences are risky or harmful and especially when the risk is removed in time. Therefore, a key issue in the study of appetite control is the relationship between hedonic and homeostatic drives (144). Historically, hedonic processes have been viewed as a function of nutritional need-state. In a state of depletion, the hedonic response (experienced palatability or pleasure) to energy providing foods is enhanced and when replete, the hedonic effect of these foods is reduced (145). This view is compatible with the association between energy density and palatability (146) and also that the consumption of fats and sugars “energy-dense nutrients“ may be under neuro-regulatory control (147). However, the idea of reward as merely serving the fulfilment of nutritional need is not sufficient to explain non-homeostatic food intake and it is perhaps more useful to try and distinguish the substrates of homeostatic and hedonic systems and to assign them separate identities (148).

Homeostasis and Hedonics: Cross-Talk and Interaction

Advances in our understanding of the molecular and neural mechanisms behind food intake regulation and appetite control are revealing how the reward system can interact with homeostatic mechanisms. For example, cannabinoid receptors and their endogenous ligands (e.g., anandamide) are implicated in the reward system. Peripheral and central administration of anandamide increased appetite in rodents, and this seemed to be related to alterations in incentive value (wanting) for palatable foods (149). However, the cannabinoid system has been shown to interact with homeostatic processes in a number of ways: Leptin signaling becomes defective when hypothalamic endocannabinoid levels are high (150); activation of CB1 receptors prevent the melanocortin system from altering food intake (151); furthermore, CB1 receptors can be found on adipocytes where they may directly increase lipogenesis (152). Opioid neurotransmission also forms part of the biological substrate mediating reward processes of consumption. For example, endogenous opioids are associated with the reinforcing effect of food (especially when palatable) (153, 154). However, there is evidence to show that in a fasted state, the reinforcing effect of food can be reinstated in enkephalin and β-endorphin knock-out mice (155). Therefore, homeostatic processes may interact with hedonic signaling to override selective reward deficit.

Hence although homeostatic and hedonic systems can be given separate identities (148), they are also - to an extent - inseparable, with neural cross-talk permitting functional interactions which may influence the organization of eating behavior. From this standpoint, the interaction of homeostatic and non-homeostatic pathways in the neuro-regulatory control of eating may be more important than the two systems studied in isolation. From behavioral and anatomical observations (156), it has been suggested that projections from the hypothalamus to the nucleus accumbens may modulate the motivation to eat via metabolic signals. Furthermore, direct and indirect projections from the accumbens to the hypothalamus may explain the ability for mesolimbic processes “activated by relevant environmental cues and incentives” to essentially hijack the homeostatic regulatory circuits and drive-up energy intake. Further research is necessary to identify the pathways that mediate such interactions; however progress has been made (157).

Liking vs. Wanting Food

The hedonic perspective on appetite control accounts for eating behavior motivated by the expectation or experience of pleasure from consuming specific foods and involves dissociable processes of “wanting” and “liking”. The “liking” component refers to the subjective experience of pleasure elicited by the sensory perception of food and is associated with the release of endogenous opioids acting on localized clusters of neurons termed “hedonic hotspots” (158) The “wanting” component of reward refers to the process by which food is assigned motivational significance or “incentive salience attribution” and is associated with the release of the neurotransmitter dopamine in the mesocorticolimbic pathway. This latter component can be activated by thoughts or cues signaling food and often precedes the actual receipt of food (159).

In human neuroimaging studies, regional differences in the neural activation to food stimuli during either anticipatory or consummatory phases of reward processing are broadly supportive of the distinction between liking versus wanting. Response to passive viewing of high- versus low-calorie foods or cues signaling the imminent receipt of a tasty food are more reliably observed in the amygdala and ventral striatum, whereas the response to the actual taste and consumption of a palatable food is associated with activation in the primary taste cortex in the insular and opercular cortices (160). Some researchers have proposed that differences between individuals who are healthy weight and those with obesity in neural activation to palatable food can be understood as a dissociation in both the direction and region of responding during the anticipatory and consummatory phases of food intake- with greater striatal activation in individuals living with obesity compared to healthy weight controls when a food is wanted but lower activation in liking-related regions when a food is actually tasted (161). However, several inconsistencies in the brain imaging literature have been noted (162), and further research is needed to substantiate this hypothesis.

Liking and wanting for food are often viewed in relation to subjective states or explicit feelings that refer to the everyday understanding of these terms in the context of food choice and food intake (163). Wanting might describe subjective states of desire, craving or perceived deprivation of pleasure, whereas liking is characteristically understood as the perceived hedonic effect of a food, the appreciation of its sensory properties or some evaluative judgment of its potential to give pleasure. As the subjective sensations of liking and wanting often overlap and are subject to interference or misinterpretation, their relationship with behavior is often difficult to discern (163). However, liking and wanting responses to food are not necessarily consciously monitored or even always accessible to the individual. Although people tend to be very good at estimating and reporting their liking for food, they are often unable to accurately gauge their implicit wanting for food (i.e., why they are unconsciously drawn to one food over another).

The hedonic aspect of eating is important in a well-functioning homeostatic system for the directing and motivating an adequate supply of nutrients and energy. Increasingly, evidence for the interplay between liking and wanting with hunger and satiety is helping to clarify the role of hedonics in the control and loss of control over food intake (164). This extension to the conventional homeostatic model recognizes that hedonic processes are affected by acute nutritional need states and might modulate food intake through their interaction with other physiological processes involved in satiation and satiety. Likewise, cognitive and sensory inputs implicated in food liking and wanting can modulate the metabolic processes associated with homeostatic control over food intake (165). In addition to the effect of liking and wanting on episodic appetite responses, more recent evidence is emerging to suggest that tonic signals of nutritional status might affect liking and wanting for food to influence food preference and the composition of the diet (164).


Physical Activity and Control of Food Intake

Some individuals believe that the energy expended during exercise will automatically drive-up hunger and food intake to compensate for the energy deficit incurred. However, evidence shows that interventions of acute exercise generate little or no immediate effect on levels of hunger or daily EI (see (166-170) for reviews). One reason that studies do not demonstrate an increase in EI following acute exercise could be that they fail to track EI for sufficiently long periods after the bout of exercise, or that the exercised-induced energy expenditure is not large enough to stimulate appetite. However, even with a high dose of exercise (gross exercise-induced increase in energy expenditure = 4.6 MJ) in a single day and following tracking of EI for the following two days, there is no automatic compensatory rise in hunger and EI (171).

Exercise training interventions have shown similar findings with regards to EI (169, 172). In a recent systematic review and meta-analysis of exercise training interventions in people living with overweight or obesity, Beaulieu et al. (172)found that among 25 exercise groups, exercise training (ranging 2-72 weeks) did not lead to any significant post-intervention differences in EI compared to non-exercise control groups. Meta-regression showed this was not affected by intervention duration. However, due to the high number of poor-quality studies (i.e., using self-reported measured of dietary intakes), further analyses were conducted in only fair/good quality studies (reduced to 5 exercise groups), and found a 102-kcal post-exercise difference between exercisers and controls. It is important to note that this is an effect observed on the average and does not illustrate inter-individual variability (discussed below). Therefore, over time, on average there may be small compensatory increases in EI in response to the increased energy demands from greater physical activity levels. Indeed, the review also found a small increase in fasting hunger after exercise training in 19 exercise groups (172). Nevertheless, depending on the daily energy expenditure accrued with exercise training, this should still lead to some degree of weight loss and favorable changes in body composition, as reviewed by Bellicha et al.(173). Small positive changes were also observed in relation to eating behavior traits, with a reduction in disinhibition (13 exercise groups) and an increase in restraint (12 exercise groups) assessed by the Three-Factor Eating Questionnaire (172). A small number of studies in the review also suggested that exercise training may reduce reward or preference for high-fat foods (172). Overall, most of the evidence indicates that exercise training in people living with overweight or obesity appears to have a relatively small but positive influence on eating behavior. This effect may be heightened with longer-term physical activity by altering the sensitivity of appetite regulation. It should be kept in mind that most exercise training studies are of modest duration (e.g. up to 72 weeks the meta-analysis of Beaulieu et al. (172)), and the long-term changes in appetite and eating behavior, and the time-course over which these occur, are yet to be fully understood. Fewer studies have examined the relationship between changes in energy expenditure and eating behavior in non-obese participants who do not have a preconceived goal associated with weight reduction.

Does Habitual Physical Activity Level Affect Appetite Sensitivity?

Appetite sensitivity refers to the capacity to detect over- or under-consumption with the potential to subsequently adjust intake accordingly. This can be achieved by compensating for a particular EI by adjusting the size of the next meal. There is some evidence to suggest that regular exercisers, or habitually physically active individuals, have a better capacity to control food intake and energy balance due to increased appetite sensitivity. Long et al. (174)demonstrated that habitual exercisers have an increased accuracy of short-term regulation of EI in comparison to non-exercisers. In this study, participants were given either a low or high energy preload for lunch, and were then asked to eat ad libitum from a test meal buffet. Energy intake did not significantly differ following the two preloads in the non-exercise group, indicating a weak compensation. However, the habitual exercisers demonstrated nearly full compensation (~90%) by reducing their EI following the high energy preload compared to the low energy preload. This study has been replicated in groups varying in objectively-assessed habitual physical activity levels (175). Similarly, Martins et al. (176) reported that the sensitivity of short-term appetite control increased in previously sedentary individuals following 6 weeks of aerobic exercise training, with participants again better able to adjust subsequent EI following high and low energy pre-loads following the exercise intervention. Furthermore, King et al. (177) examined the effects of 12 weeks of supervised aerobic exercise on hunger and satiety in 58 individuals living with overweight or obesity. Two separate processes were revealed that acted concurrently to influence the impact of exercise on appetite regulation. Post-intervention, a significant increase in fasting hunger was seen, but this increased orexigenic drive was offset by a parallel increase in post-prandial satiety (as measured in response to a fixed energy meal). Therefore, this ‘dual process’ may reflect the balance between the strength of tonic and episodic signaling following chronic exercise, and may be an important factor which determines whether individuals successfully lose weight or not. Interestingly, these improvements in appetite control appear to be related to satiety and perhaps not to satiation (178), but more research on the interplay between diet composition, satiation/satiety and physical activity level is required, as well as underlying mechanism.

Findings of improved appetite sensitivity with increased physical activity are in line with Jean Mayer’s work over 60 years ago. Mayer et al. (179) demonstrated a non-linear relationship between energy expenditure and EI in Bengali jute mill workers. Daily occupational physical activity and EI were closely matched in those performing physically demanding jobs. However, in those performing light or sedentary occupational roles, this coupling was lost such that daily EI exceeded expenditure. Such work has led Blundell et al. (180) to suggest a J relationship between physical activity and appetite regulation, with ‘regulated’ and ‘non-regulated’ zones of appetite regulation seen across the physical activity spectrum. Sedentary or low levels of physical activity coincide with an ‘unregulated zone’ of appetite in which EI and energy expenditure are disassociated (thereby promoting overconsumption of food at low levels of physical activity). At higher levels of physical activity however, stronger regulation of appetite and food intake exists such that EI better matches energy expenditure. This would promote the better maintenance of energy balance, albeit at higher levels of absolute intake and expenditure, i.e. higher ‘energy turnover/flux’ (180). This non-linear relationship has since been replicated in a systematic review (181) and in a study of over 400 participants (182). The model proposed by Blundell was recently updated by Beaulieu et al. (183), as shown in Figure 4. Furthermore, emerging evidence from a highly-controlled metabolic chamber study suggests that appetite control is indeed improved at higher levels of energy turnover (184). Longer-term studies are required to understand the impact of high energy turnover on appetite control, but it is believed that high levels of EI relative to expenditure may improve weight management via a favorable impact on physiological adaptations (185).

Chapters Archive - Endotext (15)

Figure 4. Regulated and non-regulated zones of appetite with varying levels physical activity from Beaulieu et al. (183). Model based on Jean Mayer’s study in Bengali jute mill workers (179) and previously published in Blundell (180).

While the mechanisms behind an improvement in appetite control with regular physical activity remains unclear, insulin sensitivity has been proposed as one mechanism by which activity-induced improvements in appetite regulation may occur. Exercise is known to increase insulin sensitivity (186-188), and insulin sensitivity is known to be involved in satiety induced by particular foods (189) and in the compensatory response to high energy loads (190). A further mechanism by which exercise could affect appetite is through altering gut peptide action. For example, CCK is implicated in the short-term regulation of appetite, and levels of CCK have been shown to rise after exercise (191). Interestingly, Martins et al. (192) measured fasting and post-prandial levels of orexigenic (total and acylated ghrelin) and anorexigenic (PYY, GLP-1) peptides in 15 individuals living with overweight or obesity during 12 weeks of supervised aerobic exercise. A significant increase in fasting hunger was again seen following the intervention, but this was offset by greater satiety in response to a fixed energy meal following the intervention. Interestingly, there was also a significant increase in the suppression of acylated ghrelin following the fixed energy meal, and a tendency toward an increase in the post-prandial release of GLP-1 following the exercise intervention. These hormonal responses would have acted to augment satiety during the post-prandial period. Further work by Gibbons et al. (193)revealed that exercise training had no overall impact on pre and postprandial appetite peptide release; however, differences emerged when participants were classified as ‘responders’ and ‘non-responders’. Compared to non-responders, responders had overall greater suppression of acylated ghrelin and greater increase in GLP-1 and total PYY. This effect was independent of the exercise intervention as differences were observed from baseline. Therefore, the specific role that appetite-related peptides play in activity-induced improvements in appetite regulation remains to be fully understood.

In addition to gastrointestinal mechanisms, it is important to understand how physical activity timing and patterns impact energy balance and appetite control. Physical activity can be prescribed by the FITT principle: frequency, intensity, time (duration) and type, and more recently, timing as another parameter has been proposed, as emerging evidence suggests that when exercise is performed relative to a meal or the time-of-day may influence obesity and cardiometabolic health in both adults and children (194). Indeed, physical activity is also another cue that influences circadian rhythms (195). In a systematic review and meta-analysis of exercise training interventions in individuals living with overweight or obesity (117), only 3 studies (1 RCT) on diurnal exercise timing were identified (196-198).These studies, in addition to observational studies (reviewed in (199)), suggest that early relative to late day exercise timing may lead to greater weight/fat loss. However, a recent RCT suggests no effect of exercise timing on weight loss, but this may have been due to the relatively low dose of aerobic exercise (200). The underlying mechanisms the greater weight loss in response to early exercise timing likely involve an impact on both behavioral and physiological processes regulating energy balance and appetite control (201). Performing morning exercise may lead to greater weight loss by enhancing the satiety response to food throughout the day, reducing the desire for and intake of high-energy-dense foods, and shifting food intake timing to earlier in the day, thus reducing daily energy intake (199).Evidence for this hypothesis is scarce, with an acute study showing that morning exercise led to greater post-exercise satiety; however, this was not examined in response to actual food intake (196). More rigorous research in this area is needed to clarify these proposed effects.


The control of food intake in humans is a biopsychological phenomenon. Motivation to eat is an important factor influencing food intake if the environment does not constrain behavior. Food intake is not necessarily solely related to situations of nutritional depletion and can be influenced by a number of homeostatic and non-homeostatic factors. Appetites are often learned and frequently sensory specific. The implication of asymmetric energy balance regulation is that food intake is under stronger physiological control in relation to negative energy balances, whereas in a state of energy balance or positive energy balances the linkages between physiological signaling and subjective motivation to eat are weaker. In obesogenic environments where palatable, energy dense food is ubiquitous, intake can easily exceed energy requirements due to motivation underpinned by food reward. Another consideration is how physical activity or exercise influence energy intake. Physical activity may influence the control of appetite via a number of pathways (e.g., alterations in gastric emptying, appetite-related hormones, food reward, circadian synchrony).


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Diabetes insipidus (DI) is a disorder characterized by excretion of large volumes of hypotonic urine. The underlying cause is either a deficiency of the hormone arginine vasopressin (AVP) in the pituitary gland/hypothalamus (central DI), or resistance to the actions of AVP in the kidneys (nephrogenic DI). In most circumstances, DI is also characterized by excessive consumption of water (polydipsia). A third condition called primary polydipsia can clinically show overlapping features with DI. Both DI and primary polydipsia are collectively referred to as ‘polyuria-polydipsia syndromes. Like other endocrine disorders, an accurate diagnosis of DI can be challenging. This is mainly because the results obtained from diagnostic testing can show significant overlap among the different forms of DI and primary polydipsia. When a case of DI is suspected, the initial step involves the confirmation of the presence of hypotonic polyuria, which is the hallmark of DI. Once hypotonic polyuria is established, the next step is to identify the type of polyuria-polydipsia disorder (central DI vs. nephrogenic DI vs. primary polydipsia). This can be determined either through the water deprivation test or through the hypertonic saline infusion test along with plasma AVP or plasma copeptin measurements. Lastly, a detailed history and physical examination must be performed and appropriate laboratory and imaging studies must be undertaken to identify the underlying etiology of DI. This chapter describes the diagnostic steps to be pursued to identify the presence of DI, distinguish the various forms of polyuria-polydipsia disorders, identify the underlying disorders responsible for the DI, the challenges faced with diagnostic testing for DI in clinical practice, and future prospects in the field of DI diagnosis.


Diabetes insipidus (DI) is a disorder of water homeostasis that is characterized by excretion of large volumes of hypotonic urine either due to the deficiency of the hormone arginine vasopressin [AVP, also known as antidiuretic hormone (ADH)], or due to resistance to the action of AVP on its receptors in the kidneys (1, 2). Large volumes of urine excretion, also known as polyuria (typically over 4 L per day), is the hallmark of DI. The urine of DI has been classically described as insipid (tasteless), hypotonic and dilute (3). Polyuria is defined as excretion of a urinary volume >150 ml/Kg/24 hours at birth, >100-110 ml/Kg/24 hours up to the age of 2 years, and >50 ml/Kg/24 hours in older children and adults (1). DI is typically classified into 3 forms: 1. Central DI, 2. Nephrogenic DI and 3. Gestational DI. Primary polydipsia is a disorder that is characterized by excessive intake of water which results in hypotonic polyuria. Clinically, this condition can manifest with symptoms of DI and adds to the diagnostic challenge of identifying DI. Primary polydipsia and DI together are referred to as ‘polyuria-polydipsia syndromes’, characterized by hypotonic polyuria (4, 5).

(Video) 1 Sources for Endocrinology

Central DI is caused by a variety of disorders that arise from either the pituitary or the hypothalamus. These conditions are characterized by defective production, transport or secretion of AVP (3, 6). This results in inappropriately low AVP levels in the setting of increased plasma osmolality. Nephrogenic DI is a form of DI that results from resistance by the kidney towards the action of AVP, due to AVP receptor defects or as an adverse effect of certain medications (3, 6). A third, rare form of DI occurs during pregnancy. Also known as gestational DI, this type of DI results from the enzymatic breakdown of the endogenous AVP by a placental cysteine aminopeptidase (3, 7). Therefore, this enzymatic degradation of plasma AVP during pregnancy can unmask sub-clinical DI in those women with borderline-low plasma AVP levels (8). Although primary polydipsia is not a true DI state, long-standing primary polydipsia can give rise to a DI-like picture on laboratory evaluation (described later) (4). There are several etiologies that give rise to each of these forms of polyuria-polydipsia syndromes (6). They have been listed in Table 1.

Table 1. Etiologies of the Various Polyuria-Polydipsia Syndromes

Central Diabetes Insipidus (involvement of pituitary and/or hypothalamus)

· Neoplastic:

o Craniopharyngioma

o Germinoma

o Meningioma

o Pituitary macroadenoma (invasive)

o Metastasis to the pituitary and/or the hypothalamus

· Vascular:

o Hypothalamic infarction/hemorrhage

o Cerebral infarction/hemorrhage

o Anterior communicating artery ligation

o Anterior communicating artery aneurysm

o Sheehan’s syndrome

o Sickle cell disease

· Trauma:

o Deceleration injury

o Intracranial surgery

o Transsphenoidal pituitary surgery

· Autoimmune/inflammatory:

o Lymphocytic hypophysitis

o IgG4 disease

o Xanthogranulomatous hypophysitis

o Anti-vasopressin neuron antibodies

o Guillain-Barré syndrome

· Infectious:

o Meningitis

o Encephalitis

o Tuberculosis

o Pituitary or hypothalamic abscess

· Granulomatous:

o Sarcoidosis

o Granulomatous hypophysitis

o Langerhans’ cell histiocytosis

o Erdheim-Chester disease

· Drug/toxin-induced:

o Phenytoin

o Ethyl alcohol

o Snake venom

· Congenital/genetic:

o Autosomal dominant AVP-neurophysin II gene alterations

o Wolfram (DIDMOAD) syndrome

o Septo-optic dysplasia

o Schinzel-Giedion syndrome

o Culler-Jones syndrome

o Alstrom syndrome

o Hartsfield syndrome

o Webb-Dattani syndrome

o X-linked recessive defects with subnormal AVP levels

Nephrogenic Diabetes Insipidus

· Metabolic:

o Hypokalemia

o Hypercalcemia

· Drug-induced:

o Lithium

o Demeclocycline

o Methoxyflurane

o Cisplatin

o Pemetrexed

o Aminoglycosides

o Amphotericin B

· Renal disease:

o chronic kidney disease

o Polycystic kidney disease

o Obstructive uropathy

· Systemic disease:

o Amyloidosis

o Sarcoidosis

o Sjogren’s syndrome

o Multiple myeloma

· Vascular:

o Renal infarction

o Sickle cell disease

· Congenital/genetic:

o Autosomal recessive aquaporin-2 channel gene alterations

o X-linked recessive V-2 receptor gene alterations

o Polyhydramnios, megalencephaly, and symptomatic epilepsy (PMSE) syndrome

o Type 4b Bartter syndrome

Pregnancy-Induced/Gestational Diabetes Insipidus

· Increased vasopressin metabolism induced by placental cysteine aminopeptidase

Primary Polydipsia

· Psychogenic polydipsia

· Drug-induced:

o Anticholinergics

o Phenothiazines

· Intracranial etiology:

o Hypothalamic tumors

o Tuberculous meningitis

o Intracranial surgery/trauma

o Sarcoidosis

· Lowering of hypothalamic threshold for thirst (‘Dipsogenic DI’)

The typical clinical presentation of DI is in the form of polyuria and polydipsia. On several occasions, co-existing conditions or other aspects of the patient’s history can provide clues towards the possible etiology causing DI. Patients with hypophysitis can present with concomitant symptoms of anterior pituitary hormonal dysfunction including fatigue, erectile dysfunction, weight changes, loss of libido, galactorrhea, and amenorrhea (9). Headaches, visual field defects, or cranial nerve palsies can be observed along with DI in case of central nervous system (CNS) tumors, hypophysitis, or a pituitary adenoma (1, 9). In most circumstances, despite losing large volumes of water in the urine, individuals with DI do not manifest dehydration. This is due to the activation of the thirst center in the hypothalamus that induces a strong sense of thirst with rising plasma osmolality which results in the intake of large volumes of water (6). Therefore, although an intact thirst mechanism helps to compensate for the urinary water loss, the polyuria and polydipsia persist, and this can cause considerable distress for the patient unless the underlying DI is treated. A plasma osmolality of about 285mOsm/Kg usually acts as a trigger for thirst, with further increments in plasma osmolality resulting in a linear increase in thirst (6, 10). DI becomes more challenging to manage if this thirst response is diminished or if the patient is unable to access water for drinking. Patients with an attenuated thirst response have reduced urge to drink water despite rising plasma osmolality and loss of large volumes of water in the urine, resulting in ‘adipsic DI’ (11). Although being classically associated with craniopharyngioma, adipsic DI can also manifest with CNS trauma, CNS tumors, or after CNS neurosurgical or neurovascular procedures (12). Moreover, the thirst mechanism may be lost in hypothalamic lesions, whereby DI may present acutely with signs and symptoms of dehydration. Patients who are unable to voluntarily access water include infants and young children, and individuals with altered consciousness. In young children, lack of adequate water intake can lead to dehydration, sleeplessness, irritability, enuresis, failure to thrive and impaired growth (1). Similarly, among patients with altered/reduced consciousness, rapid output of large volumes of urine can result in dehydration and acute, sometimes severe, hypernatremia can ensue (2). The shrinkage of the brain induced by excessive water loss and severe hypernatremia could potentially lead to intracranial bleeding, obtundation, convulsions or coma (13).

Diagnosing the type of DI/polyuria-polydipsia syndrome is essential for making the optimal treatment decision. A potential misdiagnosis and the resultant treatment can lead to catastrophic consequences (7). For instance, if primary polydipsia is misdiagnosed as central DI and desmopressin treatment is initiated, severe hyponatremia can occur (14, 15). There have been several tests that have been developed over the past century to diagnose the presence of DI and to differentiate the various types of polyuria-polydipsia syndromes. This chapter specifically comprises the description of different diagnostic tests utilized in the diagnosis of these conditions.


The major challenge with diagnosing and classifying the type of DI arises from the fact that the various forms of polyuria-polydipsia syndromes can show overlapping features on diagnostic testing. The water deprivation test, also known as the indirect water deprivation test should potentially be able to distinguish the various forms of DI. Deprivation of water intake should allow patients with primary polydipsia to concentrate their urine while those with central or nephrogenic DI continue to excrete dilute urine. Administration of desmopressin (Deamino-8-D-arginine vasopressin), the synthetic analogue of AVP, after water deprivation should help with differentiating patients with central DI from those with nephrogenic DI as the former should be able to concentrate the urine once the deficient action of AVP is replaced with desmopressin, while the latter should not show a significant response due to end-organ resistance to the action of AVP or its analogues. While water deprivation followed by desmopressin administration should be theoretically sufficient to identify the type of DI, in reality, the interpretation of these tests is more complicated, especially if a patient has partial central DI, partial nephrogenic DI or chronic primary polydipsia (4). Patients with partial central or nephrogenic DI retain some amount of response to water deprivation and desmopressin administration (4, 16). In the case of chronic primary polydipsia, long-standing water diuresis blunts the renal medullary concentration gradient and causes down-regulation of the aquaporin-2 channels in the proximal tubule and the collecting duct due to suppressed endogenous AVP, thus creating a state mimicking nephrogenic DI (4, 17).

The basic algorithmic approach for diagnosing any suspected case of DI usually involves the following steps: 1. Confirmation of hypotonic polyuria 2. Diagnosis of the type of polyuria-polydipsia syndrome and 3. Identification of the underlying etiology (6). Performing diagnostic testing in this order can potentially aide with establishing the appropriate diagnosis and with choosing the most relevant biochemical and imaging tests.

Confirmation Of Hypotonic Polyuria

Polyuria is defined as excretion of a urinary volume >150 ml/Kg/24 hours at birth, >100-110 ml/Kg/24 hours up to the age of 2 years, and >50 ml/Kg/24 hours in older children or adults. A hypotonic urine is typically defined as a urine with an osmolality of <300 mOsm/Kg. The primary objective in this step involves differentiating between conditions that give rise to polyuria resulting from osmotic diuresis (such as in hyperglycemia) and DI/primary polydipsia, in which polyuria predominantly involves water diuresis.

Conformation Of Polyuria

The first step is to confirm if a patient indeed has polyuria. Complaints of ‘polyuria’ can often be a misrepresentation of the actual symptoms of urinary urgency, nocturia, urinary incontinence, urinary tract infection, or prostatic hypertrophy (2). Once these symptoms have been ruled out, a 24-hour urine collection should be obtained. A 24-hour urine volume of <2.5 L could be reassuring and there is no concern for osmoregulatory disruption. Those patients without polyuria but with other above-mentioned urinary symptoms must be referred for urological evaluation. Alternatively, individuals can keep a diary for 24 hours making a note of the amount of their urine output (3). A 24-hour urine creatinine will help ensure an appropriate sample collection. Patients need not hold any medications that can cause polyuria, such as diuretics or sodium-glucose co-transporter-2 (SGLT-2) inhibitors for this step as the goal of this step is to establish the presence of polyuria.

Excluding Other Causes of Polyuria

Once polyuria is confirmed, the next step would be to assess for urinary osmolality. The urine in cases of DI/primary polydipsia is hypotonic. A urine osmolality of >800 mOsm/Kg indicates optimal plasma AVP levels and appropriate renal response to AVP, therefore ruling out any form of DI (7, 18). In most cases, polyuria with isotonic/hypertonic urine is driven by glucose, sodium, urea, or medications such as diuretics or mannitol (19). Glycosuria can result either from uncontrolled diabetes mellitus (DM), general hyperglycemia (from steroid administration, tube-feeding/parenteral nutrition), or by the use of SGLT-2 inhibitors (20). Administration of normal saline in large volumes for intravascular volume expansion can lead to sodium-induced polyuria (19). Sodium-induced polyuria is also seen with release of bilateral bladder obstruction (21). Urea-induced solute diuresis can be seen with high amounts of protein intake, tissue catabolism, steroid administration, all of which result in production of urea from breakdown of proteins (19). Administration of urea for treatment of hyponatremia, and recovery from azotemia can also result in urea-solute diuresis (19). Mannitol-induced diuresis can result from treatment of increased intracranial pressure with mannitol (19).

Initial Serum/Plasma and Urine Investigations

In individuals with established hypotonic polyuria or in individuals with urine osmolality of ≥300 mOsm/Kg and <800 mOsm/Kg, further evaluation must be undertaken through laboratory investigations. Serum sodium and plasma osmolality measurements could assist with indicating the type of the underlying polyuric state. A high serum sodium (>146 mmol/L) could point towards central or nephrogenic DI while a low normal or low sodium (<135 mmol/L) could indicate primary polydipsia as the underlying disorder (5, 22, 23). Similarly, a high plasma osmolality (≥300 mOsm/Kg) is typically seen in DI while a normal or low plasma osmolality (≤280 mOsm/Kg) is usually seen in primary polydipsia (4, 24).

As an alternative to urine osmolality, urine specific gravity is also useful in identifying a hypotonic polyuric disorder. For normal plasma osmolality, the urine specific gravity is between 1.003 to 1.030 (25). The specific gravity value depends on the number and the size of particles in the urine, unlike urine osmolality which solely depends on the number of particles in the urine. Because of this, although urine specific gravity and urine osmolality generally correlate well, presence of large molecules might elevate urine specific gravity despite the chance of the urine osmolality actually being low (26).Unlike falsely elevated urine specific gravity, false low values of urine specific gravity are uncommon and a low urine specific gravity suggests DI or primary polydipsia. Both urine osmolality and specific gravity are easily measured on a urine specimen, but urine osmolality is more widely utilized in management of DI as osmolality is not affected by the size of the particles in the urine. Situations where urine specific gravity is suggested to be useful is when facilities for urine osmolality measurement are not available or if a rapid results are required, especially in managing neurosurgical patients, and on rare occasions where DI co-exists with DM with hyperglycemia (as in craniopharyngioma, Wolfram syndrome, described later) (27). With uncontrolled DM, the urine osmolality and specific gravity should be high. But with co-existent DI and DM, the urine osmolality and the urine specific gravity can be inappropriately low (28). Some studies have also noted low urine specific gravity but normal urine osmolality with concomitant DI and DM (27). In clinical practice, some physicians rely solely on urine osmolality while others prefer to assess both urine osmolality and urine specific gravity for managing DI.

Any underlying renal dysfunction must be ruled out by measuring urine sodium, blood urea nitrogen and serum creatinine (2). Electrolyte abnormalities including hypokalemia and hypercalcemia can give rise to polyuria due to down-regulation of aquaporin-2 channels giving rise to a clinical picture of nephrogenic DI (29). Therefore, any serum potassium or calcium abnormalities must be appropriately corrected.


Probably the most challenging step in the work-up of a suspected case of DI is to assign an accurate diagnosis: central DI vs. nephrogenic DI vs. primary polydipsia. These polyuric-polydipsic states can demonstrate substantial overlap, both in their clinical presentation and in their response to diagnostic testing. It is also possible for one or more forms of polyuria-polydipsia syndrome to co-exist in a single individual. An algorithm for the diagnostic approach for DI is described in Figure 1.

Chapters Archive - Endotext (16)

Figure 1. Algorithm for Diagnosis of the Various Types of Polyuria Polydipsia Syndromes.

Under some circumstances, a diagnosis of DI or primary polydipsia is established based on clear evidence of hypotonic polyuria and based on serum sodium and plasma osmolality values, with high serum sodium/plasma osmolality being consistent with DI and a low serum sodium/plasma osmolality being consistent with primary polydipsia (see Figure 1). But in most situations, the diagnosis is still unclear. Such situations, where the initial diagnostic testing is indeterminate, include a urine osmolality value of 300 – 800 mOsm/Kg or a normal serum sodium/plasma osmolality. In these circumstances, there is a need to establish the diagnosis with more accuracy, and further testing must be considered. In this section, the various diagnostic tests specifically utilized to diagnose and identify the type of polyuria-polydipsia syndrome are discussed, along with their advantages and limitations.

Water Deprivation Test

The water deprivation test is also known by the terms ‘indirect water deprivation test’ and ‘dehydration test’. The term ‘indirect’ is utilized as this test generally does not involve ‘direct’ measurements of plasma AVP to diagnose and differentiate the various forms of DI. The water deprivation test is almost always followed with desmopressin administration to further characterize the type of polyuric polydipsic state. The basic principle behind the water deprivation test is that in individuals with normal posterior pituitary and renal function (or those with primary polydipsia), an increase in plasma osmolality from dehydration stimulates AVP release from the posterior pituitary which then leads to water reabsorption in the nephrons, thus resulting in concentration of urine and an increase in urine osmolality. In central or nephrogenic DI, the urine fails to optimally concentrate with water deprivation and there is persistent excretion of hypotonic urine. Once the diagnosis of DI is established, desmopressin administration can distinguish between central and nephrogenic DI. In central DI, once the deficient action of AVP is substituted with desmopressin administration, the urine osmolality should increase while in nephrogenic DI, as the desmopressin is ineffective due to lack of renal response to its actions, the low urine osmolality persists.


The test is performed either as an out-patient or preferably after admitting the patient to an in-patient ward in a controlled setting. For those individuals with milder polyuria (50 – 70 ml/Kg/24 hours), the test can be initiated in the evening and the dehydration can be performed overnight as an out-patient. However, for those who experience significant polyuria or nocturia, the test is better performed during the day so that the patient can be supervised (3). The individual undergoing the test must not have undergone thirsting prior to the test. Any electrolyte abnormalities (potassium, calcium) must be corrected prior to the test. The patient has to discontinue any medications that can affect urine output (diuretics, SGLT-2 inhibitors, desmopressin, carbamazepine, chlorpropamide, glucocorticoids, non-steroidal anti-inflammatory drugs) 24 hours prior to initiation of dehydration, and refrain from activities such as smoking and caffeine intake that might affect AVP release or urine output (6, 30). Baseline plasma osmolality, serum sodium, urine osmolality (and plasma AVP or plasma copeptin where available) are obtained. In case of an out-patient overnight water deprivation, these baseline measures are obtained on the morning preceding the overnight water deprivation test (31).

The dehydration phase is initiated overnight among those patients in whom this phase is performed as an out-patient. The timing of initiation of overnight water deprivation for out-patient testing is determined based on the 24-hour urine output of the patient (which would have been previously determined in order to establish the diagnosis of a polyuria-polydipsia syndrome), with a goal of achieving approximately 3% loss of body weight.

The duration of overnight water deprivation (in hours) can be determined by using the following formula: patient’s weight (Kg) x 0.03 x 1000 (ml) / urine output (ml/hour) (31). Clear, written instructions must be provided to the patient about terminating the dehydration phase in case of unseen adverse events such as nausea, diarrhea, dizziness, or syncope. Once the overnight dehydration phase is completed, the patient arrives at the hospital the following day around 07:00 – 08:00 am to obtain serum/plasma and urine studies. Patients are advised to bring along an accompanying person for their hospital visit following the overnight water deprivation. In case the overnight dehydration is prematurely terminated for any reason (dizziness, or intractable thirst leading to consumption of water), then in these patients, in-patient water deprivation must be undertaken.

The dehydration phase for in-patient testing usually begins at 08:00 am. The patient voids prior to beginning the test and the baseline weight, blood pressure, and heart rate is measured prior to initiation of dehydration. Following the initiation of the test, patient should have nothing by mouth. Weight, blood pressure and heart rate must be recorded hourly as a cautionary measure due to the risk of severe water loss and dehydration in the setting of lack of access to drinking water. Adequate supervision is advised to watch for any undisclosed drinking of water. Every voided urine is recorded and the osmolality of the urine is measured. Alternatively, for the convenience of measurement, urine output and urine osmolality can be measured once every 2 hours. Serum sodium and plasma osmolality are also obtained every 2 hours along with urine measurements. The dehydration phase should be discontinued if one of the following occur: loss of more than 3% of body weight, elevation of serum sodium to above normal limits (≥146 – 150 mmol/L as per most literature), orthostatic hypotension or orthostatic symptoms (dizziness), or intractable thirst (4).

The next phase of the test is the desmopressin phase, which involves administration of desmopressin following dehydration. This phase can be initiated if either one of the following end-points are achieved: 1. Dehydration phase is completed for 8 hours (except in those who need longer periods of dehydration), 2. Two consecutive urine osmolality measures do not differ by >10% and loss of 2% body weight, 3. Premature termination of dehydration phase due to loss of more than 3% of body weight, elevation of serum sodium to above normal limits, or intractable thirst (3). An injection of 2 µg desmopressin is administered either through intravenous or intramuscular route and the above-mentioned urine and serum/plasma measures are obtained hourly for 1 – 2 hours after injection. Administration of desmopressin through oral or intranasal route does not result in predictable absorption, and especially where assigning an accurate diagnosis is crucial, optimal desmopressin concentrations in the blood has to be ensured. So, oral or intranasal desmopressin administration is not recommended to be used as a part of water deprivation test. During this phase, the patient can eat and drink, even up to 1.5 – 2 times the volume of urine passed during the dehydration phase. The total duration of the test can vary based on the clinical presentation. In those patients with complete forms of DI, the test can be performed in less than 8 hours while in those with partial DI or a non-DI condition, the test could last longer, sometimes even over 18 hours (3).


In normal individuals, with dehydration, the urine osmolality usually increases up to 800 – 1200 mOsm/Kg (3). If the dehydration phase is begun at 12:00 am, a morning (8:00 - 9:00 am) urine osmolality of 800 – 1200 mOsm/Kg excludes DI (3). A urine osmolality of <300 mOsm/Kg with a concomitant plasma osmolality of >300 mOsm/Kg or a sodium level above upper limit of normal following dehydration (>146 mmol/L) is suggestive of either central or nephrogenic DI (3, 4, 6). Desmopressin could be administered at this time (8.00 - 9:00 am). An increase of at least>50%in urine osmolality after desmopressin administration suggests complete central DI (the increase can be up to 200% to 400%) while a <50% increase points towards complete nephrogenic DI (3). An increase in urine osmolality over 300 mOsm/Kg prior to an increase in serum sodium/plasma osmolality suggests preservation of some endogenous AVP activity/renal response to AVP, suggesting either partial central or partial nephrogenic DI (5). Thus, in patients with partial DI (central or nephrogenic) the urine osmolality after water deprivation is usually between 300 – 800 mOsm/Kg and there can be <50% increase in urine osmolality following desmopressin administration.

In primary polydipsia, water deprivation results in an increase in urine osmolality, anywhere between 300 – 800 mOsm/Kg (usually up to 600 – 700 mOsm/Kg), without a substantial increase in plasma osmolality, but the increase in urine osmolality is not as substantial as in a normal response (3, 4, 6). Following desmopressin administration, an increase of <9% in urine osmolality is usually associated with primary polydipsia with a concomitant plasma osmolality of 300 – 800 mOsm/Kg as per some literature (4). However, this amount of increase in the urine osmolality when the urine osmolality is between 300 – 800 mOsm/Kg can also be seen in partial nephrogenic DI and adds to the diagnostic conundrum, and these criteria alone should not be utilized to diagnose primary polydipsia. A graphical representation of the response of each of the polyuria-polydipsia disorders to the water deprivation test is provided in Figure 2.

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Figure 2. Graphical representation of the water deprivation test. Image courtesy: Sriram Gubbi, NIDDK, NIH.


Although widely regarded as the gold standard in literature for diagnosing DI, the water deprivation test does have its limitations (3, 22). The most common scenario being partial central or partial nephrogenic DI. Patients with partial central DI retain some degree of AVP secretory capacity that can be stimulated with dehydration leading to concentration of urine. In partial nephrogenic DI, maximal AVP secretion from water deprivation can overcome renal resistance to AVP’s action and enable water reabsorption. In addition, long-standing central DI and chronic primary polydipsia can also give rise to false results on water deprivation test. With long standing central DI, due to chronic deficiency of AVP, the aquaporin-2 channels are down-regulated as AVP is required for the synthesis and membrane translocation of these channels (32). Therefore, administration of desmopressin after water deprivation in long-standing central DI might not result in an adequate increase in urine osmolality due to lack of adequate water reabsorption from insufficient aquaporin-2 channel availability, which could misleadingly suggest a picture of nephrogenic DI. On the other hand, chronic primary polydipsia can suppress the release of endogenous AVP due to increased intravascular volume and decreased plasma osmolality from high volumes of water intake. In this situation, water deprivation might not lead to an adequate rise in plasma osmolality high enough to stimulate the release of endogenous AVP. This can result in a sub-optimal increase in urine osmolality, thus simulating a clinical picture of central/nephrogenic DI. Moreover, high volumes of water intake from chronic primary polydipsia can create a ‘wash out’ of the renal medullary osmotic gradient and also a down-regulation of the aquaporin-2 channels, and administering desmopressin in this situation may not result in adequate increase in urine osmolality (4, 17). Also, the water deprivation test must be performed in children only under the supervision of a pediatrician and the test should not be performed in infants (33). Adjunctive measurements of plasma AVP or copeptin levels can improve the diagnostic yield of the water deprivation test and are described in detail in the upcoming sections below. The protocol for the water deprivation test, interpretation of the results, and limitations are represented in Table 2.

Table 2. Summary of the Indirect Water Deprivation Test


Preparation phase:

· Test can begin either overnight (for out-patient testing) or at 08:00 am (for in-patient testing).

· No thirsting prior to the test. Smoking and caffeine intake are avoided.

· Any electrolyte abnormalities (potassium, calcium) are corrected.

· Drugs that can affect urine output (diuretics, sodium-glucose co-transporter-2 inhibitors, glucocorticoids, non-steroidal anti-inflammatory drugs) must be held 24 hours prior to dehydration.

· Baseline weight, blood pressure and heart rate are measured prior to dehydration.

· Baseline plasma osmolality, serum sodium, urine osmolality (and plasma AVP or plasma copeptin where available) are obtained (these measures can be obtained on the morning prior to overnight dehydration in cases of out-patient water deprivation test).

Dehydration phase:

· This phase usually lasts for 8 hours (can last longer in certain cases).

· For out-patient testing, the duration (hours) of overnight dehydration can be determined using the formula: patient’s weight (Kg) x 0.03 x 1000 (ml) / urine output (ml/hour).

· Patient is allowed to have nothing by mouth.

· Adequate supervision is necessary to watch for any undisclosed drinking (in case of in-patient testing).

· Weight, blood pressure and heart rate are measured every 1 hour (in case of in-patient testing).

· Urine output, urine osmolality, serum sodium and plasma osmolality are measured every 2 hours (for in-patient testing). Plasma copeptin is measured towards the end of the dehydration phase. (In case of out-patient overnight dehydration, serum/plasma and urine measures are obtained around 07:00 – 08:00 am the following morning)

· Dehydration phase is discontinued if one of the following occurs:

o Dehydration is completed for 8 hours (not applicable for those who might need longer periods of dehydration).

o Two consecutive urine osmolality measures do not differ by >10% and loss of 2% body weight.

o The total body weight reduces by more than 3%.

o Serum sodium increases to above upper limit of normal (preferably >150 mmol/L).

o Orthostatic hypotension or orthostatic symptoms.

o Intractable thirst (or if patient admits to drinking water overnight in case of out-patient testing).

Desmopressin phase:

· An injection of 2 µg desmopressin is administered either through intravenous or intramuscular route (use of oral or intranasal desmopressin is not preferred due to unpredictable absorption).

· The patient is allowed to eat and drink, even up to 1.5 – 2 times the volume of urine passed during the dehydration phase.

· Urine output, urine osmolality, serum sodium and plasma osmolality are measured hourly for 1 – 2 hours after desmopressin administration.


Central DI:

o Baseline urine osmolality of <300 mOsm/Kg and an increase in urine osmolality by >50% from baseline following desmopressin administration (complete central DI).

o Baseline plasma copeptin of <2.6 pmol/L without prior dehydration (complete central DI).

o The ratio of Δ copeptin from start till the completion of dehydration phase to serum sodium at the end of the dehydration phase of <0.02 pmol/L (indicates partial central DI).

Nephrogenic DI:

o Urine osmolality fails to rise above 300 mOsm/Kg or by <50% after desmopressin administration (complete nephrogenic DI).

o Baseline plasma copeptin of ≥21.4 pmol/L without prior dehydration (complete and partial nephrogenic DI).

o Baseline plasma AVP of ≥3 pg/ml without prior dehydration (complete and partial nephrogenic DI).

Primary Polydipsia:

o Urine osmolality usually increases (300 – 800 mOsm/Kg, usually up to 600 – 700 mOsm/Kg) without significant changes in plasma osmolality following dehydration.

o The ratio of Δ copeptin from start till the completion of dehydration phase to serum sodium at the end of the dehydration phase of ≥0.02 pmol/L


· Most extensively utilized and validated test.

· No risks of hypertonic saline administration (thrombophlebitis, need for central line).

· Plasma copeptin or AVP measurements are not necessary if the center does not have the facilities/assays to measure these peptides.


· More time consuming than hypertonic saline infusion test.

· Can give overlapping results in cases of partial central DI, partial nephrogenic DI, or chronic primary polydipsia.

· Majority of the interpretation relies on urinary measurements, which can be affected by any of the above conditions due to their modulation of aquaporin-2 channel synthesis and expression.

· More burdensome for the patients and less convenient when compared with hypertonic saline infusion test.

Measurement Of Plasma AVP

AVP is encoded by the arginine vasopressin gene (AVP, 20p13), which also encodes prepro-AVP (a single peptide), neurophysin II (NPII), and a glycoprotein, copeptin. The pro-hormone of AVP is synthesized in the magnocellular neurons of the hypothalamus (Figure 3). Due to the above mentioned limitations of the indirect water deprivation test, measurement of plasma AVP levels was suggested for its potential to be used as a ‘direct’ test in conjunction with water deprivation test to distinguish the various polyuria-polydipsia syndromes (34). In nephrogenic DI, as there is no deficiency of AVP, the plasma AVP levels is high, in order to overcome the resistance posed by the kidneys to the action of AVP. On the other hand, plasma AVP should be low or relatively low in central DI for the elevated plasma osmolality (4). In primary polydipsia, plasma AVP levels may be normal or can be suppressed in long-standing cases. Subnormal AVP levels in cases of central DI could be due to osmoreceptor dysfunction in the hypothalamus or due to defective AVP release from the neurohypophysis (35). Despite the initial promising results of its potential utility, plasma AVP is seldom utilized for diagnosing DI in clinical practice.

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Figure 3. Anatomy of the pituitary gland and the hypothalamus. The pituitary gland comprises of two developmentally and functionally distinct parts: the anterior pituitary (adenohypophysis), derived from the Rathke’s cleft and the posterior pituitary (neurohypophysis). The gland is attached to the hypothalamus through the posterior pituitary via a stalk. The posterior pituitary along with the hypothalamus and the stalk forms the functional unit of infundibuloneurohypophysis. This diagram emphasizes on the neuronal network that supplies the posterior pituitary. The vasopressinergic neurons (magnocellular neurons) are present in the supraoptic and paraventricular nuclei of the hypothalamus. These neurons synthesize arginine vasopressin (AVP), its precursors and co-peptides, and their axons project into the posterior pituitary where the AVP is stored along with oxytocin (produced by the oxytotic neurons present in the same nuclei) in the axonal terminals. These hormones are transported to the posterior pituitary through the supraoptic-hypophyseal tract. Another set of neurons of the paraventricular nucleus (also called parvocellular neurons) project into other areas of the brain and spinal cord, some of which secrete AVP. Image courtesy: Sriram Gubbi, NIDDK, NIH.

There are several limitations to measuring plasma AVP levels. AVP is rapidly cleared from the plasma, with a half-life of around 16 minutes (36). In addition, the pre-analytical instability of AVP in the plasma is high. A large amount of circulating AVP is bound to platelets through V1 receptors and failure to adequately segregate platelets from the plasma after blood sampling or prolonged storage of unprocessed blood samples can lead to wide fluctuations or even an increase in the measured plasma AVP levels (22, 37). For the AVP to be detectable by the currently available assays, an additional step of extraction/concentration of plasma is necessary, and this requires at least 1 ml of blood sample (22). Additionally, the current laboratory method employed in AVP measurement is radioimmunoassay (RIA), which has several limitations and analytical errors. Plasma concentration of AVP (measured in pg/ml) is one of the lowest among all hormones, and as AVP is a small peptide, sandwich assays cannot be effectively utilized to measure plasma AVP levels (5). The AVP levels in the plasma can be stabilized up to 2 - 4 hours after blood sampling by storing the sample at 4ºC (22, 38, 39). However, the average turn-around time for the measurement of AVP is 3-7 days in most laboratories which would make maintenance of hormonal stability even more challenging (4, 40). For these reasons, plasma AVP measurements are not routinely utilized. A related peptide to AVP, copeptin, is now emerging as a new, more stable marker to diagnose the various hypotonic polyuric states.

Measurement Of Plasma Copeptin

Copeptin (Carboxy-Terminal-Pro-vasopressin) is the C-terminal peptide of pro-vasopressin that is co-secreted with AVP in stoichiometric amounts from the posterior pituitary (4, 40). Both of these peptides are derived from a precursor molecule synthesized in the magnocellular neurons of the hypothalamus(Figure 3) (22). The post-transcription processing of AVP, copeptin, and related peptides are diagrammatically represented in Figure 4. Unlike plasma AVP measurement, which is technically challenging, copeptin measurement in the plasma is relatively less cumbersome and has several advantages: copeptin can remain stable for days after sampling of blood and can be measured relatively quickly (40). Plasma levels of copeptin strongly correlate with plasma AVP levels over a wide range of osmolalities, both in healthy individuals and those with DI or primary polydipsia (22, 41). Moreover, plasma copeptin demonstrates the same response to changes in plasma osmolality and plasma volume as does plasma AVP (5,22). Over the past 12 years, several studies have been conducted to validate the utility of plasma copeptin in the diagnosis of hypotonic polyuric states and to distinguish one form from the other (4, 5, 7, 22, 30).

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Figure 4. Post-transcription processing of vasopressin and related peptides. The pre-pro-vasopressin is a peptide consisting of 164 amino acids. This pre-pro-hormone is then converted to pro-vasopressin after the removal of the signal peptide and N-linked glycosylation of copeptin. Further processing of pro-vasopressin gives rise to the individual peptides: arginine vasopressin (AVP), neurophysin II (NP II), and copeptin. Image courtesy: Sriram Gubbi, NIDDK, NIH.

The next question logically is whether plasma copeptin is a better test when compared to plasma AVP with regards to diagnosing the various forms of polyuria-polydipsia syndromes. Without prior thirsting (without water deprivation/hypertonic saline infusion), baseline plasma copeptin of ≥21.4 pmol/L or a plasma AVP level of ≥3 pg/ml have been shown to distinguish nephrogenic DI (partial and complete) from other types of polyuria-polydipsia syndromes with 100% sensitivity and specificity (5, 42). Similarly, a single baseline plasma copeptin measurement of ≥2.9 pmol/L without prior water deprivation has demonstrated the ability to differentiate primary polydipsia from central DI with a sensitivity of 82% and specificity of 78%, while plasma AVP of ≥1.8 pg/ml can distinguish primary polydipsia from central DI with 54% sensitivity and 89% specificity (5).

Prior data has also shown that a plasma copeptin of <2.6 pmol/L can accurately distinguish between complete central DI from primary polydipsia with 95% sensitivity and 100% specificity (4, 22). Some studies have utilized a combined water deprivation/hypertonic saline infusion test to stimulate endogenous release of AVP and copeptin, and based on the results, plasma copeptin levels of ≥4.9 pmol/L can differentiate primary polydipsia from central DI (partial or complete) with 94% sensitivity and 96% specificity, while a plasma AVP of ≥1.8 pg/ml can do the same with 83% sensitivity and 96% specificity (5).In addition, the ratio of Δ copeptin from start till the completion of water deprivation (08:00 am to 04:00 pm) to the serum sodium at the end of water deprivation test has been shown to discern partial central DI (<0.02 pmol/L) from primary polydipsia (≥0.02 pmol/L) with a sensitivity of 83% and a 100% specificity, although newer data has demonstrated that the ratio of Δ copeptin to serum sodium following water deprivation to be surprisingly of lower diagnostic accuracy than water deprivation alone (4, 30). Recent data suggests plasma copeptin measurement coupled with hypertonic saline infusion test provides more accurate results with regards to differential diagnosis of DI when compared with plasma copeptin measurement with water deprivation test (30)(see below, Hypertonic saline infusion test’). There is also emerging evidence for plasma copeptin as a valuable marker to predict post-operative risk for DI after pituitary surgeries, with plasma copeptin of <2.5 pmol/L predicting the risk of DI with a positive predictive value of 81% and plasma levels of >30 pmol/L on postoperative day 1 demonstrating a negative predictive value of 95% to rule out DI (43).

Based on these data, plasma copeptin measurement appears to be overall a better test when compared with plasma AVP measurement. However, it is the technical aspects of better pre-analytical stability, less cumbersome sample handling, and quicker reporting of results that make plasma copeptin a more attractive test. As of now, copeptin assays are not yet available worldwide and its utilization is limited to a few centers. Once the test becomes more widely available in the future, plasma copeptin level measurements are likely to be frequently pursued to diagnose and distinguish the various forms of polyuria-polydipsia syndromes.

Hypertonic Saline Infusion Test

The indirect water deprivation test is the most well tested and widely utilized as the standard diagnostic test for DI. Adjunctive measurements of plasma AVP or copeptin levels can enrich the diagnostic yield of the water deprivation test. But it is quite evident that the test has several limitations and, in many situations, just does not provide an accurate diagnosis. Although relatively newer than the water deprivation test, some of the earliest reports on the potential utility of hypertonic saline infusion in the differential diagnosis of DI dates to the 1940s (44). Hypertonic saline (3% saline, 513 mOsm/L) infusion coupled with plasma copeptin measurement is an alternative test that is now being recommended by many experts in the field of DI as the preferred test to be used in place of water deprivation test.


The test overall lasts for about 3 hours and can be initiated at 08:00 am. Medications that have diuretic or anti-diuretic effects (diuretics, SGLT-2 inhibitors, desmopressin, carbamazepine, chlorpropamide, glucocorticoids, non-steroidal anti-inflammatory drugs) need to be discontinued 24 hours prior to testing (30). The patient lies in a supine position. Two intravenous cannulas are inserted, one for infusion and the other for blood sampling. Before commencing the intravenous infusion, venous sampling is performed to obtain plasma copeptin, serum sodium, glucose, urea, and plasma osmolality (30). Hypertonic saline infusion is then commenced, initially with a bolus dose of 250 ml given over 10 – 15 minutes, followed by a slower infusion rate of 0.15 ml/Kg/min. Serum sodium and osmolality are measured every 30 minutes. The infusion is terminated once the serum sodium is ≥150 mOsm/L (30). Typically, protocols allow up to a maximum of 3 hours of infusion (30,45). At this point, a plasma copeptin is measured and the patient is asked to drink water at 30ml/Kg within 30 minutes (30, 45). This is followed by intravenous infusion of 5% glucose (dextrose) at 500 ml/hour for one hour (30, 45). Serum sodium is measured once more after completing the 5% glucose infusion to ensure its return to normal values. Vital parameters, including blood pressure and heart rate must be constantly assessed, preferably on a monitor.


A plasma copeptin level of <4.9 pmol/L after hypertonic saline infusion indicates central DI (partial and complete) while a level of ≥4.9 pmol/L indicates primary polydipsia (5, 30). It is likely that this cut-off value might be changed to 6.5 pmol/L in the future due to its higher diagnostic accuracy, based on more recent data (described below) (30). Baseline copeptin value of >21.4 pmol/L is indicative of nephrogenic DI (partial and complete) and <2.6 pmol/L indicates complete central DI (4, 5, 30).

The major advantage with the hypertonic saline infusion test is that it basically excludes the renal component out of the equation and any potential down-regulation of aquaporin-2 channels will not affect the diagnosis. This test depends only on two aspects: 1. Increasing the plasma osmolality, which is a strong stimulus for endogenous AVP synthesis and release and 2. Measurement of endogenous AVP secretory capacity by measuring plasma copeptin, which is secreted in equimolar proportions from the posterior pituitary along with AVP (3, 4). Thus, unlike water deprivation test which is mainly a test based on urine output and osmolality, the hypertonic saline infusion test is free from any requirements to measure urinary indices. In fact, when compared with water deprivation test with desmopressin administration with/without plasma copeptin measurement, the hypertonic saline infusion test with plasma copeptin measurements has been demonstrated to be the superior test for diagnosing the various forms of polyuria-polydipsia syndromes (30). Hypertonic saline infusion test also takes less time to perform, causes less patient burden, and is more convenient and tolerable when compared with indirect water deprivation test (30).

A modified hypertonic saline infusion test followed by water deprivation has also been previously described when the diagnosis after water deprivation is inconclusive (46). In this scenario, administration of hypertonic saline (as described in the protocol) followed by measurements of plasma AVP, serum sodium, plasma and urine osmolality can potentially differentiate the types of DI when the plasma AVP levels are plotted on a nomogram relating plasma AVP values to either urine osmolality or serum sodium/plasma osmolality (normal plasma AVP for urine osmolality or serum sodium/plasma osmolality suggests primary polydipsia, low plasma AVP for serum sodium/plasma osmolality suggests central DI, and low urine osmolality for plasma AVP levels suggests nephrogenic DI). However, this test is more cumbersome than either indirect water deprivation test or hypertonic saline infusion test alone and requires measurements of plasma AVP levels which is also challenging (as described above. Measurement of plasma AVP’). Therefore, this combined method is not utilized in routine clinical practice. With the advent of plasma copeptin assays and with the ability to eliminate urine measurements out of the equation for the regular hypertonic saline infusion test, the plasma AVP measurement-based combined water deprivation and hypertonic saline infusion test is likely going to be obsolete in the future.

Recent data by Fenske et al. have demonstrated the diagnostic accuracy of hypertonic saline infusion test combined with plasma copeptin measurement to be higher (96.5%) than that of water deprivation test (76.6%) in correctly identifying the type of polyuria-polydipsia syndrome (30). Even when it came to differentiating those with partial central DI from patients with primary polydipsia, the hypertonic saline infusion test with plasma copeptin measurement had a higher accuracy (95.2%) as compared to water deprivation test without plasma copeptin measurement (73.3%) (30). Data from the same study has also shown that a plasma copeptin value of 6.5 pmol/L provides the best diagnostic accuracy (97.9%) for differentiating central DI (partial and complete) from primary polydipsia (vs. 96.5% for the plasma copeptin cut-off level of 4.9 pmol/L), with a value of >6.5 pmol/L being consistent with primary polydipsia. Surprisingly, in the same study, water deprivation test followed by plasma copeptin measurement demonstrated a lower diagnostic accuracy when compared to water deprivation test alone without plasma copeptin measurement (44% vs. 76.6%). These results show that combining hypertonic saline infusion and plasma copeptin measurements currently offers the best diagnostic capability with distinguishing the various forms of hypotonic polyuric states. The protocol for the hypertonic saline infusion test, interpretation of the results, and limitations are presented in Table 3.

Table 3. Hypertonic Saline Infusion Test (with plasma copeptin measurement)


Preparation phase:

· Test can begin at 08:00AM.

· Drugs that can affect urine output (diuretics, sodium-glucose co-transporter-2 inhibitors, glucocorticoids, non-steroidal anti-inflammatory drugs) must be held 24 hours prior to dehydration.

· Patient lies in a supine position. Two intravenous cannulas are placed: one for blood sampling and the other for infusion.

· Baseline serum sodium, glucose, urea, plasma osmolality and plasma copeptin are obtained prior to infusion.

Hypertonic saline infusion phase:

· Hypertonic saline infusion is commenced: Bolus dose of 250ml given over 10 – 15 minutes, followed by 0.15 ml/Kg/min.

· Serum sodium and osmolality are measured every 30 minutes.

· The infusion is stopped if:

o The serum sodium raises to >150 mmol/L.

o The infusion is completed for 3 hours.

· Plasma copeptin is measured after the infusion is stopped.

· Heart rate and blood pressure are continuously monitored throughout the phase.

Hypotonic fluid administration phase:

· Patient is asked to drink water at 30ml/Kg within 30 minutes.

· This is followed by intravenous infusion of 5% glucose at 500ml/hour for 1 hour.

· Serum sodium is measured after the completion of 5% glucose infusion to ensure its return to normal values.

· Heart rate and blood pressure are continuously monitored throughout the phase.


Central DI:

o Baseline plasma copeptin <2.6 pmol/L prior to hypertonic saline infusion (complete central DI).

o Plasma copeptin level of ≤4.9 pmol/L after hypertonic saline infusion (partial or complete). *

Nephrogenic DI:

o Baseline plasma copeptin ≥21.4 pmol/L prior to hypertonic saline infusion (partial or complete).

Primary polydipsia:

o Plasma copeptin level of >4.9 pmol/L after hypertonic saline infusion. *


· Takes less time to perform than water deprivation test.

· Eliminates the need to obtain urine studies which can be affected by aquaporin-2 channel availability, thus reducing the chances of confounding results.

· Hypertonic saline is a stronger osmotic stimulus when compared to dehydration and is therefore more potent with causing AVP release in cases of partial central DI or chronic primary polydipsia. So, this is likely to be a better test to utilize in cases of partial central DI and chronic primary polydipsia.

· Distinguishes central DI (complete and partial) from primary polydipsia with a higher accuracy when compared with water deprivation (with/without plasma copeptin) test.

· Less burdensome and more convenient for the patients.


· Several centers mandate the use of central lines for hypertonic saline administration.

· Hypertonic saline infusion has the theoretical risk of causing superficial thrombophlebitis.

· Higher risk for hypernatremia when compared to water deprivation test.

· Plasma copeptin assays are currently of limited availability.

*A higher cut-off value of 6.5 pmol/L distinguishes central DI from primary polydipsia with increased accuracy based on newer data, with a value of >6.5 pmol/L being consistent with primary polydipsia (Fenske et al., 2018).


The challenges with utilizing hypertonic saline infusion test are as follows: 1. Hypertonic saline has been claimed to cause thrombophlebitis when administered through peripheral intravenous line. However, this risk is overestimated and is rarely observed in practice (47), 2. Several institutions require placement of a central venous catheter and admission to an intensive care unit for administration of hypertonic saline. Central line insertion is a more invasive and time-consuming procedure when compared to a peripheral intravenous line and can cause more bleeding or infections, and an admission to an intensive care unit exerts substantial expenditure on the patient and the institute, and 3. Copeptin assays are not yet commercially available in several countries.


Once the type of polyuria-polydipsia syndrome is identified, efforts must be undertaken to diagnose the underlying pathology responsible for this clinical presentation. A detailed list of the potential causes for each type of DI and primary polydipsia is provided in Table 1. In cases of central DI, a thorough clinical history should be obtained, and a detailed physical exam needs to be performed to evaluate for the signs and symptoms of hormonal deficiencies (or excess in cases of hyperprolactinemia) from other pituitary axes (9, 48). Biochemical evaluation must include a morning plasma measurement of pituitary hormones (growth hormone, prolactin, ACTH, TSH, FSH, and LH), and the hormones from their target organs (insulin-like growth factor 1, cortisol, free thyroxine, total and free testosterone, estradiol). An MRI of the sella and suprasellar regions with gadolinium needs to be obtained to evaluate for any anatomical disruptions of the pituitary or hypothalamic anatomy (macroadenomas, empty sella, infiltrative diseases). The normal posterior pituitary demonstrates hyperintensity on T1 images (also known as the ‘bright spot’), suggested to be due to phospholipid-rich granules storing AVP and oxytocin (Figure 5) (49). The absence of this bright spot could indicate an absence of posterior pituitary function. However, this should not be used as a sole criterion to attribute a pituitary etiology as causing the DI, because absence of the bright spot on the pituitary MRI is also seen in up to 25% of normal individuals and may disappear with aging (50). Also, an enlarged/thickened pituitary infundibular stalk can be found on the MRI, which may be seen in cases of hypophysitis, granulomatous disorders, tuberculosis, craniopharyngioma, germinoma or a metastasis to the sella or suprasellar region (3, 9, 51). The hypothalamus is the site of AVP synthesis (Figure 3) and any pathological involvement of the region (inflammatory, infectious, vascular, or neoplastic process) can certainly result in the destruction of the hypothalamus leading to deficiency of AVP synthesis (3). This is specifically important among patients with adipsic DI as the thirst center might be disrupted due to the above-mentioned etiologies that can involve the hypothalamus. Any history of cranial trauma or intra-cranial surgery could also give rise to central DI.

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Figure 5. Magnetic resonance imaging (MRI) of the pituitary gland. The above image is a non-contrast T1 MRI image of a normal pituitary gland. The white arrow point towards the ‘bright spot’ seen in the posterior pituitary. This finding is a result of phospholipid-rich granules that store arginine vasopressin (AVP) and oxytocin. Image courtesy: NIDDK, NIH.

In cases of DI with onset during infancy or early childhood, genetic/congenital causes must be suspected. Some of the genetic conditions causing DI include AVP-neurophysin II gene alterations (AVP-NP II,autosomal dominant), Wolfram syndrome, an autosomal recessive disorder caused by alterations inWFS1(4p16.1) associated with diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (OMIM 22300, DIDMOAD syndrome), and other autosomal recessive disorders due to production of mutant, weaker forms of AVP, and X-linked recessive disorders resulting in sub-normal plasma AVP levels (52-55). Congenital malformations, such as septo-optic dysplasia (HESX1, OMIM601802, 3p14), Schinzel-Giedion midface retraction syndrome (SETBP1, OMIM 611060, 18q12), Culler-Jones syndrome (GLI2,OMIM 165230, 2q14), Alstrom syndrome (ALMS1, OMIM606844, 2p13), Hartsfield syndrome (FGFR, OMIM615465, 8p11), Webb-Dattani syndrome (ARNT2, OMIM 606036, 15q25), amongst others, can also give rise to childhood-onset DI (56-60).

Nephrogenic DI in most cases is acquired, usually in the setting of intake of certain drugs like lithium, demeclocycline, pemetrexed, cisplatin, and others (6, 7, 61). Therefore, a review of the patient’s medication intake history can lead to the identification of the potential culprit medication, which can then be discontinued. Use of osmotic diuretics or electrolyte abnormalities such as hypercalcemia or hypokalemia must be investigated in cases of nephrogenic DI (6, 29). Any underlying acute or chronic renal disease (vascular, inflammatory, or neoplastic processes, polycystic kidney disease), obstructive uropathy, and systemic diseases such as amyloidosis or sickle cell disease can also give rise to nephrogenic DI and prompt evaluation for these disorders is necessary (6, 7). Congenital causes for nephrogenic DI include mutations in the gene for aquaporin-2 receptor (autosomal recessive) and the gene for V-2 receptor (X-linked recessive inheritance), and must be suspected in childhood-onset nephrogenic DI (7). Other genetic causes of nephrogenic DI include; polyhydramnios, megalencephaly, and symptomatic epilepsy (STRADA, OMIM 608626, 17q23) and some Barter syndrome subtypes, including Bartter syndrome, type 4b, digenic forms (CLCNKA, OMIM 602024, 1p36.13) (62, 63).

Primary polydipsia or dipsogenic DI is often seen in individuals on treatment for mood disorders or schizophrenia (64). The dry mouth caused by intake of medications with strong anticholinergic properties to treat these disorders is most likely the cause for excessive water intake (64). Hypothalamic disease (sarcoidosis, tuberculosis, trauma, neoplasms) can alter the thirst response by lowering the thirst threshold, either by disruption of the thirst center or through osmoceptor dysfunction, which leads to polydipsia (3, 7). Primary polydipsia which results from this subset of cases with low thirst threshold in hypothalamus, is also referred to as ‘dipsogenic DI’. In other cases, individuals are just habitual compulsive drinkers of large volumes of water without any underlying organic or pharmacologic cause (6).

A unique presentation of DI which is worthy of note, is the one that occurs during pregnancy. Also known as gestational diabetes insipidus, this disorder occurs during pregnancy due to the enzymatic breakdown of the endogenous AVP by a placental cysteine aminopeptidase (6, 7). In women with borderline-low plasma AVP levels, pregnancy can unmask sub-clinical DI from enzymatic degradation of AVP (8). However, work-up for other etiologies of DI must be considered when appropriate in this situation.

If none of the modalities succeed in establishing an appropriate diagnosis of DI, a therapeutic trial with desmopressin can be undertaken (3, 46). The therapeutic trial can be provided in the form of desmopressin tablets of 100 µg by mouth every 8 hours for 48 hours. If this treatment abolishes polyuria, normalizes serum sodium/plasma osmolality or at least brings down serum sodium to near normal levels, resulting in elimination of thirst, the diagnosis is most certainly central DI. If there is cessation of polyuria and there is slight normalization of serum sodium/plasma osmolality without reduction in polydipsia/thirst, the most likely diagnosis is primary polydipsia. In nephrogenic DI, therapeutic trial with desmopressin does not result in any significant changes in serum sodium or plasma and urine osmolality (46). This method can distinguish between various forms of DI with 90% accuracy (46). However, caution is advised when utilizing desmopressin for diagnostic testing due to the risk of hyponatremia, which can sometimes be severe in patients with primary polydipsia and the test is preferably done in a monitored in-patient setting (46).


A major forthcoming change that could be anticipated is the renaming of the term, “diabetes insipidus/DI”. An expert panel comprising physicians from various endocrine societies around the globe, ‘The Working Group for Renaming Diabetes Insipidus’, has recommended to rename ‘central diabetes insipidus’ as ‘arginine vasopressin deficiency (AVP-D)’, and ‘nephrogenic diabetes insipidus’ as ‘arginine vasopressin resistance (AVP-R)’ (65). While gestational DI has not been specifically brought up in this proposal, it may be apt to rename the condition as ‘gestational AVP-D.’ Similarly, ‘adipsic DI’ could be renamed as ‘adipsic AVP-D.’ There are two main reasons for this proposal. One reason is that the current name (DI) is not reflective of the underlying pathophysiologic mechanism as this term was coined in the 18th century when AVP was not yet discovered (66). The second reason is that both healthcare workers (especially those in non-endocrine-related fields) as well as patients tend to confuse the term ‘diabetes insipidus’ with the more common ‘diabetes mellitus’. This confusion has led to mismanagement of DI resulting in catastrophic consequences (67, 68). In fact, a large survey with 1034 patients with central DI showed that 80% participants encountered a scenario where a healthcare professional confused DI with DM, and 85% of participants supported renaming DI. The proposed plan for the upcoming years is to start transitioning to the new terms while retaining the old terms in parenthesis till the medical community and patients get used to the new terminologies.

With regards to diagnostic testing, further refinements are implemented to the copeptin-based testing, such as utilization of non-osmotic neurohypophyseal secretagogues, to minimize adverse effects or discomfort related to iatrogenic hypernatremia induced by hypertonic saline. Arginine is a non-osmotic stimulus to the posterior pituitary, and it increases plasma copeptin levels in healthy individuals (69). Arginine-stimulated copeptin measurement is being evaluated as a potential diagnostic test for central DI and primary polydipsia. In a recent prospective study, intravenous arginine-stimulation was able to distinguish patients with central DI, and primary polydipsia, and healthy controls using the 60-minute post-stimulation plasma copeptin levels, with high diagnostic accuracy (69). Glucagon, another neurohypophyseal stimulant, was also recently evaluated in a double-blind, placebo-controlled, randomized trial for differential diagnosis of DI (70). Glucagon injection led to significant increase in plasma copeptin levels in healthy controls compared to placebo, while there was a minimal increase in plasma copeptin in central DI patients, there was a substantial increase in patients with primary polydipsia. Further studies performing head-to-head prospective comparisons with hypertonic saline infusion test are required to identify the best copeptin-based diagnostic test.


Making an accurate diagnosis of DI and ascertaining its type and the underlying etiology poses a significant challenge to this day. In clinical practice, under some circumstances, a diagnosis of DI is established based on serum/plasma and urine studies alone and based on the history and clinical presentation, investigations to identify the underlying etiology are pursued. However, an accurate diagnosis of the type of DI or any polyuria-polydipsia syndrome in general, is difficult to establish as there can be a significant overlap in the results among the various forms of polyuria-polydipsia syndromes on diagnostic testing. Specific testing protocols, such as the indirect water deprivation test or the hypertonic saline infusion test can assist with providing a diagnosis with increased accuracy. Measurement of plasma AVP levels is not routinely performed due to several pre-analytical considerations and lack of widespread availability of assays. With the promising potential of the utility of plasma copeptin levels, diagnosing DI might get less cumbersome in the future once this assay is standardized across laboratories worldwide and once the test becomes commercially available on the global market. The combination of hypertonic saline infusion coupled with plasma copeptin level measurement has achieved diagnostic accuracies that have not been previously attained by any other testing modalities with regards to differential diagnosis of polyuria-polydipsia syndromes (30). But the newer non-osmotic, neurohypophyseal secretagogue (arginine and glucagon)-based plasma copeptin measurements hold the promise of being the safer, less cumbersome testing modalities. However, randomized trials comparing these modalities with hypertonic saline infusion are necessary, with cost and regional availability of these agents also taken into consideration. Therefore, hypertonic saline infusion and plasma copeptin-based approach could potentially become the standard of practice in the future to accurately establish the diagnosis of DI and related polyuria-polydipsia syndromes.


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Hypopituitarism is described as partial or complete loss of isolated or combined deficits of pituitary hormones. The clinical presentation of this condition varies depending on the age of onset, underlying cause of the disease, severity of deficiency and number of affected hormones. Investigation to confirm the diagnosis is based on basal hormone status and dynamic stimulatory tests. In this review, we will provide a concise background knowledge regarding underlying etiologies, clinical manifestations, hormonal investigations, and related treatments which necessitate close lifelong monitoring.


Hypopituitarism refers to complete or partial failure of secretion of anterior and/or posterior pituitary hormones. It may arise as a result of congenital defects in the development of individual anterior pituitary cell types or hypothalamic function, acquired disease of the pituitary or hypothalamus, or from infundibular lesions which interfere with the hypothalamic control of the pituitary. The multiple aspects of normal pituitary function serve to predict the wide range of clinical manifestations of hypopituitarism which are determined by the severity, extent and duration of the condition. Onset may occur during childhood or adult life and is generally permanent, requiring one or more specific hormone replacements.


The major causes of hypopituitarism are shown in Table 1.

Table 1. Causes of Hypopituitarism


Isolated pituitary hormone deficiency

· Receptor mutation: GHRH, CRH, GnRH, TRH receptor mutations

· Transcription factor defect: PITX2, TBX19, DAX1, NR5A1, NR0B1

· Hormone mutation: GH1, bio-inactive GH, FSHb, LHb, TSHb, POMC, POMC processing defect

· Prader-Willi syndrome

· Bardet-Biedl syndrome

· Kallmann syndrome

Multiple pituitary hormone deficiency

· Transcription factor defect: HESX1, SOX 2/3, LHX3/4, PROP1, POU1F1, IGSF1 mutations

· Prohormone convertase enzyme mutation: PC1


Pituitary adenoma

· Functioning and Non-functioning

Peri-pituitary tumors

· Parasellar lesion: craniopharyngioma, Rathke's cleft cyst

· Non-adenomatous neoplasm: meningioma, glioma, germ cell tumor

· Metastases, especially breast, renal, bronchus


Infarction/ hemorrhage

· Sheehan's syndrome

· Pituitary apoplexy

· Aneurysms

Inflammatory /

Infiltrative / Immunological

· Hypophysitis: lymphocytic, granulomatous, xanthomatous, necrotizing, IgG4-related, immunotherapy-induced (CTLA-4 inhibitors), other immune-associated

· Hemochromatosis

· Sarcoidosis

· Wegener's granulomatosis

· Giant cell granuloma

· Langerhans cell histiocytosis


· Bacterial: tuberculosis, syphilis, leptospirosis

· Fungal: candidiasis, aspergillosis

· Viral: Herpes/Varicella infection, SARS-CoV-2 virus


· Pituitary

· Nasopharyngeal

· Cranial


· Empty sella

· Traumatic brain injury

· Medications


Formation of the normal pituitary during embryonic development depends on the juxtaposition of cells of neuroectodermal origin, which form the posterior pituitary, and endodermal cells derived from the primitive stomodeum, which form the anterior pituitary. Congenital forms of hypopituitarism are best considered as being derived either from hypothalamic or pituitary origin. Defects of RPX/HESX1, PROP1, and PIT1 are associated with varying degrees of inherited hypopituitarism in humans (1). Autosomal dominant mutations of the arginine vasopressin-neurophysin II gene give rise to familial antidiuretic hormone (ADH) deficiency (central diabetes insipidus, CDI). Congenital combined pituitary hormone deficiency (CPHD) (or multiple pituitary hormone deficiency (MPHD)) may be associated with hypoplasia of the anterior pituitary and ectopic siting of the posterior pituitary in a superior position. The underlying mechanism for this condition, once thought to be a consequence of birth trauma, remains undetermined but a congenital molecular defect is likely. Furthermore, since some genetic mutations affect early development, they may include extra-pituitary features or midline anomalies as a part of a syndrome. For instance, the transcription factors, including HESX1, SOX2, SOX3, and OTX2, are the most common genes involved in the etiology of septo-optic dysplasia. Hypogonadotropic hypogonadism is a recognized feature of both Prader-Willi and Bardet-Biedl syndromes. The salient features of the currently described transcription factor defects are indicated below.


HESX1 is a member of the homeobox gene family. It is one of the earliest markers of the pituitary primodium expressed as a prelude to the development of Rathke's pouch. Mutations of the HESX1 gene in humans are associated with septo-optic dysplasia and evolving hypopituitarism. Septo-optic dysplasia is characterized by the classical triad of optic nerve hypoplasia, midline neuroradiological abnormalities such as agenesis of the corpus callosum, and pituitary hypoplasia with consequent panhypopituitarism (2). Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and its consequences are more extensive.


The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain. Individuals with a single inactivating mutation in PROP1 have deficiencies of luteinizing hormone (LH), follicle stimulating hormone (FSH), GH, prolactin, and TSH (3). Their pituitary glands may be small, normally sized, or large with extrasellar extension. Pituitary degeneration may produce acquired deficiency of adrenocorticotropic hormone (ACTH). Growth restriction and GH responses to GHRH stimulation are more variable in children with PROP1 mutation. This variability does not seem to be dependent on the type of PROP1 mutation because variability exists within sibships. Recessive mutation in PROP1 is the most frequent genetic cause of CPHD and appears to be much more common than POU1F1 mutation (4,5). Clinical suspicion of these mutations should be high in any individual with early onset CPHD, even in those with an intrasellar or suprasellar mass lesion. However, the clinical manifestations of each hormone may vary in onset and severity. GHD is usually present in the first years of life; whereas TSH and gonadotropin deficiency might manifest at birth or later in life. Most patients do not experience adrenal insufficiency during the first years of life although this can evolve later.


PIT1, also known as POU1F1, is a pituitary specific transcription factor found in somatotrophs, lactotrophs, and thyrotrophs in the anterior pituitary gland from early fetal development and throughout life. Autosomal recessive defects of PIT1 are associated with combined deficiencies of growth hormone (GH), prolactin, and thyrotropin stimulating hormone (TSH) (6,7). Patients with PIT1 mutations (coded by POU1F1 gene) tend to firstly manifest with GHD and prolactin deficiency in the first year of life and generally do not release detectable amounts of GH after GHRH stimulation. TSH deficiency typically develops later in life (8).


SOX2 belongs to the SRY-related HMG box (SOX) family of transcription factors which are expressed in various stages of embryonic development and cell differentiation, and play critical roles from the earliest stages of development, in particular expression of anterior neuroectoderm. De novo truncating mutations of SOX2 are found in individuals with bilateral anophthalmia/microphthalmia, small corpus callosum, hippocampal abnormalities, variable mental retardation, anterior pituitary hypoplasia and often hypopituitarism (9-11)


SOX3 also belongs to the SRY-related HMG box (SOX) family of transcription factors. This dosage sensitive gene is essential for normal hypothalamic-pituitary development (12). Patients manifest with variable hypopituitarism, infundibular hypoplasia, abnormal corpus callosum, hypoplasia of anterior pituitary, absent stalk, ectopic/undescended posterior pituitary, and intellectual disability (13).


OTX2 belongs to the orthodenticle homeobox (OTX) and plays an essential role in brain, eyes, and pituitary development. Patients with OTX2 mutations manifest with variable degrees of pituitary dysfunction, intellectual disability, ocular abnormalities including microphthalmia, anophthalmia, and abnormal peripapillary pigmentation (14).


Mutations in DAX1 (coded by NR0B1), which is another X chromosome gene, cause hypogonadotropic hypogonadism in association with congenital adrenal hypoplasia in males. DAX1 encodes an orphan nuclear hormone receptor with a novel DNA-binding domain, that has a critical role in the development of the hypothalamus, pituitary, adrenal, and gonads (15). DAX1 appears to influence the maintenance of testicular epithelial integrity and spermatogenesis.


Isolated gonadotropin deficiency with hyposmia (Kallmann syndrome) may be inherited as an autosomal X-linked disorder or occur sporadically. The X-linked disorder occurs in approximately 1/10,000 to 60,000 live births (16) and a mutation in the KAL1 gene is the basis for this disease. Gonadotropin-releasing hormone (GnRH) neurons develop in the medial olfactory placode and migrate to the hypothalamus during embryonic development; the KAL protein is essential for the initiation and maintenance of this process. Patients with the disorder usually present in late adolescence with delayed pubertal initiation or progression and characteristically exhibit hyposmia. They may develop eunuchoid proportions and usually demonstrate small testes and a short penis; other features which are specific to X-linked Kallmann syndrome include unilateral renal agenesis and synkinesis (mirror movements), whereas midline facial defects, choanal atresia, short metacarpals, malrotation of the gut, and ocular coloboma are more common in autosomal and sporadic forms of the condition.


Mutations in the major GH gene, GH1, can occur as deletions of different size, resulting in severe isolated GH deficiency and may be familial with either a dominant or recessive pattern of inheritance.


TBX19, also known as TPIT, is essential for both pro-opiomelanocortin (POMC) transcription and terminal differentiation of POMC-expressing cells. A mutation of TBX19 results in congenital isolated ACTH deficiency and this mutation is discovered in 65% of patients with neonatal-onset congenital isolated ACTH deficiency (17).

Other single gene mutations affecting individual anterior pituitary hormones or pituitary receptors for hypothalamic peptides are indicated in Table 1.


Pituitary adenomas are the most common cause of adult-onset hypopituitarism (18,19). Craniopharyngiomas are numerically the next most prevalent and may present as sellar and/or suprasellar masses.


Pituitary tumors can be either non-functioning or secrete one or more anterior pituitary hormones. They are classified according to size and the presence of extrasellar extension; magnetic resonance imaging (MRI) permits accurate measurement of maximum diameter and tumors are conventionally classified as microadenomas (less than 10mm), and macroadenomas (more than 20mm in diameter) (20). The reported prevalence of pituitary tumors is approximately 10 per million with an average annual incidence of approximately 30 per million depending on tumor type, age, and gender with the highest incidence occurring in pre-menopausal women. Pituitary adenomas may cause typical clinical syndromes resulting from hypersecretion of one or more anterior pituitary hormones. Approximately 25-30% will not present with these symptoms, so they will only be detected when tumor expansion and local compression of the surrounding structures emerges. Post-mortem studies reveal a prevalence of incidental intrasellar adenomas of 10-20%.

Clinically non-functioning pituitary adenomas (NFPA) are non- or low grade-secreting tumors which frequently synthesize and secrete free alpha or beta glycoprotein subunits but do not cause clinically recognized symptomatology. Hence, the treatment of choice is surgical debulking, and sometimes followed by external radiation therapy. In contrast to patients with secreting pituitary tumor who present with clinical syndromes of acromegaly, hyperprolactinemia, Cushing's disease, and secondary hyperthyroidism non-functioning tumors present as an incidental finding, hypopituitarism, or due to mass effects of the tumor. The mainstream treatment in these diseases must be surgical therapy, except prolactinoma which is typically responsive to dopamine agonists.


Craniopharyngiomas are benign neoplastic lesions which are presumed to originate from the embryological remnants of Rathke's pouch. They may be located in the suprasellar region, within the sella, or both. Due to the infiltration of surrounding structures, they may have extensive adverse consequences. This tumor accounts for 1% of all intracranial tumors in adults and 6-13% of intracranial tumors in children (21). Peak incidence is in the first decade with a subsequent increase in incidence between 50-60 years of age. Adamantinomatous craniopharyngiomas commonly occur in children and typically contain both solid and cystic components containing a lipid rich secretion. By contrast, squamous papillary craniopharyngiomas develop in adults and are rare in younger patients. This type of tumor is predominantly solid tumor with small well-defined cavities containing less lipid rich fluid. Craniopharyngiomas in adults have a generally better prognosis in respect to endocrine, visual, and other neurological deficits than those in children (22).


Epithelial cysts known as Rathke's cysts are thought to originate from the remnants of Rathke's pouch. Their location can be intrasellar, with or without suprasellar extension and, rarely purely suprasellar. Compared to craniopharyngioma, they are more common in adults (23). Possible symptoms of Rathke's cleft cysts include pituitary hypofunction, hyperprolactinemia brought on by stalk disruption, visual disturbance, and headache. Due to the difficulty in differentiation between craniopharyngiomas and Rathke's cysts from both a clinical and radiologic standpoint, the diagnosis of an intrasellar Rathke’s cyst cannot always be made with absolute certainty. Definitive diagnosis is often only made histologically. Only 6.4% of asymptomatic patients in a recent retrospective cohort showed cystic enlargement over 41 months follow-up and no pituitary deficiency, confirming the safety of conservative management in these patients (24).


Peri-sellar meningiomas are well-circumscribed masses, originating from the sphenoid ridge and are associated with varying degrees of hyperostosis. Meningiomas are typically single, but they can occasionally be multiple. However, females are more likely to develop multiple meningiomas.

Primary intracranial germ cell tumors are neoplasms that arise from aberrantly migrated primordial germ cells. According to histology, the tumor types are similar to those occurring in the gonads and are classified into two categories: germinomas, which are comparable to seminomas and dysgerminomas, and non-germinomatous germ cell tumors, which can include teratomas, yolk sac tumors, embryonal carcinomas, and choriocarcinomas. Geographically, their incidence varies; it accounts for 2-3% of all childhood primary CNS tumors in Western countries, whereas the incidence is higher at 4-15% in Japan (25-27). Patients commonly present in the first two decades of life with diabetes insipidus, visual failure, and hypopituitarism. A recent report highlighted the favorable response to a combined chemotherapy-radiotherapy protocol (27).

Metastasis to the pituitary gland may present as an intrasellar mass resulting in hypopituitarism, diabetes insipidus, or pressure effects. In a published series of 500 consecutive autopsy examinations of cancer patients in whom the pituitary fossa and gland were examined, pituitary metastases were found in 3.6% (28). Pituitary metastases have been described in patients with primary malignant tumors of breast, lung, kidney, thyroid, bladder, uterus, pancreas, and colon.

Vascular Causes

The term pituitary apoplexy denotes the clinical consequences of hemorrhage or infarction in a pre-existing adenoma. The expansion of pituitary mass to neighboring cranial nerves, cavernous sinus, optic pathways, or diencephalon results in localizing signs or altered conscious state. It frequently manifests as a sudden onset of severe headache, a visual disturbance, or ophthalmoplegia as a result of cranial nerve III IV or VI palsies (29). The clinical syndrome of pituitary apoplexy usually evolves fully within only hours to two days. The incidence of surgically treated pituitary adenomas ranges from 0.6 to 9.1%, (30) with a broad age range of occurrence from the first to the ninth decade. Although apoplexy usually occurs spontaneously, systolic and/or diastolic hypertension was seen in 26% of patient in a retrospective analysis, making it an important predisposing factor. (31). Other reported predisposing factors include diabetes mellitus, radiation therapy, anticoagulant therapy, bleeding disorders, head trauma, sudden changes in arterial or intracranial pressure, such as during carotid angiography, the use of bromocriptine, and postpartum hemorrhage (Sheehan's syndrome). Sheehan's syndrome, which denotes pituitary necrosis after postpartum hemorrhage and hypovolemia, may result in hypopituitarism, that is either immediate or delayed over several years depending on the extent of tissue destruction. However, this condition is rare with modern obstetric care (32).

Suprasellar or intrasellar aneurysms of the carotid arteries, as well as suprasellar aneurysms of the anterior or posterior communicating arteries can manifest as an expanding mass inside the fossa. Hypopituitarism may result directly from intrasellar aneurysms. Histologically, typical degenerative sclerotic changes of the arterial wall are found along with chronic inflammatory changes. An arteriovenous fistula may occasionally develop in the cavernous sinus as a result of an aneurysm.

Immunological/Inflammatory Disease

Inflammatory lesions of the pituitary gland can clinically and radiologically mimic pituitary tumors, with varying degrees of hypopituitarism and mass effects like headaches and visual field impairment.

Lymphocytic hypophysitis classically presents as partial hypopituitarism associated with a pituitary mass lesion, particularly in relation to pregnancy (33). It has been speculated that many cases of postpartum pituitary failure, previously attributed to Sheehan's syndrome, may in fact have been caused by lymphocytic hypophysitis (34,35). In this condition, the interstitial tissue of the adenohypophysis is more or less densely infiltrated by lymphocytes and plasma cells. As a result, the anterior pituitary exhibits necrosis and subsequent fibrosis of hormone-producing cells. Since the histological findings are similar to those of other organ-specific autoimmune diseases, the primary etiology is considered to be autoimmune (36,37). Partial hypopituitarism, commonly with isolated ACTH deficiency, or with TSH deficiency, is seen in approximately 80% of patients, with LH and FSH generally remaining unaffected.

Sarcoidosis is a multisystem disorder, which is characterized by the presence of non-caseating granulomas. It most often affects lungs and reticuloendothelial system but can involve any organ. Central nervous system sarcoidosis occurs with an incidence of 5-15% (38,39) and less than 1% of patients have symptoms of a hypothalamic-pituitary disease (40). Neurosarcoidosis affects primarily the leptomeninges of the brain base and posterior fossa; the hypothalamus and/or pituitary subsequently experience local granulomatous infiltration. Coexisting optic nerve infiltration occurs in many patients causing central and peripheral visual defects. In terms of anterior pituitary function, LH, FSH and GH deficiency are frequent, but TSH and ACTH are relatively preserved.

Langerhans cell histiocytosis is a rare disease characterized by aberrant proliferation of a specific dendritic (Langerhans) cell belonging to the monocyte-macrophage system (41). Deposits occur at multiple sites within the body, and frequently involve the hypothalamo-pituitary axis (42). Diabetes insipidus is a well-recognized and common feature of this condition but anterior pituitary dysfunction also occurs frequently.

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor, a IgG1 monoclonal antibody against CTLA-4 is used for metastatic melanoma and other neoplasms. As a consequence of immune activation, secondary hypophysitis has been reported in 10-15% of ipilimumab treated patients (36). In severe hypophysitis, immune reactions induced extensive necrosis of the adenohypophyseal architecture. Pituitary autoantibodies against thyrotrophs, corticotrophs, and gonadotrophs were identified in patients with ipilimumab induced hypophysitis (37). Patients usually present with headache and fatigue, the diagnosis of hypopituitarism with multiple deficiencies is made 2-3 months or later after initiation of ipilimumab. Central hypothyroidism is the most frequent deficit, followed by central hypocortisolism, and hypogonadism.


Bacterial pituitary sepsis is a rare phenomenon which may arise as a consequence of hematogenous spread or by extension from sinus or meningeal sepsis with subsequent pituitary abscess formation. Small pituitary abscesses have been described at postmortem in patients dying from septicemia. Chronic infection predisposes to necrosis of pituitary tissue with subsequent hypopituitarism. Pituitary tuberculomas may present as space occupying lesions (43)but this appears to be a very rare phenomenon; isolated manifestation of the disease is rare and it is more likely to occur in the context of generalized tuberculous infection with meningitis. Gumma formation, as a manifestation of tertiary syphilis, may occur in the sella region but is extremely rare.

Fungal pituitary infection may occur as a complication of AIDS (44) and pituitary necrosis with hypopituitarism has been described in toxoplasmosis (45).

Radiation Therapy

Hypopituitarism is a common complication of irradiation administered for pituitary adenomas, head and neck tumors, intracranial malignancy, or as adjunctive cranial irradiation for acute lymphoblastic leukemia. Although fractionated radiotherapy with daily doses of <200cGy is well tolerated and safe in terms of neurological sequelae, the majority of patients who have undergone external irradiation will manifest some degree of pituitary failure during long term follow up. In general, this is a relatively late complication and is rarely evident in less than 3 years in patients with completely normal baseline pituitary function. Growth hormone reserve is particularly susceptible and is progressively more likely with time so that periodic assessment of residual pituitary function is mandatory; a five year follow up study after external pituitary irradiation therapy reported, 100% GH deficiency, 91% LH-FSH deficiency, 77% ACTH deficiency and 42% TSH deficiency (46).

Traumatic Brain Injury

Traumatic brain injury (TBI) is common, the prevalence of endocrine dysfunction in these patients ranges from 15-68% (47), with estimated annual incidence 30 patients per 100,000 population per year. Abnormal axes during the acute phase of injury may recover over time, but other pituitary hormone deficits may evolve later even at 6 months after the initial insult. A prospective longitudinal study looking at patients with severe TBI, found that 6% of their patients had proven severe GHD when evaluated after 12 months (48). Multiple pituitary deficiencies or isolated pituitary deficiencies (TSH or ACTH) were very rare.

Empty Sella Syndrome

An enlarged empty sella may be primary (due to a congenital diaphragmatic defect) or secondary to surgery, radiation, or pituitary infarction. The majority of patients with congenital empty sella have normal pituitary function. Hypopituitarism (and/or hyperprolactinemia) may be found in instances due to previous pituitary disease. Occasionally, a cystic pituitary mass may simulate an empty sella on CT or MRI, necessitating a cisternogram for precise delineation.


The clinical impact of pituitary insufficiency is dependent on the extent and severity of hormone deficiencies (Table 2), the duration of the disease and the age of onset; childhood onset hypopituitarism has consequences for all aspects of somatic development in addition to the pathophysiological effects of specific hormonal deficiencies. In addition, there may be clinical features relating to the mass effect of causative lesion or specific consequences attributable to hypersecretion of prolactin, GH, ACTH or TSH from individual tumor types. Hypopituitarism classically develops in sequential order with the secretion of growth hormone, then gonadotrophins being affected first, subsequently followed by TSH and ACTH. Prolactin deficiency is rarely seen, except in Sheehan's syndrome which is associated with failure of lactation. ADH deficiency is almost never seen as a primary feature of pituitary adenomas but is a usual presenting manifestation of germ cell tumors, pituitary metastases, and granulomatous disorders.

Table 2. Summary of Clinical Features of Hypopituitarism

Hormone deficiency


Symptoms and signs

Adrenocorticotrophic hormone


Fatigue, weakness, dizziness, nausea, vomiting, circulatory failure. As in Addison's disease, except lack of hyperpigmentation, absence of hyperkalemia


Tiredness, pallor, anorexia, nausea, weight loss, myalgia, hypoglycemia



Delayed puberty


Impaired fertility, impotence, reduced libido, decreased muscle mass and strength, decreased bone mass, decreased erythropoiesis and hair growth, fine wrinkles, testicular hypotrophy


Amenorrhea, oligomenorrhea, infertility, loss of libido, dyspareunia, fine wrinkles, breast atrophy, osteoporosis, premature atherosclerosis

Thyroid-stimulating hormone


Growth retardation


Fatigue, cold intolerance, constipation, weight gain, dry skin, slow relaxing reflexes

Growth hormone


Growth retardation, short stature, increased adiposity


Reduced exercise capacity, impaired psychological well­being, increased cardiovascular risk, increased central obesity, reduced lean body mass



Failure of lactation


Galactorrhea, oligo/amenorrhea, loss of libido

Antidiuretic hormone

Polyuria, polydipsia including nocturnal

The clinical features of anterior pituitary hypofunction described in this chapter will concentrate on adult-onset hypopituitarism. Many of the symptoms and signs of a specific pituitary hormone deficiency are similar to those that occur in patients with a primary deficiency of the target gland, but there are exceptions.

Growth Hormone Deficiency

GH deficiency (GHD) in adults is characterized by decreased exercise tolerance, decreased mood and general well-being, decreased quality of life, central adiposity, hyperlipidemia, increased predisposition to atherogenesis, and reduced bone remodeling activity. Fine facial wrinkles may result from a deficiency of growth hormone in addition to hypogonadism. The patient will usually have dry thin skin which contrasts with the thickened skin and increased sweating found in acromegaly. Adults with long-standing GHD are often overweight, have reduced lean body mass, increased fat mass, especially visceral fat, relative insulin resistance, and reduced total bone mass. There is a reduction in cardiac and physical performance. GHD is associated with increases in total cholesterol, LDL-cholesterol and apolipoprotein B (49-51). Studies in Sweden and the UK in patients with hypopituitarism receiving controlled thyroid and steroid hormone replacement but without growth hormone replacement, have demonstrated an approximately two fold increase in cardiovascular mortality compared to the general population. (51) and the increase in standardized mortality ratio is more striking in females (52).

The accumulating evidence suggests that the cardiovascular morbidity cannot be explained solely by suboptimal glucocorticoid, gonadal steroid, or thyroid hormone replacement and unsubstituted growth hormone deficiency is probably an important contributing factor. The decreased bone mineral density found in GHD is associated with increased fracture risk (53).

Adrenocorticotrophic Hormone Deficiency

Cortisol and adrenal androgen secretion are ACTH-dependent and are variably decreased in hypopituitarism. Patients with combined ACTH and LH deficiency are completely androgen deficient, a phenomenon that may be particularly important when considering optimum regimens for gonadal steroid replacement in females. Since aldosterone secretion is largely determined by activation of the renin-angiotensin-aldosterone system, it is relatively preserved in the hypopituitary patients. Major symptoms of ACTH deficiency are non-specific, including fatigue, weakness, headache, anorexia, weight loss, nausea, vomiting, abdominal pain, myalgia, and decreased concentration. Hypoglycemia may be present at diagnosis. Hyponatremia is common and is attributable to reduced renal free water clearance as a result of cortisol deficiency with an additional contribution from TSH deficiency if present. Hyperkalemia, which is a frequent finding in primary adrenal failure, does not occur in patients with hypopituitarism due to the relative preservation of aldosterone secretion. Over and above the effects of secondary hypogonadism, reduced adrenal androgen production further exacerbates loss of body hair, particularly in women.

Gonadotrophin Deficiency

Gonadotrophins are responsible for gonadal sex-steroid production, secondary sexual development, maintenance of secondary sexual characteristics, and fertility.

Gonadotrophin deficiency in males’ results in secondary hypogonadism with consequent testosterone deficiency. Clinical features include loss of libido, erectile impotence, oligospermia, reduced erythropoiesis, and decreased lean body mass. Testosterone, via estradiol derived by aromatization, plays an important role in the regulation of bone mineralization, therefore, hypogonadism results in decreased bone mineral density.

Clinical features of secondary hypogonadism in females include oligo/amenorrhea, breast atrophy, diminished secondary sexual hair especially when combined with ACTH deficiency, and a predisposition to osteoporosis.

Thyroid Stimulating Hormone Deficiency

The clinical features of secondary thyroid failure are similar to those of primary thyroid failure with the exception that weight gain is less likely to be a feature if ACTH deficiency is also present. Classical features include cold intolerance, fatigue, and myalgia. Physical examination may demonstrate periorbital edema and delayed reflex relaxation. Additionally, hyponatremia and normochromic normocytic anemia are observed, with the former being exacerbated by cortisol deficit while the latter is aggravated by secondary hypogonadism and GH deficiency.

Prolactin Deficiency

Prolactin deficiency in females result in puerperal alactogenesis, However, isolated prolactin deficiency is uncommon because the great majority of prolactin deficiency states develop as a result of general anterior pituitary problems or as a result of treatment intervention. On the other hand, hyperprolactinemia, which is caused by stalk disruption and results in a lack of dopaminergic regulation from the hypothalamus, is more frequent and can manifest as galactorrhea, abnormal menstruation, loss of libido, and decreased bone density.

Antidiuretic Hormone Deficiency

Antidiuretic Hormone Deficiency (AHD) previously called central diabetes insipidus (CDI) is characterized by polyuria and polydipsia due to decreased secretion of antidiuretic hormone by the neurosecretory cells terminating in the posterior pituitary. If excessive water excretion exceeds intake, patients will progressively become water-depleted and eventually have a reduction in circulating volume. It occurs more frequently in associated with tumors of the hypothalamus, pituitary metastases, lymphocytic hypophysitis, sarcoidosis, Langerhans cell histiocytosis, craniopharyngiomas, and Rathke's cleft cysts. (54). Additionally, it is commonly observed following neurosurgery and head injury. As cortisol is an essential prerequisite for normal glomerular filtration and free water clearance, it should be highlighted that AHD is masked by co-existing ACTH deficiency and only becomes clinically evident after commencement of glucocorticoid replacement. However, it is extremely unusual for AHD to be a primary manifestation of pituitary adenoma.


In hypopituitarism, basal serum hormone measurements are required to confirm the insufficiency; however, dynamic testing is mandatory for the diagnosis of some deficiencies. Pituitary function testing is required for all patients presenting with pituitary disease or in whom an evolving endocrine deficiency is anticipated, e.g., those who have undergone pituitary or cranial radiotherapy.

Baseline Investigations

Basal concentrations of the anterior pituitary hormones and hormones produced by their respective target glands should be measured. Serum samples should be taken unstressed, with no physiological or pharmacological manipulation, between 7 and 9 AM when serum cortisol and testosterone levels are highest. This is important given that the decision to proceed to

testing is based on these levels. The pituitary hormones may remain within the normal range despite low levels of the target hormones indicating that target gland failure is a consequence of inadequate pituitary stimulation. Baseline investigations (Table 3) are sufficient for the diagnosis of secondary hypothyroidism and hypogonadism and will also confirm virtually complete ACTH deficiency (55).

Table 3. Baseline investigation of pituitary function

1. Adrenocortical axis: serum cortisol (09.00 AM), ACTH
2. Thyroid axis: free T4, TSH
3. Gonadal axis:

Men - testosterone (09.00 AM), SHBG, LH, FSH;

Women - estradiol, LH, FSH, progesterone (Day 21 if menstruating)
4. Prolactin
5. Insulin-like growth factor-1, growth hormone
6. Paired plasma and urine osmolality

ACTH, adrenocorticotropic hormone, FSH, follicle stimulating hormone; LH, luteinizing hormone; SHBG, sex hormone binding globulin

It should be mentioned that the serum total testosterone measurement should be performed in the morning by a reliable assay and the test should be repeated before verifying the diagnosis of testosterone deficiency. Measuring the level of free or bioavailable testosterone level should be performed in those whose total testosterone levels are close to the lower limit of the normal range or whose SHBG is suspected to have altered (56).

Dynamic Testing

Stimulation tests are used when hypofunction is suspected and are designed to assess the reserve capacity to form and secrete hormone. In contrast, suppression tests are used when endocrine hyperfunction is suspected and are designed to determine whether negative-feedback control is intact, as in the administration of glucocorticoids to inhibit corticotrophin secretion in patients with suspected Cushing’s syndrome or in the administration of glucose in patients with suspected acromegaly. Dynamic pituitary function tests may assess the hypothalamic-pituitary unit (e.g., insulin tolerance test, glucagon, and arginine tests) or directly stimulate the anterior pituitary with pharmacological doses of synthetic hypothalamic peptides and the pituitary hormone response measured (e.g., TRH, GnRH, GHRH tests).


Baseline and dynamic tests are valuable in the diagnosis of ACTH deficiency. With virtually complete ACTH deficiency, the 0900h serum cortisol is less than 100 nmol/L (3.625 µg/dL). In contrast, if the serum cortisol is 400-500nmol/L (14.5 – 18.1µg/dL) or more, ACTH deficiency is unlikely. (57-59). Therefore, dynamic testing of ACTH reserve is required if the basal serum cortisol measured lies between 100 and 400-500nmol/L. The insulin tolerance test (ITT) or glucagon test may be used to assess the adequacy of the hypothalamic-pituitary-adrenal axis. If the patient is taking hydrocortisone this should be discontinued for 18-24 hours; while prednisolone cross reacts in the cortisol assay and therefore should not be administered within 24 hours of investigation of adrenocortical reserve. The rationale of the ITT is to produce physiological stress in a controlled environment by inducing hypoglycemia with intravenous insulin. Hypoglycemia is a powerful stimulus which stimulates GH and ACTH release and a rise in serum cortisol levels in the presence of an intact hypothalamic-pituitary axis. Although the safety of ITT has been questioned, the only absolute contraindications are ischemic heart disease, epilepsy or unexplained loss of consciousness, untreated hypothyroidism or hypoadrenalism, and glycogen storage disease (58). A decrement in plasma glucose to less than 2.2 mmol/L (40 mg/dL) is required for the test to be valid. With normal ACTH reserve the serum cortisol should rise to at least 550 nmol/L (20 µg/dL). Patients who show a normal cortisol response can withstand major surgery without corticosteroid replacement; while patients with subnormal responses but satisfactory basal values (> 250 nmol/L (9 µg/dL) may not require regular replacement therapy but should be fully informed and carry a steroid card. The ITT should only be performed by fully trained staff in designated units. Hypoglycemia may occasionally require reversal with intravenous glucose to avoid the risk of cerebral edema, which affects 50% of adults but limited to 10% of children.

The glucagon stimulation test (GST) may be used for the assessment of ACTH/cortisol and GH reserve simultaneously when the ITT is contraindicated (60). The subcutaneous injection of glucagon causes a transient rise in plasma glucose. During the subsequent fall in plasma glucose, ACTH and GH are released and measured. Serum cortisol cut-off values are similar to those described for the ITT. Glucagon is less reliable than the ITT as a test of ACTH/ cortisol reserve; it is a less powerful stress stimulus and hence false positive results are a recognized problem.

The short synacthen (Cortrosyn in the USA) test (SST) was originally introduced as a test for primary adrenal failure. It involves the intramuscular or intravenous injection of a pharmacological dose (250μg) of synthetic ACTH, with measurement of the serum cortisol response at 30 and 60 minutes later. The test does not distinguish primary from secondary adrenal insufficiency, and it cannot directly assess pituitary ACTH reserve. The 30-minute serum cortisol response is advocated as a surrogate test of ACTH reserve and has been widely used because of its simplicity. However, there is no study showing that a normal SST indicates that the hypothalamic-pituitary­-adrenal axis is capable of responding normally to major illness or stress. Demonstrations of good correlations between peak serum cortisol responses on ITT and 30-minute responses on SST have been published and are intuitively predictable. However, false negatives with the SST are well recognized although this may be partially obviated by the use of lower synacthen doses (1μg). Nonetheless, the concern about false negative results and the fact that assessment of GH reserve is also frequently required serve to limit the use of the SST in the investigation of pituitary function.

A recent study comparing the ITT, low dose ACTH, and glucagon stimulation test in the evaluation of the HPA axis and GH-IGF-1 axis in patients with pituitary disorders concluded that all three tests were well correlated in terms of peak cortisol and GH response (61). However, low dose ACTH stimulation gave a higher peak cortisol response. Therefore, the cut-off level for the diagnosis of insufficiency of the HPA axis needs to be individualized for each test. See Chapter Adrenal Insufficiency


Normal GH secretion is pulsatile, with four or six pulses per 24 hours, mostly at night in association with REM sleep. A single measurement of serum GH is rarely useful although the fortuitous coincidence of blood sampling at the time of a GH secretory peak may exclude GHD. A stimulatory test is therefore usually required to assess somatotroph reserve. Most, actions of GH are mediated through hepatically or locally-derived insulin-like growth factor-1 (IGF-1). Measurement of baseline serum IGF-1 is a specific but insensitive test of GHD in patients with pituitary disease. Importantly, IGF-1 declines with normal aging. In adult onset GHD, 30% of patients may have serum IGF-1 levels in the lower half of the age-related reference range, with the percentage increasing with age (62). Therefore, a low serum IGF-1, in an adequately nourished patient without liver dysfunction, strongly supports a diagnosis of GHD, but a normal serum IGF-1 cannot exclude it.

A rigid and precise biochemical diagnosis of GHD is required in the context of justification of growth hormone replacement. The ITT is one of the most reliable provocative tests of GH secretion and currently remains the test of choice for this purpose since it has the advantage of assessing ACTH reserve simultaneously. Severe GHD is defined as a peak GH response to insulin induced hypoglycemia of less than 3ng/mL (9mU/L) (63). Obesity may blunt the GH response to dynamic testing so that the diagnosis of GHD should only be made in the context of structural pituitary disease or previous cranial irradiation and/or additional pituitary hormone deficits.

If the ITT is contraindicated, an alternative test for assessment of GH reserve is required. Glucagon, arginine, a combination of arginine and GHRH or growth hormone-releasing peptides may all be used for this purpose. The combination of GHRH plus arginine is the most powerful provocative test of GH secretion but appropriate normative data are required (64-66). Recently, the FDA approved macimorelin, which is a non-peptidyl agonist of GH secretagogue receptor 1a as a diagnostic agent for diagnosing GH deficiency, and macimorelin stimulation test was known to have good sensitivity and specificity in comparison with ITT (67). However, this oral agent is not widely used due to its cost and limited availability.


In the appropriate clinical context, secondary hypothyroidism can be diagnosed on the basis of a low serum thyroxine (T4, total or free) in the presence of low or low normal TSH (TSH is rarely undetectable in hypopituitarism). However, it should be borne in mind that any systemic illness may produce a reversible reduction in serum T4 (sick euthyroid state).

Dynamic testing using thyrotrophin releasing hormone (TRH) has no diagnostic value for secondary hypothyroidism or predicting a risk of developing TSH deficiency. Although patients with hypothalamic disease may show a delayed response to TRH, this test is seldom used in pituitary reserve assessment. Furthermore, intravenous TRH may precipitate hemorrhagic infarction of pituitary adenomas.


Symptoms of sex steroid deficiency, menstrual disturbance, low serum estradiol or low serum testosterone levels in the presence of normal or low concentrations of FSH/LH are the mainstay of diagnosis of hypogonadotropic hypogonadism. The gonadotrophin releasing hormone (GnRH) test has virtually no diagnostic value but is used to confirm gonadotroph reserve in the setting of pulsatile GnRH therapy for infertility.


Basal investigations include plasma and urine osmolalities obtained simultaneously. In overt AHD, plasma osmolality is usually raised in the presence of inappropriately diluted urine. The diagnosis of partial AHD is confirmed by means of a water deprivation test followed by demonstration of a response to desmopressin. Copeptin is the most recent clinical diagnostic marker for AHD due to its strong correlation with plasma arginine vasopressin (AVP) (68). Copeptin is secreted in equimolar ratio to AVP, mirroring AVP concentrations in the circulation. A copeptin level of 4.9pmol/L stimulated with hypertonic saline infusion differentiates between AHD and primary polydipsia with a high diagnostic accuracy and is superior to the water deprivation test (69). See Chapters on Posterior Pituitary.

Imaging the Pituitary

Magnetic resonance imaging (MRI) is the optimum method of imaging, with computerized tomography (CT) as an acceptable alternative. The only disadvantage to MRI is its insensitivity in defining pathological calcification and lack of signal from corticated bone. CT may be required to demonstrate calcification in craniopharyngiomas and hyperostosis; it is also used by many surgeons to define skeletal anatomy prior to surgery.


Hypopituitarism, once established, is usually permanent. However, resection of pituitary tumors may, on occasion, result in resumption of normal pituitary function.

Although a seemingly straightforward clinical exercise, hormone replacement therapy cannot simulate normal physiology precisely. With the exception of GH replacement and treatment of infertility, replacement therapy is achieved by administering target hormones. The aim of hormone replacement is to safely eliminate or minimize the symptoms and clinical signs of specific hormone deficiencies. The Endocrine Society Clinical Practice Guidelines committee have issued recommended treatment option of each target hormone deficiency either primary or secondary for the management of hypopituitarism (70).


Hydrocortisone is now the most widely used form of glucocorticoid replacement in patients with primary and secondary adrenal insufficiency. There is no universal agreement regarding the appropriate dose, timing, and monitoring of hydrocortisone replacement. Normal individuals demonstrate undetectable serum cortisol and ACTH when asleep at midnight, with a rise during the early hours of the morning to reach a peak at 0800-0900h, followed by a steady decline throughout the rest of the waking day. There are variable peaks of cortisol secretion due to other factors such as stress, meals, and exercise. Since cortisone acetate requires conversion into hydrocortisone under the influence of 11ß-hydroxysteroid dehydrogenase type 1 (11β-HSD1), and the fact that the enzyme activity is altered in patients with GHD; therefore, cortisone renders intuitively less satisfactory than hydrocortisone for replacement purposes in this condition. Prednisolone has been advocated by some on account of its longer duration of action than hydrocortisone. However, because it cannot be routinely measured, it is impossible to fine tune the replacement dose. Dexamethasone, the alternative synthetic glucocorticoid replacement, is even less satisfactory because of wide interindividual variations in metabolic clearance rates.

Traditional hydrocortisone replacement utilized twice daily dosing but many patients report fatigue or headache in the afternoon on this regimen. There is evidence that many patients 'feel better' on thrice daily regimes (71). Recent cross-sectional studies did not demonstrate any superiority between three times a day versus twice daily replacement in terms of quality of life (72). The average daily requirement is approximately 20mg of hydrocortisone. This should be given as 10mg on waking, 5mg at lunchtime and 5mg in the early evening. Enzyme-inducing drugs, especially, phenytoin, carbamazepine, and rifampicin can increase the metabolism of corticosteroids and should prompt an increment in replacement doses. Doses are therefore fine-tuned according to patient’s well-being and multiple serum cortisol levels (hydrocortisone day curve, HCDC) taken during the day in many centers. (55,73). Some centers have attempted to utilize urine free cortisol measurements to adjust the hydrocortisone dose; however, this is unreliable because saturation of cortisol binding globulin (CBG) following oral hydrocortisone results in supraphysiological urine free cortisol excretion (74). The HCDC should demonstrate adequate levels of cortisol throughout the day, without excess peak (cortisol e.g., >1000 nmol/L (36.25 µg/dL)) or trough (e.g., <100 nmol/L (3.625 µg/dL)) levels before or after doses.

In clinical practice, there is no reliable measure to be certain if patients are receiving optimal glucocorticoid replacement therapy. As a result, patients may be over- or under- treated with resultant morbidity (65). There is a significant increase in 11β-HSD1 activity resulting in abnormalities in corticosteroid metabolism in patients with ACTH deficiency treated with conventional doses of hydrocortisone (66). In ACTH-deficient patients daily hydrocortisone dose exceeding 20mg/day is associated with increase waist to hip ratio. The induction of 11β-HSD1 is associated with central adiposity and has an important role in the development of the metabolically adverse hypopituitary phenotype.

Conventional hydrocortisone cannot mimic the circadian rhythms of cortisol release; in particular the early morning rise in cortisol which slowly declines throughout the day. This has led to the development of a modified release formulation of hydrocortisone (MR-HC, Chronocort®) which can be taken late at night thus allowing a delayed and sustained release (75). A study in healthy men demonstrated that MR-HC 20mg and 10mg, given at 2300 and 0700 hours respectively, could achieve a near normal cortisol circadian rhythm (76). A subsequent phase II study demonstrated that the MR-HC mimics the normal circadian pattern closer to physiological baseline (77); however, the studied preparation is no longer available. An alternative modified formulation (Plenadren®) incorporates an immediate release and delayed release components, which displays diurnal plasma cortisol levels similar to physiological profile. Once daily Plenadren® has been shown to reduce weight, blood pressure and improve glucose metabolism when compared with thrice daily dosing (78).

Another newer oral immediate-release granule formulation of HC (Alkindi®) was approved in EU in 2018. This is an immediate-release hydrocortisone preparation specifically designed for pediatric dosing, which is available in four doses: 0.5mg, 1mg, 2mg and 5mg (79).

Table 5 summarizes the glucocorticoid compounds currently available for adrenal insufficiency.

Table 5. Compounds Available for Adrenal Insufficiency





2-3 times/day; waking, early PM, and at least 6 hours from bedtime



Once in morning


Modified release hydrocortisone (Chronocort®)

2times/day; waking and before bed

10mg in AM and 20mg at bedtime

Modified release hydrocortisone (Plenadren®)

Once daily in early morning

Equal to standard HC dose or a 20% increase

Oral immediate release granules (Alkindi®)

Once daily in early morning

Equal to appropriate pediatric hydrocortisone dosing

The effects of continuous subcutaneous hydrocortisone infusion (CSHI) have been compared with conventional oral hydrocortisone in patients with Addison’s disease (80). CSHI produced a more physiological circadian rhythm, normalization of morning ACTH and restoration of nocturnal serum cortisol levels. These infusions are cumbersome and impractical outside of expert centers. Despite a more circadian pattern of cortisol exposure, subcutaneous hydrocortisone infusion was not associated with improved quality of life scores, casting doubt on the potential quality of life effects of circadian cortisol delivery (81).

There is a push to design orally active delayed or sustained release formulations of hydrocortisone to aid the physiological replacement of hydrocortisone and ultimately improve quality of life and side effect profiles in patients requiring lifelong glucocorticoid replacement.

Patients with ACTH deficiency receiving hydrocortisone therapy are unable to respond to surgery, trauma, infections, and severe illnesses by increasing their cortisol concentrations. They therefore require supplemental hydrocortisone therapy with increased oral doses during minor illness, or administration of intramuscular hydrocortisone 100mg four times per day with more severe disease or if oral intake is compromised. When intramuscular injections are contraindicated, a continuous intravenous infusion of hydrocortisone at a rate of 1-3mg per hour provides satisfactory replacement for the severely ill patient. Maintenance mineralocorticoid is not required since aldosterone secretion is usually preserved.

Patients should carry a 'steroid card' and wear a 'medic-alert' bracelet to indicate their requirement for supplemental hydrocortisone in the event of severe illness or trauma. They and their families should understand the importance of life-long compliance, be taught to double the hydrocortisone dose in the event of pyrexial illness, and understand the need for parenteral glucocorticoid replacement if vomiting or diarrhea occurs. An 'emergency' ampoule of hydrocortisone should be provided for domiciliary emergency intramuscular injection and the patient instructed on its use.

Dehydroepiandrosterone (DHEA) is an androgen produced by the adrenal cortex and is also under the regulation of corticotrophin, and is thus deficient in hypopituitarism. In hypopituitarism, DHEA supplementation (25-50mg per day) has shown benefit with respect to well-being and sexual function (82,83). Furthermore, in patients who are replaced with GH, DHEA can augment the IGF-1 response hence leading to a reduction of GH dose in females (84). Currently, there is no licensed preparation of DHEA available, and it is considered to be a food supplement rather than a bioactive drug. However, not all patients respond. Moreover, the androgenic side effects such as greasy skin, acne, and increased body hair may be a limiting factor although generally responsive to dose reduction.

Secondary Hypothyroidism

Synthetic levothyroxine sodium (for example, Synthroid) is the preferred form of replacement. It has a long half-life, allowing a once daily dose. Liothyronine (T3) displays superior gastrointestinal absorption, but its short half-life requires two to three daily doses. Use of T3 is largely restricted to thyroid cancer patients undergoing frequent isotopic imaging or treatment, and occasionally the initiation of thyroid hormone replacement when a gradual increase is desired. Although some experts consider using a T4/T3 combination as an experimental treatment for patients with persistent hypothyroid symptoms on LT4 since patients with normal thyrotropin levels may have lower T3 levels, the evidence is primarily limited to athyreotic patients.(85,86)

Commencing thyroid hormone replacement in patients with severe, untreated ACTH deficiency may result in hypoadrenal crisis. In the situation of combined deficiency, hydrocortisone should always be commenced before thyroxine. The duration of hypopituitarism and presence of co-morbidities, especially ischemic heart disease, should be considered. Young patients with a short history of hypopituitarism and TSH deficiency may commence an initial thyroxine dose of 100µg daily. On the other hand, in patients with a long history of hypopituitarism or elderly patients, a low dose of 25-50µg daily should be commenced, in order to minimize the risk of precipitation of cardiac events.

Alteration of the hypothalamic-pituitary-thyroid axis following growth hormone replacement is well documented (87-89). GHD will mask central hypothyroidism in a significant proportion of hypopituitary patients both in children and adults. It has been observed that apparently euthyroid hypopituitary patients will require commencement or an increase in thyroxine replacement following initiation of GH replacement. A higher target serum free T4 in the upper half of reference range is appropriate in the GH deficient patient who is not on GH replacement.

Unlike primary hypothyroidism, in which serum TSH is a sensitive marker of under-or over-replacement, there is no biochemical marker to indicate the optimum level of replacement in TSH deficiency. The serum free T4 is the best marker for assessing replacement adequacy in hypopituitarism. By analogy with serum T4 levels in adequately replaced primary hypothyroidism, a conventional recommendation is to maintain serum free T4 in the upper part of the reference range for normal individuals (90). Serum total T4 levels are elevated artefactually by conditions which increase serum thyronine binding globulin, especially estrogen administration.

Gonadotrophin Deficiency

Choice of replacement ranges from oral, transdermal, intramuscular, or subcutaneous administration of gonadal steroids to gonadotropin or gonadotropin-releasing hormone therapy if and when fertility is desired.


Estrogen replacement should be offered to all women with secondary hypogonadism under the age of 50 years in order to avoid immediate symptoms of estrogen deficiency and prevent premature reduction in bone mineral density. The addition of progesterone is mandatory if the uterus is intact in order to avoid unopposed estrogen stimulation of the endometrium with the attendant risk of hyperplasia and neoplasia. The standard regimen for replacement involves the daily administration of estrogen with progesterone co-administrated for 12-14 consecutive days during a 4-week cycle; menses occur cyclically after progesterone withdrawal. Alternatively, a continuous regimen may be employed in which estrogen and progesterone are combined. The latter may be preferred by older patients and there are no adverse effects described apart from unpredictable menstrual bleeding during the initial few months of therapy in a minority of patients. However, since the risk-benefit profile of estrogen therapy is influenced by numerous factors, such as age, treatment-onset since menopause, and existing comorbidities, shared decision making is essential when determining what and route to administer as well as when to stop this treatment.

Estrogen replacement can be administrated via the oral, transdermal, and subcutaneous routes. Oral estrogens undergo extensive hepatic first-pass metabolism necessitating average doses of 1-2mg estradiol per day, or equivalent.

Transdermal preparations are usually applied twice weekly and provide 50-100µg mcg of estradiol per 24 hours in a cyclical combination with a progestogen. Skin irritation may occur but transdermal therapy is the first choice in patients with complex pituitary disease since it avoids the effects of oral estrogen on other hormone binding proteins. Furthermore, in women on concomitant GH replacement, IGF-1 generation is greater when transdermal rather than oral estrogen is used, therefore decreasing the dose of GH required. Subcutaneous implants are inserted every six months, but tachyphylaxis is a frequent problem and limits the value of this regimen.

In addition to relieve vasomotor symptoms and reduce risk of bone loss, hormone therapy may be prescribed on the market to seeking for cardioprotection. According to recent research from the combined Women’s Health Initiative (WHI) studies, women who start hormone therapy closer to the period of natural menopause have a decreased risk of coronary heart disease than women who wait longer after menopause. However, this replacement is not advised for either primary or secondary cardiovascular disease prevention (91).

Concerning the negative effects of estrogen replacement, it has been well established that estrogen, particularly oral estrogen, increases the risk of venous thromboembolism; although transdermal hormone therapy does not seem to enhance this risk (92). In terms of breast cancer, risk varies depending on hormonal formulation and length of treatment. In light of this, selective estrogen receptor modulators (SERMs) have an important role and have obtained licensing for this purpose. Raloxifene has very little effect on the vasomotor symptoms of estrogen deficiency, but is beneficial in prevention and treatment of osteoporosis as well as chemoprevention of breast cancer. Tibolone, an agent with estrogenic, progestogenic, and weak androgenic activity, can provide an alternative treatment for postmenopausal symptoms and also exerts favorable effects on bone.

In some patients with combined LH/FSH and ACTH deficiency, low libido may persist despite conventional estrogen and progesterone replacement. This could be a consequence of complete androgen deficiency and some studies have shown that low dose testosterone replacement, e.g., 50-100mg subcutaneous implants every 6 months, is effective for treating hypoactive sexual desire disorder. The guideline also recommends considering a 6-month therapeutic trial of transdermal testosterone for women who fit this diagnosis (93). Oral combinations of estradiol and testosterone are also available. FSH and LH injections are necessary for fertility; recombinant forms of both are now available and this treatment is usually supervised through fertility clinics.


Apart from relief of the symptoms of hypogonadism, androgen replacement is also important in maintaining bone integrity, muscle mass, and normal erythropoiesis (94).

The most common method of androgen replacement is as an intramuscular depot injection of testosterone ester (e.g., testosterone enanthate, 250mg intramuscularly three weekly). The ensuing supraphysiological peaks and troughs of serum testosterone may lead to fluctuations in mood, libido, and energy levels but treatment is generally very well tolerated. Depot testosterone injection has become available; testosterone undecanoate (Nebido), can be administrated intramuscularly every three months (95), and maintains physiological testosterone levels without major fluctuation.

Oral testosterone undecanoate is administered two or three times a day. It is extensively metabolized to dihydrotestosterone in the intestine and is absorbed via the lymphatic system. It is generally well tolerated and is most useful in patients with partial hypogonadism or in those who are unable to tolerate depot injections.

Testosterone pellets, implanted subcutaneously at a dose between 400-600mg, will provide normal testosterone levels as well as physiological levels of estradiol and dihydrotestosterone for up to six months (96). Peak serum testosterone levels are seen 2-4 weeks after placement with a gradual decline thereafter. The main disadvantage is the need for a skin incision and the occasional complication of local infection and extrusion of the pellets.

Several other systems for testosterone delivery are available and include patches (97) and gels (98) applied to the skin and buccal bioadhesive tablets (99). Transdermal gel (Testogel, Testim) has become very popular; it must be applied daily to maintain desirable serum testosterone levels. Patches can cause skin irritation in approximately 50% of patients. Buccal testosterone (Striant) is placed on the buccal mucosa above the incisor tooth; testosterone is slowly released over 12 hours. However, gum irritation and inconvenience have been reported.

Induction of spermatogenesis requires injections of FSH and LH; our own practice is to administer FSH 300 units three times weekly and LH 1500 units twice weekly; increases in sperm density are not evident for at least 4 months.

Monitoring Testosterone Replacement

Testosterone levels can be measured in blood as a guide of the adequacy of replacement. With intramuscular depot injections, serum testosterone usually peaks at approximately one week after injection with a nadir prior to the next injection. The nadir serum testosterone concentration should approximate the lower end of the normal reference range and this may require adjustment of the frequency of injections. Random serum testosterone is often low or low normal on oral testosterone undecanoate, but the additional measurement of serum dihydrotestosterone is useful.

Published guidelines (100) addressed the concerns of the risk for either benign or malignant prostate disease. There is no evidence that the incidence of prostate carcinoma in patients on testosterone replacement is greater than background population risk. Referral to a urologist is recommended if the patient has prostatic symptoms or an abnormal digital rectal examination or elevated serum prostate specific antigen (PSA) increasing by more than 1.4ng/mL over 12 months. With the increasing use of long-acting testosterone, it is important to monitor the hematocrit to detect polycythemia. The hematocrit should return to normal before testosterone may be reinstituted at a lower dose.

The available evidence suggests that testosterone replacement should be offered cautiously in patients with a low risk of recurrence for prostatic neoplasm who have been treated with radical prostatectomy and in whom the PSA has normalized (101). Similarly, a retrospective study of patients treated with prostatectomy as primary management with a low PSA at baseline of testosterone therapy and concurrent use of 5α reductase reported that there was no increase in PSA at 15 months (56). However, patients with no definitive surgery treated with brachytherapy or external beam radiation and a raised PSA at baseline should not be offered this treatment.

Growth Hormone Deficiency

The rationale and protocol for growth hormone replacement in adults is discussed in detail in the "Adult Growth Hormone Deficiency" chapter.

Diabetes Insipidus

1-desamino-8-D-arginine-vasopressin (DDAVP) is a synthetic analogue of arginine vasopressin which produces prolonged antidiuresis after intravenous, intranasal or oral administration in patients with AHD. A therapeutic trial of DDAVP, 10-20 mcg intranasally should control polyuria for up to 16 hours. Patients with AHD must have instant improvement in symptoms. In the acute clinical setting, for example where diabetes insipidus follows pituitary surgery, DDAVP is best administrated via the subcutaneous route at a dose of 0.5-1μg. Doses of DDAVP that are too high can lead to hyponatremia if patients continue to drink inappropriately despite antidiuresis. Patient education is required to achieve optimum symptom control particularly at night and to maintain a normal serum osmolality and sodium concentration (102). Slight undertreatment, with normal water homeostasis being maintained by thirst mechanisms, is the preferred approach. Many patients demonstrate adequate control of the condition with a single bedtime dose of intranasal DDAVP but an additional morning dose may be required. Mild degrees of AHD may be treated with oral DDAVP up to 600µg daily in divided doses. If ADH deficiency is accompanied by a reduced thirst threshold, it is most important to monitor body weight and urine output on a fixed dose of DDAVP and adjust fluid intake accordingly.

A recent guideline has been developed for inpatient treatment of diabetes insipidus highlighting that this can develop into a life-threatening situation if inappropriately managed (103). This again highlights that there is an increased risk of mortality and adverse outcomes in patient with hypopituitarism admitted for acute medical conditions especially ones with diabetes insipidus (104).


Wierman ME, Arlt W, Basson R, Davis SR, Miller KK, Murad MH, Rosner W, Santoro N. Androgen therapy in women: a reappraisal: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2014 Oct;99(10):3489-510.

Fleseriu M, Hashim IA, Karavitaki N, Melmed S, Murad MH, Salvatori R, Samuels MH. Hormonal Replacement in Hypopituitarism in Adults: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016 Nov;101(11):3888-3921.

Bhasin S, Brito JP, Cunningham GR, Hayes FJ, Hodis HN, Matsumoto AM, Snyder PJ, Swerdloff RS, Wu FC, Yialamas MA. Testosterone Therapy in Men With Hypogonadism: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018 May 1;103(5):1715-1744


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The neurohypophysis is the structural foundation of a neuro-humoral system coordinating fluid balance and reproductive function through the action of two peptide hormones: vasopressin and oxytocin. Vasopressin is the principle endocrine regulator of renal water excretion, facilitating adaptive physiological responses to maintain plasma volume and plasma osmolality. Oxytocin is important in parturition and lactation. Data support a wider role for both peptides in the neuro-regulation of complex behavior. Clinically, deficits in the production or action of vasopressin manifest as diabetes insipidus. An understanding of the physiology and pathophysiology of vasopressin is also critical in approaching the diagnosis and management of hyponatremia, the most common electrolyte disturbance in clinical practice. This chapter explores the anatomy, physiology, and pathophysiology of the neurohypophysis, vasopressin and oxytocin: highlighting developments in the neural basis of osmo-sensing; the mechanism of action of vasopressin and oxytocin; together with a description of the cell and molecular biology underpinning some of the disease processes in which both the structure and functions of the two hormones are involved.


The neurohypophysis consists of three parts: the supraoptic and paraventricular nuclei of the hypothalamus; the supraoptico-hypophyseal tract; and the posterior pituitary. The neurohypophysis is one component of a complex neurohumoral system coordinating physiological responses to changes in both the internal and external environment. This chapter will concentrate on the physiology and pathophysiology of two hormones made by the hypothalamus and posterior pituitary, vasopressin (AVP) and oxytocin (OT). These hormones have key roles in water balance and reproductive function.


Anatomy Of the Neurohypophysis

The posterior pituitary is derived from the forebrain during development and is composed predominantly of neural tissue. It lies below the hypothalamus, with which it forms a structural and functional unit: the neurohypophysis. The supraoptic nucleus (SON) is situated along the proximal part of the optic tract. It consists of the cell bodies of discrete vasopressinergic and oxytotic magnocellular neurons projecting to the posterior pituitary along the supraoptico-hypophyseal tract. The paraventricular nucleus (PVN) also contains discrete vasopressinergic and oxytotic magnocellular neurons, also projecting to the posterior pituitary along the supraoptico-hypophyseal tract. The PVN contains additional, smaller parvocellular neurons that project to the median eminence and additional extra-hypothalamic areas including forebrain, brain stem, and spinal cord. Some of these parvocellular neurons are vasopressinergic. A group of those projecting via the median eminence co-secrete VP and corticotrophin releasing hormone (CRH), and terminate in the hypophyseal-portal bed of the anterior pituitary. These and other vasopressinergic parvocellular neurons terminating in the hypophyseal-portal bed have a role in the regulation of adrenocorticotrophin (ACTH) release from the anterior pituitary gland, acting synergistically with CRH produced by other hypothalamic neurons. A schematic overview of the anatomy of the neurohypophysis and its major connections is shown in Figure 1.

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Figure 1. Schematic representation of the anatomy of the neurohypophysis, and its major afferent and efferent connections.

The posterior pituitary receives an arterial blood supply from the inferior hypophyseal artery and the artery of the trabecula (a branch of the superior hypophyseal artery), derivatives of the internal carotid artery and its branches. The SON and PVN receive an arterial supply from the supra-hypophyseal, anterior communicating, anterior cerebral, posterior communicating and posterior cerebral arteries, all derived from the circle of Willis. Venous drainage of the neurohypophysis is via the dural, cavernous, and inferior petrosal sinuses.

Molecular-Cell Biology of Vasopressin and Oxytocin

AVP is a 9 amino acid peptide with a disulphide bridge between the cysteine residues at positions 1 and 6 (Figure 2). Most mammals have the amino-acid arginine at position 8, though in the Pig family arginine is substituted by lysine. The structure of OT differs from that of AVP by only 2 amino acids: isoleucine for phenylalanine at position 3; and leucine for arginine at position 8. Non-mammalian species have a variety of peptides very similar to AVP and OT, suggesting they derive from a common ancestral gene.

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Figure 2. The structural and chemical characteristics of Vasopressin and Oxytocin. The cyclical peptides differ in only 2 amino acid positions. Both contain disulphide bridges between Cysteine residues at positions 1 and 6


The genes encoding AVP and OT are in a head-to-head tandem array on chromosome 20p13 in Man, separated by 12 Kb of DNA. Each has 3 exons, and encodes a polypeptide precursor with a modular structure: an amino-terminal signal peptide; the AVP or OT peptide; a hormone-specific mid-molecule peptide termed a neurophysin (NPI and NPII for OT and AVP respectively); and a carboxyl-terminal peptide known as copeptin (Figure 3). There is considerable homology between the NP sequences of the AVP-NP and OT-NP genes, positions 10-74 of the NP sequences being highly conserved at the amino acid level.

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Figure 3. Structural organization of the Vasopressin-neurophysin II gene, and processing of its product. The AVP-NPII gene has 3 exons. Translation of the mRNA yields a larger preprohormone precursor, subsequently modified through substantial post-translational modification. The OT gene has a similar structure, and its product undergoes similar processing and post-translational modification. AVP: Vasopressin. NPII: Neurophysin II. OsRE: osmo-sensitive response element. GRE: glucocorticoid response element. ERE: estrogen response element. AP1-RE: AP1 response element.

Regulatory control of AVP gene expression is mediated through positive and negative elements in the proximal promoter. Several transcription factors bind to these elements. AP1, AP2 and CREB stimulate AVP gene expression. The glucocorticoid receptor (GR) represses expression (1, 2). The human, rat and mouse OT promoters contain half estrogen-response elements, and IL-6 response elements (3). The inter-genic region between the AVP and OT genes contains regulatory elements responsible for selective expression. The region -288 to -116 5′ upstream of the AVP gene promoter confers cell-type (magnocellular neuron) specific expression of the AVP gene (4). AVP gene expression can also be regulated at a post-transcriptional level. The length of the poly (A) tail of AVP mRNA increases in response to water deprivation, influencing mRNA stability (4). AVP mRNA also contains a dendritic localization sequence (DLS). Interaction of the DLS with a multifunctional poly(A) binding protein (PABP) may play key role in RNA stabilization, initiation of translation and translational silencing (5).

Synthesis And Release of Vasopressin and Oxytocin

Synthesis of the AVP and OT precursors occurs in the cell bodies of discrete vasopressinergic and oxytotic magnocellular neurosecretory neurons within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. Generation of the mature hormone entails post-translational modification of the large primary precursor (Figure 4). Following ribosomal translation of the respective mRNA, the carboxyl terminal domain of the precursor is glycosylated, and the product packaged in vesicles of the regulated secretory pathway. These migrate along the axons of the magnocellular neurons, during which the precursor is cleaved by basic endopeptidases into the mature hormone and the associated NP and copeptin. These are stored in secretory granules within the terminals of the magnocellular neurons in the posterior pituitary. Increased firing frequency of vasopressinergic and oxytotic neurons opens voltage-gated Ca2+ channels in these nerve terminals. This, in turn, leads to transient Ca2+ influx, fusion of the neurosecretory granules with the nerve terminal membrane, and release of the hormone and its NP and copeptin into the systemic circulation in equimolar quantities. NPs act as carrier proteins for AVP and OT during axonal migration, and appear to serve no other function. The function of copeptin remains unclear to date, it has been postulated to play a role as a prolactin-releasing factor, but never confirmed (6, 7). Another role as a chaperone-like molecule involved in the folding of the AVP precursor has been speculated (8) .In addition, it was reported to interact with the calnexin–calreticulin system (9), which prevents the export of misfolded, glucose-tagged proteins from the endoplasmic reticulum.

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Figure 4. Schematic overview of the post-translational processing of the AVP-NP II gene product. Sequential modification of 164 amino-acid AVP-NPII preprohormone in endoplasmic reticulum and golgi lead to trafficking through the regulated secretory pathway and ultimately release from neurosecretory vesicles in the posterior pituitary. AVP-NPII precursor is complexed as tetramers or high oligomers during processing. A small amount of partially processed precursor is released through the constitutive secretory pathway. OT is processed in a similar manner.

AVP and OT circulate unbound to plasma proteins, though AVP does bind to specific receptors on platelets. AVP concentrations in platelet-rich plasma are 5-fold higher than in platelet-depleted plasma (10). AVP and OT have short circulating half-lives of 5-15 minutes. Several endothelial and circulating endo- and amino-peptidases degrade the peptides. A specific placental cysteine amino-peptidase degrades AVP and OT rapidly during pregnancy and the peri-partum period.

The Physiology of The Secretion of Vasopressin and Thirst

AVP is a key component in the regulation of fluid and electrolyte balance, through direct effects on renal water handling. However, the physiology of AVP has a wider context, encompassing roles in the integrated response to changes in cardiovascular status.


There are three distinct AVP receptor (V-R) subtypes (Table 1). All have seven transmembrane spanning domains, and all are G protein coupled. They are encoded by different genes and differ in tissue distribution, down-stream signal transduction and function. The human V2-R gene maps to Xq28. Interestingly, the V2-R is up regulated by its ligand (11).

Table 1. Vasopressin Receptor Subtypes

Vasopressin receptor





Vascular smooth muscle




Pituitary corticotroph

Basolateral membrane of distal nephron

Amino acid structure

418 amino acids (human)

424 amino acids (human)

370 amino acids (human)

Second messenger system

Gq/11mediated phospholipase C activation: Ca2+, inositol triphosphate & diacyl glycerol mobilization

As V1a

Gs mediated adenylate cyclase activation: cAMP production & protein kinase A stimulation

Physiological effects

●Smooth muscle contraction

●Stimulation of glycogenolysis.

●Enhanced platelet adhesion ●Neurotransmitter & neuromodulatory function

Enhanced ACTH release

Increased synthesis & assembly of aquaporin-2


Although AVP has multiple actions, its principle physiological effect is in the regulation of water resorption in the distal nephron, the structure and transport processes of which allow the kidney to both concentrate and dilute urine in response to the prevailing circulating AVP concentration. Active transport of solute out of the thick ascending loop of Henle generates an osmolar gradient in the renal interstitium, which increases from renal cortex to inner medulla, a gradient through which distal parts of the nephron pass end route to the collecting system. AVP stimulates the expression of a specific water channel protein (aquaporin) on the luminal surface of the interstitial cells lining the collecting duct. The presence of aquaporin (AQP) in the wall of the distal nephron allows resorption of water from the duct lumen along an osmotic gradient, and excretion of concentrated urine. To date, 13 different AQPs have been identified in Man, seven of which (AQP1-4, AQP6-8) are found in the kidney. AQPs act as passive pores for small substrates and are divided into 2 families: the water only channels; and the aquaglyceroporins that can conduct other small molecules such as glycerol and urea. Most substrates are neutral. However, this is not always the case. For example, AQP6 is a gated ion channel. AQPs are involved in a variety of cell processes: small molecule permeation; gas conduction and cell-cell interaction. As with other membrane channels, specific structural arrangements within the primary, secondary, and tertiary structure convey the three functional characteristics of permeation, selectivity, and gating. The structure of AQPs involves 2 tandem repeats, each formed from 3 transmembrane domains, together with 2 highly conserved loops containing the signature motif asparagine-proline-alanine (NPA). All AQPs form homotetramers in the membrane, providing 4 functionally independent pores with an additional central pore formed between the 4 monomers. Water can pass through all the 4 independent channels of water-permeable AQPs. There are data to suggest that the central pore may act as independent channel in some AQPs (12-14). AQP1 is constitutively expressed in the apical and basolateral membranes of the proximal tubule and descending loop of Henle, where it facilitates isotonic fluid movement. Loss of function mutations of AQP1 in man lead to defective renal water conservation (15). AQP2 is expressed on the luminal surface of collecting duct cells and is the water channel responsible for AVP-dependent water transport from the lumen of the nephron into the collecting duct cells. V2-R activation in collecting duct cells produces a biphasic increase in expression of AQP2. Ligand-receptor binding triggers an intracellular phosphorylation cascade ultimately resulting in phosphorylation of the nuclear transcription factor CREB and expression of c-Fos. In turn, these transcription factors stimulate AQP2 gene expression through CRE and AP-1 elements in theAQP2gene promoter. In addition, AVP stimulates an immediate increase in AQP2 expression by accelerating trafficking and assembly of pre-synthesized protein into functional, homo-tetrameric water channels.

Maximum diuresis occurs at plasma AVP concentrations of 0.5 pmol/l or less. As AVP levels rise, there is a sigmoid relationship between plasma AVP concentration and urine osmolality, with maximum urine concentration achieved at plasma AVP concentrations of 3-4 pmol/L (Figure 5). Following persistent AVP secretion, antidiuresis may diminish. Down-regulation of both V2-R function and AQP2 expression may be responsible for this escape phenomenon.

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Figure 5. The relationship of plasma AVP concentration to urine osmolality. Shaded area represents range of normal; single line indicates representative individual. AVP has additional effects at other sites in the nephron: decreasing medullary blood flow; stimulating active urea transport in the distal collecting duct; and stimulating active sodium transport into the renal interstitium. AVP up-regulates the bumetanide-sensitive sodium-potassium-chloride cotransporter (SLC12A1) in the thick ascending loop of Henle through both a rapid acceleration of post-translational processing/trafficking and an increase in SCLC12A1 gene expression. Together, these contribute to the generation and maintenance of a hypertonic medullary interstitium, and augment AVP-dependent water resorption (16).


Osmoregulation of Vasopressin

Plasma osmolality is the most important determinant of AVP secretion. The osmoregulatory systems for thirst and AVP secretion, and in turn the actions of AVP on renal water excretion, maintain plasma osmolality within narrow limits of 284 to 295 mOsmol/kg. The relationship between plasma osmolality and plasma AVP concentration has 3 characteristics.

  • The osmotic threshold or 'set point' for AVP release.
  • The shape of the line describing changes in plasma AVP concentration with changing plasma osmolality
  • The sensitivity of the osmoregulatory mechanism coupling plasma osmolality and AVP release.

Increases in plasma osmolality increase plasma AVP concentrations in a linear manner (Figure 6). The abscissal intercept of this line indicates the mean 'osmotic threshold' for AVP release (284 mOsmol/kg): the mean plasma osmolality above which plasma AVP increases in response to increases in plasma osmolality. AVP levels increase from a basal rate through activation of stimulatory osmoreceptor afferents, and decrease to minimal values when this drive is removed and synergistic inhibitory afferents are activated. The slope of the line relating plasma osmolality to plasma AVP concentration reflects the sensitivity of osmoregulated AVP release. There are considerable inter-individual variations in both the threshold and sensitivity of AVP release. However, they are remarkably reproducible within an individual over time (17).

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Figure 6. The relationship of plasma AVP concentration to changes in plasma osmolality during controlled hypertonic stimulation. AVP concentration determined during progressive hypertonicity induced by infusion of 855 mmol/l saline in a group of healthy adults. Increases in plasma osmolality increase plasma AVP concentrations in a linear manner, defined by the function, plasma AVP = 0.43 (plasma osmolality - 284), r = +0.96. The abscissal intercept of this regression line indicates the mean 'osmotic threshold' for AVP release: the mean plasma osmolality above which plasma AVP starts to increase. The shaded area represents the range of normal response. LD represents the limit of detection of the assay, 0.3 pmol/l.

There are situations where the normal relationship between plasma osmolality and AVP concentration breaks down:

  • Rapid changes of plasma osmolality: rapid increases in plasma osmolality result in exaggerated AVP release.
  • During the act of drinking: drinking rapidly suppresses AVP release, through afferent pathways originating in the oropharynx.
  • Pregnancy: the osmotic threshold for AVP release is lowered in pregnancy.
  • Whether aging is accompanied by changes in AVP concentrations is controversial.

As befits its major function and physiological role, AVP production by the neurohypophysis is influenced by sensory signals reflecting osmotic status and blood pressure/circulating volume. The relationships of the SON and PVN with the autonomic afferents and central nervous system nuclei responsible for osmo- and baroregulation are key to the physiological regulation of AVP. Functional osmoreceptors are situated in anterior circumventricular structures: the subfornicular organ (SFO), and the organum vasculosum of the lamina terminalis (OVLT). Local fenestrations in the blood brain barrier at these sites allow neural tissue direct contact with the circulation. Subsequent sensory input to the SON and PVN is via glutaminergic afferents. Moreover, these neurons integrate osmolar status with additional endocrine signals reflecting circulating volume status through the action of angiotensin II (A-II), relaxin, and atrial natriuretic peptide (ANP). A-II and relaxin excite both OT and AVP magnocellular neurons. In contrast, ANP inhibits AVP neuron activity. AVP neurons themselves have independent osmo-sensing properties and V-Rs are present on vasopressinergic neurons of both the PVN and SON, highlighting the potential for auto-control of AVP release through direct osmoregulation and short loop feedback (18). AVP magnocellular neurons in the SON and PVN co-express the peptide Apelin and its G-protein coupled receptor. A ‘yin and yang’ relationship has been proposed between AVP and these 36 amino-acid peptides (and indeed it’s shorter active derivatives Apelin-17 and Apelin-13). Intra-cerebroventricular injection of Apelin-17 inhibits the phasic firing of AVP magnocellular neurons, reducing AVP release and stimulating aquaresis. Hypertonic stress and water loading have reciprocal effects on plasma AVP and Apelin concentrations. Apelin receptors are also co-expressed in AVP target cells in the renal collecting duct. AVP and Apelin are thus regulated in opposite directions to maintain volume and osmolar homeostasis (19-22).

Changes in the osmotic environment of osmo-sensitive neurons in the OVLT, SFO and vasopressinergic neurons of the SON and PVN result in altered cell volume. These physical changes alter the activity of the stretch-sensitive cationic channel TRPV1, expressed on the cell surface of these neurons. TRPVI thus acts as the transduction mechanism linking changes in osmolality to altered membrane potential and firing frequency. Osmoregulatory function is not lost in Trpv1−/−mice, indicating additional osmo-sensing pathways must be in operation (23).

A related, but distinct osmo-sensory input feeds additional data on peripheral osmolar status to the neurohypophysis. Hepatic portal blood vessels contain sensory neurons responsive to changes in the osmolality of peripheral blood. In contrast to central mechanisms, the key transducing element of the peripheral process is the stretch-sensitive ion channel, TRPV4. Plasma osmolality is frequently elevated in patients after liver transplant in which the donor organ is denervated, demonstrating the function of this peripheral pathway (24).

Osmosensitivity of AVP release is influenced by circadian rhythms. AVP release increases during sleep. This effect is mediated by clock neurons projecting from the suprachismatic nucleus, increasing the activity of osmosensory afferent input to the SON (25).

Baroregulation of Vasopressin

Reductions in circulating volume stimulate AVP release. Falls in arterial blood pressure of 5 to 10 per cent are necessary to increase circulating AVP concentrations in man. Progressive reduction in blood pressure produces an exponential increase in plasma AVP, in contrast to the linear increases of osmoregulated AVP release. Baroregulatory influences on neurohypophyseal AVP release derive from aortic arch, carotid sinus, cardiac atrial, and great vein stretch-sensitive afferents via cranial nerves IX and X. Ascending projections are via the nucleus tractus solitarius (NTS) in the brain stem. From the NTS, further afferents project to the SON and PVN, which also receive additional adrenergic afferents from other brain stem nuclei involved in cardiovascular control, such as the locus coeruleus. These nuclei integrate a number of afferent inputs that reflect volume status. Ascending baroregulatory pathways must affect some tonic inhibition of AVP release, as interruption increases plasma AVP levels (26, 27). Osmoregulated AVP responses can be modified by factors triggered as part of the coordinated neurohumoral response to changes in circulating volume and blood pressure. A-II amplifies the proportional relationship between osmolality and action potential firing in the SON. The peptide produces this effect through polymerization of intracellular actin filaments, resulting in altered cell shape, a mechanism that is synergistic with those mediating responses to changes in extracellular osmolality. A-II thus enhances osmosensitivity. This mechanism underpins the changes in osmo-regulated AVP release in hypo- and hypervolemia: the osmotic threshold and sensitivity of AVP release is lowered by hypovolemia; while the converse is found in hypervolemia and hypertension (19).

Additional Mechanisms Regulating Vasopressin Release

A number of other stimuli influence AVP release independent of osmotic and hemodynamic status.

  • Nausea and emesis
  • Unspecific stress
  • Pain
  • Manipulation of abdominal contents
  • Immune-response mediators and inflammatory triggers

These stimuli contribute to high plasma AVP values observed in acute illness and after surgery.


Cardiovascular Effects

AVP is a potent pressor agent; its effects mediated through a specific receptor (V1-R) expressed by vascular smooth muscle cells. Though systemic effects on arterial blood pressure are only apparent at high concentrations, AVP is important in maintaining blood pressure in mild volume depletion. The most striking vascular effects of AVP are in the regulation of regional blood flow. The sensitivity of vascular smooth muscle to the pressor effects of AVP varies according to the vascular bed. Vasoconstriction of splanchnic, hepatic and renal vessels occur at AVP concentrations close to the physiological range. Furthermore, there are differential pressor responses within a given vascular bed. Selective effects on intrarenal vessels lead to redistribution of renal blood flow from medulla to cortex. Baroregulated AVP release thus constitutes one of the key physiological mediators of an integrated hemodynamic response to volume depletion.4.2.

Effects on the Pituitary

AVP is an ACTH secretagogue, acting through pituitary corticotroph-specific V1b-Rs. Though the effect is weak in isolation, AVP and CRF act synergistically. AVP and CRF co-localize in neurohypophyseal parvocellular neurons projecting to the median eminence and the neurohypophyseal portal blood supply of the anterior pituitary. Levels of both AVP and CRF in these neurons are inversely related to glucocorticoid levels, consistent with a role in feedback regulation.

Effects of AVP on Regulation of Bone Mass

AVP exerts its action both on osteoblasts and osteoclasts thru AVPR1 and AVPR2 receptors (28). Mice studies showed that AVP upregulates osteoclast differentiation genes and inhibits osteoblast formation. Mice rendered deficient in AVPr1a (Avpr1a-/-) have a high bone mass, and they show an increase in osteoblastogenesis after additional inhibition of AVPR2 (29) .

The AVP effect is opposed by oxytocin, which stimulates osteoblast formation and inhibits mature osteoclast activation. Mice studies showed that both haploinsufficiency for oxytocin and deletion of the oxytocin receptor (Oxr-/-) result in osteopenia (30). The role of both hormones in the regulation of skeletal physiology remains to be further explored (31).

Behavioral Effects of Vasopressin

Vasopressinergic fibers and V-Rs are present in CNS neural networks anatomically and functionally independent of the neurohypophysis, including the cerebral cortex and limbic system. An increasing amount of data highlight the role of central vasopressinergic systems in mediating complex social behavior. Data in Man link V1a-R gene sequence variation with a range of normal and abnormal behavior patterns, including gender dimorphic behavior. Dysregulation of central AVP action may be a distal end point in complex conditions characterized by altered social and emotional behavior (32, 33).


Renal free water clearance can be reduced to a minimum by the antidiuretic actions of vasopressin, but water loss is not completely eliminated, and insensible water loss from respiration and sweating is a continuous process. To maintain water homeostasis, water must also be consumed to replace the obligate urinary and insensible fluid losses. This is regulated by thirst. Thirst and drinking are key processes in the maintenance of fluid and electrolyte balance. Thirst perception and the regulation of water ingestion involve complex, integrated neural and neurohumoral pathways. As with those mediating AVP release, the osmoreceptors regulating thirst are situated in the OVLT, effectively outside the blood-brain barrier and distinct from those mediating AVP release. Neural activity in and around the OVLT remains active in hyperosmolar states following immediate satiety of thirst, indicating that other centers must be involved in thirst perception. The anterior cingulate cortex and insular cortex receive input from osmo-sensitive afferents and have been implicated as key higher centers in thirst pathways (20). There is a linear relationship between thirst and plasma osmolalities in the physiological range. The mean osmotic threshold for thirst perception is 281 mOsm/kg, similar to that for AVP release. Thirst occurs when plasma osmolality rises above this threshold. As with osmoregulated AVP release, the characteristics of osmoregulated thirst remain consistent within an individual on repeated testing, despite wide inter-individual variation.

As with AVP release, there are also specific physiological situations in which the relationship between plasma osmolality and thirst breaks down.

  • The act of drinking: reduces osmotically stimulated thirst.
  • Extracellular volume depletion: this stimulates thirst through volume-sensitive cardiac afferents and the generation of circulating and intra-cerebral A-II, a powerful dipsogen.
  • Pregnancy, the luteal phase of the menstrual cycle and super ovulation syndrome: these states reduce the osmolar threshold for thirst.
  • Aging: both thirst appreciation and fluid intake can be blunted in the elderly

The act of drinking reduces thirst perception before any change in plasma osmolality. This effect is produced through three mechanisms: oropharyngeal sensory afferents; gastro-intestinal stretch-sensitive afferents; and peripheral osmoreceptors in the hepatic portal vein. Recent data have highlighted how thirst-promoting neurons in the SFO integrate sensory inputs from the oropharynx (drinking and food composition) with central osmolar status to influence thirst perception. This complex mechanism effectively explains anticipatory changes in water consumption that precede changes in plasma osmolality (34). As with AVP release, hypovolemia resets the relationship between plasma osmolality and thirst. A-II is one of the key mediators of this physiological response. Peripheral A-II generation can act on central osmoreceptors, to increase both thirst and AVP release. An independent, intra-cerebral A-II system is activated in parallel. A-II is a powerful central dipsogen.

The Physiology of Oxytocin

OT binds to specific G-protein coupled cell surface receptors (OT-Rs) on target cells to mediate a variety of physiological effects, largely concerned with reproductive function. The classical physiological roles of OT are the regulation of lactation, parturition and reproductive behavior. Data from transgenic animals with targeted disruption of the oxytocin gene (and thus lacking OT) have forced a review of this dogma (35).


In the rat, stimulation of vagal sensory afferents in the nipple by the act of suckling triggers reflex synchronized firing of oxytotic magnocellular neurons in the neurohypophysis, and corresponding pulsatile OT release. OT acts on OT-Rs on smooth muscle cells lining the milk ducts of the breast, initiating milk ejection. OT is essential for completion of this milk ejection reflex in rodent. Mice lacking OT fail to transfer milk to their suckling young. This deficit is corrected by injection of OT. In contrast, women lacking posterior pituitary function can breast-feed normally, illustrating that OT is not necessary for lactation in man. Pituitary lactotrophs express OT-R mRNA, and OT released into the hypophyseal portal blood supply from the median eminence can stimulate prolactin release. However, the role of OT in the physiology of prolactin release remains unclear.


OT is a uterotonic agent. In many mammals, there is both an increase in OT secretion and an increase in uterine responsiveness to OT during parturition (3). These data suggest a key role for the hormone in the initiation and progression of labor. Falling progesterone concentrations toward the end of pregnancy lead to up-regulation of uterine myometrial OT-Rs, enhanced contractility, and increased sensitivity to circulating OT. Stretching of the 'birth canal' during parturition leads to the stimulation of specific autonomic afferents, reflex firing of oxytotic neurons and OT release. A positive feedback loop is formed, OT stimulating uterine contraction further and enhancing the production of additional local uterotonic mediators such as prostaglandins. The difficulties of analyzing pulsatile release, and the short circulating half-life of the hormone (due to placental cysteine aminopeptidase), have made it difficult to demonstrate increased circulating OT levels in women during labor. Mice lacking OT have normal parturition. Moreover, women with absent posterior pituitary function can have a normal labor. However, the importance of OT in the birth process is highlighted by the effectiveness of OT antagonists in the management of pre-term labor (36). The role of OT in parturition is not limited to maternal responses. Maternal OT produces a switch to inhibitory GABAergic signaling in the fetal CNS. This, in turn, increases fetal neuronal resistance to damage that may occur during delivery. OT therefore mediates direct adaptive mother-fetal signaling during parturition in line with a wider-ranging role in maternal-fetal physiology (37).


OT-R expression is widespread in the CNS of many species, and OT has widespread roles as a neurotransmitter. The central oxytocinergic system and related limbic networks affect complex neural circuits of socio-emotional behavior and promote pro-social effects such as in-group favoritism and protection against social threats, interpersonal trust and attachment, empathy, and emotion recognition (38-41). In humans, brain regions such as the amygdala, hippocampus, cingulate cortex, and nucleus accumbens, which play a key role in human socio-emotional behavior, show a high density of OT-R expression. In some cases, these overlap those involving AVP (32, 33).

OT knockout (OT-KO) models have been used to identify aspects of dysfunctions in social behavior. Generally, an impairment in forming social memories and higher anxiety-related behaviors were observed (42-44). These results were substantiated by the fact that central administration of OT rescued OT-KO mice from these changes. In the context of behavior; OT facilitates both lordosis and the development of maternal behavior patterns in rat (3). However, mice lacking OT exhibit normal sexual and maternal behavior, suggesting the behavioral effects to be species-specific or the potential for considerable redundancy in neural pathways. In humans, lower endogenous OT or impaired signaling has been linked with mental disorders associated with social deficits, such as autism spectrum disorder (ASD), anxiety and depression disorder or borderline personality disorder (45-50). In these disorders, no OT deficiency per se has been proven; the evidence is largely based on observational studies with individual variations in social behavior associated with alteration in peripheral OT levels, in genes involved in OT signaling or OT-R polymorphisms (51-53). In ASD, recent data have highlighted associations with the single nucleotide polymorphisms (SNPs) rs7632287, rs237887, rs2268491 and rs2254298 (54-56). Intranasal OT has been investigated to ameliorate symptoms of these conditions. However, overall, the effect size is inconsistent, and the results of these studies are controversial. Central oxytocinergic transmission reduces anxiety behavior and hypothalamo-pituitary-adrenal stress responses in female rats. It may be that OT has a complex role in the stress response, with context-dependent differential effects. It buffers responses to social stress, reduces cortisol levels during conflict situations, improves self-representations in patients with anxiety disorder, and reduces amygdala response to emotional stimuli, consequently reducing fear reactivity and anxiety (57-63).

Only limited research has been devoted to the role of OT in patients with hypothalamic-pituitary dysfunction and focused primarily on patients with craniopharyngioma (CP). These data assume direct tumor-induced or post-surgical damage to the SON/PVN and consequent disruption of the oxytocinergic system (64-68). Results demonstrate personality changes and increased psycho-social comorbidities – including anxiety, depression, and social withdrawal (64, 69-71). This was confirmed by a systematic review showing behavioral dysfunctions in 57%, and social impairment and difficulties holding relationships in 40% (72). Interestingly, the age of onset appears to influence the type of socio-behavioral dysfunction: while adult-onset had higher levels of anxiety and depression, younger patients had more impact in the domains of social isolation. Research in patients with confirmed posterior pituitary dysfunction, i.e., CDI, demonstrated higher depression and anxiety levels, self-reported autistic traits, lower joy when socializing and worse scores on an emotional recognition task than healthy adults (73, 74).

Similar to other hormones, single basal OT levels are unreliable and insufficient in identifying a deficiency (75) with inconclusive results in patients with hypothalamic-pituitary dysfunction. Research still disagrees regarding the precise relationship between peripheral OT and centrally generated behavior. Cerebrospinal fluid (CSF) concentrations of OT are more likely to mirror the behavioral effects than plasma concentrations, and whether plasma levels may serve as a surrogate for CSF OT is controversially discussed (76, 77). Accordingly, peripheral levels of OT correlate to central levels only after stimulation but not at baseline (78). Established pituitary provocation tests do not show a consistent strong OT increase to test for a suspected deficiency (79). OT-R expression is widespread in the CNS of many species and OT has widespread roles as a neurotransmitter, including neural networks that mediate a range of complex behaviors. In some cases, these overlap those involving AVP (32, 33).


Defects in the production or action of AVP manifest as clinical problems in maintaining plasma sodium concentration and fluid balance, reflecting the key role of the hormone in these processes. A further group of related clinical conditions reflect primary defects in thirst. In some cases, the two may coincide, reflecting the close anatomical and functional relationship of both processes.

Diabetes Insipidus


Diabetes insipidus (DI) is characterized by production of dilute urine in excess of >50 ml/kg/24 hours in adults. DI arises through one of four mechanisms (Figure 7 and Table 2).

  • Deficiency of AVP: central diabetes insipidus (CDI). Also called Arginine Vasopressin Deficiency
  • Inappropriate, excessive water drinking: primary polydipsia.
  • Renal resistance to the antidiuretic action of AVP: nephrogenic diabetes insipidus (NDI). Also called Arginine Vasopressin Resistance
  • increased vasopressinase expression in pregnancy: Gestational diabetes insipidus

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Figure 7. Different forms of hypotonic polyuria (80)

Table 2. Classification of Hypotonic Polyuria (80)

Type of hypotonic polyuria

Basic defect


Central DI

Deficiency in AVP synthesis or secretion


· Trauma (surgery, deceleration injury)

Neoplastic (craniopharyngioma, meningioma, germinoma, metastases)

· Vascular (cerebral/ hypothalamic hemorrhage, infarction or ligation of anterior communicating artery aneurysm)

· Granulomatous (histiocytosis, sarcoidosis)

· Infectious (meningitis, encephalitis, tuberculosis)

· Inflammatory/autoimmune (lymphocytic infundibuloneurohypophysitis, IgG4 neurohypophysitis)

· Drug/toxin-induced

· Osmoreceptor dysfunction (adipsic DI)

· Others (hydrocephalus, ventricular/suprasellar cyst, trauma, degenerative disease)

· Idiopathic


· Autosomal dominant: AVP gene mutation

· Autosomal recessive: Wolfram Syndrome (DIDMOAD)

· X-linked recessive

Primary Polydipsia

Excessive osmotically unregulated fluid intake

· Dipsogenic (downward resetting of the thirst threshold; idiopathic or similar lesions as with central DI)

· Psychosis intermittent hyponatremia polydipsia (PIP syndrome)

· Compulsive water drinking

· Health enthusiasts

Nephrogenic DI

Reduced renal sensitivity to antidiuretic effect of physiological AVP levels


· Drug exposure (lithium, demeclocycline, cisplatin etc.)

· Hypercalcemia, hypokalemia

· Infiltrating lesions (sarcoidosis, amyloidosis, multiple myeloma etc.)

· Vascular disorders (sickle cell anemia)

· Mechanical (polycystic kidney disease, ureteral obstruction)


· X-linked AVPR2 gene mutations

· autosomal recessive or dominant AQP2 gene mutations

Gestational DI

Exaggerated enzymatic metabolism of circulating AVP hormone

Increased AVP metabolism

· Pregnancy

Central Diabetes Insipidus (CDI) (Arginine Vasopressin Deficiency)

CDI (also known as neurogenic or cranial DI) is the result of partial or complete lack of osmoregulated AVP secretion. Plasma AVP concentrations are inappropriately low with respect to prevailing plasma osmolalities. Presentation with CDI implies destruction or loss of function of more than 80% of vasopressinergic magnocellular neurons. It is rare (estimated prevalence of 1: 25000), with an equal gender distribution. Though persistent polyuria can lead to dehydration, most patients can maintain water balance through appropriate polydipsia if given free access to water.

Most cases of CDI are acquired. Trauma (head injury or surgery) can produce CDI through damage to the hypothalamus, pituitary stalk, or posterior pituitary. Pituitary stalk trauma may lead to a triphasic disturbance in water balance, an immediate polyuric phase followed within days by a more prolonged period (up to several weeks) of antidiuresis suggestive of AVP excess. This second phase can be followed by reversion to CDI, or recovery. This characteristic 'triple response' reflects initial axonal damage; the subsequent unregulated release of large amounts of pre-synthesized AVP; and either recovery or development of permanent CDI (as determined by the magnitude of initial damage to vasopressinergic neurons). All phases of the response are not apparent in all cases.

Hypothalamic tumors (e.g., craniopharyngioma) or pituitary metastases (e.g., breast or bronchus) can present with CDI. However, primary pituitary tumors rarely cause CDI. In childhood, craniopharyngioma and germinoma/teratoma are a relatively common cause (81).

When obvious causes are not present, most cases of CDI will be “idiopathic”. However, the possibility of an autoimmune process should be considered, as many idiopathic cases are considered to be autoimmune in origin (82). A well-recognized cause of autoimmune CDI is lymphocytic infundibulohypophysitis (83).

Familial forms are rare, but are a recognized cause of CDI in childhood. Most reported cases are expressed as autosomal dominant and the genetic defect is usually in the biologically inactive neurophysin or in the signal peptide of the pre-prohormone. Lack of normal cleavage of the signal peptide from the prohormone and abnormal folding of the vasopressin/neurophysin precursor are thought to produce fibrillar aggregations in the endoplasmic reticulum, which is cytotoxic to the neuron, explaining the dominant phenotype (84, 85). Wolfram syndrome is a rare autosomal recessive disease with diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD). The genetic defect is for the protein wolframin that is found in the endoplasmic reticulum and is important for folding proteins (86). Wolframin is localized to chromosome 4. It is involved in beta-cell proliferation and intracellular protein processing and calcium homeostasis, producing a wide spectrum of endocrine and central nervous system (CNS) disorders. Diabetes insipidus is usually a late manifestation and is associated with decreased magnocellular neurons in the paraventricular and supraoptic nuclei (87, 88).

Primary Polydipsia (PP)

PP is a polyuric syndrome secondary to excess fluid intake. PP can be associated with organic structural brain lesions, e.g. sarcoidosis of the hypothalamus (89) and craniopharyngioma (90). It can also be produced by drugs that cause a dry mouth or by any peripheral disorder causing an elevation of renin and/or angiotensin. However, mostly there is no identifiable pathologic etiology; in this circumstance the disorder is often associated with psychiatric syndromes. Series of patients in psychiatric hospitals have shown that as many as 42% have some form of polydipsia and for over half of those there was no obvious explanation for the polydipsia (91). It also seems to be increasingly prevalent in health conscious people who voluntarily change their drinking habits with the aim to improve their well-being, in which case it is often called habitual polydipsia (92).

Nephrogenic Diabetes Insipidus (NDI)

NDI is due to renal resistance to the antidiuretic effects of AVP. Genetic variants of NDI usually present in infancy (93). In these forms, NDI can occur as a result of mutations in the V2 receptor and mutations of the aquaporin 2 water channels. Over 90% of cases are X-linked recessive in males, and over 200 different mutations of the V2 receptor have been reported affecting all aspects of receptor function: expression; ligand binding; and G-protein coupling. Most lead to complete loss of function, though a few are associated with a mild phenotype. 10% of kindreds with familial NDI have an autosomal recessive form, with normal V2-R function. Affected individuals harbor loss of function mutations of the AQP2 gene. Most mutations occur in the region coding for the transmembrane domain of the protein. Additional rare kindreds have been described harboring a mutation in the portion of the gene encoding the carboxyl-terminal intracellular tail of AQP2. The NDI of these kindreds is inherited as an autosomal dominant trait, mutant protein sequestering the product of the wild type AQP2 allele within mixed tetramers in a dominant-negative manner.

The development of NDI in an adult is less likely to reflect a genetic cause. The commonest cause of acquired NDI in clinical practice is lithium therapy, with other causes including hypokalemia, hypercalcemia, and release of bilateral urinary tract obstruction associated with downregulation of aquaporin 2 and decreased function of vasopressin (94, 95). NDI secondary to lithium is characterized by dysregulated AQP2 expression and trafficking along the whole collecting duct as well as dysregulated expression of the amiloride-sensitive epithelial sodium channel (ENaC) in the cortical collecting duct. Lithium enters collecting duct cells through ENaC expressed on the apical cell membrane and leads to inhibition of glycogen synthase kinase type 3 (GSK-3) signaling pathways. NDI secondary to lithium toxicity can persist after drug withdrawal, and may be irreversible. Demeclocycline is another commonly recognized drug to causes NDI and is sometimes used clinically to treat SIAD. The final common pathway producing NDI in many of these cases is down-regulation of AQP2 expression (96).

Gestational Diabetes Insipidus

In normal pregnancy, physiologic adaptations include expansion of blood volume and decreased plasma osmolality and serum sodium. Thirst and increased fluid intake are commonly reported in pregnancy, but in some patients the increased thirst is driven by marked polyuria, which may point to the presence of diabetes insipidus. Two types of transient diabetes insipidus must be differentiated in pregnancy, both caused by the placental enzyme cysteine aminopeptidase, named oxytocinase, which enzymatically degrades oxytocin (97). Because of the close structural homology between AVP and oxytocin, this enzyme also metabolizes AVP. In the first type of pregnancy-associated DI, the activity of oxytocinase is abnormally elevated. This syndrome has been referred to as vasopressin resistant diabetes insipidus of pregnancy (98) and has been reported to be associated with preeclampsia, acute fatty liver, and coagulopathies. Symptoms usually develop at the end of the second or early third trimester and it is more common during multiple pregnancy (99). In the second type of pregnancy-associated DI, the accelerated metabolic clearance of vasopressin produces DI in a patient with borderline pre-existing vasopressin function from a mild nephrogenic diabetes insipidus or partial central diabetes insipidus. Vasopressin is rapidly destroyed and the neurohypophysis is unable to keep up with the increased demand. Symptoms in this second type usually appear early in pregnancy (100).


Investigations have the following three aims:

  • To confirm DI
  • To classify the DI into central or nephrogenic DI or PP (or gestational DI in case of pregnancy)
  • To establish the etiology of the specific form of DI

After establishing polyuria and polydipsia, and excluding hyperglycemia, hypokalemia, hypercalcemia, and significant renal insufficiency, attention should be focused on the AVP axis.

For many years, the indirect water deprivation test was the gold standard for differential diagnosis of diabetes insipidus. This test indirectly assesses AVP activity by measurement of the urine concentration capacity during a prolonged period of dehydration, and again after a subsequent injection of an exogenous synthetic AVP variant, desmopressin. Interpretation of the test results goes back to the publication of Miller et al (101). If upon thirsting, urinary osmolality remains <300mOsm/kg and does not increase >50% after desmopressin injection, complete nephrogenic DI is diagnosed. If the urinary osmolality increase after desmopressin injection is >50%, complete central DI is diagnosed. In partial central DI and primary polydipsia, urinary concentration increases to 300–800mosm/kg, with an increase of >9% (in partial central DI) and <9% (in primary polydipsia), respectively, after desmopressin injection. However, these published criteria are based on post hoc data from only 36 patients, who had a wide overlap in their urinary osmolalities. Furthermore, the diagnostic criteria for this test are derived from a single study with post-hoc assessment (101) and have not been prospectively validated. Consequently, the indirect water deprivation test has been shown to have considerable diagnostic limitations with an overall diagnostic accuracy of 70%, and an accuracy of only 41% in patients with primary polydipsia (102).

Several reasons exist for this limited diagnostic outcome of the indirect water deprivation test. First, chronic polyuria itself can affect renal concentration capacity, through renal washout (103) (104, 105) or downregulation of AQP2 expression in the kidneys (106). This may lead to a reduced renal response to osmotic stimulation or exogenous desmopressin in different forms of chronic polyuria. Second, in patients with AVP deficiency, urine concentration can be higher than expected (107, 108), especially in those patients with impaired glomerular function, or can result from a compensatory increase in AVPR2 expression in patients with chronic central DI (109). Finally, patients with acquired nephrogenic DI are often only partially resistant to AVP, resulting in a clinical presentation that is similar to partial central DI.

To overcome these limitations, direct measurement of AVP levels has been proposed. In a study published in 1981, patients with central DI were reported to have AVP levels below a calculated normal area (defining the normal relationship between plasma osmolality and AVP levels), whereas AVP levels were above the normal area in patients with nephrogenic DI and within the normal area in patients with primary polydipsia (110). However, despite these promising initial results, direct measurement of AVP levels failed to enter routine clinical use for various reasons. First, several technical limitations of the AVP assay result in a high preanalytical instability of AVP in samples (111). Second, the accuracy of diagnoses using commercially available AVP assays has been disappointing, with correct diagnoses in only 38% of patients with DI, and particularly poor differentiation between partial central DI and primary polydipsia (102, 112). Third, an accurate definition of the normal physiological area defining the relationship between plasma AVP levels and osmolality is still lacking, especially for commercially available assays (113, 114), which is a crucial prerequisite for the identification of AVP secretion outside the normal range in patients suspected of having DI (102).

Copeptin, the C-terminal segment of the AVP prohormone, is an easy-to- measure AVP surrogate that is very stable ex vivo (111), mirroring AVP concentrations. Two studies have shown that a basal copeptin level >21.4pmol/l without prior thirsting unequivocally identifies nephrogenic DI, rendering a further water deprivation test unnecessary in these patients (102, 115). For the differentiation between patients with primary polydipsia and central DI, results of a study including 144 patients (116) showed that an osmotically stimulated copeptin level >4.9pmol/l after infusion of 3% saline (aiming at a sodium level >150mmol/l) had an overall diagnostic accuracy of 96.5% (93.2% sensitivity and 100% specificity) in distinguishing between patients with primary polydipsia and those with central DI. The classic water deprivation test had an overall diagnostic accuracy of only 76.5%. Importantly, the hypertonic saline infusion test requires close monitoring of sodium levels to ascertain a diagnostically meaningful increase of plasma sodium within the hyperosmotic range while preventing a marked increase. Addition of copeptin measurement did not improve the diagnostic performance of the indirect water deprivation test, most likely due to the lack of osmotic stimulus by thirsting alone. Another study suggests that non-osmotically stimulated copeptin levels upon arginine infusion also provide a high diagnostic accuracy in differentiating central DI from PP (117). These data indicate that plasma copeptin is a promising biomarker to distinguish between different forms of polyuria–polydipsia syndrome. A possible diagnostic algorithm with and without the availability of copeptin measurement is shown in Figure 8.

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Figure 8. Algorithm for the differential diagnosis of diabetes insipidus (Rev Christ-Crain, Nat Rev Primer)

In establishing the underlying mechanisms of central DI once the diagnosis is confirmed, imaging of the hypothalamus, pituitary and surrounding structures with MRI is essential. If no mass lesion is identified, imaging should be repeated after 6-12 months so that slow growing germ cell tumors are not missed. Idiopathic and familial central DI are often associated with loss of the normal hyper-intense signal of the posterior pituitary on T1-weighted images (Figure 9). Signal intensity is correlated strongly with AVP content of the gland (118). In the absence of appropriate history and diagnostic testing, the loss of a posterior pituitary bright spot does not make the diagnosis of central DI. Importantly, presence of an appropriate bright spot does not exclude the diagnosis of central DI.

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Figure 9. Loss of the posterior pituitary 'bright spot' on T1 weighted MRI in hypothalamic diabetes insipidus. The normal posterior pituitary can be demonstrated as a 'bright spot' within the sella turcica on T1-weighted MRI (a). This increased signal intensity can be lost in HDI (b). An ectopic posterior pituitary 'bright-spot' can be seen some cases of childhood onset hypopituitarism, implying failure to complete normal developmental migration. Function can be normal despite the aberrant position

Evidence of anterior pituitary dysfunction should be looked for in central DI, though it is relatively uncommon in the adult population. Interestingly, evidence of organ-specific autoimmune disease is relatively common in adult patients with isolated central DI, consistent with an autoimmune basis for the condition (119).


Treatment of Central DI

The primary aim of treatment in patients with diagnosed central DI should be to reduce polyuria and polydipsia to levels that allow maintenance of a normal lifestyle. The treatment of choice for those with significant symptoms is the synthetic, long-acting AVP analogue DDAVP. The long half-life, selectivity for AVPR2 and availability of multiple preparations renders desmopressin an ideal treatment for central DI. Optimal dosage and dosing intervals should be determined for each patient. The available treatment options are the intranasal spray, oral tablets, sublingual tablets or a parenteral injection, in divided doses. Oral preparations provide greater convenience and are usually preferred by patients. However, starting with a nasal spray initially is preferable because of greater consistency of absorption and physiological effect, after which the patient can be switched to an oral preparation. After trying both preparations, the patient can then choose which they prefer for long-term treatment. A satisfactory schedule can generally be determined using modest doses of desmopressin. The maximum dose of desmopressin required rarely exceeds 0.2 mg orally, 120 µg sublingually or 10 µg (one nasal spray) given 2–3 times daily. These doses usually produce plasma desmopressin levels higher than those required to cause maximum antidiuresis but reduce the need for more frequent treatment (120).

Hyponatremia is the major complication of desmopressin therapy — a 27% incidence of mild hyponatremia (serum sodium 131–134 mmol/l) and a 15% incidence of more severe hyponatremia (serum sodium ≤130 mmol/lm) have been reported after long-term follow-up of patients with chronic central DI (121). Hyponatremia usually occurs if the patient is antidiuretic while continuing normal fluid intake. Severe hyponatremia in patients with central DI who are treated with desmopressin can be avoided first by monitoring serum electrolyte levels frequently during initiation of therapy and second, patients should be instructed to delay a scheduled dose of desmopressin once or twice weekly until polyuria recurs, thereby allowing excess retained fluid to be excreted. A recent publication showed that patients using this approach had a significantly lower the risk of hyponataemia compared to those who did not follow this approach (OR 0.4, 95%CI 0.3-0.7, p<0.01) (122).

Mostly, Desmopressin is a lifelong treatment. An exception is treatment of postsurgical central DI. If the patient is awake and responds to thirst, thirst is a sufficient guide for water replacement. If the duration of diabetes insipidus is transient, it is therefore acceptable to treat simply with fluid replacement, parenterally or orally. However, most patients who develop diabetes insipidus require desmopressin 0.5 to 2 μg subcutaneously, intramuscularly, or intravenously. Urine output will be reduced in 1 to 2 hours and the duration of effect is 6 to 24 hours. Care should be taken that hypotonic intravenous fluids are not given excessively after administering desmopressin, as the combination can lead to profound hyponatremia. Because there is always the possibility of developing the triphasic response due to pituitary stalk damage, it is recommended that polyuria should be recurrent before a decision to administer subsequent doses of desmopressin is made (123).

Patients with hypernatremia due to osmoreceptor dysfunction (adipsic central DI) should be treated acutely with the same treatment as any hyperosmolar patient. The long-term management of osmoreceptor dysfunction syndromes requires a thorough search for potentially treatable causes, combined with measures to prevent dehydration. Because hypodipsia cannot be cured, the focus of management is based on education of the patient and family about the importance of regulating their fluid intake according to their hydration status (124). This can be accomplished most efficaciously by establishing a daily schedule of fluid intake regardless of the patient's thirst, which can be adjusted in response to changes in body weight (125). As these patients will not drink spontaneously, daily fluid intake must be fixed and prescribed. If the patient has polyuria, desmopressin should also be prescribed, as in any patient with central DI. The success of the fluid prescription should be monitored periodically by measuring serum Na+concentration. In addition, periodic recalculation of the target weight (at which hydration status and serum Na+concentration are normal) might be required.

Treatment of NDI

Treatment of acquired nephrogenic DI should target the underlying cause (e.g., correction of hypercalcemia or hypokalemia), if possible. If not, different approaches are possible.

  • Low salt diet to minimize the osmotic load
  • For patients on long-term lithium therapy, amiloride prevents uptake of lithium in the collecting duct epithelial cells and thus the inhibitory effects of intracellular lithium on water transport (126).
  • Hydrochlorothiazide has been shown to reduce urine output in both central and nephrogenic DI (127, 128). Thiazides decrease salt reabsorption by inhibiting the thiazide-sensitive co-transporter SLC12A3 in the distal tubule. The loss of sodium reduces plasma volume, so that less water is presented to the collecting duct and lost in the urine.
  • In an animal model of nephrogenic DI, use of the NSAID indomethacin reduced water diuresis independently of AVP (129). A similar effect of prostaglandin synthesis inhibitors was later reported in patients with nephrogenic DI (130). Since these early studies, prostaglandin synthesis inhibitors have become an essential component of the treatment of nephrogenic DI
  • High dose DDAVP can produce a response in partial NDI
Treatment of PP

Treatment of primary polydipsia entails reduction of excessive fluid intakes, best done in a graded fashion to allow patients to slowly achieve a level of intake that reduced urine volume below polyuric levels (50 mL/kg BW). Measures to reduce mouth dryness (e.g., ice chips, hard candy to stimulate salivary flow) are useful adjuncts to reduce thirst. Pharmacologic therapies have been tried but without consistent evidence of success. A recent study suggests that GLP-1 analogues reduce fluid intake, urine output, and thirst perception(131).

Treatment of Gestational DI

Desmopressin is the only therapy recommended for treatment of diabetes insipidus during pregnancy. Desmopressin is reported to be safe for both the mother and the child (132, 133). During delivery, patients can maintain adequate oral intake and are therefore safe to continue administration of desmopressin. Physicians should be cautious about over administration of fluid parenterally during delivery because these patients will not be able to excrete the fluid and can develop water intoxication and hyponatremia. After delivery, plasma oxytocinase decreases and patients can recover completely or be asymptomatic with regard to fluid intake and urine excretion.

Syndrome Of Inappropriate Antidiuresis


Hyponatremia (serum sodium <135 mmol/l) is a clinical feature in some 15–20% of non-selected emergency admissions to hospital. It is associated with increased morbidity and mortality across a range of conditions. Moreover, data support the association of hyponatremia correction with improvements in clinical outcome. The relationship of serum sodium and outcome is not straightforward. Co-morbidity and disease severity, rather than hyponatremia per se, may make a significant contribution to adverse outcome in these patients. Further data are needed to clarify whether the relationship between sodium levels and outcome is causal or the association of two variables linked with disease severity (134-137).

Hyponatremia is not invariably associated with a low serum osmolality; high concentrations of other circulating osmolytes (e.g., glucose) can lead to a fall in plasma sodium that is appropriate to maintain normal osmolar status. A reduced plasma aqueous phase secondary to dyslipidemia can result in artefactual hyponatremia with normal plasma osmolality, even when using an ion-specific electrode. This is consequent to the use of a standard dilution step in most clinical biochemistry laboratories. This type of artefactual hyponatremia is not seen when a direct potentiometric method is used, such as when using a blood-gas analyzer. In many clinical situations, hyponatremia is multifactorial (Table 4).

Table 4. Causes of Hyponatremia


Reduced renal free water clearance

Hyperglycemia, Hyperlipidemia, Non-physiological osmolyte, Elevated paraprotein

Cardiac failure
Nephrotic syndrome
Nephrogenic syndrome of antidiuresis

Renal failure
Portal hypertension & ascites

Fluid sequestration

Sodium depletion

Renal loss

Salt wasting nephropathy Hypoadrenalism
Central salt wasting

Extra-renal loss

Gut loss

Excess water intake

Dipsogenic DI
Sodium-free, hypo-osmolar irrigant solutions
Dilute infant feeding formula
Exercise-associated hyponatremia

AVP plays a key role in many pathophysiological situations of which hyponatremia is a feature. Importantly however, even when AVP plays a role in the development of hyponatremia, AVP production may not be inappropriate. Hyponatremia may reflect an appropriate physiological response to volume depletion. To maintain circulating volume in hypovolemia, baroregulated AVP release may proceed despite plasma osmolalities below the osmotic threshold for AVP release. This can result in hyponatremia, which can become persistent. Though clinical assessment can identify the extracellular volume status of some patients, it is unreliable and has poor sensitivity and specificity (138).


An individual with hypoosmolar plasma, a normal circulating volume, and a plasma AVP concentration high for the prevailing osmolality, has the syndrome of inappropriate antidiuresis (SIAD). The clinical criteria to diagnose SIAD go back to Schwartz and Bartter (139) in 1967 and are summarized in Table 5.

Excretion of urine that is not maximally dilute in the context of dilute plasma (i.e., urine concentration greater than 100mOsm/Kg) indicates the action of AVP on renal water resorption. Importantly however, it does not define whether this action is appropriate (for instance in the context of hypovolemia and baro-stimulated AVP release) or inappropriate. Measurement of urinary sodium concentration is key in the differential diagnosis of SIAD from hypovolemia. Renal sodium excretion should be above 30mol/L to make a diagnosis of SIAD. Below this value, volume depletion needs to be considered more likely and below 20 mmol/L, hypovolemia is the likely cause of hyponatremia. SIAD is often associated with urine sodium concentrations of 60 mmol/L or more. SIAD is a volume-expanded state and there is evidence of mild sodium loss as other homeostatic regulators of volume homeostasis attempt to minimize volume expansion. The utility of urinary sodium concentration in defining the etiology of hyponatremia is limited by concurrent use of drugs that produce a natriuresis: diuretics, angiotensin converting enzyme inhibitors, and angiotensin II antagonists. In this situation, a serum urate <4 mg/dl, or a fractional urate excretion >12% can help differentiate SIAD from mild hypovolemia (140, 141).

Cortisol deficiency is a key differential diagnosis as secondary adrenal deficiency can present a biochemical picture identical to SIAD. Therefore, formal biochemical exclusion of adrenal insufficiency is mandatory before diagnosing SIAD.

Four patterns of abnormal AVP secretion have been identified (table 6). The same pattern has also been shown for copeptin (142). Absolute plasma AVP or copeptin concentrations may not be strikingly high and in fact AVP and copeptin measurement is not helpful in establishing the diagnosis (143). The key feature is that they are inappropriate for the prevailing plasma osmolality (10).

Table 5. Criteria for Diagnosis of SIAD

● Low serum osmolality <275 mOsmol/kg H20

● Elevated urine osmolality >100 mOsmol/kg H20 (inappropriately concentrated)

● Clinical euvolemia (absence of signs of hypovolemia or hypervolemia)

● Elevated urinary sodium excretion >30mEq/L with normal salt and water intake

● Absence of other potential causes of euvolemic hypo-osmolality (glucocorticoid insufficiency, severe hypothyroidism)

● Normal renal function and absence of diuretic use

Table 6. Classification of SIAD




Wide fluctuations in plasma AVP concentration independent of plasma osmolality



Osmotic threshold for AVP release subnormal
Osmoregulation around subnormal osmolar set point



Failure to suppress AVP release at low plasma osmolality
Normal response to osmotic stimulation


Normal osmoregulated AVP release
Unable to excrete water load.


Etiology of SIAD

Many conditions have been reported to cause SIAD, though the mechanism(s) of inappropriate AVP release are not clear in many cases (Table 7). SIAD is a non-metastatic manifestation of small cell lung cancer and other malignancies. Some tumors express AVP ectopically. However, excessive posterior pituitary AVP secretion also occurs in association with malignancy. The normal osmoregulated AVP release found in the Type D syndrome suggests an increase in renal sensitivity to AVP, or the action of an additional antidiuretic factor.

Table 7. Causes of SIAD

Neoplastic disease

Chest disorders

Carcinoma (bronchus, duodenum, pancreas, bladder, ureter, prostate)
Lymphoma, leukemia
Ewing's sarcoma
Bronchial adenoma

Cystic fibrosis

Neurological disorders


Head injury, neurosurgery
Brain abscess or tumor
Meningitis, encephalitis
Guillain-Barré syndrome
Cerebral hemorrhage
Cavernous sinus thrombosis
Cerebellar and cerebral atrophy
Shy-Drager syndrome
Peripheral neuropathy
Subdural hematoma
Alcohol withdrawal

Alkylating agents & Vinca alkaloids
Thiazide diuretics
Dopamine antagonists
Tricyclic antidepressants
3,4-MDMA ("Ecstasy")


Abdominal surgery
Diverse non-osmotic stimuli (e.g., nausea, stress, pain)

SIAD is a common mechanism of drug-induced hyponatremia, and can reflect direct stimulation of AVP release from the hypothalamus; indirect action on the hypothalamus; or aberrant resetting of the hypothalamic osmostat (table 8). The prevalence of hyponatremia in patients taking high dose dopamine antagonists is greater than 25%, and is not restricted to one class of these drugs. Hyponatremia secondary to antidepressants is well recognized, occurring with most SSRIs, and the related drug Venlafaxine. It can arise in the first few weeks of treatment. Anticonvulsants are another common cause of SIAD and hyponatremia. The frequency in patients treated with carbamazepine (CBZ) ranges from 4.8 to 40%. Increased sensitivity of central osmoreceptors and increased renal responses to AVP have both been described with CBZ.

Table 8. Mechanisms of Drug Induced Hyponatremia

Reduction in free water clearance

Sodium depletion


Dopamine antagonists
Tricyclic antidepressants
Sodium valproate
3,4-MDMA ('ecstasy')


Loop diuretics

AVP-like activity


ACE inhibitors/Angiotensin II receptor antagonists

Potentiation of AVP action


Direct renal toxicity


Exercise Associated Hyponatremia

Extreme endurance exercise is a profound physiological stressor. While the magnitude of the physiological stress is likely to reflect a number of factors, duration of the event and the effort entailed are likely to be major contributors. Non-osmoregulated AVP release is a feature of extreme endurance exercise: a reflection of the stressed state. When combined with reduced renal blood flow, another feature of extreme endurance exercise, this can lead to a marked antidiuretic state. If endurance athletes maintain a fluid intake in excess of water loss, hyponatremia will ensue. This can be further complicated if there is aggressive fluid resuscitation in the event of collapse. There is a positive correlation between the odds ratio for developing hyponatremia during extreme endurance exercise and the length of time taken to complete the event. Athletes developing hyponatremia also demonstrate weight gain over the course of the event, clearly implicating water intake in excess of water and electrolyte loss as the cause. Occasional runners should be advised to follow their thirst as they run and avoid rigid, time-based fluid intake. Health professionals attending endurance events need to be aware of the problem of exercise-associated hyponatremia. In addition, they should avoid attempting resuscitation with large volumes of hypotonic fluid in the absence of appropriate indications and without biochemical monitoring (144).

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Figure 10. Exercise associated hyponatremia in triathletes. 1089 triathletes were studied. The mean plasma sodium level at the finish was 140.5±4.2 mmol/L (range 111-152). Among athletes completing the study events, 10.6% had documented hyponatremia: 8.7% mild; 1.6% severe; and 0.3% critical (3 athletes in total with plasma sodium levels 120, 119, and 111 mmol/L respectively). Multivariate analysis showed a significant association between development of hyponatremia and the following factors: female gender; longer times to complete a race. Critical hyponatremia occurred in participants who finished in the 12th and 14th hours of the race (145).

Nephrogenic Syndrome of Inappropriate Antidiuresis

The G-protein-coupled V2-R mediates the action of AVP on renal water excretion. Rare kindreds have been found that harbor constitutively activating mutations in the V2-R that led to AVP-independent, V2-R mediated, antidiuresis associated with persistent hyponatremia (Figure 11). This nephrogenic syndrome of inappropriate antidiuresis (NSIAD) was initially described in male infants with persistent hyponatremia in keeping with the haploinsufficiency associated with the V2-Rgene being on the X chromosome. However, subsequent studies have found the condition is not limited to males, expression of the condition being clearly identified in heterozygous females. The true prevalence of NSIAD is not known. However, as some 10% of patients with SIAD have been described as having undetectable AVP, it seems likely that at least some of these cases may be due to activating mutations of the V2-R (146, 147).

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Figure 11. In vitro bioactivity of different V2-R constructs relative to wild type (WT) in a cAMP-dependent luciferase reporter system in the absence of AVP. The R137H construct is the V2-R found in X-linked NDI. R137C and R137L are receptor variants found in NSIAD. Constructs differ only by the amino acid at position 137. R137C and R137L found in NSIAD demonstrate constitutive, AVP-independent activity. R: arginine. H: histidine. C: cysteine. L: leucine.

Cerebral Salt Wasting

This acquired, primary natriuretic state remains a subject of controversy. It has been characterized as a combination of hyponatremia with hypovolemia associated with neurological or (more often) neurosurgical pathologies. The underlying mechanism(s) remain unclear, but increased release of natriuretic peptides and/or reduced sympathetic drive have been proposed. The diagnosis of cerebral salt wasting (CSW) hinges on the natural history: the development of hyponatremia being preceded by natriuresis and diuresis with ensuing clinical and biochemical features of hypovolemia. In contrast to SIAD, urea and creatinine are elevated and there may be postural hypotension. The simple observation of weight loss over the period in question can be helpful.

SIAD can occur in the same group of patients in whom CSW has been reported. Both CSW and SIAD are associated with urine sodium concentrations greater than 40mmols/L. However, the natriuresis of CSW is much more profound than that of SIAD and precedes the development of hyponatremia. CSW is a particular concern for the neurosurgical patient in whom autoregulation of cerebral blood flow is disturbed and in whom small reductions in circulating volume can reduce cerebral perfusion. The management of CSW is volume replacement with 0.9% saline; while the cause-directed approach to SIAD would often involve restriction of fluid. The clinical and practice context, together with the opposed cause-directed management approaches to CSW and SIAD can lead to significant tension. A cause-independent approach to the management of the neurosurgical patient with hyponatremia is often the practical and pragmatic approach to take: balancing management of hyponatremia with the need to avoid threatening cerebral perfusion and avoidable vasospasm (148). Importantly, in a prospective, single center study of 100 patients developing hyponatremia after subarachnoid hemorrhage not a single case of CSW was identified: hyponatremia was attributable to SIAD, glucocorticoid deficiency, or inappropriate fluid administration (Figure 12). Therefore, CSW is probably a very rare condition.

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Figure 12. Hyponatremia after subarachnoid hemorrhage. Prospective study of 100 patients. Demographics, outcomes and etiology of hyponatremia noted. SAH: subarachnoid hemorrhage (149).


The morbidity and mortality of hyponatremia secondary to SIAD are the result of disturbance in central nervous system (CNS) function: the combined impact of cerebral oedema and direct neuronal dysfunction (Table 9). While it is intuitive that the greater the derangement in serum sodium should be associated with the most profound neurological disturbance, the relationship between serum sodium and neurological function is not simple. Patients with marked biochemical disturbance may have mild symptoms if hyponatremia develops over a prolonged period. This reflects CNS adaptation: brain edema being limited by efflux of organic solutes. This adaptation can complicate the management of hyponatremia. Rapid correction of serum sodium following the gradual development of hyponatremia (which has resulted in a degree of CNS adaptation) can lead to significant changes in brain volume as the osmolar gradient across the blood-brain barrier alters. This can trigger CNS demyelination (osmotic demyelination syndrome, ODS). This is a rare but serious complication of hyponatremia and its treatment. ODS develops within 1-4 days of rapid correction of serum sodium, irrespective of the method employed to achieve it. It can even occur when sodium levels are corrected slowly. Risk factors for ODS are severe liver disease, potassium depletion, malnutrition and profound hyponatremia, especially if <105mmol/L. Neurological manifestations include quadriplegia, opthalmoplegia, pseudo-bulbar palsy and coma.

Table 9. Clinical Features of Hyponatremia Secondary to SIAD

• Headache

• Nausea

• Vomiting

• Muscle cramps

• Lethargy

• Disorientation

• Seizure

• Coma

• Brain-stem herniation

Management of Hyponatremia Associated with Severe or Moderately Severe Symptoms

Hyponatremia associated with severe or moderately severe symptoms requires urgent management as an emergency. Treatment is cause-independent. Treatment should be given priority over establishing the etiology of hyponatremia. The aim of treatment is to reduce immediate risk through increasing serum sodium to a level that decreases morbidity and mortality. Importantly, the rate of increase in serum sodium must be at a rate that does not result in harm through precipitating ODS (Figure 13). The immediate target should not be to normalize serum sodium. Once the patient is stable, investigations to establish the cause of hyponatremia can begin, the outcome of which subsequently guiding cause-directed therapy (140, 150, 151).

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Figure 13. Immediate, cause-independent management of hyponatremia associated with severe or moderately severe symptoms.

Previous strategies for the emergency management of hyponatremia have stratified patients based on presumed duration of the electrolyte disturbance. Furthermore, some have advocated hypertonic fluid administration volume and rates based on calculated deficits in serum sodium using a standard formula (152). A more pragmatic and reliable approach to patient stratification is to base the decision on use of hypertonic fluid on patient symptoms and signs; while the determining hypertonic fluid prescription by calculated serum sodium deficit is associated with a significant risk of over-correction (153). Over-correction of hyponatremia (increase in serum sodium above the recommended rate) should prompt review of the fluid regimen and consideration of active management with hypotonic fluid, with or without concurrent use of DDAVP (140, 154).

Two recent studies have compared bolus versus infusion hypertonic saline, both in favor of a bolus regimen. An Irish observational study compared outcomes for 22 hyponatremic patients treated with bolus 100ml 3% NaCl, compared to a historical cohort of 28 patients managed with 20ml/hour 3% NaCl infusion (155). Bolus hypertonic saline was associated with a faster elevation of serum sodium at 6 hours and significantly greater improvement in Glasgow Coma Scale at 6 hours. There were no cases of ODS in either group. A larger randomized trial from Korea included 178 patients with hyponatremia who were randomized to either bolus or infusion NaCl 3% (156). The results showed that both therapies were effective and safe, with no significant difference in overcorrection risk, but there was less need for therapeutic relowering in the bolus group compared to the slow continuous infusion. Bolus therapy is therefore currently recommended in guidelines.

Management of SIAD Associated with Mild to Moderate Symptoms

Fluid restriction: Fluid restriction of 0.5–1L/day is the first-line treatment recommended by EU and US guidelines (157). It is a reasonable initial intervention when the clinical condition is not critical. All fluids need to be included in the restriction. As SIAD is associated with a degree of natriuresis, sodium intake should be maintained. Fluid restriction may need to be maintained for several days before sodium levels normalize and it is important that a negative fluid balance is confirmed during this period. As cause-directed therapy progresses (e.g., treatment of underlying infection or removal of drug causing SIAD), fluid restriction may be relaxed. A recent randomized controlled trial evaluated the efficacy of fluid restriction in comparison to no treatment showing a modest early rise in serum sodium of 3mmol/L in fluid restricted patients compared to 1mmol/L with no treatment. Similar results were seen in other recent trials (158).

Of note, fluid restriction is not always effective, and a clinically useful response is only seen in around half of hyponatremic patients. In a large hyponatremia registry fluid restriction was unsuccessful in 55% of cases (159). Factors predicting a poor response to fluid restriction include high urine osmolality (>500 mosm/L), and high urine sodium concentration (or high urine/serum electrolyte ratio i.e. (UNa + UK)/pNa > 1, i.e., ‘Furst equation’) (150, 160, 161).

Drug treatment in SIAD: If patients do not respond to fluid restriction, a second-line treatment has to be initiated. There are several different possibilities, which are discussed in the following.

Urea is a product of hepatic nitrogen metabolism which is renally excreted and has an osmotic effect to promote free water excretion. It is recommended as a second line treatment in the European and US guidelines(140, 150). The evidence base for the use of urea is limited due to the retrospective observational nature of most current studies. In fact, there are several observational studies of urea, but no randomized trials to date. A retrospective observational trial reported a mean increase in sodium levels of 7 mmol/L over 4 days, with no instances of rapid correction (162). Another retrospective study reported that the majority of patients normalized sodium levels within 48 hours despite fluid intake >2 L/d (163). Finally, in patients with SIAD in the context of subarachnoid hemorrhage urea treatment led to sodium normalization within a mean time of 3 days (164).

Although overcorrection has been described, there have been no published cases of ODS due to urea therapy. The main limitations are the difficult access and poor palatability due to bitter taste, which can be ameliorated by combining it with a small volume of orange juice. Urea therapy can cause an increase in serum urea concentration and blood urea nitrogen, but this does not necessarily represent deterioration in renal function or hypovolemia (162).

Tolvaptan is an oral vasopressin V2-receptor antagonist that blocks AVP action in the kidney, inducing water diuresis and thereby raising sodium levels. An intravenous alternative conivaptan is also approved, but its use is limited to a maximum duration of 4 days because of drug interaction effects with other agents metabolized by the CYP3A4 hepatic isoenzyme.

The efficacy of tolvaptan versus placebo was investigated in the randomized placebo-controlled SALT-1 and SALT-2 trials in patients with chronic eu- or hypervolemic hyponatremia (165). The results showed that tolvaptan increased sodium levels within 4 days by 4 mmol/L compared to only 1 mmol/L in the placebo group and an even higher increase within 30 days. The American expert panel recommends its use as a second-line treatment, whereas the European clinical practice guidelines recommend against the use of tolvaptan in moderate to profound hyponatremia, mainly due to lack of proven outcome benefit aside from increase in sodium levels and the concern of overcorrection. Importantly, discontinuing fluid restriction when vaptans are started, initiation of treatment in the hospital and regular sodium monitoring limit the risk for overcorrection. A lower initial dose of tolvaptan, 7.5 mg, which has been associated with lower rates of overcorrection, should also be considered (166). Common side effects of tolvaptan include dry mouth, thirst, and urinary frequency.

Sodium-glucose co-transporter 2 inhibitors are approved for treatment of diabetes and heart failure, but may also be beneficial in SIAD. The sodium-glucose co-transporter 2 in the proximal renal tubule is responsible for 90% of renal glucose resorption (167). Inhibition of SGLT2 leads to glycosuria, accompanied by an osmotic diuresis. A randomized trial of inpatients with SIAD compared 4 days of empagliflozin 25mg or placebo, in addition to fluid restriction (168). Plasma sodium increased by 10mmol/L over 4 days in the empagliflozin group, compared to 7mmol/L with placebo (p = 0.04). There was no hypotension or hypoglycemia observed. The relatively high sodium increment in the placebo group suggests that at least in some patients, reversible stimuli to AVP secretion may have been present. A second, randomized controlled cross-over study in 14 patients’ outpatients with chronic SIAD compared a 4-week treatment with empagliflozin 25mg/day to placebo, without concomitant fluid restriction. Empagliflozin treatment resulted in a serum sodium increase of 4.1mmol/L (p=0.004) under empagliflozin as compared to placebo. Treatment with empagliflozin was generally well tolerated with no events of sodium overcorrection, hypoglycemia, hypotension, urinary tract or genital infection occurring during the observation period (169). Future larger studies are needed to validate these findings.

Loop diuretics block the Na+-K+-2CL- cotransporter in the thick ascending limb of the loop of Henle and decrease delivery of sodium and chloride to the kidney medulla, decreasing the medullary gradient necessary for water reabsorption in the collecting duct. Salt tablets are added to replace loop diuretic-induced sodium losses. They are listed in the European practice guidelines as a second line therapy for SIAD in combination with salt tablets. However, a recent randomized controlled trial comparing the effect of FR alone versus FR plus loop diuretics versus FR plus loop diuretics plus salt tablets did not show a significant additive effect of loop diuretics or salt tablets (158). Acute kidney injury and hypokalemia were more common in patients receiving furosemide, therefore caution should be taken when adopting this strategy.

Demeclocycline has been used for many years in management of hyponatremia of SIAD. It produces a form of NDI and so increases renal water loss even in the presence of AVP. There is a lag time of some 3–4 days in onset of action and the effect is unpredictable occurring in 33-60% of patients. Photosensitive skin reactions are a significant additional adverse effect. There are very limited data to support long-term efficacy and a systematic review did not find good quality evidence for its use (170).

Lithium also induces nephrogenic diabetes insipidus and has therefore been tried as treatment for SIAD, however, due to adverse effects and questionable efficacy it is not recommended in European guidelines.

Adipsic And Hypodipsic Syndromes

Adipsic and hypodipsic disorders are characterized by inadequate spontaneous fluid intake due to defects in osmoregulated thirst. Patients deny thirst and do not drink, despite dehydration and hypovolemia. If the defect is mild, the resultant hypernatremia is often well tolerated. Severe disorders can lead to somnolence, seizures, and coma. Because of the close anatomical relationship of the osmoregulatory centers for thirst and AVP release, adipsic syndromes are often associated with defects in osmoregulated AVP release and HDI.


Four patterns of adipsic/hypodipsic syndrome are recognized (Table 10, Figure 14). Causes are outlined in Table 11. Patients with the Type A syndrome osmoregulate around a supra-normal osmolar set point and are protected from extreme hypernatremia, as are those with the Type B syndrome. In Type C adipsia, osmoregulated thirst and AVP release are absent. Patients present with adipsic diabetes insipidus. Precipitants include rupture and repair of anterior communicating artery (ACA) aneurysm, as the osmoreceptors mediating both thirst and AVP release receive a blood supply from perforating branches of the anterior cerebral artery and ACA. Some patients with the Type C syndrome have constitutive low level AVP release, and are at risk of dilutional hyponatremia.

Table 10. Classification of Adipsic and Hypodipsic Syndromes

Adipsia/hypodipsia Syndrome

Osmoregulated Thirst

Osmoregulated AVP release

Type A (essential hypernatremia)

Osmotic threshold increased
Normal sensitivity

Osmotic threshold increased
Normal sensitivity Normal non-osmotic stimulation

Type B

Normal osmotic threshold
Reduced sensitivity

Normal osmotic threshold
Reduced sensitivity
Normal non-osmotic stimulation

Type C

No response to osmotic stimulation

Persistent low level AVP release
No response to osmotic stimulation
Normal non-osmotic stimulation

Type D

No response to osmotic stimulation


Table 11. Causes of Adipsic and Hypodipsic Syndromes

Neoplastic (50%)


Germ cell tumor


Pituitary tumor
Bronchial carcinoma
Breast carcinoma

Granulomatous (20%)

Miscellaneous (15%)
Ventricular cyst

Toluene poisoning

Vascular (15%)
Internal carotid artery ligation
Anterior communicating artery aneurysm
Intra-hypothalamic hemorrhage

Chapters Archive - Endotext (34)

Figure 14. Patterns of plasma AVP and thirst responses to hypertonic stress in patients with adipsic syndromes. Normal range responses to osmolar stimulation are shown by the shaded areas. The 4 types of adipsic syndrome are demonstrated. Patients with the Type A syndrome osmoregulate around a higher osmolar set point, while those with the Type B syndrome mount AVP and thirst responses but with reduced sensitivity. Patients with the Type C syndrome have much reduced or absent AVP and thirst responses to osmolar stimulation. Those with the Type D syndrome demonstrate normal AVP responses to osmolar stimulation but much reduced thirst responses.


As those with Type A and Type B adipsia are protected from extreme hypernatremia, treatment is to recommend an obligate fluid intake of about 2L/24 hours with appropriate adjustment for climate and season. If fluid balance cannot be maintained during intercurrent illness, hospital in-patient management may be required. The adipsic central diabetes insipidus of the Type C syndrome can be difficult to manage. The structural and vascular problems producing the syndrome often led to associated defects in short term memory and task organization, complicating long-term management. A pragmatic approach is to effectively dictate an acceptable urine output (1-2L/24 hours) with regular DDAVP (producing a fixed obligate antidiuresis). Together with an estimation of standard insensible fluid loss (approximately 0.4L), this can be set to create a fixed net fluid loss of some 2Ls. In turn, this can be balanced by a daily fluid intake that varies in response to depending on day-to-day fluctuations from a target weight at which the patient is euvolemic and normonatremic.

Daily fluid intake = 2L + (daily weight in Kg- target weight in Kg).

Plasma sodium should be checked weekly, to avoid the creeping development of hyper- and hyponatremia as dry weight changes. This approach can result in stable fluid balance and successful independent living (171).


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Adrenal insufficiency (AI) is an uncommon clinical condition resulting from inadequate glucocorticoid secretion or action, either at the basal state or during stress. Hypothalamic-pituitary-adrenal axis disturbance or primary adrenal failure itself is responsible for this condition. AI presentation can range from asymptomatic hormonal dysfunction to adrenal crisis. Prompt diagnosis and management are crucial since AI may be fatal if unrecognized or untreated. Among the various investigations, the appropriate tests should be performed in a timely manner in order to reverse hormonal and metabolic disturbances, treat associated conditions, and prevent acute crises. In this review, we will provide background knowledge regarding pathophysiology, clinical manifestations, underlying etiologies, hormonal investigations, and related treatments in AI.


Adrenal insufficiency (AI) is a life-threatening condition characterized by the failure of adrenocortical function. This failure results in impaired secretion of glucocorticoids (GCs) only or of GCs and mineralocorticoids (MCs) and adrenal androgens, which are crucial for energy, salt and fluid homeostasis, and androgenic activity. The disorder may be caused by adrenocortical disease, primary adrenal insufficiency (PAI), known as Addison’s disease, which is a rare disease with reported prevalence in Europe ranging from 39 cases/million in England in 1968 (1) to 221 cases/million in Iceland in 2016 (2), the highest prevalence reported. Depending on the study, the annual incidence of AI in Europe is estimated to range between 4.4 and 6.2 new cases per million people (3,4). AI may be secondary in the setting of conditions affecting the pituitary gland and the secretion of adrenocorticotropic hormone (ACTH) (secondary AI) and/or the hypothalamus and the secretion of corticotropin-releasing hormone (CRH) and/or other ACTH secretagogues such as vasopressin (tertiary AI). Central AI is estimated to have a prevalence between 150 and 280 per million, making it more prevalent than PAI (5-7). A prevalence of AI after pituitary surgery varies, with higher rates of up to 90% after craniopharyngioma surgery, while hypopituitarism has high prevalence after cranial radiation for non-pituitary tumors, but may take several years to develop.

The clinical manifestations of AI depend upon the extent of loss of adrenal function, and whether MCs and androgen production are preserved, whereby the renin-angiotensin-aldosterone system (RAAS) is intact in central AI. The presentation can be acute or insidious, depending on the underlying cause of the adrenal failure. The diagnosis may be delayed until an intercurrent illness, such as serious infection, acute stress, bilateral adrenal infarction, or hemorrhage, precipitates a life-threatening adrenal crisis. The symptoms of PAI are pleiotropic and non-specific, except for salt craving (Table 1).

Adrenal crisis is an acute deterioration in health which is associated with hypotension, acute abdominal symptoms, and marked laboratory abnormalities, which resolve after parenteral glucocorticoid administration. In PAI, the patients may have more severe symptoms which are due to concomitant MCs deficiency. This condition is commonly presented in PAI, but less common in central AI, in which a typical example would be pituitary apoplexy. Retrospective and prospective analysis revealed a prevalence of adrenal crises 6.6-8.3 cases/100 patient-years, with mortality 0.5/100 patient-years, mainly due to gastrointestinal and other infectious diseases (8). A recent retrospective case-control analysis of the European Adrenal Insufficiency Registry (EU-AIR) reported an incidence of adrenal crisis of 6.53 cases per 100 patient-years for PAI and 3.17 cases per 100 patient-years for central AI (9).

A significant feature to clinically differentiate PAI from central AI is skin pigmentation, which is nearly always present in long-standing PAI. The most probable cause of the pigmentation seems to be the increased stimulation of the melanocortin-1 receptor (MC1R) by elevated levels of ACTH itself with its intrinsic α-melanocyte stimulating hormone activity. The rest of the clinical features of secondary and tertiary AI are similar to those of PAI type (Table 1). Rarely, the presentation may be more acute in patients with pituitary apoplexy. Hyponatremia and compensatory water retention may be the result of an “inappropriate” increase in vasopressin secretion. The clinical manifestations of a pituitary or hypothalamic tumor, such as symptoms and signs of deficiency of other anterior pituitary hormones, headache or visual field defects, may also be present.

Another condition with a dissociation in GCs and MCs secretion presenting as AI is congenital adrenal hyperplasia (CAH), with a frequency of adrenal crisis of 5.8 cases/100 patient-years (4.9 cases/100 patient-years after correction for a neonatal salt-wasting crisis) and often respiratory infections, firstly in early childhood, followed by gastrointestinal infections at older ages (10).

Table 1. Symptoms, Physical Findings, and Laboratory Findings Associated with Adrenal Insufficiency


Adrenal crisis: hypotension (< 110mmHg systolic) and syncope/ shock (> 90%); volume depression

Non-specific symptoms:

· Gastrointestinal symptoms: abdominal pain, flank pain, back pain, or lower chest pain: 86%- may mimic acute abdomen

· Fever (66%)

· Anorexia (early feature), nausea, vomiting (47%)

· Abdominal rigidity or rebound tenderness (22%)

· Diarrhea, which may alternate with constipation

· Neuropsychiatric symptoms: Confusion, lethargy, disorientation, coma (42%)


Psychiatric symptoms: memory impairment, depression, anxiety, psychosis,reduced consciousness, delirium

Chronic AI

General malaise, weakness, fatigue, lassitude, generalized weakness


Hypoglycemia; increased risk in children, thin women, alcohol abuse, GH deficiency


Sudden severe headache, loss of vision or visual field defect

SAI (pituitary apoplexy)


· Hyperpigmentation: sun-exposed or pressure areas, recent scars (after AI manifestation), axillae, nipples, palmar creases, mucous membranes as buccal mucosa

· Vitiligo (as marker of autoimmune disease)

Chronic PAI

Postural hypotension due to volume depletion, or improvement in blood pressure control in previously hypertensive patients, postural dizziness


Salt craving (22%)


Autoimmune manifestations: vitiligo, autoimmune thyroid disease, type 1 diabetes, primary ovarian failure, autoimmune gastritis


Weight loss

Chronic PAI/SAI

Decreased axillary and pubic hair, loss of libido in females (DHEA deficiency), amenorrhea in women (in 25% due to chronic illness, weight loss or associated premature ovarian failure)


Auricular calcification

Low grade fever


Associated endocrinopathies in the context of autoimmune polyglandular syndrome



Electrolyte abnormalities:

· Hyponatremia: 85-90% (PAI: MCs deficiency; CAI: dilutional effect)

· Hyperkalemia: 60-65% due to MCs deficiency

· Metabolic acidosis

· Mild hypercalcemia (uncommon)






Liver enzymes abnormalities: may be observed in autoimmune hepatitis


Changes in blood count:

· Mild anemia (normocytic normochromic)

· Eosinophilia

· Lymphocytosis

TSH with normal or low normal T4 (transient with ACTH; permanent with autoimmune thyroiditis)


 Erythrocyte Sedimentation Rate

ACTH: adrenocorticotropic hormone; AI: adrenal insufficiency; DHEA: dehydroepiandrosterone); GCs: glucocorticoids; GH: growth hormone; MCs: mineralocorticoids; PAI: primary AI, SAI: secondary AI, T4: thyroxine; TAI: tertiary AI; TSH: thyrotropin stimulating hormone


The majority of cases of PAI are the result of gradual destruction of all three layers of the adrenal cortex. Clinical manifestations of this condition appear when the loss of the adrenocortical tissue of the combined glands is greater than 90%. In the initial phase of chronic gradual destruction, adrenal reserve is decreased, and although the basal steroid secretion is normal, the secretion in response to stress is suboptimal, resulting in inadequate GCs, MCs and androgen production, leading to partial ΑΙ; this is manifested by an inadequate cortisol response during stress. Any major or even minor stressor can precipitate an acute adrenal crisis, followed by a complete AI, since with further loss of adrenocortical tissue, even basal steroid secretion is decreased, leading to the clinical manifestations of the disease. Adrenal hemorrhage or infarction may lead to adrenal crisis, a medical emergency manifesting as hypotension and acute circulatory failure crisis due to MCs deficiency when the appropriate doses of GCs are not met to cover MCs requirements. On the other hand, GCs deficiency may also contribute to hypotension by decreasing vascular responsiveness to angiotensin II, norepinephrine/noradrenaline, and other vasoconstrictive hormones, reducing the synthesis of renin substrate, and increasing the production and effects of prostacyclin and other vasodilatory hormones. Combined GCs and MCs deficiency leads to increased urinary sodium loss and hypovolemia resulting in hypotension and electrolyte imbalance including hyponatremia and hyperkalemia. ‘Inappropriate’ anti-diuretic hormone (ADH) release and action on the renal tubule due to GCs deficiency contributes to the hyponatremia, although it could be argued that this attempt at volume maintenance is far from inappropriate. Low plasma cortisol concentrations reduce GCs negative feedback, which in turn increases the production and secretion of ACTH and other POMC-peptides. These mechanisms are responsible for the well-recognized hyperpigmentation by acting on the MC1R in the skin.

Conversely, ACTH deficiency in central AI leads to decreased secretion of cortisol and adrenal androgens, while MCs production remains normal, as MCs are principally regulated by the RAAS. In the early stages, basal ACTH secretion is normal, while its stress-induced release is impaired. With further loss, there is atrophy of zonae fasciculata and reticularis of the adrenal cortex. Therefore, basal cortisol secretion is decreased but aldosterone secretion by the zona glomerulosa is preserved. However, hypotension in central AI may still occur due to decreased vascular tone as a result of reduced vascular responsiveness to angiotensin II and noradrenaline.


Table 2. Etiology of Primary Adrenal Insufficiency (11-17)


· Sporadic: not associated with other autoimmune disorders

· APS type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dysplasia (APECED): Addison’s disease, chronic mucocutaneous candidiasis, hypoparathyroidism, dental enamel hypoplasia, pernicious anemia, alopecia, primary gonadal failure

· APS type 2 or Schmidt’s syndrome: Addison’s disease, autoimmune thyroid disease, primary gonadal failure, type 1 diabetes, celiac disease, pernicious anemia, myasthenia gravis, vitiligo

· APS type 4: Addison’s disease with other autoimmune diseases excluding autoimmune thyroid disease and type 1 diabetes


· Tuberculosis

· Fungal infections: histoplasmosis, cryptococcosis, candidiasis, African trypanosomiasis, paracoccidioidomycosis (South America)

· Syphilis

· Cytomegalovirus, HIV (up to 5% patients with AIDS)


From lung, breast, kidney, colon cancers, melanoma, lymphoma


· Sarcoidosis

· Amyloidosis

· Haemochromatosis

Intra-adrenal hemorrhage

· Drugs: anticoagulant, tyrosine kinase inhibitor

· Trauma

· Waterhouse-Friderichsen syndrome: mostly associated with meningococcal septicemia


Anti-phospholipid syndrome

Hematological disorders


Adrenoleukodystrophy (ABCD1 and ABCD2 gene mutations): X-linked disorder of very long chain fatty acid (VLCFA) metabolism, presents in childhood, may progress to severe spinal cord problems, adrenomyeloneuropathy (AMN), and cerebral demyelination causing dementia.

Kearns-Sayre syndrome (mitochondrial DNA deletions): progressive external ophthalmoplegia, bilateral pigmentary retinopathy, cardiac conductions, CNS dysfunction, endocrine abnormalities (Addison’s disease, hypogonadism, hypothyroidism, hypoparathyroidism, diabetes mellitus)

Wolman’s disease (LIPA gene mutations): inherited disorder of lysosomal enzyme, presented with abdominal distension from accumulation of foamy lipid droplet in various organs, along with adrenal calcification and malabsorption.

Congenital adrenal hyperplasia (CAH)

Autosomal recessive disorders of enzyme deficiency in adrenal steroidogenesis pathway, which can have various manifestations depending on their subtype and severity. Hence, this disease may be diagnosed in older individuals.

· 21-Hydroxylase deficiency (CYP21A2 gene mutation): the most common type, may be presented with salt wasting form in infancy, or simple virilizing form in later life if neonatal screening is not performed

· 11β-hydroxylase deficiency (CYP11B1 gene mutation): hyperandrogenism with hypertension in older children and adults

· 3β-hydroxysteroid dehydrogenase 2 deficiency (CYP3B2 gene mutation): neonatal wasting, ambiguous genitalia in boys, hyperandrogenism in girls

· P450 oxidoreductase deficiency (POR gene mutation): abnormal genitalia with or without skeletal malformations, and with or without maternal virilization

· P450 side-chain cleavage deficiency (CYP11A1 mutations): may be presented with neonatal salt wasting with 46,XY under-androgenization, or later onset of PAI and ambiguous genitalia

· Congenital lipoid adrenal hyperplasia (StAR gene mutations): may be presented with varied severity of PAI and 46,XY DSD

Congenital adrenal hypoplasia

· X-linked form (NR0B1 mutations or deletion): variable manifestations, hypogonadotropic hypogonadism, impaired spermatogenesis, hypoaldosteronism, shock

· Xp21 contiguous gene syndrome (deletion of NR0B1, glycerol kinase and genes for Duchenne muscular deficiency): with psychomotor retardation, hepatic iron deposition

· SF-1 linked (NR5A1 mutations or deletions): range from isolated adrenal failure to isolated gonadal failure, XY sex reversal, 46,XX DSD, gonadoblastoma, gonadal insufficiency

· IMAGe syndrome (CDKN1C pathogenic variant): Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genital abnormalities in males

· MIRAGE syndrome (SAMD9 pathogenic variant): Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes, and Enteropathy)

· Familial steroid-resistant nephrotic syndrome with AI (SGPL1 mutations)

Inherited unresponsiveness to ACTH syndromes

Familial glucocorticoid deficiency (FGD): autosomal recessive cause of childhood onset AI, hyperpigmentation, hypoglycemia with normal MCs activity.

· Type 1 variant (MC2R gene mutations)

· Type 2 variant (MRAP gene mutations)

· Other variants (MCM4, NNT, TXNRD2, GPX1, PRDX3, partial mutation of StAR and CYP11A1)

· Triple A or Allgrove syndrome (AAAS gene mutations): Addison’s disease, Achalasia, Alacrima, along with neurodegenerative change with or without mental retardation


Bilateral adrenalectomy


· Inhibition of steroidogenesis: ketoconazole, fluconazole, etomidate, aminoglutethimide, suramin

· Acceleration of cortisol metabolism: phenytoin, phenobarbital, thyroxine, rifampicin, St John’s Wort (Hypericum perforatum)

· Promotion of adrenolytic activity: mitotane

· Enhancement of autoimmunity: CTLA-4 inhibitors

Table 3. Etiology of Central Adrenal Insufficiency (18,19)

Pituitary, parasellar and hypothalamic masses

· Pituitary adenomas, rarely carcinomas

· Parasellar lesions: cysts, craniopharyngioma

· Nonadenomatous neoplasms: meningioma, chordoma, glioma, germinoma, pituicytoma

· Metastases: breast, lung, prostate, colon, lymphoma


· Pituitary abscess: Gram-positive cocci

· Tuberculosis

· Syphilis, leptospirosis

· Fungal infections: candidiasis, aspergillosis

· Herpes/Varicella infection, SARS-CoV-2 virus


· Hypophysitis: lymphocytic, granulomatous, xanthomatous, necrotizing, IgG4-related, immunotherapy-induced, other autoimmune-associated

· Hemochromatosis

· Sarcoidosis

· Histiocytosis X

· Wegener’s granulomatosis


· Pituitary apoplexy


· Sheehan’s syndrome


· Pituitary surgery

· Pituitary irradiation


· Steroid

· Mifepristone: impaired GCs signal transduction

· Somatostatin analogues

· Opiates

· Antipsychotics and antidepressants


· Traumatic brain injury

Transcription factor mutations

Hereditary ACTH deficiency can manifest as an isolated pituitary defect or as a combination of pituitary hormone deficiencies.

· HESX1: panhypopituitarism, cognitive change, septo-optic dysplasia

· OTX2: panhypopituitarism, neonatal hypoglycemia, pituitary hypoplasia, ectopic posterior pituitary

· LHX4: panhypopituitarism

· PROP1: panhypopituitarism

· SOX3: panhypopituitarism, infundibular hypoplasia, mental retardation

· TBX19: isolated ACTH deficiency

POMC and related processing

· POMC gene mutations: AI, severe early-onset obesity, hyperphagia, red hair, pale skin

· PC1 mutations: AI, abnormal glucose metabolism, early-onset obesity, hypogonadotropic hypogonadism, neonatal-onset persistent malabsorptive diarrhea

Prader-Willi syndrome: hypotonia, failure to thrive, obesity, multiple endocrine abnormalities (GH deficiency, central hypothyroidism, hypogonadotropic hypogonadism, central AI)

Familial corticosteroid binding-globulin deficiency: unexplained fatigue, hypotension

Idiopathic hypopituitarism

ACTH, adrenocorticotropic hormone; AI, adrenal insufficiency; APS, autoimmune polyglandular syndrome; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; DSD, disorders of sexual development; GCs, glucocorticoids;GH, growth hormone; HPA, hypothalamic-pituitary-adrenal; MCs, mineralocorticoids; PAI, primary AI; POMC, pro-opiomelanocortin; TSH, thyrotropin stimulating hormone.

When an adrenal crisis is present, there is no need for immediate investigation to confirm AI but treatment should be initiated without delay as soon as clinically suspected. Clinical suspicion could be the case of a cancer patient under treatment with immunotherapy, particularly with the anti-CTLA-4 agent ipilimumab and signs and/or symptoms suspicious for AI, where the differential diagnosis of the rare metastatic involvement of adrenal or pituitary gland has to be also considered (20,21). In any case, confirmatory testing should be deferred until the patient has been stabilized; however, a blood sample taken at this time for cortisol and ACTH levels is extremely helpful for later assessment. In cases of insidious presentation, clinical suspicion of AI should be followed by diagnostic dynamic tests to confirm the inappropriately low cortisol secretion and whether cortisol deficiency is dependent or independent of ACTH deficiency by measuring ACTH levels (Table 4). In PAI, cortisol deficiency results in decreased feedback to the HPA axis, leading to increased secretion of ACTH to stimulate the adrenal cortex. Simultaneously, MCs deficiency causes increased release of renin by the juxtaglomerular apparatus of the kidneys.

In a non-acute setting, the diagnosis should be suspected based on the patient’s history and physical examinationalong with low morning cortisol levels, and confirmed by an ACTH stimulation test. Basal morning cortisol levels lower than 3μg/dL suggest ACTH deficiency. Conversely, morning cortisol higher than 15μg/dL indicates sufficient ACTH reserve. However, random cortisol levels are not advised for the diagnosis of AI. Additional tests are required to establish the diagnosis of AI if the cortisol levels are in the range of these cut-offs. Based on the pathophysiology, different dynamic tests have been proposed to diagnose this condition.

The classic short Synacthen test (SST; 250μg of ACTH [1-24], i.m. or i.v.) is considered the standard diagnostic method to detect AI, with a sensitivity of 92% (95% confidence interval, 81–97%) for the diagnosis of AI (22). On the other hand, no statistically significant difference was found between low-dose and high-dose ACTH stimulation tests (23,24). Low levels for age and sex of dehydroepiandrosterone sulphate (DHEAS) concentration (or less frequently, dehydroepiandrosterone, DHEA) represents an additional marker to increase the level of suspicion of PAI, but it is not per se diagnostic. A CRH test may also be used, but is less common and has limited availability, while a prolonged ACTH stimulation test is rarely required other than to distinguish secondary or tertiary deficiency (Table 4).

The insulin-induced hypoglycemia or insulin tolerance test (ITT), also previously known as the gold standard test, is performed by injecting 0.1 or 0.15u/kg of short-acting insulin intravenously after overnight fast, followed by serial measurements of venous glucose and cortisol over 2 hours. Importantly, this procedure must only be used if there is adequate supervision and experience. However, the ITT is beneficial in assessing growth hormone (GH) response simultaneously in patients with suspected co-existing deficiency in secondary AI, in which GH may not exceed 3-5μg/dL. It must be emphasized that all normative values quoted are assay-dependent, and using immunoassays or liquid chromatography with tandem mass spectrometry (LC-MS/MS) may result in a lower cut-off than the historic value of 18μg/dL derived from polyclonal antibody assay (25). Further investigations such as imaging studies, auto-antibodies, or microbiological screening should be arranged accordingly to identify the underlying cause of AI.

Table 4. Diagnostic Tests Used to Diagnose and Differentiate AI

Test/ procedure

Interpretation of the result/ comments

Cortisol physiologic response

Adrenal testing

Morning serum cortisol levels at 8-9am in combination with plasma ACTH

· 8-9am cortisol levels < 3μg/dL (80nmol/L) suggest AI (26).

· In recent onset central AI within 4-6 weeks or severe stress such as sepsis, a ‘normal’ level may still indicate AI.

· ACTH levels > 300ng/L (66pmol/L) or > 2-fold the ULN confirms PAI.

· Cortisol levels > 14.5μg/dL (400nmol/L) indicates normal HPA axis (27).

· Morning cortisol levels in early postoperative pituitary surgery higher than 10µg/dL (275nmol/L) are a predictor of corticotroph reserve (28,29).

SST or cosyntropin test or ACTH test; sampling at 8-9am for cortisol and ACTH level following by 250μg Synacthen for adults, children ≥ 2y of age (15μg/ kg for infants, 125μg for children < 2y of age) ACTH i.v. or i.m.; collect samples at 0, 30 and 60min for cortisol levels.

· Peak cortisol levels < 18μg/dL indicate AI (depending on assay) (26).

· Indicated when morning cortisol levels 3-15μg/dL.

· Recent-onset central AI may produce a normal response.

· SST can be performed at any time of the day but testing for cortisol levels should be collected at least 18–24 h after the last HC dose or longer for GCs.

· Peak cortisol levels > 18µg/dL (430-500nmol/L) at 30 or 60 minutes (depending on assay).

· Pregnancy: higher diagnostic cortisol cut-offs of 25μg/dL (700 nmol/L), 29μg/dL (800 nmol/L), and 32μg/dL (900nmol/L) for the first, second, and third trimesters, respectively (30).

Low-dose SST; 1μg ACTH i.v.; collect samples at 0, 30min for cortisol levels (31).

· Peak cortisol levels < 18μg/dL indicate AI (depending on assay).

· Indicated when suspected recent-onset central AI or a shortage of Synacthen itself.

· Insufficient response to low-dose SST should be considered for other dynamic tests.

· Peak cortisol level > 18µg/dL (500nmol/L)

Pituitary testing

CRH stimulation test; 1µg/kg or 100µg ovine or human CRH i.v.; collect samples at -5, -1, 0, 15, 30, 60, 90, and 120min for cortisol and ACTH levels (32).

· Central AI demonstrates low ACTH levels that do not respond to CRH.

· PAI shows high ACTH levels that rise after administration of CRH.

· Limited use due to wide variation of responses.

· Peak ACTH response should be 2-4 folds above baseline at 15 or 30min.

· Peak cortisol level > 20µg/dL between 30 and 60min or incremental cortisol > 10µg/dL above baseline (33).

Prolonged ACTH stimulation test;

(A) 8-h protocol - 1mg depot Synacthen i.m. or 250μg cosyntropin i.v. infusion over 8h; collect serum samples hourly for cortisol and additional samples at 0, 1, 7, 8h for plasma ACTH levels.

(B) 24-h protocol - cosyntropin 250μg i.v. infusion over 24h on 2 or 3 consecutive days; take blood sample for 9am cortisol and ACTH level; then blood sample for serum cortisol levels at 30min, 60min, 120min, 4h, 8h, 12h and 24h.

· Central AI illustrates a delayed response and typically has a much greater value at 24 and 48 hours than at 4 hours.

· PAI shows no response at either time.

· Useful in differentiating primary from central AI when ACTH level is equivocal

· Helpful in detecting more subtle degrees of AI than standard SST.

A: serum cortisol levels > 20μg/dL (550nmol/L) at 30min, 60min; 25μg/dL (695nmol/L) at 6-8h after initiation of infusion (34,35).

B: serum cortisol levels at 4h > 36μg/dL (1000nmol/L) with no further increase beyond this time (36).

Hypothalamic testing

ITT; insulin 0.1-0.15 U/kg i.v. is administrated after overnight to achieve symptomatic hypoglycemia and blood glucose level lower than 40mg/dL (<2.2mmol/L); collect blood samples at -15, 0, 15, 30, 45, 60, 90 and 120min for glucose, cortisol, ACTH.

Repeat insulin dose if blood glucose does not fall to level of less than 40mg/dL at 45 min

· AI is confirmed when a fall of glucose to less than 40mg/dL with corresponding an inability to demonstrate a cortisol response higher than 18μg/dL (37). Contraindications in patients with

basal cortisol < 3μg/dL, untreated hypothyroidism, electrocardiographic evidence or history of ischemic heart disease, or seizure.

· Useful in diagnosis of coexisting GH deficiency.

Peak cortisol levels > 18μg/dL (500nmol/L).

Overnight metyrapone test; 30mg/kg (max 3g) metyrapone is given at midnight; serum cortisol, 11-deoxycortisol, and ACTH levels are measured at 8am the following day.

· Alternative test when ITT is contraindicated.

· Test is valid only when cortisol levels fall to lower than 10μg/dL.

· Peak ACTH response > 200ng/L

· 11-deoxycortisol level > 7mg/dL

· Sum of cortisol and 11-deoxycortisol should exceed 16.5μg/dL (38).

17-OHCS, 17-hydroxycorticosteroids; ACTH, adrenocorticophic hormone; AI, adrenal insufficiency; CBG, cortisol-binding globulin; CRH, corticotrophin releasing hormone; HC, hydrocortisone; h-CRH, human CRH; GCs, glucocorticoids; GH, growth hormone; i.m., intramuscular; ITT, insulin tolerance test; i.v., intravenous; MCs, mineralocorticoids; PAI, primary AI; SST, short Synacthen test; ULN, upper limit of normal.

The notion of Critical Illness-Related Corticosteroid Insufficiency (CIRCI) emerged to describe impairment of the HPA axis during critical illness that is characterized by the dysregulated systemic inflammation caused by the inadequate intracellular GC-mediated anti-inflammatory activity. The Society of Critical Care Medicine (SCCM) and European Society of Intensive Care Medicine (ESICM) have updated the guideline for the diagnosis and management of CIRCI, which was originally proposed in 2008 (39). The task force makes no recommendation on whether to use delta cortisol after SST or random plasma cortisol levels of less than 10μg/dL to diagnose CIRCI. However, some cohort studies found that patients with CIRCI had poorer outcomes than patients without CIRCI when total cortisol levels are less than 10μg/dL or delta cortisol after SST < 9μg/dL. This evidence may be helpful in deciding upon replacement treatment when CIRCI is suspected (Table 5) (40,41). Other diagnostic tests, such as salivary cortisol or serum ACTH, are not recommended to diagnose this condition. Additionally, markers of inflammation and coagulation, morbidity, length of intensive care unit (ICU) stay, and mortality should be taken in consideration. Nevertheless, this whole concept has been questioned, especially since cortisol-binding globulin and albumin are invariably decreased in severe critical illness, implying that simple measurement of total cortisol is likely to be an inaccurate reflection of the true state of the HPA axis. However, the guideline cautions against using plasma free cortisol levels rather than plasma total cortisol levels to diagnose CIRCI. Most recent evidence suggests that CIRCI is not a useful diagnostic or therapeutic description, and that during the acute phase of the response to severe sepsis there is no failure of the HPA axis and no need for corticosteroid therapy, although in the recovery phase this may become important.

Table 5. Tests Used in Adrenal Insufficiency in Critical Illness (but see text above)


Indicators for poorer outcome


Random levels < 10μg/dL (275nmol/L)


Delta peak/basal levels < 9μg/dL (250nmol/L)

SST: short Synacthen test

In the context of the different diseases associated with AI, additional investigations may be necessary (Table 4). CT scanning of adrenals should be performed to identify infectious diseases such as tuberculosis, tumors, or adrenal hemorrhage.

Table 6. Additional Studies Used in Patients with AI

Primary AI

Specific tests for autoimmune antibodies

· Autoantibodies against CYP21A2 for the vast majority of autoimmune PAI.

· Other antibodies against 17-hydroxylase and side-chain-cleavage enzyme are also identified.

· It should be note that tests for autoantibodies are not standardized.

Other autoimmune markers and hormonal assays for evidence of APS

· Autoantibodies against IFNa and IFNw for APS-1.

· Serum calcium and PTH for hypoparathyroidism.

· Autoantibodies for autoimmune thyroid disease and thyroid function test.

· Autoimmune for insulin, GAD65, ICA, and ZnT8 along with blood glucose for autoimmune diabetes.

· Antibody against parietal cell with or without intrinsic factor antibody for pernicious anemia.

· Antibodies to tissue transglutaminase and anti-endomysial IgA antibody for celiac disease.

· Liver function along with ANA, SMA, and anti-LKM1 antibodies for autoimmune hepatitis in those with abnormal liver biochemical tests.

Microbial and serological tests

· Tuberculosis (tuberculin testing, early morning urine samples cultured forMycobacterium tuberculosis)

· Other infective cause.

CT / MRI scan

· Calcified adrenals can be found in infection, hemorrhage, and malignancy.

· Large adrenals with or without calcification can be seen in metastatic deposits and early phase of infection.

· Small atrophic glands with calcified foci are commonly illustrated in chronic stage of infection.

· Adrenal hemorrhage shrinks and attenuates gradually on CT and has a variable appearance on MRI depending on the stage of blood products.

· Adrenal venous thrombosis, in turn raises internal pressure, resulting in the same hemorrhagic finding.

Chest radiograph

· Clue for pulmonary manifestation of tuberculosis or fungal infection.

CT guided adrenal biopsy

· Useful in confirming the diagnosis of adrenal metastasis from extra-adrenal malignancy, or certain infiltrations such as histoplasmosis.


· Circulating levels of VLCFA.

17-OH progesterone and 24-hour urine steroid profile

· Classic CAH.

Secondary AI

Pituitary hormonal assessment

· Pituitary hormone producing tumor or co-existing pituitary hormone deficiency.

Pituitary MRI scans

· Pituitary or parasellar lesion.

Biopsy of pituitary

· Occasionally necessary such as hypophysitis.

Other Investigations

Measurement of plasma renin and aldosterone in PAI to determine MCs reserve

· High renin levels in combination with an inappropriately normal or low aldosterone concentration suggests PAI.

· In early phase of evolving PAI, MCs deficiency may predominate and may be the only sign.

Measurement of serum DHEAS

· Decreased DHEAS levels in women in both PAI (direct effect on adrenal cortex) and central AI (decrease in ACTH stimulus).

AI, adrenal insufficiency; ANA, antinuclear antibodies; anti-LKM, anti-liver microsomal; APS, autoimmune polyglandular syndrome; CAH, congenital adrenal hyperplasia; CT, computerized tomography; DHEAS, dehydroepiandrosterone sulphate; MRI, magnetic resonance imaging; SMA, anti-smooth muscle antibodies; VLCFA, very long-chain fatty acids.

As previously stated, laboratory tests should be interpreted with caution in specific situations, such as those affecting CBG concentration. Low CBG levels are commonly found in conditions characterized by inflammation, nephrotic syndrome, liver disease, the immediate postoperative period or requiring intensive care, or rare genetic disorders, whereas estrogen, pregnancy and mitotane can raise CBG levels. Systemic estrogen-containing drug prescriptions should be discontinued at least four weeks prior to testing. However, using an estrogen patch has no effect on CBG levels. Different diagnostic criteria should be considered according to the cortisol assay. If patients are currently on GCs replacement, omitting the steroid dose before testing is advised. The evening and morning dose of HC or prednisolone should be omitted, whereas the duration of drug withdrawal in patients taking other synthetic GCs may be longer. In pregnancy, higher diagnostic cortisol cut-offs of 25μg/dL (700nmol/L), 29μg/dL (800nmol/L), and 32μg/dL (900nmol/L) should be considered for the first, second, and third trimesters, respectively (30). In pituitary diseases, testing of GCs reserve is suggested before and after initiation of GH replacement or upon documentation of an unexplained improvement in co-existing diabetes insipidus (DI).


Acute Adrenal Insufficiency (Adrenal Crisis)

Adrenal insufficiency is a potentially life-threatening medical emergency when presented as an adrenal crisis, which requires prompt treatment with HC and fluid replacement. When clinical suspicion exists, treatment should be initiated without any delay while awaiting definitive proof of diagnosis. Blood samples should be obtained for later cortisol concentration measurements, and the management approach should be similar to that of any critically ill patient (Table 7, 8).

Table 7. Treatment of Acute Adrenal Insufficiency (Adrenal Crisis) - Management During Resuscitation of Critically Ill Patient.

Maintain airway and breathing.

· Correct hypovolemia and reverse electrolyte abnormalities; caution should be taken in correcting chronic hyponatremia (not more than 12mmol in 24h, preferably < 8mmol) to prevent central pontine myelinolysis.

· Replace glucocorticoid; clinical improvement especially blood pressure should be seen within 4-6h.

· The half-life of HC is 90min after i.v. injection, and more prolonged after i.m. administration; switch to oral HC 40mg in the morning and 20mg in the afternoon if oral intake is resumed; taper to a standard dose of 10-20mg on awakening and 5-10mg in the early afternoon if there is no other major illness.

· Some experts recommend dexamethasone while dynamic tests are awaited, as dexamethasone does not interfere with the assay, but HC is preferred for its MCs activity.

· Regarding the electrolyte imbalances, no need for fludrocortisone replacement in an acute crisis since the MCs activity of HC and 0.9% NaCl infusion is sufficient.

Establish i.v. access with two large bore cannulas.

Collect venous blood samples for urea and electrolytes, glucose, full blood count, bicarbonate, infection screen, and store samples (plasma cortisol and ACTH measurement). Do not wait for blood results.

Rapid infusion of 1L isotonic saline solution (0.9% NaCl) within the 1st hour, followed by continuous i.v. isotonic saline solution guided by individual patient needs; usually infuse 2-3 L of normal saline solution within 12 hours; after this, fluid management should be guided by volume status, urine output and biochemical results; 50g/L (5%) dextrose in saline solution if there is evidence of hypoglycemia.

Inject i.v. 100mg of HC immediately (50-100mg/m2 for children) and then followed by 200mg/day (50–100mg/m2/d for children divided q 6h) of HC (via continuous i.v. therapy or 6-8 hourly i.v. injection) for 24h, reduce to HC 100mg/day on the following day; i.m. administration should be used if venous access is not possible; prednisolone may be used as an alternative drug if unavailable HC; dexamethasone is the least preferred and should be given only if no other glucocorticoid is available.

Use additional supportive measures as needed

For hypoglycemia

· Dextrose 0.5-1g/kg of dextrose or 2-4mL/kg of D25W should be infused slowly at rate of 2-3mL/min; standard initial glucose dose is 25g.

Cardiac monitoring.

MCs replacement is not required if the HC dose exceeds 50mg/day.

ACTH, adrenocorticotropic hormone; HC, hydrocortisone; i.m., intramuscular; i.v., intravenous; MCs, mineralocorticoid.

Table 8. Treatment of Acute Adrenal Insufficiency (Adrenal Crisis) - After Patient Stabilization

Continue i.v. 0.9%NaCl; rate may be slower and maintained for 24-48h.

Search for and treat possible infectious precipitating causes of adrenal crisis; treat any associated condition(s).

Perform SST to confirm the diagnosis.

Differential diagnosis if needed.

Taper parenteral glucocorticoids over 1-3 days, depending on precipitating illness.

After the first 24 hours, HC dose can be reduced to 50mg q 6h and switched to oral HC 40mg in the morning and 20mg in the afternoon, then tapered to a standard dose of 10mg on awakening, 5mg at lunchtime and 5-10mg in the early afternoon.

In aldosterone deficiency, begin MCs replacement with fludrocortisone (100μg by mouth daily) when saline infusion ceased to prevent sodium loss, intravascular volume depletion and hyperkaliemia.

However, MCs replacement is not required if the HC dose exceeds 50mg/day.

ACTH, adrenocorticopic hormone; HC, hydrocortisone; i.m., intramuscular; i.v., intravenous; MCs, mineralocorticoids; SST, short Synacthen test.

Management of Chronic or Insidious Onset of Adrenal Insufficiency

The aim of replacement treatment in AI is to mimic the normal cortisol secretion rate, which is around 5-8mg/m2/day(42,43). Previously this rate was thought to be approximately 25-30mg/day of HC, but normal cortisol production rates seem to be about 8-15mg/day. Most patients can cope with less than 30mg/day (usually 15-25mg/day in divided doses). Doses are usually given upon waking with a smaller dose at lunchtime and then one in late afternoon. Weight-adjusted dosing may be associated with a better safety profile. Despite the various types of cortisol replacement regimens, no head–to-head comparison data is available to advocate one over the other. Decisions regarding the form and dose of GCs replacement therapy are based on crude end-points such as weight, well-being, and blood pressure,as well as on local availability, cost and clinical need (Table 9). Bone mineral density may be reduced on conventional doses of 30mg/day HC, highlighting the importance of aiming for effective but safe doses. Long-duration GCs can be administered once daily but may be associated with higher risk of side effects. Patients should be monitored for clinical symptoms.

Table 9. Glucocorticoid Replacement Schemes

Drug profile

Commonly used doses

Immediate-release HC (Hydrocortisone)

Short acting, given in 2-3 divided doses; this biologically active GCs approximately mimics the endogenous diurnal rhythm; obese individuals may require more GCs replacement than lean individuals; higher frequency regimes and size-based dosing may be beneficial in individual cases; high doses in the evening may disturb sleep and alter metabolism.

15-25 mg or 5-8 mg/m2/day; the highest dose in the morning on

awakening, the next in the early afternoon (2h after lunch) (2-dose regime) or at lunch and afternoon but not later than 4-6h before bedtime) (3-dose

regime); usually 10mg upon awakening, 5mg at lunchtime and 5mg in the late afternoon.

Dual-release HC (combination of immediate-release HC in the

outer-layer coat and extended-release core, PlenadrenÒ) (44,45).

Given once daily in the morning; resulting in higher morning and lower evening cortisol levels with no overnight cortisol rise. This may be advantageous in patients with high risk of metabolic comorbidities and in patients with poor administrative compliance.

20-30mg; lower dose may be sufficient in patients with some remaining endogenous cortisol production; identical total daily dose may be given when switching and clinical response needs to be monitored due to lower bioavailability than HC.

Modified-release HC

(delayed and sustained release in multiple microcrystals with polymer sheathing,

ChronocortÒ) (46).

Given twice daily as a “toothbrush regime”, with 2/3 of the total daily dose before bedtime (11p.m.) and 1/3 administered in the morning (7a.m.), resulting in overnight rise with morning peak of cortisol and near physiological cortisol levels throughout the day. This is beneficial for CAH patients since it prevents the ACTH-driven excess production of adrenal androgens.

Usual dose of HC; then titrated based on symptoms and 17OHP along with androstenedione measurement. (Phase 3 study)

Cortisone acetate (47).

Short acting but longer than HC; the peak of serum cortisol level is delayed compared to HC since the oral form requires a conversion to cortisol at liver to become active; available in oral preparation only.

20-35mg in 2-3 divided doses.

Prednisolone /prednisone

Long-acting, once-daily dose is sufficient; some may need additional 2.5mg in the evening; does not mimic diurnal rhythm of endogenous cortisol; better choice in patients with poor administrative compliance or in patients with poor quality of life on HC replacement; prednisone must be processed in liver to become prednisolone which is then able to cross the cellular membrane; cross-reaction occurs in most cortisol assays.

3-5mg once daily on waking.


Inter-individual variable metabolism makes it difficult to predict the adequate dose; dose needs to be titrated if patient is on hepatic enzyme inducing medications; it is not recommended in PAI because of risk of Cushingoid side effects; concurrent fludrocortisone replacement is necessary in PAI patients if dexamethasone is undeniable.

0.25-0.75mg once daily.

MCs replacement

(Fludrocortisone) (48).

Required only in PAI; doses may need to temporary increase by 50-100% in hot weather or conditions that promote excessive sweating; available in oral preparation only; if parenteral action required, use DOCA if available.

50-200μg (median 100μg) once daily in the morning with starting dose at 50-100μg.

Androgen replacement (DHEA) (49,50).

A trial of replacement can be offered to PAI women with low energy, low mood, and low libido, despite otherwise optimized GCs and MCs replacement; some evidence of benefit; not generally available on prescription, but obtained as a ‘health food’ supplement.

25–50mg as a single oral dose in the morning; replacement should be discontinued if no clinical benefit after initial 6-month trial.

AI, adrenal insufficiency; DHEA, dehydroepiandrosterone; DOCA, deoxycorticosterone acetate; GCs, glucocorticoids; i.m., intramuscular; i.v., intravenous; HC, hydrocortisone; MCs, mineralocorticoids; PAI, primary adrenal insufficiency.

It should be noted that while HC and prednisolone are active GCs, cortisone acetate and prednisone require activation via hepatic 11β-hydroxysteroid dehydrogenase type 1 to become biologically active.

Another novel aspect on the management of the chronic AI is the development in glucocorticoid formulation. A dual-release HC preparation that can be administered once daily upon awakening, Plenadren® (developed by Duocort, Viropharma, Shire Pharmaceuticals), which was approved for treatment of AI in adults since 2011. Moreover, another delayed- and sustained-release preparation of HC, ChronocortÒ, given twice daily before bedtime and in the morning, is currently undergoing the approval process for treatment of CAH patients (Table 9). Advances in understanding of the normal cortisol circadian rhythm in the setting of endogenous clock genes, as well as its importance in controlling innate immunity, metabolism, and stress may imply that some of these GCs replacement formulas will be superior over the traditional ones in the future.

One important aspect of the management of chronic PAI is patient and family education. Careful instructions should be given to up-titrate the daily dose in the event of intercurrent febrile illness, accident, or severe mental stress, such as an important examination (Table 10). If the patient is vomiting and cannot take medication by mouth, parenteral HC must be given urgently. Ideally, patients should wear a ‘medical-alert’ bracelet or necklace and carry the Emergency Medical Information Card, which should provide information on the patient’s diagnosis, prescribed medications and daily doses, and the physician involved in the patient’s care. Patients should also have supplies of HC for emergencies, and should be educated about how and when to administer them by the subcutaneous route. Rectal suppositories of prednisolone 100 mg, enemas of prednisolone 20mg/100mL, or HC acetate enema 10% have also been used, but they are clearly ineffective when diarrhea is present.

Table 10. Glucocorticoid Replacement Schemes During Illness

Examples of commonly used schemes

During minor illness

Increase dose by 2-3 times of usual

dosage for 3 days; do not change MC dose.

HC 25-75mg/day twice daily per oral, then taper rapidly to maintenance dose as patient recovers (usually 1-2 days); HC 50mg/m2/day i.m. or double/triple HC replacement doses in children.

During minor-to-moderate surgery

HC 50-100mg/day twice daily per oral or i.v., then taper rapidly to maintenance dose as patient recovers.

During major illness

Increase dose up to 10 times of usual dosage with continuous infusion of HC, or equivalent dexamethasone dosage, then decrease dose by half per day, to usual maintenance dose. Half dose 2nd postoperative day, maintenance dose 3rd postoperative day.

HC 100mg i.v., followed by continuous i.v. infusion of 200mg HC in 24h, then taper rapidly by half per day regarding course of illness; alternative administration of HC 50mg iv. or i.m. q 6h may be considered.

Major surgery with general anesthesia

HC 100mg i.v. just before induction of anesthesia, followed by continuous i.v. infusion of 200mg HC in 24h (alternatively 50mg every 6h i.v. or i.m.); then taper rapidly by half each day and switch to oral regime depending on clinical state; children: HC 50mg/m2 i.v. followed by HC 50–100mg/m2/d divided q 6h. Weight-appropriate continuous i.v. fluids with 5% dextrose and 0.2 or 0.45% NaCl.

Uncomplicated, outpatient dental procedures under local anesthesia and most radiologic studies

No extra supplementation is required.

Severe stress or trauma

Inject prefilled dexamethasone (4-mg) syringe.

Moderately stressful procedures (barium enema, endoscopy, or arteriography)

Extra supplementation is required only before the procedure.

100mg i.v. dose of HC just before the procedure


HC should be increased 5-10mg/day in the last trimester (20–40% from the 24th week onward) to mimic physiologic increase in free cortisol; fludrocortisone dose adjustment may need in response to serum sodium and potassium levels; HC is preferred over the other GCs, whereas dexamethasone is warned as a contraindication since it is not inactivated in the placenta and can transfer to the fetus.

· Last trimester: 5-10mg/day of HC usually (20–40% from the 24th week onward)

· Laboratory: HC stress dosing with HC 100mg i.v. bolus, followed by continuous i.v. infusion of 200mg HC in 24h, and rapidly taper to pre-pregnancy doses after delivery.

HC, hydrocortisone; i.m., intramuscularly; i.v., intravenously; GCs, glucocorticoids

Regarding MC replacement therapy, the dose of fludrocortisone is titrated individually based on clinical examination, mainly body weight and blood pressure, and the levels of plasma renin activity (PRA).

Hydrocortisone is generally preferred for use in patients with AI because it has both GCs and MCs activity. Patients receiving prednisone or prednisolone, on the other hand, may require additional fludrocortisone due to their relatively low MCs activity. Notably, dexamethasone should not be used to treat PAI patients because its lack of MCs activity (51). After resolution of an adrenal crisis, the adequacy of MC replacement should be assessed by measuring supine and erect blood pressure, electrolytes and PRA. Inadequate fludrocortisone dose may cause postural hypotension with elevated PRA, whereas excessive administration results in the opposite. Mineralocorticoid replacement therapy is frequently neglected in patients with adrenal failure. The dose may be temporarily increased in the summer along with an increase in salt intake, particularly if patients are exposed to temperatures above 30ºC (85ºF) or other conditions causing increased sweating. Newborns and children may also require higher fludrocortisone because MCs sensitivity is lower.

In chronic central AI, GCs replacement is similar to that in PAI; however, measurement of plasma ACTH concentrations cannot be used to titrate the optimal GCs dose. Replacement of other anterior pituitary deficits may be also necessary, while changing doses of growth hormone and thyroxine may affect the GCs requirements. More specifically, the HPA axis should be evaluated prior to and following the initiation of GH replacement therapy since GH inhibits the conversion of cortisone to cortisol. Consequently, patients receiving GCs replacement may require higher doses once GH treatment is introduced (18,52).

For patients with either primary or central AI, the beneficial effects of adrenal androgen replacement therapy with 25 to 50mg/day of DHEA have been reported. To date, the reported benefit is principally confined to female patients and includes improvement in sexual function and well-being, but the effects are variable.

In children with PAI, HC in three or four divided doses with total starting daily dose of 8mg/m2 body surface area are preferred over synthetic long-acting GCs, such as prednisolone or dexamethasone. In the case of documented aldosterone deficiency, fludrocortisone treatment with a typical dosage of 100μg/day without body surface area adjustment is suggested, while sodium chloride supplements are needed in the newborn period until the age of 12 months. Most experience has been gained from the treatment of children with CAH; however, since in this case under treatment is confirmed by hyperandrogenism higher GCs therapy is usually given (22).

Other therapies have been studied such as rituximab with or without depot tetracosactide in newly diagnosed autoimmune PAI patients (53). The regenerative potential of adrenocortical stem cells combined with immunomodulatory treatment to stop the autoimmune destruction, adrenal transplantation, or gene therapy in forms of monogenic PAI may be a useful option in the future.

For CIRCI with septic shock, it has been suggested to use i.v. HC < 400mg/day for at least 3 days at full dose (rather than high-dose and short-course regimes) when they are not responsive to fluid and moderate- to high-dose vasopressor therapy (> 0.1μg/kg/min of norepinephrine or equivalent) (39). In hospitalized adults with community-acquired pneumonia a daily dose of < 400mg i.v. HC or equivalent has been suggested. GCs are also suggested in patients suffering from meningitis, cardiac arrest (methylprednisolone given during resuscitation or HC given for post-resuscitation shock), and cardiopulmonary bypass surgery (CPB) (250mg i.v. of methylprednisolone at anesthesia induction and at onset of CPB) or dexamethasone (1mg/kg perioperatively). Corticosteroids are not suggested for patients after major trauma or suffering from influenza. Nevertheless, it should be emphasized that this is a rapidly changing situation, and the use of corticosteroids in the ICU with severely ill patients remains controversial.

Despite concerns that patients with AI were more vulnerable to infection during the recent COVID-19 pandemic,adequately treated and well-trained AI patients demonstrated the same incidence of COVID-19-suggestive symptoms and disease severity as the people without AI.


Patients with chronic AI should be closely followed-up by an endocrinologist or a healthcare provider with endocrine expertise at least annually (for infants every 3 to 4 months) to ensure the adequacy of their GCs and/or MCsreplacement dose (Table 11, 12), to reduce the risk of adrenal crisis, to provide necessary education to patients, and to confirm that they have a prompt updated emergency pack with HC to treat an emergency. Clinical symptoms including body weight, postural blood pressure and energy levels should be monitored in order to avoid signs of frank GCs excess and adjust accordingly the dose of steroids. The ‘mapping’ of the dose has been suggested to assess the compliance and also help decide when tablets should be taken in problematic cases of reduced quality of life where the detailed questioning for daily habits, working patterns, general feelings of energy, mental concentration, daytime somnolence, and energy dips. Hormonal monitoring of GCs replacement, such as serum or salivary cortisol ‘day-curves’ is not routinely recommended, but can be useful in specific situations where the clinical response cannot be reliably used to adjust treatment or when malabsorption is suspected (54,55). In addition, the use of ACTH levels to assess GCs replacement is not suggested since it leads to over-replacement.

Similarly, MCs replacement should be monitored based on clinical assessment, which includes postural hypotension as measured by lying and standing blood pressure and pulse, symptoms of salt craving, oedema, blood electrolyte measurements. Reported well-being, normal blood pressure without orthostatic hypotension and electrolytes within the normal range indicate adequate replacement. PRA in the upper reference range has been found to be a useful predictor for a correct MCs dose (56,57). Liquorice and grapefruit juice enhance the MC effect of HC and should be avoided. Phenytoin potentiates hepatic clearance of GCs and increase fludrocortisone metabolism; therefore, and higher doses are needed when it is co-administered.

It is of note that in patients who develop hypertension while receiving fludrocortisone, the dose of fludrocortisone should be reduced and HC replacement should be adjusted. If blood pressure remains uncontrolled, anti-hypertensive treatment should be initiated without fludrocortisone discontinuation. First-line anti-hypertensive drugs to be selected are the angiotensin II receptor blockers or angiotensin-converting enzyme blockers to counterbalance the vasoconstrictive effects of elevated angiotensin II, second-line a dihydropyridine calcium blocker, while diuretics should be avoided and aldosterone receptor blockers are contraindicated.

Since AI might mask the presence of partial DI, urine output should be monitored after starting GCs replacement, or conversely, when DI has unreasonably improved, patients have to be tested for HPA reserve. With DHEA replacement, morning serum DHEAS levels should be measured before the intake of the daily dose aiming at the mid-normal range. An annual screening for autoimmune diseases, which includes autoimmune thyroid disease, type 1 diabetes, premature ovarian failure, coeliac disease, and autoimmune gastritis should be performed in PAI patients without other obvious cause of adrenal failure.

Special populations, like pregnant females, should be surveyed for clinical symptoms and signs of GCs over- and under-replacement including weight gain, fatigue, postural hypotension or hypertension, hyperglycemia, with at least once per trimester. Only sodium and potassium can be reliably monitored in blood and urine for the adequacy of replacement. In contrast, PRA monitoring is not advised since plasma renin physiologically increases during this time.

In children, monitoring of GCs replacement includes clinical assessment such as growth velocity, body weight, blood pressure, and energy levels (58).

Although familial autoimmune PAI is less frequent, genetic counselling is suggested in particular situations due to monogenic disorders.

Table 11. Assessment of Glucocorticoid Replacement

Under replacement

Lethargy, tiredness, nausea, poor appetite, weight loss, hyperpigmentation.
Low serum cortisol level on cortisol day curve (useful in specific cases for HC or cortisone replacement only)

Over replacement

Cushingoid appearance, weight gain, insomnia, peripheral edema, low bone mineral density.
High 24-h UFC, high serum cortisol on hydrocortisone day curve (useful in specific cases for HC or cortisone replacement only)

Table 12. Assessment of Mineralocorticoid Replacement

Inadequate replacement

Postural hypotension, light-headedness
High PRA (plasma renin activity, should be at the upper limit of normal)

Over replacement

Hypertension, peripheral oedema, hypernatremia, hypokalemia

Patient Education

This is very crucial in the management of AI and for the prevention of adrenal crisis. All patients and their relatives should be educated about their condition and the emergency measures they should take at home to avoid crises, particularly concerning GCs adjustments in stressful events and preventive strategies including parenteral self- or lay-administration of emergency GCs particularly in situations of intercurrent illness, fever, or any type of stress. This information should be reinforced during annual follow-up visits by clinicians and if possible, through a structured patient education program. All patients should be given a steroid emergency card and medical-alert identification to inform health personnel of the need for increased GCs doses to avert or immediately treat adrenal crisis. Every patient should be equipped with a GCs injection kit for emergency use and be educated on how to use it (Table 13).

Table 13: Information and Equipment for Patients with AI

Steroid Sick Day Rules

Sick day rule 1: Patients should be advised to increase the oral GCs when the patient experiences fever or illness requiring bed rest, or when requiring antibiotics for an infection. For instance, the total oral GCs dose should be doubled (> 38°C) or tripled (> 39°C) for at least 72 hours; if the patient remains unwell after 72 hours, they should contact their caring physician. There is no need to increase the MCs dose.

Sick day rule 2: There should always be a supply of additional oral GCs on prescription for sick days and HC emergency injection kit prescription.

Sick day rule 3: Every patient should carry a medical alert bracelet or leaflet with information stating their conditions, treatment, physician, emergency phone number of endocrine specialist team, and proposed GCs regimen during adrenal crisis.

Sick day rule 4: Parenteral injection of GCs preparation, either i.m. or i.v. should be provided in case of severe illness, trauma, persistent vomiting, before moderately stressful procedure (i.e., barium enema endoscopy, arteriography), or before surgical intervention.

Steroid Emergency Pack

· Every patient should be provided with this pack to keep at home.

· The pack contains a vial of 100mg HC or 4mg dexamethasone, syringes, needles, also oral HC or prednisolone suppositories.

· The patient and/ or any responsible family member should be educated to administer this medication i.m. or s.c. during an emergency situation including severe accident, significant hemorrhage, fracture, unconsciousness, diarrhea and vomiting, and they should call the emergency medical personal immediately (adults, i.m. or s.c. HC 100mg; children, i.m. HC 50mg/m2; infants, 25mg; school-age children, 50mg; adolescents, 100mg).

· The expiry date on the pack should be checked regularly and replaced with a new pack if expired.

· The patient should be advised to take the pack when travelling.

Medical-Alert bracelet or pendant and emergency steroid card

· Every patient should wear or carry these in which the diagnosis and daily medication should be clearly documented.

Follow up

· A regular visit in order to reinforce education and confirm understanding should be scheduled at least annually in a patient without specific problems or recent crises. Other circumstances, such as monitoring during infancy, may necessitate more frequent visits.

GCs, glucocorticoids; HC, hydrocortisone; i.m., intramuscularly; i.v., intravenously; HC, hydrocortisone; MCs, mineralocorticoids; s.c., subcutaneously


The mortality of patients with PAI was increased in some studies and adrenal crisis was a significant cause of death, emphasizing the importance of educating patients with AI to prevent crises. Recent large cohorts confirmed the increased mortality of patients with ΑΙ, especially PAI. Although cardiovascular disease (CVD) was the leading cause of death, infectious diseases were found to pose the greatest risk in comparison to controls. Adrenal crisis was also found to be a common contributor, particularly in those with co-existing CVD. In addition, despite an adequate replacement dose, the quality of life of PAI patients remains impaired. This appears to be related to the delay in diagnosis.


Bornstein SR, Allolio B, Arlt W, Barthel A, Don-Wauchope A, Hammer GD, Husebye ES, Merke DP, Murad MH, Stratakis CA, Torpy DJ. Diagnosis and Treatment of Primary Adrenal Insufficiency: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016 Feb;101(2):364-89.

Fleseriu M, Hashim IA, Karavitaki N, Melmed S, Murad MH, Salvatori R, Samuels MH. Hormonal Replacement in Hypopituitarism in Adults: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016 Nov;101(11):3888-3921.

Annane D, Pastores SM, Rochwerg B, Arlt W, Balk RA, Beishuizen A, Briegel J, Carcillo J, Christ-Crain M, Cooper MS, Marik PE, Umberto Meduri G, Olsen KM, Rodgers SC, Russell JA, Van den Berghe G. Guidelines for the Diagnosis and Management of Critical Illness-Related Corticosteroid Insufficiency (CIRCI) in Critically Ill Patients (Part I): Society of Critical Care Medicine (SCCM) and European Society of Intensive Care Medicine (ESICM) 2017. Crit Care Med. 2017 Dec;45(12):2078-2088.

Pastores SM, Annane D, Rochwerg B. Corticosteroid Guideline Task Force of SCCM and ESICM. Guidelines for the Diagnosis and Management of Critical Illness-Related Corticosteroid Insufficiency (CIRCI) in Critically Ill Patients (Part II): Society of Critical Care Medicine (SCCM) and European Society of Intensive Care Medicine (ESICM) 2017. Crit Care Med. 2018 Jan;46(1):146-148.

Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, Meyer-Bahlburg HFL, Miller WL, Muraf MH, Oberfield SE, White PC. Congenital Adrenal Hyperplasia Due to Steroid 21-Hydroxylase Deficiency: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018 Nov;103(11):4043-4088.


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Congenital Adrenal Hyperplasia (CAH) is a term used to describe a group of genetically determined disorders of defective steroidogenesis that result in variable deficiency of the end products cortisol and/or aldosterone and their deleterious, including life-threatening, effects on metabolism and electrolytes with simultaneous diversion to the accumulation of androgens and their virilizing effects. Although we discuss the various enzymatic defects that are involved, we focus on the most common enzyme deficiency, 21-hydroxylase. Depending on the residual enzymatic activity governed by the genetic mutation, 21-hydroxylase deficiency CAH is classified as either classical (salt wasting or simple virilizing) or non-classical. In classical 21-hydroxylase deficiency CAH, there is an accumulation of 17-hydroxyprogesterone which is shunted into the intact androgen pathway and may lead to prenatally virilized external genitalia in females as early as 9 weeks of gestation. Inadequately treated patients may develop progressive penile or clitoral enlargement, premature adrenarche, precocious puberty, rapid linear growth accompanied by premature epiphysis maturation leading to compromised final adult height and impaired fertility. Moreover, inadequately treated salt loss increases the risk for adrenal crises. In contrast, over treatment with cortisol results in “Cushingoid” effects including retarded bone development. We describe the various defects, their manifestations and goals for therapy, and emerging newer therapies for CAH to both correct the deficiency in cortisol and aldosterone secretion while suppressing overproduction of ACTH.


Congenital adrenal hyperplasia (CAH) refers to a group of disorders that arise from defective steroidogenesis. The production of cortisol in the zona fasciculata of the adrenal cortex occurs in five major enzyme-mediated steps. CAH results from deficiency in any one of these enzymes. Impaired cortisol synthesis leads to chronic elevations of ACTH via the negative feedback system, causing overstimulation of the adrenal cortex and resulting in hyperplasia and over-secretion of the precursors to the enzymatic defect. The five forms of CAH are summarized in Table 1. Impaired enzyme function at each step of adrenal cortisol biosynthesis leads to a unique combination of elevated precursors and deficient products. The most common enzyme

deficiency that accounts for more than 90% of all CAH cases is 21-hydroxylase deficiency (1).


Data from close to 6.5 million newborn screenings worldwide indicate that classical CAH occurs in about 1:13,000 to 1:15,000 live births (2). It is estimated that 75% of patients have the salt-wasting phenotype(1). Non-classical 21OHD CAH (NC21OHD) is more common. The incidence in the heterogeneous population of New York City is about 1 in 100, making NC21OHD the most frequent autosomal recessive disorder in humans. NC21OHD is particularly prevalent in certain populations, showing a high ethnic specificity. In the Ashkenazi Jewish population, 1 in 3 are carriers of the allele, and 1 in 27 are affected with the disorder (3-5). CAH resulting from 11β-hydroxylase deficiency (11β-OHD) is the second most common cause of CAH, accounting for 5-8% of all cases (6). It occurs in about 1 of every 100,000 live births in the general population (7) and is more common in some populations of North African origin (8). In Moroccan Jews, for example, the disease incidence was initially estimated to be 1 in 5,000 live births (9); subsequently, it was shown to occur less frequently (10), but remains more common than in other populations. The other forms of CAH are considered rare diseases and their incidence is unknown in the general population.


Adrenal steroidogenesis occurs in three major pathways: glucocorticoids, mineralocorticoids, and sex steroids as shown in Figure 1. The adrenal gland architecture suggests that the adrenal acts as three separate glands: zona glomerulosa, zona fasciculate, zona reticularis (11). The hypothalamic-pituitary-adrenal feedback system is mediated through the circulating level of plasma cortisol by negative feedback of cortisol on CRF and ACTH secretion. (Figure 2) Therefore, any CAH condition that results in a decrease in cortisol secretion leads to increased ACTH production, which in turn stimulates (1) excessive synthesis of adrenal products in those pathways unimpaired by the enzyme deficiency and (2) a build-up of precursor molecules in pathways blocked by the enzyme deficiency.

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Figure 1. The classical and backdoor pathways of adrenal steroidogenesis: The classical pathway is highlighted in blue and the backdoor pathway is highlighted in orange. In the classical pathway, five enzymatic steps are necessary for cortisol production. In the first step of adrenal steroidogenesis, cholesterol enters mitochondria via a carrier protein called steroidogenic acute regulatory protein (StAR). ACTH stimulates cholesterol cleavage, the first and rate limiting step of adrenal steroidogenesis. The five enzymes required for cortisol production are cholesterol side chain cleavage enzyme (SCC), 17α-hydroxylase, 3β-hydroxysteroid dehydrogenase (3βHSD2), 21-hydroxylase, and 11β-hydroxylase. The backdoor pathway is an alternative pathway producing dihydrotestosterone. The enzymes include 5α-reductase 1, aldo keto reductases, retinol dehydrogenase RoDH, 17β-hydroxysteroid dehydrogenases, 17α-hydroxylase.

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Figure 2. The hypothalamic-pituitary-adrenal (HPA) axis. Corticotropin-releasing factor produced in the hypothalamus stimulates the secretion of adrenocorticotropic hormone (ACTH) from the anterior lobe of the pituitary gland. ACTH stimulates the production of cortisol and androgens in the adrenal cortex. There is negative feedback on the hypothalamus and pituitary gland by cortisol.

The clinical symptoms of the five different forms of CAH result from the specific hormones that are deficient and those that are produced in excess as outlined in Table 1. In the most common form 21OHD-CAH, the function of 21-hydroxylating cytochrome P450 is deficient, creating a block in the P450 cortisol production pathway. This leads to an accumulation of 17-hydroxyprogesterone (17-OHP), a precursor of the 21-hydroxylation step. Excess 17-OHP is then shunted into the intact androgen pathway, where the 17,20-lyase enzyme converts 17-OHP to Δ4-androstenedione, the major adrenal androgen. Mineralocorticoid (aldosterone) deficiency is a feature of the most severe form of the disease and hence named salt wasting CAH. The enzyme defect in the non-classical form of 21OHD CAH is mild and salt wasting does not occur. The analogy of all other enzyme deficiencies in terms of precursor retention and product deficiencies are shown in Table 1.

Table 1. Summary of the Clinical, Hormonal, and Genetic Features of Steroidogenic Defects (1)





Mineralocorticoid Effect

Typical Features


Lipoid CAH


StAR Protein

Female, with no sexual development

Salt wasting

All steroid products low

StAR 8p11.2

Lipoid CAH



Female, with no sexual development

Salt wasting

All steroid products low


3β-HSD deficiency, classic



Females virilized, males under-virilized

Salt wasting

Elevated DHEA, 17-pregnenolone, low androstenedione, testosterone, elevated K, low Na, CO2

HSD3B2 1p13.1

3β-HSD deficiency, non-classic



Normal genitalia with mild to moderate hyperandrogenism postnatally


Elevated DHEA, 17-pregnenolone, low androstenedione, testosterone

Absent or unknown

17α-OH deficiency



Variable sexual development

Hypokalemic low-renin hypertension

Normal or decreased androgens and estrogen, elevated DOC, corticosterone

CYP17 10q24.3

17,20-Lyase deficiency



Infantile female genitalia


Decreased androgens and estrogens

CYP17 10q24.3

Combined 17α-OH/17,20-lyase deficiency



Infantile female genitalia

Hypokalemic low-renin hypertension

Decreased androgens and estrogens

CYP17 10q24.3

Combined 17α-OH/17,20-lyase deficiency



Infertility, Infantile female genitalia


Decreased follicular estradiol and increased progesterone

CYP17 10q24.3

Classic 21-OH deficiency, salt wasting



Females prenatally virilized, normal male genitalia, hyperpigmentation

Salt wasting

Elevated 17-OHP, DHEA, and androstenedione, elevated K, low Na, CO2

CYP21 6p21.3

Classic 21-OH deficiency, simple virilizing



Females prenatally virilized, normal male genitalia


Elevated 17-OHP, DHEA, and androstenedione, normal electrolytes

CYP21 6p21.3

Non-classic 21-OH deficiency



Males and females with normal genitalia at birth, hyperandrogenism postnatally


Elevated 17-OHP, DHEA, and androstenedione on ACTH stimulation

CYP21 6p21.3

Classic CAH 11β-deficiency



Females virilized with atypical genitalia, males unchanged

Low-renin hypertension

Elevated DOC, 11-deoxycortisol (S); androgens, low K, elevated Na, CO2

CYP11B1 8q24.3

Non-classic CAH 11β-deficiency



Males and females with normal genitalia at birth, hyperandrogenism postnatally


Elevated 11-deoxycortisol ± DOC, elevated androgens

CYP11B1 8q24.3

P450 Oxido-Reductase Deficiency



Females virilized with atypical genitalia, males under-virilized


Partial, combined and variable defects of P450c21, P450c17 and P450aro activity



CAH, Congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; DOC, deoxycorticosterone; 3β-HSD, 3β-hydroxysteroid dehydrogenase; OH, hydroxylase; 17-OHP, 17-hydroxyprogesterone. Adapted from: Wajnrajch MP and New MI. Chapter 103: Defects of Adrenal Steroidogenesis. Endocrinology, Adult and Pediatric. 6th Edition. 2010. pp 1897-1920. Permission obtained.


External Genitalia


Females with classical CAH owing to 21OHD and 11β-hydroxylase deficiency generally present at birth with virilization of their genitalia. Adrenocortical function begins at the 7th week of gestation (12); thus, a female fetus with classical CAH is exposed to adrenal androgens at the critical time of sexual differentiation (between 9 to 15 weeks gestational age). Androgens masculinize external female genitalia causing physical changes including: clitoral enlargement, fusion and scrotalization of the labial folds, and rostral migration of the urethral/vaginal perineal orifice, placing the phallus in the male position. The degree of genital virilization may range from mild clitoral enlargement alone to, in rare cases, a penile urethra (Prader V genitalia). Degrees of genital virilization are classified into five Prader stage (13) (see Figure 3).

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Figure 3. Different degrees of virilization according to the scale developed by Prader (15). Stage I: clitoromegaly without labial fusion Stage II: clitoromegaly and posterior labial fusion Stage III: greater degree of clitoromegaly, single perineal urogenital orifice, and almost complete labial fusion Stage IV: increasingly phallic clitoris, urethra-like urogenital sinus at base of clitoris, and complete labial fusion Stage V: penile clitoris, urethral meatus at tip of phallus, and scrotum-like labia (appear like males without palpable gonads).

Internal Genitalia

In contrast to the virilization of the external genitalia, internal female genitalia, the uterus, fallopian tubes and ovaries, develop normally. Females with CAH do not produce anti-Müllerian hormone (AMH), which is produced by the testicular Sertoli cells. Internal female structures are Müllerian derivatives and are not androgen responsive. Therefore, the affected female born with virilized external genitalia but normal female internal genitalia has the possibility of normal fertility (1). Surgical correction of the external genitalia (genitoplasty) may be considered in severely virilized females. This is further discussed in a later section, Treatments.

Postnatal Effects and Growth

Deficient postnatal treatment in boys and girls results in continued exposure to excessive androgens, causing progressive penile or clitoral enlargement, the development of premature pubic hair (pubarche), axillary hair, acne, and impaired fertility. Advanced somatic and epiphyseal development occurs with rapid growth during childhood. This rapid linear growth is usually accompanied by premature epiphyseal maturation and closure, resulting in a final adult height that is below that expected from parental heights (on average -1.1 to -1.5 SD below the mid-parental target height) (14). This is on average 10 cm below the mid-parental height (15). On the other hand, poor growth can occur in patients with 21OHD as a result of excess glucocorticoid treatment. Short stature occurs even in patients with good hormonal adrenal control. A study of growth hormone therapy alone or in combination with a GnRH analog and aromatase inhibitors in CAH patients with compromised height prediction showed improvement in short- and long-term growth to reduce the height deficit (15). Aromatase inhibitor as a sole adjunct treatment reduces bone age advancement without adverse effects on bone mineral density or visceral adipose tissue (16).


In the majority of patients treated adequately from early life, the onset of puberty in both girls and boys with classical 21OHD occurs at the expected chronological age. A careful study showed that the mean ages at onset of puberty in both males and females were somewhat younger than the general population, but did not differ significantly among the three forms of 21OHD (17).

In those who are inadequately treated, advanced epiphyseal development can lead to central precocious puberty. In those with advanced bone maturation at the initial presentation, such as in simple virilizing males, the exposure to elevated androgens followed by the suddenly decreased androgen levels after initiation of glucocorticoid treatment may cause an early activation of the hypothalamic-pituitary-gonadal axis. Studies suggest that excess adrenal androgens (aromatized to estrogens) inhibit the pubertal pattern of gonadotropin secretion by the hypothalamic-pituitary axis. This inhibition, via a negative feedback effect, can be reversed by glucocorticoid treatment (17, 18).

Following the onset of puberty, in a majority of successfully treated patients, the milestones of further development of secondary sex characteristics in general appear to be normal (17). In adolescents and adults, signs of hyperandrogenism may include male-pattern alopecia (temporal balding) and acne. Female patients may develop hirsutism and menstrual irregularities. Although the expected age of menarche may be delayed in females with classical CAH (18), when adequately treated many have regular menses after menarche (19, 20). Menstrual irregularity and secondary amenorrhea with or without hirsutism occur in a subset of post-menarchal females, especially those in poor hormonal control. Primary amenorrhea or delayed menarche may occur if a female with classical CAH is untreated, inadequately treated, or over treated with glucocorticoids (20). In addition, women with CAH may develop polycystic ovarian syndrome (PCOS), likely as a complication of poorly controlled CAH (21, 22).

Gender Role Behavior and Cognition

Prenatal androgen exposure in females affected with the classic forms of 21OHD CAH not only has a masculinizing effect on the development of the external genitalia, but also on childhood behavior. Both physical and behavioral masculinization were related to genotype, indicating that behavioral masculinization in childhood is a consequence of prenatal androgen exposure. Further, changes in childhood play behavior is correlated with reduced female gender satisfaction. Prenatal androgen exposure is related to a decrease in self-reported femininity in dose response manner in adulthood (23) . Affected adult females are more likely to have gender dysphoria, and experience less heterosexual interest and reduced satisfaction with the assignment to the female sex (24). In contrast to females, males affected with CAH do not show a general alteration in childhood play behavior, core gender identity and sexual orientation (24). The rates of bisexual and homosexual orientation were increased in women with all forms of 21OHD CAH. They were found to correlate with the degree of prenatal androgenization (24). Of interest, bisexual/homosexual orientation was correlated with global measures of masculinization of nonsexual behavior and predicted independently by the degree of both prenatal androgenization and masculinization of childhood behavior (25). Among 46,XX CAH patients raised as girls, reported rates of gender dysphoria varied from 6.3%to 27.2%(26). Most expressed gender dysphoria in late adolescence and adulthood. Cultural views may determine serious biases in reports of gender dysphoria coming from countries with disproportional societal advantages for males and/or religious restrictions. Male gender raised patients were approximately 10% of CAH cohorts, mostly from underdeveloped countries, with a high proportion of late diagnoses (26).With regards to cognitive abilities, such as visuospatial/motor ability and handedness, the effect of prenatal androgen exposure continues to be elucidated. A study found males and females with CAH scored higher than their siblings of the same sex in measures of visual special processing suggesting that androgens affect spatial ability (27). The effect of CAH on intelligence is controversial. One study showed no evidence of intellectual deficit in either females or males with CAH. Intelligence was not significantly associated with disease characteristics (28).


Difficulty with fertility in females with CAH may arise for various reasons, including anovulation,secondary polycystic ovarian syndrome, irregular menses, non-suppressible serum progesterone levels, or an inadequate introitus. Fertility is reduced in salt-wasting 21OHD (29). In a retrospective survey of fertility rates in a large group of females with classical CAH, simple virilizers were shown to be more likely to become pregnant and carry the pregnancy to term than salt-wasters. Adequate glucocorticoid therapy is an important variable with respect to fertility outcome. The development of PCOS in CAH patients is not uncommon and may be related to both prenatal and postnatal excess androgen exposure, which can affect the hypothalamic-pituitary-gonadal axis. An inadequate vaginal introitus can affect up to a third of classical CAH adult females. Since vaginal dilation is needed to maintain good patency, vaginoplasty is delayed until sexual intercourse is regular or when the patient can assume responsibility for vaginal dilatation (30).

Males with CAH, particularly if poorly treated, may have reduced sperm counts and low testosterone as a result of small testes due to suppression of gonadotropins and sometimes intra-testicular adrenal rests(31, 32). All of these complications may result in diminished fertility. In male patients with classical CAH, several long-term studies indicate that those who have been adequately treated undergo normal pubertal development, have normal testicular function, and normal spermatogenesis and fertility (32, 33). However, small testes and aspermia can occur in patients as a result of inadequately controlled disease (34, 35). Testicular adrenal rest tumor can lead to end stage damage of testicular parenchyma, most probably as a result of longstanding obstruction of the seminiferous tubules (36). In contrast, some investigators have reported normal testicular maturation as well as normal spermatogenesis and fertility in patients who had never received glucocorticoid treatment (37).

Studies demonstrate that post pubertal males with inadequately treated CAH are at a very high risk to develop testicular adrenal rest tumors (TARTs) In one study, almost all these patients were found to have adenomatous adrenal rests within the testicular tissue, as indicated by the presence of specific 11β-hydroxylated steroids in the blood from gonadal veins (38). These tumors have been reported to be ACTH dependent and to regress following adequate steroid therapy (39-43). These testicular adrenal rests are more frequent in males with salt-wasting CAH and are associated with an increased risk of infertility(30, 44) . Regular testicular examination and periodic testicular ultrasonography are recommended for early detection of testicular lesions. If present, dexamethasone treatment can be considered to suppress TARTs.

Salt-Wasting 21-Hydroxylase Deficiency

When the deficiency of 21-hydroxylase is severe, adrenal aldosterone secretion is not sufficient for sodium reabsorption by the distal renal tubules, and individuals suffer from salt wasting in addition to cortisol deficiency and androgen excess. Infants with renal salt wasting have poor feeding, weight loss, failure to thrive, vomiting, dehydration, hypotension, hyponatremia, and hyperkalemic metabolic acidosis progressing to adrenal crisis (azotemia, vascular collapse, shock, and death). Adrenal crisis can occur as early as age one to four weeks. The salt wasting is presumed to result from inadequate secretion of salt-retaining steroids, primarily aldosterone. In addition, hormonal precursors of the 21-OH enzyme may act as antagonists to mineralocorticoid action in the sodium-conserving mechanism of the immature newborn renal tubule (45-47).

Affected males who are not detected in a newborn screening program are at high risk for a salt-wasting adrenal crisis because their normal male genitalia do not alert medical professionals to their condition. They may be discharged from the hospital after birth without diagnosis and experience a salt-wasting crisis at home. On the other hand, salt wasting females are born with atypical genitalia that trigger the diagnostic process and appropriate treatment. It is important to recognize that the extent of genital virilization may not differ among the two forms of classical CAH, the simple virilizing and the salt-wasting form. Thus, even a mildly virilized newborn with 21OHD should be observed carefully for signs of a potentially life-threatening crisis within the first few weeks of life. The difference between salt-wasting and simple virilizing form of 21OHD is the quantitative difference in activity of the 21-hydrozylase enzyme, which results from specific mutations. In vitro expression studies show that as little as 1% of 21-hydroxylase activity is sufficient to synthesize enough aldosterone to prevent significant salt wasting (48). It has been observed that an aldosterone biosynthetic defect apparent in infancy may ameliorate with age (49, 50).A spontaneous partial recovery of aldosterone biosynthesis in an adult patient with a homozygous deletion of the CYP21A2 gene who had documented severe salt wasting in infancy has been reported (51). Therefore, it is desirable to follow the sodium and mineralocorticoid requirements carefully by measuring plasma renin activity (PRA) in patients who have been diagnosed in the neonatal period as salt wasters.

Simple-Virilizing 21-Hydroxylase Deficiency

The salient features of classical simple virilizing 21OHD are prenatal virilization and progressive postnatal masculinization with rapid somatic growth and advanced epiphyseal maturation leading to early epiphyseal closure and likely short stature. There is usually no evidence of mineralocorticoid deficiency in this disorder.

Diagnosis at birth of a female with simple virilizing CAH is usually made immediately because of the apparent genital ambiguity. Since the external genitalia are not affected in newborn males, hyperpigmentation and an enlarged phallus may be the only clues suggesting increased ACTH secretion and cortisol deficiency. Diagnosis at birth in males thus rests on prenatal or newborn screening. If a female is not treated with glucocorticoid replacement therapy early post-natally, her genitalia may continue to virilize due to continued excess adrenal androgens, and pseudo precocious puberty may occur. In patients with salt-wasting 21OHD, signs of hyperandrogenism in children affected with CAH include early onset of facial, axillary, and pubic hair, adult body odor, and rapid somatic growth and bone age advancement, leading to short stature in adulthood. The same issues as discussed above related to puberty, fertility, behavior and cognition apply to patients with simple-virilizing 21OHD (1).

Non-Classical 21-Hydroxylase Deficiency

Non-classical 21OHD (NC-21OHD), previously known as late-onset 21OHD, is much more common than the classical form, with an incidence as high as 1:27 in Ashkenazi Jews (3). Individuals with the non-classical (NC) form of 21OHD have only mild to moderate enzyme deficiency and present postnatally, eventually developing signs of hyperandrogenism. Females with NC-CAH do not have virilized genitalia at birth.

NC-CAH may present at any age after birth with a variety of hyperandrogenic symptoms. This form of CAH results from a mild deficiency of the 21-hydroxylase enzyme. Table 2 summarizes the main clinical characteristics of all forms of 21OHD CAH. While serum cortisol concentration is typically low in patients with the classic form of the disease, it is usually normal in patients with NC 21OHD. Similar to classical CAH, NC-CAH may cause premature development of pubic hair, advanced bone age and accelerated linear growth velocity in both males and females. Severe cystic acne has also been attributed to NC-CAH (52, 53).

Table 2. Clinical Features in Individuals with Classic and Non-Classic 21-Hydroxylase Deficiency in the Untreated Form


21-OH Deficiency



Prenatal virilization

Females only


Postnatal virilization (hyperandrogenism)

Females and Males


Salt wasting

~75% of all individuals


Women may present with a variety of symptoms of androgen excess which may be highly variable and organ-specific, including hirsutism, temporal baldness, acne and infertility. Menarche in females may be normal or delayed, and secondary amenorrhea is a frequent occurrence. Further masculinization may include hirsutism, male habitus, deepening of the voice, or male-pattern alopecia (temporal recession). Polycystic ovarian syndrome may also be seen as a secondary complication in these patients. Possible reasons for the development of PCOS include reprogramming of the hypothalamic-pituitary-gonadal axis from prenatal exposure to androgens, or chronic levels of excess adrenal androgens that disrupt gonadotropin release and have direct effects on the ovary, ultimately leading to the formation of cysts. Because of the overlap of hyperandrogenic symptoms, it is important to consider NC 21OHD in a patient diagnosed with PCOS (54, 55).

In adult males, early balding, acne, or impaired fertility may prompt the diagnosis of NC-CAH. A highly reliable constellation of physical signs of adrenal androgen excess is the presence of pubic hair, enlarged phallus, and relatively small testes. Males may have small testes compared to the phallus, which results from suppression of the hypothalamic-pituitary-gonadal axis from adrenal androgens. They may also develop TARTs, which can cause infertility, although some untreated men have been fertile(31, 33). Signs of NC-CAH in adult males may be limited to short stature, oligo-zoospermia and impaired fertility.

A subset of individuals with NC-21OHD are completely asymptomatic when detected (usually as part of a family study or evaluation for infertility), but it is thought, based on longitudinal follow-up of such patients, that symptoms of hyperandrogenism may wax and wane with time. The presence of 21OHD can also be discovered during the evaluation of an incidental adrenal mass (56). One study showed that an increased incidence of adrenal incidentalomas has been found, which was reported as high as 82% in patients with 21OHD and up to 45% in subjects heterozygous for 21OHD mutations. This probably arises from hyperplastic tissue areas and does not require surgical intervention (57). Overall, however, CAH is an uncommon cause of incidentalomas, accounting for less than 1% in one series (58, 59).


11-β Hydroxylase Deficiency

Virilization and low renin hypertension are the prominent clinical features of 11β hydroxylase deficiency (11β-OHD) (60). The virilizing signs and symptoms of this disorder are similar to or more severe than classical 21OHD. Despite failure of aldosterone production, overproduction of deoxycorticosterone (DOC), in vivo a less potent mineralocorticoid, causes salt retention and hypertension. Elevated blood pressure is usually not identified until later in childhood or in adolescence, although its appearance in an infant 3 months of age has been documented (61). In addition, hypertension correlates variably with biochemical values, and clinical signs of mineralocorticoid excess and the degree of virilization are not well correlated. Some severely virilized females are normotensive, whereas mildly virilized patients may experience severe hypertension leading to fatal vascular accidents (62, 63). Complications of long-standing uncontrolled hypertension, including cardiomyopathy, retinal vein occlusion and blindness have been reported in 11β-OHD patients (64, 65). Potassium depletion develops concomitantly with sodium retention, but hypokalemia is variable. Renin production is suppressed secondary to mineralocorticoid-induced sodium retention and volume expansion.

A mild non-classical form of 11β-OHD CAH has been reported. Unlike the common non-classical form of 21OHD, this form is very rare. Non-classical 11β-OHD has been diagnosed in normotensive children with mild virilization or precocious pubarche (6) and in adults with signs of hyper-androgen effect (66) as well as a woman with infertility (67)(69). Despite a hormonal profile consistent with 11β-OHD, mutations in the CYP11B1 gene may not always be present (66). The best biochemical marker of 11β-OHD is elevated serum 11-deoxycortisol concentration.

3-β Hydroxysteroid Dehydrogenase Deficiency

There are two forms of the 3 β -hydroxysteroid dehydrogenase enzyme (3 β -HSD): type I and type II. Type II 3 β -HSD enzyme is expressed in the adrenal cortex and gonads and is responsible for conversion of Δ5 (delta 5) to Δ4 (delta 4) steroids (1). This enzyme is essential for the formation of progesterone, which is the precursor for aldosterone, and 17-OHP, which is the precursor for cortisol in the adrenal cortex as well as for androstenedione, testosterone, and estrogen in the adrenal cortex and gonads (68, 69). Therefore, deficiency of 3ß-HSD in the classic form of 3ß-HSD deficiency CAH results in insufficient cortisol synthesis, salt-wasting in the most severe form, and virilization of external genitalia in females due to androgen effect from the peripheral conversion of circulating Δ5 precursors to active Δ4 steroids. Simultaneous type II 3ß-HSD deficiency in the gonads results in incomplete virilization of the external genitalia in males. Thus, genital ambiguity can result in both sexes (70).

17 α -Hydroxylase/17,20 Lyase Deficiency

Steroid 17 α-hydroxylase/17,20 lyase deficiency accounts for approximately 1% of all CAH cases and affects steroid synthesis in both the adrenals and gonads(71). Patients have impaired cortisol synthesis, leading to ACTH over secretion, which increases serum levels of deoxycorticosterone and especially corticosterone, resulting in low renin hypertension, hypokalemia, and metabolic alkalosis. Affected females are born with normal external genitalia, however affected males are born with under-virilized genitalia due to their deficient gonadal testosterone production. 17 α-Hydroxylase/17,20 lyase deficiency is often recognized at puberty in female patients who fail to develop secondary sex characteristics (72).

Congenital Lipoid Adrenal Hyperplasia

Congenital lipoid adrenal hyperplasia is an extremely rare and severe form of CAH which is caused by mutations in the steroidogenic acute regulatory protein (StAR). Both the adrenal glands and the gonads exhibit a severe defect in the conversion of cholesterol to pregnenolone (73, 74). More specifically, StAR mediates the acute steroidogenic response by moving cholesterol from the outer to inner mitochondrial membrane (the rate-limiting step of steroidogenesis), and when this does not occur, cholesterol and cholesterol esters accumulate (75). In the most severe form, males with congenital lipoid hyperplasia are born with female-appearing external genitalia. Females have a normal genital phenotype at birth but remain sexually infantile without treatment. Salt wasting occurs in both males and females. If not detected and treated, the severe form of lipoid CAH is usually fatal (76). Several cases have been reported that demonstrate that lipoid CAH has a spectrum of clinical presentation, with varying degrees of genital ambiguity (including normal male genitalia in a 46, XY male) and adrenal insufficiency. Mutations in the StAR protein have been reported that retain partial protein function, leading to variable phenotype (77).

The cholesterol side-chain cleavage enzyme (P450scc) is a very slow enzyme located on the inner mitochondrial membrane and catalyzes the conversion of cholesterol to pregnenolone in the first and rate-limiting step in the production of corticosteroids (78). The CYP11A1 gene encoding P450cc lies on chromosome 15q23-24. Mutations in this gene are rare with approximately 20 patients reported (77-79).

Cytochrome P450 OxidoReductase Deficiency

Cytochrome P450 oxido-reductase (POR) deficiency is another rare form of CAH that is caused by a mutation on 7q11.2(80-82) P450 oxidoreductase is an important cofactor for electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) to several enzymes of steroidogenesis including 21-hydroxylase and 17α-hydroxylase/17,20-lyase.The various constellations of partial enzymatic deficiency of 21-hydroxylase, 17α-hydroxylase/17,20-lyase, and aromatase in the developing fetus account for the broad range of genital anomalies seen in both sexes (80).Female newborns may have severe virilization, and males may have under-virilized genitalia due to combined partial 21-hydroxylase and 17α-hydroxylase/17,20-lyase deficiencies. Maternal virilization and low maternal estriol levels are common findings during pregnancies with affected male and female fetuses. Maternal gestational virilization is likely due to the P450 oxidoreductase effect on placental P450 aromatase (82). Many affected patients also have Antley-Bixler syndrome (type 2) characterized by craniosynostosis, radio-humeral or radioulnar synostosis, arachnodactyly and bowing of the femur (82).


In general, all forms of CAH are transmitted in an autosomal recessive mode of inheritance as a monogenic disorder. However, there have been reports of cases where none or only one mutation in the responsible gene was identified, including in cases of 21 OHD CAH (83, 84), deficiency of the cholesterol side chain cleavage enzyme (85) and POR deficiency (80). The genes responsible for each form of CAH are shown in Table 1.

21-Hydroxylase Deficiency

The gene encoding the enzyme 21-hydroxylase, CYP21A2, is a microsomal cytochrome P450 located on the short arm of chromosome 6 (86) in the human lymphocyte antigen (HLA) complex (87). CYP21A2 and its homologue, the pseudogene CYP21P, alternate with two genes called C4B and C4A (87, 88) that encode the two isoforms of the fourth component (C4) of serum complement (89). CYP21A2 and CYP21P, which each contain 10 exons, share 98% sequence homology in exons and approximately 96% sequence homology in introns (90, 91).

More than 140 mutations have been described including point mutations, small deletions, small insertions, and complex rearrangements of the gene (92). The most common mutations appear to be the result of either of two types of meiotic recombination events between CYP21 and CYP21P: 1) misalignment and unequal crossing over, resulting in large-scale DNA deletions, and 2) apparent gene conversion events that result in the transfer to CYP21A2 of smaller-scale deleterious mutations present in the CYP21P pseudogene (1, 93).

Both classical and non-classical 21-hydroxylase deficiency are inherited in a recessive manner as allelic variants of the CYP21A2 gene. Classical 21-hydroxylase deficiency tends to result from the presence of two severely affected alleles and non-classical 21-hydroxylase deficiency tends to result from the presence of either two mild 21-hydroxylase deficiency alleles or one severe and one mild allele (compound heterozygote). It is important to note, however, that the 10 most common mutations observed in CYP21A2 cause variable phenotype effects and are not always concordant with genotype. One study demonstrated that the genotype-phenotype concordance was as high as 90.5% for salt-wasting CAH, 85.1% for simple-virilizing CAH, and 97.8% for non-classical CAH (94). In a study of 1,507 subjects with CAH by New et al, a direct genotype–phenotype correlation was noted in less than 50% of the genotypes studied. However, in the salt wasting and non-classical forms of 21OHD CAH, a phenotype was strongly correlated to a genotype (95). Moreover, it was shown in 2008 that CAG repeats in the androgen receptor have a great influence on variability in virilization of external genitalia of CAH women (96).

Chapters Archive - Endotext (38)

Figure 4. Common mutations in CYP21A2 gene and their related phenotypes. The numbers indicated exons of the gene. From: Forest MG. Recent advances in the diagnosis and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (97).


Hormonal Diagnosis

Potential diagnosis of CAH must be suspected in infants born with atypical genitalia. The physician is obliged to make the diagnosis as quickly as possible to initiate therapy. The diagnosis and rational decision of sex assignment must rely on the determination of genetic sex, the hormonal determination of the specific deficient enzyme, and an assessment of the patient’s potential for future sexual activity and fertility. Physicians are urged to recognize the physical characteristics of CAH in newborns (e.g., atypical genitalia) and to refer such cases to appropriate clinics for full endocrine evaluation. As indicated in Table 1, each form of CAH has its own unique hormonal profile, consisting of elevated levels of precursors and elevated or diminished levels of adrenal steroid products. Traditionally, laboratories measured urinary excretion of adrenal hormones or their urinary metabolites (e.g., 17-ketosteroids). However, collection of 24-hour urine excretion is difficult, particularly in neonates. (98) Therefore, simple and reliable immunoassays are utilized now for measuring circulating serum levels of adrenal steroids (99). Alternatively, a non-invasive random urine collection in the first days of life for steroid hormone metabolites and precursor/product ratio assessments can be measured simultaneously. It can be used independently or in conjunction with serum steroid assays to increase accuracy and confidence in making the diagnosis and distinguishing the separate enzymatic forms of the disorder (100, 101).

Diagnosis of the 21OHD CAH can also be confirmed biochemically by a hormonal evaluation in blood or serum. In a randomly timed blood sample, a very high concentration of 17-hydroxyprogesterone (17-OHP), the precursor of the defective enzyme, is diagnostic of classical 21OHD. Such testing is the basis of the newborn-screening program developed to identify classically affected patients who are at risk for salt wasting crisis (102). Only 20µl blood, obtained by heel prick and blotted on microfilter paper, is used for this purpose to provide a reliable diagnostic measurement of 17-OHP. The simplicity of the test and the ease of transporting microfilter paper specimens by mail have facilitated the implementation of CAH newborn screening programs worldwide. As of 2009, all 50 states in the United States screen for CAH. Now, more than 35 countries worldwide have also established newborn screening programs. (103) False-positive results are, however, common with premature infants (104). Appropriate references based on weight and gestational age are therefore in place in many screening programs (105). The majority of screening programs use a single screening test without retesting of questionable 17-OHP concentrations. To improve efficacy, a small number of programs perform a second screening test of the initial sample to re-evaluate borderline cases identified by the first screening. Current immunoassay methods used in newborn screening programs yield a high false positive rate. To decrease this high rate, liquid-chromatography- tandem mass spectrometry measuring different hormones (17-OHP, Δ4-androstenedione, and cortisol) has been suggested as a second-tier method of analyzing positive results (106).

The gold standard for hormonal diagnosis is the corticotropin stimulation test (250 μg cosyntropin intravenously), measuring levels of 17-OHP and Δ4-androstenedione at baseline and 60 min. These values can then be plotted in the published nomogram (Figure 5) to ascertain disease severity (107). It is important to note that the corticotropin stimulation test should not be performed during the initial 24 hours of life as samples from this period are typically elevated in all infants and may yield false-positive results. Testing is typically performed between 48 to 72 hours of life in newborns suspected of classical CAH and glucocorticoid treatment is initiated while awaiting results. In newborns who are hemodynamically unstable and adrenal crisis is suspected, stimulation testing may delay life-saving treatment. Screening measurements of 17-OHP, cortisol, and adrenal androgen along with ACTH can be obtained before emergent glucocorticoid administration. The corticotropin stimulation test is crucial in establishing hormonal diagnosis of non-classical form of the disease since early-morning values of 17-OHP may not be sufficiently elevated to allow accurate diagnosis.

Chapters Archive - Endotext (39)

Figure 5. Nomogram relating baseline to ACTH-stimulated serum concentrations of 17-hydroxyprogesterone (17-OHP). The scales are logarithmic. A regression line for all data points is shown. Data points cluster as shown into three nonoverlapping groups: classic and non-classic forms of 21-hydroxylase deficiency are readily distinguished from each other and from those that are heterozygotes and unaffected. Distinguishing unaffected from heterozygotes is difficult. (107) Adapted from: New MI, Lorenzen F, Lerner AJ, et al. 1983 Genotyping steroid 21-hydroxylase deficiency: hormonal reference data. J Clin Endocrinol Metab 57:320-6. Permission obtained.

Prenatal Diagnosis of 21OHD

A number of approaches to prenatal identification of affected fetuses have been used. In 1965, Jeffcoate et al first reported a successful prenatal diagnosis of 21OHD, based on elevated levels of 17-ketosteroids and pregnanetriol in the amniotic fluid (108). The hormonal diagnostic test for 21OHD is amniotic fluid 17-OHP. Hormonal diagnosis is rarely used and considered only when molecular diagnosis is unavailable.

Advances in genotyping of the CYP21A2 gene have made molecular genetic studies of extracted fetal DNA the ideal method to diagnose 21OHD CAH in the fetus. Approximately 95% to 98% of the mutations causing 21OHD have been identified through a combination of molecular genetic techniques to study large gene rearrangement and arrays of point mutations (109, 110). Of the currently available methods for prenatal diagnosis of CAH, chorionic villus sampling (CVS), rather than amniocentesis, with molecular genotyping is the preferred diagnostic method in use. CVS is performed between the 9th and 11th week of gestation, while amniocentesis is usually performed in the second trimester. The timing of prenatal diagnosis is particularly important when deciding to treat the fetus at risk for CAH with dexamethasone prenatally to prevent virilization of the genitalia (see Prenatal Treatment below). As we only wish to treat affected females until term and only 1/8 of the fetuses will be affected and 1/2 will be males, 7 out of 8 fetuses do not require treatment. Thus, amniocentesis, which is performed later in gestation, results in treatment of unaffected fetuses for a longer period of time than CVS. However, amniocentesis can be used as a reliable alternative method of prenatal diagnosis when CVS in unavailable. In such instances, the supernatant is used for hormonal measurement and the cells are cultured to obtain a genotype through DNA analysis. The supernatant hormone measurements distinguish affected status from unaffected status only in SW patients. Nonetheless, pitfalls do occur in a small percentage of the patients undergoing prenatal diagnosis utilizing genetic diagnosis, such as undetectable mutations (111), allele drop outs (112), or maternal DNA contamination. Commercially available genetic testing utilizing short range PCR to detect common mutations may miss rare de novo mutations and thus the ACTH stimulation test remains vital to the evaluation. Determination of satellite markers may increase the accuracy of molecular genetic analysis (113).

Non-Invasive Prenatal Diagnosis of CAH

Virilization of the genitalia in a female fetus affected with CAH owing to 21OHD and 11B-OHD can be treated prenatally with dexamethasone administered to the mother (see Prenatal Treatment below). Because CAH is an autosomal recessive disorder, the risk is 1/4 of the fetus being affected with the disease and 1/8 of the fetus being a female with atypical genitalia. Therefore, 7 out of 8 pregnancies will receive unnecessary treatment until the sex and the affection status of the fetus are known. Treatment with dexamethasone must begin before the 9th week of gestation, yet chorionic villous sampling can only be done at the 9-11th week, with karyotype and DNA results available 2-3 weeks later. Non-invasive prenatal diagnosis would eliminate unnecessary treatment and invasive procedures such as CVS and amniocentesis. Dennis Lo et al. in 1997 discovered the presence of fetal DNA in the maternal circulation (113). Fetal DNA has been extracted and enriched with high accuracy and yield in fetal Rh factor identification (114), aneuploidy and monogenic disorders such as thalassemia and cystic fibrosis (115). Identification of the SRY sequence in maternal blood, performed in multiple academic centers and now available in commercial laboratories, has also achieved excellent accuracy in several studies (116, 117). In non-invasive prenatal diagnosis of CAH, by extracting fetal DNA from the maternal blood as early as 4-5 weeks gestation, the SRY sequence can be identified to determine sex (118). If the fetal genetic sex is deduced to be female (SRY sequence not identified), DNA analysis on extracted fetal DNA can be used to determine CAH affected status. Targeted massive parallel sequencing of cell-free fetal DNA in maternal plasma was used for the noninvasive prenatal diagnosis of CAH due to 21OHD(119). In the fourteen expectant families studied, each with a previous child affected with classical CAH (proband) and parents with at least one mutant CYP21A2 gene, the fetal CAH affection status was correctly deduced using this method from maternal plasma drawn as early as 5 weeks and 6 days (119).

Preimplantation Diagnosis

Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer, so only unaffected embryos established from IVF are transferred. The procedure has been utilized in many monogenic recessive disorders such as cystic fibrosis, hemoglobinopathies, spinal muscular atrophy and Tay Sach’s disease. PGD is being used for a growing number of genetic diseases (120). There is only one report of PGD utilized in a family whose offspring is at risk for CAH (121), however we know from experience that families are seeking PGD with greater frequency. It would be desirable to have further studies of preimplantation diagnosis in CAH families.

Prenatal Treatment

In 21OHD, prenatal treatment with dexamethasone was introduced in France in 1978 (122) and in the United States in 1986 (123). Institution of therapy before the 9th week of gestation, prior to the onset of adrenal androgen secretion, effectively suppresses excessive adrenal androgen production and prevents virilization of external female genitalia. Dexamethasone is used because it binds minimally to cortisol binding globulin (CBG) in the maternal blood, and unlike hydrocortisone, it escapes inactivation by the placental 11-dehydrogenase enzyme. Thus, dexamethasone crosses the placenta from the mother to the fetus and suppresses ACTH secretion with longer half-life compared to other synthetic steroids (123).

When dexamethasone administration begins as early as the 8th week of gestation, the treatment is blind to the disease status and sex of the fetus. If the fetus is later determined to be a male upon karyotype or an unaffected female upon DNA analysis, treatment is discontinued. Otherwise, treatment is continued to term. A simplified algorithm of management of potentially affected pregnancies is shown in Figure 6. The optimal dosage is 20 µg/kg/day of dexamethasone per maternal pre-pregnancy body weight, in three divided daily doses (124). It is recommended to start the treatment as soon as pregnancy is confirmed, and no later than 9 weeks after the last menstrual period (125, 126). The mother’s blood pressure, weight, glycosuria, HbA1C, symptoms of edema, striae and other possible adverse effects of dexamethasone treatment should be carefully observed throughout pregnancy (127) . Prenatal dexamethasone treatment remains controversial and should be administered under an IRB approved research protocol(104).

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Figure 6. Algorithm of treatment, diagnosis and decision-making for prenatal treatment of fetuses at risk for 21-hydroxylase deficiency congenital adrenal hyperplasia. Mercado AB, Wilson RC, Cheng KC, Wei JQ, New MI 1995 Extensive personal experience: Prenatal treatment and diagnosis of congenital adrenal hyperplasia owing to steroid 21-hydroxylase deficiency. J Clin Endocrinol Metab 80:2014-2020. (125) Permission obtained.

Outcome of Prenatal Treatment of 21OHD

Prenatal treatment of 21OHD has proven to be successful in significantly reducing genital virilization in affected females. Our group has performed prenatal diagnosis in over 685 pregnancies at risk for 21OHD, and 59 affected female fetuses have been treated to term (128). Treatment was highly effective in preventing genital ambiguity when the mother was compliant until term. In all the female fetuses treated to term, the degree of virilization was on average 1.69, as measured using the Prader scoring system (Figure 3). Late treatment as well as no treatment resulted in much greater virilization, and the average Prader score was 3.73 for those female fetuses not treated. 124 and unpublished data Not only does prenatal treatment effectively minimize the degree of female genital virilization in the patients, it also lessens the high-level androgen exposure of the brain during differentiation. The latter is thought to cause a higher tendency to gender ambiguity in some females with CAH (129, 130). Genital virilization in female newborns with classical 21OHD CAH has potential adverse psychosocial implications that may be alleviated by prenatal treatment (124).

Prenatal dexamethasone treatment has continued to be a subject of controversy (104). Some uncertainties and concerns have been expressed about the long-term safety of prenatal diagnosis and treatment (131, 132). Concerns have been raised in regards to the glucocorticoid effects on the fetal brain, which arise from studies of other conditions rather than direct studies on prenatal treatment of 21OHD CAH. These include studies whereby much higher doses of dexamethasone were given to the human subjects at the later part of pregnancy (133) or to animals (134, 135) and therefore hold little relevance to using dexamethasone prenatally in CAH. In a small-sample study of children prenatally treated with dexamethasone, Lajic and her colleagues found no effects on intelligence, handedness, memory encoding, or long-term memory, but short-term treated CAH-unaffected children had significantly poorer performance than controls on a test of verbal working memory. These patients also had lower questionnaire scores in self-perceived scholastic competence and social anxiety (136). However, parents described these children as more sociable than controls, without significant difference in psychopathology, school performance, adaptive functioning or behavioral problems (137). A larger multi-center study (US and France) indicated no adverse cognitive effects of short-term prenatal DEX exposure, including no adverse influence on verbal working memory; a small sample of dexamethasone treated girls affected with CAH showed lower scores on two of eight neuropsychological tests, however given the variability of cognitive findings in dexamethasone unexposed CAH-affected patients, this result cannot be linked to dexamethasone with certainty (138). This indicates that further studies are needed.

Compelling data from large cohorts of pregnancies with prenatal diagnosis and treatment of 21OHD CAH prove its efficacy and safety (126, 139). All of the mothers who received prenatal treatment (partial or full-term) stated that they would take dexamethasone again for a future pregnancy (140). Rare adverse events have been reported in treated children, but no harmful effects have been documented that can be clearly attributed to the treatment (141). Another long-term follow-up study in Scandinavia showed that 44 children who were variably treated prenatally demonstrated normal prenatal and postnatal growth compared to matched controls. Further, there was no observed increase in fetal abnormalities or fetal death (142). Although some abnormalities in postnatal growth and behavior were observed among dexamethasone exposed offspring, none could logically be explained by the present knowledge of teratogenic effects of glucocorticoids.

Published studies of almost 600 pregnancies, 80 of which were prenatally treated until term and 27 who were male and received dexamethasone for a short period of time, the newborns in the dexamethasone treated group did not differ in weight, length or head circumference from untreated, unaffected siblings. No significant or enduring side effects were noted in either the mothers or the fetuses. Greater weight gain in treated versus untreated mothers did occur, as well as the presence of striae and edema. Excessive weight gain was lost after birth. No differences were found regarding gestational diabetes or hypertension (127). No cases have been reported of cleft palate, placental degeneration or fetal death, which have been observed in the rodent model of in utero exposure to high-dose glucocorticoids (143). One explanation for the safety of human versus rodent is that glucocorticoid receptor-ligand systems in human differ from that of rodents (144). A comprehensive long-term outcome study looking at 149 male and female patients 12 years of age and older, affected and unaffected with CAH, who were treated with dexamethasone partially or to term was conducted. To date, this is the largest study evaluating the long-term effects of dexamethasone. No adverse effects such as increased risk for cognitive defects, disorders of gender identity and behavior, sexual function in adulthood, hypertension, diabetes, and osteopenia were found (127).

Prenatal Diagnosis and Treatment of 11β-OHD CAH

Several approaches to prenatal identification by measuring steroid precursors in affected fetuses have been used (145-147). Advances in genotyping of the CYP11B1 gene have made molecular genetic studies of fetal DNA extracted from maternal blood, the ideal method to diagnose 11β-OHD CAH in the fetus (148, 149). The established protocol of prenatal diagnosis and treatment in 21OHD CAH can be applied to 11β-OHD CAH. Reduced virilization of affected females in prenatal diagnosis and treatment in 11β-OHD CAH have been reported (128, 148).


Hormone Replacement

The goal of therapy in CAH is to both correct the deficiency in cortisol secretion and to suppress ACTH overproduction (104). Proper treatment with glucocorticoid reduces stimulation of the androgen pathway, thus preventing further virilization and allowing normal growth and development. The usual requirement of hydrocortisone (or its equivalent) for the treatment of classical 21OHD form of CAH is about 10-15 mg/m2/day divided into 2 or 3 doses per day. Conventionally, oral hydrocortisone tablets with the smallest available dose of 5 mg have been preferred. To administer small doses to infants and young children, 10 mg and 5 mg tablets have to be cut and crushed. Hydrocortisone granules and tablets at smaller doses are becoming more widely available allowing for ease of administration and avoidance of excess dosing (150).

Dosage requirements for patients with NC-21OHD CAH are typically less. Adults may be treated with the longer-acting dexamethasone or prednisone, alone or in combination with hydrocortisone. A small dose of dexamethasone at bedtime (0.25 to 0.5 mg) is usually adequate for androgen suppression in non-classical patients. Anti-androgen treatment may be useful as adjunctive therapy in adult women who continue to have hyperandrogenic signs despite good adrenal suppression. Females with concomitant PCOS may benefit from an oral contraceptive, though this treatment would not be appropriate for patients trying to get pregnant. Treatment of adult males with NC-21OHD may not be necessary, though our group has found that it may be helpful in preventing adrenal rest tumors and preserving fertility. Optimal corticosteroid therapy is determined by adequate suppression of adrenal hormones balanced against normal physiological parameters. The goal of corticosteroid therapy is to give the lowest dose required for optimal control. Adequate biochemical control is assessed by measuring serum levels 17-OHP and androstenedione; serum testosterone can be used in females and prepubertal males (but not in newborn males). It is recommended that hormone levels are measured at a consistent time in relation to medication dosing, usually 2 hours after the morning corticosteroid. Titration of the dose should be aimed at maintaining androgen levels at age and sex-appropriate levels and 17-OHP levels of <1000 ng/dL. Analysis of diurnal and 24-hour urine collection of 17-OHP metabolites can be utilized to further assess adrenal control. (151) Concurrently, over-treatment should be avoided because it can lead to Cushing syndrome. Depending on the degree of stress, stress dose coverage may require doses of up to 50-100 mg/m2/day (1).

Patients with salt-wasting CAH have elevated plasma renin in response to the sodium-deficient state, and they require treatment with the salt-retaining 9α-fludrocortisone acetate. The average dose is 0.1 mg daily, ranging from 0.05 mg to 0.2 mg daily. Infants should also be started on salt supplementation, as sodium chloride, at 1-2 g daily, divided into several feedings. Although patients with the SV and NC form of CAH can make adequate aldosterone, the aldosterone to renin ratio (ARR) has been found to be lower than normal, though not to the degree seen in the salt-wasting form (152)(155). It has not been customary to supplement conventional glucocorticoid replacement therapy with the administration of salt-retaining steroids in the SV and NC forms of CAH, though there has been some suggestion that adding fludrocortisone to patients with elevated PRA may improve hormonal control of the disease (153). The requirement for fludrocortisone appears to diminish with age, and over-suppression of the PRA should be avoided, to prevent complications from hypertension and excessive mineralocorticoid activity. Measurements of plasma renin and aldosterone are used to monitor the efficacy of mineralocorticoid therapy in all patients with the salt wasting form of the disease (1). The use of systemic cortisol injection for impending adrenal crises is discussed below.

In managing 11β-OHD, glucocorticoid administration provides cortisol replacement and decreases ACTH, as it does in 21OHD. This in turn removes the drive for over secretion of deoxycorticosterone (DOC) and 11-deoxycortisol and, in most cases, normalizes blood pressure. A thorough examination undertaken by endocrine challenge and suppression studies to evaluate zonal differences has shown that in 11β-OHD CAH, the zona fasciculata exhibits reduced 11β-hydroxylation and 18-hydroxylation, while both functions appear to be spared in the zona glomerulosa (154). This demonstrates that the zona glomerulosa and the zona fasciculata function as two physiologically, and likely genetically, separate glands. Glucocorticoid treatment produces natriuresis and diuresis, normalizes plasma volume and thus increases the plasma renin to levels that stimulate aldosterone production in the zona glomerulosa. In addition to normalizing blood pressure, the goal of treatment is to replace deficient steroids and in turn minimize adrenal sex hormone excess, prevent virilization, optimize growth, and protect potential fertility. Serum DOC and 11-deoxycortiol are thus the principal steroid index of the 11β-OHD. Plasma renin activity is useful as a therapeutic index as well. In poorly controlled 11β-OHD patients, DOC is elevated, whereas plasma renin is suppressed; both are normal in well-controlled patients. As in patients with 21OHD, oral hydrocortisone at a dose of 10-15 mg/m2 divided into two to three daily doses is the preferred treatment. Long-acting glucocorticoids may be used at or near the completion of linear growth. In patients who have had ongoing hypertension for some time before diagnosis is made, adding spironolactone, calcium channel blockers or amiloride may be necessary (60).

In patients with 3β-HSD deficiency, glucocorticoid administration also reduces the excess production of androgens. In addition, these patients have mineralocorticoid deficiency and require treatment with the salt-retaining 9α-fludrocortisone acetate. Patients with the StAR protein deficiency or SCC deficiency (lipoid form of CAH) classically have severe adrenal insufficiency with mineralocorticoid deficiency and salt wasting; they require both glucocorticoid and mineralocorticoid replacement. Patients with 17 α -hydroxylase/17,20 lyase deficiency typically have excess DOC and low-renin hypertension, and treatment with a glucocorticoid should normalize serum DOC level and lead to normalization of blood pressure. In several conditions, such as StAR protein deficiency, 3β-HSD, 17 α -hydroxylase/17,20 lyase deficiency and cytochrome P450 oxidoreductase deficiency, patients require sex steroid replacement. Sex steroids should be added at a developmentally appropriate time to allow patients to resemble their peers.

Because patients with CAH are at risk for short stature as adults, other adjunct therapies are being utilized. Two studies have demonstrated significant improvement in growth velocity, final adult height prediction (17) and final adult height (15) with the use of growth hormone in conjunction with a GnRH analogue.

In non-life-threatening periods of illness or physiologic stress, the corticosteroid dose should be increased to 2 or 3 times the maintenance dose for the duration of that period. Each family should be given injection kits of hydrocortisone for emergency use (25 mg for infants, 50 mg for children, and 100 mg for adults). In the event of a surgical procedure, a total of 5 to 10 times the daily maintenance dose may be required during the first 24-48 hours, which can then be tapered over the following days to the normal preoperative schedule. Stress doses of dexamethasone should not be given because of the delayed onset of action. It is not necessary for increased mineralocorticoid doses during these periods of stress (1, 104).

It is imperative for all patients who are receiving corticosteroid replacement therapy, such as patients with CAH, to wear a Medic-Alert bracelet, medallion, or equivalent identification that will enable correct and appropriate therapy in case of emergencies. Additionally, all responsible family members should be trained in the intramuscular administration of hydrocortisone.

Bone Mineral Density

In order to adequately suppress androgen production in patients with CAH, the usual requirement of hydrocortisone is generally higher than the endogenous secretory rate of cortisol. Chronic therapy with glucocorticoids at supraphysiologic levels can result in diminished bone accrual and lead to osteopenia and osteoporosis. Glucocorticoid induced bone loss is a well-known phenomenon and is the most prevalent form of secondary osteoporosis (155, 156).

Unlike other diseases treated with chronic glucocorticoid therapy, however, the effect of glucocorticoid replacement in CAH on BMD is unclear. Previous studies of patients with 21OHD have reported increased, normal, or decreased BMD (157-160). It has been postulated that the elevated androgens typically found in patients may have a protective effect on bone integrity, but the precise mechanism is unknown. The increased adrenal androgens, which are converted to estrogens, may counteract the detrimental effects of glucocorticoids on bone mass. This may explain why older CAH women, particularly those who are post-menopausal, are at higher risk for osteoporosis than younger CAH patients. It has been proposed that the inhibitory effect of corticosteroid therapy on bone formation is counteracted by estrogen’s effect on bone resorption through the RANK-L/osteoprotegerin (OPG) system (161).


The decision for genital surgery in females with virilizing forms of CAH and males with forms of CAH associated with under virilization should be made by parents or patients themselves with the guidance of a multidisciplinary team involving pediatric endocrinology, urology, genetics, and psychology. As part of a case-by-case approach, attention should be given to gender identity, quality of life and potential for fertility. The aim of surgical repair in females with atypical genitalia caused by CAH, if the decision is made, is generally to preserve the sexually sensitive glans clitoris, decrease frequency of urinary tract infections and provide a vaginal orifice that functions adequately for menstruation, intromission, and delivery. A medical indication for early surgery other than for sex assignment is recurrent urinary tract infections as a result of pooling of urine in the vagina or urogenital sinus.

In the past, it was routine to recommend early corrective surgery for neonates born with atypical genitalia. However, in recent years, the implementation of early corrective surgery has become increasingly controversial due to lack of data on long-term functional outcome. Data show that genital sensitivity is impaired in areas where feminizing genital surgery had been done, leading to difficulties in sexual function (162). Another study showed that patients with more severe mutations in the CYP21A2 gene, i.e., those with the null genotype and thus those more severely virilized, had more surgical complications that those less severely virilized and were less satisfied with their sexual life (163). Because of the scarcity of these data, the role of the parents in sex assignment becomes crucial in all aspects of the decision-making process, and should include full discussion of the controversy and all possible therapeutic options for the intersex child, particularly early versus delayed surgery. A large repository of data concerning long-term outcomes in patients affected by CAH, including psychosexual well-being, has been enhanced by the establishment of disease registries (164).

In a study of intersex individuals ≥ 16 years old, 66% of individuals with CAH thought that the appropriate age of genital surgery was infancy or childhood (165). In a study of caregivers of female infants with CAH who underwent genitoplasty, 2/3 of caregivers reported no regret over their decision-making. However, 1/3 reported some level of regret in the process (165).

Other Treatment Strategies and Novel Therapies

Glucocorticoid replacement has been an effective treatment for CAH for over 50 years and remains its primary therapy; however, the management of these patients presents a challenge because both inadequate treatment, as well as over suppression, can cause complications. In forms of CAH with decreased cortisol production, increased ACTH production stimulates excessive synthesis of adrenal products in those pathways unimpaired by the enzyme deficiency such as androgens. The goal of therapy in CAH is to both correct the deficiency in cortisol secretion and to suppress ACTH overproduction which drives androgen production (104).

Often, doses of hydrocortisone higher than physiologic replacement or dosing in reverse diurnal pattern are required to suppress androgen production driven by ACTH. Alternatives to oral glucocorticoid administration 2-3 times a day include continuous subcutaneous pump and newer modified release formulations. Physiologic cortisol secretion patterns can be mimicked with varied infusion rates of hydrocortisone via continuous subcutaneous pump administration. This more intense method of administration should be considered in those with rapid cortisol metabolism or poor gastrointestinal absorption (166). Modified release and delayed release formulations of hydrocortisone were developed recently (167). When taken at bedtime, these formulations approximate the diurnal pattern of physiological cortisol production with an early morning peak and aim to prevent the ACTH surge at dawn that drives androgen production (168).

Adjunct treatments targeting hypothalamic and pituitary signaling to the adrenal glands are in development. To suppress ACTH production, corticotrophin-releasing factor (CRF) antagonists have been developed. CRF produced in the hypothalamus stimulates the release of ACTH from the pituitary gland. CRF antagonism can reduce ACTH production and reduce ACTH driven androgen production in patients with CAH. With the adjunct use of CRF antagonists, lower doses of hydrocortisone can be used for physiologic replacement only. Two oral agents are currently undergoing clinical trials in patients with classical CAH (169, 170).

To reduce androgen production, the use of abiraterone acetate indicated for prostate cancer was proposed for patients with CAH. (171) Abiraterone acetate is a potent inhibitor of steroid enzyme 17-hydroxylase/17,20-lyase (CYP17A1) needed for androgen production and is being studied in clinical trials as adjunct therapy in adult and soon pediatric subjects. (164, 172)

Bilateral adrenalectomy is a radical measure that can be effective in some cases. A few patients who were extremely difficult to control with medical therapy alone showed improvement in their symptoms after bilateral adrenalectomy (164, 165). Because this approach renders the patient completely adrenal insufficient, however, it should be reserved for extreme cases and is not a good treatment option for patients who have a history of poor compliance with medication.


The pathophysiology of the various types of CAH (the most common being 21OHD) can be traced to specific, inherited defects in the genes encoding enzymes for adrenal steroidogenesis. Clinical presentation of each form is distinctive and depends largely on the underlying enzyme defect, its precursor retention and deficient end products. Treatment of CAH is targeted to replace the insufficient adrenal hormones, notably cortisol and salt retaining hormones, and to suppress the excess precursors. With proper hormone replacement therapy, normal and healthy development may be expected. Glucocorticoid and, if necessary, mineralocorticoid replacement, has been the mainstay of treatment for CAH, but new treatment strategies continue to be developed and studied to improve care. Molecular genetic techniques used postnatally and prenatally along with well described genotype phenotype correlations can help guide clinical management.


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Congenital hypopituitarism refers to a deficiency of one or more pituitary hormones resulting from issues in fetal development. Embryological pituitary development involves a complex interplay of transcription factors, extrinsic and intrinsic to the oral ectoderm and neuroectoderm which develop to form the mature pituitary during early embryogenesis. Disruption of this process can result in isolated pituitary dysfunction, or effect nearby structures, such as the eye, olfactory bulbs, midline structures, and forebrain. Genetic causes make up an important portion of cases and have varying phenotypes even within identical mutations. This chapter provides an overview of pituitary development, and various causes of hypopituitarism including septo-optic dysplasia, syndromic and non-syndromic causes, isolated pituitary deficiencies, and syndromes associated with hypopituitarism. It also provides guidance on the investigation of genetic causes of hypopituitarism in the current genetic landscape.


The pituitary gland can be thought of as the “hormonal control center” from which most endocrine organs are regulated. It is located in the sella turcica, posterior to the sphenoid sinus, inferior to the optic chiasm and the hypothalamus. The pituitary comprises 3 lobes, the anterior, intermediate and posterior.

The posterior lobe or neurohypophysis contains antidiuretic hormone (ADH or AVP) and oxytocin, contained in vesicles, as part of axonal projections from hypothalamic cells, known as the hypothalamic-hypophyseal tract. The intermediate lobe contains melanotrophs which produce alpha-melanocyte stimulating hormone (αMSH), although in humans typically an embryological remnant and may not be present.

The anterior lobe or adenohypophysis contains 5 cell lines, producing 6 different hormones regulated by hypothalamic hormones via the hypophyseal portal system. Somatotrophs produce growth hormone (GH) while lactotrophs produce prolactin making up the somatomammotroph class of hormones. The glycoprotein hormone class consists of hormones with identical alpha subunits and different beta subunits, with thyroid stimulating hormone (TSH) produced by thyrotroph cells, and gonadotroph cells producing follicle stimulating hormone (FSH) and luteinizing hormone (LH). Corticotroph cells produce adrenocorticotrophic hormone (ACTH).

Growth hormone mediates its effect through induction of insulin like growth factor 1 (IGF-1) from the liver and is critical for linear growth in the child. Prolactin stimulates development of the mammary gland and lactation and is typically quiescent outside of pregnancy or childrearing. TSH stimulates production of thyroid hormone from the thyroid gland, which regulates metabolic rate and is critical for growth and cognitive development in early childhood. FSH and LH support gonadal development, production of testosterone or estrogen, and maturation of gamete cells. ACTH stimulates production of cortisol which is an essential stress hormone and ADH regulates plasma osmolality by inducing resorption of water in the collecting duct of the kidney.

Congenital hypopituitarism refers to a deficiency of one or more pituitary hormones resulting from events during fetal development. This may be the result of genetic mutation, antenatal insult, or as is commonly the case, be idiopathic. Pituitary deficiencies may be detected during the neonatal period; however, in some individuals’ pituitary deficiencies may not manifest until later in childhood. Furthermore, the severity can be variable.

Hypopituitarism may be isolated affecting one cell line (isolated hormone deficiency), or multiple, termed combined or multiple pituitary hormone deficiency (CPHD, MPHD). Pan-hypopituitarism is typically used to describe deficiency of all anterior pituitary hormones; however, in some cases this term may also include the presence of AVP deficiency. AVP (or ADH) deficiency is termed as central diabetes insipidus (DI) but is undergoing a formal process to change the name to AVP deficiency. The new name is preferred as it highlights both etiology and treatment, while avoiding confusion with diabetes mellitus. AVP deficiency may coexist with other deficiency’s or be isolated.

The incidence of isolated growth hormone deficiency (IGHD) is reportedly 1 in 4000 live births (3), with CPHD also seen in 1 in 4000 live births (4), thus presenting an uncommon but important chronic condition.

Embryological pituitary development involves a complex interplay of transcription factors, extrinsic and intrinsic to the oral ectoderm and neuroectoderm which develop to form the mature pituitary. Disorders occurring during early development may affect nearby structures commonly the eye, olfactory bulbs, midline structures, and forebrain, whereas later events are typically isolated to pituitary abnormalities. Transcription factors gene defects have been demonstrated to cause different biochemical and structural forms of congenital hypopituitarism, although the majority of cases remain idiopathic. There is considerable phenotypic variability within known genetic causes of hypopituitarism, with some forms having incomplete penetrance, and presentation ranging from asymptomatic, to severe neonatal onset forms. This review discusses the clinical phenotypes of various genetic anomalies associated with hypopituitarism. Clinical manifestations and investigation of hypopituitarism are discussed in the “Hypopituitarism” chapter of Endotext.

This chapter will present an overview of the embryology of pituitary development before discussing the genetic causes of hypopituitarism. It will firstly discuss genes that cause septo-optic dysplasia, before discussing genes that cause other clinical phenotypes and hypopituitarism. After describing the genes associated with non-syndromic hypopituitarism, and isolated hormone deficiencies we will discuss other syndromes where hypopituitarism may be part of the phenotype. Each gene is described within the subheading that they most typically present; however, due to phenotypic variability some genes can present in multiple categories. For example, PROKR2 mutation classically causes isolated hypogonadotrophic hypogonadism, but can result in non-syndromic CPHD or septo-optic dysplasia in some cases (5-8). The information within this review is up to date as of May 2022; however, understanding of these genes continues to progress.

Chapters Archive - Endotext (41)

Figure 1. Stages of rodent pituitary development. (a) Oral ectoderm. (b) Rudimentary pouch. (c) Definitive pouch. (d) Adult pituitary gland. I infundibulum; NP neural plate; Nnotochord; PP pituitary placode; OM oral membrane; H heart; F forebrain; MB midbrain; HB hindbrain; RP Rathke's pouch; AN anterior neural pore; O oral cavity; PL posterior lobe; OC optic chiasm; P pontine flexure; PO pons; IL intermediate lobe; AL anterior lobe; DI diencephalon; SC sphenoid cartilage. Adapted from Sheng and Westphal, Trends in Genetics 1999;15:236-240, with permission (2).


Pituitary gland development is classically expressed based on timing with murine development. The pituitary gland forms from the hypophyseal placode on the anterior neural ridge of the neural plate (evident embryonic (E) day 7.5 in the mouse model). Ventral displacement leads to the formation of the oral ectoderm and the roof of the mouth. A portion of this ectoderm thickens (E8.5) before invaginating rostrally at E9. At E9.5, corresponding with 4 weeks gestation, this migrates dorsally resulting in a cone shape, known as the rudimentary Rathke’s pouch. This continues to thicken with increased rostral mitosis spreading into the mesenchyme. Following ongoing proliferation, it separates from the oral ectoderm at E12.5 (approximately 6 weeks gestation). Hormonal cell progenitors arise from ventral proliferation forming the anterior pituitary. (Figure 1) The somatotrophs arise caudomedially, gonadotrophs rostroventrally and corticotrophs ventrally. The dorsal aspect later adjoins the descending infundibulum and remains thin (Intermediate lobe) (9).

Throughout this process the tissue is in contact with the neural plate neuroectoderm, at the base of the developing diencephalon. This neuroectoderm evaginates and progress dorsally to form the posterior pituitary. The tissue immediately ventral to this evagination forms the optic chiasm. The diencephalon develops throughout this process to form the hypothalamus (9).

The olfactory placode forms immediately lateral to the hypophyseal placode on the neural plate. These cells form the olfactory bulb but also the GnRH secreting cells which migrate to the forebrain from 6 weeks gestation, progressing to the hypothalamus by week 15. Axons from these cells extend into the hypothalamic portal system allowing regulation of gonadotroph cells (10-12). The optic nerve origin and optic chiasm develop from the neuroectoderm immediately anterior to the posterior pituitary, which has a complex interplay with some causes of hypopituitarism (13).

Correlation of pituitary development in mice to humans is difficult. Whilst pluripotent stem cells have been induced to form hypothalamic and anterior pituitary cells, the complex interplay of transcription factors, as well as interaction from other anatomical structures, mean these methods cannot give us a clear timeline in humans (14). Historical embryo samples demonstrate the formation of Rathke’s pouch by week 4, with interruption of connection to the oral cavity by week 6 with the anterior pituitary fully differentiated by 16 weeks (15).

Using hormone expression to correlate these two embryonic timelines, humans start producing GH, FSH, LH and ACTH at 8 weeks gestation, with TSH and prolactin at 13 weeks (9). Somatotrophs make up 50% of the cellular composition of the anterior pituitary, with 15-20% of cells being lactotrophs, 5% thyrotropes, 10-15% gonadotrophs and 15-20% corticotrophs (16) (Figure 2).

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Figure 2. Structural development of pituitary. Lateral representation of process of oral and neuroectoderm folding. Cross-sectional representation of hormone producing pituitary cell groups. Adapted from Larkin S. et al, Endotext (1).

Various transcription factors are involved in coordinating these processes and gene mutation can result in underdevelopment or arrest of pituitary development. Figure 3 summarizes the temporal expression of various transcription factors within the pituitary. Abnormalities in genes expressed early in development such as SOX2, HESX1 and GLI2 more frequently effect other nearby structures, whereas those expressed later such as PROP1 and POU1F1 typically have localized effects. Transcription factors may be induced, upregulated or downregulated by multiple other transcription factors and the degree and site of expression can vary across the embryological development. For a diagrammatic overview of the role each transcription factor plays in pituitary development please see figure 1 of the 2020 JCEM review by Gregory L and Dattani M (4).

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Figure 3. Timing of gene expression of transcription factors implicated in combined hypopituitarism.

Disruptions to this cascade of transcription factors causes a range of structural phenotypes, varying from a normal pituitary appearance, to anterior pituitary hypoplasia or agenesis. The infundibulum/pituitary stalk may be thin or absent and the posterior pituitary may be hypoplastic, absent, or ectopic. The combination of anterior pituitary hypoplasia (APH), Infundibular thinning/absence, and ectopic posterior pituitary (EPP), is known as pituitary stalk interruption syndrome (PSIS) (Figure 4).

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Figure 4. Lateral appearance of structural pituitary phenotypes. Adapted from Larkin et al, Endotext (1).


Septo-optic dysplasia (SOD) is defined by the presence of two or more of: optic nerve hypoplasia, hypopituitarism, and midline defects (agenesis of the corpus callosum or septum pellucidum) forming a clinical triad. SOD is a rare condition with incidence estimates between 1 in 9,000 to 1 in 40,000 (17,18). The association between hypopituitarism and optic nerve hypoplasia relates to the embryological origin of the optic chiasm being immediately anterior to the infundibular tissue, thus an insult at this location can interrupt both pathways. The phenotype is highly variable even in familial SOD (13). Although several genetic causes have been identified, these make up less than 10% of cases of SOD, and are summarized in Table 1 (19). Septo-optic dysplasia is commonly seen in young primiparous mothers (20) and while antenatal exposure to alcohol and drug use have been suggested, there is a lack of evidence to support causality (21).

Optic nerve hypoplasia (ONH) is typically bilateral (80-90%) with midline defects typically being septum pellucidum (85%) or corpus callosum (30%) hypoplasia or aplasia (21). 55-80% of cases have pituitary hormone deficiency, with growth hormone (GHD) and TSH deficiency (TSHD) being the most common deficiencies, followed by ACTH. Diabetes insipidus and hypogonadotrophic hypogonadism (HH) are found in less than 30% of hormone deficient cases (22-25). Approximately half of these hormone deficiencies are detected in the first 2 years of life and can present with severe neonatal hypoglycemia and cardiovascular instability, with the remainder of cases presenting across childhood to adolescence. The degree of deficiency is also noted to progress over time (22). Children with SOD typically have at least some degree of visual impairment, with 44- 80% being legally blind (22,26), while epilepsy and behavioral issues can be seen in approximately a quarter of patients, and cognitive impairment in 42.5% (22). Recently, 31% of cases were found to be obese despite therapy, suggesting the contribution of hypothalamic obesity (22).


HESX1 is a member of the paired-like class of homeobox genes, located on chromosome 3p14.3 with autosomal dominant and recessive mutations described, making up approximately 1% of septo-optic dysplasia cases (19,27).

It appears from E8.5 through to E13.5 in the ventral diencephalon and oral ectoderm (28). HESX1 regulates the structural organization of the pituitary and interacts with the infundibulum and surrounding tissues through repressing transcription. Murine studies suggest absence typically does not prevent Rathke’s pouch/anterior pituitary formation (5%) but can result in polypituitary. It is suggested HESX1 regulates the regional expression of LHX3, PROP1, FGF8 and FGF10, preventing excessive expansion of the pituitary (19). HESX1 is upregulated by OTX2 and LHX3 transcription factors while being repressed by FGF8 and PROP1 (28,29).

Dattani et al. described a sibling pair with consanguineous parents, with a homozygous (R160C) HESX1 mutation in the late 1990’s. The SOD phenotype comprised optic nerve hypoplasia, midline defects (absent corpus callosum (CC) and septum pellucidum (SP)), ectopic posterior pituitary and anterior pituitary hypoplasia with hypopituitarism (30). Subsequent reports indicate that HESX1 mutation results in the complete SOD triad in approximately 30% of cases with ophthalmic changes including anophthalmia, microphthalmia, optic nerve hypoplasia and optic nerve aplasia (28,31). The majority of HESX1 pathogenic variants manifest GHD, with variability in deficiency of the other anterior hormones and ADH deficiency being uncommon. MRI appearance varies from normal to posterior pituitary ectopia and anterior pituitary hypoplasia in affected individuals (19). It is also associated with hypoplastic nasal cavities/olfactory bulbs and underdevelopment of the forebrain (28).

Autosomal dominant forms typically cause a milder phenotype, with undescended posterior pituitary and GHD, but phenotype-genotype correlation is poor (19). Penetrance is variable with asymptomatic parents and siblings being reported and midline defects and optic nerve anomalies absent in some cases (31,32). Testing of 217 patients with Kallmann syndrome (hypogonadotrophic hypogonadism (HH) and anosmia) revealed 1.4% had a mutation of HESX1 and normal pituitary on imaging (33).


SOX2 and SOX3 are from the SOX (SRY-related high mobility group HMG Box) family of transcription factors and are both single exon genes that are expressed early in development and decline as cells differentiate. SOX 2 is located on chromosome 3q26.33 with autosomal dominant expression and cause 10-15% of microphthalmia and anophthalmia cases (19,34).

SOX2 is expressed during the morula stage, E2.5 but from gastrulation is restricted to the presumptive neuroectoderm, and E9.5 is found in the brain, CNS, sensory placodes, in the branchial arches, esophagus and trachea (28). At E11.5 it is expressed throughout Rathke’s pouch but from E18.5 is restricted to the intermediate lobe(35). In humans it is expressed within the developing anterior pituitary from weeks 3.5-9 and in the diencephalon but not in the neurohypophysis or infundibulum (19).

In 2006 Williamson et al. described heterozygous de novo mutations of SOX2 causing bilateral anophthalmia or severe microphthalmia associated with developmental delay, esophageal atresia and genital anomalies (36). Later that year, Kelberman et al. reported the association of SOX2 with HH, GHD and anterior pituitary hypoplasia (37). A cohort of 18 participants with SOX2 mutation showed 33% (6/18) had short stature or GHD, and 44% (8/18) had genital anomalies or hypogonadism (38).

Alongside hypoplastic anterior pituitary, hypothalamic hamartomas and corpus callosum abnormalities are often seen, as are hippocampus anomalies, sensorineural hearing loss, learning difficulties and cognitive impairment. The vast majority have anophthalmia in at least one eye, but milder cases have microphthalmia, coloboma and optic nerve hypoplasia, with normal ocular structures seen very rarely (34). SOX2 mutations are associated with esophageal/tracheal abnormalities and underdeveloped genitalia known as Anophthalmia-esophageal-Genital syndrome (AEG). Micropenis and cryptorchidism are commonly seen, and while genital anomalies are noted less commonly in females, they can be severe, with some cases having vaginal agenesis (28).

Anophthalmia or microphthalmia with esophageal abnormalities, sensorineural hearing loss or GHD and HH should raise the question of a SOX2 mutation for a clinician. Given the autosomal dominant inheritance, detection would be critical for family planning for both the patient and their parents.


SOX3 gene is found at Xq27 causing X-linked recessive inheritance found to cause 0.2% of all GH deficient cases (28,39). SOX3 is expressed throughout the central nervous system during embryonic development.

It is first expressed at E6.5 but is rapidly restricted to the anterior ectoderm, and to the presumptive neuroectoderm. It is highly expressed in the infundibulum and presumptive hypothalamus but not in Rathke’s pouch (28).

SOX3 supports development of the hypothalamus and infundibulum as well as the development of the AP. Absence results in thinning of the infundibulum, which is thought to reduce expression of other genes involved with supporting pituitary proliferation. As a result, Rathke’s pouch is initially expanded but eventually the anterior pituitary becomes hypoplastic. It is important in confining the expression of FGF8 and BMP4, preventing excessive activity. Interestingly, duplications of SOX3 are also associated with similar phenotypes to those with non-functional mutations (typically polyalanine expansions). This suggests that correct dose of SOX3 is required for optimal embryonic development (28).

In 2002, Laumonnier et al. described a boy with IGHD and learning difficulties associated with a SOX3 mutation (40). Subsequently, GHD has been found in affected males and is associated with developmental delay or cognitive impairment of varying severity. One family of 5 affected females with short stature and language/ hearing impairment has been described (28). Other concurrent pituitary hormone deficiencies are present with variable frequency with panhypopituitarism described in some cases (28).

MRI findings include absent corpus callosum and absent or hypoplastic infundibulum and ectopic posterior pituitary, with cognitive impairment and learning difficulties also reported. SOX3 is not associated with optic nerve hypoplasia and has not been reported in patients with the complete SOD triad. Craniofacial abnormalities were reported frequently in murine models but are rare in human populations and 1 case of spina bifida has been reported (28).

Patients with an x-linked pattern of inheritance, and isolated GHD or CPHD should raise clinical suspicion for SOX3 mutation.


Orthodentic Homeobox 2 (OTX2) is critical for anterior structure development and forebrain maintenance (28). It is located on chromosome 14q22.3 with an autosomal dominant inheritance that has been detected in 2-3% of cases of anophthalmia/microphthalmia and can be associated with hypopituitarism (29,38,41).

From E10.5 OTX2 is strongly expressed in the neurohypophysis and hypothalamus but the expression in Rathke’s pouch is minimal. Expression continues until E16.5 but is silenced by E12.5. Knockout within Rathke’s pouch caused no abnormalities in pituitary development or function (42). OTX2 deficiency results in decreased posterior lobe and pituitary stalk development and secondary anterior lobe hypoplasia. This is mediated through failure of FGF10 production, resulting in the hypoplastic anterior pituitary, but with normal cell differentiation (42). It is also important for upregulating HESX1 and POU1F1 (29).

Isolated GHD is classical for OTX2 mutations but CPHD, typically TSHD or HH and panhypopituitarism have been described. Imaging typically demonstrates ectopic or absent posterior pituitary with hypoplastic or normal anterior pituitary but may be normal (28,41). Ocular manifestations vary from Anophthalmia/microphthalmia, to optic nerve hypoplasia or retinal dystrophy, with normal ocular phenotype also described. Additional anomalies are rarely reported (41).

OTX2 abnormalities should be considered in cases of anophthalmia/microphthalmia and hypopituitarism. Posterior pituitary anomalies and absence of extracranial features may differentiate from SOX2 mutations (28,41).


Other genes known to be associated with anophthalmia/microphthalmia have been shown to causes septo-optic dysplasia and hypopituitarism. PAX6 is located on chromosome 11p13 and is inherited in autosomal dominant pattern with incomplete penetrance (43). In murine pituitary development, PAX6 is expressed in the dorsal side of Rathke’s pouch from E9-12.5 and establishes the ventral-dorsal cell boundaries (44). It has a major role in early eye development, classically causing aniridia, with optic nerve anomalies including hypoplasia or microphthalmia or anophthalmia also described (43). Over 500 cases of PAX6 mutation have been described with only four cases of hypopituitarism, including isolated ACTH deficiency in one, GH deficiency in the remainder, two of which had plausible HH. Two were noted to have a hypoplastic pituitary (45-47). Thus, PAX6 is a rare cause of hypopituitarism in patients with eye abnormalities and may present with SOD.


The RAX gene found on chromosome 18q21.32 is involved in forebrain and eye development and is inherited in an autosomal recessive nature. In humans, it is associated with anophthalmia, microphthalmia and palatal anomalies and is present in approximately 3% of anophthalmia/microphthalmia cases (38). One patient to date with a severe mutation had anterior and posterior pituitary agenesis on MRI resulting in panhypopituitarism, but other patients have not been found to have pituitary anomalies (48).


The transcription factor 7-like 1 (TCF7L1) gene on chromosome 2p11.2 is a transcription regulator gene, important for brain development through WNT/β-catenin signaling pathway. Heterozygous mutations of TCF7L1 have been identified in 2 SOD patients, 1 of whom had GHD and ACTHD and MRI demonstrating ONH, anterior pituitary hypoplasia and absent posterior pituitary (49).

Other Genes

GLI2, BMP4, TBC1D32, PROKR2, FGF8, FGFR1 mutations are also associated with SOD but are described later as this is not their typical phenotype. Genes causing SOD are summarized in Table 1.


Table 1. Genes Associated with Septo-Optic Dysplasia


Inheritance/ prevalence

Ocular Phenotype

Pituitary appearance

Pituitary deficiencies

Other Features


Chr 3p14.3

AD/AR (incomplete penetrance)

1% of SOD




ACTH + ADH less common

CC/SP changes, hypoplastic olfactory, underdeveloped forebrain


Chr 3q26.33


10-15% of AO/MO


APH, hypothalamic hamartoma


CC changes, SNHL, ID,
esophageal anomalies, genital anomalies


Chr Xq27


0.4% of IGHD


Thin infundibulum, EPP, APH, PSIS


CPHD uncommon

CC changes, ID


Chr 14q22.3


3% of AO/MO

AO/MO, ONH, retinal dystrophy




ACTH less common


Chr 11p13



Aniridia, ONH, AO/MO





ASD, ADHD, obesity, diabetes mellitus




3% of AO/MO




Palate changes


Chr 2p11.2




Normal, APH, absent posterior pituitary


CC/SP changes




1-13% of CPHD





SP/CC changes, polydactyly, midface hypoplasia, cleft palate/lip, HPE


Chr 14q22-23






Myopia, cleft palate/lip, polydactyly


Chr 6q22.31




APH, EPP or absent pituitary


Oro-facial-digital syndrome, CC agenesis


Chr 20p12.3


5-23% of HH





Chr 10q24


1% of HH





Chr 8p11.2p12


5-11% of HH




Split hand/foot malformation

AD – Autosomal Dominant, ADHD – Attention Deficit Hyperactivity Disorder, AO – Anophthalmia, APH – Anterior Pituitary Hypoplasia, AR – Autosomal Recessive, ASD – Autism Spectrum Disorder, CC– Corpus Callosum, CPHD - Combined Pituitary Hormone Deficiency, EPP – Ectopic Posterior Pituitary, HH – Hypogonadotrophic Hypogonadism, HPE – Holoprosencephaly, ID – Intellectual Disability, MO – Microphthalmia, ONH – Optic Nerve Hypoplasia, PSIS – Pituitary Stalk Interruption Syndrome, SP – Septum Pellucidum, SNHL – Sensorineural Hearing Loss, VACTERL – Vertebral defects, Anal atresia, Cardiac defects, Tracheo-esophageal fistula, Renal anomalies, and Limb abnormalities.


Septo-optic dysplasia is the most common syndromic presentation of hypopituitarism. Other genes causing hypopituitarism, which do not typically cause optic nerve hypoplasia are discussed below, although GLI2 and BMP4 may present with SOD. Syndromic causes of hypopituitarism are summarized in Table 3.


The LIM homeobox proteins LHX3 and LHX4 are expressed in early development and have a co-dependent role in supporting pituitary development. The LHX3 gene located on chromosome 9q34.3 has autosomal recessive expression and is seen in 0.5-1.2% of hypopituitarism cases (39,50,51).

LHX3 is first expressed in Rathke’s pouch at E9.5 following induction by FGF8 and by E12.5 is predominantly expressed in the dorsal aspect of the pouch. LHX3 is also expressed in the ventral hindbrain, inner ear and spinal cord, and is upregulated by LHX4 in the pituitary and SOX2 in the inner ear. LHX3 is expressed in the anterior and intermediate pituitary throughout development, persisting into adulthood and is thus thought to have a role in maintaining some of the cell types. In LHX3 null mice Rathke’s pouch is initially formed, but regresses resulting in aplasia of the anterior and intermediate lobes, which is at least in part due to failure to support HESX1 and POU1F1 production. LHX3 is critical for maturation of Rathke’s pouch into the anterior pituitary and regulates differentiation of all anterior cell lines, although corticotrophs are somewhat preserved (52).

As a result, LHX3 mutations commonly cause CPHD or panhypopituitarism, with ACTH production normal in approximately half. Children typically present with CPHD at birth but milder phenotypes of developing hypopituitarism throughout childhood are described. A variable appearance is seen on imaging, ranging from normal (10%) to aplasia or hyperplasia, with one case having a microadenoma type appearance (53). Sensorineural hearing loss is seen in up to 50% of cases as LHX3 is present in a restricted region of the inner ear in tandem with SOX2. Intellectual or learning difficulties are reported in 38% of cases (51). A short stiff neck with limited rotation is seen in approximately 70% of cases, with other vertebral anomalies such as scoliosis seen in approximately 50% of cases. Respiratory distress has occasionally been described (54,55).

LHX3 should be suspected in patients with early-onset CPHD with a stiff neck and/or hearing impairment. Neonatal respiratory distress is a rare but important association that may also point to LHX3 mutation.


The LHX4 gene is found at chromosome 1q25.2 with autosomal dominant expression causing 0.9-1.4% of hypopituitarism cases (50,56).

LHX4 is expressed in Rathke’s pouch from E9.5 like LHX3; however, by E12.5 it is restricted to the anterior lobe. It also expressed in hindbrain, cerebral cortex and spinal cord tissues. LHX4 and LHX3 work in tandem to support specialization of pituitary cells and LHX4 is important for upregulating LHX3 and POU1F1 and ensuring correct timing of this induction. Thus, LHX4 deficiency may result in an element of LHX3 deficiency and causes a hypoplastic anterior pituitary with appropriate cell differentiation but reduced numbers of hormone producing cells (2,57-59).

Like in LHX3, LHX4 also typically results in CPHD, but milder presentations, including transient isolated GHD, have been reported (56). One family of recessive lethal LHX4 has been reported with severe combined hypopituitarism and fatal respiratory distress thought to be secondary to their hypopituitarism (60). Appearances are again variable on imaging; ectopic posterior pituitary is common with the anterior pituitary ranging from normal to aplasia. Chiari 1 malformation is also associated but likely represents a minority of cases (56). Cardiac defects have been reported in 2 cases and respiratory distress in 4 cases (56,60-62). Penetrance is variable with asymptomatic effected parents being a common occurrence (56).

LHX4 mutation should be considered in patients with Chiari malformation or respiratory/cardiac defects in a patient with hypopituitarism.


The GLI2 gene is a zinc finger transcription factor found on chromosome 2q14.2 and has an autosomal dominant inheritance with incomplete penetrance (63). Whilst prevalence has been reported in as many of 13.6% of cases of hypopituitarism, other cohorts have found it to be rare (<1%) (39,63).

GLI2 is expressed in the ventral diencephalon, where it induces BMP4 and FGF8 and is present in the oral ectoderm and Rathke’s pouch from E8.5, where it is important for mediating SHH (Sonic Hedgehog) response. GLI2 is important for supporting pituitary progenitor proliferation and its absence results in reduced pituitary cell numbers, particularly corticotrophs, somatotrophs and lactotrophs, but appropriate differentiation of cells in general (64).

GLI2 has been shown to mediate SHH effect and causes of holoprosencephaly (HPE) are commonly known to affect SHH. In 2003, Roessler et al. identified GLI2 gene mutations in 7 out of 390 patients meeting HPE criteria (65); however, this has since been shown to be an uncommon manifestation GLI2 (66). A study of 112 cases with GLI2 mutations found only three children with holoprosencephaly, and one of whom had an alternative causative gene detected. This study showed that of those with a truncated GLI2 mutation, 62% had pituitary abnormalities and polydactyly and only 15% did not have either anomaly. 58% of those with non-truncated variants had hypopituitarism. 13% had midface hypoplasia with 16% having cleft palate/lip (66). Unlike other causes of holoprosencephaly, GLI2 typically manifests with anterior pituitary anomalies, particularly GH and ACTH deficiency (63).

MRI findings vary, including optic nerve hypoplasia, septum pellucidum or corpus callosum hypoplasia/agenesis, anterior pituitary hypoplasia, ectopic posterior pituitary or PSIS. Thus SOD, PSIS, and rarely HPE are all plausible manifestations of GLI2 mutation (67).

The presence of anterior pituitary deficiency and polydactyly or midface abnormalities/central incisor should raise suspicion for GLI2 and trigger further investigation.


Heterozygous mutation of another zinc finger transcription factor GLI3 found on chromosome 7p14.1, results in Pallister-Hall syndrome which presents with mesoaxial polydactyly and hypothalamic hamartoma (68). In mouse studies GLI3 is not critical for pituitary or hypothalamic development or differentiation, but if combined with GLI2 mutation, the phenotype is more severe than GLI2 anomalies alone (64,69). Mutation in the amino-terminal third of the GLI3 gene results in Greig Cephalosyndactyly syndrome, associated with polydactyly, macrocephaly and hypotelorism, without hypothalamic abnormalities, with Pallister Hall being caused by mutations in the middle third of the gene (70).

In 1980 Hall and Pallister et al. described 6 cases with hypothalamic hamartoma, panhypopituitarism, postaxial polydactyly and imperforate anus, with lethality in all cases (71). Since this time, milder/non-lethal familial forms have been denoted, with the pituitary phenotype varying from normal pituitary function to isolated GHD and panhypopituitarism, thus all cases should be assessed for pituitary dysfunction (72). As with other causes of hypothalamic hamartoma, central precocious puberty can also be associated and should be monitored for (68).

Hypopituitarism Associated with Holoprosencephaly

The prosencephalon cleaves into right and left hemispheres between day 18 and 28 gestation in humans. Failure of this process results in holoprosencephaly (HPE) of varying degrees with lobar holoprosencephaly having separated 3rd ventricles with a connection within the frontal cortex, semilobarholoprosencephaly being partially separated and alobarholoprosencephaly having a continuous single anterior ventricle. (Figure 5) Facial development is affected by the same process and can cause hypotelorism or cleft palate, but also severe cases may have anophthalmia or cyclopia with a superiorly displaced proboscis. Holoprosencephaly is present in 1 in 250 embryos’ but there is a high rate of fetal demise. It is estimated to occur in 1 in 8,000-16,000 live births (73).

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Figure 5. Types of holoprosencephaly.

Other associated midline defects include underdevelopment of olfactory bulbs and corpus callosum, single midline maxillary incisor and hypoplastic pituitary or ectopic posterior pituitary. Holoprosencephaly is associated with anterior hypopituitarism in 5-10% of cases, with 70% having diabetes insipidus (DI) (74).

Many cases are caused by trisomy 13 making up 40-60% of all causes with a genetic anomaly, trisomy 18 and triploidy are also associated. Sonic hedgehog and ZIC2 mutations make up 5% of non-syndromic cases each. Importantly, structural chromosomal anomalies in almost all chromosomes have been shown to cause holoprosencephaly (75,76).

The SHH gene on chromosome 7q36 is critical for early development of the CNS, particularly the forebrain and is present in the notochord, neural tube and posterior limb buds of the gut. It is a major gene implicated in holoprosencephaly and its effect is mediated through other transcription factors, many of which have been demonstrated to cause HPE shown in Table 2 (73).

There is limited data regarding the rates of anterior pituitary dysfunction with each gene type although cases in SHH and ZIC2 have been reported (67,77).

Table 2. Genes Associated with Holoprosencephaly and Hypopituitarism



% of HPE

Other Features




Renal-urinary anomalies




Renal-urinary anomalies




Typically, severe HPE phenotype




Wide spectrum of severity




CHD, renal dysplasia, radial defects, gallbladder agenesis

CHD – Congenital Heart Disease, HPE – Holoprosencephaly. Adapted from Tekendo-Ngongang C, et al. Holoprosencephaly Overview. GeneReviews. 2020 (76).

Pituitary Stalk Interruption Syndrome

Pituitary stalk interruption syndrome (PSIS) is a triad of a thin/absent pituitary stalk, ectopic posterior pituitary and aplasia/hypoplasia of the anterior pituitary (Figure 6). SHH and other downstream genes of the SHH signaling pathway, including SIX3, TGIF, CDON and GPR161 have all been reported in 1-2 cases with PSIS and CPHD, without holoprosencephaly (76,78-82). Other causes of hypopituitarism that have been associated include POU1F1, PROP1, LHX3, LHX4, HESX1, SOX3, OTX2, PROKR2 and FOXA2. 100% of patients have been GHD in large case series with gonadotrophin, ACTH and TSH deficiencies in the majority and hyperprolactinemia in a minority (83).

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Figure 6. Lateral appearance of pituitary stalk interruption syndrome (PSIS). Adapted from Larkin S. et al, Endotext (1).


Heterozygous ROBO1 gene mutations (found on chromosome 3p12.3) has been described in 5 patients with PSIS, with a 6th case of homozygous mutation (84,85). Ocular anomalies, including hypermetropia and ptosis were also seen, but penetrance was incomplete with unaffected parents in some cases. Combined GHD and TSHD, and a case of panhypopituitarism have been described (84).


The forkhead box A2 (FOXA2) gene is found on chromosome 20p11.21 with dominant or de novo inheritance (86). FOXA2 regulates expression of GLI2 and SHH and mutations of the FOXA2 gene and 20p11 deletions have been reported to cause hypopituitarism (87-89). Imaging demonstrates anterior pituitary hypoplasia, absent or ectopic posterior pituitary and absent or thin pituitary stalk (86,90). FOXA2 is also involved in regulation of the ABCC8 and KCNJ11 genes, critical for the K-ATP channels in pancreatic beta-cells, and mutation has been shown to cause congenital hyperinsulinism (91). Other phenotypic features include craniofacial dysmorphism, choroidal coloboma and malformations of the liver, lung and heart (90,92,93).


Mutations in the EIF2S3 gene found on chromosome Xp22.11, result in the MEHMO syndrome (Mental retardation, Epileptic seizures, Hypogenitalism, Microcephaly and Obesity). EIF2S3 is expressed in the pituitary and hypothalamus during embryological development (94). Mutation typically results in GHD, and panhypopituitarism has been described (94,95). Imaging typically demonstrates corpus callosum thinning and either normal, or hypoplastic anterior pituitary (94).


Bone morphogenetic protein 4 (BMP4) gene is found on chromosome 14q22-q23 with dominant inheritance and incomplete penetrance (70). BMP4 is expressed in the diencephalon and infundibulum from E8.5, and absence results in failure of pituitary placode and Rathke’s pouch development in mouse studies (96). Mutations are frequently associated with anophthalmia/microphthalmia, cleft lip/palate and polydactyly/syndactyly. In 2018 Rodríguez-Contreras et al. reported a pathogenic BMP4 mutation in a boy with GH and TSH deficiency and myopia, but further cases have not yet been described (97).


Chromosome 15q25.1 is the locus for the aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) gene and has autosomal recessive inheritance. ARNT2 is expressed in the developing anterior and posterior pituitary and hypothalamus and mutations result in CPHD with diabetes insipidus. Pituitary imaging demonstrates absent posterior pituitary bright spot with stalk thinning and a hypoplastic anterior pituitary. It is also associated with postnatal microcephaly, hypoplastic frontal and temporal lobes, renal anomalies and vision impairment (98).


The TBC1 domain family member 32 (TBC1D32) gene is found at Chromosome 6q22.31 and results in disease with an autosomal recessive inheritance. TBC2D32 is expressed in the developing hypothalamus and Rathke’s pouch and is involved in SHH signaling and ciliary functioning. Isolated GHD or panhypopituitarism were found in the limited cases reported to date. Anterior pituitary hypoplasia or aplasia is described with ectopic or absent posterior pituitary as is partial agenesis of the corpus callosum and optic chiasm hypoplasia. Other phenotypic features include oral anomalies (cleft palate), facial dysmorphism (hypertelorism/cleft lip) and limb anomalies (polydactyly/syndactyly), consistent with oro-facial-digital syndrome (99).

Table 3. Syndromic Causes of Hypopituitarism


Inheritance/ prevalence




Pituitary appearance

Pituitary deficiencies

Other Features


Chr 9q34.3




APH, Normal, hyperplasia

ACTH in 50%

SNHL, ID, short stiff neck, scoliosis, rarely RDS








CC hypoplasia, Chiari 1 malformation, rarely CHD/RDS









ONH, SP/CC changes, polydactyly, midface hypoplasia, cleft palate/lip


Chr 7p14.1




Hypothalamic hamartoma



Postaxial polydactyly, imperforate anus. CPP


Chr 3p12.3







Hypermetropia, ptosis


Chr Xp22.11



Normal, APH



CC hypoplasia, ID, epilepsy, gonadal failure, microcephaly, obesity


Chr 14q22-23






AO/MO, myopia, cleft palate/lip, polydactyly


Chr 20p11.21

AD/De novo





Hyperinsulinism, craniofacial dysmorphism, choroidal coloboma, and liver, lung and heart malformations


Chr 15q25.1



APH, thin stalk, absent PP

Pan-hypopituitarism, DI

Microcephaly, fronto-temporal hypoplasia, renal anomalies and vision impairment.


Chr 6q22.31



APH, EPP or absent pituitary

GH, pan-hypopituitarism

Oro-facial-digital syndrome, CC agenesis

HPE Genes


- ZIC2

- SIX3







Rarely PSIS


Rarely CPHD

AO – Anophthalmia, APH – Anterior Pituitary Hypoplasia, CC – Corpus Callosum, CHD – Congenital Heart Disease, CPP – Central Precocious Puberty, EPP – Ectopic Posterior Pituitary, FTT – Failure To Thrive, HPE – Holoprosencephaly, ID – Intellectual Disability, MO – Microphthalmia, PP – Posterior Pituitary, PSIS – Pituitary Stalk Interruption Syndrome, RDS - Respiratory Distress, SOD – Septo-optic Dysplasia, SNHL – sensorineural hearing loss, SP – Septum Pellucidum, XR – X-linked Recessive



Chapters Archive - Endotext (47)

Figure 7. Cross-sectional representation of pituitary cell groups in POU1F1. Adapted from Larkin S. et al, Endotext (1).

POU1F1 was the first genetic cause of CPHD detected, initially described in mice (known as PIT-1) and 2 years later in humans. It is a member of the POU family of transcription factors, and the gene is located at 3p11.2. Approximately 65% of cases are autosomal recessive (homozygous/compound heterozygous) with 35% having an autosomal dominant form, the most common being the R271W mutation. In a review of 15 cases, the majority had affected parents, with only 3 cases of hormonally intact parents (100). POU1F1 is present in 2.8% of CPHD, with up to 21% of familial CPHD (50). There is considerable variability between countries, with one cohort of 23 CPHD patients in India detected 6 POU1F1 mutations and many other studies having low numbers of detected mutations (50,100,101).

Expression of POU1F1 is triggered by PROP1 and is seen within the pituitary from E13.5, persisting through to adulthood. It is critical for supporting the differentiation and proliferation of thyrotroph, lactotroph and somatotroph cells, and stimulates the GH1, prolactin and TSHB genes, required for hormone synthesis (102). LHX3, LHX4 and OTX2 upregulate POU1F1 but PROP 1 is essential for its function (52,57,59).

Tatsumi et al. detected the mutation in a case of “cretinism” with cognitive impairment and short stature, with GH, TSH and prolactin deficiency, reported in 1992 (103). 100% of cases in the literature have GH deficiency with 87.5% having TSH deficiency and 95.6% having prolactin deficiency (demonstrated in Figure 7). TSH deficiency is typically detected in infancy, either prior to, or simultaneously with GH and prolactin deficiency, with ~13.5% detected after GHD. 70% have anterior pituitary hypoplasia, but other midline and peripheral defects are rarely seen. Autosomal dominant forms are less likely to have an absent pituitary (63.6%) and typically have less severe GHD with 20% having an undetectable GH peak on stimulation testing, compared to 48%. Apart from hypopituitarism, there are no other phenotypic features known to be associated with POU1F1 (100).

Patients with severe short stature, GHD, prolactin deficiency and TSH deficiency only and isolated anterior pituitary findings on MRI should raise suspicion of POU1F1 mutation, especially if there is a known family history.


The prophet of POU1F1 (PROP1) is a paired like homeodomain transcription factor on chromosome 5q35 with autosomal recessive inheritance (104). It has been shown to cause 11% of all CPHD patients, with up to 50% of familial forms and approximately 7% of sporadic forms (50). This is highly variable across different populations and ethnicities with no cases found in studies in Japan, Germany, the United Kingdom, Spain or Australia and numbers in Lithuania being as high as 65% of CPHD cases and >90% of familial cases (50).

Prop1 is initially expressed in Rathke’s pouch at E10, with levels rising until E12 and decreasing to E15.5 when it ceases. It stimulates stem cell transformation to mesenchymal cells and supports cell migration and differentiation, without which cells remain static. It is essential for POU1F1 initiation and failure of this results in further issues with cell differentiation. It is involved in the stimulation and differentiation of all cell lines including gonadotrophins and to a lesser extent, corticotrophs (104).

Most cases have CPHD, and is typically progressive, with the number of diagnosed deficiencies and severity of deficiency increasing over time. Onset of GHD, TSHD and HH can occur antenatally with diagnosis at birth, but often present later, and in rare cases, some cell lines are normal. ACTH deficiency has been described from the age of 6 and typically develops across late childhood and adolescence. Imaging demonstrates a normal or hypoplastic anterior pituitary (occasionally hyperplastic) with normal posterior pituitary structures, and aside from features secondary to hypogonadism (micropenis and undescended testis) no extracranial phenotype has been described (105).

The presentation is typically milder than POU1F1 mutation and the presence of features of hypogonadism and adrenal insufficiency in a non-syndromic child with CPHD should raise suspicion for PROP1 over POU1F1.


The presence of any pituitary hormone deficiency raises the question of other hormone deficiencies. While isolated forms of pituitary deficiency occur, there is the potential for other deficiencies to develop over time as is seen in POU1F1 or LHX, and routine monitoring is required (53,100). It is also true that genes classically causing isolated hormone deficiencies, like GH1, can cause CPHD, and thus screening may be required (106,107).

Isolated Growth Hormone Deficiency

Chapters Archive - Endotext (48)

Figure 8. Cross-sectional representation of pituitary cell groups in isolated growth hormone deficiency. Adapted from Larkin S. et al, Endotext (1).

Isolated growth hormone deficiency (IGHD) is reported to have a prevalence between 1 in 4,000-10,000. Whilst most cases are sporadic, suggesting an in-utero insult to development of a specific cell-line, 3-30% have a first degree relative, supporting the possibility of genetic causes (108). Approximately 6.5% of patients have a genetic cause currently detectable (39) with rates as high as 38% in those with a family history (109). Growth Hormone 1 (GH1) and Growth Hormone Releasing Hormone Receptor (GHRHR) genes are the most commonly implicated, although genes implicated in CPHD, such as HESX1, SOX3, OTX2, LHX4 and GLI2 can also present as isolated GHD, (appearance shown in figure 8). Genetic causes are classified into 4 categories, listed in table 4. Type 1A which has undetectable GH levels caused by autosomal recessive GH1 gene deletions, Type 1B caused by GH1 gene splice site mutation or other genes (such as listed above), Type 2 caused by exon skipping mutations in GH1 cause an autosomal dominant phenotype, and Type 3 is X-linked recessive, and is associated with agammaglobulinemia (108).

The GH1 gene located on 17q22-24consists of 5 exons and 4 introns and encodes for a 191 amino acid peptide with a molecular weight of 22kDa. Transcription of GH1 is triggered by cAMP release in response to GHRH binding to the GHRHR, and is down-regulated by somatostatin via inhibiting cAMP release, in response to receptor binding (108). This pathway is demonstrated in Figure 9. GH1 mutations cause 22.7% of familial isolated GHD, the majority being type 2 autosomal dominant mutations (109).

Chapters Archive - Endotext (49)

Figure 9. Cellular function of somatotroph cell. cAMP- Cyclic Adenosine Monophosphate, GH – Growth Hormone, GHRH – Growth Hormone Releasing Hormone, GHRHR – Growth Hormone Releasing Hormone Receptor.


In 1981 Phillips et al. described GH1 mutation in Swiss children with a homozygous deletion causing Type 1A GHD (110). Type 1A GHD is caused by a GH1 gene deletion or less commonly, by a frameshift or nonsense mutation and makes up <15% of GH1 mutation related disease (109). The resulting phenotype is of severe GHD, with undetectable levels and can present with hypoglycemia neonatally as well as growth failure in the first 6 months. Complicating therapy is the frequent occurrence of anti-GH antibodies requiring therapy with recombinant insulin-like growth factor 1 (IGF1) (108).


Type 1B GHD caused by GH1 mutations are typically the result of gene splice site mutations and are again autosomal recessive. They have a variable phenotype ranging for an IGHD Type 1A phenotype to mid-childhood growth failure (108).

GHRHR anomalies also cause a Type 1B phenotype. GHRHR is found on chromosome 7p14.3 and was reported in 1996 by Wajnrajch et al. (111). Patients have normal GHRH levels but undetectable GH and low IGF1 levels. The phenotype is of proportional short stature, with anterior pituitary hypoplasia (108). GHRHR mutations are responsible for 16-19% of familial IGHD but are rarely the cause of sporadic IGHD (109). No GHRH mutations causing short stature have been reported to date (108).


Type 2 GHD originates from mutations that result in loss of exon 3 from the final protein. This causes a dominant negative effect on secretion of normal GH from the other allele, through retention in the endoplasmic reticulum, impairing Golgi apparatus activity (108). Type 2 GHD has varying degrees of severity as well as of pituitary hypoplasia and represents approximately 70% of GH1 mutation related disease (109).


IGHD has been reported in 10 cases of X-linked agammaglobulinemia, known as IGHD type 3; however, an individual gene causing both associations has not been well characterized (112).

Sporadic IGHD does not typically have a genetic basis; however, familial forms have a high likelihood of detecting a mutation (38%) (107). It is important in patients with IGHD to always consider the possibility of a developing CPHD, with this occurring in 5.5% without structural anomalies, and 20.7% with structural anomalies(106,107(113)). This should be considered even in patients with GH1 mutation’s, in particular Type 2 mutations have been found to develop other deficiencies, in particular TSH and ACTH deficiency (106,107).

Table 4. Summary of Types of Isolated Growth Hormone Deficiency.

IGHD type

Genetic abnormalities


Pituitary imaging

Percentage of familial cases



GH1 gene deletions and nonsense mutations


Normal/ hypoplastic


GH levels undetectable, anti-GH antibodies, transient response to GH therapy,


GH1 gene splice site mutations


Normal/ hypoplastic


GHRHR gene mutations


Normal/ hypoplastic



GH1 gene splice site and missense mutations and intronic deletions


Normal/ hypoplastic


Variable height, CPHD may occur







AD – Autosomal Dominant, AR – Autosomal Recessive, CPHD – Combined Pituitary Hormone Deficiency, EPP – Ectopic Posterior Pituitary, GHRHR – Growth Hormone Releasing Hormone Receptor, IGHD – Isolated Growth Hormone Deficiency, XR – X-li linked Recessive. Adapted from Alatzoglou K. et al, JCEM;94:3191-3199 and Casteras A. et al, Endocrinology, Diabetes and Metabolic Case Reports (109,114).

Central Hypothyroidism

Neonatal screening studies using T4 testing determined neonatal central hypothyroidism to occur in 1 in 16000-30000 infants (115,116). Up to 40% of these cases have isolated central hypothyroidism, with neonatal rates between 1 in 40,000 to 75,000, while some cases of isolated central hypothyroidism develop after the neonatal period (117). Thyroid hormone receptors (THR) are present on both the hypothalamus and pituitary and downregulate hormone secretion. Thyrotropin releasing hormone (TRH) is released from the hypothalamus and binds the TRH receptor (TRHR) upregulating TSH production (pathway demonstrated in Figure 10). 5 different genes have been implicated but there is limited data on the frequency of genetic mutations in patient with isolated central hypothyroidism (118).

Chapters Archive - Endotext (50)

Figure 10. Cellular function of thyrotroph cell. cAMP- Cyclic Adenosine Monophosphate, TRH – Thyrotropin Releasing Hormone, TRHR – Thyrotropin Releasing Hormone Receptor, TSH – Thyroid Stimulating Hormone.


Thyroid stimulating hormone beta subunit (TSHB) gene is found on chromosome 1p13.1 and is associated with autosomal recessive inheritance (118). Hayashizaki et al. described the mutation in 1989 in a patient with severe neonatal onset hypothyroidism associated with prolonged jaundice, failure to thrive and developmental delay (119). Subsequent cases have also had severe deficiencies presenting neonatally (118).


The thyrotropin releasing hormone receptor (TRHR) gene is found on chromosome 8q23.1 and again has autosomal recessive inheritance. The phenotype is milder than for TSHB with detection ranging from neonatal period to late childhood, and other patients being asymptomatic in adulthood. This is likely due to the presence of thyroid hormone receptors on the thyrotroph cells, upregulating TSH release independently of TRH (118).


Immunoglobulin superfamily 1 (IGSF1) gene is found on chromosome Xq26.1 and is associated with X-linked recessive central hypothyroidism. Hypothyroidism is seen in all cases, but the degree of deficiency can be variable, with many presenting in the neonatal period with jaundice and failure to thrive, others are detected asymptomatically on family screening (118).

IGSF1 is expressed in Rathke’s pouch and the adult pituitary gland and mutation results in low TSHB and TRHR mRNA, thus it is proposed IGSF1 has a role in regulating expression of these genes. 60% of patients have prolactin deficiency, supporting the involvement of decreased TRHR activity. GHD has been reported in a few cases, and 1 patient has been reported to have a hypoplastic anterior pituitary and ectopic posterior pituitary (118).

Other known associations include macroorchidism in 80% with low adulthood testosterone levels but preserved fertility. Follow up shows that despite thyroxine replacement, 25% of children and 75% of adults are overweight and 65% of cases have ADHD (attention deficit hyperactivity disorder) (118).


Transducing b-like protein X-linked (TBL1X) gene is found at chromosome Xp22.2 and mutations are thought to prevent upregulation of TSHB and TRHR transcription, causing central hypothyroidism. It has a mild hypothyroid phenotype and is associated with hearing loss, and ADHD (118).

Insulin receptor substrate 4 (IRS4) is found on chromosome Xq22.3 is often described as a cause of TSHD; however, mutations to date have resulted in low T4 but normal T3 and TSH, which does not suggest true central hypothyroidism (118).

Central hypothyroidism is rare and while genetic testing may be appropriate, the prevalence of individual causative genes is unknown. If genetics are not performed, siblings of patients should have formal thyroid function tests including a T4 arranged in the newborn period.

Table 5. Genetic Causes of Isolated Central Hypothyroidism



Degree of hypothyroidism

Other pituitary deficiency

Other features






Jaundice, FTT, developmental delay









Variable (mild to severe)

Prolactin, (rarely GH)

Macroorchidism, low testosterone, obesity, ADHD






Hearing loss, ADHD

AD – Autosomal Dominant, ADHD – Attention Deficit Hyperactivity Disorder, AR – Autosomal Recessive, XR – X-linked Recessive. Adapted from Tajima T. et al, Clin Pediatr Endocrinol;28(3):69-79 (118).

Isolated ACTH Deficiency

Isolated ACTH deficiency (IAD) is an extremely rare but serious condition with mortality rates of up to 25% in severe neonatal-onset forms (120).

Corticotrophin releasing hormone (CRH) is produced by the hypothalamus in response to low cortisol levels and binds to corticotroph cells. CRH stimulates cAMP release triggering protein kinase A (PKA) which binds CRH response element-binding protein (CREB) which in turn stimulates TBX19 which stimulates production of pro-opiomelanocortin (POMC). This is converted by prohormone convertase 1/3 (PCSK1 gene) to ACTH, which stimulates glucocorticoid production in the adrenal gland (Figure 11) (121). Abnormalities in TBX19, POMC and PCSK1 genes have all been shown to cause IAD, and NFKB2 mutation causes IAD and immunodeficiency. Genetic causes are summarized in Table 6.

Chapters Archive - Endotext (51)

Figure 11. Cellular function of the corticotroph cell. ACTH – Adrenocorticotrophic Hormone, cAMP- Cyclic Adenosine Monophosphate, CRH – Corticotropin Releasing Hormone, CRHR – Corticotropin Releasing Hormone Receptor, PC 1/3 – Prohormone Convertase 1/3, POMC – Pro-opiomelanocortin, TBX19 – T-Box Transcription Factor 19.


T-box transcription factor 19 (TBX19) previously known as TPIT is located on chromosome 1q24.2 and causes severe neonatal onset IAD. It has an autosomal recessive inheritance and has been detected in 65% of cases of severe IAD, with 100% penetrance reported to date (120). As reported above, TBX19 is an important part of the pathway from CRH stimulation to ACTH release but is also critical for terminal differentiation of corticotrophs and can result in absence or severely hypoplastic cells (120).

All patients present with severe neonatal hypoglycemia and >50% have hypoglycemic seizures, with a mortality rate of 25%. >60% have prolonged cholestatic/ conjugated jaundice and if diagnosis is delayed, intellectual impairment and developmental delay have been reported (120). Other abnormalities have occasionally been reported including Chiari type 1 malformation, and subtle dysmorphic features (120,122).

Cortisol assessment should be performed in any neonate with recurrent hypoglycemia and cholestatic jaundice, and consideration of TBX19 gene assessment on confirmation of the diagnosis. Low levels of maternal estriol on antenatal testing has been shown to herald adrenal steroidogenesis issues and seen in a case of IAD caused by TBX19, but estriol is no longer routinely used in antenatal screening (123).


Proprotein convertase of Subtisilin/Kexin 1 gene (PCSK1) found on chromosome 5q15-21, codes for prohormone convertase 1/3 (PC 1/3) and has an autosomal recessive pattern of inheritance. It is found throughout the body including the pituitary, hypothalamus and endocrine pancreas. It plays a critical role in processing POMC, proinsulin and proglucagon but multiple other hormones as well (124).

PC 1/3 is required for conversion of POMC to ACTH in corticotroph cells. Deficiency thus results in low levels or absence of ACTH production. Despite this, there is typically a mild clinical phenotype, only 75% of patients have ACTH deficiency and not all are associated with hypocortisolemia. It is unclear whether this is due to partial ACTH binding capacity of POMC or other ACTH precursors, or whether an alternative enzyme compensates (124).

PC1/3 deficiency can also cause CPHD. HH, TSHD, and GHD are seen in approximately 50%, 70% and 35% of cases respectively and have been demonstrated or proposed to be secondary to failure of hypothalamic conversion of pro-releasing hormones (proGnRH to GnRH (suspected), proTRH to TRH, proGHRH to GHRH). Like with the corresponding adrenal insufficiency, thyroid and GH deficiency are often phenotypically mild. DI, presumed due to ineffective provasopressin conversion to vasopressin is also reported in a subset of patients (124).

Other associated features include obesity, occurring in nearly all patients, associated with hyperphagia and develops from approximately 2 years of age, the cause of which is multifactorial. Like in Prader-Willi syndrome, initially children present with failure to thrive; however, this is likely due to the presence of malabsorptive diarrhea. All cases to date have had severe neonatal diarrhea with demonstrated fat, amino acid and monosaccharide malabsorption, with the majority requiring hospitalization and parenteral nutrition. The underlying mechanism is not understood, and the intestinal architecture is typically normal on biopsy. The malabsorptive state typically displays partial improvement after the first year of life (124).

PC1/3 is also critical for cleavage of proinsulin to insulin, resulting in undetectable insulin levels. To compensate, much higher quantities of proinsulin are produced which typically prevents diabetes mellitus as it is maintains a 2-5% activity at the insulin receptor. The most common symptom is in fact postprandial hypoglycemia, resulting from proinsulins four to six times longer half-life, causing a relative hyperinsulinism after meals. Later in life some patients proceed to diabetes mellitus likely due to b-cell exhaustion (124).

PCSK1 mutation should be considered in patients with early onset malabsorptive diarrhea and later onset obesity. Other features may include post-prandial hypoglycemia, gonadal abnormalities and reduced linear growth. CPHD and DI should be screened for but are typically mild (124).


Pro-opiomelanocortin gene (POMC) is found on chromosome 2p23.3 and deficiency manifests in an autosomal recessive inheritance pattern (125). It causes a classic triad of adrenal insufficiency, hypopigmented skin with red hair, and obesity associated with hyperphagia, that can commence within infancy (125,126).

Children typically present with neonatal hypoglycemia (~70%) and adrenal insufficiency is detected at this point. Convulsions, hyperbilirubinemia and hyponatremia were present in over 30% of presenting cases. All known cases to date have been reported to have adrenal insufficiency and obesity, with 75% having red or Reddish-brown hair (125).

Other endocrinopathies have been reported, TSHD being the most common. Melanocyte stimulating hormones (MSH) bind the melanocortin 4 receptor which has been shown to regulate TRH release and it is hypothesized that the limited production of MSH’s drives the hypothyroidism. TSHD is reported in 35% of cases to date and is often subclinical. GHD and HH have been reported in 10-20% of patients with mechanisms not well understood. Type 1 diabetes has also been seen in 10-20% of cases (double antibody positive) it is thought this may be secondary to the increased inflammatory state in the absence of melanocortin which have been demonstrated to have an anti-inflammatory effect (125).

DAVID Syndrome

DAVID syndrome (Deficient Anterior pituitary with Variable Immune Deficiency) was first described in 2011 and resultsfrom a mutation of NFKB2 gene on chromosome10q24.32. Classically it presents with common variable immunodeficiency (CVID) due to hypogammaglobulinemia, typically resulting in recurrent sinopulmonary infections. IAD typically develops years or decades later; however, initial presentation with IAD has been reported in 1 case (127). GHD has been reported in 2 cases and TSHD in a single case, and imaging may demonstrate a normal or hypoplastic anterior pituitary (127,128).

Neonatal onset isolated adrenal deficiency should be screened for TBX19 mutations, and if absent, especially in a child with red hair, POMC gene testing should be considered, similarly the possibility of PCSK1 mutation in children with malabsorptive diarrhea and adrenal insufficiency.

Table 6. Summary of Genetic Causes of Isolated ACTH Deficiency (IAD).



Onset of IAD


Other pituitary deficiencies

Other phenotype

Chr 1q24.2

AR, 100% penetrance

65% of severe neonatal onset IAD

Neonatal hypoglycemia, cholestatic jaundice


Intellectual impairment if delayed diagnosis, Chiari type 1 malformation

Chr 5q15-21



Infantile malabsorptive diarrhea,

FSH, LH, GH, TSH, Rarely ADH

Obesity in early childhood


Chr 2p23.3



Neonatal hypoglycemia, jaundice

TSH (35%), GH, LH/FSH (10%)

Obesity, reddish brown hair. Type 1 diabetes


Chr 10q24.32



Recurrent sinopulmonary infections

GH, TSH (rare)

Combined variable immunodeficiency

AD -- Autosomal Dominant, AR – Autosomal Recessive.

Central Hypogonadism

Congenital hypogonadotrophic hypogonadism (HH), also known as Kallmann syndrome when seen in association with anosmia, occurs in 1 in 10,000 – 50,000 people, with a 4 to 1 male predominance (12,129).

Gonadotrophin releasing hormone (GnRH) is secreted in a pulsatile manner from the hypothalamus and has a complex pattern of regulation by estradiol and other factors. It binds GnRH receptors on gonadotroph cells leading to secretion of LH and FSH. In fetal development, the cells responsible for GnRH release migrate to the hypothalamus from the olfactory placode, and thus mutations triggering HH are often associated with olfactory dysgenesis and anosmia (12).

There are over 25 gene defects implicated in HH, and a mutation is found in up to 50% of cases. No gene is implicated in more than 10% of familial cases. Oligogenicity is also described in multiple genes which further complicates diagnosis. Broadly speaking, HH is a result of one of four mechanisms, defects in GnRH fate specification, GnRH neuron migration, abnormal neuroendocrine secretion/homeostasis or gonadotroph cell defects (130).

55% of cases have anosmia/hyposmia (129). The majority of anosmic cases have an autosomal dominant inheritance, except for FEZF1, PROK2 and PROKR2, while norosmic cases may be autosomal dominant or recessive. However, incomplete penetrance, oligogenicity and variability of anosmia and norosmia of some genes makes delineating likely causative genes difficult. X-linked recessive inheritance is seen in KAL1 mutations only to date (12,130).

While individual genes have specific associations, larger studies demonstrate between 5 and 15% of cases have hearing loss, palate anomalies or unilateral renal agenesis. Up to 30% have synkinesia or eye movement disorders and one study found 5-15% of patients had dental agenesis, scoliosis or finger anomalies (129).

In the absence of anosmia or congenital anomalies, HH can be difficult to separate from constitutional delay (normal late onset puberty). Adolescents require pubertal induction to support bone, metabolic and sexual health and to limit the effect on mental health, and input from fertility specialists maybe required when starting a family (129).

Multiple genes associated with HH have been implicated in combined hypopituitarism including PROKR2, FGF8, FGFR1 and CHD7 and summarized in Table 7.

CHARGE Syndrome (CHD7)

CHARGE syndrome results from mutation of the CHD7 gene on chromosome 8q12.2 with autosomal dominant inheritance, although typically resulting from de novo mutation and occurs in 1 in 15,000 to 17,000 live births (131). The acronym CHARGE is of Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital hypoplasia and Ear and hearing abnormalities, and was coined by Pagon et al. in 1981 (132).

HH, typically presenting with cryptorchidism and micropenis in males is present in 60-88% of patients, most of whom will not enter puberty spontaneously (131,133). Growth failure is present in 60-72% of cases, with approximately 10% being due to GHD (131,134-136). Other pituitary abnormalities are rare with one study reporting 2 out of 25 patients with TSHD, and while other studies report hypothyroidism, these are typically primary, or the etiology is not reported. Imaging typically demonstrates olfactory bulb anomalies, but the pituitary gland is typically normal (134,136). Thus, while hypogonadism is a well-known consequence of CHARGE syndrome, GHD and TSHD must also be considered.


Prokineticin-2 Receptor (PROKR2) gene is found on chromosome 20p13 and typically has dominant inheritance with incomplete penetrance, but recessive mutations are also described (70). PROKR2 prevalence within HH varies greatly, ranging from 5.1-23.3% of mutations in different populations(137). PROKR2 is highly expressed in the olfactory placode and GnRH neurons in the mouse model but is not seen in the pituitary or infundibulum. It typically causes HH and individuals often harbor mutations in other HH genes, demonstrating it sometimes has an oligogenic effect (70). CPHD has been described in 20 patients, 13 of whom had SOD and 4 with pituitary stalk interruption syndrome (PSIS) (5-8). The pathogenicity of the mutations in these cases is uncertain (6,70).


The fibroblast growth factor 8 (FGF8) gene is found on chromosome 10q24 and mutations can have either autosomal dominant or recessive inheritance, making up approximately 1% of HH cases (70,137). FGF8 is first expressed at E9.25 in the diencephalon, prospective hypothalamus and infundibulum, and activates LHX3 (70,96,138,139). FGF8 plays a role in coordinating anterior pituitary development, with excess in mice resulting in a dysplastic, hyperplastic pituitary and absence results in pituitary hypoplasia. Most mutations are associated with Kallmann syndrome; however, multiple cases of CPHD have been identified as well as a case of Holoprosencephaly, and SOD with GH deficiency. VACTERL (Vertebral defects, Anal atresia, Cardiac defects, Tracheo-esophageal fistula, Renal anomalies, and Limb abnormalities) or VACTERL like phenotype has also been described (70).


The Fibroblast Growth Factor Receptor 1 (FGFR1) gene is found on chromosome 8p11.2-p12 with autosomal dominant inheritance making up 5-11.7% of mutations associated with HH (138,140). It is expressed in the olfactory placode where is ensures fate specification, proliferation and migration of GnRH neurons (140). It typically causes HH, with or without anosmia but also been reported in CPHD. 9 cases have been described, 3 associated with SOD, 3 with PSIS and 3 without (5,7,141,142). While some variants have demonstrable pathogenicity for this phenotype, it is unclear in others. Split hand/foot malformation is frequently part of the FGFR1 phenotype and the mutation is also seen in Pfeiffer syndrome, Hartsfield syndrome and Jackson-Weiss syndrome (142).

Table 7. Causes of Hypogonadotrophic Hypogonadism Associated with Combined Pituitary Hormone Deficiency.


Inheritance/ prevalence



Other pituitary deficiencies

Other Features


Chr 20p12.3


5-23% of HH




Chr 10q24


1% of HH





Chr 8p11.2-p12


5-11% of HH



Split hand/foot malformation



Chr 8q12.2

AD/de novo

6% of HH



Coloboma, CHD, choanal atresia, short stature, ID, genital hypoplasia, ear abnormalities

AD – Autosomal Dominant, AR – Autosomal Recessive, CHD – Congenital Heart Disease HPE – Holoprosencephaly, ID – Intellectual Disability, PSIS – Pituitary Stalk Interruption Syndrome, SOD – Septo-optic Dysplasia, VACTERL- Vertebral defects, Anal atresia, Cardiac defects, Tracheo-esophageal fistula, Renal anomalies, and Limb abnormalities.

Central Diabetes Insipidus

Isolated diabetes insipidus (DI) is rare, with a reported prevalence of 1 in 25000, including both nephrogenic and central forms (143). Arginine vasopressin (AVP) or antidiuretic hormone (ADH) is produced from pre-pro-AVP in the supraoptic and paraventricular hypothalamic nuclei, and their axonal projections extend into the posterior pituitary. AVP mediates its affect through the AVPR2 receptor which increases urine concentration in the collecting duct. Apart from cases related to holoprosencephaly, genetic causes are rare (144).


The AVP gene is located on chromosome 20p13 and has an autosomal dominant inheritance except for a few recessive mutations. The AVP mutant proteins are thought to accumulate in the endoplasmic reticulum resulting in cell death. As a result, it usually manifests between 1 to 6 years of age with gradual progression of deficiency but there is wide variation even within affected families. Along with polyuria and polydipsia classically seen in DI, failure to thrive is commonly seen and resolves with AVP replacement (144).

Wolfram Syndrome

The WFS1 gene, located on chromosome 4p16.1 encodes for wolframin which is an endoplasmic reticulum channel and has an autosomal recessive inheritance (144). Prevalence is estimated between 1 in 500,000 – 770,000 children with rates higher in areas of increased consanguinity (1 in 68,000 in Lebanon) (145). Wolfram syndrome causes diabetes mellitus, usually diagnosed at 6 years of age, optic nerve atrophy at around age 11 and sensorineural hearing loss typically progressing over time. DI is seen in approximately 70% of cases and is typically diagnosed in the 2nd or third decade of life (143). A case DI without other manifestations of Wolfram syndrome has been reported (146). The mechanism of DI in Wolfram syndrome is not well understood (144).


Hypopituitarism has been reported in a wide variety of syndromes as an associated feature. These syndromes are reported separately as hypopituitarism is a less common association.

Prader-Willi Syndrome

Prader Willi Syndrome (PWS) is a complex condition with major neurological and endocrine challenges. It results from a lack of the paternally imprinted genes on chromosome 15q11.2-q13 which can result from paternal gene deletion, maternal uniparental disomy, or imprinting defects (147). Children typically have hypotonia and failure to thrive in infancy, but later progress to hyperphagia and obesity. Obesity and pituitary hormone deficiencies result from hypothalamic dysfunction. Growth failure and GHD are commonly seen but CPHD can also occur, and the anterior pituitary can be hypoplastic in approximately 70% of patients (148,149).

Growth failure in Prader-Willi Syndrome occurs due to failure within the GH-IGF-1 axis, with GHD reported in 40-100% of patients and IGF-1 deficiency in nearly 100% of cases. Growth hormone replacement early in life (<1 year of age) is recommended regardless of GHD and is shown to improve lean muscle mass, motor development and cardiovascular health (150-153).

Primary gonadal failure is present in most cases, with underdeveloped gonads typical in both genders. HH may compound this in some cases (154). TSHD has been reported in up to 30% cases, but the true prevalence is unclear. High rates were reported in the population younger than 2 years of age but in older children, rates did not exceed the general population, suggesting CNS maturation may result in this early childhood phenotype being transient. It is unclear if there is an association between PWS and ACTH deficiency with reported rates ranging from 0-60%, with rates likely being 5-14%. Routine screening of 8 AM cortisol is recommended (147).

The mechanism for hypopituitarism in PWS is not well understood. The MAGEL2 gene, located within the PWS locus causes Schaaf-Yang Syndrome and presents similarly to PWS but with arthrogryposis. MAGEL2 is highly expressed in the developing hypothalamus and pituitary between 7-8 weeks gestation and mutations can result in IGHD or CPHD (155).

Genetic testing is indicated in children with a history of neonatal hypotonia and failure to thrive that progress to obesity, with or without short stature.

Axenfield-Rieger Syndrome (PITX2)

Axenfield-Rieger syndrome involves dysgenesis of the anterior segment of the eye, typically resulting in glaucoma. The cause of many cases is unknown; however, 40% are due to either the PITX2 or FOXC2 genes. PITX2 is a pituitary homeobox gene located on chromosome 4q25 with autosomal dominant inheritance and variable penetrance (156). PITX2 is expressed early in the oral ectoderm (E8.5), and later in the anterior pituitary where it induces POU1F1 amongst other factors (156,157). Some cases of PITX2 mutation present with GHD, with dental and cardiac issues also associated (156).

Johanson-Blizzard Syndrome (UBR1)

Mutation of the UBR1 gene on 15q15-21 results in Johanson-Blizzard syndrome (JBS) and is inherited autosomal recessively, occurring in 1 in 250,000 live births, with 70 patients reported in the literature (158). It is characterized by exocrine pancreatic insufficiency, nasal alae hypoplasia/aplasia and cutis aplasia of the scalp. Along with developmental delay, failure to thrive and hearing impairment, hypothyroidism, genitourinary abnormalities and hypopituitarism are sometimes reported (159).

In 2007, Hoffman et al. reported a case of a 4-year-old deceased boy with ACTH, GH and TSH deficiency, with undescended testicles and hypoplastic pituitary gland with an absent stalk (160). 3 cases of IGHD and isolated TSHD have also been reported, with microgenitalia reported in 4 patients, who have not been evaluated for HH. Other known endocrine associations include diabetes mellitus and primary hypothyroidism (158,159).

Oliver-McFarlane Syndrome (PNPLA6)

Oliver McFarlane syndrome is caused by a recessive mutation of chromosome 19p13 encoding for the PNPLA6 gene. This encodes for neuropathy target esterase (NTE) and is found in the human eye, brain and pituitary, which also results in other syndromes including Gordon-Holmes, Lawrence-Moon and Boucher–Neuhäuser syndrome, which are associated with HH and occasionally hypopituitarism (161). A 2021 systematic review found of the 31 cases in the literature, 90% had hypopituitarism, with 67% having GHD or HH and 50% having TSHD (162). Other features include chorioretinal atrophy, trichomegaly, alopecia, and spinocerebellar involvement (161,162).

Wiedemann-Steiner Syndrome (KMT2A)

Wiedemann-Steiner syndrome results from heterozygous mutation of the KMT2A gene on chromosome 11q23.3. It classically presents with intellectual disability, facial dysmorphism, hypertrichosis of the elbow and short stature. A study of 104 individuals found short stature in 57.8% (163) and of the 12 patients tested within a cohort of 33, 6 were found to have GHD. The mechanism for hypopituitarism is not well understood (164).

Kabuki Syndrome (KMT2D)

Mutations of KMT2D gene on chromosome 12q13.12 are responsible for 55-80% of cases of Kabuki syndrome and result in autosomal dominant inheritance (165). Kabuki syndrome is characterized by distinctive facial features, intellectual disability, short stature and cardiac disease. 10 cases of hypopituitarism have been reported in the literature, 6 with GHD, 2 with DI and precocious puberty, 1 with ACTH deficiency and 1 with GHD and DI. Imaging showed 2 had PSIS and 1 had an absent posterior pituitary bright spot (166).

Williams Syndrome

Deletions of the 7q11.23 region result in Williams or Williams-Beuren syndrome, which has autosomal dominant inheritance. Williams-syndrome occurs in between 1 in 7,500 and 20,000 live births and is characterized by congenital heart disease (supravalvular aortic stenosis), intellectual impairment, short stature and facial dysmorphism (167). 40% of patients have short stature, with hypercalcemia, precocious puberty and primary hypothyroidism commonly reported (168). Growth hormone deficiency is less frequently reported(168-170), and a case of combined ACTH and TSH deficiency has also been described (167). MRI imaging is typically normal, with one patient having an absent posterior-pituitary bright spot but normal ADH production (170), and another with ONH and normal pituitary function has been described (171).


PHACES syndrome is a rare constellation of signs including posterior fossa brain malformations, cervicofacial infantile Hemangiomas, Arterial anomalies, Cardiac defects, Eye anomalies and midline/ ventral defects. It is typically diagnosed following the detection of large, segmental facial hemangiomas and no gene defect has been identified. Over 300 cases have been reported to date (172). Goddard et al. reported a child with partially empty sella and GHD and TSHD in 2006 (173). Since then, 11 cases with hypopituitarism have been reported, 7 with abnormal pituitary MRI, 2 unreported, and 2 with normal pituitary appearance. The majority had GH (9/11) or TSH deficiency (7/11) with adrenal insufficiency and HH occurring in 2 out of 11 each. There is also an association with thyroid development issues and primary hypothyroidism (172). Thus, patients with PHACES syndrome should be monitored for growth and development with consideration of endocrine assessment if there are concerns. Abnormal pituitary appearance on MRI requires routine endocrine follow up (174).

Table 8. Syndromes Associated with Hypopituitarism.


Inheritance/ prevalence

Pituitary appearance

Pituitary deficiencies

Other Features

Prader Willi






Infantile FTT, obesity, hyperphagia, ID, typically primary hypogonadism, dysmorphic features

Axenfield-Rieger PITX2

Chr 4q25




Eye changes, glaucoma, dental issues, CHD

Johansson Blizzard




APH, absent stalk



Exocrine pancreas insufficiency, nasal anomalies, cutis aplasia, ID, genitourinary anomalies



Chr 19p13




Chorioretinal atrophy, trichomegaly, alopecia, spinocerebellar involvement.



Chr 11q23.3




ID, facial dysmorphism, elbow hypertrichosis, short stature



Chr 12q13.12


Normal, PSIS / absent PP



Facial dysmorphism, ID, short stature, CHD






Facial dysmorphism, ID, short stature, CHD, hypercalcemia, precocious puberty, primary hypothyroidism


(gene unknown)




Posterior fossa brain malformations, hemangiomas, arterial anomalies, CHD, eye anomalies and midline/ ventral defects.

ACTH – Adrenocorticotrophic Hormone, AD – Autosomal Dominant, APH – Anterior Pituitary Hypoplasia, CHD – Congenital Heart Disease, FTT – Failure To Thrive, ID – Intellectual Disability, PP – Posterior Pituitary, PSIS – Pituitary Stalk Interruption Syndrome.


Known genetic causes make up only a small proportion of the current hypopituitarism population, thus routine broad screening will not be appropriate at most centers and currently remains in the realms of research. Testing should be targeted to patients with consistent features, such as anophthalmia and SOX2 or a short stiff neck in LHX4. Individual genes can have a wide range of presentations and thus presence of a family history of hypopituitarism, whether similar in presentation or varied, should raise a high index of suspicion and screening should be strongly considered. Autosomal recessive causes are frequently seen, and dominant causes often have incomplete penetrance thus even relatively distant family associations should cause consideration for testing. Finally, presence of hypopituitarism in the context of a consanguineous parents should again raise suspicion of a likely genetic cause. Genetic testing typically is through next generation sequencing (NGS), although some mutations may be detectable on chromosomal microarray. Deletions on chromosomal microarray may include associated genes causing hypopituitarism. All the genes described above are summarized in table 9, in order of gene location, to assist in determining which mutations may be associated in such cases.

Septo-Optic Dysplasia

Current evidence suggests genetic causes of SOD make up <10% of cases thus routine screening will not be appropriate in most centers (19). In the absence of family history, the main indication for testing is in patients with microphthalmia or anophthalmia, where testing for SOX2 and OTX2 would be appropriate, and if negative, testing could be extended to PAX6, RAX and BMP4 (38). In patients with facial midline defects or polydactyly, GLI2 testing should also be considered (66).

Syndromic Causes

Syndromic causes should be tested for based on clinical suspicion, with family history and consanguinity again increasing the likelihood. Respiratory distress syndrome and a short stiff neck or Chiari I malformation should raise suspicion of LHX3 and LHX4 mutations respectively (53,56). Polydactyly and either midline facial changes or hypothalamic hamartoma suggest GLI2 and GLI3 mutations respectively (66,68). In cases of holoprosencephaly with anterior pituitary dysfunction, outside of GLI2, there are only case reports of other known genes causing hypopituitarism (66).


POU1F1 testing may be appropriate in patients with severe short stature, GHD, TSHD and prolactin deficiency alone, especially in Western-Indian populations where rates of up to 25% of the CPHD cohort have been reported (100). PROP1 typically has progressive hypopituitarism with late onset ACTH deficiency and routine screening in European countries, particularly Lithuania, may be appropriate (50).

Isolated Hormone Deficiencies

Unless familial, isolated GH deficiency rarely has a genetic basis thus genetic testing is not currently indicated with the exception of those with absent GH levels on stimulation testing as seen in type 1a GH1 mutations (109). The frequency of genetic causes within the TSH deficient cohort is unknown. Macroorchidism points to IGSF1 mutation but otherwise family history is the main indicator for genetic testing (118). Cases with isolated ACTH deficiency in the neonatal period should be tested for TBX19 mutations, and if absent, especially in a child with red hair, POMC gene testing should be performed (120,124). Children with malabsorptive diarrhea and adrenal insufficiency should be screened for PCSK1 mutation, and NFKB2 if there is known immunodeficiency (125,127). Hypogonadotrophic hypogonadism testing is well described and typically involves panel testing, but targeted testing based on inheritance pattern may be appropriate (12,130). Isolated diabetes insipidus is rare and testing should be based on a family history or presence of other features consistent with Wolfram syndrome, such as childhood onset optic atrophy or diabetes mellitus (144).

Syndromes Associated with Hypopituitarism

Testing for Prader-Willi, Williams, PITX2, UBR1, PNPLA6, KMT2A and KMT2D gene mutations should be considered based on their clinical features as listed in table 8, and in patients known to have these mutations, pituitary screening may be necessary, especially in the context of growth failure.

Table 9. Summary of Genes Associated with Hypopituitarism



Pituitary deficiencies


1p13.1 - TSHB



Jaundice, FTT, developmental delay

1q24.2 - TBX19



Intellectual impairment if delayed diagnosis, Chiari type 1 malformation

1q25.2 - LHX4



CC hypoplasia, Chiari 1 malformation, rarely CHD/RDS

2p11.2 - TCF7L1



SOD, CC/SP changes

2p21 - SIX3




2p23.3 - POMC


ACTH, TSH (35%), GH, LH/FSH (10%)

Obesity, reddish brown hair. Type 1 diabetes

2q14.2 - GLI2



SOD, PSIS, SP/CC changes, polydactyly, midface hypoplasia, cleft palate/lip, HPE

3p11.2 - POU1F1


GH, TSH, prolactin (typically CPHD)

3p12.3 - ROBO1



PSIS, hypermetropia, ptosis

3p14.3 - HESX1


GH, CPHD, (ACTH, ADH uncommon)

SOD, CC/SP changes, hypoplastic olfactory, underdeveloped forebrain

3q26.33 - SOX2



SOD, AO/MO, hypothalamic hamartoma, CC changes, SNHL, ID,
esophageal anomalies, genital anomalies

4p16.1 - WFS1 Wolfram



Diabetes mellitus, optic nerve atrophy, SNHL

4q25 - PITX2 Axenfield-Rieger



Eye changes, glaucoma, dental issues, CHD

5q15-21 - PCSK1



Obesity in early childhood

5q35 - PROP1



6q22.31 - TBC1D32



SOD, Oro-facial-digital syndrome, CC agenesis

7p14.1 - GLI3 Pallister-Hall



Hypothalamic hamartoma, Postaxial polydactyly, imperforate anus. CPP

7p14.3 - GHRHR



7q11.23 - Williams


GH, (TSH, ACTH uncommon)

Facial dysmorphism, ID, short stature, CHD, hypercalcemia, precocious puberty, primary hypothyroidism

7q36 - SHH



HPE, PSIS, renal-urinary anomalies

8p11.2-p12 - FGFR1


FSH/LH, (GH/CPHD uncommon)

SOD, PSIS, split hand/foot malformation

8q12.2 – CHD7 CHARGE

AD/de novo

FSH/LH, (GH/TSH uncommon)

Coloboma, CHD, choanal atresia, short stature, ID, genital hypoplasia, ear abnormalities

8q23.1 - TRHR


TSH, Prolactin

9q34.3 - LHX3


CPHD, ACTH in 50%

SNHL, ID, short stiff neck, scoliosis, rarely RDS

10q24.32 - NFKB2


ACTH, (GH, TSH uncommon)

Combined variable immunodeficiency

10q24.32 - FGF8


FSH/LH, (GH/CPHD uncommon)


11p13 - PAX6



SOD, AO/MO, ASD, ADHD, obesity, diabetes mellitus

11q23.3 - KMT2A Wiedemann-Steiner



ID, facial dysmorphism, elbow hypertrichosis, short stature

11q24.2 - CDON



HPE, PSIS, CHD, renal dysplasia, radial defects, gallbladder agenesis

12q13.12 - KMT2D Kabuki



Facial dysmorphism, ID, short stature, CHD

13q32 - ZIC2



HPE, PSIS, renal-urinary anomalies

14q22.3 - OTX2


(ACTH uncommon)


14q22-23 - BMP4



myopia, cleft palate/lip, polydactyly

15q11.2-q13 - Prader Willi



Infantile FTT, obesity, hyperphagia, ID, primary hypogonadism, dysmorphic features

15q15-21 - UBR1 Johansson Blizzard


GH, TSH, (ACTH uncommon)

Exocrine pancreas insufficiency, nasal anomalies, cutis aplasia, ID, genitourinary anomalies

15q25.1 - ARNT2



Microcephaly, fronto-temporal hypoplasia, renal anomalies and vision impairment.

17q22-24 - GH1


GH, (CPHD uncommon)

Anti-GH antibodies in some forms

18p11.3 - TGIF




AD – Autosomal Dominant, ADHD – Attention Deficit Hyperactivity Disorder, AO – Anophthalmia, AR – Autosomal Recessive, ASD – Autism Spectrum Disorder, CC– Corpus Callosum, CHD – Congenital Heart Disease, CPHD – Combined Pituitary Hormone Deficiency, CPP – Central Precocious Puberty, FTT – Failure To Thrive, HH – Hypogonadotrophic Hypogonadism, HPE – Holoprosencephaly, ID – Intellectual Disability, MO – Microphthalmia, PSIS – Pituitary Stalk Interruption Syndrome, RDS – Respiratory Distress Syndrome, SOD – Septo-optic dysplasia, SP – Septum Pellucidum, SNHL – Sensorineural Hearing Loss, VACTERL- Vertebral defects, Anal atresia, Cardiac defects, Tracheo-esophageal fistula, Renal anomalies, and Limb abnormalities, XR – X-linked Recessive.


ACTH – Adrenocorticotrophic Hormone

AD – Autosomal Dominant

ADH – Antidiuretic Hormone (also known as AVP)

ADHD – Attention Deficit Hyperactivity Disorder

APH – Anterior Pituitary Hypoplasia

αMSH – Alpha-Melanocyte Stimulating Hormone

AR – Autosomal Recessive

AVP – Arginine Vasopressin (also known as ADH)

CC – Corpus Callosum

CHD – Congenital Heart Disease

CPHD – Combined Pituitary Hormone Deficiency

CRH – Corticotrophin Releasing Hormone

CVID – Common Variable Immunodeficiency

DI – Diabetes Insipidus

EPP – Ectopic Posterior Pituitary

FSH – Follicle Stimulating Hormone

GH – Growth Hormone

GHD – Growth Hormone Deficiency

HH – Hypogonadotrophic Hypogonadism

HPE – Holoprosencephaly

IAD – Isolated ACTH Deficiency

ID – Intellectual Disability

IGF1 – Insulin-like Growth Factor 1

IGHD – Isolated Growth Hormone Deficiency

LH – Luteinizing Hormone

MPHD – Multiple Pituitary Hormone Deficiency

NGS – Next Generation Sequencing

ONH – Optic Nerve Hypoplasia

PSIS – Pituitary Stalk Interruption Syndrome

SOD – Septo-Optic Dysplasia

SP – Septum Pellucidum

THR – Thyroid Hormone Receptor

TRH – Thyrotropin Releasing Hormone

TRHR – Thyrotropin Releasing Hormone Receptor

TSH – Thyroid Stimulating Hormone

TSHD – Thyroid Stimulating Hormone Deficiency

XR – X-linked Recessive


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The pineal gland was described as the “Seat of the Soul” by Renee Descartes and it is located in the center of the brain. The main function of the pineal gland is to receive information about the state of the light-dark cycle from the environment and convey this information by the production and secretion of the hormone melatonin. Changing photoperiod is indicated by the duration of melatonin secretion and is used by photoperiodic species to time their seasonal physiology. The rhythmic production of melatonin, normally secreted only during the dark period of the day, is extensively used as a marker of the phase of the internal circadian clock. Melatonin itself is used as a therapy for certain sleep disorders related to circadian rhythm abnormalities such as delayed sleep phase syndrome, non-24h sleep wake disorder and jet lag. It might have more extensive therapeutic applications in the future, since multiple physiological roles have been attributed to melatonin. It exerts physiologic immediate effects during night or darkness and when suitably administered has prospective effects during daytime when melatonin levels are undetectable. In addition to its role in regulating seasonal physiology and influencing the circadian system and sleep patterns, melatonin is involved in cell protection, neuroprotection, and the reproductive system, among other possible functions. Pineal gland function and melatonin secretion can be impaired due to accidental and developmental conditions, such as pineal tumors, craniopharyngiomas, injuries affecting the sympathetic innervation of the pineal gland, and rare congenital disorders that alter melatonin secretion. This chapter summarizes the physiology and pathophysiology of the pineal gland and melatonin.


Pineal Anatomy and Structure

The pineal gland in humans is a small (100-150 mg), highly vascularized, and a secretory neuroendocrine organ (1). It is located in the mid-line of the brain, outside the blood-brain barrier and attached to the roof of the third ventricle by a short stalk. In humans, the pineal gland usually shows a degree of calcification with age providing a good imaging marker (for a speculative discussion of pineal calcification see reference(2)). The principal innervation is sympathetic, arising from the superior cervical ganglia (3). Arterial vascularization of the pineal gland is supplied by both the anterior and posterior circulation, being the main artery supplying the lateral pineal artery, which originates from the posterior circulation (4). In mammals, the main cell types are pinealocytes (95%) followed by scattered glial cells (astrocytic and phagocytic subtypes) (5). Pinealocytes are responsible for the synthesis and secretion of melatonin.

Main Function of the Pineal Gland

The main function of the pineal gland is to receive and convey information about the current light-dark cycle from the environment via the production and secretion of melatonin cyclically at night (dark period) (6, 7). Although in cold-blooded vertebrates (lower-vertebrate species), the pineal gland is photosensitive, this property is lost in higher vertebrates. In higher vertebrates, light is sensed by the inner retina (retinal ganglion cells) that send neural signals to the visual areas of the brain. However, a few retinal ganglion cells contain melanopsin and have an intrinsic photoreceptor capability that sends neural signals to non-image forming areas of the brain, including the pineal gland, through complex neuronal connections. The photic information from the retina is sent to the suprachiasmatic nucleus (SCN), the major rhythm-generating system or “clock” in mammals, and from there to other hypothalamic areas. When the light signal is positive, the SCN secretes gamma-amino butyric acid, responsible for the inhibition of the neurons that synapse in the paraventricular nucleus (PVN) of the hypothalamus, consequently the signal to the pineal gland is interrupted and melatonin is not synthesized. On the contrary, when there is no light (darkness), the SCN secretes glutamate, responsible for the PVN transmission of the signal along the pathway to the pineal gland. However, it is important to note that in continuous darkness the SCN continues to generate rhythmic output without light suppression since it functions as an endogenous oscillator (master pacemaker or clock). The rhythm deviates from 24h and ‘free-runs’ in the absence of the important light time cue. Light-dark cycles serve to synchronize the rhythm to 24h.

The PVN nucleus communicates with higher thoracic segments of the spinal column, conveying information to the superior cervical ganglion that transmits the final signal to the pineal gland through sympathetic postsynaptic fibers by releasing norepinephrine (NE). NE is the trigger for the pinealocytes to produce melatonin by activating the transcription of the mRNA encoding the enzyme arylalkylamine N-acetyltransferase (AA-NAT), the first molecular step for melatonin synthesis (8) (Figure 1).

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Figure 1. Melatonin synthesis in the pineal gland

Melatonin Synthesis

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized within the pinealocytes from tryptophan, mostly occurring during the dark phase of the day, when there is a major increase in the activity of serotonin-N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT), responsible for the transformation of 5-hydroxytryptamine (5HT, serotonin) to N-acetylserotonin (NAS) (Figure 1). Finally, N-acetylserotonin is converted to melatonin by acetylserotonin O-methyltransferase. The rapid decline in the synthesis with light treatment at night appears to depend on proteasomal proteolysis (9). Both AA-NAT and serotonin availability play a role limiting melatonin production. AA-NAT mRNA is expressed mainly in the pineal gland, retina, and to a lesser extent in some other brain areas, pituitary, and testis. Melatonin synthesis is also described in many other sites. AA-NAT activation is triggered by the activation of β1 and α1b adrenergic receptors by NE (9). NE is the major transmitter via β-1 adrenoceptors with potentiation by α-1 stimulation. NE levels are higher at night, approximately 180 degrees out of phase with the serotonin rhythm. Both availability of NE and serotonin are stimulatory for melatonin synthesis. Pathological, surgical or traumatic sympathetic denervation of the pineal gland or administration of β-adrenergic antagonists abolishes the rhythmic synthesis of melatonin and the light-dark control of its production.

There is evidence that melatonin can be synthesized in other sites of the body (skin, gastrointestinal tract, retina, bone marrow, placenta and others) acting in an autocrine or paracrine manner (10). Very recently it has been demonstrated that in the mouse brain melatonin is exclusively synthesized in the mitochondrial matrix. It is released to the cytoplasm, thereby activating a mitochondrial MT1signal-transduction pathway which inhibits stress-mediated cytochromecrelease and caspase activation: these are preludes to cell death and inflammation. This is a new mechanism whereby locally synthesized melatonin may protect against neurodegeneration. It is referred to as automitocrine signaling (11). Except for the pineal gland, other structures contribute little to circulating concentrations in mammals, since after pinealectomy, melatonin levels are mostly undetectable (12). Importantly several other factors, summarized in Table 1, have been related to the secretion and production of melatonin (13, 14).

Table 1. Factors Influencing Human Melatonin Secretion and Production (11),(12)


Effect(s) on melatonin




>30 lux white 460-480 nm most effective


Phase-shift/ Synchronization

Short wavelengths most effective

Sleep timing


Partly secondary to light exposure


­ standing (night)


­ phase shifts

Hard exercise


¯ synthesis



­ fluvoxamine

Metabolic effect


­ change in timing



­ may change phase



¯ alpha-1, ­ alpha-2


Variable¯ diazepam, alprazolam

GABA mechanisms







¯? Not clear

Menstrual cycle


­ amenorrhea


Possible changes ­¯ ?



Dose dependent



Delays clearance (exogenous)

Aspirin, Ibuprofen




Metabolic effect


Possible phase change, Parkinson patients

Aromatic amino-acid decarboxylase-I

Abbreviations: A: antagonist, U: uptake, I: inhibitor, MAO: monoamine oxidase, OC: oral contraceptives, 5HT: 5-hydroxytryptamine.

Control of Melatonin Synthesis: A Darkness Hormone

The rhythm of melatonin production is internally generated and controlled by interacting networks of clock genes in the bilateral SCN (15). Damage to the SCN leads to a loss of the majority of circadian rhythms. The SCN rhythm is synchronized to 24 hours mainly by the light-dark cycle acting via the retina and the retinohypothalamic projection to the SCN; the longer the night the longer the duration of secretion is, and the ocular light serves to synchronize the rhythm to 24h and to suppress secretion at the end of the dark phase, as explained above. Light exposure is the most important factor related to pineal gland function and melatonin secretion. A single daily light pulse of suitable intensity and duration in otherwise constant darkness is enough to phase shift and to synchronize the melatonin rhythm to 24h (16). The amount of light required at night to suppress melatonin secretion varies across species. In humans, intensities of 2500 lux full spectrum light (domestic light is around 100 to 500 lux) or light preferably in the blue range (460 to 480 nm) are required to completely suppress melatonin at night, but lower intensities < 200 lux can suppress secretion and shift the rhythm (17-21). Previous photic history influences the response: living in dim light increases sensitivity. Furthermore, the degree of light perception between individuals is related to the incidence of circadian desynchrony; along these lines, blind people with no conscious or unconscious light perception show free-running or abnormally synchronized melatonin and other circadian rhythms (22-24). Some blind subjects retain an intact retinohypothalamic tract and therefore a normal melatonin response despite a lack of conscious light perception (25, 26). It seems clear that an intact innervated pineal gland is necessary for the response to photoperiod change (27). Melatonin functions as a paracrine signal within the retina, it enhances retinal function in low intensity light by inducing photomechanical changes and provides a closed-loop to the pineal-retina-SCN system. All together, they are the basic structures to perceive and transduce non-visual effects of light, and to generate the melatonin rhythm by a closed-loop negative feedback of genes (Clock, “Circadian locomotor output cycles kaput” and Bmal, “Brain and muscle ARNT-like” genes), positive stimulatory elements (Per, “period and Cry, “Cryptochrome” genes), and negative elements (CCG, clock-controlled genes) of clock gene expression in the SCN (Figure 2).

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Figure 2. Diagrammatic representation of the control of production and the functions of melatonin, regarding seasonal and circadian timing mechanisms. Abbreviations: SCN: suprachiasmatic nucleus, PVN: paraventricular nucleus, SCG: superior cervical ganglion, NA: norepinephrine (noradrenalin), RHT: retino-hypothalamic-tract, CCG: clock-controlled genes. Based on an original diagram by Dr Elisabeth Maywood, MRC Laboratory of Molecular Biology, Neurobiology Division, Hills Road Cambridge, CB2 2QH, UK.

Melatonin Metabolism

Once synthesized, melatonin is released directly into the peripheral circulation (bound to albumin) and to the CSF without being stored. In humans, melatonin’s half-life in blood is around 40 minutes and it is metabolized within the liver, converted to 6-hydroxymelatonin mainly by CYP1A2 and conjugated to 6-sulfatoxymelatonin (aMT6s) for subsequent urinary excretion. The measurement of urinary aMT6s is a good marker of melatonin secretion, since it follows the same pattern with an approximate 2-hours offset (28, 29). Overall, women have slightly higher values of plasma melatonin at night than men (30). On average, the maximum levels of plasma melatonin in adults occur between 02.00 and 04.00 hours and are on average about 60 to 70 pg/mL when measured with high-specificity assays. Concentrations in saliva, like other hormones, are three times lower than in plasma. Minimum concentrations detected are below 1 pg/mL. The plasma melatonin rhythm (timing and amplitude) strongly correlates with urinary aMT6s. Although there is a large variability in amplitude of the rhythm between subjects, the normal human melatonin rhythm is reproducible from day to day intraindividually (Figure 3 and 4).

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Figure 3. Average concentrations of melatonin in human plasma (black, N=133), saliva (blue, N=28) and 6-sulphatoxymelatonin (aMT6s) in urine (red, N=88) using radioimmunoassay measurements. Diagrammatic representation of mean normal values (healthy men and women over 18 years old) from Dr. J. Arendt. Stockgrand Ltd., University of Surrey, UK.

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Figure 4. Plasma melatonin and urinary aMT6s in hourly samples to show the delay in the rhythm of urinary aMT6s compared to plasma melatonin (mean/SEM, N=14). Red lines show typical urine sample collection times over 24-48h to determine timing of the rhythm in out-patient or field studies. Redrawn from R. Naidoo, Thesis, University of Surrey, UK, 1998.

Melatonin Production During Development and Across Life

At birth, melatonin levels are almost undetectable, the only fetal source of melatonin being via the placental circulation. Melatonin levels in fetal umbilical circulation reflect the day-night difference as seen in the maternal circulation. Maternal melatonin sends a temporal circadian signal to the fetus (called “maternal photoperiodic adaptative programming”), preparing the CNS to properly deal with environmental day/night fluctuations after birth. A melatonin rhythm appears around 2 to 3 months of life (31), levels increasing exponentially until a lifetime peak on average in prepubertal children; melatonin concentrations in children are associated with Tanner stages of puberty (32). Thereafter, a steady decrease occurs reaching mean adult concentrations in late teens (33, 34). Values are stable until 35 to 40 years, followed by a decline in amplitude of melatonin rhythm and lower levels with ageing, associated with fragmented sleep-wake patterns (35). In people >90 years, melatonin levels are less than 20% of young adult concentrations (36). The decline in age-related melatonin production is attributed to different reasons; calcification of the pineal gland starting early in life and an impairment in the noradrenergic innervation to the gland or light detection capacity (ocular mydriases, cataracts) (2, 37). Interestingly, pinealectomy accelerates the aging process and several reports suggest that melatonin has anti-aging properties (38).


Melatonin Target Sites and Receptors

Melatonin’s target sites are both central and peripheral. Binding sites have been found in many areas of the brain, including the pars tuberalis and hypothalamus, but also in the cells of the immune system, gonads, kidney, and the cardiovascular system (39, 40). Melatonin binding sites in the brain might vary according to species. Melatonin exerts both non-receptor and receptor-mediated actions. Non-receptor-mediated actions are due to amphipathic properties (of both lipo- and hydrophilic structure) that allows melatonin to freely cross the cell and nuclear membranes. It can be easily detected in the nucleus of several cells in the brain and peripheral organs (41). Antioxidant properties are one example of non-receptor-mediated actions of melatonin. On the other hand, melatonin has receptor-mediated actions.

Two types of a new family of G protein coupled melatonin receptors (MT) have been cloned in mammals (42). Melatonin receptors are widely expressed, often with overlapping distributions. MT1 is primarily expressed in the pars tuberalis, the SCN together with other hypothalamic areas, pituitary, hippocampus, and adrenal glands, suggesting that circadian and reproductive effects are mediated through this receptor. MT2 is mainly expressed in the SCN, retina, pituitary and the other brain areas, and is associated with phase shifting (43). The affinity of melatonin is five-fold greater for MT1 than MT2. Melatonin administration induces (1) an acute suppression of neuronal firing in the SCN via the MT1 receptor, and (2) a phase-shifting of SCN activity through the MT2 receptor. However, agonists that exclusively act on one or another receptor have not been identified as yet, and the understanding of the role of each receptor in most of the tissues in which both receptors are present is difficult. Also, melatonin receptors are transiently expressed in neuroendocrine tissues during development, suggesting that melatonin has a role as a neuroendocrine synchronizer in developmental physiology (44).

Chronobiotic Effects of Melatonin

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Figure 5. Phase shifting of circadian rhythms. From data in Middleton B. et al., J. Sleep Res, 2002, 11, Suppl 1, p 154, Melatonin phase shifts all measured rhythms abstr. No. 309. Rajaratnam SW, et al., J Clin Endocrinol Metab 2003;88:4303-9. Rajaratnam SW, et al., J Physiol 2004; 561:339-351.

Phase shifting of circadian rhythms by melatonin (Figure 5) was first described in the 1980s (45). Melatonin has chronobiotic effects and is able to synchronize and reset biological oscillations. Melatonin acts on oscillators according to a well-defined phase-response curve (PRC). PRC is characterized by phase-advance zone (early in the evening before the beginning of the nocturnal melatonin production), phase-delay zone (in the late night/early morning hours), and a non-responsive dead. zone (when melatonin levels are high) (46). The melatonin PRC is useful for the clinical administration of melatonin as a chronobiotic agent and treatment of sleep circadian and mood disorders (47). In addition to the circadian chronobiotic effects, melatonin importantly has chronobiotic seasonal effects and acts as a circannual synchronizer, one season determined by increasing duration of the nocturnal melatonin production (in the direction of the winter solstice) and the other season defined by the reduction of nocturnal melatonin production (in the direction of the summer solstice) (8, 48). Interestingly, most of the clock genes are expressed in the pars tuberalis with a 24h rhythmicity different from their expression in the SCN (49), and these clock genes are influenced by melatonin with numerous potential seasonal effects. However, whether melatonin modulates the activity of the SCN via regulation of clock genes is unclear. Also, a central clock, independent of the SCN, can be entrained by food availability, temperature variations, forced activity and rest, drugs (melatonin itself), and timing (50).


As previously stated, melatonin can act through several mechanisms and at almost all levels of the organism. Therefore, it has multiple and diversified actions with immediate (endogenous melatonin, during the night) or prospective effects (exogenous melatonin, during the previous day).

Hypomelatoninemia is more common, and it can be due to factors that affect directly the pineal gland, innervation, melatonin synthesis as a result of congenital disease; or secondary as a consequence of environmental factors and/or medications (shift work, spinal cord cervical transection, sympathectomy, aging, neurodegenerative diseases, genetic diseases, β-blockers, calcium channel blockers, ACE inhibitors). Hypermelatoninemia is less common, and except for pharmacological effects, few conditions have been associated with high melatonin pro