38th International Symposium on Growth Hormone and Growth Factors in Endocrinology and Metabolism: Granada, Spain, April 7-8, 2006 (Hormone Research)

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HORMONE RESEARCH

38th International Symposium on Growth Hormone and Growth Factors in Endocrinology and Metabolism Granada, Spain, April 7–8, 2006

Guest Editors

Hans P.F. Koppeschaar, Utrecht Torsten Tuvemo, Uppsala Peter Trainer, Manchester Philip Zeitler, Denver, Colo.

80 figures, 33 in color, and 35 tables, 2007

Basel • Freiburg • Paris • London • New York • Bangalore • Bangkok • Singapore • Tokyo • Sydney

Disclaimer Please note that while Pfizer is the sponsor of the 38th International Symposium on Growth Hormone and Growth Factors in Endocrinology and Metabolism, the organization of the program was developed by a Scientific Board of counselors (the Board). The Board is a technical advisory body comprised of specialists who provide primary scientific oversight to the overall program. Specifically, the Board advises on matters of scientific program content. Its members are recognized authorities in the fields of endocrinology. The opinions expressed in the Symposium are the opinions of the individual presenters and may not necessarily reflect the opinions of Pfizer Inc., or any of its subsidiaries, partners or employees.

S. Karger Medical and Scientific Publishers Basel • Freiburg • Paris • London New York • Bangalore • Bangkok Singapore • Tokyo • Sydney

Disclaimer The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the journal is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

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All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center (see ‘General Information’). © Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISBN 978–3–8055–8255–1

Vol. 67, Suppl. 1, 2007

HORMONE RESEARCH

Contents KIGS Highlights

1 Foreword

43 KIGS Highlights

Plenary Lecture 1

Darendeliler, F. (Istanbul); Ferrández Longás, Á. (Zaragoza)

2 Impact of Sleep and Sleep Loss on Neuroendocrine and

Metabolic Function Van Cauter, E.; Holmbäck, U.; Knutson, K.; Leproult, R.; Miller, A.; Nedeltcheva, A.; Pannain, S.; Penev, P.; Tasali, E. (Chicago, Ill.); Spiegel, K. (Brussels) Plenary Lecture 2

45 Noonan Syndrome: Genetics and Responsiveness to

Growth Hormone Therapy Binder, G.; Wittekindt, N.; Ranke, M.B. (Tübingen) 50 Idiopathic Short Stature: Reflections on Its Definition

and Spontaneous Growth Wit, J.M. (Leiden) 58 Hormonal Treatment of Idiopathic Short Stature

10 The Genomic Approach to Growth Prediction

Reiter, E.O. (Springfield, Mass.)

Clayton, P.E.; Whatmore, A.J. (Manchester) Pediatric Workshop 1 Plenary Lecture 3

64 How Proinflammatory Cytokines May Impair Growth

16 Factors Predisposing to Osteoporosis in Childhood:

New Concepts in Diagnostics Schoenau, E.; Fricke, O. (Cologne) 23 Skeletal Health in Adulthood

Eastell, R. (Sheffield)

and Cause Muscle Wasting Thissen, J.-P. (Brussels) Pediatric Workshop 2 71 Disorders of Salt and Water Balance in Children

Maghnie, M.; Ambrosini, L.; di Iorgi, N.; Napoli, F. (Genova)

Plenary Lecture 4 28 Use of Embryonic Stem Cells for Endocrine Disorders

Trounson, A. (Clayton, Vic.)

Pediatric Workshop 3 77 ABCs of Natriuretic Peptides: Cardiac Aspects

Nir, A. (Jerusalem)

Plenary Lecture 5

81 ABCs of Natriuretic Peptides: Growth

32 Controversial Debate: Growth Hormone and Glucose

Metabolism Hardin, D.S. (Columbus, Ohio) 33 Growth Hormone and Insulin Resistance

Jørgensen, J.O.L.; Larsen, R.L.; Møller, L.; Krag, M.; Jessen, N.; Nørrelund, H.; Christiansen, J.S.; Møller, N. (Aarhus) 37 Growth Hormone Effects on Glucose Metabolism

Dunger, D. (Cambridge); Yuen, K. (Portland, Oreg.); Salgin, B. (Cambridge)

Espiner, E.A.; Prickett, T.C.; Yandle, T.G.; Barrell, G.K.; Wellby, M.; Sullivan, M.J.; Darlow, B.A. (Christchurch) Pediatric Workshop 4 91 Disorders of Sexual Differentiation

Hughes, I.A. (Cambridge) Pediatric Clinical Case Sessions 96 Pediatric Clinical Case Sessions

Dacou-Voutetakis, C. (Athens); Latronico, A.C. (São Paulo) 98 Diagnosis and Long-Term Human Growth Hormone

Treatment of a Boy with Noonan Syndrome de Lima Jorge, A.A. (São Paulo)

© 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Access to full text and tables of contents, including tentative ones for forthcoming issues: www.karger.com/hre_issues

102 Isolated Growth Hormone Deficiency due to GH1 Gene

Deletion: Central Nervous System Hypertension during Growth Hormone Treatment Antonini, S.R.; Faleiros, L.; Machado, H.R.; Carlos dos Santos, A.; de Castro, M. (São Paulo) 106 Hepatic Enzyme Abnormalities in Turner Syndrome:

A Case Report Pervanidou, P.; Xekouki, P.; Dacou-Voutetakis, C. (Athens) 109 PROP1 Gene Mutations and Pituitary Size: A Unique

Case of Two Consecutive Cycles of Enlargement and Regression Xekouki, P.; Sertedaki, A.; Livadas, S. (Athens); Argyropoulou, M.; Voutetakis, A. (Ioannina) Hot Topics in Pediatric Endocrinology 114 Hot Topics in Pediatric Endocrinology

Deal, C. (Montreal) 115 Leptin Reversal of the Metabolic Phenotype:

Evidence for the Role of Developmental Plasticity in the Development of the Metabolic Syndrome Gluckman, P.D.; Beedle, A.S. (Auckland); Hanson, M.A.; Vickers, M.H. (Southampton) 121 Gain-of-Function Mutations in the V2 Vasopressin

Receptor Rosenthal, S.M.; Gitelman, S.E.; Vargas, G.A.; Feldman, B.J. (San Francisco, Calif.) Clinical Practice in Adult Growth Hormone Deficiency: Learnings from KIMS 126 Clinical Practice in Adult Growth Hormone Deficiency:

Learnings from KIMS Fideleff, H. (Buenos Aires)

Adult Workshop 4 165 Ten Essential Points about Body Water Homeostasis

Verbalis, J.G. (Washington, D.C.) Adult Clinical Case Sessions 173 Adult Clinical Case Sessions

Casanueva, F.F. (Santiago de Compostela); Ghigo, E. (Turin) 174 Positive Metabolic Impact of Treatment

with Pegvisomant in an Acromegalic Patient Grottoli, S.; Gasco, V.; Mainolfi, A.; De Giorgio, D.; Ghigo, E. (Turin) 177 Depression following Traumatic Brain Injury

Associated with Isolated Growth Hormone Deficiency: Two Case Reports Maric, N.; Pekic, S.; Doknic, M.; Jasovic-Gasic, M.; Zivkovic, V.; Stojanovic, M.; Djurovic, B.; Popovic, V. (Belgrade) 180 Hypodipsic Hypernatremia after Hypothalamic Infarct

Marazuela, M.; López-Gallardo, G.; López-Iglesias, M.; Manzanares, R. (Madrid) Hot Topics in Adult Endocrinology 184 Hot Topics in Adult Endocrinology

Johannsson, G. (Gothenburg) 186 The Endocannabinoid System in the Physiopathology

of Metabolic Disorders Pagotto, U.; Vicennati, V.; Pasquali, R. (Bologna) 191 Klotho, an Aging-Suppressor Gene

Rosenblatt, K.P.; Kuro-o, M. (Dallas, Tex.) Abstract Winners 204 Vitamin D Stimulates Growth Hormone-Insulin-Like

Adult Workshop 1 128 T4 versus T3 and T4: Is It a Real Controversy?

Weetman, A.P. (Sheffield) 132 Recombinant Human Thyroid-Stimulating Hormone:

Use in Papillary and Follicular Thyroid Cancer Schlumberger, M.; Borget, I.; De Pouvourville, G. (Villejuif); Pacini, F. (Siena) Adult Workshop 2 143 Novel Medical Approaches to the Treatment of

Pituitary Tumors van der Lely, A.J. (Rotterdam) Adult Workshop 3

Growth Factor (GH-IGF) Gene Axis Expression and Potentiates GH Effect to Reverse the Inhibition Produced by Glucocorticoids in Human Growth Plate Chondrocytes Fernández-Cancio, M.; Andaluz, P.; Torán, N.; Esteban, C.; Carrascosa, A.; Audí, L. (Barcelona) 206 Abnormalities of Pituitary Function after Traumatic

Brain Injury in Children Niederland, T.; Makovi, H.; Gál, V.; Andréka, B. (Győr); Ábrahám, C.S. (Debrecen); Kovács, J. (Szeged) TBI Monograph 208 Hypopituitarism in Adults and Children following

Traumatic Brain Injury Casanueva, F.F. (Santiago de Compostela); Ghigo, E. (Turin); Polak, M. (Paris); Savage, M.O. (London)

149 Genetics of Hypogonadotropic Hypogonadism

Simoni, M.; Nieschlag, E. (Münster) 155 Management of Glucocorticoid Replacement in Adult

Growth Hormone Deficiency Filipsson, H.; Johannsson, G. (Gothenburg)

IV

222 Author Index 223 Subject Index

Contents

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):1 DOI: 10.1159/000097542

Published online: February 15, 2007

Foreword

Pfizer’s 38th International Symposium on Growth Hormone and Growth Factors in Endocrinology and Metabolism was held on April 7 and 8, 2006, in Granada, Spain. Endocrinologists from around the world were welcomed to this beautiful historic city by Fernando Escobar-Jiménez, Professor of Medicine-Endocrinology, University Hospital San Cecilio in Granada. This conference exemplified Pfizer Endocrine Care’s ongoing commitment to providing an educational forum for both pediatric and internist endocrinologists. The broad scope of topics and the structure of the programme were designed to provide opportunities for interaction and debate. The symposium was accredited by the European Accreditation Council for Continuing Medical Education. The first of five plenary lectures addressed the role of sleep in neuroendocrine and metabolic functions. Other lectures considered the genomic approach to growth prediction, the role of growth hormone in glucose metabolism, skeletal health, and use of embryonic stem cells for endocrine applications. Parallel workshops were conducted on issues specifically pertinent to pediatric and internal medicine endocrinology. Pediatric topics included the endocrinology of critical illness, disorders of salt and water balance in children, new advances in natriuretic peptides and disorders

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0001$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

of sexual differentiation. Internal medicine topics included thyroid replacement therapy, treatment of pituitary tumors, disorders of salt and water balance in adults, genetics of hypogonadotropic hypogonadism and management of glucocorticoid replacement. Presentations also included pediatric and adult clinical cases, highlights from KIGS (Pfizer International Growth Study Database), and the impact of KIMS (Pfizer International Metabolic Database) on clinical practice for patients with adult growth hormone deficiency. ‘Hot’ topics of current interest to the pediatric endocrine community included leptin and related strategies for preventing metabolic disease and the nephrogenic syndrome of inappropriate antidiuresis: a paradigm for activating mutations causing endocrine dysfunction. ‘Hot’ topics for internal medicine endocrinology included the relations between the cannabinoid receptor and weight loss and the involvement of the Klotho gene in the process of aging. Sincere thanks go to the outstanding symposium faculty who so willingly shared their insights and expertise. Their excellent presentations provided invaluable information for the international community of clinicians and researchers contending with the challenges of pediatric and internal medicine endocrinology on a daily basis. The Scientific Planning Committee

Plenary Lecture 1

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):2–9 DOI: 10.1159/000097543

Published online: February 15, 2007

Impact of Sleep and Sleep Loss on Neuroendocrine and Metabolic Function Eve Van Cauter a Ulf Holmbäck a Kristen Knutson a Rachel Leproult a Annette Miller a Arlet Nedeltcheva a Silvana Pannain a Plamen Penev a Esra Tasali a Karine Spiegel b a

Departments of Medicine and Health Studies, University of Chicago, Chicago, Ill., USA; b Laboratory of Physiology, Université Libre de Bruxelles, Brussels, Belgium

Key Words Ghrelin Leptin Insulin resistance Obesity Diabetes

Abstract Background: Sleep exerts important modulatory effects on neuroendocrine function and glucose regulation. During the past few decades, sleep curtailment has become a very common behavior in industrialized countries. This trend toward shorter sleep times has occurred over the same time period as the dramatic increases in the prevalence of obesity and diabetes. Aims: This article will review rapidly accumulating laboratory and epidemiologic evidence indicating that chronic partial sleep loss could play a role in the current epidemics of obesity and diabetes. Conclusions: Laboratory studies in healthy young volunteers have shown that experimental sleep restriction is associated with a dysregulation of the neuroendocrine control of appetite consistent with increased hunger and with alterations in parameters of glucose tolerance suggestive of an increased risk of diabetes. Epidemiologic findings in both children and adults are consistent with the laboratory data. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0002$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Hormones and Sleep: An Introduction

It has been known for several decades that sleep exerts profound modulatory effects on hormones and metabolism. The secretion of growth hormone (GH) and prolactin (PRL) is markedly increased during sleep, whereas the release of cortisol and thyrotropin (TSH) is inhibited. Conversely, awakenings interrupting sleep inhibit nocturnal GH and PRL secretions and are associated with increased cortisol and TSH concentrations. Modulatory effects of sleep on endocrine release are not limited to the hormones of the hypothalamic-pituitary axis. Indeed the hormonal control of carbohydrate metabolism and water and electrolyte balance is also different during total sleep deprivation as compared with normal sleep. The release of GH is particularly dependent on the occurrence and quality of sleep. In the late 1960s it was recognized that the most reproducible GH pulse occurs shortly after sleep onset [1]. In men, the sleep-onset GH pulse is generally the largest, and often the only, secretory pulse observed over the 24-hour span. In women, daytime GH pulses are more frequent, and the sleep-associated pulse, although still present, does not account for the majority of the 24-hour secretory output. As illustrated in figure 1, stimulation of GH release during sleep is evident in children as well. Sleep onset elicits a pulse in

Eve Van Cauter, MD Department of Medicine University of Chicago, 5841 S. Maryland Ave. Chicago, IL 60637 (USA) Tel. +1 773 702 0169, Fax +1 773 702 7686, E-Mail [email protected]

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Fig. 1. 24-hour profiles of plasma GH in prepubertal children, pubertal children and adults. The black bar represents the sleep period. Note that a large pulse of GH release consistently follows sleep onset, irrespective of age or gender. Data in children were obtained courtesy of Professor Zvi Zadik (Kaplan Medical Center, Rehovot, Israel).

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GH secretion whether sleep is advanced, delayed or interrupted and reinitiated. Current evidence is consistent for a combined and probably synergic role of GH-releasing hormone stimulation and decreased somatostatinergic tone in the control of GH secretion during sleep. Nocturnal levels of ghrelin, a powerful GH secretagogue, are also higher during sleep than during wake, but it is unclear whether the nighttime elevation of ghrelin levels plays a role in the control of sleep-related GH release. Hormonal events during sleep are dependent upon sleep quality. Sleep involves two states of distinct neuronal activity that are each actively generated in specific brain regions, and in the course of a normal night of sleep, brain activity oscillates between non-rapid eye movement (NREM) stages and REM stages. The periodicity of this oscillation is approximately 90 min and is normally repeated four to six times per night. During REM sleep, a cortical electroencephalogram (EEG) resembles that of active waking, with mixed high-frequency, low-amplitude waveforms; muscle tone is inhibited and bursts of REMs are present. NREM sleep is subdivided into stages I, II, III and IV, with the higher stages corresponding to deeper sleep that requires stronger stimuli for arousal.

During deep NREM sleep (stages III and IV), the EEG becomes synchronized with low frequency (in the 0.5– 4 Hz range), high-amplitude waveforms, referred to as slow waves or delta waves. Stages III and IV are therefore referred to as ‘slow-wave sleep’ (SWS). During a normal night in healthy young subjects, approximately 20% of the night is spent in SWS, 25% in REM, 50% in stages I and II NREM and only 5% awake. In adults over 60 years of age, SWS is generally reduced to only 5–10% and REM sleep to 10–15% while the proportion of time awake may occupy as much as 30% of the night. The quantification of EEG recordings by spectral analysis provides useful information regarding sleep depth or sleep intensity that is not captured by stage scoring because, in contrast to stage scoring, spectral analysis is more sensitive to the amplitude of the waveform. The EEG signal is digitalized and, after appropriate filtering, spectral power is estimated in standard frequency bands. The low-frequency waves that are apparent during SWS are reflected in an increase in spectral power in the delta range (typically 0.5–4 Hz), often referred to as slow-wave activity (SWA). Higher SWA reflects more intense, deeper NREM sleep.

Impact of Sleep and Sleep Loss

Horm Res 2007;67(suppl 1):2–9

3

Self-reported sleep duration in adolescents 8.5

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weekdays in American adolescents from 6th grade (11–12 years old) to 12th grade (17–18 years old). Data from the National Sleep Foundation ‘2006 Sleep in America Poll’.

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Several of the more robust peripheral effects of sleep occur during SWS and are dependent on the intensity of SWS, as quantified by SWA. In particular, the stimulation of GH release occurs during SWS and is proportional to SWA [1]. Pharmacological stimulation of SWA results in a dose-dependent stimulation of nocturnal GH secretion. But sleep, and particularly SWS, is associated with multiple peripheral and central effects besides stimulation of GH release, including stimulation of PRL release, inhibition of corticotropic and thyrotropic activity, decreased heart rate, decreased blood pressure, decreased sympathetic nerve activity, increased vagal tone and decreased cerebral glucose utilization. While sleep deprivation has been clearly demonstrated to be associated with profound reductions in neurobehavioral performance, the multiple peripheral effects of sleep suggest that sleep loss might be associated with deleterious health effects. Until recently, nearly all studies of the peripheral impact of sleep loss examined the effects of acute total sleep deprivation, a condition that is necessarily of short duration in humans and invariably followed by sleep recovery. Alterations evidenced during acute total sleep deprivation are readily corrected following sleep recovery and therefore the possibility that sleep loss may result in longterm adverse effects appeared unlikely. Most individuals experience a full night of sleep loss only occasionally, if ever at all. As indicated below, a much more common condition that appears to have become increasingly prevalent in both adults and children is chronic partial sleep curtailment, i.e., having too little sleep night after night. 4

Horm Res 2007;67(suppl 1):2–9

Chronic Partial Sleep Loss: An Endemic Condition of Modern Society Sleep curtailment is a behavior that seems to have developed during the past few decades and has become highly prevalent, particularly among Americans. In 1960, the American Cancer Society conducted a survey study in adults that found modal sleep duration to be 8.0 to 8.9 h [2]. In 1995, a survey conducted by the National Sleep Foundation concluded that the mean had dropped to 7 h [3]. In 2004, more than 30% of adult men and women between the ages of 30 and 64 years reported sleeping less than 6 h/night [4]. Sleep need varies between individuals and is likely to be influenced by age. Several authors have distinguished between ‘sleep need’ and ‘sleep capacity or ability’, particularly in older populations [5–7]. Sleep ‘capacity’ may be estimated as the stable total sleep time achieved after several consecutive nights of extended bedtimes. A month-long experimental extension of the bedtime period to 14 h/day has provided evidence that a ‘normal’ 8hour night does not meet the sleep capacity of healthy young adults who may carry a substantial sleep debt even in the absence of obvious efforts at sleep curtailment [8]. This study estimated ‘sleep capacity’ in young adults to be 8 h, 14 min with an SD of 51 min, suggestive of large interindividual differences. Several independent studies have consistently indicated that the average sleep capacity of young adults is between 8 and 9 h/night [9, 10]. Using a similar approach, Carskadon and Acebo [11] showed that sleep ‘need’, operationally defined as the amount of Van Cauter et al.

4 Hours in Bed 3h48' of Sleep

Leptin (ng/ml)

8 Hours in Bed 6h52' of Sleep

12 Hours in Bed 8h52' of Sleep

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30 HOMA 20 (Insulin (mU/L)* Glucose (mmol/L)/22.5) 10 0 9

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Fig. 3. 24-hour profiles of plasma leptin (upper panels) and ho-

meostatic model assessment (HOMA; lower panels) in 11 healthy lean young men under three bedtime conditions. The black bars represent the bedtime period. The shaded areas in the lower pro-

sleep obtained in a 10-hour sleep opportunity, does not change across the adolescent span (aged 10 to 17 years) and is about 9 h. In 2005 the National Sleep Foundation conducted a poll regarding sleep duration among US children and adolescents. This 25-min telephone survey of 1,602 individuals aged 11–17 years assessed sleep habits based on responses to questions addressed to the children as well as to the parent or caregiver. The sample was representative of the distribution of home telephones in the U.S. and included similar proportions of boys and girls. The margin of error was estimated at 82.5%. As shown in figure 2, modern-day US adolescents do not satisfy their sleep need, as mean self-reported sleep duration is under 9 h at all ages and decreases markedly from 11 to 18 years of age. Adolescents 16 to 18 years of age appeared to have an average sleep deficit of roughly 2 h during the week. The poll further revealed that the adolescents were well aware that they had insufficient sleep. An astounding 28% of high school students admitted falling asleep at school at least once a week. Those reporting that they had sufficient sleep (69 h) were more likely to have better grades than those with insufficient sleep.

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files represent the area under the curve for the first 90 min after the morning meal. The vertical line at each time point represents the SEM. Note the progressive increase in leptin levels and decrease in breakfast response with increasing bedtime duration.

Neuroendocrine and Metabolic Implications of Short Sleep: Clinical Studies While the concept that ‘sleep is for the brain, not for the rest of the body’ has long prevailed, recent studies of

subjects submitted to repeated curtailment of the bedtime period in the laboratory have demonstrated that chronic partial sleep loss is associated with deleterious hormonal and metabolic alterations that are consistent with an increased risk of obesity and diabetes. The first detailed laboratory study that examined the neuroendocrine and metabolic effects of recurrent partial sleep deprivation on glucose metabolism involved healthy young men who were subjected to 6 nights of 4 h in bed (‘sleep debt’) followed by 7 nights of 12 h in bed (‘sleep recovery’) [12]. The subjects ate identical carbohydrate-rich meals and were on continuous bed rest on the last 2 days of each condition. They underwent an intravenous glucose tolerance test (ivGTT) followed by a 24hour period of frequent blood sampling to assess hormonal levels [12]. A control condition with 8-hour bedtimes was performed on a separate occasion and involved similar experimental procedures. Figure 3 illustrates the highlights of the findings. The levels of leptin, a secretory product of the adipocytes that signals energy balance to the brain and promotes satiety, were clearly and markedly dependent on sleep duration (upper panels). When the subjects had only 4 h in bed for 6 nights, mean leptin levels were 19% lower, the nocturnal acrophase was 26% lower and the amplitude of the diurnal variation was 20% lower than when sleep had been extended to 12 h in bed for 7 nights [13]. These changes occurred despite similar levels of caloric intake

Impact of Sleep and Sleep Loss

Horm Res 2007;67(suppl 1):2–9

5

and physical activity with no change in body mass index (BMI) [13]. Maximal leptin levels between the state of sleep debt and the fully rested state differed on average by 1.7 ng/ml, which is somewhat larger than the decrease reported in young adults after 3 days of dietary restriction by approximately 900 kcal/d [13]. The leptin profiles observed during the 8-hour bedtime condition were intermediate. The lower panels of figure 3 illustrate the profiles of homeostatic model assessment (HOMA) levels, an index of insulin resistance directly proportional to the product of insulin ! glucose. It can be seen that the area under the HOMA curve for the breakfast meal decreased with increasing sleep duration and was more than 50% higher after 6 days of sleep restriction than when the subjects were fully rested [13, 14]. Although the HOMA has only been validated as a measure of insulin resistance under fasting conditions, these results suggest that insulin resistance may develop progressively with increasing exposure to partial sleep loss. The results of the ivGTT, analyzed using the minimal model [15], revealed a nonsignificant trend for reduced insulin sensitivity (SI) during the sleep debt condition. When the short sleep condition was compared with the fully rested condition, the rate of glucose clearance during the initial phase of the test was 40% lower; glucose effectiveness, a measure of the ability of glucose to mediate its own disposal, was 30% lower; and the acute insulin response to glucose (AIRG) was also 30% lower. The disposition index (DI) is the product of AIRG ! SI, and it is a marker of diabetes risk. Low DI values represent a higher risk of type 2 diabetes. DI values of 2,000 and above are typical of subjects with normal glucose tolerance while DI values under 1,000 have been reported in populations at high risk for type 2 diabetes, such as Hispanic women with prior gestational diabetes [16]. In the short sleep condition, the DI was 40% lower than after sleep recovery (p ! 0.001), and three of the 11 subjects had DI values under 1,000. In summary, the findings of this first ‘sleep debt study’ revealed that recurrent partial sleep loss results in a clear-cut dysregulation of a key component of appetite regulation as well as in important alterations of carbohydrate metabolism. A second study that examined the impact of sleep restriction (4 h/night for 2 nights) as compared with sleep extension (10 h/night for 2 nights) used a randomized crossover design and confirmed the findings of the ‘sleep debt’ study in a similar subject population. In both bedtime conditions, daytime levels of plasma leptin, ghrelin, glucose and insulin were measured at frequent intervals following the second night of sleep restriction or exten6

Horm Res 2007;67(suppl 1):2–9

sion [17]. Caloric intake was replaced by iv glucose infusion at a constant rate. Hunger and appetite were assessed hourly using standardized scales. Table 1 summarizes the major findings [17]. Morning glucose levels were higher, and insulin levels tended to be lower, after 2 nights with 4 h in bed as compared to 2 nights with 10 h in bed [14]. Daytime levels of leptin were decreased in the short sleep condition while ghrelin levels were higher. Ghrelin is a peptide released primarily from the stomach that increases appetite and food intake [18]. Thus, leptin and ghrelin exert opposing effects on hunger and appetite. Importantly, the change in the ghrelin-to-leptin ratio between the two conditions was strongly correlated with the change in hunger ratings, suggesting that the changes observed in these appetite hormones were partially responsible for the increase in appetite and hunger. These observed changes suggest that these subjects, if allowed food ad libitum, would have increased their food intake. Very similar findings regarding associations between leptin, ghrelin and sleep duration were obtained in a population study that involved more than 1,000 men and women [19]. This study collected sleep diaries from which average nightly sleep duration was calculated, and each subject underwent one night of sleep recording in the laboratory. In the morning following the sleep study, a single blood sample was obtained for the measurement of leptin and ghrelin. After controlling for BMI, having a usual sleep time of 5 h as compared with 8 h was associated with leptin levels 18% lower and ghrelin levels 15% higher [19]. In ongoing studies from our laboratories that involve longer periods of bedtime restriction (8 days of bedtime restriction to 5 h/night or 15 days of bedtime restriction by 1.5 h/night) than the initial sleep debt study, a consistent finding that emerges from preliminary data is a marked reduction in insulin sensitivity. Thus, more prolonged exposure to chronic partial sleep restriction appears associated with increased insulin resistance. Epidemiologic Studies of Sleep Loss and Risk of Obesity and Diabetes Between 2000 and 2006, ten publications have reported an association between short sleep and high BMI in adults based on epidemiologic data [19–28]. The various studies originated from Spain, France, Germany, Switzerland and the U.S. and involved different BMI ranges. Only two studies involved a longitudinal design [27, 28]; all others were cross-sectional. Sleep duration was obtained by self-report in all of these studies. Despite these Van Cauter et al.

Table 1. Levels of morning (9:00 to 10:00) glucose and insulin, daytime (9:00 to 21:00) leptin and appetite and afternoon and early evening (12:00-21:00) ghrelin in 12 healthy lean young men after 2 days of 4- or 10-hour bedtimes

Mean (8SEM) levels

After 2 days of 4-hour bedtimes

After 2 days of Change 10-hour bedtimes (%)

P value

Glucose, mg/dl Insulin, pM Leptin, ng/ml Ghrelin, ng/ml Ghrelin:leptin ratio Hunger, 0–10 cm Global appetite, 0–70 cm Appetite for high-carbohydrate food, 0–30 cm* Appetite for other food types (0–40 cm)*

12383 133822 2.180.4 3.380.2 2.380.4 7.280.4 47.783.4

11683 154823 2.680.5 2.680.2 1.680.3 6.080.5 39.783.0

+6% –4% –18% +28% +71% +24% +23%

<0.05 <0.12 0.04 <0.04 <0.07 <0.01 0.01

20.681.4

16.381.3

+32%

<0.02

27.182.2

23.481.8

+18%

<0.2

*Hunger and appetite were measured on visual analogue scales.

Table 2. Summary of findings from studies examining the association between sleep and obesity in children

Authors

Country

Study design

Subjects

Age, years

Results

Locard et al., 1992 [33]

France

Case-control

1,031

5

Odds ratio (OR) for obesity was 4.9 (95% CI 1.9–12.7) for <10 h/night; 2.8 (95% CI 1.2–6.3) for 10–11 h/night vs. >12 h/night.

Gupta et al., 2002 [34]

US

Cross-sectional

383

11–16

Total sleep time-adjusted OR was 0.20 (95% CI 0.11– 0.34) predicting obesity.

von Kries et al., Germany 2002 [35]

Cross-sectional

6,862

5–6

Adjusted OR for being overweight was 0.77 (95% CI 0.59–0.99) for sleep times 10.5–11 h/night; 0.54 (95% CI 0.40–0.73) for ≥11.5 h/night relative to ≤10 h/night. Adjusted OR for being obese was 0.53 (95% CI 0.35– 0.80) for 10.5–11 h/night and 0.45 (95% CI 0.28–0.75) for ≥11.5 h/night.

Sekine et al., 2002 [36]

Japan

Cross-sectional

8,274

6–7

OR for obesity relative to ≥10 h sleep/night was 3.06 (95% CI 1.72–5.36) for <8 h; 2.01 (95% CI 1.43–2.91) for 8–9 h.

Agras et al., 2004 [37]

US

Prospective

150

Sleep at 3–5; weight at 9.5

The difference in mean sleep at ages 3–5 years between those who became overweight and those who did not was 30 min, most of which was daytime sleep.

Reilly et al., 2005 [38]

UK

Prospective

8,234

Sleep at 3.2; weight at 7

OR for obesity was 1.45 (95% CI 1.10–1.89) for <10.5 h and 1.32 (95% CI 1.02–1.79) for 10.5–11.4 h relative to ≥12 h per night.

Chaput et al., 2006 [39]

Canada

Cross-sectional

422

5–10

OR for overweight/obesity was 3.45 (95% CI 2.61–4.67) for 8–10 h and 1.42 (95% CI 1.09–1.98) for 10.5–11.5 h relative to 12–13 h per night.

Knutson, 2005 [40]

US

Cross-sectional

4,486

Mean 16

OR for overweight among males was 0.90 (95% CI 0.82–1.00). Not significant among females.

Impact of Sleep and Sleep Loss

Horm Res 2007;67(suppl 1):2–9

7

limitations, the consistency of the findings is remarkable. A limited number of prospective studies have examined the association between sleep duration and the development of diabetes. Results from the US Nurses Health Study, which included only women, found an increased risk of incident symptomatic diabetes over 10 years among those reporting sleep durations of 5 h or less instead of 7–8 h, even after controlling for many covariates such as BMI, shiftwork, hypertension, exercise and depression [29]. A study conducted in Sweden followed 1,187 men and women free of diabetes at baseline for 12 years [30]. Men who reported difficulty maintaining sleep or who reported sleep duration of 5 h or less had a significantly greater risk of developing diabetes, but no significant associations between sleep and diabetes risk was observed in women [30]. Another study from Sweden suggested that the impact of sleep loss on diabetes risk may be gender-dependent as the incidence of diabetes in more than 600 women followed for 32 years beginning in 1968 was not found to be associated with sleep duration at baseline [31]. Finally, the Massachusetts Male Aging Study observed that among men without diabetes at baseline, a sleep duration of 6 h or less per night was associated with twice the risk of developing diabetes after adjustment for a large number of covariates [32]. Thus, there is some epidemiologic evidence to indicate that short sleep may increase the risk of developing type 2 diabetes, particularly in men. Table 2 lists the eight epidemiologic studies to date linking sleep duration and risk of overweight and obesity in children [33–40]. Again, the studies originated from a variety of industrialized countries. The age range of the

participants varies widely from one study to another, but the findings are quite consistent. Of particular interest are the two prospective studies showing a relationship between short sleep at baseline and the development of overweight/obesity [37, 38].

Conclusions

A rapidly growing body of evidence suggests that chronic partial sleep loss, a behavior that is specific to the human species and appears to have become more and more prevalent during the past few decades, may increase the risk of obesity and type 2 diabetes. The major neuroendocrine and metabolic alterations associated with short sleep are an upregulation of appetite, with lower leptin and higher ghrelin levels, and a peculiar disturbance of glucose regulation that involves both reduced -cell responsiveness and lower insulin sensitivity. Mechanisms underlying these adverse effects of sleep loss remain to be identified and are likely to be multifactorial. Acknowledgments We are grateful for financial support from the US National Institutes of Health (Eve Van Cauter: PO1 AG-11412, RO1 HL72694, Karine Spiegel: RO1 HL-75025), the National Sleep Foundation Pickwick Fellowship and the Johan and Henning Throne Holst Foundation (Ulf Holmbäck), the Belgian Fonds National de la Recherche Scientifique and the Belgian Fonds National de la Recherche Scientifique Médicale (Karine Spiegel), the University of Chicago Diabetes Research and Training Center (Eve Van Cauter and Silvana Pannain: DK-20595) and the University of Chicago General Clinical Research Center (MO1 RR-00055).

References 1 Van Cauter E, Plat L, Copinschi G: Interrelations between sleep and the somatotropic axis. Sleep 1998;21:553–566. 2 Kripke D, Simons R, Garfinkel L, Hammond E: Short and long sleep and sleeping pills. Is increased mortality associated? Arch Gen Psychiatry 1979;36:103–116. 3 Gallup Organization: Sleep in America. Princeton, NJ: Gallup Organization, 1995. 4 National Center for Health Statistics: QuickStats: percentage of adults who reported an average of ^6 h of sleep per 24-hour period, by sex and age group 8 United States, 1985 and 2004. MMWR. Morbidity and Mortality Weekly Report 2005.

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5 Ancoli-Israel S: Sleep problems in older adults: putting myths to bed. Geriatrics 1997;52:20–30. 6 Buysse DJ, Hubbard R, Ombao H, Houck P, Monk TH: Sleep-dependent and circadian influences on normal nocturnal sleep. Sleep 2001;24:A1. 7 Buysse DJ, Monk TH, Carrier J, Rose L, Begley A, Ombao H: Sleep ability and sleep need in aging: insights from a 90-minute day study. Sleep 2001;24:A2–A3. 8 Wehr T, Moul D, Barbato G, Giesen H, Seidel J, Barker C, Bender C: Conservation of photoperiod-responsive mechanisms in humans. Am J Physiol 1993;265:R846–R857.

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9 Andersen C, Maislin G, Van Dongen H, Rogers N, Powell J, Carlin M, Mullington J, Dinges D: Effect of chronically reduced nocturnal sleep, with and without daytime naps, on neurobehavioral performance. Sleep 2000;Abstract Supplement 2:A74–A75. 10 Wright K Jr, Hughes R, Hull J, Czeisler C: Cumulative neurobehavioral performance deficits on a 24-hour day with 8-hour of scheduled sleep. Sleep 2000;23:A21. 11 Carskadon MA, Acebo C: Regulation of sleepiness in adolescents: update, insights, and speculation. Sleep 2002;25:606–614. 12 Spiegel K, Leproult R, Van Cauter E: Impact of sleep debt on metabolic and endocrine function. Lancet 1999;354:1435–1439.

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13 Spiegel K, Leproult R, L’Hermite-Baleriaux M, Copinschi G, Penev P, Van Cauter E: Leptin levels are dependent on sleep duration: relationships with sympatho-vagal balance, carbohydrate regulation, cortisol and TSH. J Clin Endocrinol Metab 2004; 89: 5762–5771. 14 Spiegel K, Knutson K, Leproult R, Tasali E, Van Cauter E: Sleep loss: a novel risk factor for insulin resistance and type 2 diabetes. J Appl Physiol 2005; 99:2008–2019. 15 Bergman RN, Ader M, Huecking K, Van Citters G: Accurate assessment of beta-cell function: the hyperbolic correction. Diabetes 2002;51(suppl 1):S212–S220. 16 Xiang AH, Peters RK, Kjos SL, Marroquin A, Goico J, Ochoa C, Kawakubo M, Buchanan TA: Effect of pioglitazone on pancreatic beta-cell function and diabetes risk in Hispanic women with prior gestational diabetes. Diabetes 2006;55:517–522. 17 Spiegel K, Tasali E, Penev P, Van Cauter E: Sleep curtailment in healthy young men is associated with decreased leptin levels, elevated ghrelin levels and increased hunger and appetite. Ann Intern Med 2004; 141: 846–850. 18 van der Lely AJ, Tschop M, Heiman M, Ghigo E: Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–457. 19 Taheri S, Lin L, Austin D, Young T, Mignot E: Short sleep duration is associated with reduced leptin, elevated ghrelin, and increased body mass index. PLoS Med 2004;1:e62. 20 Vioque J, Torres A, Quiles J: Time spent watching television, sleep duration and obesity in adults living in Valencia, Spain. Int J Obes Relat Metab Disord 2000; 24: 1683– 1688. 21 Shigeta H, Shigeta M, Nakazawa A, Nakamura N, Yoshikawa T: Lifestyle, obesity, and insulin resistance. Diabetes Care 2001; 24: 608.

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22 Kripke DF, Garfinkel L, Wingard DL, Klauber MR, Marler MR: Mortality associated with sleep duration and insomnia. Arch Gen Psychiatry 2002;59:131–136. 23 Patel SR, Ayas NT, Malhotra MR, White DP, Schernhammer ES, Speizer FE, Stampfer MJ, Hu FB: A prospective study of sleep duration and mortality risk in women. Sleep 2004; 27: 440–444. 24 Cournot M, Ruidavets JB, Marquie JC, Esquirol Y, Baracat B, Ferrieres J: Environmental factors associated with body mass index in a population of Southern France. Eur J Cardiovasc Prev Rehabil 2004;11:291–297. 25 Vorona R, Winn M, Babineau T, Eng B, Feldman H, Ware J: Overweight and obese patients in a primary care population report less sleep than patients with a normal body mass index. Arch Intern Med 2005; 165: 25– 30. 26 Singh M, Drake CL, Roehrs T, Hudgel DW, Roth T: The association between obesity and short sleep duration: a population-based study. J Clin Sleep Med 2005;1:357–363. 27 Gangwisch JE, Malaspina D, Boden-Albala B, Heymsfield SB: Inadequate sleep as a risk factor for obesity: analyses of the NHANES I. Sleep 2005;28:1289–1296. 28 Hasler G, Buysse D, Klaghofer R, Gamma A, Ajdacic V, Eich D, Rössler W, Angst J: The association between short sleep duration and obesity in young adults: a 13-year prospective study. Sleep 2004;27:661–666. 29 Ayas NT, White DP, Al-Delaimy WK, Manson JE, Stampfer MJ, Speizer FE, Patel S, Hu FB: A prospective study of self-reported sleep duration and incident diabetes in women. Diabetes Care 2003;26:380–384. 30 Mallon L, Broman JE, Hetta J: High incidence of diabetes in men with sleep complaints or short sleep duration: a 12-year follow-up study of a middle-aged population. Diabetes Care 2005;28:2762–2767. 31 Bjorkelund C, Bondyr-Carlsson D, Lapidus L, Lissner L, Mansson J, Skoog I, Bengtsson C: Sleep disturbances in midlife unrelated to 32-year diabetes incidence: the prospective population study of women in Gothenburg. Diabetes Care 2005;28:2739–2744.

32 Yaggi HK, Araujo AB, McKinlay JB: Sleep duration as a risk factor for the development of type 2 diabetes. Diabetes Care 2006; 29: 657–661. 33 Locard E, Mamelle N, Billette A, Miginiac M, Munoz F, Rey S: Risk factors of obesity in a five year old population. Parental versus environmental factors. Int J Obes Relat Metab Disord 1992;16:721–729. 34 Gupta NK, Mueller WH, Chan W, Meininger JC: Is obesity associated with poor sleep quality in adolescents? Am J Hum Biol 2002; 14:762–768. 35 von Kries R, Toschke AM, Wurmser H, Sauerwald T, Koletzko B: Reduced risk for overweight and obesity in 5- and 6-y-old children by duration of sleep – a cross-sectional study. Int J Obes Relat Metab Disord 2002;26:710– 716. 36 Sekine M, Yamagami T, Hamanishi S, Handa K, Saito T, Nanri S, Kawaminami K, Tokui N, Yoshida K, Kagamimori S: Parental obesity, lifestyle factors and obesity in preschool children: results of the Toyama Birth Cohort Study. J Epidemiol 2002;12:33–39. 37 Agras WS, Hammer LD, McNicholas F, Kraemer HC: Risk factors for childhood overweight: a prospective study from birth to 9.5 years. J Pediatr 2004;145:20–25. 38 Reilly J, Armstrong J, Dorosty A, Emmett P, Ness A, Rogers I, Steer C, Sherriff A: Early life risk factors for obesity in childhood: cohort study. BMJ 2005;doi:10.1136/bmj.38470. 670903.E0. 39 Chaput JP, Brunet M, Tremblay A: Relationship between short sleeping hours and childhood overweight/obesity: results from the ‘Quebec en Forme’ Project. Int J Obes (Lond) 2006;30(7):1080–1085. Epub 2006 Mar 14. 40 Knutson KL: Sex differences in the association between sleep and body mass index in adolescents. J Pediatr 2005;147:830–834.

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Plenary Lecture 2

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):10–15 DOI: 10.1159/000097544

Published online: February 15, 2007

The Genomic Approach to Growth Prediction P.E. Clayton A.J. Whatmore Endocrine Science Research Group, Division of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK

Key Words Insulin-like growth factor I Growth hormone deficiency Growth hormone receptor Turner syndrome Small-for-gestational age

Abstract Background: A major objective when using recombinant human growth hormone (rhGH) in childhood is to attain the maximum sustained growth response that can be achieved in an individual child while avoiding adverse events and ensuring that biomarkers of response, such as insulin-like growth factor I (IGF-I), remain within acceptable limits. Conclusion: In practice, this is currently achieved by titrating GH dose against growth rate and serum IGF-I concentration. Copyright © 2007 S. Karger AG, Basel

Prediction Models

It is possible to estimate how children with growth hormone deficiency (GHD), Turner syndrome (TS) and those born small-for-gestational age (SGA) will respond during the first years of recombinant human (rh) GH treatment [1–3]. Such prediction models are based on anthropometric and biochemical parameters. Models based on pretreatment variables can predict up to 61% of the variability in the first-year growth response in GHD, 46%

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in TS and 52% in SGA (table 1), and lesser percentages in subsequent years. Up to 89% of first-year variability can be explained in models that also include early growth response to rhGH, insulin-like growth factor I (IGF-I) and bone marker changes [4]. An observed growth response can be expressed as a ‘studentised residual’ with regard to the expected growth response derived from the prediction model [1]. One could infer from a low studentised residual that the growth response has failed to meet expectation and that could alert the clinician to further pathology or poor compliance. These models, which were derived from large cohorts of patients reported to KIGS (Pfizer International Growth Study Database), have been validated in other populations and generally perform appropriately. As with all such models, there is a tendency to ‘overoptimism’, where the observed growth rate is higher at low predicted rates and lower at high predicted rates, when applied to an independent cohort [5]. This indicates that the prediction model may need calibration for a given population, and to some extent complicates its use. Nevertheless, the value of these models is contingent upon the clinician’s ability to individualise treatment, to make the most efficient use of GH and to present the family with a realistic expectation of treatment outcome. However, as a significant percentage of response is unexplained by these models, there is a need to explore other factors that may influence growth response.

P.E. Clayton, MD Endocrine Science Research Group, Division of Human Development The Medical School, University of Manchester Stopford Building, Oxford Road, M13 9PT Manchester (UK) Tel. +44 161 275 5593, Fax +44 161 275 5958, E-Mail [email protected]

Table 1. Prediction models for first-year growth response to rhGH

Table 2. Examples of gains in height (mean, range [cm or SDS])

based on auxological and biochemical parameters in GHD, TS and SGA [1–3]

to final adult height in various growth disorders treated with rhGH

Prediction model parameter

rhGH dose (g/kg/day)

Gain in height SDS, range

TS [6] 45 45]67.5 45]67.5]90

12, 2.8–17.8 cm 15.7, 8.1–20.4 cm 19.9, 7.2–28.7 cm

Maximum GH response* Age at treatment Ht – MPH SDS (MPH SDS for SGA)** Body weight at start of treatment GH dose Birth weight Oxandrolone Number of injections/week Total R2, %

GHD 1 (–) 2 (–) 3 (–) 4 (+) 5 (+) 6 (+) – – 58

TS

SGA

– – 2 (–) 2 (–) 5 (–) 4 (+) 3 (+) 3 (+) 1 (+) 1 (+) – – 4 (+) – 6 (+) – 46 52

The numbers represent the rank order for each parameter in the prediction model (with the direction of effect in brackets). * Maximum GH response during a stimulation test. ** Ht – MPH SDS = Height SDS at start of treatment minus midparental height SDS (i.e., a measure of height discrepancy from target height).

Chronic renal insufficiency [7] 50 16 cm; +2.6 SDS* SGA [8] 33 67 ISS [9] 43

12 cm; +1.9, +0.5 to +4 SDS 14 cm; +2.2, 0 to +6 SDS 9.2, 5.5–12.8 cm (boys)** 5.7, 2.1–9.4 cm (girls)**

* Over 8 years of rhGH treatment. ** Values are means and 95% confidence intervals.

In all conditions treated with rhGH, the range of growth responses observed over the short-, mid- and long-term can be considerable. For instance, in SGA children treated with GH at a dose between 20 and 40 g/kg/ day, the lowest first-year growth response was 3 cm/ year while the highest response was 12 cm/year, generating a 4-fold range of response [3]. Over the course of childhood, this range of sensitivity to rhGH can make an appreciable difference in height gained. Although the group mean for height gained up to final height would suggest that rhGH has been successful in non-GHD conditions such as TS, SGA and chronic renal failure (table 2) [6–9], the range of actual height gained around this mean can vary widely, with some children having no significant response. Therefore, there is a need to improve our ability to distinguish between those children who will benefit from rhGH and those who will not even after many years of treatment. As a general rule, poor responders to rhGH in the first year remain poor responders thereafter. A recent cost-effectiveness analysis of GH treatment in treated and untreated patients with idiopathic short stature (ISS) estimated that the cost for each inch (2.54 cm) of height gained was approximately USD 53,000 [10]. The amount of time spent receiving GH treatment and the need for dose increases at puberty did not have a major impact on this figure, but cost was influenced by the variability in response. It was concluded that ‘targeting treatment of

children with ISS with the greatest potential for growth appears critical for maximising cost-effectiveness of GH treatment’ [10].

Genomic Approach to Growth Prediction

Horm Res 2007;67(suppl 1):10–15

Influence of a GH Receptor Gene Polymorphism on Response to rhGH

At present, examination of genetic background is one avenue being explored in an attempt to better predict response to rhGH. At the simplest monogenic level, we recognise that a child with a GH-releasing hormone-receptor (GHRH-R) gene mutation resulting in severe GHD will respond very well to rhGH, while a child with mutations in the GH-receptor gene (congenital GH insensitivity, Laron syndrome) will not. Such genes also carry changes in their DNA sequence that do not completely abolish generation or function of the protein, but that can slightly alter their function. These changes are termed polymorphisms rather than mutations. They are not sufficient to cause a disease but do affect function enough to give rise to natural variation. Polymorphisms occur at variable frequencies in a population. One individual polymorphism may have an impact on a given characteristic, but more likely it is the combined effect of polymorphisms in a number of genes that generates a variation. In terms of response to rhGH, polymorphisms in genes that control response to GH (e.g., the GH receptor [GHR]), 11

GH signaling molecules, IGF-I and its binding proteins) could all contribute to growth outcome. One example of a single polymorphism in a growthrelated gene that has a major impact on response to rhGH is the exon 3 deletion within the GHR. Individuals may carry one or two copies of this deletion, with the incidence of the deletion varying between populations, but in general being common (50% carriage rate). In in vitro assays, cells transfected with an exon 3-deleted (d3-) GHR and a GH-responsive reporter gene respond better to rhGH than cells transfected with full-length (fl) GHR [11]. Cells transfected with both fl- and d3-receptors show an intermediate response. These findings indicate that this GHR polymorphism is functional. In the first report on the effect of this polymorphism on growth response to rhGH, children born SGA and those with ISS who carried at least one copy of the d3receptor had a first-year response to GH that was 1.7-fold greater than those homozygous for the fl GHR [11]. In linear regression models that examine factors influencing growth response in both cohorts, GHR genotype was the most significant variable in both the first and second years of treatment. This work has been followed by other reports on the effect of the d3-GHR genotype. In severe GHD [12], carriage of at least one d3-allele improves first-year growth response to GH treatment by a mean of 1.7 cm/ year and final adult height by 0.9 standard deviation score (SDS). In TS, first-year growth response was dependent on d3-allele dose: d3-homozygotes grew better than heterozygotes, who grew better than fl-GHR homozygotes [13]. Interestingly, increments in IGF-I and insulin-like growth factor binding protein 3 were not influenced by GHR genotype. Not all reports have confirmed the initial findings. Pilotta et al. [14] found that first-year response in prepubertal children with GHD was not influenced by the d3-allele. This group also genotyped the GHR for other polymorphisms in exon 6 and exon 10, and found that all patients carried at least one polymorphism and that growth response did not differ among the seven combinations of polymorphisms identified. This indicates that ethnic differences are likely regarding the extent to which a given polymorphism will affect function. Nevertheless, this one polymorphism in the GHR appears to be a significant factor in response to rhGH in many children.

12

Horm Res 2007;67(suppl 1):10–15

Pharmacogenomics and Analysis of Changes in Gene Expression with GH

Pharmacogenomics involves analysis of the effect of multiple genetic polymorphisms on drug efficacy or toxicity. Based on assessment of the presence of single nucleotide polymorphisms (Snps) in an individual’s genome, technology has been developed using a small postagestamp-sized ‘chip’ that could theoretically identify all genes that mediate the growth response to rhGH, given a sufficiently large cohort. Such an approach could also be used to identify individuals who might be more at risk of developing side effects to GH, such as insulin resistance and glucose intolerance. An alternative approach to assessing rhGH response is to examine changes in gene expression using messenger ribonucleic acid (mRNA) before and during intervention. This allows identification of genes whose expression is significantly altered by treatment. If such gene expression changes can be related to outcomes, such as growth response to rhGH, then this gene could be used for prediction. This approach requires access to an appropriate tissue that can reflect GH-induced changes in gene expression. In adult studies, this might involve analysing mRNA expression in fat or muscle before and while on treatment with rhGH. However, in paediatric studies, the most readily accessible tissue is peripheral blood mononuclear cells (PBMCs). Many factors may influence mRNA expression in PBMCs, such as interindividual variation, time of day and infection status, but studies do indicate that these cells can reflect differences induced by disease [15]. We have therefore started to explore the profile of gene expression, both in vitro and in vivo, in PBMCs in normal individuals and in those with growth disorders. We have compared gene expression in freshly harvested PBMCs taken, with ethical permission and informed consent, from a healthy adult and two prepubertal children with isolated GHD (peak GH levels during two stimulation tests !7.5 g/l and an IGF-I SD score !–1.5). InvitrogenTM gene filters were used to assess levels of mRNA expression. Embedded on these filters were 5,760 gene sequences, and significant gene expression was defined as a signal 11.6 times higher than background. There was a marked difference between the adult and the two children and between the two children in the number of genes significantly expressed (table 3). However, when comparing those genes coexpressed across the three samples relative to the GHD child with the lowest number of significantly expressed genes (patient 1), nearly all these Clayton/Whatmore

Table 3. Basal gene expression in PBMCs from an adult control

Table 6. GH-regulated genes by function in adult PBMCs

and two children with GHD measured using GF211 gene filters (InvitrogenTM)

Gene function

Increased expression

Decreased expression

Metabolic Signaling Transcription/translation Structural Other

11 9 6 3 15

1 6 3 3 5

Expression status

Adult

Patient 1

Patient 2

Genes expressed Coexpressed in adult Coexpressed in patient 2

1,722

283 276 (98%) 272 (96%)

718 694 (97%)

Table 4. Analysis of significantly regulated genes by function in a prepubertal child with GHD treated with rhGH

Gene function

Total genes changed

Increased expression

Decreased expression

Signaling Metabolic Structural Transcription Immune Other

18 17 15 14 11 14

12 14 12 7 4 7

6 3 3 7 7 7

Treatment of PBMCs from a normal adult, cultured in 10% FCS and phytohaemoglutinin, then treated with rhGH (200 ng/ ml) for 24 h resulted in the expression level of 62 genes being changed >2-fold (44 increased and 18 decreased). These genes were categorised by function.

genes were expressed in all samples. When genes were ranked by level of expression, 97 of the top 100 in patient 2 were also expressed in patient 1, and all were expressed in the adult. In one of these children, PBMCs were harvested after treatment for 3 months with rhGH (25 g/kg/day). Results showed that 89 genes were altered 11.5-fold (56 in-

creased and 33 decreased) between the two filters with an assumption that rhGH had contributed at least in part to this change (table 4). Examination of the function of these genes revealed that there was a modest bias towards signaling and metabolic genes compared with structural and transcription genes. Using an Affymetrix HG-U133A chip, which has 22,283 probe sets representing 14,500 genes, we have also shown that PBMCs, cultured in phytohaemoglutinin and fetal calf serum (FCS), from a normal statured adult significantly expressed 11,949 of these genes in the basal state (82%). After stimulation with rhGH (200 ng/ml) for 24 h, 11,712 genes (81%) were significantly expressed, with 11,310 genes (78%) expressed in both states (table 5). Only 62 of these genes were altered more than 2-fold by rhGH; the expression of 44 was increased with a maximum change of 3.4-fold and the expression of 18 was decreased by a maximum of 4.1-fold (table 6). Those genes that changed in response to rhGH treatment were mainly involved in signaling (24%) and metabolism (19%) and less in transcription/translation (15%) and in structural functions (10%), a similar pattern to that seen in the child treated with rhGH in vivo. These preliminary experiments have indicated that genes in PBMCs do change significantly in response to rhGH, both in vivo and in vitro, with a bias towards those involved in signaling and metabolism. In addition, these responses appear to be independent of the technique used for array analysis. Despite overall significant interindividual differences in gene expression, those genes with the highest basal expression levels are consistently present. Further evaluation of GH-induced gene expression in larger numbers of children with a range of growth dis-

Genomic Approach to Growth Prediction

Horm Res 2007;67(suppl 1):10–15

GH treatment resulted in a >1.5-fold change in 89 genes [56 increased (maximum 2.3-fold) and 33 decreased (maximum 1.9fold)]. Significantly changed genes were then categorised by function.

Table 5. Number of genes expressed in adult PBMCs cultured for 72 h in medium containing 10% FCS, 5 g/ml phytohaemoglutinin and either 0 or 200 ng/ml rhGH for the final 24 h

Condition

Genes expressed

Total, %

Basal GH Coexpression

11,949 11,712 11,310

82 81 78

mRNA expression levels were determined following hybridisation of labelled cDNA to HG-U133A oligonucleotide chips (Affymetrix).

13

orders is required to define whether PBMCs are an appropriate model with which to assess GH sensitivity and hence to build a prediction model. Gene array has also been used to examine GH-induced changes in gene expression both in mouse 3T3 F442A fibroblasts in vitro [16] and in the hypophysectomised rat model [17]. In the 3T3 cells, cluster analysis on gene ontology categories has been used to examine time-dependent gene expression. Thirty minutes after treatment with GH, there is an early wave of increased expression of genes that regulate transcription. After 4 h, transcription regulators are still the main category of genes expressed both at increased and decreased levels compared with untreated controls. At 48 h, six categories of genes are expressed, including those affecting immune response, carboxylic acid metabolism, transcription, carbohydrate and alcohol metabolism and lipid biosynthesis [16]. In the hypophysectomised rat model [17], genes expressed in liver, heart and kidney tissues were examined. In the basal state, 1,600 genes were significantly expressed in heart tissue, 966 in kidney tissue and 720 in liver tissue, with 524 expressed in all three. When compared with normal tissues, 16% of the genes in the heart, 23% in the kidney and 29% in the liver were differentially expressed in the hypophysectomised state. Small numbers of genes were affected by GH treatment in hy-

pophysectomised animals (58 in the liver, 32 in the heart and 16 in the kidney). The majority of differentially regulated transcripts were related to metabolism, signal transduction, protein turnover and cell structure. However, the function of a large number of regulated genes was unknown.

Conclusion

Gene array technology is a very powerful tool for exploring the contribution of genes to human growth. The magnitude and complexity of the data generated mean that substantial bioinformatic support is required to identify important messages. The number of patients required for gene array studies in growth disorders is likely to be substantial, and thus major collaborative programmes will be required. Nevertheless, the use of genomics to help manage GH treatment is a realistic, achievable goal within the next few years. We will then be in a position to combine established auxology-based prediction models and well-characterised biomarkers such as IGF-I with key gene polymorphisms and changes in gene expression in response to GH to identify those children who will derive the greatest benefit from GH treatment with the lowest risk of adverse effects.

References 1 Ranke MB, Lindberg A, Chatelain P, Wilton P, Cutfield W, Albertsson-Wikland K, Price DA: Derivation and validation of a mathematical model for predicting the response to exogenous recombinant human growth hormone (GH) in prepubertal children with idiopathic GH deficiency. KIGS International Board. Kabi Pharmacia International Growth Study. J Clin Endocrinol Metab 1999;84:1174–1183. 2 Ranke MB, Lindberg A, Chatelain P, Wilton P, Cutfield W, Albertsson-Wikland K, Price DA, KIGS International Board: Kabi International Growth Study. Prediction of longterm response to recombinant human growth hormone in Turner syndrome: development and validation of mathematical models. KIGS International Board. Kabi International Growth Study. J Clin Endocrinol Metab 2000;85:4212–4218. 3 Ranke MB, Lindberg A, Cowell CT, Wikland KA, Reiter EO, Wilton P, Price DA, KIGS International Board: Prediction of response to growth hormone treatment in short children

14

born small for gestational age: analysis of data from KIGS (Pharmacia International Growth Database). J Clin Endocrinol Metab 2003;88:125–131. 4 Schonau E, Westermann F, Rauch F, Stabrey A, Wassmer G, Keller E, Bramswig J, Blum WF, German Lilly Growth Response Study Group: A new and accurate prediction model for growth response to growth hormone treatment in children with growth hormone deficiency. Eur J Endocrinol 2001; 144: 1– 20. 5 de Ridder MA, Stijnen T, Hokken-Koelega AC: Validation and calibration of the Kabi Pharmacia International Growth Study prediction model for children with idiopathic growth hormone deficiency. J Clin Endocrinol Metab 2003; 88:1223–1227. 6 Van Pareren YK, de Muinck Keizer-Schrama SM, Stijnen T, Sas TC, Jansen M, Otten BJ, Hoorweg-Nijman JJ, Vulsma T, StokvisBrantsma WH, Rouwe CW, Reeser HM, Gerver WJ, Gosen JJ, Rongen-Westerlaken C, Drop SL: Final height in girls with Turner

Horm Res 2007;67(suppl 1):10–15

syndrome after long-term growth hormone treatment in three dosages and low dose estrogens. J Clin Endocrinol Metab 2003; 88: 1119–1125. 7 Hokken-Koelega A, Mulder P, De Jong R, Lilien M, Donckerwolcke R, Groothof J: Longterm effects of growth hormone treatment on growth and puberty in patients with chronic renal insufficiency. Pediatr Nephrol 2000;14:701–706. 8 Van Pareren Y, Mulder P, Houdijk M, Jansen M, Reeser M, Hokken-Koelega A: Adult height after long-term, continuous growth hormone (GH) treatment in short children born small for gestational age: results of a randomized, double-blind, dose-response GH trial. J Clin Endocrinol Metab 2003; 88: 3584–3590. 9 Hintz RL, Attie KM, Baptista J, Roche A: Effect of growth hormone treatment on adult height of children with idiopathic short stature. Genentech Collaborative Group. N Engl J Med 1999;340:502–507.

Clayton/Whatmore

10 Lee JM, Davis MM, Clark SJ, Hofer TP, Kemper AR: Estimated cost-effectiveness of growth hormone therapy for idiopathic short stature. Arch Pediatr Adolesc Med 2006;160:263–269. 11 Dos Santos C, Essioux L, Teinturier C, Tauber M, Goffin V, Bougneres P: A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone. Nat Genet 2004;36:720–724. 12 Jorge AA, Marchisotti FG, Montenegro LR, Carvalho LR, Mendonca BB, Arnhold IJ: Growth hormone (GH) pharmacogenetics: influence of GH receptor exon 3 retention or deletion on first-year growth response and final height in patients with severe GH deficiency. J Clin Endocrinol Metab 2006; 91: 1076–1080.

Genomic Approach to Growth Prediction

13 Binder G, Baur F, Schweizer R, Ranke MB: The d3-growth hormone (GH) receptor polymorphism is associated with increased responsiveness to GH in Turner syndrome and short small-for-gestational-age children. J Clin Endocrinol Metab 2006;91:659– 664. 14 Pilotta A, Mella P, Filisetti M, Felappi B, Prandi E, Parrinello G, Notarangelo LD, Buzi F: Common polymorphisms of the growth hormone (GH) receptor do not correlate with the growth response to exogenous recombinant human GH in GH-deficient children. J Clin Endocrinol Metab 2006;91:1178–1180.

15 Whitney AR, Diehn M, Popper SJ, Alizadeh AA, Boldrick JC, Relman DA, Brown PO: Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci USA 2003;100:1896–1901. 16 Huo JS, McEachin RC, Cui TX, Duggal NK, Hai T, States DJ, Schwartz J: Profiles of growth hormone (GH)-regulated genes reveal time-dependent responses and identify a mechanism for regulation of activating transcription factor 3 by GH. J Biol Chem 2006;281:4132–4141. 17 Flores-Morales A, Stahlberg N, Tollet-Egnell P, Lundeberg J, Malek RL, Quackenbush J, Lee NH, Norstedt G: Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat. Endocrinology 2001; 142: 3163–3176.

Horm Res 2007;67(suppl 1):10–15

15

Plenary Lecture 3

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):16–22 DOI: 10.1159/000097545

Published online: February 15, 2007

Factors Predisposing to Osteoporosis in Childhood: New Concepts in Diagnostics E. Schoenau O. Fricke Children’s Hospital, University of Cologne, Cologne, Germany

Key Words Osteoporosis Functional muscle-bone unit Bone density Densitometry Muscle force

Abstract Background: Bone densitometric data are often difficult to interpret in children and adolescents because of large interand intraindividual variations in bone size. We propose a functional approach to bone densitometry that addresses two questions: is bone strength normally adapted to the largest physiological loads represented by muscle force, and is muscle force adequate for body size? Methods and Results: The theoretical background for this approach is provided by the mechanostat theory, which proposes that bones adapt their strength to keep the strain caused by physiological loads close to a set point. Because the largest physiological loads are caused by muscle contractions, there should be a close relationship between bone strength and muscle force or size. The proposed two-step diagnostic algorithm requires a measure of muscle force or size and a measure of bone mineral content at a corresponding location. Conclusion: This approach justifies the hope that more detailed insights into this matter could help devise targeted strategies for the prevention and treatment of pediatric bone diseases. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0016$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

It is generally accepted that fractures result from low bone mass. Bone mass accounts for 75–85% of the variance in the ultimate strength of bone tissue, and measurements of it also provide an accurate indication of whole bone strength [1]. Bone mass in adulthood is a result of bone gained during growth and what is lost thereafter. Peak bone mass is most likely achieved by 18 years of age. Total body calcium increases from about 30 g at birth to 2,000 g by 15–20 years of age [2–8]. Most fractures occur late in life, and patients with fractures often have low bone density compared with agematched controls [9, 10]. As bone loss is associated with aging, investigators have reasoned that people who have fractures and low bone density probably have lost more bone mass than people who do not have fractures [11–13]. This hypothesis may be correct, but an alternative explanation may be true as well: people with fractures may have achieved a lower peak bone mass as young adults and subsequently lost bone mass at the same rate as the rest of the population [14]. The second hypothesis has direct implications for pediatricians. If low peak bone density is an important contributor to the reduced bone density of fracture patients, then the development of optimal peak bone mass during childhood and adolescence is of crucial importance to prevent osteoporosis. Based on this hypothesis, there is

E. Schoenau, MD Pediatric Endocrinology and Diabetes, Children’s Hospital University of Cologne, Kerpener Strasse 62, DE–50924 Cologne (Germany) Tel. +49 221 478 4360/4361, Fax +49 221 478 373/4635 E-Mail [email protected]

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200 BMC 4% (mg/mm)

evidence that bone mass measurements can stratify patients on the basis of fracture risks. This has been recommended strongly by the World Health Organization (WHO) [15]. However, there is ongoing discussion about whether the WHO definition of osteoporosis, which is based on measurement of bone density, is oversimplistic and needs to include indices representing distribution of bone, mineral composition and other risk factors such as fall biomechanics [16, 17].

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What Are the Predictors of Bone Development?

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Factors Predisposing to Osteoporosis in Childhood

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It is standard procedure in both pediatrics and adult medicine to relate many diagnostic parameters to age. Hence, the WHO recommends comparing individual bone densitometry data with either age-related reference data (z-score) or results of 20- to 30-year-old healthy persons, who presumably are at peak bone mass (t-score) [15]. Such recommendations are based on epidemiological studies of osteoporotic fracture risk [1]. However, these recommendations differ from those of Mazess and Cameron [3, 4], the developers of single-photon absorptiometry. More than 30 years ago, in one of the first bone densitometric studies ever performed in children, study investigators compared bone mineral content (BMC) (i.e., the mass of bone mineral) to bone width in order to eliminate the effects of skeletal size. As well as relating BMC to age, the investigators provided data relative to height and weight, indices that are independent of body size [4]. This was necessary as BMC was highly correlated with bone width (r = 0.85), height and weight (r = 0.83). The partial correlations between age and BMC fell (from r = 0.75 to r !0.10) after adjustment for body size. These early observations have been confirmed by a number of later studies, which have added more information regarding the determinants of bone mass development (fig. 1) [18–22]. The strongest predictors of bone mass development are height and muscle mass (fig. 1). There is a moderate correlation between age and BMC during childhood, but it is quite different between boys and girls. In contrast, the relationship between muscle and bone mass varies little with age and sex. Using agerelated reference data would yield a lot of ‘healthy’ children with osteopenia (fig. 2a). In healthy children, these data illustrate that short-normal or subnormal stature is associated with ‘osteopenia’ or ‘osteoporosis,’ using WHO standards. Figure 2b demonstrates that BMC is similarly dependent on body height after cessation of bone growth length in adults. Actually, this relationship has not been

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[BMC], distal radius) analyzed by peripheral computed tomography and age (a), height (b), and local muscle mass (c) studied in healthy children and their parents. Methods and probands have been described previously [21]. Reproduced with permission from Schoenau et al. [39].

Horm Res 2007;67(suppl 1):16–22

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Fig. 2. SD or Z-score of bone mass (BMC at the distal radius) in relationship to SD or Z-score of height in healthy children (a) and adults (b) [21]. Reproduced with permission from Schoenau et al. [39].

Mice have a lower bone mass than elephants. Therefore, mice will be diagnosed as osteoporotic when age-matched elephant standards are used as a reference. However, there is no evidence that mice have more fractures than elephants. As Galileo Galilei commented in the seventeenth century, small bones perform the same function in small animals as large bones in large animals [28]. The same should apply to short and tall children, adolescents and adults. Based on such simple ideas, it can be reasonably suggested that analyses of bone mass (and bone structure) should focus on the question of whether they are adequate for bone function. The main purpose of bones is to provide enough strength (not merely enough mass) to keep voluntary physical loads from causing spontaneous fractures, regardless of whether these loads are chronically subnormal (as in inactivity or muscle disorders), normal or supranormal. Achieving that ‘mechanical competence’ would be the ultimate test of a bone’s health and the main goal of its biological mechanisms [29]. This concept has been recommended recently by Ward and Glorieux [30]: ‘Osteoporosis will be said to exist when the skeleton has not been able to withstand its mechanical challenges (growth and muscle force) due to inadequate bone mass and/or structure, resulting in traumatic fractures. This means that a child will not be labeled with osteoporosis unless there is a history of fractures that occur with minimal trauma. With such an approach, the term osteopenia will not be employed in the assessment of a child’s bone health.’

The ‘Functional Muscle-Bone Unit’ in Clinical Practice

taken into consideration in analyses of osteoporosis in adulthood. The clinical relevance of this problem in young adults has been demonstrated in patients with delay in constitutional growth and puberty. These patients have a final height in the lower normal range. A study using linear absorption techniques in combination with age-related reference data suggested a high risk for osteoporosis [23]. However, as reported by Bertelloni et al. [24], the purported bone mass deficit of these patients disappears when results are corrected for body height or bone size. Similar effects have been described in children with renal failure and kidney transplantation [25–27]. These considerations may appear somewhat abstract, but a simple example from nature should clarify the point. 18

Horm Res 2007;67(suppl 1):16–22

Previously, we proposed a diagnostic algorithm for applying the muscle-bone relationship to clinical practice (fig. 3) [21]. It requires muscle force or size and BMC at a corresponding location to be measured. If BMC is lower than expected for muscle force or size, a ‘primary bone defect’ is diagnosed. In the second situation, muscle force or size is too low for height. Even if BMC is adapted adequately to the decreased mechanical challenge, this means that bone mass and, presumably, strength are still too low for body height. Therefore, a ‘secondary bone defect’ is diagnosed. If muscle force or size is abnormally low and BMC is even lower than expected from a normal muscle-bone relationship, a ‘mixed bone defect’ (primary and secondary) is present. Schoenau/Fricke

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Reproduced, with permission, from Schoenau et al. [21].

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Fig. 5. Correlation between muscle area and bone mass in cystic

area (MA) and bone-muscle interaction (BMC/MA, SSI/MA) in cystic fibrosis.

fibrosis (CF).

Height-dependent reference ranges for muscle crosssectional area at the forearm and muscle-related reference data for radial BMC at the same site have been published [31]. Although this investigation was performed using peripheral quantitative computed tomography, the algorithm should be sufficiently simple to be adaptable to other densitometric methods. Indeed, Schiessl et al. found identical correlations between muscle and bone mass using dual-energy X-ray absorptiometry [32]. Therefore, we used BMC as an indicator of bone strength because this probably is the most basic densitometric parameter. These data were used to test the proposed diag-

nostic approach in children and adults with cystic fibrosis (CF). Twenty-two patients with CF (9 females and 13 males, aged 8 to 36 years) were examined (fig. 4). Bone mass (BMC), bone strength (SSI) and muscle area (MA) at the radius analysed by pQCT were decreased. The analysis of bone adaptation on muscle development (BMC/MA, SSI/MA) showed normal results. Figure 5 describes the correlation between MA and BMC in patients with CF in comparison to healthy controls. The data show that bone problems in CF are due to impaired muscle development (secondary bone defect; fig. 6). In a similar way, other chronic diseases in childhood should

Factors Predisposing to Osteoporosis in Childhood

Horm Res 2007;67(suppl 1):16–22

19

Bone Normals

Primary or true bone disorder

Fig. 6. The ‘functional muscle-bone unit’. Primary bone disease: bone structure/ mass not adapted on muscle development. Secondary bone disease: disturbed muscle development but normal adapted skeleton.

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10

tive and retired soccer players compared with controls. Reproduced with permission from Johnston et al. [36].

be analysed. ‘Osteopenia’ and ‘osteoporosis’ are symptoms. Diagnosis is necessary for selecting the optimal therapy. Physiotherapy and physical activity should include aspects of muscle training to optimize prevention of osteoporosis in patients with CF. These data also illustrate the utility of a new diagnostic approach to pediatric bone diseases, which is based on analysis of the balance between bone strength and the physiological challenge to bone strength. This approach allows a new classification of bone disorders in children and adolescents.

20

Horm Res 2007;67(suppl 1):16–22

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Peak Bone Mass and Long-Term Effects Based on the generally accepted concept of optimal bone mass acquisition, many studies have analyzed the influence of calcium intake on bone mass development. A study of twins, in which only one twin was given calcium supplements, indicated a positive but modest effect of higher calcium intake on bone mass in children [33]. Interestingly, calcium intake did not have any impact on bone mass during puberty, although calcium supply is widely believed to be critically important during the pubertal growth period. Further follow-up in these twins showed that the significant differences in bone mineral Schoenau/Fricke

density between the calcium-supplemented and -unsupplemented twins disappeared after withdrawal of calcium tablets. Another study found that 18 months of controlled calcium supplementation led to higher bone mineral mass, but these gains disappeared within 18 months of the trial [34]. Studies on biochemical markers that reflect bone modeling and remodeling showed that calcium intake reduced remodeling rates but did not have any influence on modeling [35]. These data are in agreement with the hypothesis that calcium supplementation decreases remodeling activity. Lower remodeling rates might occur via decreased parathyroid hormone levels and lead to lower cortical porosity. However, this apparent gain in bone mass is entirely reversible once remodeling rates increase. It is important to note that calcium supplementation does not seem to stimulate modeling, which is the main process for increasing bone strength (cortical thickness, cortical area) during childhood and adolescence. The long-term effects of physical activity may be similarly reversible. A study on soccer players showed that exercise in youth confers a high and clinically relevant benefit on peak bone mineral density, but that cessation of exercise results in accelerated loss (fig. 7) [36]. Thus, those individuals who stopped playing for more than 35 years and who were 60 years or older did not have any significant residual benefit in bone mineral density. The fracture rate was not lower than expected for age. Similar data were reported by Pajamaki et al. in a rat experiment [37]. Exercise-induced bone benefits are lost if exercise is completely stopped. Thus continued training is probably needed to maintain the positive effects of exercise in youth into adulthood. On the other hand, animal experiments have shown that a period of disturbed bone mass acquisition in early

life may not result in an altered peak bone mass [38]. The same study demonstrated that areas of the juvenile skeleton are not completely remodeled but actually replaced by skeletal growth in toto. As the juvenile bone enlarges and the medullary cavity expands, bone formed early in life, regardless of its quality, is gradually resorbed and replaced by new bone due to modeling. This mechanism ‘repairs’ insufficient bone development after cessation of negative influences. In summary, short-term effects on bone mass, whether positive or negative, do not seem to have any effect in the long run. The skeletal system seems to adapt to current requirements rather than to those of an earlier developmental phase.

Conclusion

Is the peak bone mass concept still relevant? The answer to that question seems to be ‘no’ if bone mass analysis and interpretation are performed in the ‘standard way’. The term ‘peak’ bone mass as reference would wrongly label many short children as osteopenic (children with short stature, short children with renal insufficiency, etc.) during development. Bone mass is not a function of age: it is a function of muscle forces, height and bone size. It remains to be seen if the peak bone mass concept can undergo a revival by using more appropriate reference data (e.g., based on height, bone size or muscle force). It is possible that peak bone mass can be replaced by peak bone strength as a more relevant diagnostic parameter. However, ‘maximizing peak bone mass’ (or peak bone strength) is not a useful concept for the prevention of fractures in old age. A short-term ‘peak’ of bone strength due to physical activity in youth is easily cancelled out by a lifetime of sloth.

References 1 Kanis JA, Melton LJ 3rd, Christiansen C, Johnston CC, Khaltaev N: The diagnosis of osteoporosis. J Bone Miner Res 1994;9:1137– 1141. 2 Garn SM, Wagner B: The adolescent growth of the skeletal mass and its implications to mineral requirements; in Heald FP (ed): Adolescent Nutrition and Growth. New York, Appleton-Century Crofts, 1969, pp 139– 161.

Factors Predisposing to Osteoporosis in Childhood

3 Mazess RB, Cameron JR: Skeletal growth in school children: maturation and bone mass. Am J Phys Anthropol 1971;35:399–407. 4 Mazess RB, Cameron JR: Growth of bone in school children: comparison of radiographic morphometry and photon absorptiometry. Growth 1972;36:77–92. 5 Gilsanz V, Gibbens DT, Carlson M, Boechat MI, Cann CE, Schulz EE: Peak trabecular vertebral density: a comparison of adolescent and adult females. Calcif Tissue Int 1988;43:260–262.

6 Gilsanz V, Gibbens DT, Roe TF, Carlson M, Senac MO, Boechat MI, Huang HK, Schulz EE, Libanati CR, Cann CC: Vertebral bone density in children: effect of puberty. Radiology 1988;166:847–850. 7 Glastre C, Braillon P, David L, Cochat P, Meunier PJ, Delmas PD: Measurement of bone mineral content of the lumbar spine by dual energy x-ray absorptiometry in normal children: correlations with growth parameters. J Clin Endocrinol Metab 1990;70:1330– 1333.

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8 Bonjour JP, Theintz G, Buchs B, Slosman D, Rizzoli R: Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991;73:555–563. 9 Mazess RB: On aging bone loss. Clin Orthop Relat Res 1982;May:239–252. 10 Riggs BL, Melton LJ: Involutional osteoporosis. N Engl J Med 1986;314:1676–1686. 11 Riggs BL, Melton LJ: Evidence for two distinct syndromes of involutional osteoporosis. Am J Med 1983;75:899–901. 12 Hansen MA, Overgaard K, Riis BJ, Christiansen C: Role of peak bone mass and bone loss in postmenopausal osteoporosis: 12 year study. BMJ 1991;303:961–964. 13 Seeman E, Tsalamandris C, Formica C: Peak bone mass, a growing problem? Int J Fertil Menopausal Stud 1993;38(suppl 2):77–82. 14 Seeman E, Hopper JL, Bach LA, Cooper ME, Parkinson E, McKay J, Jerums G: Reduced bone mass in daughters of women with osteoporosis. N Engl J Med 1989; 320: 554– 558. 15 Genant HK, Cooper C, Poor G, Reid I, Ehrlich G, Kanis J, Nordin BE, Barrett-Connor E, Black D, Bonjour JP, Dawson-Hughes B, Delmas PD, Dequeker J, Eis SR, Gennari C, Johnell O, Johnston CC, Lau EM, Liberman UA, Lindsay R, Martin TJ, Masri B, Mautalen CA, Meunier PJ, Miller PD, Mithal A, Morii H, Papapoulos S, Woolf A, Yu W, Khaltaev N: Interim report and recommendations of the World Health Organization task-force for osteoporosis. Osteoporos Int 1999;10:259–264. 16 Pinilla TP, Boardman KC, Bouxsein ML, Myers ER, Hayes WC: Impact direction from a fall influences the failure load of the proximal femur as much as age-related bone loss. Calcif Tissue Int 1996;58:231–235. 17 Sandor T, Felsenberg D, Brown E: Comments on the hypotheses underlying fracture risk assessment in osteoporosis as proposed by the World Health Organization. Calcif Tissue Int 1999;64:267–270. 18 Molgaard C, Thomsen BL, Michaelsen KF: Influence of weight, age and puberty on bone size and bone mineral content in healthy children and adolescents. Acta Paediatr 1998;87:494–499.

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19 Kroger H, Vainio P, Nieminen J, Kotaniemi A: Comparison of different models for interpreting bone mineral density measurements using DXA and MRI technology. Bone 1995; 17:157–159. 20 Lu PW, Cowell CT, Lloyd-Jones SA, Briody JN, Howman-Giles R: Volumetric bone mineral density in normal subjects, aged 5–27 years. J Clin Endocrinol Metab 1996; 81: 1586–1590. 21 Schoenau E, Neu CM, Beck B, Manz F, Rauch F: Bone mineral content per muscle crosssectional area as an index of the functional muscle-bone unit. J Bone Miner Res 2002;17: 1095–1101. 22 Rauch F, Schoenau E: The developing bone: slave or master of its cells and molecules? Pediatr Res 2001;50:309–314. 23 Finkelstein JS, Neer RM, Biller BM, Crawford JD, Klibanski A: Osteopenia in men with a history of delayed puberty. N Engl J Med 1992;326:600–604. 24 Bertelloni S, Baroncelli GI, Ferdeghini M, Perri G, Saggese G: Normal volumetric bone mineral density and bone turnover in young men with histories of constitutional delay of puberty. J Clin Endocrinol Metab 1998; 83: 4280–4283. 25 Klaus G, Paschen C, Wuster C, Kovacs GT, Barden J, Mehls O, Scharer K: Weight-/ height-related bone mineral density is not reduced after renal transplantation. Pediatr Nephrol 1998; 12:343–348. 26 Sanchez CP, Salusky IB, Kuizon BD, Ramirez JA, Gales B, Ettenger RB, Goodman WG: Bone disease in children and adolescents undergoing successful renal transplantation. Kidney Int 1998;53:1358–1364. 27 Reusz GS, Szabo AJ, Peter F, Kenesei E, Sallay P, Latta K, Szabo A, Tulassay T: Bone metabolism and mineral density following renal transplantation. Arch Dis Child 2000; 83: 146–151. 28 Galilei G: Dialogues concerning two new sciences (de Slavio A, Crew H, transl. 1939). Italian original 1638. 29 Carter DR, Wong M, Orr TE: Musculoskeletal ontogeny, phylogeny, and functional adaptation. J Biomech 1991; 24(suppl 1):3–16.

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30 Ward L, Glorieux F: The spectrum of pediatric osteoporosis; in Glorieux F, Pettifor J, Juepper H (eds): Pediatric Bone: Biology and Disease. San Diego, Academic Press, 2003. 31 Neu CM, Rauch F, Manz F, Schoenau E: Modeling of cross-sectional bone size, mass and geometry at the proximal radius: a study of normal bone development using peripheral quantitative computed tomography. Osteoporos Int 2001;12:538–547. 32 Schiessl H, Frost HM, Jee WS: Estrogen and bone-muscle strength and mass relationships. Bone 1998;22:1–6. 33 Johnston CC, Miller JZ, Slemenda CW, Reister TK, Hui S, Christian JC, Peacock M: Calcium supplementation and increases in bone mineral density in children. N Engl J Med 1992;327:82–87. 34 Lee WT, Leung SS, Leung DM, Cheng JC: A follow-up study on the effects of calciumsupplement withdrawal and puberty on bone acquisition of children. Am J Clin Nutr 1996; 64:71–77. 35 Slemenda CW, Peacock M, Hui S, Zhou L, Johnston CC: Reduced rates of skeletal remodeling are associated with increased bone mineral density during the development of peak skeletal mass. J Bone Miner Res 1997; 12:676–682. 36 Karlsson MK, Linden C, Karlsson C, Johnell O, Obrant K, Seeman E: Exercise during growth and bone mineral density and fractures in old age. Lancet 2000; 355:469–470. 37 Pajamaki I, Kannus P, Vuohelainen T, Sievanen H, Tuukkanen J, Jarvinen M, Jarvinen TL: The bone gain induced by exercise in puberty is not preserved through a virtually life-long deconditioning: a randomized controlled experimental study in male rats. J Bone Miner Res 2003;18:544–552. 38 Gafni RI, McCarthy EF, Hatcher T, Meyers JL, Inoue N, Reddy C, Weise M, Barnes KM, Abad V, Baron J: Recovery from osteoporosis through skeletal growth: early bone mass acquisition has little effect on adult bone density. FASEB J 2002;16:736–768. 39 Schoenau E, Neu CM, Beck B, Manz F, Rauch F: Bone mineral content per muscle crosssectional area as an index of the functional muscle-bone unit. J Bone Miner Res 2002; 17:1095–1101.

Schoenau/Fricke

Plenary Lecture 3

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):23–27 DOI: 10.1159/000097546

Published online: February 15, 2007

Skeletal Health in Adulthood Richard Eastell Academic Unit of Bone Metabolism, Section of Medicine, Division of Clinical Sciences (North), University of Sheffield, Sheffield, UK

Key Words Osteoporosis Bone mineral density Fracture risk Treatment Alendronate Risedronate Calcitonin Raloxifene Teriparatide

Abstract Introduction: Osteoporosis is a major public health problem. We now have an approach to case finding that involves the measurement of bone mineral density in people at high risk of fractures. Diagnosis: Diagnostic evaluation includes assessing risk factors, measuring bone mineral density, establishing the fracture status of the individual and monitoring treatment effects with either bone mineral density or bone turnover markers. Management: Successful management of the individual requires selection of optimal therapy and encouragement of long-term adherence with the planned treatment. The drugs available for the prevention of fractures are classified as either anticatabolic or anabolic. The efficacy of these agents can be evaluated in the individual by monitoring changes in bone mineral density or bone turnover markers. Copyright © 2007 S. Karger AG, Basel

Introduction

Osteoporosis is a major public health problem, which makes attainment and maintenance of good bone health in the adult of paramount importance. Assessments of bone health include the use of risk factors and measure-

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ment of bone mineral density. A practical approach to the use of these tools is given in figure 1. At present, there are also a number of treatments that are safe and effective in reducing the risk of subsequent fracture for subjects known to have osteoporosis [1]. Guidelines have been developed in the United Kingdom (UK) for measurement of bone density using dual-energy X-ray absorptiometry and subsequent treatment. These evidence-based guidelines were developed by the Royal College of Physicians and were updated in 2000 in conjunction with the Bone and Tooth Society [2]. This approach is likely to be superseded in the next few years by the use of several risk factors to estimate absolute fracture risk [3]. Continued study is still needed in response to the substantial public health challenges posed by osteoporosis.

Background

Osteoporosis is defined as a bone mineral density Tscore below –2.5, with the T-score being the number of standard deviations from the mean of healthy young women. In evaluating a subject with osteoporosis, it is important to consider the risk of fracture in the near future and to look for secondary osteoporosis. The risk of fracture can be increased by many factors (table 1). Common risk factors include low bone mineral density, occurrence of fracture after the age of 40 years, family history of fracture (particularly a maternal history of hip fracture before the age of 80), slender habitus (weight less than 57 kg) and current smoking habit. Fracture risk is also

Richard Eastell, MD Academic Unit of Bone Metabolism, Division of Clinical Sciences (North) Clinical Sciences Centre, Northern General Hospital, Herries Road Sheffield S5 7AU (UK) Tel. +44 114 271 4705, Fax +44 114 261 8775, E-Mail [email protected]

Risk factors Referred by GP for bone densitometry Measure bone density Identify any fracture Radiograph of the spine

Questionnaire Search for causative factors

Biochemical evaluation

Clinical examination

Initiate treatment plan – if criteria are met Identify diseases that modify choice of treatment

Identify diseases that merit treatment Treat

Monitor eﬀect

Fig. 1. Evaluation of bone health.

Table 1. Common risk factors for osteoporosis

Risk factor Low-trauma fracture since age 40 Maternal history of osteoporotic fracture Age 665 years Thin body build (body weight ~57 kg) Prolonged amenorrhea Early menopause Long-term corticosteroid use (`6 months) Disease predisposing to osteoporosis Thyrotoxicosis Coeliac disease

Bone turnover markers can be assessed using automated immunoassay analysers and used to evaluate bone resorption and bone formation. High levels of bone turnover markers in older women are associated with an increase in the risk of fracture. The levels of bone turnover markers decrease within a few weeks of starting anticatabolic therapy, and their decrease reflects a decrease in the risk of fractures. Thus bone turnover markers are useful alternatives to bone mineral density for monitoring therapy.

General Measures

greater among those at increased risk of falling and those taking glucocorticoids. Secondary osteoporosis can develop as a potential complication of treatment for an underlying condition, even though the treatment may result in either a reduction or an increase in fracture risk. For example, treatment of primary hyperparathyroidism results in a decrease in bone turnover, an increase in bone mineral density and a reduced risk of fracture. Other conditions may impair the response to treatment for osteoporosis. It has been reported that vitamin D deficiency impairs the response to bisphosphonates. Consequently, it is important to evaluate subjects for the presence of secondary osteoporosis. 24

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General measures for managing the subject with osteoporosis include advising the subject to stop smoking, to be moderate in alcohol consumption and to regularly perform weight-bearing exercise. These recommendations are good for general health and can result in small gains in bone density, but they have not been shown to reduce the risk of fracture. Similarly, subjects at risk of falling should undergo a falls assessment, and they should take measures to prevent subsequent falls. However, there is no evidence that prevention of falls reduces the risk of fractures. For instance, the use of hip protectors has been proposed to reduce the risk of hip fractures. However, there is no strong evidence to support their use: results of clinical trials showing an apparent beneficial effect of their use were not balanced in terms of the active and control group characteristics [4]. Eastell

For subjects who present after sustaining a vertebral fracture, it is important to ensure that they receive adequate analgesia and to consider use of other measures such as a lumbar support corset or transcutaneous electrical nerve stimulation. Subjects also may benefit from joining a support group such as those affiliated with the International Osteoporosis Foundation (http://www. osteofound.org/).

Drug Therapy

Drug treatments for osteoporosis [5] fall into two major categories – anticatabolic and anabolic [6]. Anticatabolic drugs that reduce the rate of bone turnover include bisphosphonate, hormone replacement therapy (HRT) and raloxifene. Anabolic drugs that stimulate the rate of bone formation include parathyroid hormone and its 1– 34 amino acid peptide, teriparatide. Some drugs such as strontium ranelate fall into a third category. They have weak anticatabolic actions, and though we do not know for sure, they might work by increasing the material strength of bone. Ultimately, we do not know how these treatments reduce the risk of fracture. It was thought that the main mechanism was an increase in bone mineral density. However, it has become apparent that the increase in bone mineral density associated with anticatabolic drugs such as risedronate, alendronate and raloxifene accounts for only 10% of the fracture benefit. It is more likely that the most important effect of these treatments is to decrease the rate of bone turnover to levels found in healthy premenopausal women. Indeed, the change in bone turnover markers accounts for up to 70% of the fracture risk reduction with risedronate [7]. The way in which anabolic agents reduce fracture risk is less well studied, but it might include qualitative improvement to bone such as increases in cortical bone thickness and better connectivity of trabecular bone.

osteoporosis. Indeed, in certain populations such as the housebound elderly this may reduce the risk of hip fracture without the need for further therapy [8]. Bisphosphonates The most common treatments for osteoporosis in the UK are the bisphosphonates, particularly alendronate and risedronate. These drugs are poorly absorbed and so must be taken in the fasting state at least half an hour before breakfast. In addition, there have been reports of oesophagitis with alendronate, so the treatment must be taken with a full glass of water and the subject should be advised to remain upright until after breakfast. Apart from occasional skin rash, these drugs are very well tolerated. They are effective in reducing the risk of vertebral fractures by 40–50% and show an effect within the first year of therapy [9]. They also are effective in reducing the risk of nonvertebral fracture, including hip fracture, by 30–50%, and in reducing the incidence of fractures among women with a history of vertebral fractures and those with bone density T-scores below –2.5. There is no evidence for efficacy among subjects with T-scores above this threshold, or among subjects with fractures other than vertebral fracture. One concern about bisphosphonates is that the complicated instructions for taking the drugs may reduce compliance. However, both agents are now available as preparations that can the taken once a week, and this form of administration has proved popular with subjects. Alendronate results in bigger increases in bone mineral density and bigger decreases in bone turnover markers [10], but we cannot be sure that this translates into a bigger reduction in fracture risk compared with risedronate. Currently there are forms of bisphosphonate that are being evaluated for intravenous injection once every 3 months (ibandronate) or annually (zoledronate) [11]. These dosing schedules would further minimize obstacles to subject compliance. However, we still do not know how long to treat subjects. Suppression of bone turnover markers persists 3–5 years after discontinuing alendronate therapy; thus, it might be worth stopping alendronate after 5 years of therapy and watching for bone loss or an increase in bone turnover before reinstating therapy [12]. We do not have much information yet about the long-term effects of risedronate therapy.

Calcium and Vitamin D In most of the clinical trials conducted to date, calcium and vitamin D were administered along with these drug treatments. Typically, older women have low dietary calcium levels and vitamin D insufficiency. Vitamin D insufficiency may impair the response to antiresorptive therapy. Because it is difficult to assess accurately dietary calcium or vitamin D status in clinical practice, it may therefore be sensible to recommend calcium and vitamin D supplementation in all subjects receiving therapy for

Hormone Replacement Therapy (HRT) HRT has been used for many years for the prevention of fractures. The evidence for fracture risk reduction is

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based on cohort and case-control studies. Results of the Women’s Health Initiative study [13] showed HRT was associated with a clear reduction in all fractures, including hip fracture, to the same extent as bisphosphonates. However, this large randomized, controlled trial also showed that HRT was associated with increased risks of cardiovascular events and stroke, as well as the known side effects of increased risks of breast cancer and venous thromboembolic disease. Consequently, HRT is not commonly recommended for the prevention of fracture risk. Raloxifene Raloxifene binds to the oestrogen receptor, like the oestrogen in HRT, but in some tissues it acts as an oestrogen agonist, and in others it acts as an oestrogen antagonist. Long-term treatment with raloxifene results in a decrease in the risk of vertebral fractures but not in the risk of nonvertebral fractures. It reduces the risk of breast cancer, particularly oestrogen-receptor-positive cancers – and this effect lasts for up to 8 years after discontinuing therapy [14]. It has minor side effects such as hot flushes and leg cramps, and rare but important side effects such as venous thromboembolic disease. However, raloxifene does not increase the risk of cardiovascular disease or stroke; indeed, in women at high risk of these conditions, it might reduce the risk of these events. Calcitonin Calcitonin is a peptide hormone made by the thyroid glands that acts directly on osteoclasts to reduce bone resorption. However, the overall effect of long-term therapy on bone resorption is quite small. This peptide has to be given by subcutaneous injection or nasal spray. It may cause nausea and a metallic taste when given subcutaneously, and it may also have an analgesic effect. Calcitonin has been used in the setting of acute vertebral fracture and for long-term treatment to reduce fracture risk [15]. Teriparatide Parathyroid hormone is a peptide hormone made by the parathyroid glands. The hormone itself (84 amino acids) or the 1–34 amino acid peptide (teriparatide) is anabolic to bone when injected subcutaneously once a day. In clinical trials teriparatide has been shown to reduce the risk of additional vertebral fractures in women with a history of vertebral fracture prior to treatment and to reduce the incidence of nonvertebral fractures [16]. Teriparatide may be more potent at lowering the risk of nonvertebral fractures compared with the antire26

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sorptive drugs, but there has been no side-by-side comparison. The side effects of teriparatide are mainly those of hypercalcaemia, such as nausea and headache. The optimum duration of therapy is not clear (the trials for teriparatide lasted 1–2 years), nor is the best treatment to use after stopping teriparatide therapy. Previous use of bisphosphonates, such as alendronate, may attenuate the increase in bone mineral density associated with teriparatide therapy [17]. Strontium Strontium ranelate appears to have weak antiresorptive and anabolic effects, but its mechanism of action is not clear. Also, the apparent increase in bone mineral density associated with this compound may be misleading in that strontium replaces calcium in the hydroxyapatite crystal and attenuates X-rays to a greater extent than calcium, thus giving a false reading of a great increase in bone mass. Treatment with strontium reduces the risk of vertebral [18] and nonvertebral fractures [19].

Discussion

Currently, there are a number of treatments available that are effective in reducing the risk of fractures associated with osteoporosis, but it is unclear how best to use them. Typical therapy consists of administering one pharmacological agent alone along with calcium and vitamin D supplements. Yet various questions arise: Would it be better to combine treatments? Since some treatments appear to exert their effects long after cessation (e.g., alendronate), would it be better to provide intermittent treatment? Finally, should we monitor therapy? In clinical trials, it is likely that subjects have a favourable reduction in fracture risk. However, in clinical practice it is possible that not all subjects benefit from treatments either because they do not fully comply with dosing instructions or because they have some coexistent disease. Treatment can be monitored by measuring bone mineral density. Bone mineral density of the spine is usually measured after 1–2 years, with an increase of more than 3–6% probably indicating a favourable response. Treatment also can be monitored using bone turnover markers. Bone resorption markers (such as N- or C-telopeptide fragments of type I collagen) are usually measured, with a decrease in the need for antiresorptive therapy of more than 30–50% probably indicating a favourable response [20]. It is also possible that monitoring may Eastell

improve subjects’ long-term adherence with the treatment regimen [21]. The major challenge in the field of osteoporosis is to identify those subjects at high risk of fracture and to treat them appropriately. There are groups of subjects who are likely to need treatment, such as subjects attending a fracture clinic or those receiving long-term glucocorticoid therapy. However, experience with the use of treatments in these groups is very limited. Almost no subjects receive treatment following a fracture of the hip or forearm, and

only one third of subjects with spine fractures are treated with these drugs [22]. More study is needed to evaluate use of these agents in the management of osteoporosis.

Acknowledgment This paper is an update of a prior publication by the author [Eastell R: Management of bone health in postmenopausal women. Horm Res 2005;64(suppl 2):76–80].

References 1 Eastell R: Treatment of postmenopausal osteoporosis. N Engl J Med 1998; 338:736–746. 2 Compston J: Prevention and treatment of osteoporosis. Clinical guidelines and new evidence. J R Coll Physicians London 2000;34: 518–521. 3 Kanis JA, Johnell O, Oden A, Dawson A, De Laet C, Jonsson B: Ten year probabilities of osteoporotic fractures according to BMD and diagnostic thresholds. Osteoporos Int 2001;12:989–995. 4 Birks YF, Porthouse J, Addie C, Loughney K, Saxon L, Baverstock M, Francis RM, Reid DM, Watt I, Torgerson DJ: Randomized controlled trial of hip protectors among women living in the community. Osteoporos Int 2004;15:701–706. 5 Delmas PD: Treatment of postmenopausal osteoporosis. Lancet 2002;359:2018–2026. 6 Riggs BL, Parfitt AM: Drugs used to treat osteoporosis: the critical need for a uniform nomenclature based on their action on bone remodeling. J Bone Miner Res 2005;20:177– 184. 7 Eastell R, Barton I, Hannon RA, Chines A, Garnero P, Delmas PD: Relationship of early changes in bone resorption to the reduction in fracture risk with risedronate. J Bone Miner Res 2003;18:1051–1056. 8 Chapuy MC, Arlot ME, Delmas PD, Meunier PJ: Effect of calcium and cholecalciferol treatment for three years on hip fractures in elderly women. Br Med J 1994; 308: 1081– 1082. 9 Sambrook P, Cooper C: Osteoporosis. Lancet 2006;367:2010–2018. 10 Rosen CJ, Hochberg MC, Bonnick SL, McClung M, Miller P, Broy S, Kagan R, Chen E, Petruschke RA, Thompson DE, de Papp AE, Fosamax Actonel Comparison Trial Investigators: Treatment with once-weekly alendronate 70 mg compared with once-weekly risedronate 35 mg in women with postmenopausal osteoporosis: a randomized doubleblind study. J Bone Miner Res 2005; 20: 141– 151.

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11 Reid IR, Brown JP, Burckhardt P, Horowitz Z, Richardson P, Trechsel U, Widmer A, Devogelaer JP, Kaufman JM, Jaeger P, Body JJ, Brandi ML, Broell J, Di Micco R, Genazzani AR, Felsenberg D, Happ J, Hooper MJ, Ittner J, Leb G, Mallmin H, Murray T, Ortolani S, Rubinacci A, Sääf M, Samsioe G, Verbruggen L, Meunier PJ: Intravenous zoledronic acid in postmenopausal women with low bone mineral density. N Engl J Med 2002; 346: 653–661. 12 Ensrud KE, Barrett-Connor EL, Schwartz A, Santora AC, Bauer DC, Suryawanshi S, Feldstein A, Haskell WL, Hochberg MC, Torner JC, Lombardi A, Black DM: Randomized trial of effect of alendronate continuation versus discontinuation in women with low BMD: results from the Fracture Intervention Trial long-term extension. J Bone Miner Res 2004;19:1259–1269. 13 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 2002;288:321–333. 14 Martino S, Cauley JA, Barrett-Connor E, Powles TJ, Mershon J, Disch D, Secrest RJ, Cummings SR: Continuing outcomes relevant to Evista: breast cancer incidence in postmenopausal osteoporotic women in a randomized trial of raloxifene. J Natl Cancer Inst 2004;96:1751–1761. 15 Chesnut C III, Silverman S, Andriano K, Genant HK, Gimona A, Harris S, Kiel D, LeBoff M, Maricic M, Miller PD, Moniz C, Peacock M, Richardson P, Watts NB, Baylink DJ, PROOF Study Group: A randomized trial of nasal spray salmon calcitonin in postmenopausal women with established osteoporosis: the Prevent Recurrence of Osteoporotic Fractures study. Am J Med 2000;109:267–276.

16 Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH: Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 2001; 344: 1434– 1441. 17 Black DM, Greenspan SL, Ensrud KE, Palermo L, McGowan JA, Lang TF, Garnero P, Bouxsein ML, Bilezikian JP, Rosen CJ: The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 2003;349: 1207–1215. 18 Meunier PJ, Roux C, Seeman E, Ortolani S, Badurski JE, Spector TD, Cannata J, Balogh A, Lemmel EM, Pors-Nielsen S, Rizzoli R, Genant HK, Reginster JY: The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis. N Engl J Med 2004; 350: 459– 468. 19 Reginster JY, Seeman E, de Vernejoul MC, Adami S, Compston J, Phenekos C, Devogelaer JP, Díaz CM, Sawicki A, Goemaere S, Sorensen OH, Felsenberg D, Meunier PJ: Strontium ranelate reduces the risk of nonvertebral fractures in post-menopausal women with osteoporosis: Treatment of Peripheral Osteoporosis (TROPOS) study. J Clin Endocrinol Metab, 2005; 90(5):2816–2822. 20 Delmas PD, Eastell R, Garnero P, Seibel MJ, Stepan J: The use of biochemical markers of bone turnover in osteoporosis. Committee of Scientific Advisors of the International Osteoporosis Foundation. Osteoporos Int 2000;11(suppl 6):S2–S17. 21 Clowes JA, Peel NF, Eastell R: The impact of monitoring on adherence and persistence with antiresorptive treatment for postmenopausal osteoporosis: a randomized controlled trial. J Clin Endocrinol Metab 2004; 89:1117–1123. 22 Torgerson DJ, Dolan P: Prescribing by general practitioners after an osteoporotic fracture. Ann Rheum Dis 1998;57:378–379.

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Plenary Lecture 4

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):28–31 DOI: 10.1159/000097547

Published online: February 15, 2007

Use of Embryonic Stem Cells for Endocrine Disorders Alan Trounson Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Vic., Australia

Key Words Preimplantation embryos Embryonic stem cells Diabetes Pancreatic differentiation

Abstract Embryonic stem cells (ESCs) can be derived from unused cultured human embryos, provided by patients undergoing treatment for infertility, that would otherwise be disposed of. The inner cell mass cells form immortal pluripotent cell colonies that may be directed into a wide range of cell lineages and end-differentiated cell types. Endocrine lineages are now being actively explored, particularly for their potential to differentiate into pancreatic insulin-producing cells. The majority of published research involving stem cell-directed differentiation into pancreatic islet cells has been conducted using mouse ESCs. The evidence shows that cells that are glucose-responsive for insulin release can be produced using differentiation strategies involving a sequence of growth factor-directed steps. However, because of the close association between neural and pancreatic cell types and markers, it is possible that many of the cells produced by these strategies may be more neural than pancreatic. Thus, it is important to replicate the normal developmental pathways that include the sequential expression of appropriate endoderm and pancreatic progenitor markers. Human pancreatic differentiation from ESCs may require instruction from embryonic mesenchyme to enable the complete maturation of early pancreatic cells and the formation of true islet cells. Copyright © 2007 S. Karger AG, Basel

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Background

Embryonic stem cells (ESCs) are formed from the developing preimplantation embryo by isolation of the inner cell mass (ICM) from the trophectoderm of the blastocyst using immunosurgery or mechanical dissection [1–3]. ESCs are derived from pluripotential embryonic epiblast and are capable of forming all of the organism’s tissue types. ESCs are considered immortal, renewing continuously when grown under carefully defined conditions in the presence of selected feeder cells. The particular property of ESCs that enables their unlimited expansion is a consequence of a checkpoint in development that exists in such species as human, monkey and mouse at the epiblast stage. This enables their expansion as undifferentiated colonies that can be instructed to maintain this phenotype indefinitely. When subjected to the appropriate culture conditions, ESCs will spontaneously differentiate as flat colonies [2] or embryoid bodies [4] with a wide range of cell types representing the germline derivatives of ectoderm, mesoderm and endoderm [5].

Derivation of Human ESCs

Patients treated for infertility are given exogenous gonadotrophins to stimulate multiple follicular development for superovulation [6, 7]. These patients generally develop a number of embryos, but only one or two are returned to the patient at any one time to control the in-

Alan Trounson, MD Monash Immunology and Stem Cell Laboratories (MISCL) Monash University Clayton Campus, STRIP Building 75, Wellington Road 3800 Clayton, Vic. (Australia), Tel. +61 3 9905 0771, Fax +61 3 9905 0780 E-Mail [email protected]

cidence of multiple births. The remainder are frozen for later use by the patients. In Australia, some embryos from 40–60% of patients remain frozen at the completion of the couple’s infertility treatment. These may be discarded, donated to other couples or provided for research. The relative proportions are around 20, 7 and 70%, respectively. With informed consent, patients who are treated for infertility can provide embryos from which human ESCs can be derived. Embryos also may be provided by patients who are treated for preimplantation genetic diagnosis of chromosome errors or disease mutations. These embryos are typically aneuploid, or they have either recessive mutations such as cystic fibrosis or thalassemia or dominant mutations such as Huntington disease. The mutated embryos can be used to produce disease-specific ESCs. These mutated embryos are being studied to better understand the physiology and cause of the diseases and the possibilities for retarding or preventing expression of the disease phenotype using drug-screening methods. ESCs are generally considered an epiblast derivative, or even a type of germ stem cell [8] that can be maintained in the presence of basic fibroblast growth factor (bFGF) as an immortal and pluripotential cell type under strict culture conditions. Self-renewal of human ESCs (hESCs) involves the Wnt family signaling pathway [9]. Recently it has been shown that hESCs have receptors from the tyrosine receptor kinase (TRK) family that are responsive to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4 [10]. The presence of these neurotrophins in culture medium for hESCs reduces apoptosis, stabilizes chromosome ploidy under high throughput passaging and enables clonal derivation of hESCs in coculture and under feeder-free conditions. ESCs are prepared by culturing embryos to the morula or blastocyst stage on irradiated feeder cells after removal of the zona pellucida, the glycoprotein shell surrounding the embryo. The embryonic cells attach to the feeder layer, and the ICM, which is the source of the embryonic epiblast and ESCs, grows as a flattened colony. Human trophoblast cells generally do not grow well in culture, so the original ESC colony is mechanically separated from trophoblast and any differentiating cells. The dissected colonies are cut into small pieces and grown on new feeder cell carpets and passaged as dissected pieces or in bulk culture reseeded onto mitotically inactivated feeders after light trypsinization. There is a wide range of feeder cells that are appropriate for the maintenance of hESCs, including murine fetal fibroblasts (e.g., STO cells) [1, 2] and human cell lines, including human embryonic Use of Embryonic Stem Cells for Endocrine Disorders

fibroblasts [11], human uterine endometrium [12], human foreskin fibroblasts [13] and human adult bone marrow cells [14]. Other commercially available human cells are also in use for the maintenance of hESC and may be appropriate for deriving hESC from human embryos. Optimizing culture conditions for hESCs is extremely important and is discussed in considerable detail by Hoffman and Carpenter [15]. The selection criteria used for choosing human embryos for deriving hESCs will determine the eventual success rates for their production. In parallel to developmental potential, it is more efficient to derive hESCs from fresh rather than previously frozen embryos. Small numbers of selected fresh blastocyst-stage embryos, grown in coculture with human oviductal epithelial cells, were used by Reubinoff et al. [2] to produce six hESC lines after preliminary experiments involving around 30 embryos [6]. The six hESC lines were derived from 12 healthy blastocysts. This very high success rate can be compared with much larger numbers of frozen embryos (blastocysts) commonly used to derive hESCs donated by patients who have finished their in vitro fertilization treatments.

Endocrine Differentiation – Insulin-Producing Pancreatic Islet Cell Types

ESCs grown under nonadhesive conditions form embryoid bodies (EBs). After 7 days in the human, EBs consist of approximately 10,000 spontaneously differentiating cells, including progenitors of a wide range of cell types from mesoderm, endoderm and ectoderm lineages. In the mouse, EBs can be grown on plastic tissue culture plates in medium containing insulin, transferrin, selenium, glutamine and fibronectin and nestin+ cells can be selected after 4 days. These cells are expanded by replating onto poly-L-ornithine/laminin or poly-D-lysine/laminin in N2 medium supplemented with insulin, transferrin, progesterone, putrescine and selenite and with bFGF and epidermal growth factor for a further 7 days. Pancreatic differentiation is finally achieved in N2 medium containing nicotinamide after 13–19 days. This basic differentiation program was used by Lumelsky et al. [16], Rajagopal et al. [17] and Sipione et al. [18] to produce insulin- and C-peptide-expressing cells, but not cells that coexpress insulin and C-peptide. Retinoic acid induces the homeodomain-containing transcription factor Pdx1+, which is a critical regulator for formation of the developing pancreas in endoderm cells when added to difHorm Res 2007;67(suppl 1):28–31

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ferentiating mouse ESCs on day 4 of differentiation as EBs [19]. The genes expressed by these cells include the early pancreatic endoderm markers Ptf1a, Foxa2, Hnf1- and Hnf6. Miyazaki et al. [20] used another approach to regulate expression of Pdx-1. They inserted the Tet-off system for Pdx-1 expression into the ROSA26 locus and used doxycycline (Dox) to control Pdx-1 transgene expression during differentiation. When Dox is absent, the cells express Pdx-1. They tested the Tet-off system of Pdx-1 expression during EB formation and thereafter, and from the expansion stage for nestin selection. Results showed that Pdx-1 induces the expression of genes specific to pancreatic cells (insulin 2, somatostatin, prohormone convertase 2, Kir6.2, glucokinase, P48, Nkx2.2, Hnf6, Pax4, Pax6 and neurogenin3) when activated during EB formation. These genes were not enhanced when Pdx-1 was activated after EB formation. However, expression of insulin 1, glucagon, Pp gene or Glut2 genes was not detected, which suggests that the differentiated cells either were of the neural lineage or were immature compared with adult pancreatic islet cells. Neuronal cells express only insulin 2, whereas pancreatic cells express both insulin 1 and insulin 2. Liver and yolk sac express mostly insulin 2 and very small amounts of insulin 1. Culture of EBs in high concentrations of monothioglycerol and longer exposure to 15% fetal calf serum favours the development of Flk-1+ cells, diminishing the neuronal potential. ESC-derived EBs grown under these conditions, followed by serum-free conditions, result in cells that express insulin 1 and insulin 2, Pdx-1 and Sox17 (endoderm marker) and coexpress C-peptide and glucagon [21]. It was also reported that the P13K inhibitor is necessary to induce coexpression of insulin and C-peptide in the final maturation stage of pancreatic differentiation using the conventional differentiation protocol [22]. There is a close relationship between neural and pancreatic cell types, so given the variety of responses observed in insulin and C-peptide coexpression, and the incomplete induction of pancreatic islet functional characteristics, Kania et al. [23] argue that selection or enrichment of nestin-positive cells be avoided because these are already committed to their fate as neural cells. The constitutive expression of Sox2 inhibits neuronal differentiation but apparently preserves neural progenitor properties [22]. Pancreatic differentiation can also be obtained without FGF-2 [24, 25]. It has also been recommended [23] to delete FGF-2 from the induction conditions because it drives proliferation and differentiation of neural progenitors and the formation of neurospheres and Nes30

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tin+ cells in vitro. Transplantation of these ESC-derived cells in the mouse failed to reverse diabetes and resulted in teratomas [18, 26]. Human ESCs and -Islet Cells

Similar differentiation strategies using human ESCs result in insulin-producing cells that coexpress insulin and C-peptide or insulin and glucagon or insulin and somatostatin, and express a number of pancreatic genes that appear to be similar to immature pancreatic -islet cells [27]. In a study by Brolen et al. [28], human ESCs were allowed to differentiate spontaneously in prolonged (34 day), adherent, two-dimensional cultures on mitotically inactivated mouse embryonic fibroblasts. After 14–19 days, they produced a heterogeneous population of cells, some of which expressed PDX-1, FOXA2 and ISL1. These cells did not produce insulin. These regions of the cultured ESCs were mechanically dissected and transplanted, together with dorsal pancreases of E11.5 or E13.5 mouse embryos, beneath the kidney capsule of severe combined immunodeficient mice for 8 weeks. Insulin+ human cells organized into islet-like 5–25 cell clusters. These insulin+ cells coexpressed proinsulin and C-peptide and the key cell transcription factors FOXA2, PDX-1 and ISL1. No insulin+ cells were found if the embryonic mouse tissue was not cotransplanted, but when hESCs were transferred alone, glucagon+ and amylase+ cells of hESC origin were found localized in ducts. These data indicate that to induce development of mature pancreatic islet cells, components of the embryonic foregut mesenchyme may need to be included to provide instructions for differentiation. This situation is analogous to that observed for differentiation of human ESCs into mature prostate tissue [29]. Similar approaches may be productive in a wide range of secretory tissues to treat endocrine disorders.

Conclusions

There is a close association between neural and pancreatic cell types and markers, and many of the cells produced by current strategies involving ESCs may be more neural than pancreatic. When using ESCs to induce development of mature pancreatic islet cells, components of the embryonic foregut mesenchyme may need to be included to provide the instructions for differentiation. Similar approaches may be productive in a wide range of secretory tissues to treat endocrine disorders. Trounson

References 1 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145–1147. 2 Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A: Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404. 3 Trounson A: The production and directed differentiation of human embryonic stem cells. Endocr Rev 2006;27:208–219. 4 Conley BJ, Trounson AO, Mollard R: Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn Ther 2004;19:218–223. 5 Sathananthan AH, Trounson A: Human embryonic stem cells and their spontaneous differentiation. Ital J Anat Embryol 2005; 110:151–157. 6 Trounson AO: The derivation and potential use of human embryonic stem cells. Reprod Fertil Dev 2001;13:523–532. 7 MacLachlan V, Besanko M, O’Shea F, Wade H, Wood C, Trounson A, Healy DL: A controlled study of luteinizing hormone-releasing hormone agonist (buserelin) for the induction of folliculogenesis before in vitro fertilization. N Engl J Med 1989; 320: 1233– 1237. 8 Zwaka TP, Thomson JA: A germ cell origin of embryonic stem cells? Development 2005; 132:227–233. 9 Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH: Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63. 10 Pyle AD, Lock LF, Donovan PJ: Neurotrophins mediate human embryonic stem cell survival. Nat Biotechnol 2006;24:344–350. 11 Richards M, Fong CY, Chan WK, Wong PC, Bongso A: Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.

Use of Embryonic Stem Cells for Endocrine Disorders

12 Rossant J: Stem cells from the mammalian blastocyst. Stem Cells 2001;19:477–482. 13 Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L: A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409. 14 Cheng L, Hammond H, Ye Z, Zhan X, Dravid G: Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 2003; 21: 131–142. 15 Hoffman LM, Carpenter MK: Characterization and culture of human embryonic stem cells. Nat Biotechnol 2005;23:699–708. 16 Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R: Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001;292:1389–1394. 17 Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA: Insulin staining of ES cell progeny from insulin uptake. Science 2003;299:363. 18 Sipione S, Eshpeter A, Lyon JG, Korbutt GS, Bleackley RC: Insulin expressing cells from differentiated embryonic stem cells are not beta cells. Diabetologia 2004;47:499–508. 19 Micallef SJ, Janes ME, Knezevic K, Davis RP, Elefanty AG, Stanley EG: Retinoic acid induces Pdx1-positive endoderm in differentiating mouse embryonic stem cells. Diabetes 2005;54:301–305. 20 Miyazaki S, Yamato E, Miyazaki J: Regulated expression of pdx-1 promotes in vitro differentiation of insulin-producing cells from embryonic stem cells. Diabetes 2004; 53: 1030–1037.

21 Ku HT, Zhang N, Kubo A, O’Connor R, Mao M, Keller G, Bromberg JS: Committing embryonic stem cells to early endocrine pancreas in vitro. Stem Cells 2004;22:1205–1217. 22 Hansson M, Tonning A, Frandsen U, Petri A, Rajagopal J, Englund MC, Heller RS, Hakansson J, Fleckner J, Skold HN, Melton D, Semb H, Serup P: Artifactual insulin release from differentiated embryonic stem cells. Diabetes 2004;53:2603–2609. 23 Kania G, Blyszczuk P, Wobus AM: The generation of insulin-producing cells from embryonic stem cells – a discussion of controversial findings. Int J Dev Biol 2004; 48: 1061–1064. 24 Blyszczuk P, Czyz J, Kania G, Wagner M, Roll U, St-Onge L, Wobus AM: Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci USA 2003;100:998–1003. 25 Blyszczuk P, Asbrand C, Rozzo A, Kania G, St-Onge L, Rupnik M, Wobus AM: Embryonic stem cells differentiate into insulinproducing cells without selection of nestinexpressing cells. Int J Dev Biol 2004; 48: 1095–1104. 26 Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, Petersen BE: Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am J Pathol 2005; 166: 1781– 1791. 27 Segev H, Fishman B, Ziskind A, Shulman M, Itskovitz-Eldor J: Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 2004;22:265–274. 28 Brolen GK, Heins N, Edsbagge J, Semb H: Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-celllike cells. Diabetes 2005;54:2867–2874. 29 Taylor RA, Cowin PA, Cunha GR, Pera M, Trounson AO, Pedersen J, Risbridger GP: Formation of human prostate tissue from embryonic stem cells. Nat Methods 2006; 3: 179–181.

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Plenary Lecture 5

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):32 DOI: 10.1159/000097548

Published online: February 15, 2007

Controversial Debate: Growth Hormone and Glucose Metabolism Dana S. Hardin Department of Pediatrics, Ohio State University, Columbus, Ohio, USA

In children, growth hormone (GH) plays a significant physiological role by promoting linear growth; in adults its effects are more metabolic in nature. However, the precise metabolic effects of GH therapy are unclear. This plenary session presented opposing views regarding the impact of GH on glucose metabolism. Jens O.L. Jørgensen of Aarhus University Hospital, Aarhus, Denmark, argues the case that GH does not improve glucose metabolism. GH induction of insulin resistance during periods of fasting is associated with enhanced lipid oxidation and protein conservation, constituting an apparently favorable metabolic adaptation. In addition, GH replacement seems to afford safe glycemic control when administered according to standard regimens. However, in acromegalic subjects GH levels are el-

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evated during the postprandial period, which may result in glucose intolerance and overt diabetes mellitus. Thus, he examines the implications of GH replacement therapy in GH-deficient patients. The opposing view is presented by David Dunger, of Addenbrooke’s Hospital, Cambridge, UK, who argues that very low doses of GH can improve postabsorptive insulin sensitivity in direct relation to increased free insulin-like growth factor I, reduce fasting glucose levels and potentially improve -cell secretory capacity in normal subjects and in those with GH deficiency. Both experts build persuasive cases and call for further studies to improve our understanding of GH’s role in glucose metabolism.

Dana Sue Hardin, MD Department of Pediatrics Ohio State University, 700 Children’s Drive ED543 Columbus, OH 43205 (USA) Tel. +1 614 722 4436, Fax +1 614 722 4440, E-Mail [email protected]

Viewpoint 1

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):33–36 DOI: 10.1159/000097549

Published online: February 15, 2007

Growth Hormone and Insulin Resistance Jens O.L. Jørgensen Rune L. Larsen Louise Møller Morten Krag Niels Jessen Helene Nørrelund Jens S. Christiansen Niels Møller Medical Department M (Endocrinology and Diabetes) and Institute of Experimental Clinical Research, Aarhus University Hospital, Aarhus, Denmark

Key Words Growth hormone Growth hormone deficiency Insulin resistance Growth hormone replacement

Abstract Background: Experimental data in human subjects demonstrate that growth hormone (GH) acutely inhibits glucose disposal in skeletal muscle. The insulin-antagonistic effects are clinically relevant since active acromegaly is accompanied by glucose intolerance, whereas children with GH deficiency may develop fasting hypoglycemia. At the same time, GH stimulates the turnover and oxidation of free fatty acids (FFAs), and there is experimental evidence to suggest a causal link between elevated FFA levels and insulin resistance in skeletal muscle. During fasting, the induction of insulin resistance by GH is associated with enhanced lipid oxidation and protein conservation, which seems to constitute a favorable metabolic adaptation. Conclusions: Observational data in GH-deficient adults do not indicate that GH replacement is associated with significant impairment of glucose tolerance; however, care should be taken to avoid overdosing and to monitor glycemic control. Copyright © 2007 S. Karger AG, Basel

Introduction

A link between the pituitary gland and glucose metabolism was originally observed by Houssay, who in 1936 recorded increased sensitivity to insulin in hypo-

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physectomized animals, which was reversed by administration of anterior pituitary extracts [1]. In 1965, Rabinowitz and colleagues showed that infusion of high-dose growth hormone (GH) into the brachial artery of healthy adults reduced forearm glucose uptake in both muscle and adipose tissue [2]. This was paralleled by increased muscular uptake and oxidation of free fatty acids (FFAs). Moreover, GH completely blocked the effects of insulin when the two hormones were administered together. Despite years of subsequent research, the mechanisms whereby GH causes insulin resistance remain unclear. This review will focus on in vivo data obtained from subjects with abnormal GH status using the euglycemic glucose clamp to assess insulin sensitivity. In addition, recent data regarding the interference of GH with insulin signaling in skeletal muscle of healthy subjects exposed acutely to GH will be discussed.

Acromegaly before and after Treatment

Møller and associates studied substrate metabolism and insulin sensitivity in six newly diagnosed acromegalic patients before and several months after successful transsphenoidal adenomectomy [3]. The methods included indirect calorimetry, a glucose tracer, measurement of substrate exchanges across the forearm and a hyperinsulinemic, euglycemic glucose clamp. In the basal state, plasma levels of insulin and glucose were significantly elevated before surgery, and this was associated with reduced forearm uptake of glucose and increased

Jens O.L. Jørgensen, MD Medical Department M Aarhus University Hospital, Aarhus Sygehus DK–8000 C Aarhus (Denmark) Tel. +45 8949 2025, Fax +45 8949 2010, E-Mail [email protected]

hepatic glucose output. Moreover, the rate of lipid oxidation was also increased in active acromegaly, which normalized after surgery. The glucose infusion rate during clamping was abnormally low in active acromegaly and became normalized with surgery. The latter implies GHinduced resistance to insulin-stimulated glucose uptake in skeletal muscle compatible with the observations made in the basal state. In addition, failure of insulin to suppress lipid oxidation was also observed in the untreated state. More recently, pegvisomant, a specific GH antagonist, has been developed and approved for treating acromegaly [4]. Numerous studies have reported that administration of pegvisomant improves glucose tolerance in acromegalic patients [5], and recent observations using the glucose clamp technique have shown that pegvisomant also improves insulin sensitivity in acromegaly [Lindberg et al., unpublished data].

Acute Effects of GH on Insulin Sensitivity in GH-Deficient Patients

The impact of replacing once daily (evening) injection with a 10-hour intravenous infusion of either saline or low-dose GH (35 g/hour) beginning the evening before the study was evaluated in a study of adolescent GH-deficient patients receiving GH replacement therapy [6]. Continued GH infusion was associated with reduced basal rates of glucose as compared to saline-treated and healthy, untreated control subjects. Reciprocal changes were observed regarding lipid oxidation. Insulin sensitivity was increased relative to control subjects during saline infusion and became reduced during GH infusion to a level comparable to the control group.

Long-Term Discontinuation of GH Replacement in GH-Deficient Patients in the Transition Phase

The impact of discontinuing GH replacement after completion of longitudinal growth on body composition and insulin sensitivity was assessed in a double-blind, placebo-controlled, parallel study [7]. The patients were randomized to receive either continued GH replacement or placebo for 12 months followed by 12 months of openlabel GH therapy in both groups. Body composition and insulin sensitivity were measured at baseline and after 12 and 24 months of therapy, respectively. In the group receiving continued GH therapy, no significant changes were recorded in either body composition or insulin sen34

Horm Res 2007;67(suppl 1):33–36

sitivity. By contrast, subjects receiving placebo treatment experienced an increase in insulin sensitivity despite a concomitant increase in fat mass. Following resumption of GH treatment in the placebo group, fat mass decreased together with insulin sensitivity. These data imply that the direct insulin-antagonistic effects of GH are not fully balanced by the favorable long-term effects on body composition.

Impact of GH on Substrate Metabolism and Insulin Sensitivity during Fasting

Endogenous GH secretion in normal subjects is stimulated during fasting and is accompanied by low levels of insulin. Concomitantly, substrate metabolism is shifted towards lipid oxidation. When glycogen stores are depleted, glucose oxidation relies on gluconeogenesis from protein. A series of studies by Nørrelund and colleagues have demonstrated that GH in the fasting state (42 h) is essential for the ongoing release and oxidation of FFA [8]. At the same time, insulin sensitivity in terms of muscle uptake of glucose is reduced by GH. These effects translate into reduced protein breakdown in muscle and at the whole-body level. The lipolytic effects of GH during fasting, which could be considered the natural domain of GH, thus seem to result in protein sparing.

Studies on the Mechanisms whereby GH Inhibits Muscle Glucose Uptake

Several studies performed in normal subjects and subjects with type 2 diabetes mellitus (T2DM) suggest a causal role of FFA for inhibiting insulin-stimulated glucose disposal in skeletal muscle [9]. It is therefore tempting to hypothesize that stimulation of lipolysis also influences the insulin-antagonistic effects of GH. In support of this theory, Nielsen and colleagues demonstrated that administration of acipimox, a nicotinic acid derivative which inhibits the hormone-sensitive lipase, was able to suppress GH-induced FFA release in GH-deficient patients [10]. At the same time, use of the glucose clamp technique showed that acipimox abrogated GH-induced insulin resistance. Studies by Shulman indicate that these putative effects of FFA involve inhibition of the activity of insulin signaling proteins, especially IRS1-associated PI3 kinase, important for the translocation and activation of GLUT4 in skeletal muscle [9]. Somewhat surprisingly, Jessen and colleagues were not able to demonstrate Jørgensen/Larsen/Møller/Krag/Jessen/ Nørrelund/Christiansen/Møller

GH

Fasting Lipolysis IGF-I

Glucose Oxidation Protein Sparing

Fig. 1. Model of GH action on substrate metabolism during fast-

ing: A hypothetical and simplified model of GH action on substrate metabolism during fasting. Hepatic IGF-I production is suppressed by fasting and the low circulating IGF-I levels amplify pituitary GH release. Subsequently, GH actions are partitioned towards lipolysis and lipid oxidation. This ultimately translates into protein sparing by impeding the demand for glucose oxidation and thus gluconeogenesis from protein.

any significant suppressive effect of GH on PI3 kinase activity in normal subjects despite the fact that GH significantly stimulated lipolysis and induced insulin resistance [11]. That study provided evidence of GH signaling in muscle biopsies in terms of signal transducer and activator of transcription 5b tyrosine phosphorylation and suppressor of cytokine signaling 3 mRNA expression [Nielsen et al., unpublished data].

matter. Growth hormone is usually administered as daily subcutaneous injections in the evening, and the dose is tailored to maintain insulin-like growth factor I levels within the normal range for age and sex [12]. With this regimen, GH replacement therapy seems to be safe in regards to glycemic control [13]. Whether the favorable effects of GH on body composition and physical fitness may fully balance the metabolic effects remains controversial. During fasting the insulin-antagonistic effects of GH on glucose disposal may constitute an important and favorable adaptation by impeding the demand of gluconeogenesis from protein [8]. As such, by combining lipolysis and protein sparing, GH is a unique counterregulatory hormone (fig. 1). The underlying molecular mechanisms remain unclear and they seem to differ from those observed in subjects with T2DM. Based on the study by Nielsen and associates, elevated FFA levels play a causal role [10]; however, there is no experimental human in vivo data to support the belief that insulin-signaling pathways are affected [11]. Many more studies are needed with particular attention focused on the dosing and timing of GH and insulin and the optimal time to obtain a muscle biopsy. It is also likely that newer methods such as gene arrays and proteomics may be important supplements to current assays, which only measure the activity of single proteins. Further research about the interaction of GH with glucose metabolism is clinically important not only for optimizing the treatment of GH-related disorders, but also for broadening our general understanding of the role of GH and other cytokines in such important conditions as critical illness and T2DM.

Discussion

Based on a continuing number of experiments since Houssay, Zierler and Rabinowitz, there is little doubt that GH quickly and strongly antagonizes the effects of insulin on glucose disposal in skeletal muscle. In subjects with acromegaly, GH levels are also elevated during the postprandial period, which may result in glucose intolerance and overt diabetes mellitus. Whether these effects of GH also have clinically important implications for GH replacement therapy in GH-deficient patients is another Growth Hormone and Insulin Resistance

Horm Res 2007;67(suppl 1):33–36

35

References 1 Houssay BA: The hypophysis and metabolism. N Engl J Med 1936;214:961–985. 2 Rabinowitz D, Klassen GA, Zierler KL: Effects of human growth hormone on muscle and adipose tissue metabolism in the forearm of man. J Clin Invest 1965;44:51–61. 3 Møller N, Schmitz O, Jørgensen JO, Astrup J, Bak JF, Christensen SE, Alberti KG, Weeke J: Basal- and insulin-stimulated substrate metabolism in patients with active acromegaly before and after adenomectomy. J Clin Endocrinol Metab 1992;74:1012–1019. 4 Kopchick JJ, Parkinson C, Stevens EC, Trainer PJ: Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly. Endocr Rev 2002; 23: 623– 646. 5 Jørgensen JO, Feldt-Rasmussen U, Frystyk J, Chen JW, Kristensen LO, Hagen C, Orskov H: Cotreatment of acromegaly with a somatostatin analog and a growth hormone receptor antagonist. J Clin Endocrinol Metab 2005;90:5627–5631.

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6 Jørgensen JOL, Møller J, Alberti KGMM, Schmitz O, Christiansen JS, Møller N: Marked effects of sustained low growth hormone (GH) levels on day-to-day fuel metabolism: studies in GH deficient patients and healthy untreated subjects. J Clin Endocrinol Metab 1993;77:1589–1596. 7 Nørrelund H, Vahl N, Juul A, Møller N, Alberti KGMM, Skakkebæk NE, Christiansen JS, Jørgensen JOL: Continuation of growth hormone (GH) therapy in GH-deficient patients during transition from childhood to adulthood: impact on insulin sensitivity and substrate metabolism. J Clin Endocrinol Metab 2000;85:1912–1917. 8 Nørrelund H: The metabolic role of growth hormone in humans with particular reference to fasting. Growth Horm IGF Res 2005; 15:95–122. 9 Shulman GI: Cellular mechanisms of insulin resistance. J Clin Invest 2000;106:171–176.

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10 Nielsen S, Møller N, Christiansen JS, Jørgensen JO: Pharmacological antilipolysis restores insulin sensitivity during growth hormone exposure. Diabetes 2001; 50: 2301– 2308. 11 Jessen N, Djurhuus CB, Jørgensen JO, Jensen LS, Møller N, Lund S, Schmitz O: Evidence against a role for insulin-signaling proteins PI 3-kinase and Akt in insulin resistance in human skeletal muscle induced by shortterm GH infusion. Am J Physiol Endocrinol Metab 2005;288:E194–E199. 12 Jørgensen JOL: Human growth hormone replacement therapy: pharmacological and clinical aspects. Endocr Rev 1991; 12: 189– 207. 13 Gotherstrom G, Svensson J, Koranyi J, Alpsten M, Bosaeus I, Bengtsson B, Johannsson G: A prospective study of 5 years of GH replacement therapy in GH-deficient adults: sustained effects on body composition, bone mass, and metabolic indices. J Clin Endocrinol Metab 2001;86:4657–4665.

Jørgensen/Larsen/Møller/Krag/Jessen/ Nørrelund/Christiansen/Møller

Viewpoint 2

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):37–42 DOI: 10.1159/000097550

Published online: February 15, 2007

Growth Hormone Effects on Glucose Metabolism David Dunger a Kevin Yuen b Burak Salgin a a

University Department of Paediatrics, Addenbrooke’s Hospital, Cambridge, UK; b Division of Endocrinology, Oregon Health and Science University, Portland, Oreg., USA

Key Words Growth hormone Insulin-like growth factor I Growth hormone deficiency Insulin sensitivity Insulin secretion

Abstract Background: The increased risk for development of type 2 diabetes mellitus (T2DM) in adults with growth hormone deficiency (GHD) has been attributed to insulin resistance arising from increased visceral fat accumulation and the putative effects of low insulin-like growth factor I (IGF-I) levels on pancreatic -cell mass and insulin secretion. Failure of GH replacement to reverse these abnormalities may reflect nonphysiological GH replacement or inability of the -cell to recover. Methods and Results: We have demonstrated in normal subjects and in those with GHD that very low doses of GH can improve postabsorptive insulin sensitivity in direct relation to increased free IGF-I, reduce fasting glucose levels and potentially improve -cell secretory capacity. Conclusions: These low doses of GH should prevent the development of T2DM in adult subjects, but this needs to be confirmed by long-term studies. Copyright © 2007 S. Karger AG, Basel

Introduction

Growth hormone (GH) is traditionally thought to contribute significantly to linear growth in childhood [1], but its metabolic actions predominate in adulthood. The

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Accessible online at: www.karger.com/hre

metabolic effects of GH on glucose homeostasis are complex and involve increased lipolysis and insulin antagonism resulting in glucose intolerance and insulin resistance [2–4]. In children with Turner syndrome [5, 6] and those born small for gestational age (SGA) [7–9], repeated administration of pharmacologic doses of recombinant GH leads to the induction of insulin resistance; in acromegalic adults chronic exposure to high GH levels predisposes to the development of T2DM [10, 11]. However, the adult GH deficiency (GHD) syndrome is also associated with insulin resistance and an increased risk for T2DM [12]. Low serum GH levels may contribute to the accelerated accumulation of central adiposity [12], while the chronically low serum insulin-like growth factor I (IGF-I) levels are probably responsible for increasing the risk of developing T2DM [13]. This is supported by the findings of Sandhu et al. [14], who reported, in a normal population, a predictive value of low serum IGF-I levels for the risk of development of glucose intolerance, independent of the variation in insulin sensitivity. Furthermore, low IGF-I levels have been associated with a reduction in the insulin disposition index [15]. In addition, there is evidence to suggest that IGF-I signaling through its receptor on the pancreatic -cells is important in maintaining -cell mass and insulin secretion [16, 17]. If we hypothesise that the risk of developing T2DM in adult GHD is associated with low serum levels of IGF-I, then GH replacement should improve glucose metabolism in these patients. However, a number of studies have

David Dunger, MD Department of Paediatrics, Addenbrooke’s Hospital University of Cambridge, Hills Road, Level 8, Box 116 Cambridge, CB2 2QQ (UK) Tel. +44 1223 336 886, Fax +44 1223 336 996, E-Mail [email protected]

shown a deterioration of glucose metabolism in adult GHD following GH replacement therapy [4, 18–20], which questions the suitability of current replacement GH doses.

Normal Physiology of the GH/IGF-I Axis

In the newborn, linear growth is largely regulated by nutrition and insulin secretion [21]. Serum GH levels are elevated in the newborn, particularly in those born SGA, but they are probably more important in the regulation of lipolysis and other non-IGF-I-mediated metabolic effects than in the regulation of endogenous IGF-I generation and linear growth [22]. Over the first 6 months of life, there is a gradual induction of GH-regulated growth that coincides with the appearance of growth hormonebinding protein in the circulation [23, 24], and by 18 months of age, linear growth is largely regulated through the GH/IGF-I axis. During childhood, serum GH and IGF-I levels continually rise, but with the onset of puberty, there is a dramatic increase in GH pulse amplitude with a concomitant increase in IGF-I generation that peaks at 1 year after peak height velocity [25–28]. The increased GH pulse amplitude during puberty induces insulin resistance, which is specific to muscle [29], thereby leading to a state of compensatory hyperinsulinaemia [30]. As the insulin resistance is specific to the muscle, other tissues are exposed to elevated serum insulin levels and this reduces the likelihood of GH-induced lipolysis and protein degradation [31]. The elevated serum insulin levels in turn progressively suppress insulin-like growth factor binding protein-1 (IGFBP-1) and sex hormone binding globulin generation during puberty [32, 33], thus enhancing the bioavailability of IGF-I and sex steroids [33]. The three hormones (GH, IGF-I and insulin) appear to act in concert during puberty to enhance anabolism and growth. However, at the end of puberty, levels of these hormones steadily decline, reaching prepubertal levels by the mid-20s. The mechanism whereby GH secretion is switched off remains unknown. It is possible that during puberty a threshold level of ‘free IGF-I’ is reached, which down-regulates the system, although GH, IGF-I and insulin probably continue to play an important role in later gains of lean body mass in males and in bone mass in both sexes after peak height velocity. In childhood and adolescence, the relationship between GH and IGF-I is linear, but there is evidence for a continuation of the nutritional regulation of growth [34]. Portal insulin levels have a role in regulation of the he38

Horm Res 2007;67(suppl 1):37–42

patic GH receptor [35, 36], and reduced portal insulin levels during fasting or in type 1 diabetes are partly responsible for reductions in hepatic IGF-I generation [35, 37]. Portal insulin concentrations also regulate the circulating levels of IGFBP-1, an inhibitor of IGF-I bioactivity [37, 38], and low serum levels of insulin reduce IGF-I bioavailability, thus activating the feedback drive for pituitary GH hypersecretion. During an extended fast, GH levels increase while GH pulse periodicity is decreased [39], which leads to enhanced lipolysis and survival in a way analogous to that seen in the neonate. Conversely, when portal insulin levels are elevated in obese individuals, hepatic IGF-I generation is enhanced while IGFBP-1 production is suppressed [40, 41], thus activating the feedback drive to reduce pituitary GH secretion [42, 43]. This pivotal role of insulin and nutrition in regulating the GH/IGF-I axis is responsible for the reciprocal changes in GH and IGF-I levels observed during starvation and in obesity. Therefore, very high serum GH levels are normally only seen when IGF-I concentrations are low, whilst low GH levels are only seen when IGF-I levels are high. The combinations of high GH and IGF-I or low GH and IGF-I, as in acromegaly and GHD, respectively, are never encountered in a normal physiological setting. In growing children and adults with normal nutritional intakes, variations in GH secretion and IGF-I generation are likely to be important determinants of growth and anabolism, respectively, and may determine postabsorptive insulin secretion and insulin sensitivity. Overnight GH effects on hepatic IGF-I mRNA generation and bioavailability may be partially masked by increases in IGFBP-1, but in the postabsorptive period the rise in insulin levels will suppress IGFBP-1 and potentially increase IGF-I bioavailability [44]. IGF-I is a potent insulin sensitiser [45] and it may also have an important role in sustaining insulin secretion [17] in addition to its direct effects on anabolism and muscle mass development [46– 49].

Metabolic Effects of GH in Normal Subjects and Adults with GHD

The relationships among variation in GH levels, IGF-I generation and metabolism have rarely been studied in normal subjects. We have examined the effects of 7 days of standard adult GHD replacement doses on glucose metabolism in normal subjects, and compared these to the effects of sequential GH dose reductions [50]. To our surprise, daily administration of the lowest GH dose Dunger /Yuen /Salgin

Growth Hormone Effects on Glucose Metabolism

0.5 Change in glucose from baseline (mmol/l)

(1.7 g/kg/day), which approximates the daily adult physiological GH production rate [51], reduced fasting glucose levels and improved insulin sensitivity. We hypothesised that this low GH dose improved insulin sensitivity by increasing postabsorptive IGF-I bioavailability, without exerting any direct effects on lipolysis. In another study, we demonstrated that this particular dose of GH suppressed hepatic glucose production and enhanced peripheral glucose uptake without inducing lipolysis, and that the improvement in insulin sensitivity was linked to an increase in IGF-I bioavailability [52]. These data supported the hypothesis that whereas high doses of GH will lead to elevated plasma free fatty acid levels [53, 54], which cause insulin resistance, lower doses of GH might enhance postabsorptive IGF-I bioavailability and thus improve insulin sensitivity. Our data in young healthy adults [50, 52] led us to hypothesise that low-dose GH replacement in adults with GHD should improve insulin action and reduce the risk of T2DM. Although GH replacement doses have been decreased in recent years, in line with the Growth Hormone Research Society recommendations [55], the GH doses used in recent studies are probably still too high [18, 19, 56]. We studied 25 adults with GHD who were randomised to receive either a fixed low GH dose (n = 13; dose 0.1 mg/d) or the standard GH dose (n = 12; dose titrated to normalise serum IGF-I levels according to sex and age; mean GH dose 0.5 mg/d), and 8 subjects (4 males) who agreed to act as untreated controls [57]. At baseline, no significant differences were observed in the three groups with respect to body mass index, waist circumference, fat and lean body mass. During the 12month treatment, serum IGF-I levels doubled in the standard-dose group, reaching levels of about 300 g/l by 3 months, which then remained stable over the next 9 months. No significant increases in total IGF-I and IGFBP-3 levels were seen in the low-dose GH group, with levels identical to those of the untreated controls. However, free IGF-I concentrations were elevated in both groups receiving GH; they increased by around 50% in the low-dose GH group and by 100% in the standarddose group. The effects of low-dose GH therapy on glucose homeostasis and insulin sensitivity were striking. Fasting blood glucose levels declined in all subjects, reaching a nadir at around 3 months and remaining there, at about 0.5 mmol/l below baseline, throughout the entire 12-month study period. There was no change in fasting glucose levels in the standard-dose group or the untreated controls (fig. 1). This apparent improvement in insulin sensitivity was confirmed using a one-step hyperinsu-

0

*

–0.5

*

*

*

–1.0 Baseline

1

3 6 Time (months)

9

12

Fig. 1. Changes in fasting glucose levels over 12 months with LGH (low GH dose; j), SGH (standard titrating GH dose; +) and in untreated controls (d). Symbols represent mean 8 SE where error bars are 1 SE. p ! 0.03 for LGH vs. untreated controls from baseline to month 6. * p ! 0.01 for LGH vs. untreated controls, and LGH vs. SGH at each time point. Reproduced with permission from Yuen et al. [57].

linaemic euglycaemic clamp at baseline and 12 months. In the low-dose GH group, we observed a significant improvement in insulin sensitivity without any changes in free fatty acid levels or body composition; whereas, insulin sensitivity remained unchanged in the standard-dose GH group, despite improvements in body composition, and free fatty acid levels were increased. Thus, it would appear that low-dose GH replacement, which avoids increases in lipolysis, can indeed improve glucose metabolism in GH-deficient adults.

Implications for GH Replacement in Adults with GHD

Chronic administration of standard GH replacement doses in GH-deficient adults promotes insulin antagonism and may increase the risk for development of T2DM. From our studies, we conclude that low GH doses of 1.7 g/kg/day or 0.1 mg/day are both physiological and more efficacious in improving glucose metabolism than standard GH doses titrated to normalise serum IGF-I levels. Low GH doses induced modest increments in free IGF-I without promoting lipolysis and thus contributed Horm Res 2007;67(suppl 1):37–42

39

to the beneficial effects on postabsorptive insulin sensitivity. However, it is yet to be determined whether -cells are able to recover in patients with GHD, and risk for T2DM may persist even with low-dose therapy. Unfortunately, we did not have sufficient subject numbers for a complete long-term follow-up [57], but we were surprised to note that subjects treated with the standard GH dose rapidly reverted to baseline. In contrast, in the low-dose group, low fasting glucose levels and improved insulin sensitivity and secretion were still present 6 months after discontinuing GH therapy [58]. In previous studies of normal subjects, we noted that low-dose GH therapy leads to time-dependent changes in total and free IGF-I [52]. After an initial week of treatment, total IGF-I levels rose but then declined in the second week, whereas free IGF-I levels continued to increase. When treatment was discontinued, the changes were reversed over the next 2 weeks. Thus, the effects of IGF-I on insulin secretion and insulin action may be much more complex than originally thought since subtle increases in IGF-I levels may also lead to alterations in IGF-I bioavailability and perhaps IGF-I action. In adults, GH and IGF-I levels decline with age, with individuals in the lowest tertile of IGF-I levels at increased risk for developing T2DM [14]. Recent animal data have suggested that IGF-I and insulin signaling, through their

respective receptors on the -cell, may be important in maintaining -cell mass [17]. In a double-blind crossover pilot study, we examined the effects of low-dose GH therapy in GH-sufficient subjects with impaired glucose tolerance. We observed reductions in glucose excursion and improvements in insulin secretion when the subjects were challenged with a standard 75 g oral glucose tolerance test [59]. These data not only suggest that a low GH dose, by modestly increasing free IGF-I levels, may have a role in enhancing postabsorptive insulin sensitivity, but also may improve long-term insulin secretory capacity, a feature critical to evaluating the risk of T2DM in GHtreated GH-deficient subjects.

Conclusion

GH has a significant physiological effect of promoting linear growth in childhood, but its metabolic effects are more pronounced in adulthood. High GH doses in GHD will increase lipolysis and lead to deterioration of insulin sensitivity. In contrast, we have shown that low doses of GH in normal subjects and in those with GHD may increase postabsorptive insulin sensitivity and could promote and preserve insulin secretion, reducing the risk for development of T2DM.

References 1 Isaksson OG, Lindahl A, Nilsson A, Isgaard J: Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 1987;8:426–438. 2 Davidson MB: Effect of growth hormone on carbohydrate and lipid metabolism. Endocr Rev 1987;8:115–131. 3 Bramnert M, Segerlantz M, Laurila E, Daugaard JR, Manhem P, Groop L: Growth hormone replacement therapy induces insulin resistance by activating the glucose-fatty acid cycle. J Clin Endocrinol Metab 2003;88: 1455–1463. 4 Giavoli C, Porretti S, Ronchi CL, Cappiello V, Ferrante E, Orsi E, Arosio M, Beck-Peccoz P: Long-term monitoring of insulin sensitivity in growth hormone-deficient adults on substitutive recombinant human growth hormone therapy. Metabolism 2004;53:740– 743. 5 Caprio S, Boulware SD, Press M, Sherwin RS, Rubin K, Carpenter TO, Plewe G, Tamborlane WV: Effect of growth hormone treatment on hyperinsulinemia associated with Turner syndrome. J Pediatr 1992; 120: 238– 243.

40

6 Sas T, de Muinck Keizer-Schrama S, Aanstoot HJ, Stijnen T, Drop S: Carbohydrate metabolism during growth hormone treatment and after discontinuation of growth hormone treatment in girls with Turner syndrome treated with once or twice daily growth hormone injections. Clin Endocrinol (Oxf) 2000;52:741–747. 7 Rosenfeld RG, Wilson DM, Dollar LA, Bennett A, Hintz RL: Both human pituitary growth hormone and recombinant DNA-derived human growth hormone cause insulin resistance at a postreceptor site. J Clin Endocrinol Metab 1982;54:1033–1038. 8 Piatti PM, Monti LD, Caumo A, Conti M, Magni F, Galli-Kienle M, Fochesato E, Pizzini A, Baldi L, Valsecchi G, Pontiroli AE: Mediation of the hepatic effects of growth hormone by its lipolytic activity. J Clin Endocrinol Metab 1999;84:1658–1663.

Horm Res 2007;67(suppl 1):37–42

9 Woods KA, van Helvoirt M, Ong KK, Mohn A, Levy J, de Zegher F, Dunger DB: The somatotropic axis in short children born small for gestational age: relation to insulin resistance. Pediatr Res 2002;51:76–80. 10 Hansen I, Tsalikian E, Beaufrere B, Gerich J, Haymond M, Rizza R: Insulin resistance in acromegaly: defects in both hepatic and extrahepatic insulin action. Am J Physiol 1986; 250:E269–E273. 11 Wasada T, Aoki K, Sato A, Katsumori K, Muto K, Tomonaga O, Yokoyama H, Iwasaki N, Babazono T, Takahashi C, Iwamoto Y, Omori Y, Hizuka N: Assessment of insulin resistance in acromegaly associated with diabetes mellitus before and after transsphenoidal adenomectomy. Endocr J 1997; 44: 617–620. 12 Simpson H, Savine R, Sonksen P, Bengtsson BA, Carlsson L, Christiansen JS, Clemmons D, Cohen P, Hintz R, Ho K, Mullis P, Robinson I, Strasburger C, Tanaka T, Thorner M: Growth hormone replacement therapy for adults: into the new millennium. Growth Horm IGF Res 2002;12:1–33.

Dunger /Yuen /Salgin

13 Sesti G, Sciacqua A, Cardellini M, Marini MA, Maio R, Vatrano M, Succurro E, Lauro R, Federici M, Perticone F: Plasma concentration of IGF-I is independently associated with insulin sensitivity in subjects with different degrees of glucose tolerance. Diabetes Care 2005;28:120–125. 14 Sandhu MS, Heald AH, Gibson JM, Cruickshank JK, Dunger DB, Wareham NJ: Circulating concentrations of insulin-like growth factor-I and development of glucose intolerance: a prospective observational study. Lancet 2002;359:1740–1745. 15 Hart ‘t LM, Fritsche A, Rietveld I, Dekker JM, Nijpels G, Machicao F, Stumvoll M, van Duijn CM, Haring HU, Heine RJ, Maassen JA, van Haeften TW: Genetic factors and insulin secretion: gene variants in the IGF genes. Diabetes 2004;53(suppl 1):S26–S30. 16 van Haeften TW, Twickler TB: Insulin-like growth factors and pancreas beta cells. Eur J Clin Invest 2004;34:249–255. 17 Ueki K, Okada T, Hu J, Liew CW, Assmann A, Dahlgren GM, Peters JL, Shackman JG, Zhang M, Artner I, Satin LS, Stein R, Holzenberger M, Kennedy RT, Kahn CR, Kulkarni RN: Total insulin and IGF-I resistance in pancreatic beta cells causes overt diabetes. Nat Genet 2006;38:583–588. 18 Spina LD, Soares DV, Brasil RR, da Silva EM, Lobo PM, Conceicao FL, Vaisman M: Glucose metabolism and visceral fat in GH deficient adults: 1 year of GH replacement. Growth Horm IGF Res 2004;14:45–51. 19 Spina LD, Soares DV, Brasil RR, Lobo PM, Lucia Conceicao F, Vaisman M: Glucose metabolism and visceral fat in GH deficient adults: two years of GH-replacement. Pituitary 2004;7:123–129. 20 Boguszewski CL, Meister LH, Zaninelli DC, Radominski RB: One year of GH replacement therapy with a fixed low-dose regimen improves body composition, bone mineral density and lipid profile of GH-deficient adults. Eur J Endocrinol 2005;152:67–75. 21 Ong KK, Emmett PM, Noble S, Ness A, Dunger DB, ALSPAC Study Team: Dietary energy intake at the age of 4 months predicts postnatal weight gain and childhood body mass index. Pediatrics 2006; 117:E503– E508. 22 Ogilvy-Stuart AL, Hands SJ, Adcock CJ, Holly JM, Matthews DR, Mohamed-Ali V, Yudkin JS, Wilkinson AR, Dunger DB: Insulin, insulin-like growth factor I (IGF-I), IGFbinding protein-1, growth hormone, and feeding in the newborn. J Clin Endocrinol Metab 1998;83:3550–3557. 23 Silbergeld A, Lazar L, Erster B, Keret R, Tepper R, Laron Z: Serum growth hormone binding protein activity in healthy neonates, children and young adults: correlation with age, height and weight. Clin Endocrinol (Oxf) 1989;31:295–303.

Growth Hormone Effects on Glucose Metabolism

24 Low LC, Tam SY, Kwan EY, Tsang AM, Karlberg J: Onset of significant GH dependence of serum IGF-I and IGF-binding protein 3 concentrations in early life. Pediatr Res 2001; 50:737–742. 25 Mauras N, Blizzard RM, Link K, Johnson ML, Rogol AD, Veldhuis JD: Augmentation of growth hormone secretion during puberty: evidence for a pulse amplitude-modulated phenomenon. J Clin Endocrinol Metab 1987;64:596–601. 26 Mauras N, Rogol AD, Veldhuis JD: Increased hGH production rate after low-dose estrogen therapy in prepubertal girls with Turner’s syndrome. Pediatr Res 1990; 28: 626– 630. 27 Smith CP, Dunger DB, Williams AJ, Taylor AM, Perry LA, Gale EA, Preece MA, Savage MO: Relationship between insulin, insulinlike growth factor I, and dehydroepiandrosterone sulfate concentrations during childhood, puberty, and adult life. J Clin Endocrinol Metab 1989;68:932–937. 28 Martha PM Jr, Goorman KM, Blizzard RM, RogoI AD, Veldhuis JD: Endogenous growth hormone secretion and clearance rates in normal boys as determined by deconvolution analysis: relationship to age, pubertal status and body mass. J Clin Endocrinol Metab 1992;74:336–344. 29 Arslanian SA, Kalhan SC: Correlations between fatty acid and glucose metabolism. Potential explanation of insulin resistance of puberty. Diabetes 1994;43:908–914. 30 Caprio S, Plewe G, Diamond MP, Simonson SC, Boulware SD, Sherwin SS, Tamborlane WV: Increased insulin secretion during puberty: a compensatory response to reductions in insulin sensitivity. J Pediatr 1989; 114:963–967. 31 Arslanian SA, Kalhan SC: Protein turnover during puberty in normal children. Am J Physiol 1996;270:E79–E84. 32 Holly JM, Smith CP, Dunger DB, Edge JA, Biddlecombe RA, Williams AJ, Howell R, Chard T, Savage MO, Rees LH, et al.: Levels of the small insulin-like growth factor-binding protein are strongly related to those of insulin in prepubertal and pubertal children but only weakly so after puberty. J Endocrinol 1989;121:383–387. 33 Holly JM, Smith CP, Dunger DB, Howell RJ, Chard T, Perry LA, Savage MO, Cianfarani S, Rees LH, Wass JA: Relationship between the pubertal fall in sex hormone binding globulin and insulin-like growth factor binding protein-I. A synchronised approach to pubertal development? Clin Endocrinol (Oxf) 1989;31:277–284. 34 He Q, Karlberg J: BMI in childhood and its association with height gain, timing of puberty, and final height. Pediatr Res 2001; 49: 244–251.

35 Taylor AM, Dunger DB, Preece MA, Holly JM, Smith CP, Wass JA, Patel S, Tate VE: The growth hormone independent insulin-like growth factor-I binding protein BP-28 is associated with serum insulin-like growth factor-I inhibitory bioactivity in adolescent insulin-dependent diabetics. Clin Endocrinol (Oxf) 1990;32:229–239. 36 Massa G, Dooms L, Bouillon R, Vanderschueren-Lodeweyckx M: Serum levels of growth hormone-binding protein and insulin-like growth factor I in children and adolescents with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1993; 36: 239–243. 37 Katz LE, DeLeon DD, Zhao H, Jawad AF: Free and total insulin-like growth factor (IGF)-I levels decline during fasting: relationships with insulin and IGF-binding protein-1. J Clin Endocrinol Metab 2002; 87: 2978–2983. 38 Lee PDK, Conover CA, Powell DR: Regulation and function of insulin like growth factor-binding protein-1. Proc Soc Exp Biol Med 1993;204:4–29. 39 Ho KY, Veldhuis JD, Johnson ML, Furlanetto R, Evans WS, Alberti KGMM, Thorner MO: Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone secretion in man. J Clin Invest 1988;81:968–975. 40 Conover CA, Lee PDK, Kanaley JA, Clarkson JT, Jensen MD: Insulin regulation of insulin-like growth factor binding protein-1 in obese and nonobese humans. J Clin Endocrinol Metab 1992;74:1355–1360. 41 Frystyk J, Vestbo E, Skjaerbaek C, Mogensen CE, Orskov H: Free insulin-like growth factors in human obesity. Metabolism 1995;44: 37–44. 42 Veldhuis JD, Iranmanesh A, Ho KK, Waters MJ, Johnson ML, Lizarralde G: Dual defects in pulsatile growth hormone secretion and clearances subserve the hyposomatotropism of obesity in man. J Clin Endocrinol Metab 1991;72:51–59. 43 Scacchi M, Pincelli AI, Cavagnini F: Growth hormone in obesity. Int J Obes Relat Metab Disord 1999;23:260–271. 44 Frystyk J, Grofte T, Skjaerbaek C, Orskov H: The effect of oral glucose on serum free insulin-like growth factor-I and -II in health adults. J Clin Endocrinol Metab 1997; 82: 3124–3127. 45 Simpson HL, Jackson NC, Shojaee-Moradie F, Jones RH, Russell-Jones DL, Sonksen PH, Dunger DB, Umpleby AM: Insulin-like growth factor I has a direct effect on glucose and protein metabolism, but no effect on lipid metabolism in type 1 diabetes. J Clin Endocrinol Metab 2004;89:425–432. 46 DeVol DL, Rotwein P, Sadow JL, Novakofski J, Bechtel PJ: Activation of insulin-like growth factor gene expression during workinduced skeletal muscle growth. Am J Physiol Endocrinol Metab 1990; 259:E89–E95.

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47 Yarasheski KE: Growth hormone effects on metabolism, body composition, muscle mass, and strength. Exerc Sport Sci Rev 1994;22:285–312. 48 Adams GR, Haddad F: The relationships between IGF-I, DNA content, and protein accumulation during skeletal muscle hypertrophy. J Appl Physiol 1996; 81:2509–2516. 49 Butler AA, LeRoith D: Control of growth by the somatropic axis: growth hormone and the insulin-like growth factors have related and independent roles. Annu Rev Physiol 2001;63:161–164. 50 Yuen K, Ong K, Husbands S, Chatelain P, Fryklund L, Gluckman P, Ranke M, Cook D, Rosenfeld R, Wass J, Dunger D: The effects of short-term administration of two low doses versus the standard GH replacement dose on insulin sensitivity and fasting glucose levels in young healthy adults. J Clin Endocrinol Metab 2002;87:1989–1995. 51 van den Berg G, Veldhuis JD, Frolich M, Roelfsema F: An amplitude-specific divergence in the pulsatile mode of growth hormone (GH) secretion underlies the gender difference in mean GH concentrations in men and premenopausal women. J Clin Endocrinol Metab 1996;81:2460–2467.

42

52 Yuen K, Frystyk J, Umpleby M, Fryklund L, Dunger D: Changes in free rather than total insulin-like growth factor-I enhance insulin sensitivity and suppress endogenous peak growth hormone (GH) release following short-term low-dose GH administration in young healthy adults. J Clin Endocrinol Metab 2004;89:3956–3964. 53 Moller N, Jorgensen JO, Schmitz O, Moller J, Christiansen J, Alberti KG, Orskov H: Effects of a growth hormone pulse on total and forearm substrate fluxes in humans. Am J Physiol 1990;258:E86–E91. 54 Fowelin J, Attvall S, von Schenck H, Smith U, Lager I: Characterization of the insulin-antagonistic effect of growth hormone in man. Diabetologia 1991;34:500–506. 55 GHRS: Consensus guidelines for the diagnosis and treatment of adults with growth hormone deficiency: summary statement of the Growth Hormone Research Society (GHRS) workshop on adult growth hormone deficiency. J Clin Endocrinol Metab 1998; 83: 379–381.

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56 Bulow B, Link K, Ahren B, Nilsson AS, Erfurth EM: Survivors of childhood acute lymphoblastic leukaemia, with radiationinduced GH deficiency, exhibit hyperleptinaemia and impaired insulin sensitivity, unaffected by 12 months of GH treatment. Clin Endocrinol (Oxf) 2004;61:683–691. 57 Yuen KCJ, Frystyk J, White DK, Twickler TB, Koppeschaar HP, Harris PE, Fryklund L, Murgatroyd PR, Dunger DB: Improvement in insulin sensitivity without concomitant changes in body composition and cardiovascular risk markers following fixed administration of a very low growth hormone dose in adults with severe growth hormone deficiency. Clin Endocrinol (Oxf) 2005; 63: 428– 436. 58 Yuen K, Dunger D: Persisting effects on fasting glucose levels and insulin sensitivity after 6 months of discontinuation of a very low dose GH therapy in adults with severe GH deficiency. Clin Endocrinol (Oxf) 2006; 64: 549–555. 59 Yuen K, Wareham N, Frystyk J, Hennings S, Mitchell J, Fryklund L, Dunger D: Shortterm low-dose growth hormone administration in subjects with impaired glucose tolerance and the metabolic syndrome: effects on beta-cell function and post-load glucose tolerance. Eur J Endocrinol 2004;151:39–45.

Dunger /Yuen /Salgin

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):43–44 DOI: 10.1159/000097551

Published online: February 15, 2007

KIGS Highlights

The KIGS database (Pfizer International Growth Study Database) is a repository for information relating to growth hormone (GH) treatment in children. KIGS is the largest database of its kind in the world, containing data collected over the last 20 years from over 51,000 patients. Information from the KIGS database has greatly facilitated investigations of GH treatment for numerous indications. This workshop focused on use of GH as treatment in patients with Noonan syndrome (NS) and idiopathic short stature (ISS). NS is a syndrome characterized by facial dysmorphism, short stature and cardiovascular diseases and features such as pectus deformity, cryptorchidism and webbed neck. GH has been used in the treatment of short stature in NS for some years. Barto Otten of the University Medical Center Nijmegen in the Netherlands presented the results of somatropin therapy in patients with NS registered in the KIGS database. Full results of Dr. Otten’s presentation are to be published elsewhere. Gerhard Binder of University Children’s Hospital in Tübingen, Germany, presented data on genetics and responsiveness to GH in NS [1]. Corroborating the results of others, he found that children with PTPN11 mutations had lower insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) levels at onset of therapy and a lower response to GH after 2 years of therapy. Numerous attempts have been made to understand and clarify the condition referred to as ISS. Biomolecular studies have made it possible to know the etiology of some

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0043$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

cases of ISS, with the result that fewer cases are actually idiopathic. Since the first description by Goddard et al. in 1995 [2], many cases of ISS have been determined to be the result of errors in various steps of the growth factor cascade [3]. In 1996, a consensus meeting subclassified ISS according to two main groups: familial and nonfamilial short stature [4]. Each group contained two subpopulations: those who entered puberty late, and those who entered puberty at a normal time. Using such a scheme, it is obviously necessary for a patient to enter puberty before he or she can be classified. These topics were addressed in the presentation by Jan-Maarten Wit of Leiden University Medical Center in the Netherlands. ISS does not fit the recognised classic and nonclassic indications for treatment with GH, so GH was not officially approved for its treatment until 2003. However, some attempts were made prior to 2003 to use GH in ISS because paediatricians were very concerned about the lack of treatment options for ISS patients with short height and very poor prognosis and because ISS was comparable to other conditions such as Turner syndrome, for which GH was approved as therapy. The results of two studies, one a placebo-controlled randomised trial and a second a study of height gain based on GH dosage in children with ISS, prompted the United States Food and Drug Administration to approve GH therapy for ISS [5]. Edward O. Reiter of Baystate Children’s Hospital and Tufts University School of Medicine in Springfield, Mass., presented an update on GH therapy for ISS.

Michael Ranke of University Children’s Hospital in Tübingen, Germany, presented data on the growth response of children with ISS registered in the KIGS database. These data, which showed that long-term treatment with GH improved final height in patients with ISS and that the age of the patient, dose of GH used, and first-year responsiveness to GH were the main factors that predict a better final height outcome, are to be published in detail elsewhere. Feyza Darendeliler Department of Paediatrics, Istanbul Medical Faculty Istanbul University Istanbul, Turkey Ángel Ferrández Longás Paediatrics and Paediatric Endocrinology University Children’s Hospital Salud Miguel Servet Zaragoza, Spain

44

Horm Res 2007;67(suppl 1):43–44

References

1 Binder G, Neuer K, Ranke MB, Wittekind NE: PTPN11 mutations are associated with mild growth hormone resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5377–5381. 2 Goddard AD, Covello R, Luoh SM, Clackson T, Attie KM, Gesundheit N, Rundle AC, Wells JA, Carlsson LM: Mutations of the growth hormone receptor in children with idiopathic short stature. The Growth Hormone Insensitivity Study Group. N Engl J Med 1995;333:1093–1098. 3 Rosenfeld RG, Hwa V: Toward a molecular basis for idiopathic short stature. J Clin Endocrinol Metab 2004;89:1066–1067. 4 Ranke MB: Towards a consensus on the definition of idiopathic short stature. Consensus of Round Table Discussions. Horm Res 1996;45(suppl 2):64–66. 5 FDA Talk Paper: FDA approves Humatrope for short stature. Available at: http://www. fd a . gov/ bb s /topic s /A NS W E R S /2 0 03 / ANS01242.html. Accessed: October 20, 2006.

Darendeliler /Ferrández Longás

KIGS Highlights

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):45–49 DOI: 10.1159/000097552

Published online: February 15, 2007

Noonan Syndrome: Genetics and Responsiveness to Growth Hormone Therapy Gerhard Binder Nicola Wittekindt Michael B. Ranke Pediatric Endocrinology Section, University Children’s Hospital, Tübingen, Germany

Key Words PTPN11 Mutation SHP2 JAK2-STAT5 Noonan syndrome

Abstract Background: The autosomal-dominant Noonan syndrome (MIM 163950) is characterized by short stature, heart defects, characteristic facial dysmorphic features and other major and minor anomalies. Its incidence has been estimated to be 1 in 1,000 to 2,500 live births. Familial cases are frequent. Methods and Results: Recently, molecular data have suggested that deregulation of signaling through the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway was the main molecular basis of Noonan syndrome. The frequently detected upstream defects of this pathway are gain-of-function mutations of PTPN11, which are associated with a mild form of growth hormone (GH) resistance and insulin-like growth factor I (IGF-I) deficiency, presumably due to interference with the Janus kinase 2 and signal transducer and activator of transcription 5b (JAK2-STAT) signaling of the GH receptor. Present data suggest reduced GH responsiveness in these cases. Conclusions: Downstream defects of the RasMAPK pathway (like K-ras mutations) do not affect the JAK2STAT pathway, and therefore response to GH therapy is likely to be better in these cases. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0045$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

In 2001, Tartaglia et al. reported that approximately 50% of all individuals with Noonan syndrome carry heterozygote missense mutations of the gene PTPN11 (protein tyrosine phosphatase [PTP], nonreceptor type 11; MIM 176876), which is located on 12q24.1 [1]. This gene encodes the protein SHP-2 (for Src-homology 2 domaincontaining PTP), which contains two SH2 domains and one phosphotyrosine phosphatase domain. SHP-2 is a ubiquitously expressed cytoplasmic phosphatase that regulates signaling pathways by binding to and dephosphorylating phosphotyrosines [1]. Most of the missense mutations of PTPN11 described lie within exons 3, 7, 8 and 13 and are responsible for changing amino acids located at the binding interface between the N-terminal SH2 domain (N-SH2) and the single catalytic domain of phosphotyrosine phosphatase [1]. Experimental evidence indicates that these changes cause a breakdown in the close interaction between N-SH2 and the phosphatase and confer a gain of function to this protein, which is usually inactive when unbound [1–3]. Protein tyrosyl phosphorylation is regulated through the antagonistic interplay between cytoplasmic phosphatases like SHP-2 and tyrosine kinases like Janus kinase (JAK) 2 [4]. In vitro experiments indicate that JAK2 and signal transducer and activator of transcription (STAT)

Gerhard Binder, MD Paediatric Endocrinology Section, University Children’s Hospital Hoppe-Seyler-Strasse 1, DE–72076 Tübingen (Germany) Tel. +49 7071 298 3781, Fax +49 7071 294 157 E-Mail [email protected]

Membrane P JAK2 P

STATs SHP-2

P JAK2 P

Cytoplasm

Fig. 1. Two GH-signaling pathways and the predicted consequences of the PTPN11 mutations leading to a gain in function for SHP-2. SHP-2 deactivates JAK2 and STAT5b in vitro, and therefore may decrease signaling through this pathway, which induces IGF-I expression. In contrast, signaling through the Ras-MAPK pathway is upregulated by activation of SHP-2. Although the mechanism of action is still unknown, it possibly occurs through GAP binding.

Shc

GEF

P

Ras

Grb2

SHP-2 GAP

Raf

STAT5b MEK MAPK

P STAT5b P STAT5b

Nucleus

Insulin-Like Growth Factor I and Growth Hormone Axis

Previously, we predicted that JAK2-STAT5b signaling of the growth hormone (GH) receptor is suppressed in the presence of SHP-2 mutations. Thus, we prospectively studied the insulin-like growth factor-I (IGF-I)/GH axis in children with Noonan syndrome [7]. Twenty-nine Horm Res 2007;67(suppl 1):45–49

Gene transcription

Gene transcription (IGF-I)

5b are substrates of SHP-2, which deactivates these molecules [5]. Therefore, it has been postulated that SHP-2 is involved in a negative deregulation of JAK2-STAT signaling. In contrast, signaling through the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway is upregulated by activated SHP-2, though the mechanism of action is still unknown [4] (fig. 1). Recently, mutations of the KRAS gene, which encodes a specific Ras isoform (K-Ras), were detected in approximately 5% of individuals with Noonan syndrome who were negative for PTPN11 mutations [6]. These newly found mutations were shown to cause a slowing of the naturally occurring intrinsic inactivation of Ras (defective hydrolysis of guanosine triphosphate) and therefore to result in an upregulation of the Ras pathway downstream to SHP-2. The phenotypes associated with K-Ras and SHP-2 mutations are similar [6]. Therefore, deregulation of the Ras-MAPK pathway is very likely to be the central functional defect at the cellular level in Noonan syndrome (fig. 1).

46

Grb2

children were recruited for study on the basis of their phenotype. Subsequently their encoding PTPN11 exons were sequenced: 55% were positive for the SHP-2 mutation (mut+), and their IGF-I, insulin-like growth factor binding protein-3 (IGFBP-3) and GH levels were measured using radioimmunoassay [7]. IGF-I levels, expressed as age-related standard deviation score (SDS) for each patient, were 1 SDS lower in mut+ patients compared with individuals negative for the SHP-2 mutation (mut–) (fig. 2). The same was true for IGFBP-3 levels (fig. 2). In contrast, GH secretion trended toward higher levels in mut+ individuals (fig. 3). These findings suggest that a new form of mild GH resistance is associated with these PTPN11 mutations [7].

GH Responsiveness

Individuals with GH resistance due to SHP-2 mutations would be expected to show little response to a pharmacological GH therapy. Table 1 summarizes data on prepubertal children with Noonan syndrome from three GH therapy studies that grouped patients according to the presence or absence of SHP-2 mutations [7–9]. Two studies were prospective [7, 8] and the other was retrospective [9]. Overall, the numbers of children studied were small, and in the study by Ferreira et al. [9] only data for the prepubertal subjects were used to permit better comparison. All three studies evaluated change in height velocity (mean and SDS), which is considered to Binder/Wittekindt/Ranke

p = 0.005

SDS

p = 0.006

0

3

–0.5

2 1

–1.0 –1.07

+0.39

0

–1.5 –0.45

–1 –2.0 –2.06

–2

–2.5

Fig. 2. Summary of IGF-I and IGFBP-3 se-

rum levels at first presentation for children with Noonan syndrome with (mut+) and without (mut–) SHP-2 mutations, given as age-related SDS and median values. The median level of GH-dependent factors was significantly lower in the mut+ group [7].

–3

–3.0

–4

–3.5

mut+ IGF-I

ng/ml 30

p = 0.14

25 20 15

Fig. 3. Stimulated and spontaneous GH se-

10

cretion by children with Noonan syndrome with (mut+) and without (mut–) SHP-2 mutations. The GH mean and peak levels tended to be higher in the mut+ group, though the difference was not significant [7].

5

Table 1. Age and height of prepubertal children with Noonan syndrome and GH dose administered in three studies reporting the effect of SHP-2 mutations on GH responsiveness [7–9]

0

10.5

6.2

mut+ mut– Peak GH (arginine)

Study Binder 2005 [7] Limal 2006 [8] Ferreira 2005 [9]

Mut+ Mut– Mut+ Mut– Mut+ Mut–

–5

mut–

mut+ IGFBP-3

p = 0.091 40 35 30 25 20 15 10 5 0

12.6 7.1

mut+ Peak

mut–

mut–

p = 0.075 8 7 6 5 4 3 2 1 0

3.79 2.56

mut+ Mean

mut–

GH (spontaneous)

n

Age years

Height SDS

Dose mg/kg/week

8 3 15 10 5 3

7.4 6.3 10.4 10.3 11.2 8.0

–3.46 –3.80 –3.50 –3.00 –3.44 –3.00

0.29 0.35 0.30 0.30 0.33 0.32

For the study by Ferreira et al. only data for the prepubertal children were used for better comparison.

Noonan Syndrome

Horm Res 2007;67(suppl 1):45–49

47

12

SHP-2 mut+

Ferreira 2005

SHP-2 mut– 10

8

6

4

2 Height velocity during GH therapy (cm/year)

0

1

2

*

Binder 2005

12

be one of the best parameters for assessing GH responsiveness because it integrates all effects on growing bone before and after initiation of GH therapy. Baseline height and GH dose (which were dose levels recommended for individuals with Turner syndrome) did not differ significantly among the three studies, though the children from our study in Tübingen [7] were the youngest. The data shown in figure 4 suggest that SHP-2 mut– individuals had a better growth response than did mut+ subjects. The only statistically significant difference in growth velocity between mut+ and mut– subjects occurred after 1 year of therapy in the children from the Tübingen study. However, data on changes in height SDS and (near) final height are still too scarce to draw any definitive conclusions.

10

Noonan Syndrome and Tumorigenesis 8

6

4

2 0

1

2

12 Limal 2006

Defects of the Ras-MAPK pathway have been implicated in tumorigenesis for many years [10]. Recently, somatic mutations of SHP-2 were found in different forms of leukemia [2, 3]. Nearly all these missense mutations are different from those found as germline mutations in Noonan syndrome [2, 3]. In vitro data indicate that the gain-of-function due to these somatic mutations is more extensive than that due to the mutations found in Noonan syndrome [2, 3].

10

Conclusions

8

6

4

2 0

1 Year of therapy

2

Fig. 4. Comparison of height velocity from three studies before

and during the first 2 years of GH therapy in prepubertal children with Noonan syndrome both with (mut+) and without (mut–) the SHP-2 mutation [7–9]. Mut– patients performed better than mut+ patients, but the only statistically significant difference was detected after the first year of therapy among patients in the Tübingen study by Binder et al. [7].

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A primary molecular mechanism of action underlying the Noonan phenotype is deregulation of signaling through the Ras-MAPK pathway. Defects in the Ras pathway upstream to SHP-2 are found in approximately 50% of individuals with Noonan syndrome and are associated with a mild form of GH resistance and IGF-I deficiency. Present data suggest that a consequence of this mutation is reduced GH responsiveness, but this has yet to be proven. Downstream defects of the Ras pathway, such as K-ras defects present in approximately 2.5% of all individuals with Noonan syndrome and in 5% of mut– individuals, do not affect the JAK2-STAT pathway. Therefore, response to GH therapy among patients with these mutations is likely to be better.

Binder/Wittekindt/Ranke

References 1 Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H, van der Burgt I, Crosby AH, Ion A, Jeffery S, Kalidas K, Patton MA, Kucherlapati RS, Gelb BD: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468. 2 Keilhack H, David FS, McGregor M, Cantley LC, Neel BG: Diverse biochemical properties of SHP2 mutants. Implications for disease phenotypes. J Biol Chem 2005; 280: 30984– 30993. 3 Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, Cordeddu V, Zampino G, Burgt I, Palleschi A, Petrucci TC, Sorcini M, Schoch C, Foa R, Emanuel PD, Gelb BD: Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006; 78: 279–290.

Noonan Syndrome

4 Neel BG, Gu H, Pao L: The ‘Shp’ing news: SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem Sci 2003;28:284–293. 5 Stofega MR, Herrington J, Billestrup N, Carter-Su C: Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:1338–1350. 6 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, Bollag G, van der Burgt I, Musante L, Kalscheuer V, Wehner LE, Nguyen H, West B, Zhang KY, Sistermans E, Rauch A, Niemeyer CM, Shannon K, Kratz CP: Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006;38:331–336.

7 Binder G, Neuer K, Ranke MB, Wittekindt NE: PTPN11 mutations are associated with mild growth hormone resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5377–5381. 8 Limal JM, Parfait B, Cabrol S, Bonnet D, Leheup B, Lyonnet S, Vidaud M, Le Bouc Y: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin Endocrinol Metab 2006;91:300–306. 9 Ferreira LV, Souza SA, Arnhold IJ, Mendonca BB, Jorge AA: PTPN11 (protein tyrosine phosphatase, nonreceptor type 11) mutations and response to growth hormone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005; 90:5156–5160. 10 Kahn S, Yamamoto F, Almoguera C, Winter E, Forrester K, Jordano J, Perucho M: The cK-ras gene and human cancer (review). Anticancer Res 1987;7:639–652.

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KIGS Highlights

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):50–57 DOI: 10.1159/000097553

Published online: February 15, 2007

Idiopathic Short Stature: Reflections on Its Definition and Spontaneous Growth J.M. Wit Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands

Key Words Idiopathic short stature Small for gestational age Familial short stature Non-familial short stature

Abstract Background: Despite clarification of the term idiopathic short stature (ISS) provided by an international consensus group, several points of discussion remain. Methods and Results: Various cut-off limits can be used for the definition of ‘short’, and in the absence of recent population-based references, decisions have to be taken about which reference to use, whether corrections for secular trend have to be made, and which references should be used for ethnic minorities. For the definition of ‘idiopathic’, decisions have to be taken regarding which disorders should be excluded and by which tools; how to deal with the fluid border between ISS and persistent short stature after being born small for gestational age; and which limit of disproportion is acceptable. Conclusions: Distinguishing between the two subclasses of ISS, familial short stature (FSS) versus non-familial short stature (non-FSS), requires a decision as to which of the various formulas for the parent-specific lower limit of height standard deviation score should be used, particularly when comparing final height with prepubertal height, predicted adult height, and target height. The distinction between FSS and non-FSS is also important for evaluating spontaneous growth in children with ISS. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0050$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

In most diagnostic classifications of short stature, three main groups are distinguished: primary growth disorders (conditions thought to be intrinsic to the growth plate), secondary growth disorders (conditions that change the milieu of the growth plates), and a remaining group in which no recognizable cause is found. In the field of pediatric endocrinology, this last group is currently known as idiopathic short stature (ISS), in accordance with the findings of an international consensus meeting [1]. However, in general pediatric textbooks, a wide variety of terms are still used, including constitutional short stature, normal-variant short stature, familial short stature (FSS), small/delay, constitutional delay of growth and adolescence and familial slow maturation [1]. This review considers the existing consensus definition, the open-ended terms that still require further definition, and the phenomenon of spontaneous growth.

Definition of Short Stature

ISS is in essence a descriptive term and can only be used if a child is short for the population he or she belongs to and if a thorough history, physical examination and additional investigations have not resulted in a definite diagnosis. Thus, it is a diagnosis that is not based on positive findings in the diagnostic workup, but rather on exclusion

Jan Maarten Wit, MD Department of Pediatrics, Room J6S-211 Leiden University Medical Center, PO Box 9600 NL–2300 Leiden RC (The Netherlands) Tel. +31 71 526 2824, Fax +31 71 524 8198, E-Mail [email protected]

of other recognizable conditions. Therefore, the cornerstones of the definition of ISS are the definition of shortness (how short must one be to be classified as ISS and what population is appropriate for comparison), the description of the diseases that have to be excluded, and the investigations necessary to make such a determination. In terms of the cut-off limit that separates ‘short’ from ‘not short’, the consensus statement is clear. Short is defined as: ‘2 SD below the corresponding mean height for a given age, sex and population group’ [1]. This proposal conforms with current practice in many countries. Thus, short stature is ‘abnormal’ from a purely statistical point of view, which obviously does not automatically imply that it is abnormal in the sense of a pathological condition. While this cut-off is generally accepted for clinical purposes, it should be noted that in the context of growth hormone (GH) therapy, lower cut-off limits are being used in selecting patients for treatment, such as 2.25 or 2.5 SD below the mean in children with persistent short stature who were born small for gestational age (SGA) and for children with ISS. The question of which population should be used for comparison is more difficult. Basically, there are three points of contention. First, which reference population should be used if no recent large, nationwide population study is available? Second, how should one correct for secular trend? Third, which references should be used for children belonging to ethnic minorities? The height of a population is influenced by (1) genetic factors, part of which are associated with ethnic origin, and (2) environmental factors, of which socioeconomic conditions are most influential. In the last 150 years a secular trend toward increased height has been observed in Western countries. It has generally been assumed that this positive trend is primarily a result of better environmental conditions (more and better food, fewer infections). However, part of the trend may be associated with differences in the procreative success of tall and short individuals [2]. In the Netherlands we are fortunate to have reference data from four subsequent nationwide growth studies conducted in 1955, 1965, 1980 and 1997 [3]. The studies were designed to cover a relatively homogeneous population – people who were considered to be ‘Dutch’ in the 1950s – and who were therefore suitable for studying the phenomenon of secular trend. We have calculated that even in our country, which apparently harbors the tallest population on earth, there is still a secular trend toward increasing height, which is presently close to 4.5 cm per generation, or 1.5 cm per decennium [3]. Population data

on this secular trend in other parts of the world have shown that a height increase of similar magnitude is present in virtually all industrialized countries with the possible exception of the Scandinavian countries, where socioeconomic circumstances appear to have been good for so long that height seems to be close to its maximum for ethnic origin and environmental conditions [4]. Growth reference diagrams primarily serve as a tool for screening (e.g., detecting growth disorders) and for follow-up (e.g., determining whether the individual growth curve follows the expected pattern). In most countries the population used for reference originates either from other countries or from their own population but collected several decades previously. Ideally, in these countries the growth references should be updated at regular intervals based on growth studies in representative population samples. As the shape of the mean growth curve remains very similar, and the ratio between the mean and SD of height distribution at each age is stable too, a reliable growth reference can be obtained even with rather small population samples [5]. If the reference charts are not updated, it could be erroneously concluded that the percentage of ‘short’ children has gradually decreased from 2.3% (2 SD below the mean) to lower percentages due to positive secular change in the population. While adaptations to the secular trend are still relatively simple, as indicated above, the situation with regard to changes in ethnic population in almost all industrialized countries is far more complex. For example, in the Netherlands, the ethnic composition has changed considerably in the last 50 years, with immigrants coming from many parts of the world, especially Indonesia, Turkey, Morocco, the Netherlands Antilles, Surinam, the Indian subcontinent and China. Currently a large number of immigrants come into the country each year either seeking asylum or for economic reasons. While first-generation immigrants might still be considered ‘non-Dutch’, there is little reason to consider subsequent generations as a separate subgroup of the Dutch population. Furthermore, it is quite possible that the immigrant subpopulation will show a more rapid secular trend than the autochthonous host population, although data supporting this scenario are presently lacking. So the question arises: how does one screen children from ethnic minorities for growth disorders? In principle, there are three approaches for handling this challenge. First, a nationwide growth study can be performed with a sample comprised of a proper representation of the ethnic mix in the society. In traditional immigrant countries, such as the United States, this seems

Idiopathic Short Stature

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51

to have been the goal, though it is unlikely that this goal has been fully achieved. The advantage of this approach is that there is just one growth reference for all children. The disadvantage is that the percentage of children with short stature varies considerably between ethnic groups: less than 2.3% of Caucasian and African-American children will have a height below the –2 standard deviation score (SDS) cut-off, whereas many more children of Indian, East-Asian, Chinese, and Latin-American descent will fall below this cut-off. Assuming that the percentage of children with a known pathological cause does not differ between ethnic groups, a general screening procedure for short stature (growth monitoring) will pick up many more children from relatively short ethnic groups than from relatively tall groups. This implies that the sensitivity and specificity of growth screening rules – at least when using height SDS or a certain percentile as a cutoff – will be quite different for each group, which is clearly undesirable. In the second approach, different growth reference charts are prepared for the indigenous population and for several relatively large immigrant groups; for other ethnic minorities the best available reference is sought. The advantage of this scenario is that the screening parameters are then similar for all ethnic groups. Disadvantages are that several growth references must be prepared, the situation becomes more complex for health workers, and uncertainty remains for children born from mixed couples or parents from countries that lack growth references. In the Netherlands, the large differences in body stature between the autochthonous Dutch population and the immigrant Moroccan and Turkish populations resulted in a final height difference of about 10 cm. Nevertheless, we in the Netherlands have decided to take this approach. We have prepared separate growth charts for the largest immigrant populations (Turks and Moroccans) [6, 7], which are to be used if a child from these immigrant populations is short in comparison to the growth diagrams based on the indigenous Dutch. For other ethnic groups one can search for the appropriate growth references in the World Health Organization database [8]. A third approach is to use the national growth reference only as a way to follow up on a child’s growth, but not to base referral criteria on a child’s height in comparison with this reference. Thus, for screening purposes one could look at the difference between the child’s height SDS and the target height SDS, the midparental height corrected for gender. In fact, we have shown that the difference between height SDS and target height is the best parameter for detecting Turner syndrome [9]. The ad52

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vantage of this approach is that it is relatively simple. The disadvantages are that in many cases the height of at least one parent is unknown or uncertain and that one or both parents can have a dominantly inherited pathological growth disorder (e.g., hypochondroplasia). Furthermore, it is likely that the range around the target height will be wider if there is a great difference in height SDS of the parents. For screening and diagnostic purposes, many clinicians evaluate three parameters of growth: (1) child’s height compared with the best reference available for the pertinent ethnic group, leading to a parameter of the height position (SDS or percentile) of the child compared with the population reference; (2) child’s height SDS compared with target height SDS based on parental height, leading to a parameter of the difference with target height, and (3) child’s growth curve based on all previous growth data in terms of deflection, crossing the SD or percentile lines, leading to a parameter of growth velocity either expressed as cm/year, height velocity SDS, or delta height SDS) [10].

Definition of Idiopathic

In ISS, the term ‘idiopathic’ describes short stature for which no underlying pathology or aetiology is known [1]. However, the ability to find underlying pathology is completely dependent on the completeness of the medical history, the thoroughness of the physical examination, and the choices made with respect to additional investigations. Relevant points in the medical history include: • birth characteristics: weight, length, head circumference, and gestational age; • symptoms suggestive of chronic organic disease such as abnormal food intake, recurrent respiratory infections, loose stools, distended abdomen, symptoms associated with thyroid or adrenal insufficiency, and • symptoms suggestive of psychiatric disease, severe emotional disturbance or adverse psychosocial family conditions. As part of a complete physical examination one should specifically look for dysmorphic features; measure body proportions; and look for signs associated with hypothyroidism, celiac disease, cystic fibrosis, Leri-Weill syndrome, etc. While this should all be considered as standard pediatric practice, two issues warrant special emphasis. First, the boundary between children with ISS and those laWit

Fig. 1. Histogram of (a) birth weight SDS and (b) birth length SDS after removing the cases of birth weight SDS

of !–2.0. (Reproduced, with permission, from Wit [11].)

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Sitting height/height ratio SDS

8

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–6

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Fig. 2. A nomogram to assess the normal range of sitting height/ height SDS for a given height SDS. The relationship is indicated by an ellipse, the regression line, and two lines at 2 SD units away from the regression line. Circles indicate hypochondroplasia, triangles indicate Marfan syndrome. (Reproduced with permission from Fredriks et al. [15].)

beled persistently short after being born SGA (if status is known) is subject to dispute. There are several different references for birth weight and length for gestational age. In some instances, these references are based on small numbers of cases, and they differ substantially from each other. Furthermore, the distribution of birth weight as well as birth length of children with ISS is shifted to the left by approximately 1 SD, so that children with a birth weight or length below –2 SDS can be considered as the left tail of the distribution for children with ISS. If we accept that stature is mainly determined by genetic factors, then some of these factors already exercise their influence in utero. If one selects children diagnosed as ISS from KIGS (Pfizer International Growth Study Database), and distinguishes ISS from SGA on the basis of birth weight only, the distribution of birth weight shows the expected sharp cut-off at –2 SDS, but birth length shows an almost perfect Gaussian curve with many birth lengths below –2 SDS (fig. 1) [11]. Thus, a short child with a birth weight of –1.9 SDS and no known birth length could be diagnosed as ISS, while the same child would be labeled SGA 54

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if birth length was known and was below –2 SDS. These and similar issues with respect to the definition of SGA are discussed in greater detail elsewhere [12, 13]. Another important issue is determination of the cutoff limit with respect to body proportions. Even if no specific diagnosis can be made (e.g., hypochondroplasia or short stature homeobox-containing gene haplo-insufficiency), short stature associated with abnormal body proportions should be considered as a form of osteochondrodystrophy rather than as ISS. In Europe sitting height is usually measured, and from this the sitting height/height (SH/H) ratio is used to assess proportionality. In a few countries, age references have been prepared for this ratio, but several of these are relatively old. Outdated agelinked references are no longer suitable as SH/H is dependent on total height, and strongly influenced by the secular trend. It has been shown that changes in the secular trend are mainly the result of changes in leg length, so that the SH/H ratio diminishes over time and with the mean increased height of the population [14]. The relationship of SH/H ratio SDS with height SDS is shown in figure 2, illustrating that a short child, on average, has a higher SH/H ratio [15]. For a child with a height !–2 SDS, a SH/H ratio between +2 and +2.5 SDS can still be considered normal. Even if no abnormalities are detected during the medical history and physical examination, there can be organic disorders such as GH deficiency, Turner syndrome, celiac disease, and renal acidosis that underlie the finding of short stature. Therefore, it is common practice to perform laboratory investigations to screen for these disorders. Although various textbooks provide lists of possible investigations, the only parameter that critical appraisal has shown to have diagnostic value is screening for celiac disease [16]. An inquiry of pediatric endocrinologists also found little consensus about the investigations that should be performed and different opinions about which growth parameters are most relevant [10]. Table 1 summarizes the investigations that are part of the Dutch consensus guidelines on the workup for individuals with short stature [17].

Categorization of ISS

At the 1996 consensus meeting on ISS, a proposal was formulated to divide ISS into two subgroups: FSS and non-FSS. According to this proposal, the child with FSS is short in comparison with the relevant population, but remains within the expected range of height relating to Wit

Table 1. Laboratory investigations in the diagnostic work up according to the Dutch Consensus Guidelines

Laboratory investigations

Test used to diagnose

Blood Hb, Ht, leukocytes, cell indices, leukocyte differentiation, ESR (ferritin) ALAT, ASAT, ␥ GT Albumin, creatinine, sodium, potassium, calcium phosphate, alkaline phosphatase, acid-base equilibrium IgA-antiendomysium, antitissue glutaminase*, total IgA TSH, FT4 IGF-I FSH**

Celiac disease Hypothyroidism Growth hormone deficiency Turner syndrome

Urine pH, glucose, protein, blood, sedimentation

Renal diseases

Anemia, infections, celiac disease, cystic fibrosis Liver diseases Renal diseases

* At the time of the consensus meeting, antitissue glutaminase had not yet been introduced as a diagnostic tool for celiac disease. ** Only in girls <2 years of age and >9 years of age.

his or her family. In contrast, the child with non-FSS is short for the population and is below the range of height relating to his or her family [1]. The tempo of growth and maturation varies among individuals and families, and a slow tempo may lead to short stature, particularly when a child has relatively short parents. However, the two main markers for the tempo of development, pubertal development and bone age, do not always show the same pattern. Thus, the consensus group proposed that in the categorization of ISS, only the onset of puberty should be used. Thus, FSS and non-FSS can be further subdivided only after the onset of puberty as either occurring with normal timing of puberty or with delayed onset of puberty. This implies that information about bone age, while useful as a rough measure of maturation of the epiphyseal plates and often used as a tool in clinical judgment, is not made part of the definitions for either FSS or non-FSS. Clinicians will undoubtedly continue to use the term constitutional delay of growth and adolescence to refer to a rather common clinical syndrome often associated with a positive family history of delayed puberty in one or both parents and consisting of growth delay, delayed bone maturation, and late pubertal onset. However, according to the consensus definition, this condition falls in the category of non-FSS with delayed onset of puberty. Thus, for this part of the definitions, one has to decide which cut-off limit must be used to separate FSS from non-FSS ISS, and which cut-off must be used to separate normal from delayed-onset puberty. For the parent-speIdiopathic Short Stature

cific lower limit of height SDS, several formulas have been developed. At the consensus meeting, a rather complicated formula was proposed to calculate midparental height SDS that included the correlation between parental heights due to assertive mating (0.3) [1]. The parentspecific lower limit of height SDS formula is given by: r ! midparental height SDS –21–r2,

which is [0.5 ! mother’s height SDS + father’s height SDS/1.61] – 1.73,

where r is the correlation between the child’s height SDS and the midparental height SDS. This correlation depends on the child’s age; it is close to 0.5 between the ages of 2 and 9 years, but it is lower during puberty. Subsequently, an alternative target height (TH) formula was published that corrects for assertive mating and regression bias and is independent of sex [18]. This formula is: cTH = 0.72 times the average of father’s and mother’s height SDS.

Notably, with this formula parental height SDS should be calculated from growth references dating back one generation. If more recent growth references are used, one should add the secular trend over 30 years. Height SDS for a given age and birth year can be calculated using another formula based on secular trend over time and accounting for shrinking with age [19]. Horm Res 2007;67(suppl 1):50–57

55

0

Height SDS

–0.5 –1.0 –1.5 –2.0 –2.5

Fig. 3. Height SDS of children with ISS. Boys are indicated by a continuous line, girls by an interrupted line (adapted from Rekers-Mombarg et al. [22]).

–0.3 0

2

In the Netherlands, we chose an alternative solution and use a formula for target height adapted from the one proposed by Tanner correcting for secular trend (4.5 cm per 30 years) and assuming that the target range spans the range between TH 8 1.3 SD (approximately 9 cm). Thus, the target height of a boy is: [(father’s height + mother’s height +13)/2] + 4.5,

and the target height of a girl is: [(father’s height + mother’s height – 13)/2] + 4.5.

For calculating target height SDS, the present growth references can be used [3]. To determine the cut-off for the onset of puberty, recent reference data should be used – preferably from the same population. In the Netherlands, puberty is considered delayed for a girl if she reaches breast development stage 2 later than age 13 years and for a boy if his testicular volume is !4 ml after age 14 years [20].

Spontaneous Growth

Several publications have described the natural history of growth for children with ISS. We have summarized the studies published before 1995 [21], and performed a cross-sectional and longitudinal study of spontaneous growth of European children with ISS in the 1990s [22, 23]. Figure 3 is a graphic representation of height SDS for age derived from the results obtained in the cross-sectional analysis for boys and girls. In a mixed group of FSS and non-FSS children, final height SDS is approximately 1 SD higher than height SDS before puberty. 56

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4

6

8

10 12 Age (years)

14

16

18

20

22

The natural histories of FSS and non-FSS are quite different with respect to their relationship to height SDS at onset, predicted adult height and TH. For instance, final height correlates strongly with height SDS at onset of therapy and with bone age delay. Final height minus initial height SDS is lower in individuals with FSS than in those with non-FSS and correlates with bone age delay. Final height minus predicted adult height is inversely correlated with bone age delay. Finally, final height minus target height is lower in non-FSS and SGA compared with FSS [24]. This must be considered when evaluating the results of growth-promoting therapy.

Conclusions

As long as there is limited understanding of the genetic factors involved in determining body stature, the group of children labeled as ISS will remain large. In the coming decade, more causes of short stature are likely to be discovered, so that children now considered ISS shall be diagnosed otherwise. Still, the pediatric community shall have to deal with the term ISS for quite some time. The least we can do is to be clear about our definitions and informed about its natural history.

Acknowledgments Thanks are due to Lyset T.M. Rekers-Mombarg, Gerdine A. Kamp, A.M. Fredriks, D. Mul, F.K. Grote and W. Oostdijk for their involvement in ISS and growth studies and to the members of the Dutch Growth Hormone Advisory Group and the European NVSS Study Group for sharing data for analysis.

Wit

References 1 Ranke MB: Towards a consensus on the definition of idiopathic short stature. Summary. Horm Res 1996;45(suppl 2):64–66. 2 Pawlowski B, Dunbar RI, Lipowicz A: Tall men have more reproductive success. Nature 2000;403:156. 3 Fredriks AM, Van Buuren S, Burgmeijer RJF, Meulmeester JF, Beuker RJ, Brugman E, Roede MJ, Verloove-Vanhorick SP, Wit JM: Continuing positive secular growth change in The Netherlands 1955–1997. Pediatr Res 2000;47:316–323. 4 Bodzsar EB, Suzanne C: Secular growth changes in Europe. 1998. Budapest, Eotvos University Press. 5 Hermanussen M, Burmeister J: Synthetic growth reference charts. Acta Paediatr 1999; 88:809–814. 6 Fredriks AM, Van Buuren S, Jeurissen SE, Dekker FW, Verloove-Vanhorick SP, Wit JM: Height, weight, body mass index and pubertal development reference values for children of Turkish origin in the Netherlands. Eur J Pediatr 2003;162:788–793. 7 Fredriks AM, Van Buuren S, Jeurissen SE, Dekker FW, Verloove-Vanhorick SP, Wit JM: Height, weight, body mass index and pubertal development references for children of Moroccan origin in The Netherlands. Acta Paediatr 2004;93:817–824. 8 WHO. Global Database on Child Growth and Malnutrition. 2006. www.who.int/nutgrowthdb/en. 9 Van Buuren S, Van Dommelen P, Zandwijken GR, Grote FK, Wit JM, Verkerk PH: Towards evidence based referral criteria for growth monitoring. Arch Dis Child 2004;89: 336–341.

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10 Grote FK, Oostdijk W, De Muinck KeizerSchrama SM, Dekker FW, Verkerk PH, Wit JM: Growth monitoring and diagnostic work-up of short stature: an international inventorization. J Pediatr Endocrinol Metab 2005;18:1031–1038. 11 Wit JM: Growth hormone treatment of idiopathic short stature in KIGS; in Ranke MB, Wilton P (eds): Growth Hormone Therapy – 10 Years’ Experience. Heidelberg, Johann Ambrosius Barth Verlag, 1999, pp 225–243. 12 Wit JM, Finken MJ, Rijken M, Walenkamp MJ, Oostdijk W, Veen S: Confusion around the definition of small for gestational age. Pediatr Endocrinol Rev 2005;3:52–53. 13 Wit JM, Finken MJ, Rijken M, de Zegher F: Preterm growth restraint: a paradigm that unifies intrauterine growth retardation and preterm extrauterine growth retardation, and has implications for the small-for-gestational-age indication in growth hormone therapy. Pediatrics 2006; 117:e793–e795. 14 Yun DJ, Yun DK, Chang YY, Lim SW, Lee MK, Kim SY: Correlations among height, leg length and arm span in growing Korean children. Ann Hum Biol 1995; 22:443–458. 15 Fredriks AM, Van Buuren S, van Heel WJ, Dijkman-Neerincx RH, Verloove-Vanhorick SP, Wit JM: Nationwide age references for sitting height, leg length, and sitting height/ height ratio, and their diagnostic value for disproportionate growth disorders. Arch Dis Child 2005;90:807–812. 16 van Rijn JC, Grote FK, Oostdijk W, Wit JM: Short stature and the probability of coeliac disease, in the absence of gastrointestinal symptoms. Arch Dis Child 2004; 89: 882– 883.

17 De Muinck Keizer-Schrama SM: [Consensus ‘diagnosis of short stature in children’. National Organization for Quality Assurance in Hospitals]. Ned Tijdschr Geneeskd 1998; 142:2519–2525. 18 Hermanussen M, Cole J: The calculation of target height reconsidered. Horm Res 2003; 59:180–183. 19 Niewenweg R, Smit ML, Walenkamp MJ, Wit JM: Adult height corrected for shrinking and secular trend. Ann Hum Biol 2003; 30: 563–569. 20 Mul D, Fredriks AM, Van Buuren S, Oostdijk W, Verloove-Vanhorick SP, Wit JM: Pubertal development in The Netherlands 1965–1997. Pediatr Res 2001;50:479–486. 21 Wit JM, Kamp GA, Rikken B: Spontaneous growth and response to growth hormone treatment in children with growth hormone deficiency and idiopathic short stature. Pediatr Res 1996;39:295–302. 22 Rekers-Mombarg LTM, Wit JM, Massa GG, Ranke MB, Buckler JMH, Butenandt O, Chaussain JL, Frisch H, Leiberman E: Spontaneous growth in idiopathic short stature. Arch Dis Child 1996;75:175–180. 23 Rekers-Mombarg LT, Cole TJ, Massa GG, Wit JM: Longitudinal analysis of growth in children with idiopathic short stature. Ann Hum Biol 1997; 24:569–583. 24 Wit JM, Rekers-Mombarg LT: Final height gain by GH therapy in children with idiopathic short stature is dose dependent. J Clin Endocrinol Metab 2002;87:604–611.

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HORMONE RESEARCH

Horm Res 2007;67(suppl 1):58–63 DOI: 10.1159/000097554

Published online: February 15, 2007

Hormonal Treatment of Idiopathic Short Stature Edward O. Reiter Tufts University School of Medicine, Baystate Children’s Hospital, Springfield, Mass., USA

Key Words Growth hormone Short stature Insulin-like growth factor deficiency

Abstract Background and Objective: The classification idiopathic short stature (ISS) represents a heterogeneous group of children who, as a group, are similar in height to patients with growth hormone (GH) deficiency, Turner syndrome, and short stature as a consequence of being born small for gestational age. GH therapy has been an effective treatment for enhancing height in children with ISS. Methods: A large body of literature – including data from KIGS (Pfizer International Growth Study Database) and the National Cooperative Growth Study database – shows that non-placebo-controlled GH treatment programs generally, though not uniformly, are associated with substantial increments in height gain. Placebo-controlled trials conducted in several countries report significant gains of 5 to 8 cm in final height. Conclusions: Long-term data are needed to assess the behavioral consequences, if any, of GH treatment for children with ISS. As the pool of children with ISS is potentially large, it would be highly desirable if clinical trials enabled accurate prediction of likely height gain and could result in greater individualization of treatment. The use of aromatase inhibitors to slow estrogen-mediated skeletal maturation is a new treatment modality, but it has only been min-

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imally tested. The lower cost and ease of oral administration are attractive considerations, but long-term safety data are needed. Copyright © 2007 S. Karger AG, Basel

Introduction

The heterogeneous group of children with the array of conditions referred to as idiopathic short stature (ISS) has been very difficult to characterize. The ISS population includes a substantial number of individuals with varying degrees of growth hormone (GH) secretory insufficiency and insulin-like growth factor (IGF) deficiency that results in poor growth. Children with ISS are as short as those in treatment trials for GH deficiency, Turner syndrome, chronic renal failure and poor growth following intrauterine growth retardation. Often included among the causes of poor growth in children with ISS are the more well-known – though certainly not etiologically unraveled – syndromes of constitutional growth delay and genetic short stature, along with unidentified syndromes and as-yet undiagnosed chronic illnesses or endocrine disorders. In these cases, ‘idiopathic’ becomes a relative term. By definition, these children have normal provocative GH responses. However, they are often IGF-insufficient, suggesting both a universe of conditions with variable insensitivity to the ac-

Edward O. Reiter, MD Baystate Medical Center Children’s Hospital Tufts University School of Medicine Springfield, MA 01199 (USA) Tel. +1 413 794 5060, Fax +1 413 794 3623, E-Mail [email protected]

tion of GH and the inadequacies of GH testing. The most important diagnoses in the whole group may be the newly described mutations of the pathways from GH binding to IGF action [1, 2]. The growing appreciation of and investigations into this latter series of potential diagnoses, with the central role of the nuclear transcription factor STAT5 [3], will likely lead to a list of definable abnormalities, and with luck will result in development of rational treatments.

Treatment of ISS with Growth Hormone

Children with ISS often experience stressful behavioral circumstances, such as being teased or having academic difficulties, but studies suggest that the relationships between psychosocial problems and short stature vary widely [4–8]. Nonetheless, hormonal intervention to enhance growth has been used in hopes of diminishing such difficulties. In fact, GH treatment has been used even when specific etiologies are unknown [9]. Important questions have been raised about the financial, ethical and psychosocial impact of GH therapy for short children who are otherwise ‘normal’ [7, 10–13]. Given the cost of GH, the financial implications of treating such children (whether at the bottom 5, 3, 1, or 0.1%) are considerable. By definition, 5% of the population will always be below the 5th centile, whether we treat with GH or not, and undue focus upon short stature potentially handicaps an otherwise normal child, psychologically or socially. No convincing data have been presented yet showing that GH treatment of short children definitively improves psychological, social or educational function [13–15], with the possible exception being the improved intellectual function observed in children born small for gestational age who were treated with GH [16]. Furthermore, among the subset of children with constitutional delay of growth and maturation – which is probably a frequent diagnosis, though not really ISS as defined by the US Food and Drug Administration – final adult height will be adequate without any treatment [17–19]. Finally, there is legitimate concern regarding the potential treatment risks (however minimal) of GH therapy when treating otherwise normal children, even if the risks are exceedingly small [20]. In such a situation, there can be no discernible risk associated with therapy. However, assessment of treatment response data has been confounded by failure to report levels of IGF-I, insulin-like growth factor binding protein-3, and growth hormone binding protein in many studies; differing interpretaHormonal Treatment of Idiopathic Short Stature

tions of the results of endogenous GH secretion studies (assay variance, control group size, etc.); and the heterogeneity of the patient groups.

GH Treatment Trials

Review of published literature shows that previous clinical trials often did not contain long-term control groups, and reported results show variable growth responses [21–25]. Most short children treated with GH experience growth acceleration (‘catch-up’ growth) that typically is sustained over the first several years of therapy [9], though attenuation of the treatment response occurs in all other populations treated with GH. It appears that a slower pretreatment growth velocity and a higher weight/height ratio, factors suggestive of GH deficiency and to a lesser degree bone age retardation, are associated with better early-growth responses [26]. Longer-term data are now available that will enable investigators to determine the impact of therapy on adult height. As of 1999, nearly 3,000 children were classified as having ISS according to KIGS (Pfizer International Growth Study Database), with 153 having reached final height [27]. GH treatment (0.2–0.25 mg/kg/week) resulted in achievement of target height in patients with familial short stature (FSS), though they continued to have short stature as adults (–1.7 standard deviation score [SDS] in males and –2.2 SDS in females), with a mean gain during therapy of 0.6 to 0.9 SDS. In non-FSS children, the mean final height was greater for males (–1.4 SDS), though not for females (–2.3 SDS), with average gains of 1.3 and 0.9 SDS, respectively. Nonetheless, these latter attained heights are quite different from the midparental target heights that were near 0 SDS. Hintz et al. [28] assessed adult height in 80 North American children with ISS who were treated for up to 10 years at a GH dose of 0.3 mg/kg/week. At the conclusion of the study, the mean height SDS was –1.4 SDS with a gain of 1.3 SDS, which was quite similar to the broader KIGS experience. Although the Hintz study was not placebo-controlled, the data were compared with predicted and actual final heights of two groups of untreated short children followed for similar periods. Treated boys achieved a mean height gain of 9.2 cm and treated girls attained a mean height gain of 5.7 cm versus predicted heights compared with the untreated children.

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Controlled GH Treatment Trials

In a meta-analysis looking at an aggregate group of 1,089 children, there were four controlled trials that presented adult height data showing treatment benefits ranging from 0.54 to 0.84 SDS, corresponding to a mean effect of 5–6 cm [29]. McCaughey et al. [30] found that 8 prepubertal girls treated with GH at a dose of approximately 0.34 mg/kg/week experienced a mean height SDS of –1.14 after 6.2 years of treatment. This was a 7.6-cm greater gain than that for the control groups whose mean height SDS (–2.55) did not change during the study. The seminal studies that led to approval of GH treatment in the United States included the long-term, randomized, doubleblind, placebo-controlled trial at the National Institutes of Health (NIH) by Leschek and associates [31] and the randomized dosing trial of the European Idiopathic Short Stature Study Group [32]. In the NIH trial, a less optimal regimen of 0.22 mg/kg/week was administered in partial doses thrice-weekly to subjects with a mean starting height of nearly –3 SDS: treated children had a mean final height gain 3.7 cm greater than that of the control group. In the European randomized study, GH doses of 0.24 and 0.37 mg/kg/week were given according to a 6-dose per week regimen (the weekly dose was subdivided and administered over 6 injections) in children with mean starting heights ranging from –3.0 to –3.4 SDS. Children in the higher dose group had a mean final height gain that was 3.6 cm greater than those in the lower dose group, achieving a final height of –1.12 SDS. Combining the data from the two trials showed a mean gain of 7.3 cm in the group treated with 0.37 mg/kg/week compared with placebo-treated children. Taken together, these data show that GH treatment of prepubertal children with ISS does increase growth velocity and final height. Concerns have been raised that GH treatment might accelerate pubertal onset and progression, resulting in failure to improve height SDS for bone age [33, 34] and thereby offsetting the positive responses observed during the early years of GH treatment for ISS [35]. Results so far are somewhat contradictory. Among the population treated at the higher dose of 0.37 mg/kg in the study by Wit et al. [32, 36], there was no evidence of accelerated pubertal or skeletal maturation. By contrast, Kamp et al. [37] reported accelerated pubertal development and skeletal maturation at a 30% greater GH dose. This concern of advancing skeletal maturation has not been substantiated by additional studies [27, 30, 38–40]. Safety, along with efficacy, is of paramount importance when treating children with ISS with GH. In the 60

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study by Hintz et al. [28], careful monitoring over 5 years did not reveal any discernible metabolic side effects [39]. In two recent studies of GH-treated ISS children, no evidence of increased adverse events relative to other GHtreated groups were noted [41, 42]. Nonetheless, the longterm safety profile of GH use for ISS deserves special monitoring in view of the heterogeneity of the patient population and the inclusion of children whose heights are barely below the normal range [20].

Other Attempts at Growth Modulation

As the biology of the growth plate has become better understood, it has become apparent that the hormonal milieu, as well as the intrinsic and finite capacity of chondrocytes to proliferate, ultimately lead to growth cessation [43]. Bone age is a reflection of the degree of growth plate senescence and thus is a useful adjunct in estimating growth opportunity. Estrogen is known to accelerate the rate of bone age maturation. Thus, the role of estrogen in the maturation (or senescence) of the epiphyseal growth plates and its presumed central role in cessation of growth has prompted consideration of the possibility that by blocking estrogen production or action one could prolong the growth stage [44]. Studies in patients with mutations of the gene for the estrogen receptor [45] or for the aromatase enzyme [46–48] have demonstrated that estrogen is primarily responsible for epiphyseal fusion [49]. However, it seems unlikely that estrogen is solely responsible for all aspects of skeletal maturation. Accordingly, attempts have been made to modulate estrogen availability in children with ISS. Several small studies assessed the value of delaying puberty with a gonadotropin-releasing hormone agonist in GH-treated ISS children [50–53]. Although two trials showed a considerable gain (6–10 cm) between predicted and final height [51, 53], results of two others trials did not show any benefit [50, 52]. Data from both KIGS [54] and the National Cooperative Growth Study [55, 56] have not demonstrated any benefit from the addition of gonadotropin-releasing hormone (GnRH) agonists to GH treatment regimens among a population largely comprised of GH-deficient patients, but probably with an intermingling of ISS patients. Although it is just as important to achieve substantial height gain during the prepubertal years among children with ISS as it is for GH-deficient patients [57], the added cost of additional therapy, along with the potentially negative impact associated with halting pubertal progression in a child already shorter and less mature Reiter

than his/her peers, further diminish enthusiasm for such a delaying regimen. Aromatase inhibition [44, 58–61] may be a more effective regimen for enhancing growth, with growth stimulated by the continued presence of androgen and bone age advancement slowed by elimination of the presence of estrogen. In a double-blind, placebo-controlled trial by Dunkel et al., the potent aromatase inhibitor letrozole was used to slow the rate of skeletal maturation in boys with short stature and delayed puberty who were also receiving testosterone [58, 62]. Predicted adult heights were significantly enhanced by this regimen (mean, 182.1 cm in treated boys vs. 175.2 cm in placebo-treated boys). In a double-blind, randomized, placebo-controlled trial by Hero and associates, a group of prepubertal boys with ISS who were treated for 2 years with letrozole demonstrated a mean increase in predicted adult height of 5.9 cm and 0.7 SDS in height for bone age [63]. Results of another retrospective study of children with short stature of diverse etiologies found generally similar efficacy after aromatase inhibition [64]. In boys, short-term treatment with an aromatase inhibitor has neither impaired skeletal mineralization [63,

65] nor diminished spermatogenesis [66]. Altering aromatase activity in girls has not been systematically studied. In an uncontrolled study, the selective estrogen receptor modulator tamoxifen slowed skeletal maturation and increased height prediction when used in conjunction with GH in pubertal males [67]. The long-term safety of this method of decreasing aromatase activity, however, remains to be demonstrated.

Implications

Once the decision is taken to treat a child for ISS, attempts to enhance growth outcomes require early (i.e., prepubertal) and aggressive initiation of GH treatment and close monitoring of treatment effects. GH dosing should be altered in response to growth velocity or IGF production. Clinical trials should be designed to permit possible use of drugs to modulate estrogen bioactivity. Ultimately, it seems unlikely that current treatment regimens will prove to be strikingly beneficial in producing substantial height SDS increments during the pubertal years, in contrast to findings for treatment during prepuberty.

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Pediatric Workshop 1

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):64–70 DOI: 10.1159/000097555

Published online: February 15, 2007

How Proinflammatory Cytokines May Impair Growth and Cause Muscle Wasting Jean-Paul Thissen Diabetes and Nutrition Unit, School of Medicine, Université Catholique de Louvain, Brussels, Belgium

Key Words Cytokines Insulin-like growth factor I Skeletal muscle Growth hormone resistance Growth retardation

Abstract Background: Cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6, that are released in response to injury are thought to inhibit growth and cause muscle wasting, at least in part by inhibiting anabolic hormones such as insulin-like growth factor I (IGF-I). Because critical illness in humans is accompanied by high circulating concentrations of growth hormone (GH), which is the main stimulus for IGF-I production by the liver, resistance to GH is thought to contribute to the IGF-I decline observed in catabolic diseases. While TNF- seems to cause GH resistance mainly through downregulation of liver GH receptor expression, IL6 may inhibit the GH-stimulated Janus kinase and signal transducer and activator of transcription pathways by induction of suppressors of cytokine signaling proteins. Elevations in circulating IGF binding protein-1 levels, as observed in many catabolic situations, may play a role in the decline in muscle mass by decreasing the rate of protein synthesis in skeletal muscle. Furthermore, the increase in local muscle cytokines produced during inflammation makes the muscle GH-resistant and reduces its own IGF-I production. Finally, not only decreased IGF-I production by muscle, but also decreased muscle sensitivity to the anabolic effects of IGF-I,

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may contribute to muscle wasting observed in response to severe stress. Conclusions: Taken together, proinflammatory cytokines may contribute to the growth retardation and muscle wasting that occur after injury by impairing the GH/IGF-I axis at several levels. Copyright © 2007 S. Karger AG, Basel

The systemic inflammatory response syndrome induced by injury can cause a catabolic reaction leading to accelerated nitrogen loss and severe muscle wasting. This catabolic response cannot be solely explained by reduced caloric or protein intake [1]. Various lines of evidence indicate proinflammatory cytokines are mediators of the catabolic reaction [2]. Cytokines are thought to exert their catabolic actions at least partially by inhibiting anabolic hormones, such as insulin-like growth factor I (IGF-I). Endocrine IGF-I is synthesized and released primarily by the liver, although most tissues synthesize IGFI that also acts as an autocrine/paracrine factor. IGF-I is essential for normal postnatal growth and development as demonstrated by the severe growth retardation observed in IGF-I knockout (KO) animals [3]. It is also evident that IGF-I plays an important role in the control of muscle mass [4]. Therefore, the decline in IGF-I bioactivity caused by injury is believed to permit the stunted growth and muscle wasting observed in these situations.

Jean-Paul Thissen, MD Diabetes and Nutrition Unit, School of Medicine Université Catholique de Louvain, Avenue Hippocrate 54 BE–1200 Brussels (Belgium) Tel. +32 2 764 5469, Fax +32 2 764 5418, E-Mail [email protected]

Growth Hormone Secretion Alterations

Pituitary growth hormone (GH) is the main physiological stimulus for IGF-I production. Impaired GH secretion might be the first cause of decreased IGF-I during critical illness. One model commonly used to assess the GH secretion changes caused by acute inflammation is administration of endotoxin or lipopolysaccharide (LPS). Using this model, investigations in sheep revealed that LPS increases GH secretion [5]. This induction does not appear to involve changes in portal GH-releasing hormone or somatostatin levels. The effects of LPS on GH levels in sheep can be reproduced by administration of cytokines, such as tumor necrosis factor (TNF)- and interleukin (IL)-1, and prevented by intravenous administration of antagonists of these cytokines [6]. Moreover, these cytokines, in particular IL-1, can stimulate GH release in cultured pituitary cells [7]. Taken together, these observations indicate that endotoxin stimulates GH secretion in the sheep through pituitary presentation of TNF- and IL-1 from peripheral origins. By contrast, in rats, LPS causes GH secretion to decline [8]. This inhibition of GH release has been documented at the level of the hypothalamus and is the result of the mediation of IL-1 [9], which acts to stimulate somatostatin secretion and decrease GH-releasing hormone [10]. Although ghrelin levels are reduced after LPS injection [11], this decrease occurs later than the decline in GH secretion. This observation suggests that the reduction of plasma ghrelin is not involved in the inhibitory effect of a single endotoxin injection on the somatotrophic axis.

Hepatic GH Resistance

It is unlikely that impaired GH secretion alone causes the decrease in circulating IGF-I in catabolic conditions because critical illness in humans is often accompanied by high circulating concentrations of GH that fail to maintain IGF-I in the normal range [12]. Therefore, resistance to GH-mediated induction of IGF-I contributes to the IGF-I decline in catabolic diseases. Because the liver is believed to be the principal source of circulating IGFI, these observations support the existence of a state of hepatic GH resistance. Since its recognition in humans [13], GH resistance has been described in several animal models characterized by the release of proinflammatory cytokines (endotoxinemia [14], sepsis [15, 16], colitis [17] and chronic renal failure [18]).

Proinflammatory Cytokines

The mechanisms responsible for the induction of this acquired GH resistance have only recently been partially unraveled. The weight of evidence suggests that transcription is the major locus for IGF-I downregulation by inflammation. Stimulation of IGF-I gene transcription by GH involves the phosphorylation, dimerization and nuclear translocation of the signal transducer and activator of transcription (STAT)-5b transcription factor [19], which is necessary and sufficient to stimulate liver IGF-I gene transcription in response to GH [20, 21]. Recent studies showed that several inflammatory conditions, such as LPS injection, sepsis, colitis and renal failure, impair GH-stimulated STAT-5b phosphorylation and DNA binding activity in liver [15–18, 22]. The reduced phosphorylation of the GH receptor-associated Janus kinase (JAK)-2 tyrosine kinase, which phosphorylates STAT-5b [18, 23], suggests that the reduction of its kinase activity might be responsible for inhibition of the GH-induced transcription of several STAT-5b-dependent genes (IGFI, acid labile subunit, or ALS, and Spi2.1) observed in inflammatory conditions. Proinflammatory cytokines such as TNF- and IL1, which are released in inflammatory conditions, have been demonstrated to cause liver GH resistance. These cytokines have been reported to blunt GH stimulation of IGF-I expression in primary cultured hepatocytes [15, 24–26]. By contrast, IL-6 action on IGF-I expression in cultured hepatocytes is not settled: both stimulation [25] and inhibition [27] have been reported. Hepatocyte exposure to TNF- and IL-1, which are released after LPS injection in vivo [15, 26], inhibits the STAT-5b phosphorylation and activity induced by GH [15, 22]. Furthermore, infusion of IL-1 or TNF- antagonists in vivo attenuates the IGF-I decline observed in sepsis, most often without changes in GH levels [28, 29]. Collectively, these observations strongly suggest that the GH resistance observed in catabolic conditions is mediated through the release of the proinflammatory cytokines TNF- and IL-1. Because GH receptors are essential for normal liver IGF-I production, the possibility that GH resistance is caused by a decrease in the number of GH receptors in the liver must be considered. Available data indicate that liver GH receptor (GHR) protein levels, as assessed by membrane binding assay or immunoblot, are decreased in some catabolic conditions [14, 16, 17] but not all [18, 22, 23]. Nevertheless, liver GH mRNA abundance is reduced in almost all catabolic conditions tested so far [14, 16–18, 22, 23]. The mechanism for downregulation of GHR expression has been studied in endotoxinemia and Horm Res 2007;67(suppl 1):64–70

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colitis, and appears to be inhibition of the Sp3 transcription factor binding to adjacent cis-acting elements of the L2 transcript promoter of the GHR gene [17, 30]. TNF- is integral to this response, as inflammation does not decrease GHR expression in TNF-receptor 1-deficient mice [30] or after TNF- blockade [17]. In this last experiment, TNF neutralization was also associated with restoration of normal levels of liver STAT-5b and circulating IGF-I. These observations support the role of TNF- in decreasing levels of liver GH receptors mediating the GH resistance observed in several catabolic conditions. However, the possibility that TNF- contributes to GH resistance independent of decreased GH receptor levels has also been proposed [22]. Additional evidence indicates that decreased GH receptor levels are not mandatory for the induction of GH resistance by inflammation. The recently described suppressor of cytokine signalling (SOCS) proteins, mainly SOCS 1–3 and cytokine-inducible SH2 protein (CIS), inhibit GH signaling [31], in particular STAT-5 activation, through interactions with the cytoplasmic portion of GHR and/or JAK-2. The observation that SOCS liver expression is commonly induced in catabolic conditions [18, 32] suggests that this induction might play a role in inhibiting the JAK-STAT pathway responsible for the GH resistance [33]. Evidence in vitro showed that cytokines, such as TNF- and IL-1, potentiate the induction of SOCS-3 and CIS [26, 32], suggesting that these SOCS proteins have a role in GH resistance, at least in vitro. However, LPS injection in vivo induces SOCS-3 and CIS in the liver [16, 32] through an IL-6-dependent mechanism. Furthermore, inhibition of GH-induced STAT-5b activation and LPS-induced upregulation of SOCS-3 and CIS are abolished in IL-6 KO, but not in TNF-R1 KO, mice [16]. IL-6 may also contribute to the GH resistance caused by LPS injection, potentially by induction of SOCS-3 and CIS. This agrees with a previous observation that the growth retardation observed in IL-6 transgenic mice is associated with GH resistance and decreased levels of circulating IGF-I [34]. Thus cytokine-mediated induction of SOCS represents another mechanism by which inflammation could inhibit GH signaling. While TNF- seems to cause GH resistance mainly through downregulation of GH receptor expression, IL-6 may inhibit the JAK-STAT pathway by induction of SOCS proteins, in particular SOCS-3 and CIS.

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Changes in Circulating IGFBPS

Less than 1% of IGF-I circulates as free peptide [35] and most (190%) is bound to the 150-kDa complex, which consists of IGF-I, IGF binding protein (IGFBP)-3 or IGFBP-5 and an ALS. This complex is not believed to cross the capillary endothelium [36] and is credited with prolonging the half-life of IGF-I in circulation. IGFBP-3 probably serves as a storage pool for IGF-I. In contrast, IGFBP-1, IGFBP-2 and IGFBP-4 associate with IGF-I in smaller complexes (30–40 kDa) that can cross the capillary endothelium [37]. Because they control the bioavailability of IGF-I to tissues [38], IGFBPs are believed to exert both stimulatory [39–41] and inhibitory [42–44] effects on IGF-I actions. Posttranslational modifications of IGFBPs (partial proteolytic degradation by specific IGFBP proteases, selective dephosphorylation) may result in IGFBPs with reduced affinity for IGF-I. Changes in IGFBPs similar to those produced by food deprivation have been observed in response to catabolic states. The most dramatic changes are an increase in circulating IGFBP-1 levels and the presence of IGFBP-3 proteolytic activity [45, 46]. After major surgery [47] and in severe catabolic states [48], proteolytic activity that specifically degrades IGFBP-3 occurs. The enzymatic alteration of IGFBP-3 results in decreased affinity for IGF-I and is associated with increased free IGF-I [49]. Although the mechanism of IGFBP-3 degradation is not yet completely unravelled, it might increase the bioavailability of IGF-I for the tissues. Despite the decline in circulating levels of IGFBP-3, which is the main carrier of IGF-I, LPS injection does not alter the [125I]-IGF-I half-time for whole blood clearance [50], in contrast to protein restriction [51]. The induction of IGFBP-1 in response to inflammatory stress is likely to be caused by proinflammatory cytokines, as TNF-, IL-1 and IL-6 directly stimulate IGFBP-1 gene transcription in cultured hepatocytes [52]. Confirmation of the role of cytokines in the elevation of circulating IGFBP-1 levels comes from the observation that pretreatment with anti-TNF Ab or IL-1ra attenuated the infection-induced increase in circulating IGFBP-1 levels [29, 51], but with no increase in skeletal muscle. While the effects of IGFBP-1 on IGF-I action in vitro are still controversial, acute infusion of IGFBP-1 decreased the rate of protein synthesis in skeletal muscle, accompanied by a 50% decrease in circulating free IGF-I levels [53]. Hence, elevations in circulating IGFBP-1, as observed in many catabolic situations, may play a role in muscle mass decline. Increases in IGFBP-2 and IGFBP-4 and declines in Thissen

ALS levels are also observed in these situations [29, 54], but the consequences of these changes have not yet been established.

Muscle IGF-I Production

It is increasingly evident that the autocrine/paracrine actions of IGF-I are critical in controlling protein balance in skeletal muscle [4]. Besides the decrease in hepatic IGF-I production, which accounts for most of the fall in circulating IGF-I levels, decreases in IGF-I mRNA and peptide in several inflammatory conditions have also been demonstrated in skeletal muscle [55]. Not only do sepsis and LPS injection cause muscle IGF-I to decrease [29, 51, 56], but they also blunt the ability of GH to stimulate muscle IGF-I mRNA [57], indicating that GH resistance induced by inflammation is not limited to the liver. However, in contrast to liver, the GH-induced increase in STAT-5b in muscle was not blocked in septic rats, which suggests that the mechanism of GH resistance in muscle differs from that in liver [57]. The use of specific cytokine antagonists has demonstrated that TNF- and IL-1 play a major role in the sepsis- or LPS-induced decrease in muscle IGF-I levels [28, 29]. Furthermore, myocytes themselves may mount an immune response and therefore produce diverse proinflammatory cytokines (TNF, IL-1, IL-6) in response to LPS [58] and in other catabolic conditions [59]. When myocytes are exposed to these cytokines, in particular TNF-, expression of IGFI mRNA is downregulated [56] and no longer inducible by GH [60]. This effect is mediated through the Jun-Nterminal kinase pathway. Taken together, these observations suggest that the increase in local muscle cytokines produced during inflammation makes muscle GH-resistant and reduces its IGF-I production. That this net decrease in IGF-I availability to skeletal muscle may impair protein synthesis is suggested by the observation that the decrease in muscle IGF-I is proportional to the reduction in protein synthesis in various catabolic states [29].

of protein synthesis and inhibition of protein degradation by IGF-I [61]. This IGF-I resistance is caused by a postreceptor defect characterized by inhibition of the autophosphorylation of the type 1 IGF receptor -subunit and insulin-receptor substrate-1 (IRS-1) [61]. In more severe catabolic states, such as sepsis, the antiproteolytic action of IGF-I also can be blunted. In rats made septic by caecal ligature and puncture, IGF-I failed to inhibit muscle proteolysis despite suppressing gene expression of several components of the ubiquitin-proteasome proteolytic pathway [62, 63]. By contrast, IGF-I retains the ability to stimulate protein synthesis and inhibit proteolysis in burned rats [64]. In this situation, inhibition of protein breakdown involves multiple mechanisms, including PI3kinase/Akt-mediated inactivation of GSK-3 and FOXO [65], leading to downregulation of several proteolytic pathways (lysosomes, ubiquitin-proteasome, caspase-3). In light of these observations in vivo, recent data indicate that proinflammatory cytokines, in particular TNF and IL-1, induce a state of IGF-I resistance in muscle cells [66, 67]. When myoblasts were exposed, even for short time periods, to small concentrations of TNF- or IL-1, the ability of IGF-I to stimulate protein synthesis was blunted [66–68]. This resistance was attributed to inhibition of IRS-1 phosphorylation without any changes in autophosphorylation of the IGF-I receptor [66]. Ceramide appears to be a key intermediate by which these cytokines impair induction of protein synthesis by IGF-I [69]. Furthermore, the fact that TNF- and IL-1 decrease expression of MyoD and myogenin [70], two transcription factors crucial for muscle differentiation, supports the hypothesis that proinflammatory cytokines inhibit myogenesis [71].

Conclusions

In addition to a decrease in IGF-I production by muscle, proinflammatory cytokines may also impair the anabolic action of IGF-I on muscle. The loss of anabolic response to IGF-I has been described in some catabolic states characterized by the release of cytokines, such as uremia [61] and sepsis [62]. Uremia impairs stimulation

Various evidence indicate that proinflammatory cytokines impair growth and cause muscle wasting by inhibiting the production and action of IGF-I. Impaired GH secretion alone is unlikely to cause a decrease in circulating IGF-I in catabolic conditions because critical illness in humans is often accompanied by high circulating concentrations of GH that fail to maintain IGF-I in the normal range. Therefore, resistance to GH-mediated induction of IGF-I contributes to the IGF-I decline in catabolic diseases. While TNF- seems to cause liver GH resistance mainly through downregulation of GH receptor expression, IL-6 may inhibit the JAK-STAT pathway by

Proinflammatory Cytokines

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Impairment of Muscle IGF-I Action

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induction of SOCS proteins, in particular SOCS-3 and CIS. Besides the decrease in hepatic IGF-I production, which accounts for most of the fall in circulating IGF-I levels, the increase in local muscle cytokines produced during inflammation makes muscle GH-resistant and reduces its IGF-I production. Finally, in addition to decreasing IGF-I production by muscle, proinflammatory cytokines may also directly impair the anabolic action of IGF-I on muscle. Collectively, these observations suggest that not only decreased IGF-I production by muscle, but also decreased

muscle sensitivity to the anabolic effects of IGF-I, may contribute to the muscle wasting observed in response to severe stress. Nevertheless, this does not exclude the possibility that IGF-I reduces muscle atrophy in some catabolic situations, as has been demonstrated in such conditions as burns [72], congestive heart failure [73] and exposure to glucocorticoids [74] or angiotensin-II [75]. Further work is needed to delineate the factors promoting IGF-I sensitivity and the catabolic situations that may benefit from IGF-I treatment.

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71 Langen RC, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM: Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factorkappa B. FASEB J 2001;15:1169–1180. 72 Fang CH, Li BG, Wray CJ, Hasselgren PO: Insulin-like growth factor-I inhibits lysosomal and proteasome-dependent proteolysis in skeletal muscle after burn injury. J Burn Care Rehabil 2002;23:318–325. 73 Schulze PC, Fang J, Kassik KA, Gannon J, Cupesi M, Macgillivray C, Lee RT, Rosenthal N: Transgenic overexpression of locally acting insulin-like growth factor-1 inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction. Circ Res 2005;97:418–426. 74 Schakman O, Gilson H, de Coninck V, Lause P, Verniers J, Havaux X, Ketelslegers JM, Thissen JP: Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats. Endocrinology 2005; 146:1789–1797. 75 Song YH, Li Y, Du J, Mitch WE, Rosenthal N, Delafontaine P: Muscle-specific expression of IGF-1 blocks angiotensin II-induced skeletal muscle wasting. J Clin Invest 2005; 115: 451–458.

Thissen

Pediatric Workshop 2

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):71–76 DOI: 10.1159/000097556

Published online: February 15, 2007

Disorders of Salt and Water Balance in Children Mohamad Maghnie Linda Ambrosini Natascia di Iorgi Flavia Napoli Department of Pediatrics, IRCCS G. Gaslini, University of Genova, Genova, Italy

Key Words Diabetes insipidus Vasopressin Magnetic resonance imaging Posterior pituitary Anterior pituitary

Abstract Background: In past decades, our understanding of the vasopressin-mediated renal concentration mechanism has improved considerably due to the discovery of new endocrinerelated neurotransmitters and the elucidation of the roles of crucial molecular players in this process. The identification of disease-causing genes in hereditary disorders of water balance has been extremely helpful in identifying these pivotal molecules. Acquired diseases, however, are the most frequent causes of impaired water homeostasis. Diagnosis: The clinical and biochemical diagnosis of hormonal deficit is confirmed by standard laboratory tests, but recent advances in imaging techniques have shed new light on the pathophysiology of many of these diseases. Management: Magnetic resonance imaging (MRI) permits identification of the posterior pituitary in vivo and can clearly delineate the shape, size and enhancement pattern of the pituitary stalk, as well as its functional integrity. Thickening of the pituitary stalk is a common finding on MRI scans in several pituitary stalk pathologies, but it is not specific to any single pathological subtype. However, biopsy of an enlarged pituitary stalk should be reserved for selected patients with hypothalamic-pituitary mass or with progressive thickening of the pituitary stalk. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0071$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Physiology of Water Homeostasis

Over the last two decades, our understanding of the mechanisms that control water balance in health and disease has increased substantially. Following the establishment of reliable assay techniques to measure circulating arginine vasopressin peptide (AVP), vasopressin-cell antibodies (AVPc-Abs), tumor markers and monoclonal antibodies; the application of molecular biology to define hormonal, receptor and gene abnormalities; a greater knowledge of intracellular events within renal tubular cells; and the improvement of imaging techniques, it is now possible to characterize disorders of water balance more accurately. The maintenance of normal water balance is achieved principally by three interrelated determinants: thirst, vasopressin and the kidneys. Small changes in blood solute (mainly sodium) concentration (plasma osmolality) regulate vasopressin release. An increase in plasma osmolality, usually indicating a loss of extracellular water, stimulates vasopressin secretion and, conversely, a decrease in plasma osmolality inhibits its release into the systemic circulation. Vasopressin then acts on its major target organ, the kidneys. The hormone binds to its V2 receptor on the basal aspect of the renal-collecting tubular cell to activate an adenyl cyclase system that stimulates intracellular protein kinases. Cytoplasmic vesicles carrying the water channel proteins (aquaporin 2) are fused to the luminal membrane in response to vasopressin, thereby in-

Mohamad Maghnie, MD Department of Pediatrics, IRCCS, Giannina Gaslini University of Genova, Largo G. Gaslini 5 IT–16147 Genova (Italy) Tel. +39 0105 636 574, Fax +39 0105 538 265, E-Mail [email protected]

creasing the water permeability of this membrane, allowing the water to pass from the lumen of the nephron into the cells of the collecting duct, and consequently concentrating the urine. As early as the middle of the nineteenth century, the question was asked: how does water get through the lipid bilayer of the cell membrane? The answer used to be that membranes have pores. It was not until 1988 that Peter Agre isolated a membrane protein that he realized must be the long-sought-after water channel. He called it aquaporin [1]. The discovery opened the door to a whole series of biochemical, physiological and genetic studies of water channels in bacteria, plants and mammals. Today, researchers can follow in detail a water molecule on its way through the cell membrane and understand why only water and not other small molecules or ions can pass through the membrane. In 2000, Agre reported the first high-resolution images of the 3-dimensional structure of aquaporin. Now, it is possible to construct a detailed map of water channel functions. Currently, ten mammalian aquaporins (AQP0–AQP10) have been identified [2, 3]. Recently, apelin – a bioactive peptide – was isolated from bovine stomach extracts (like ghrelin, another stomach-hypothalamus association). Apelin is expressed in the supraoptic and the paraventricular nuclei, and it acts on specific receptors located on vasopressinergic neurons. Apelin acts as a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of AVP neuron activity and AVP release. The coexistence of apelin and AVP in magnocellular neurons, and their opposite biological effects and regulation, are likely to play key roles in maintaining body fluid homeostasis [4]. Vasopressin Gene The AVP-neurophysin II (AVP-NPII) gene is located on chromosome 20p13 and contains three exons. Exon 1 encodes the signal peptide (SP), the nonapeptide AVP and the N-terminal portion of NPII; exon 2 encodes the central portion of the NPII peptide, and exon 3 the C-terminal portion of NPII and a 39-amino-acid glycopeptide of unknown function, known as copeptin. The AVP-NPII gene directs the synthesis of a precursor polypeptide, preprohormone, which includes the AVP-peptide, its carrier protein NPII and the copeptide. The precursor is cotranslationally targeted to the endoplasmatic reticulum, where the signal is cleaved off by signal peptidase and the copeptide is core glycosylated. Vasopressin and NPII associate after cleavage and then form a tetramer, which increases the binding affinity of vasopressin for NPII. After formation of seven disulfide bonds within NPII and one 72

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within AVP, and after glycosylation of the copeptide, the proprecursor is packaged into neurosecretory granules and cleaved into the product peptides during axonal transport to the posterior pituitary [5]. Neurophysin serves to stabilize the hormone during its transport and storage. At present, more than 50 different mutations have been identified in familial neurohypophyseal diabetes insipidus [6, 7] and all except one show an autosomal dominant pattern of inheritance [8]. Most familial neurohypophyseal diabetes insipidus mutations reside within the neurophysin-II. Disorders of Salt and Water Balance Diabetes insipidus is a disease in which large volumes of urine are excreted due to vasopressin deficiency (central diabetes insipidus [CDI]) or vasopressin resistance (nephrogenic diabetes insipidus). In young children, polyuria may be the presenting symptom of primary polydipsia. CDI is a heterogeneous condition characterized by the excretion of abnormally large volumes of dilute urine, in excess of 2 l/m2/24 h or approximately 150 ml/kg/24 h at birth, 100–110 ml/kg/24 h at 2 years and 40–50 ml/kg/ 24 h in the older child and adult, due to a deficiency of AVP. X-linked (Xq28) nephrogenic diabetes insipidus is secondary to AVP receptor-2 mutations, which result in a loss of function or dysregulation of the V2 receptor. Mutations in the collecting duct AQP2 water channel explain two additional types of nephrogenic diabetes insipidus: familial autosomal recessive and dominant nephrogenic diabetes insipidus. Primary polydipsia is characterized by excessive water drinking. The ingested water causes an increase in body fluids and a modest dilution of serum osmolality. The decrease in serum osmolality removes the stimulation for vasopressin release with a consequent excretion of a large volume of dilute urine. Psychogenic primary polydipsia is more common in infancy, while frank psychiatric compulsive drinking are uncommon causes in children.

Central Diabetes Insipidus

Tumors CDI, especially in children and young adults, is caused by the destruction or degeneration of the neurons that originate in the supraoptic and paraventricular nuclei of the hypothalamus (table 1). The known causes of these lesions include germinoma or craniopharyngioma, Langerhans cell histiocytosis (LCH), local inflammatory, Maghnie/Ambrosini/di Iorgi/Napoli

Table 1. Causes of CDI

Familial Autosomal dominant (AVP-NPII gene mutations) Autosomal recessive (very rare)/reduced biological activity of the mutant vasopressin peptide X-linked recessive (very rare) Unknown gene(s) DIDMOAD syndrome (Wolframin gene mutation) Cerebral malformations Midline brain developmental defects (septo-optic dysplasia, holoprosencephaly) Associated with ectopic posterior pituitary, anterior pituitary hypoplasia and congenital hypopituitarism Acquired Idiopathic Brain tumors: germinoma, craniopharyngioma, optic glioma Inflammatory/autoimmune: lymphocytic hypophysitis/ lymphocytic infundibulo-neurohypophysitis/lymphocytic infundibulo-hypophysitis Autoimmune (antibodies against vasopressin-producing cells, T-cell damage) Granulomatosis (tuberculosis, sarcoid, Langerhans cell histiocytosis, Wegener) Infections/postviral (varicella, congenital cytomegalovirus and toxoplasmosis, encephalitis, meningitis) Traumatic brain injury Vascular impairment Metastases

autoimmune or vascular diseases, and trauma from surgery or accident. Midline cerebral and cranial malformations are other possible causes of CDI. However, 20–50% of cases are apparently idiopathic [9]. Intracranial germ tumors comprise 7.8% of primary pediatric brain tumors. Partial or complete pituitary stalk thickening (PST) is detectable in 78–100% of cases at presentation and may be the only finding in small germinomas [10]. Intracranial biopsy becomes mandatory in cases where there is a progressive thickening of the pituitary stalk up to or beyond 6.5–7 mm or an enlargement of the anterior pituitary (AP), either of which may be associated with positive tumor markers, such as -fetoprotein and -human chorionic gonadotropin. Growth arrest and multiple pituitary hormone deficiency are common early findings in pituitary germinomas, but hormone deficiency is not predictive of the presence of these tumors [9]. The frequency of presurgery CDI in craniopharyngioma varies from 8–35%, while postsurgical permanent CDI is a common finding, accounting for up to 80% of cases [7]. Disorders of Salt and Water Balance in Children

Langerhans Cell Histiocytosis CDI is the most frequent endocrine sequela of LCH, occurring in 10–20% of all patients [11–17]. PST is found in approximately 50–70% of patients with LCH at presentation or at follow-up, and may even be present before CDI onset [11]. Serial magnetic resonance imaging (MRI) scans show a range of changes, from spontaneous resolution to further enlargement or stability of the lesion. Anterior pituitary size can be normal, reduced or, rarely, enlarged. The risk of neurodegenerative LCH appears to be related more to pituitary involvement than to growth hormone therapy [12]. The search for extracranial lesions suggestive of LCH in patients with PST (dermatological and bone survey, chest X-ray and ear, nose and throat examinations) is recommended and could reduce the need for intracranial biopsies [11]. Idiopathic CDI Common MRI findings in idiopathic CDI include lack of posterior pituitary hyperintensity. This occurs in 94% of cases at presentation, but this signal invariably disappears at follow-up [9]. Pituitary stalk size at presentation is variable and changes over time. In two large pediatric studies of idiopathic CDI, PST was found in approximately 50–60% of subjects. Spontaneous evolution of the lesion was similar in both reports with stability (about 30%), regression or reduction (30–50%) or further enlargement (10–20%) in stalk size. Among subjects with idiopathic CDI and PST, 90–94% developed AP hormone deficits with isolated growth hormone deficiency accounting for 60% of cases. Multiple pituitary hormone deficits were present in 30–50% of subjects with PST, while, in one study, only 10% of the 19 subjects with a normal pituitary stalk had an additional hormonal deficit. AP hormone deficits were strongly associated with reduced AP size [9]. The underlying PST process in ‘idiopathic’ CDI is not completely understood. Preliminary data from a pediatric cohort of idiopathic CDI subjects [18] showed a high prevalence of AVPc-Abs in these subjects, although their precise role in the pathogenesis of the disease is still unclear. Diagnosis of autoimmune CDI is based on the presence of autoantibodies to AVP-secreting cells or the coexistence of other autoimmune polyendocrine syndromes [9, 19]. Histologic findings are suggestive of autoimmune involvement as well [20]. Vascular CDI Dynamic MRI studies after contrast medium injection showed no enhancement of the posterior pituitary Horm Res 2007;67(suppl 1):71–76

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Fig. 1. A Sagittal MRI in normal subject showing posterior pituitary hyperintensity (arrow), normal anterior pituitary size (double arrows) and normal pituitary stalk (arrowhead). B Sagittal MRI in a patient with idio-

pathic CDI. Posterior pituitary hyperintensity is absent (arrow); anterior pituitary size is normal (double arrows), and pituitary stalk size is normal (arrowhead). C Sagittal MRI in a patient with germinoma-dependent CDI. Note the thick proximal pituitary stalk (arrow) and pineal mass (double arrows).

lobe in patients with idiopathic CDI, whereas normal enhancement of the AP was present. Using this technique, an abnormal pituitary blood supply has been demonstrated in idiopathic CDI [21] as well as a delayed enhancement of the anterior lobe in patients with evolving pituitary hormone deficiency [22]. The lack of contrast enhancement of the posterior lobe suggests that a vascular injury to the inferior hypophyseal artery could be causally linked to CDI. Diagnosis of Diabetes Insipidus The age at which symptoms develop, together with the pattern of fluid intake, may influence subsequent investigation of diabetes insipidus. Young children may have severe dehydration, vomiting, constipation, fever, irritability, failure to thrive and growth retardation. Clinical examination may provide important clues to possible underlying diagnoses. A range of baseline investigations including plasma electrolytes, random plasma osmolality and urine osmolality, as well as an assessment of kidney function, may assist in a correct diagnosis. In the absence of an immediate diagnosis, the child’s fluid intake and output should be studied in greater detail. The ability of the central nervous system to produce, and of the kidney to respond to, vasopressin should be established by means of a formal water deprivation test.

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A 7-hour (or less) deprivation test is usually sufficient for diagnosis, except in cases of primary polydipsia, where a longer dehydration period is sometimes required. The test must be discontinued if weight loss exceeds 5% of starting weight or if thirst becomes intolerable. The administration of desmopressin will help to make a differential diagnosis between central and nephrogenic diabetes insipidus. Recently, the measurement of aquaporin-2 has also been used in the differential diagnosis of central versus nephrogenic diabetes insipidus [23]. Once the biochemical diagnosis of diabetes insipidus has been established, other investigations are mandatory, including tumor markers and MRI of the brain. On an MRI, the posterior pituitary can be seen as a hyperintense signal on sagittal T1-weighted imaging under basal conditions; the absence of this hyperintensity may be a nonspecific hallmark of CDI [9]. Thickening of the pituitary stalk or infundibulum, defined as exceeding 3 mm, although not specific, is observed in approximately one third of children with CDI (fig. 1 and 2). Dynamic MRI, in particular, could help identify cases of CDI associated with abnormal blood supply to the posterior pituitary. Clinical, radiological and endocrine studies are needed during follow-up. MRI follow-up is recommended in all patients with PST (every 3–6 months). Enlargement of a pituitary lesion (16.5–7 mm) or enlargement of the AP gland are indications for a pituitary stalk biopsy [9, 17]. Maghnie/Ambrosini/di Iorgi/Napoli

Fig. 2. Sagittal (A , C) and coronal (B, D) MRI in a patient with idiopathic CDI before (A , B) and after (C , D) gadolinium administration. Posterior pituitary hyperintensity is absent (arrow in A) and pituitary stalk is thick

(arrowhead); anterior pituitary size is normal (double arrows).

Treatment of CDI The drug of choice for the treatment of diabetes insipidus is desmopressin (DDAVP), a synthetic analogue of the endogenous hormone arginine vasopressin, but with a 2,000- to 3,000-fold lower vasopressor effect. Desmopressin may be administered orally, intranasally or parenterally. Given intranasally or orally, maximum plasma concentrations are reached in 40–55 min. The drug’s half-life is 3.5 h. Generally, urine output will decrease 1 or 2 h after administration and the duration of action will range from 6 to 18 h. There is broad individual variation in the dosage required to control diuresis. Daily dosages for oral preparations vary from 100 to 1,200 g in one to three divided doses, for the intranasal preparation approximately from 2 to 40 g, and for the

parenteral, from 0.1 to 1 g. A low dose should be used initially and increased as necessary. Oral desmopressin has been shown to be particularly helpful in children. Its positive characteristics include better absorption, fewer complications and, due to the easy route of administration, good compliance in children and adolescents. Symptomatic dilutional hyponatremia is the only potential hazard if desmopressin is administered in excess over a prolonged time period. Symptoms of hyponatremia include headache, nausea, vomiting and seizure. Untreated, these symptoms can lead to coma and death. However, asymptomatic hyponatremia can also occur. Particular attention is required in cases of multidrug therapy because of the risk of extrapontine myelinolysis [24].

Disorders of Salt and Water Balance in Children

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Rare side effects with intranasal delivery of DDAVP include eye irritation, headache, dizziness, rhinitis or epistaxis, cough, flushing, nausea, vomiting, abdominal pain, chest pain, palpitations and tachycardia. Evidence to date indicates that the use of DDAVP during pregnancy is safe and is not associated with adverse effects in the mother or fetus/child [25]. In the presence of adipsia or

hypodipsia, diabetes insipidus presents a difficult challenge and initially is best managed by adjusting the DDAVP dosage and fluid intake in a hospital setting. Daily weight can be used as an index of fluid balance, but regular monitoring of electrolytes will be required as well.

References 1 Agre P: Nobel lecture. Aquaporin water channels. Biosci Rep 2004; 24:127–163. 2 Engel A, Fujiyoshi, Agre P: The importance of aquaporin water channel protein. EMBO J 2000;19:800–806. 3 Agre P: Aquaporin water channels in kidney. J Am Soc Nephrol 2000; 11:764–777. 4 De Mota N, Reaux-Le Goazigo A, El Messari S, Chartrel N, Roesh D, Dujardin C, Kordon C, Vaudru H, Moos F, Llorens-Cortes C: Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release. Proc Natl Acad Sci USA 2004;101:10464–10469. 5 Ito M, Yu RN, Jameson JL: Mutant vasopressin precursors that cause autosomal dominant neurohypophyseal diabetes insipidus retain dimerization and impair the secretion of wild-type proteins. J Biol Chem 1999; 274: 9029–9037. 6 Christensen JH, Siggaard C, Corydon TJ, Robertson GL, Gregerson N, Bolund L, Rittig S: Differential cellular handling of defective arginine-vasopressin (AVP) prohormones in cells expressing mutations of the AVP gene associated with autosomal dominant and recessive familial neurohypophyseal diabetes insipidus. J Clin Endocrinol Metab 2004;89:4521–4531. 7 Maghnie M, Ghirardello S, Genovese E: Magnetic resonance imaging of the hypothalamus-pituitary unit in children suspected of hypopituitarism: who, how and when to investigate. J Endocrinol Invest 2004; 27: 496–509. 8 Willcuts MD, Felner E, White PC: Autosomal recessive familial neurohypophyseal diabetes insipidus with continued secretion of mutant weakly active vasopressin. Hum Mol Genet 1999;8:1303–1307. 9 Maghnie M, Cosi G, Genovese E, Manca-Bitti ML, Cohen A, Zecca S, Tinelli C, Gallucci M, Bernasconi S, Boscherini B, Severi F, Arico M: Central diabetes insipidus in children and young adults. N Engl J Med 2000; 343: 998–1007. 10 Mootha SL, Barkovich AJ, Grumbach MM, Edwards MS, Gitelman SE, Kaplan SL, Conte FA: Idiopathic hypothalamic diabetes insipidus, pituitary stalk thickening, and the

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occult intracranial germinoma in children and adolescents. J Clin Endocrinol Metab 1997;82:1362–1367. Prosch H, Grois N, Prayer D, Waldhauser F, Steiner M, Minkov M, Gadner H: Central diabetes insipidus as presenting symptom of Langerhans cell histiocytosis. Pediatr Blood Cancer 2004;43:594–599. Donadieu J, Rolon MA, Pion I, Thomas C, Doz F, Barkaoui M, Robert A, Deville A, Mazingue F, David M, Brauner R, Cabrol S, Garel C, Polak M, for the French LCH study group: Incidence of growth hormone deficiency in pediatric-onset Langerhans cell histiocytosis; efficacy and safety of growth hormone treatment. J Clin Endocrinol Metab 2004;89:604–609. Maghnie M, Aricò M, Villa A, Genovese E, Beluffi G, Severi F: MR of the hypothalamic-pituitary axis in Langerhans cell histiocytosis. Am J Neuroradiol 1992; 13: 1365– 1371. Maghnie M, Bossi G, Klersy C, Cosi G, Genovese E, Aricò M: Dynamic endocrine testing and magnetic resonance imaging in the long-term follow-up of childhood Langerhans cell histiocytosis. J Clin Endocrinol Metab 1998;83:3089–3094. Maghnie M, Villa A, Aricò M, Larizza D, Pezzotta S, Beluffi G, Genovese E, Severi F: Correlation between magnetic resonance imaging of posterior pituitary and neurohypophyseal function in children with diabetes insipidus. J Clin Endocrinol Metab 1992;74: 795–800. Grois N, Prayer D, Prosch H, Minkov M, Pötschger U, Gadner H: Course and clinical impact of magnetic resonance imaging findings in diabetes insipidus associated with Langerhans cell histiocytosis. Pediatr Blood Cancer 2004;43:59–65. Leger J, Velasquez A, Garel C, Hassan M, Czernichow P: Thickened pituitary stalk on magnetic resonance imaging in children with central diabetes insipidus. J Clin Endocrinol Metab 1999;84:1954–1960. Maghnie M, Ghirardello S, De Bellis AM, di Iorgi N, Ambrosini L, Secco A, De Amici M, Tinelli C, Bellastella A, Lorini R: Idiopathic central diabetes insipidus in children and young adults is commonly associated with

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vasopressin-cell antibodies and markers of autoimmunity. Clin Endocrinol 2006, accepted for publication. De Bellis A, Colao A, Bizzarro A, Di Salle F, Coronella C, Solimeno S, Vetrano A, Pivonello R, Pisano G, Lombardi G, Bellastella A: Longitudinal study of vasopressin-cell antibodies and of hypothalamic-pituitary region on magnetic resonance imaging in patients with autoimmune and idiopathic complete central diabetes insipidus. J Clin Endocrinol Metab 2002;87:3825–3829. Maghnie M, Genovese E, Sommaruga MG, Aricò M, Locatelli D, Arbustivi E, Pezzotta S, Severi F: Evolution of childhood central diabetes insipidus into panhypopituitarism with a large hypothalamic mass: Is ‘lymphocytic infundibulo-neurohypophysitis’ in children a different entity? Eur J Endocrinol 1998;139:635–640. Maghnie M, Altobelli M, di Iorgi N, Genovese E, Meloni G, Manca-Bitti ML, Cohen A, Bernasconi S: Idiopathic central diabetes insipidus is associated with abnormal blood supply to the posterior pituitary gland caused by vascular impairment of the inferior hypophyseal artery system. J Clin Endocrinol Metab 2004;89:1891–1896. Maghnie M, Genovese E, Aricò M, Villa A, Beluffi G, Campani R, Severi F: Evolving pituitary hormone deficiency is associated with pituitary vasculopathy: dynamic MR study in children with hypopituitarism, diabetes insipidus, and Langerhans cell histiocytosis. Radiology 1994;193:493–499. Kanno K, Sasaki S, Hirata Y, Fushimi K, Nakanishi S, Bichet DG, Marumo F: Urinary excretion of aquaporin-2 in patients with diabetes insipidus. N Engl J Med 1995; 332: 1540–1545. Maghnie M, Genovese E, Lundin S, Bonetti F, Aricò M: Iatrogenic extrapontine myelinolysis in central diabetes insipidus: are cyclosporine and 1-desamino-8-D-arginine vasopressin harmful in association? J Clin Endocrinol Metab 1997;82:1749–1751. Kim RJ, Malattia C, Allen M, Moshang T, Maghnie M: Vasopressin and desmopressin in central diabetes insipidus: adverse effects and clinical considerations. Pediatr Endocrinol Rev 2004;2(suppl 1):115–123.

Maghnie/Ambrosini/di Iorgi/Napoli

Pediatric Workshop 3

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):77–80 DOI: 10.1159/000097557

Published online: February 15, 2007

ABCs of Natriuretic Peptides: Cardiac Aspects Amiram Nir The Unit of Pediatric Cardiology, Shaare Zedek Medical Center, Jerusalem, Israel

Key Words Natriuretic peptides Cardiac effects Heart disease

Abstract Background: The natriuretic peptides are a family of structurally similar peptides. Atrial natriuretic peptide and Btype natriuretic peptide (BNP) are cardiac hormones secreted from the heart in response to cardiac chamber distention. Their biological actions include diuresis, natriuresis and vasodilatation, as well as suppression of the renin-angiotensin-aldosterone and endothelin systems. The hemodynamic actions of the secreted natriuretic peptide unload the heart in response to increased intravascular volume. Knowledge regarding the biological actions of the natriuretic peptides continues to evolve into clinical use. A synthetic BNP analogue has shown a beneficial short-term effect in acute decompensated heart failure, while plasma levels of natriuretic peptides have been shown to be elevated in patients with heart disease. Conclusions: BNP and the N-terminal segment of its prohormone, NT-pro-BNP, are very sensitive markers for many forms of heart disease. Future research will likely allow greater use of these peptides in the diagnosis and treatment of heart diseases in adults and children. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0077$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Background

The natriuretic peptides are a family of structurally similar peptides. These peptides play an important role in the regulation of extracellular fluid volume and blood pressure. They induce natriuresis, diuresis and vasodilatation and specifically act to counter the effects of stress hormones such as those produced by the renin-angiotensin-aldosterone and adrenergic systems and endothelin [1]. The cardiac members of this family are the atrial or A-type natriuretic peptide (ANP) and the B-type natriuretic peptide (BNP). ANP is secreted primarily from the cardiac atria in response to increased left and right atrial pressure and volume loads, whereas BNP is secreted primarily from the ventricles in response to increased left and right ventricular pressure and volume loads. The primary stimulus for natriuretic peptide release is myocyte stretch [1, 2]. Other stimuli for the production and secretion of the natriuretic peptides are stress hormones, growth factors and proinflammatory cytokines [3]. The third member of the family is C-type natriuretic peptide (CNP), a paracrine factor secreted from endothelial and other cell types. The natriuretic peptides are synthesized as pre-prohormones, are cleaved to prohormones and, upon release from the cells, are cleaved to C-terminal biologically active peptides and N-terminal prohormones that have no known biological actions. Both segments are found in plasma. The natriuretic peptides bind to specific mem-

Amiram Nir, MD Pediatric Cardiology Shaare Zedek Medical Center, The Heart Institute Hadassah University IL–94142 Jerusalem (Israel) Tel. +972 2 677 7111, Fax +972 2 641 1028, E-Mail [email protected]

brane receptors on the surface of target cells; ANP and BNP bind to the natriuretic peptide receptor type A (NPR-A) while CNP has highest affinity to the natriuretic peptide receptor type B (NPR-B). Activation of the receptors leads to generation of cyclic guanosine monophosphate (cGMP), which mediates most of the biological effects [4]. Data from cell culture and knockout mouse experiments revealed that natriuretic peptides have direct effects on the heart. ANP improves cardiac muscle relaxation, and BNP plays a predominant role in the inhibition of cardiac hypertrophy and fibrosis. Evidence for negative feedback of the natriuretic peptides on their own secretion has been reported [5]. Degradation of the natriuretic peptides occurs by binding to a clearance receptor (NPR-C) and by enzymatic degradation, mainly by neutral endopeptidase [4].

Clinical Applications

The physiological properties of the natriuretic peptides made them attractive candidates for heart failure therapy. Indeed BNP (in its recombinant form marketed as nesiritide) was approved for treatment of acute decompensated congestive heart failure in the United States in 2001. There are reports of its beneficial short-term effects [6]; however, there are also reports that its use is associated with increased renal impairment and mortality [7]. Plasma levels of the natriuretic peptides are elevated in many cardiac diseases, and they have been found to be markers for heart disease. In comparative studies, BNP and its amino terminal prohormone fragment, NT-proBNP, were superior to ANP as markers in this setting [8]. Plasma levels of BNP and NT-pro-BNP are elevated in adult patients with left ventricular dysfunction [1, 2], ischemic heart disease, diastolic dysfunction [9] and hypertrophic cardiomyopathy [10]. BNP levels correlate with the severity of left ventricular dysfunction and congestive heart failure, as well as with prognosis [4]. BNP is also elevated in right heart failure [11]. Thus, BNP may be thought of as the ‘sedimentation rate of the heart’. It is not specific, but it is a very sensitive marker for overt or subtle cardiac dysfunction. Heart failure syndrome is characterized by activation of the natriuretic peptide system as well as of stress hormones such as renin-angiotensin-aldosterone and endothelin [12]. Stress hormones inhibit the actions of natriuretic peptides, and thus, in heart failure, there are high levels of natriuretic peptides but their hemodynamic effects are blunted.

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In recent years, commercial kits for the measurement of BNP and NT-pro-BNP have become available for commercial laboratory systems. The availability of these tests enables BNP and NT-pro-BNP to serve as candidate markers for heart disease in everyday practice. BNP and NT-pro-BNP are now in use to screen for ventricular dysfunction in asymptomatic people at risk [13], to identify heart failure in patients with symptoms suggestive of heart disease [14], to monitor the effectiveness of heart failure therapy [15] and to predict prognosis [1, 16, 17]. A number of well-conducted studies have shown that both BNP and NT-pro-BNP can differentiate between patients with dyspnea due to lung disease and those with dyspnea due to heart disease. The capacity to make this distinction can improve clinical judgment and practice as well as reduce economic cost [18]. Notably, use of BNP as a marker for heart disease is complicated by the effects of various factors on plasma BNP levels. These include patient age, gender [19], and the effects of various concurrent noncardiac disease states, such as renal, liver and endocrine dysfunction, on normal values. Data on the use of natriuretic peptides in the pediatric population are limited. In normal infants, most studies show an elevated level of natriuretic peptides immediately after birth [20, 21], with a decrease during the first week of life. The reason for this neonatal surge is not clear. The transition from fetal to neonatal circulation is accompanied by an increase in pulmonary blood flow as the newborn takes its first breaths. These perinatal circulatory changes lead to an increase in ventricular volume and pressure load, and this may stimulate ANP and BNP synthesis and secretion. After the neonatal period, most studies show that peptide levels remain constant throughout childhood [22] and that there is no difference between males and females in the pediatric age group [22]. Results of studies addressing the natriuretic peptide level in children with heart disease are similar to those of studies in adults with heart failure. In general, pediatric studies found a correlation between the clinical heart failure score and BNP and NT-pro-BNP levels [23]. Elevated peptide levels were found in patients with heart failure from structural heart disease as well as in patients with dilated cardiomyopathy, though the latter group tended to have higher BNP levels than patients with structural heart disease and a similar functional class or left ventricular function [24]. Studies have also shown that infants with respiratory distress due to heart disease had significantly higher plasma NT-pro-BNP levels than infants with respiratory distress due to respiratory disease Nir

or than control children [25]. These findings suggest that peptide levels can differentiate between infants with respiratory distress due to heart disease and those with respiratory distress due to lung disease. Patients with congenital heart lesions causing left-toright shunt have increased pulmonary blood flow – with or without elevated pulmonary artery pressure. In children with ventricular septal defect who had both volume and pressure loads measured, plasma BNP levels correlated with the pulmonary-to-systemic flow ratio, the mean pulmonary artery pressure and the pulmonary-tosystemic vascular resistance ratio [26]. Thus, BNP levels are able to identify patients with significant left-to-right shunts or pulmonary hypertension. Available data suggest that both pressure and volume loads in the right and left ventricles induce elevation of plasma BNP and that BNP levels correlate with the hemodynamic severity of the defect. Other lesions for which BNP has known or potential value as a marker for the presence or severity of disease

include obstructive lesions [27], pure right heart disease or right ventricular dysfunction secondary to pulmonary hypertension [28] and inflammatory heart diseases. BNP was shown to identify allograft disease in infants and children following cardiac transplantation [29]. In one study, patients with acute Kawasaki disease had higher BNP levels than patients with Kawasaki disease in the convalescent phase or patients with acute viral illness [30].

Conclusion

The natriuretic peptides are cardiac hormones that participate in the control of water and sodium balance. They are potentially useful in the treatment of heart failure. BNP and NT-pro-BNP are important markers for heart disease in the adult. Available data suggest that the natriuretic peptides have similar clinical value for pediatric patients with heart diseases.

References 1 Mair J, Hammerer-Lercher A, Puschendorf B: The impact of cardiac natriuretic peptide determination on the diagnosis and management of heart failure. Clin Chem Lab Med 2001;39:571–588. 2 Cowie MR, Mendez GF: BNP and congestive heart failure. Prog Cardiovasc Dis 2002; 44: 293–321. 3 Forero McGrath M, Kuroski de Bold ML, de Bold AJ: The endocrine function of the heart. Trends Endocrinol Metab 2005;16:469–477. 4 Abassi Z, Karram T, Ellaham S, Winaver J, Hoffman A: Implications of the natriuretic peptide system in the pathogenesis of heart failure: diagnostic and therapeutic importance. Pharmacol Ther 2004;102:223–241. 5 Kuhn M: Cardiac and intestinal natriuretic peptides: insights from genetically modified mice. Peptides 2005;26:1078–1085. 6 Publication Committee, for the VMAC Investigators. Intravenous nesiritide vs nitroglycerin for treatment of decompensated congestive heart failure: a randomized controlled trial. JAMA 2002;287:1531–1540. 7 Sackner-Bernstein DJ, Kowalski M, Fox M, Aaronson K: Short-term risk of death after treatment with nesiritide for decompensated heart failure. A pooled analysis of randomized controlled trials. JAMA 2005;293:1900– 1905.

ABCs of Natriuretic Peptides: Cardiac Aspects

8 McCullough PA, Sandberg KR: Sorting out the evidence on natriuretic peptides. Rev Cardiovasc Med 2003;4(suppl 4):S13–S19. 9 Lubien E, DeMaria A, Krishnaswamy P, Clopton P, Koon J, Kazanegra R, Gardetto N, Wanner E, Maisel AS: Utility of B-natriuretic peptide in detecting diastolic dysfunction: comparison with Doppler velocity recordings. Circulation 2002;105:595–601. 10 Nakamura T, Sakamoto K, Yamano T, Kikkawa M, Zen K, Hikosaka T, Kubota T, Azuma A, Nishimura T: Increased plasma brain natriuretic peptide level as a guide for silent myocardial ischemia in patients with non-obstructive hypertrophic cardiomyopathy. J Am Coll Cardiol 2002; 39:1657– 1663. 11 Kucher N, Printzen G, Goldhaber SZ: Prognostic role of brain natriuretic peptide in acute pulmonary embolism. Circulation 2003;107:2545–2547. 12 Emdin M, Passino C, Prontera C, Iervasi A, Ripoli A, Masini S, Zucchelli GC, Clerico A: Cardiac natriuretic hormones, neurohormones, thyroid hormones and cytokines in normal subjects and patients with heart failure. Clin Chem Lab Med 2004; 42: 627– 636. 13 Galasko GIW, Lahiri A, Barnes SC, Collinson P, Senior R: What is the normal range for N-terminal pro-brain natriuretic peptide? How well does this normal range screen for cardiovascular disease? Eur Heart J 2005;26: 2269–2276.

14 Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, Wu AHB, Clopton P, Steg PG, Westheim A, Knudsen CW, Perez A, Kazanegra R, Herrmann HC, McCullough PA: Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002; 347: 161–167. 15 Troughton RE, Farmpton CM, Yandle TG, Espiner EA, Nicholls MG, Richards AM: Treatment of heart failure guided by plasma aminoterminal brain natriuretic peptide (NBNP) concentrations. Lancet 2000; 355: 1126–1130. 16 Anand IS, Fisher LD, Chiang YT, Latini R, Masson S, Maggioni AP, Glazer RD, Tognoni G, Cohn JN, Val-HeFT Investigators: Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in the Valsartan Heart Failure Trial (Val-HeFT). Circulation 2003; 107: 1278– 1283. 17 Doust JA, Pietrzak E, Dobson A, Glasziou P: How well does B-type natriuretic peptide predict death and cardiac events in patients with heart failure: systematic review. BMJ 2005;330:625–634. 18 Mueller C, Scholer A, Laule-Kilian K, Martina B, Schindler C, Buser P, Pfisterer M, Perruchoud AP: Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea. N Engl J Med 2004;350:647– 654.

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19 Redfield MM, Rodeheffer RJ, Jacobsen SJ, Mahoney DW, Bailey KR, Burnett JC Jr: Plasma brain natriuretic peptide concentration: impact of age and gender. J Am Coll Cardiol 2002;40:976–982. 20 Yoshibayashi M, Kamiya T, Saito Y, Nakao K, Nishioka K, Temma S, Itoh H, Shirakami G, Matsuo H: Plasma brain natriuretic peptide concentrations in healthy children from birth to adolescence: marked and rapid increase after birth. Eur J Endocrinol 1995;133: 207–209. 21 Nir A, Bar-Oz B, Perles Z, Brooks R, Korach A, Rein AJJT: N-terminal pro-B-type natriuretic peptide: reference plasma levels from birth to adolescence. Elevated levels at birth and in heart diseases. Acta Paediatr 2004;93: 603–607. 22 Mir TS, Flato M, Falkenberg J, Haddad M, Budden R, Weil J, Albers S, Laer S: Plasma concentrations of N-terminal brain natriuretic peptide in healthy children, adolescents, and young adults: effect of age and gender. Pediatr Cardiol 2006;27:73–77.

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23 Ohuchi H, Takasugi H, Ohashi H, Okada Y, Yamada O, Ono Y, Yagihara T, Echigo S: Stratification of pediatric heart failure on the basis of neurohormonal and cardiac autonomic nervous activities in patients with congenital heart disease. Circulation 2003; 108:2368–2376. 24 Westerlind A, Wahlander H, Lindstedt G, Lundberg PA, Holmgren D: Clinical signs of heart failure are associated with increased levels of natriuretic peptide types B and A in children with congenital heart defects or cardiomyopathy. Acta Paediatr 2004; 93: 340–345. 25 Cohen S, Springer C, Perles Z, Rein AJJT, Avital A, Argaman Z, Nir A: Amino-terminal pro-brain-type natriuretic peptide: heart or lung disease in pediatric respiratory distress? Pediatrics 2005;115:1347–1350. 26 Suda K, Matsumura M, Matsumoto M: Clinical implication of plasma natriuretic peptides in children with ventricular septal defect. Pediatr Int 2003;45:249–254.

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27 Gerber IL, Stewart RAH, Legget ME, West TM, French RL, Sutton TM, Yandle TG, French JK, Richards AM, White HD: Increased plasma natriuretic peptide levels reflect symptom onset in aortic stenosis. Circulation 2003;107:1884–1890. 28 Reynolds EW, Ellington JG, Vranicar M, Bada HS: Brain-type natriuretic peptide in the diagnosis and management of persistent pulmonary hypertension of the newborn. Pediatrics 2004;114:1297–1304. 29 Claudius I, Lan YT, Chang RK, Wetzel GT, Alejos J: Usefulness of B-type natriuretic peptide as a noninvasive screening tool for cardiac allograft pathology in pediatric heart transplant recipients. Am J Cardiol 2003;92: 1368–1370. 30 Kawamura T, Wago M, Kawaguchi H, Tahara M, Yuge M: Plasma brain natriuretic peptide concentrations in patients with Kawasaki disease. Pediatr Int 2000;42:241–248.

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Pediatric Workshop 3

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):81–90 DOI: 10.1159/000097558

Published online: February 15, 2007

ABCs of Natriuretic Peptides: Growth E.A. Espiner a T.C. Prickett a T.G. Yandle a G.K. Barrell b M. Wellby b M.J. Sullivan c B.A. Darlow c a Department of Medicine, Christchurch School of Medicine and Health Sciences, b Agricultural and Life Sciences Division, Lincoln University, and c Department of Pediatrics, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand

Key Words C-type natriuretic peptide Amino-terminal pro-CNP Linear growth Growth plate Chondrocytes

Abstract Background: Although they are better known for their cardiovascular and antiproliferative effects, natriuretic peptides also have been found to produce skeletal overgrowth in transgenic rodents. This finding has unmasked a novel paracrine role in endochondral bone growth for this family of hormones. Genetic studies in which key components in the C-type natriuretic peptide (CNP) signaling pathway are disrupted or overexpressed and spontaneous mutations in the CNP receptor in humans indicate that CNP plays a crucial role in determining postnatal skeletal elongation. CNP appears to promote expansion within several areas of the growth plate, but its effects are most obvious in the hypertrophic zone. Since its actions are largely paracrine in nature, new approaches are needed to study CNP’s role in vivo. CNP itself degrades rapidly, but amino-terminal pro-CNP (NT pro-CNP) escapes rapid degradation and can be readily detected in plasma. Methodology: Studies have been made of changes in CNP synthesis in humans and sheep by measuring NT pro-CNP levels. Plasma NT pro-CNP levels are markedly elevated in newborns, but fall progressively as growth velocity declines in both children and lambs. In keeping with these growth-related changes, CNP levels are inhibited by

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0081$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

glucocorticoids and malnutrition in lambs and are stimulated by growth hormone and testosterone in adolescent boys. Further, levels fall abruptly in children receiving growth-suppressing chemotherapy regimens. Conclusions: Plasma NT pro-CNP levels may be an indicator of CNP synthesis within skeletal tissues and provide a much needed biomarker of linear growth, with applications for diagnosis and management of growth disorders. Copyright © 2007 S. Karger AG, Basel

Introduction

Within 10 years of the discovery of natriuretic peptides, a consensus had emerged on their regulation and integrated actions [1]. Briefly stated, in response to increased cardiac wall tension, plasma concentrations of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are raised by enhanced release and synthesis from atrial and ventricular myocytes, respectively. By acting on multiple tissues regulating sodium status and vascular pressure, these cardiac hormones reduce vascular volume and pressure, and assist in restoring intracardiac pressure to normal. C-type (C)NP was not considered to be a product of cardiac secretion, and in keeping with this view, circulating levels were low – often at the limits of detection [2–5].

E.A. Espiner, MD Department of Medicine Christchurch School of Medicine and Health Sciences 14 Hidden Hollow Trail, Madison, WI 53717 (USA) Tel. +1 608 836 1220, Fax +1 608 265 5421, E-Mail [email protected]

The physiological consequences of the well-documented antiproliferative effects of all three natriuretic peptides within cardiac and vascular tissues were much less clear [6–9]. Clarifying these actions required powerful new tools of genetic engineering. In keeping with antiproliferative and other actions, transgenic (tg) mice that overexpressed the ANP [10] or BNP [11] gene in the liver showed a cardiovascular phenotype characterized by cardiac hypoplasia and systemic hypotension. Surprisingly, however, the BNP tg mice also exhibited marked skeletal overgrowth [12]. Growth plates in these mice were expanded by increased cellular proliferation and hypertrophy – in strong contrast to the antimitogenic effects observed in cardiac fibroblasts [7]. In hindsight, it is now clear that this genetic manipulation had unmasked an entirely new paracrine pathway regulating endochondral bone growth that was not based on BNP but on a close family relative: CNP. This review focuses on the evidence implicating CNP as a crucial new growth factor in skeletal biology. The first half covers the genetic evidence linking NPs to skeletal growth and briefly summarises the skeletal actions of CNP. Because CNP’s actions are largely paracrine in nature, new approaches are needed to study regulation of CNP and its role in vivo. The second half of this review covers these issues together with possible clinical applications in disorders of linear growth.

Genetic Evidence Linking Natriuretic Peptides with Skeletal Growth

Despite the obvious skeletal overgrowth shown by the BNP tg mice, neither the BNP knockout [13] nor mice with disrupted NPR-11 (the receptor for ANP and BNP) exhibited a skeletal or growth abnormality, though a cardiovascular phenotype was evident [14]. Moreover, when the BNP tg mouse was crossed with the NPR-1 knockout mouse, the offspring retained the skeletal overgrowth phenotype [15], indicating that BNP must be signaling in skeletal tissues via receptors other than NPR-1. The CNP signaling pathway was implicated in a series of elegant studies chiefly conducted by Nakao and colleagues in Kyoto, Japan. These workers showed that disrupting CNP expression in all tissues markedly reduced postnatal linear growth without evidence of obvious cardiovas-

1 Also known as NPR-A. According to this notation, NPR-B is the CNP receptor, and NPR-C is the clearance receptor.

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CNP

NPR-2

cGMP

cGKII

Bone actions

BNP

Fig. 1. CNP signaling pathway. The large arrow indicates crossactivation of the CNP receptor (NPR-2) by BNP at high plasma concentrations.

cular phenotype, in contrast to what is seen in the ANP [16] or BNP knockout [13] models. Long bones and vertebra in mice with disrupted CNP expression were 50– 80% the size of their wild-type litter mates. Convincingly, this dwarfed phenotype could be reversed by crossing the knockout mice with mice overexpressing the CNP gene targeted to a component of growth plate collagen [17]. Independent studies conducted previously showed that disrupting the clearance receptor (NPR-3) also produced mice with postnatal skeletal overgrowth strongly resembling that seen in the BNP tg mice [18, 19]. Taken together, these studies provide at least two possible mechanisms for the skeletal overgrowth observed in the BNP tg model. As shown in figure 1, greatly elevated circulating levels of BNP in the BNP tg mouse [12] (compared with normal mice) may be sufficient to cross-activate the CNP receptor, NPR-2 [20, 21]. Second, such high BNP blood levels may saturate clearance sites (NPR-3), raising local levels of CNP within growth plate tissues and thereby mimicking the skeletal pathology of the NPR-3 knockout. Subsequent work has shown that genetic manipulations or spontaneous mutations at each point in the CNP signaling pathway yield the expected disorders of endochondral bone growth (table 1). In several instances bone formation and thickness were also affected. Recent studies have dramatically confirmed the relevance of these findings in humans by showing that loss-of-function mutations in NPR-2 cause extremely short stature in subjects with the rare autosomal recessive disorder, acromesomelic dysplasia, Maroteaux type [22]. In this syndrome, affected subjects are normal at birth, develop shortEspiner /Prickett /Yandle /Barrell /Wellby / Sullivan /Darlow

limbed (disproportionate) short stature in the subsequent 1–2 years and attain a final adult height of approximately 95–125 cm (3–4 feet) (fig. 2) [23, 24]. Apart from the skeletal system, no other organ system abnormality has been identified so far in humans [22]. However, aplasia of reproductive organs has been found in female mice with disrupted NPR-2 [25], and cardiac hypertrophy has been recently reported in rats with impaired NPR-2 function [26], indicating the need for more focused studies in patients with impaired CNP/NPR-2 signaling. Of special interest is the finding of reduced adult height in obligate carriers with NPR-2 functional haplo insufficiency [22]. Olney et al. recently confirmed this observation in a large affected kindred [27]. As shown in figure 3, carriers with one copy of the functionless allele are 9–10 cm shorter than noncarrier family relatives, suggesting that 3–4% of children presenting with idiopathic short stature may be carriers of NPR-2 loss-of-function mutations. Clearly these genetic manipulations and spontaneous mutations in rodents as well as in humans provide compelling evidence of the importance of the CNP signaling pathway in postnatal growth. Further study is required to define other effects within the mature skeleton or within reproductive, cardiac and possibly other tissues in humans.

Fig. 2. Three affected siblings with acromesomelic dysplasia (foreground), their mother (left) and five of the unaffected siblings. (Reprinted with permission from Kant et al. [24].)

Table 1. Growth plate histology in genetic models of natriuretic peptide action

Model

Proliferative zone

Hypertrophic zone

Other effects

BNP transgenic [12]

Increased

Increased

ALP and bone formation rate increased

CNP knockout [17]

Reduced

Greatly reduced

Prehypertrophic zone reduced

NPR-3 knockout [18]

Unaffected

Increased

Trabeculae thicker, longer Osteoblasts 3-fold increased Bone turnover increased

NPR-2 knockout [25]

Reduced

Reduced

Prehypertrophic zone reduced

CNP overexpression targeted to the growth plate [32]

Increased

Increased Chondrocyte volume greater

Subchondral trabeculae longer and more abundant Bone mineral density increased in proximal tibia

cGMP-dependent protein kinase II loss of function mutation [32]

ABCs of Natriuretic Peptides: Growth

Growth plate expanded by abnormal accumulation of ‘postmitotic’ chondrocytes After fracture, increased uncalcified callus (impaired endochondral ossification)

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2 Female 1

Male

tion. The heights of carriers and noncarrier family members were measured and values were transformed into z-scores. The points show the individual results (circles, female subjects; squares, male subjects; bars, mean for each group; error bars, SD; horizontal line, population mean [z-score of 0]). For the carriers, n = 16, and for the noncarriers, n = 23. * p ! 0.0005 versus noncarrier family members. † p ! 0.0005 versus general population (analysis of variance with Holm’s t test post hoc comparisons). (Reprinted with permission from Olney et al. [27].)

Height (z-score)

Fig. 3. Stature of carriers of NPR-2 muta0

*† –1 –2 –3 –4 Carriers

Noncarriers

Growth hormone Thyroid hormone Estrogen Insulin-like growth factor

Resting

Fig. 4. Schematic depiction of an endo-

chondral growth plate, with resting, proliferative, prehypertrophic and hypertrophic zones noted. Paracrine factors known to regulate endochondral growth are shown at left. Sites of expression of CNP and associated receptors (NPR-2 and the clearance receptor NPR-3) are shown. Systemic growth factors are noted above. (Modified with permission from Bartels et al. [22].)

CNP BMPs FGFs

Prehypertrophic

VEGF

CNP transcripts are present in osteoblasts [28] and chondrocytes [29] as well as in a diverse range of other tissues [30]. However, because of the striking effects of CNP on linear growth, most studies of its actions in the skeleton have focused on growth plate chondrocytes. Growth plates represent areas of cartilaginous tissue at each end of long bones, spatially situated between the epiphysis above and the newly formed metaphyseal bone below. Growth occurs as chondrocytes are recruited from Horm Res 2007;67(suppl 1):81–90

NPR-2

Ihh

CNP Actions within Skeletal Tissues

84

Proliferative

PthrP IGFs

NPR-3

Hypertrophic

resting cells to form proliferating columns, later exiting the mitotic phase to become differentiated enlarged hypertrophic cells that eventually apoptose to form a template for new bone formation and trabeculation [31]. Increases in cell size of the hypertrophic zone contribute up to 70% of the increase in linear height from infancy to adulthood. Each of these processes (fig. 4) is carefully coordinated by a variety of local growth factors interlinking with systemic hormones at different stages of growth and maturation. Just how the CNP pathway fits into this complex milieu is not yet known, but in experiments conEspiner /Prickett /Yandle /Barrell /Wellby / Sullivan /Darlow

CNP - Increases chondrocyte proliferation

- Via cGMP, controls exit from mitotic to prehypertrophic phase

cGKII

- Stimulates hypertrophy

- Increases matrix synthesis

- Is reduced by NPR-3 in hypertrophic zone

NPR-3

Fig. 5. Actions of CNP in the growth

plate.

-ss1

1

pro-CNP(1–103)

50 NT-CNP(1–50)

51

103 CNP-53

-ss103

-ss-

Fig. 6. Hypothetical processing pathways

of pro-CNP. Bioactive forms are CNP-53 and CNP-22.

1

81 NT-CNP(1–81)

82

103 CNP-22

ducted in fetal tibial explants, the growth-promoting effect of CNP exceeded that of any of the better known paracrine or systemic factors (fig. 4) on a molar basis [32]. In vitro and ex vivo studies show that CNP acts at several levels within the growth plate (fig. 5). First, CNP, which is presumably synthetised by proliferating chondrocytes [17], acts locally to stimulate further proliferation, as shown in the fetal tibial explant [33]. Some evidence suggests that this action may be mediated indirectly by opposing fibroblast growth factor (FGF) signaling,

which in cartilage tissue acts to constrain growth [34]. Second, CNP-activated cGMP-dependent protein kinase II serves as a molecular switch coupling the cessation of proliferation and the beginning of hypertrophic differentiation, possibly by inhibiting Sox 9 nuclear entry (an inhibitory regulator of hypertrophic differentiation) [35]. Third, and perhaps most importantly, CNP stimulates the hypertrophic cell to increase in size and enlarge the hypertrophic zone of the growth plate. Again, this action may be indirect in part, inhibiting the normally constraining effect of FGF signaling [32]. For example, Ya-

ABCs of Natriuretic Peptides: Growth

Horm Res 2007;67(suppl 1):81–90

85

100 80 60 NT pro-CNP (pmol/l)

40 20

CNP (pmol/l)

0

Adults (n = 117)

Children (n = 58)

Fig. 7. Plasma NT pro-CNP and CNP levels in children (5–18 years) compared with healthy adults (20–80 years). Mean and SEM (bars) are shown. (Reprinted with permission from Pricket et al. [39].)

soda et al. have shown that CNP counteracts the inhibitory effects of FGFR-3 constitutive activation, which is the basis of a murine model of achondroplasia. Remarkably, the severely dwarfed phenotype of this murine model could be largely rescued by crossing affected mice with mice overexpressing the CNP gene targeted to the growth plate [32]. This result indicates that CNP must act downstream of FGF signaling. Fourth, by increasing matrix secretion and expanding the intercellular space, CNP promotes growth-plate enlargement in a manner both dependent [32] and independent [34] of the constraining effects of FGF-signaling activity. Together these actions largely account for the prominent hypertrophic zone and generally increased ratio of hypertrophic to proliferative tissue within the growth plate as reported in several genetic models (table 1). The importance, and the mechanism, underlying the increased bone turnover and prominent trabeculation noted in some studies have yet to be clarified.

New Approaches to Assessing CNP Synthesis in vivo

The crucial role of CNP in skeletal growth highlights the need for studies of the hormone’s day-to-day regulation and role in growing mammals. However, the paracrine nature of CNP secretion, its low levels in plasma 86

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[2–5] and its rapid half-life [5, 36] hinder attempts to define the factors regulating CNP secretion in vivo. Mindful that (1) CNP is first synthetised as the prohormone pro-CNP(1–103), which is processed to yield either bioactive CNP-22 or CNP-53 (fig. 6), and that (2) unlike bioactive forms, any amino-terminal forms generated would not be degraded by the clearance receptor or by neprilysin [37] and could therefore enter the circulation in measurable quantities, we raised antisera to the first 15 amino acid residues of human pro-CNP. This led to the successful identification of the amino-terminal 5-kD fragment (consistent with pro-CNP 1–50) in ovine and human plasma [38], indicating that CNP-53 (and not CNP-22) was the dominant form produced from pro-CNP in tissues [39]. Using a specific radioimmunoassay for aminoterminal pro-CNP (NT pro-CNP), we found that plasma levels of NT pro-CNP were 10- to 50-fold higher than those of CNP. Furthermore, in keeping with the presence of active growth plates, NT pro-CNP levels were 2- to 3fold higher in children aged 5–18 years compared with fully mature adults (fig. 7). Cross-sectional studies have found levels of NT proCNP are extremely high in umbilical vein plasma but progressively fall to much lower – and more stable – values in mature adults [39]. In studies involving 58 children (aged 5–18 years), the NT pro-CNP level was strongly correlated with alkaline phosphatase (ALP) activity (r = 0.55, p ! 0.001). In 23 of these children, height was measured at intervals of at least 3 months and within 6 months of blood sampling, which allowed calculation of concurrent growth velocity. In these 23 children, the NT proCNP level was strongly correlated with growth velocity (r = 0.57, p ! 0.005). No distinct increase in plasma NT pro-CNP levels was detected during the adolescent years, but this cross-sectional study took no account of pubertal status and was not designed to examine changes in the peripubertal growth phase [39]. While these data in children were consistent with the view that plasma NT pro-CNP reflects growth plate activity and concurrent growth velocity, they were limited by the small numbers in each age group and by the necessity to sample from children attending a diverse range of outpatient clinics. Therefore, we conducted serial (longitudinal) studies in normal healthy lambs from 1 week of age to 30 weeks when linear growth was largely completed [39]. As shown in figure 8, levels of both NT pro-CNP and the much lower concentration of CNP were strongly correlated with plasma ALP level (r = 0.94, p ! 0.001) and with metacarpal growth velocity as measured by vernier calipers (r = 0.55, p ! 0.001). Espiner /Prickett /Yandle /Barrell /Wellby / Sullivan /Darlow

Possible Clinical Applications

NT pro-CNP

pmol/l

60

40

20 CNP 0 0

5

10

15

1,000

20

25

30

ALP

800

IU/l

600 400 200 0 0

5

1.0

10

15

20

25

30

Growth velocity

0.8 0.6 mm/day

Taken together, these findings in children and lambs are consistent with the hypothesis that NT pro-CNP reflects CNP synthesis within growth plates. Moreover, the decline in the concentration of NT pro-CNP from birth to maturation is likely to be linked to the progressive decrease in both hypertrophic and proliferative chondrocytes as observed in human studies over the same time period [40]. These results also raise the possibility that NT pro-CNP may constitute a unique marker of growth plate activity and concurrent growth velocity. Next, we studied the effects of interventions known to impair linear growth (e.g., high-dose glucocorticoids and malnutrition) to gain better insight into CNP synthesis. As shown in figure 9, compared with saline injections, administration of 0.25 mg/kg/day dexamethasone for 15 days abruptly and reversibly reduced plasma NT proCNP in 4-week-old lambs and induced similar but delayed changes in plasma ALP and growth velocity [39]. Similarly, plasma NT pro-CNP levels fell abruptly (30%) and reversibly in 4-week-old lambs fed a restricted diet containing 25% of normal calories and protein for 6 days when compared with lambs fed a normal diet [41]. There was a similar fall in plasma ALP (31%). Metacarpal growth velocity was also reduced, but the difference between the lambs on the restricted diet and those on the normal diet did not achieve statistical significance within the 6-day study period. Lambs given either dexamethasone or a restricted diet had similar temporal patterns of response in both plasma NT pro-CNP and ALP levels, as well as similar degrees of inhibition. These findings suggest that the responses could be based on depletion of proliferating chondrocytes in catabolic states, as reported by others [42, 43]. Though the underlying mechanisms remain to be clarified, the studies clearly indicate that CNP synthesis is dynamic and subject to day-to-day regulation.

0.4 0.2 0

0

5

10

15 20 Age (weeks)

25

30

35

Fig. 8. Plasma CNP forms (top panel), ALP activity (middle panel) and metacarpal growth velocity (lower panel) in 24 lambs (12 male, 12 female) age 1 to 30 weeks. Values are mean 8 SEM. Filled symbols for males are shown where it was possible to distinguish male from female values.

Linear growth rate can be an important index of a child’s overall health, yet it is not readily measurable at any one point in time. Currently no markers of growth plate activity are available for clinical use. Thus, impaired linear growth, which frequently accompanies chronic illness or drug usage, often goes unrecognized. Plasma NT pro-CNP level, which reflects changes in bone formation rate and growth velocity, could provide the clinician with an early indication of changing growth velocity months before changes could be detected using conventional

measurements of height by stadiometry. To explore this potential application, we studied children receiving chemotherapy for acute lymphoblastic leukaemia (ALL), because chemotherapy is a well-recognized cause of growth failure and predictably halts linear growth during and after the inductive phase of treatment [44, 45]. As shown in figure 10, standard induction treatment in a 7-year-old girl with ALL was associated with a precipitous fall in

ABCs of Natriuretic Peptides: Growth

Horm Res 2007;67(suppl 1):81–90

87

80 70

40

** * * * * *

20

Saline Dexamethasone

NT pro-CNP (pmol/l)

Plasma NT pro-CNP (pmol/l)

Treatment period 60

60 50 40 30 20 10

0 –6

0

6

18

12

24

30

Vincristine dexamethasone

0 –5

Treatment period

0

5

10

15 20 Time (days)

25

30

35

40

ALP (IU/l)

800

Fig. 10. Response of plasma NT pro-CNP to chemotherapy induction in a 7-year-old girl with acute lymphoblastic leukemia. The horizontal bar with dashes indicates the provisional age reference range. Treatment included twice-daily injections of dexamethasone (3 mg/m2/day) for 28 days and weekly injections of vincristine over 3 weeks.

600

400

* *

200

*

0 –6

0

6

12

18

24

30

Treatment period Metacarpal length (mm)

150

140

* *

*

130

*

120 0 –6

0

6

12 Days

18

24

30

Fig. 9. Effect of dexamethasone (filled symbols, n = 8) or saline (open symbols, n = 8), administered to 4-week-old lambs for 15 days, on plasma NT pro-CNP (top panel), ALP activity (middle panel) and metacarpal length (lower panel). Values are mean 8 SEM. Significant differences between dexamethasone and saline control time-matched data are indicated by asterisks (* p ! 0.05). (Reproduced with permission from Prickett et al. [39].)

plasma NT pro-CNP from levels considered normal for age prior to treatment. After completion of chemotherapy, her plasma NT pro-CNP level increased, but it was still below pretreatment levels. This child showed no lin88

Horm Res 2007;67(suppl 1):81–90

ear growth during the 9 weeks of observation. Five children with ALL have now been studied, and all have shown very low levels of plasma NT pro-CNP at the completion of the induction phase of treatment. It remains to be seen whether lesser doses of glucocorticoids such as those used for treating persistent asthma also affect NT pro-CNP levels. It is conceivable that monitoring plasma NT proCNP levels could provide an early warning of growth impairment – or harmful skeletal effects – in at-risk children. Other important areas in which assays of NT proCNP may assist in clinical management are situations characterized by increases in linear growth in response to treatment interventions. Not all short children respond predictably to growth-inducing treatments such as growth hormone, insulin-like growth factor I or sex steroids. In collaborative studies with Nelly Mauras and Robert Olney in Jacksonville, Fla., USA, we explored the possibility that NT pro-CNP levels may reflect early skeletal response to growth stimulants. Prompt and significant increases (22–25% above basal) in plasma NT proCNP levels have been observed within 2 months of starting conventional doses of growth hormone treatment in poorly growing prepubertal adolescents, with a significant correlation between increased NT pro-CNP levels and growth velocity. Among poorly growing prepubertal Espiner /Prickett /Yandle /Barrell /Wellby / Sullivan /Darlow

boys, even greater (65–70% above basal) and more rapid increases were observed within 4 weeks of receiving a single intramuscular injection of depot testosterone (50 mg). If these preliminary results are confirmed by larger studies, it opens up the prospect of using plasma NT pro-CNP as an early predictor of skeletal response to growth-inducing treatments, with consequential savings in time, inconvenience and cost. These findings suggest that plasma NT pro-CNP may uniquely reflect growth plate activity, but a number of questions remain. Not the least of these is how much of a contribution do tissues outside the skeletal system make to plasma NT pro-CNP levels, and what is the source of elevated levels in adult subjects with severe heart disease [46] or moderate-to-severe renal impairment [47]? In addition, there is an urgent need for a well-defined age- and growth-related reference range for NT pro-CNP levels in healthy children along with profiles of pubertal responses in boys and girls. Finally, our recent demonstration of markedly elevated levels of both plasma CNP and NT pro-CNP in a subject with a homozygous loss-of-function mutation in NPR-2 [27] strongly suggests that NT pro-CNP levels will be elevated in other states of ‘CNP resistance’ – some of which may exhibit growth impairment. Such paradoxical relationships must be considered when interpreting individual levels in some subjects.

Conclusions

CNP synthesis and action are essential for endochondral bone growth in mammals, including humans. CNP appears to promote expansion within several areas of the growth plate, but its effects are most obvious in the hypertrophic zone. A new approach to assessing CNP synthesis and regulation in vivo is to assay NT pro-CNP, a fragment of pro-CNP that is presumed to be inactive. Unlike CNP, NT pro-CNP escapes rapid degradation and thus can be readily measured in plasma. Plasma NT proCNP levels are strongly correlated with growth velocity and markers of bone formation and may therefore reflect CNP synthesis within skeletal tissues. As such, NT proCNP may serve as a biomarker of linear growth with applications for the diagnosis and management of growth disorders. The possible roles CNP plays in bone formation and skeletal biology in the adult remain to be defined.

Acknowledgments Laboratory studies cited in this review were supported by grants from the Health Research Council of New Zealand, the Canterbury Medical Research Foundation, the Lotteries Board of New Zealand and the New Zealand Child Health Research Foundation.

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6 Furuya M, Yoshida M, Hayashi Y, Ohnuma N, Minamino N, Kangawa K, Matsuo H: Ctype natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells. Biochem Biophys Res Commun 1991; 177:927–931. 7 Cao L, Gardner DG: Natriuretic peptides inhibit DNA synthesis in cardiac fibroblasts. Hypertension 1995;25:227–234. 8 Gardner DG: Natriuretic peptides: markers or modulators of cardiac hypertrophy? Trends Endocrinol Metab 2003;14:411–416. 9 Silberbach M, Roberts CT Jr: Natriuretic peptide signalling: molecular and cellular pathways to growth regulation. Cell Signal 2001;13:221–231. 10 Steinhelper ME, Cochrane KL, Field LJ: Hypotension in transgenic mice expressing atrial natriuretic factor fusion genes. Hypertension 1990;16:301–307. 11 Ogawa Y, Itoh H, Tamura N, Suga S, Yoshimasa T, Uehira M, Matsuda S, Shiono S, Nishimoto H, Nakao K: Molecular cloning of the complementary DNA and gene that encode mouse brain natriuretic peptide and generation of transgenic mice that overexpress the brain natriuretic peptide gene. J Clin Invest 1994;93:1911–1921.

12 Suda M, Ogawa Y, Tanaka K, Tamura N, Yasoda A, Takigawa T, Uehira M, Nishimoto H, Itoh H, Saito Y, Shiota K, Nakao K: Skeletal overgrowth in transgenic mice that overexpress brain natriuretic peptide. Proc Natl Acad Sci USA 1998;95:2337–2342. 13 Tamura N, Ogawa Y, Chusho H, Nakamura K, Nakao K, Suda M, Kasahara M, Hashimoto R, Katsuura G, Mukoyama M, Itoh H, Saito Y, Tanaka I, Otani H, Katsuki M: Cardiac fibrosis in mice lacking brain natriuretic peptide. Proc Natl Acad Sci USA 2000; 97: 4239–4244. 14 Oliver PM, Fox JE, Kim R, Rockman HA, Kim HS, Reddick RL, Pandey KN, Milgram SL, Smithies O, Maeda N: Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. Proc Natl Acad Sci USA 1997;94:14730–14735. 15 Chusho H, Ogawa Y, Tamura N, Suda M, Yasoda A, Miyazawa T, Kishimoto I, Komatsu Y, Itoh H, Tanaka K, Saito Y, Garbers DL, Nakao K: Genetic models reveal that brain natriuretic peptide can signal through different tissue-specific receptor-mediated pathways. Endocrinology 2000; 141: 3807– 3813.

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26 Langenickel TH, Buttgereit J, Pagel-Langenickel I, Lindner M, Monti J, Beuerlein K, Al-Saadi N, Plehm R, Popova E, Tank J, Dietz R, Willenbrock R, Bader M: Cardiac hypertropy in transgenic rats expressing a dominant-negative mutant of the natriuretic peptide receptor B. Proc Natl Acad Sci USA 2006;103:4735–4740. 27 Olney RC, Bukulmez H, Bartels CF, Prickett TCR, Espiner EA, Potter L, Warman ML: Heterozygous mutations in natriuretic peptide receptor-B (NPR2) are associated with short stature. J Clin Endocrinol Metab 2006; 91:1229–1232. 28 Suda M, Tanaka K, Fukushima M, Natsui K, Yasoda A, Komatsu Y, Ogawa Y, Itoh H, Nakao K: C-type natriuretic peptide as an autocrine/paracrine regulator of osteoblast. Biochem Biophys Res Commun 1996; 223:1–6. 29 Hagiwara H, Sakaguchi H, Itakura M, Yoshimoto T, Furuya M, Tanaka S, Hirose S: Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-B. J Biol Chem 1994; 269:10729–10733. 30 Minamino N, Aburaya M, Kojima M, Miyamoto K, Kangawa K, Matsuo H: Distribution of C-type natriuretic peptide and its messenger RNA in rat central nervous system and peripheral tissue. Biochem Biophys Res Commun 1993;197:326–335. 31 Kronenberg HM: Developmental regulation of the growth plate. Nature 2003; 423: 332– 336. 32 Yasoda A, Komatsu Y, Chusho H, Miyazawa T, Ozasa A, Miura M, Kurihara T, Rogi T, Tanaka S, Suda M, Tamura N, Ogawa Y, Nakao K: Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nat Med 2004; 10:80–86. 33 Yasoda A, Ogawa Y, Suda M, Tamura N, Mori K, Sakuma Y, Chusho H, Shiota K, Tanaka K, Nakao K: Natriuretic peptide regulation of endochondral ossification. Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway. J Biol Chem 1998; 273:11695–11700. 34 Krejci P, Masri B, Fontaine V, Mekikian PB, Weis M, Prats H, Wilcox WR: Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis. J Cell Sci 2005;118:5089–5100. 35 Chikuda H, Kugimiya F, Hoshi K, Ikeda T, Ogasawara T, Shimoaka T, Kawano H, Kamekura S, Tsuchida A, Yokoi N, Nakamura K, Komeda K, Chung UI, Kawaguchi H: Cyclic GMP-dependent protein kinase II is a molecular switch from proliferation to hypertrophic differentiation of chondrocytes. Genes Dev 2004;18:2418–2429.

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36 Charles CJ, Espiner EA, Richards AM, Nicholls MG, Yandle TG: Comparative bioactivity of atrial, brain, and C-type natriuretic peptides in conscious sheep. Am J Physiol Regul Integr Comp Physiol 1996;270:R1324– R1331. 37 Yandle TG: Biochemistry of natriuretic peptides. J Intern Med 1994;235:561–576. 38 Prickett TCR, Yandle TG, Nicholls MG, Espiner EA, Richards AM: Identification of amino-terminal pro-C-type natriuretic peptide in human plasma. Biochem Biophys Res Commun 2001;286:513–517. 39 Prickett TCR, Lynn AM, Barrell GK, Darlow BA, Cameron VA, Espiner EA, Richards AM, Yandle TG: Amino-terminal proCNP: a putative marker of cartilage activity in postnatal growth. Pediatr Res 2005;58:334–340. 40 Byers S, Moore AJ, Byard RW, Fazzalari NL: Quantitative histomorphometric analysis of the human growth plate from birth to adolescence. Bone 2000;27:495–501. 41 Prickett TCR, Yandle TG, Barrell GK, Wellby M, Richards AM, Espiner EA: Plasma NTproCNP reflects the skeletal response to anabolic and catabolic interventions in growing lambs. Proceedings from the Endocrine Society 88th Annual Meeting, June 24–27, 2006, Boston, Massachusetts, USA. 42 Mushtaq T, Bijman P, Ahmed SF, Farquharson C: Insulin-like growth factor-I augments chondrocyte hypertrophy and reverses glucocorticoid-mediated growth retardation in fetal mice metatarsal cultures. Endocrinology 2004;145:2478–2486. 43 Farnum CE, Lee AO, O’Hara K, Wilsman NJ: Effect of short-term fasting on bone elongation rates: an analysis of catch-up growth in young male rats. Pediatr Res 2003;53:33–41. 44 Dalton VK, Rue M, Silverman LB, Gelber RD, Asselin BL, Barr RD, Clavell LA, Hurwitz CA, Moghrabi A, Samson Y, Schorin M, Tarbell NJ, Sallan SE, Cohen LE: Height and weight in children treated for acute lymphoblastic leukemia: relationship to CNS treatment. J Clin Oncol 2003;21:2953–2960. 45 Yamashita N, Tanaka H, Moriwake T, Nishiuchi R, Oda M, Seino Y: Analysis of linear growth in survivors of childhood acute lymphoblastic leukemia. J Bone Miner Metab 2003;21:172–178. 46 Wright SP, Prickett TC, Doughty RN, Frampton C, Gamble GD, Yandle TG, Sharpe N, Richards M: Amino-terminal pro-C-type natriuretic peptide in heart failure. Hypertension 2004;43:94–100. 47 Obineche EN, Pathan JY, Fisher S, Prickett TCR, Yandle TG, Frampton CM, Cameron VA, Nicholls MG: Natriuretic peptide and adrenomedullin levels in chronic renal failure and effects of peritoneal dialysis. Kidney Int 2006;69:152–156.

Espiner /Prickett /Yandle /Barrell /Wellby / Sullivan /Darlow

Pediatric Workshop 4

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):91–95 DOI: 10.1159/000097559

Published online: February 15, 2007

Disorders of Sexual Differentiation I.A. Hughes Department of Paediatrics, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK

Key Words Sexual differentiation Disorders of sex development Gender dysphoria Gender identity disorder

Abstract In the context of sex development, sex differentiation defines the phenotype of the internal and external genitalia that logically follows sex determination. In 2006, a consensus statement was published summarizing results of a worldwide expert meeting that proposed, among other things, alternative and more precise nomenclature on management of disorders of sex development coupled with a new diagnostic classification. Key principles for management of DSD are: (1) avoid instantaneous gender assignment before expert evaluation, (2) assessment and longer-term management must be performed in a centre with an experienced multidisciplinary team, and (3) full and open communication with affected families is mandatory, with the families encouraged to participate in the decision-making. Copyright © 2007 S. Karger AG, Basel

Introduction

When considering the clinical problem of disorders of sexual differentiation, it is customary to refer to infants born with ambiguous genitalia in whom there is some doubt about sex assignment at birth. The term differen-

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0091$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

tiation in relation to anatomical structures refers to the acquisition of one or more characteristics or functions that differ from the primordial structure. Consequently, in the context of sex development, sex differentiation defines the phenotype of the internal and external genitalia that logically follows sex determination, which defines the gonad as being a testis or an ovary.

Control of Male and Female Development

The components of fetal male and female sex development from an embryological and genetic perspective are illustrated in figure 1 and are detailed in exemplar reviews [1–3]. Key elements include migration of primordial germ cells from the yolk sac endoderm to the urogenital ridge, the mesonephros as the source of somatic cells that form Sertoli and Leydig cells in the male and the close proximity of the anlage of gonad, renal and adrenal development. The principal testis-determining gene is SRY (sex-related gene, Y chromosome), but numerous other genes have been identified in the mouse. The homologues most relevant in the human testis are shown in figure 1. Once formed, the Leydig cells of the testis produce androgens in high concentrations that subsequently stabilise Wolffian duct development and virilise the external genitalia. The action of androgens is mediated by the ligand-activated androgen receptor, a member of the nu-

Ieuan A. Hughes, MD Department of Paediatrics, Addenbrooke’s Hospital University of Cambridge, Hills Road Box 116, Level 8 Cambridge CB2 2QQ (UK) Tel. +44 1223 336 885, Fax +44 1223 336 996, E-Mail [email protected]

Male development Somatic cells

Germ cells

Genital ridge WT1 SF1 Bipotential gonad SRY SOX9

AMH/R

Testis

AR

AR

Wolﬃan ducts Insl3/ Lgr8

Mullerian regression

External genitalia Descent

Female development Somatic cells

Genital ridge

Germ cells

WT1 SF1 Bipotential gonad SRY SOX9

FOXL2

DAX1 WNT4

Ovary Wolﬃan ducts regress

Mullerian ducts

External genitalia

Fig. 1. Components of male and female sex development.

clear receptor superfamily. Sertoli cells produce antiMüllerian hormone, which, via its receptor expressed in Müllerian duct mesenchyme, causes regression of the Müllerian ducts and hence prevents the formation of a uterus and fallopian tubes in the male. The female is devoid of a similar panoply of ovariandetermining genes and factors essential for differentiation of a female phenotype during fetal and prepubertal development. Thus, a functioning ovary or the presence of estrogens is not an essential requirement for early fetal development, in keeping with the constitutive sex in females being female. However, as indicated in figure 1, overexpression of gonadal genes such as DAX-1 and WNT4 due to chromosomal duplication causes XY sex reversal. This suggests that they function in an ‘antitestis’ manner. The forkhead transcription factor, FOXL2, is key to follicular development in the ovary and for suppression of somatic testis determination in genetic fe92

Horm Res 2007;67(suppl 1):91–95

males [4]. An autosomal dominant syndrome comprising ocular anomalies (blepharophimosis, ptosis and epicanthus inversus) and ovarian dysfunction has been ascribed to mutations in the FOXL2 gene [5]. The main components in the pathway of androgen production, metabolism and action are illustrated in figure 2. Each of the steps is encoded by genes that, when subject to mutational inactivation, can lead to partial or complete loss of androgen function. When this occurs in an XY male, the consequences are either ambiguity of the external genitalia at birth or a deficiency so severe that the constitutive female sex is apparent. The final step in androgen action is mediated by ligand activation of the androgen receptor (AR), a nuclear receptor that is encoded by a gene on chromosome Xq11–12 [6]. The AR is a member of a large family of nuclear receptors that share a common functional domain structure comprising a large N-terminal domain involved in transactivation, a central DNA-binding domain (the most highly conserved domain within the family) and a C-terminal ligand-binding domain to which androgens bind on entering target cells (fig. 3). The AR also contains subdomains (AF1 and AF2) that mediate intrareceptor N- and C-terminal interaction, a process unique to the AR. A variable number of glutamine and glycine repeats are located in the N-terminal domain, the lengths of which appear to influence the transcriptional activity of the AR.

Terminology and Nomenclature

The subject of disorders of sexual differentiation is bedevilled with terms that are imprecise, confusing and lacking in descriptive definitions of pathophysiology. In terms of normal fetal sex development, sex determination refers to the gonad type (a testis or an ovary) and sex differentiation defines the phenotype as being male or female with respect to the appearance of the internal and external genitalia. Sex assignment (used interchangeably with gender assignment) is the instant assignation of male or female sex at birth, although this information is increasingly known to parents now before birth. Gender role is the aspect of one’s behaviour as being male or female. In childhood, this would refer to, for example, male-typical behaviour of boys preferring to choose vehicles and weapons as toys in contrast to girls preferring dolls. Gender role is influenced by exposure to prenatal androgens and is best manifest in the observations recorded in girls with congenital adrenal hyperplasia (CAH) who generally show increased male-typical beHughes

LH/hCG

LHR Cholesterol StAR CYP11A1 3␤-HSD CYP17/POR

DHA

A’dione

17␤ HSD3 Testosterone 5␣ RD2

CYP19

DHT

Fig. 2. Main components of the pathway of androgen production, metabolism and action.

AR

haviour [7]. Gender identity is the sense of self as being male or female; based on studies in individuals with CAH, there is less evidence for gender identity being influenced by prenatal testosterone [8]. Sexual orientation refers to the target of sexual arousal, which can be heterosexual, bisexual or homosexual. Gender dissatisfaction denotes unhappiness with assigned sex for which the terms gender dysphoria or gender identity disorder are used. The term intersex has traditionally been used to define an abnormality of the genitalia at birth (ambiguous genitalia) that leads to difficulty with the instantaneous sex assignment expected of normal births. There has been increasing concern expressed by affected families that the term intersex is potentially pejorative. Furthermore, terms such as pseudohermaphroditism (with prefixes, male or female), true hermaphroditism and sex reversal are confusing and are neither descriptive nor definitive with respect to causation. Consequently, a consensus meeting of worldwide experts charged with producing a statement on management of disorders of sex development included amongst its remit proposals to define alternative nomenclature coupled with a new diagnostic classification [9]. To replace the word intersex, the term disorders of sex development (DSD), defined as any congenital condition in which development of chromosomal, gonadal or anatomical sex is atypical, was proposed. Disorders of Sexual Differentiation

E2

ER␣/␤

(CAG)n

(GGT)n

Hinge

1

919 AF1 Transactivation domain

DBD

AF2 LBD

Fig. 3. The androgen receptor comprises a large N-terminal domain, a central DNA-binding domain and a C-terminal ligandbinding domain.

Classification and Causes of DSD

The traditional terminology linked to the new proposals is summarised in table 1. The karyotype is key to the classification and sets a pathophysiologic framework. It is then possible to derive a matrix of causes as shown in table 2. This is not an exhaustive list of causes of DSD but does offer a more practical approach for the clinician when asked to see an infant in the newborn nursery with ambiguous genitalia. The three most common causes of ambiguous genitalia in the newborn represent each of the classification categories listed in table 2: 45,X/46,XY mixed gonadal dysgenesis, partial androgen insensitivity syndrome (PAIS) and CAH secondary to 21-hydroxylase deficiency. Horm Res 2007;67(suppl 1):91–95

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Table 1. New terminology recommended for disorders of sex development

Table 3. Genital anomalies in newborns

Previous

Proposed

Intersex

DSD

Male pseudohermaphrodite Undervirilisation of an XY male Undermasculinisation of an XY male

46,XY DSD

Female pseudohermaphrodite Overvirilisation of an XX female Masculinisation of an XX female

46,XX DSD

Ambiguous genitalia Severe hypospadias 8 Undescended testes Micropenis Bifid scrotum Male with nonpalpable testes Female with bilateral inguinal herniae Isolated clitoromegaly Isolated labial fusion Genital anomalies in syndromes

True hermaphrodite XX male or XX sex reversal XY sex reversal

that merit investigation

Ovotesticular DSD 46,XX testicular DSD 46,XY complete gonadal dysgenesis

Table 2. A suggested classification of DSD

Sex chromosome DSD

46,XY DSD

46,XX DSD

A: 45,X (Turner syndrome and variants) B: 47,XXY (Klinefelter syndrome and variants) C: 45,X/46,XY mixed gonadal dysgenesis D: 46,XX/46,XY (chimeric)

A: Disorders of gonadal (testicular) development 1. Complete gonadal dysgenesis (Swyer syndrome) 2. Partial gonadal dysgenesis 3. Gonadal regression 4. Ovotesticular DSD

A: Disorders of gonadal (ovarian) development 1. Ovotesticular DSD 2. Testicular DSD (e.g., SRY+, dup SOX9) 3. Gonadal dysgenesis

B: Disorders in androgen synthesis or action 1. Androgen biosynthesis defect (e.g., 17␤-hydroxysteroid dehydrogenase deficiency, 5␣-reductase deficiency, StAR mutations 2. Defect in androgen action (e.g., CAIS, PAIS)

B: Androgen excess 1. Fetal (e.g., 21-hydroxylase deficiency, 11 hydroxylase deficiency) 2. Fetoplacental (aromatase deficiency, POR) 3. Maternal (luteoma, exogenous, etc.)

C: Other (e.g., severe hypospadias, cloacal extrophy, persistent Müllerian duct syndrome, other syndromes)

C: Other (e.g., cloacal extrophy, vaginal atresia, MURCS, other syndromes)

Clinical Assessment and Investigation of DSD in the Newborn

It is axiomatic that a relevant history is taken in the case of ambiguous genitalia, particularly since so many of the causes are genetic. Examination of the external genitalia should include measurements of the dimensions of the phallus, a description of the external orifices and their sites, the position of palpable gonads and any other congenital anomalies. Some quantitative assessment of the degree of masculinisation, such as the Prader score in CAH [10], or the degree of undermasculinisation, such as 94

Horm Res 2007;67(suppl 1):91–95

the external masculinisation score [11], in mixed gonadal dysgenesis or PAIS should be attempted. There are published norms for genital anatomy [9]. Table 3 includes clinical scenarios that merit investigation, together with a suggested list of tests. The latter is not exhaustive and is influenced by local resources. Useful diagnostic algorithms are also available [12]. It has been estimated that genital anomalies occur in 1 in 4,500 births. Not all will require a full investigative approach, but investigations are required for those whose genitalia are ambiguous, for an undermasculinised male with severe hypospadias, undescended testes and a micropenis Hughes

or for an apparent female with significant virilisation. The apparent male newborn with nonpalpable testes must be investigated, as these may be signs of a fully masculinised (Prader V) female infant with CAH. In such circumstances it is indefensible not to check the karyotype. Inguinal herniae are uncommon in girls, but this may be the clinical presentation in a significant number of infants with complete androgen insensitivity syndrome (CAIS) due to herniation of the testes. This raises the question of whether infant girls with bilateral inguinal herniae should have a routine karyotype. A survey of surgeons and paediatric endocrinologists indicated that the majority would consider a possible diagnosis of CAIS and investigate appropriately [13]. Fluorescence in situ hybridization to identify a Y chromosome is a rapid and reliable method for checking the sex chromosomes. Biopsy of a gonad found in the hernial sac may also be indicated. Isolated clitoromegaly warrants investigation; whereas, isolated apparent labial fusion may just be labial adhesions, a common finding in infant girls.

Management

Detailed discussion of the management of DSD is beyond the scope of this article. The principles to emphasise include the following. Avoid instantaneous gender assignment before expert evaluation has occurred. Assessment and longer-term management must be performed in a centre with an experienced multidisciplinary team. Full and open communication with affected families is mandatory, with the families encouraged to participate in the decision-making. Encourage patients and family to be involved with patient support groups where available. Considerable progress has been made in the diagnosis of DSD through advances in molecular genetics and hormone assays and in the raised awareness of ethical issues and patient advocacy concerns. Longer-term outcome data are accumulating now in patients with XX,DSD due to CAH, while similar data remain to be collated in XY,DSD individuals assigned male.

References 1 Federman DD: The biology of human sex differences. N Engl J Med 2006; 54: 1507– 1514. 2 Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation; in Larsen PR, Kronenberg HM, Melmed S, et al (eds): Williams Textbook of Endocrinology, 10th edition. Philadelphia, WB Saunders, 2003, pp 842–1002. 3 Ahmed SF, Hughes IA: The genetics of male undermasculinisation. Clin Endocrinol 2002;56:1–18. 4 Ottolenghi C, Omari S, Garcia-Ortiz JE, Uda M, Crisponi L, Forabosco A, Pilia G, Schlessinger D: Foxl2 is required for commitment to ovary differentiation. Hum Mol Genet 2005;15:2053–2062.

Disorders of Sexual Differentiation

5 Raile K, Stobbe H, Trobs RB, Kiess W, Pfaeffle R: A new heterozygous mutation of the FOXL2 gene is associated with a large ovarian cyst and ovarian dysfunction in an adolescent girl with blepharophimosis/ptosis/epicanthus inversus syndrome. Eur J Endocrinol 2005;153:353–358. 6 Quigley CA, DeBellis A, Marschke KB, ElAwady MK, Wilson EM, French FS: Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 1995:16: 271–321. 7 Hines M, Kaufman FR: Androgen and the development of human sex-typical behaviour: rough-and-tumble play and sex of preferred playmates in children with congenital adrenal hyperplasia (CAH). Child Dev 1994: 65:1042–1053. 8 Hines M, Brook C, Conway GS: Androgen and psychosexual development: core gender identity, sexual orientation and recalled childhood gender role behaviour in women and men with congenital adrenal hyperplasia (CAH). J Sex Res 2004;41:1–7.

9 Hughes IA, Houk C, Ahmed SF, Lee PA: Consensus statement on management of intersex disorders. Arch Dis Child 2006; 91: 554–563. 10 von Prader A: Der Genitalbefund beim Pseudohermaphroditus feminus des kongenitalen adrenogenitalen Syndromes. Helv Pediatr Acta 1954;9:231–248. 11 Ahmed SF, Khwaja O, Hughes IA: The role of a clinical score in the assessment of ambiguous genitalia. BJU Int 2000; 85: 120– 124. 12 Ogilvy-Stuart AL, Brain CE: Early assessment of ambiguous genitalia. Arch Dis Child 2004;89:401–407. 13 Deeb A, Hughes IA: Inguinal hernia in female infants: a cue to check the sex chromosomes? BJU Int 2005;96:401–403.

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95

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Horm Res 2007;67(suppl 1):96–97 DOI: 10.1159/000097560

Published online: February 15, 2007

Pediatric Clinical Case Sessions

The presentation of interesting clinical cases usually serves as a mental exercise for the audience, requiring thoughtful participation in resolving the issues presented. In the hopes of achieving this objective, the following four pediatric cases were presented. Case 1: ‘Diagnosis and Long-Term Human Growth Hormone Treatment of a Boy with Noonan Syndrome’ Noonan syndrome is a pleiomorphic, autosomal-dominant disorder with features that may include short stature, facial dysmorphia, a webbed neck and congenital heart disease. It is a genetically heterogeneous disorder, with nearly half of the cases caused by gain-of-function mutations in protein tyrosine phophatase, nonreceptor type 11 (PTPN11), the gene encoding the protein tyrosine phosphatase SHP-2. This case illustrates the efficacy and safety of recombinant human growth hormone (rhGH) administration in a boy with Noonan syndrome. The rhGH therapy promoted growth in this case, although its effect was not as pronounced as it would be in the treatment of GH-deficient short stature. Case 2: ‘Isolated Growth Hormone Deficiency due to GH1 Gene Deletion: Central Nervous System Hypertension during Growth Hormone Treatment’ Several genetic abnormalities of GH production and secretion have been described. Isolated GH deficiency IA is inherited as an autosomal-recessive trait caused by deletions or mutations of the GH-1 gene. Affected individuals have profound congenital GH deficiency. Since GH is

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not produced, even in fetal life, patients are immunologically intolerant of GH, typically acquiring anti-GH antibodies when treated with GH. A girl with severe short stature due to GH-1 deletion developed central nervous system hypertension during GH replacement therapy. Despite an adequate growth response, GH therapy was interrupted, and a rare and unsuspected central nervous system disorder was identified by cranial magnetic resonance imaging (MRI). Case 3: ‘Hepatic Enzyme Abnormalities in Turner Syndrome’ It remains puzzling that abnormal liver enzymes, especially -glutamyl transpeptidase, occur more often in patients with Turner syndrome than in the general population. This patient presented the difficulties in providing a valid interpretation of such findings. Specifically, no correlation was found between fluctuations in the liver enzyme values and the therapeutic regimen applied, namely GH and estrogens, either orally or transdermally administered. Nevertheless, this case, as well as others reported to date, points to an intrinsic defect in liver function, which is exaggerated by advancement in age and possibly by adiposity. The prognosis for this abnormality is also uncertain. The overall data on liver disease in Turner syndrome yield an important question: how should adolescents with Turner syndrome who have abnormal liver enzymes be treated to prevent severe liver disease such as cirrhosis?

Case 4: ‘PROP1 Gene Mutations and Pituitary Size: A Unique Case of Two Consecutive Cycles of Enlargement and Regression’ Understanding of the pathophysiology of the PROP1 gene mutation has greatly advanced since the mutation was first recognized as a causative factor in combined pituitary hormone deficiency (CPHD) in 1998. Patients with PROP1 gene mutations exemplify the fact that changes in pituitary size occur, irrespective of the therapy regimen used or the occurrence of corticotroph failure. Based on MRI findings, it has been proposed that the pituitary enlargement in PROP1-deficient patients originates in the intermediate lobe. Nevertheless, the cellular alterations responsible for variations in size of the pituitary gland in patients with PROP1 gene mutations have not been defined. This case confirms that such patients

are potential candidates for Addison disease, and longterm follow-up is required to prevent Addisonian crises, especially during stressful situations. Moreover, the case also illustrates how clinical puzzles, such as the occurrence of CPHD associated with supposed pituitary adenoma in siblings, were interpreted differently after the unraveling of an analogous prototype in the Ames dwarf mouse and, shortly thereafter, in humans with PROP1 gene mutations.

Pediatric Clinical Case Sessions

Horm Res 2007;67(suppl 1):96–97

Catherine Dacou-Voutetakis Pediatric Endocrinology Athens University Medical School, Athens, Greece Ana Claudia Latronico São Paulo University, São Paulo, Brazil

97

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HORMONE RESEARCH

Horm Res 2007;67(suppl 1):98–101 DOI: 10.1159/000097561

Published online: February 15, 2007

Diagnosis and Long-Term Human Growth Hormone Treatment of a Boy with Noonan Syndrome Alexander Augusto de Lima Jorge Developmental Endocrinology Unit and Laboratory of Hormones and Molecular Genetics, Discipline of Endocrinology, Hospital das Clinicas of the Medical School of the University of São Paulo, São Paulo, Brazil

Key Words Noonan syndrome Short stature Growth velocity PTPN11 mutations Growth hormone insensitivity

Abstract Noonan syndrome (NS) is a relatively common, clinically heterogeneous disorder, with an estimated incidence of 1:1,000 to 1: 2,500 live births. It is characterized by proportionate postnatal short stature, dysmorphic facial features, chest deformities and congenital heart disease. This report describes a child with NS who harbors the N308D mutation in protein tyrosine phosphatase, nonreceptor type 11 (NM_002834; PTPN11) gene. Because of his short stature, poor growth rate and low levels of insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3), recombinant human growth hormone (rhGH) therapy was initiated at 11.5 years. During the first year of therapy, the child remained prepubertal and had a significant increase in growth velocity (from 3.0 to 7.8 cm/year). Treatment was maintained throughout puberty, and after approximately 5 years of therapy, the subject still has a good growth velocity (7.6 cm/year) and his height is 0.5 standard deviation score (SDS) above pretreatment height. When the subject’s growth evolution is analyzed using an NS-specific growth chart, it is evident that his height increased almost 2 SD. During treatment, IGF-

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I and IGFBP-3 levels increased progressively and reached normal ranges for sex and age. Data strongly suggest that PTPN11 mutations identified in NS patients cause partial GH insensitivity. Long-term treatment with rhGH doses in this NS child with a PTPN11 mutation, in whom a partial postreceptor GH insensitivity was expected, normalized IGF-I and IGFBP-3 levels, improved growth velocity and increased his final height prediction. Copyright © 2007 S. Karger AG, Basel

Introduction

The patient is an apparently healthy Brazilian boy of mixed ethnicity, with short stature noticed by his parents at 5 years of age. He was born at term by caesarean section after an uneventful pregnancy, with normal birth weight and length. He is the second child born to nonconsanguineous parents. His father is of normal height, 170 cm (–0.7 SD), whereas his mother’s height is 150 cm (–2 SD). The patient’s target height is 166.5 cm (–1.2 SD). The patient came to our service at 10.6 years of age. At that time, he was prepubertal and presented with delayed bone age (7 years), short stature (height standard deviation score [SDS] = –3.2 below the normal mean) and nor-

Alexander Augusto de Lima Jorge, MD, Developmental Endocrinology Unit and Laboratory of Hormones and Molecular Genetics Hospital das Clinicas of the Medical School of the University of São Paulo Rua Trajano Reis 155, Apt 44A, 05541-030 São Paulo (Brazil) Tel. +55 11 3772 1143, Fax +55 11 3069 7519, E-Mail [email protected]

190

CA: 10 years 8 months

180

BA: 7 years

170

Height: 118.5 cm (–3.3 SD)

+2.0 SD +1.0 SD

160

+0.0 SD –1.0 SD –2.0 SD

Height (cm)

150 140 130 120

G2,T4

110

GH

100

CA: 16 years 1 month

90

BA: 13 years 6 months

80

Height: 152.5 cm (–2.8 SD) GV: 7.6 cm/year

70 1

2

3

4

5

6

7

8

9

Fig. 1. Subject’s follow-up during rhGH

10 11 12 13 14 15 16 17 18 19 20 21 Age (years)

treatment in Noonan syndrome-specific growth chart.

Diagnosis and Long-Term rhGH Treatment in Noonan Syndrome

6

500 IGF-I

IGFBP-3

4 300 3 200

2

100

Puberty hGH

0 Basal

Basal

1st

2nd

3rd

IGFBP-3 (mg/l)

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mal weight (body mass index SDS = –0.14) and body proportions (sitting height/height SDS for age = –0.7) (fig. 1). Physical examination disclosed a triangular face; low-set, posteriorly rotated ears; hypertelorism; epicanthal folds; deeply grooved philtrum; high, arched palate; micrognathia and mild pectus excavatum. No heart murmur or webbed neck was observed and the subject had normal prepubertal genitalia and normal neuropsychomotor development. Results of routine laboratory assessments were normal, as were function tests, karyotype, echocardiogram and electrocardiogram. Gonadotropin and testosterone levels were adequate for his prepubertal stage. Insulinlike growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) levels were below the normal reference values (fig. 2). The subject’s facial features were typical as described in patients with NS. He presented with short stature and mild pectus excavatum, which are often noted in patients with NS. NS is a relatively common, clinically heterogeneous disorder, with an estimated incidence of 1:1,000 to 1:2,500 live births, characterized by proportionate postnatal short stature, dysmorphic facial features, chest deformities and congenital heart disease (most commonly, pulmonary valve stenosis and hypertrophic cardiomyopathy) [1]. In our service, the diagnosis of NS is based on

1 0 4th year

Fig. 2. Changes in IGF-I and IGFBP-3 levels during rhGH treat-

ment.

van der Burgt criteria [2]. This subject presented with typical facial features, short stature and chest deformity as the major criteria, indicating a clinical diagnosis of NS. At that time, molecular studies were not available, and because he presented with low levels of IGF-I and IGFBP3, we decided to investigate his growth hormone (GH)/ IGF-I axis. Horm Res 2007;67(suppl 1):98–101

99

Evaluation and Treatment

Clonidine testing showed a GH peak of 6.1 ng/ml after 60 min, which ruled out GH deficiency in this subject according to our cut-off based on GH response in normal children [3]. The subject also underwent IGF-I and IGFBP-3 generation testing, which involved daily injections of recombinant human growth hormone (rhGH) 0.1 U/kg subcutaneously at bedtime for 10 consecutive days. Fasting blood samples for IGF-I and IGFBP-3 measurements were drawn in the morning before the first injection and 4 and 10 days after. The subject demonstrated an increase in IGF-I and IGFBP-3 levels comparable to other patients without GH insensitivity: IGF-I increased from 86 ng/ml to 211 and 251 ng/ml and IGFBP-3 from 1.5 mg/l to 2.0 and 4.0 mg/l, after 4 and 10 days of rhGH use, respectively. The subject was followed for 10 months without any intervention, and his growth velocity was 3.0 cm/year during this period. Because of his short stature, poor growth rate and low levels of IGF-I and IGFBP-3, rhGH therapy was initiated at that time. The rhGH dose was 50 g/kg/day, 7 days a week. During the first year of therapy, the subject remained prepubertal and had a significant increase in growth velocity (from 3.0 to 7.8 cm/year). In the beginning of the second year of therapy, the subject started puberty. The growth velocity observed in this subject during the first year of rhGH therapy was comparable to that observed in other patients with NS (8.0 8 2 cm/year) [4]. This growth rate is similar to that observed in individuals with Turner syndrome (7.8 8 1.8 cm/year) [5] but lower than the rate seen in patients with GH deficiency (9.2 8 2.3 cm/year) [6, 7] or children born small-for-gestational age (8.7 8 1.8 cm/year) [8]. Treatment was maintained throughout puberty, and after approximately 5 years of therapy, the subject still shows good growth velocity (7.6 cm/year) and his height is 0.5 SD above pretreatment height. When the subject’s growth evolution is analyzed using an NS-specific growth chart (fig. 1), it is evident that his height increased almost 2 SD. During treatment, IGF-I and IGFBP-3 levels increased progressively (fig. 2) and reached normal ranges for sex and age.

Molecular Study

Recently, the entire PTPN11 gene was sequenced and a heterozygous missense mutation was identified in exon 8 at the first nucleotide of codon 308 (AAT r GAT). It is 100

Horm Res 2007;67(suppl 1):98–101

a de novo mutation and the parents do not harbor it. This mutation causes a substitution of asparagine by aspartic acid in SHP-2 protein.

Discussion

Recently, PTPN11, which encodes for Src homology region 2-domain phosphatase 2 (SHP-2), was identified as one of the genes involved in NS [9]. Missense mutations in PTPN11 have been demonstrated in 33 to 60% of patients with NS [9–13]. SHP-2 is a protein tyrosine phosphatase that is involved in signal transduction of growth factors and cytokines. The mutations identified in PTPN11 in NS patients are predicted to be gain-of-function changes [14] and are expected to cause a decrease in GH [15] and IGF-I actions [16]. The mutation identified in the present case, N308D, is the most frequently identified mutation in NS patients. Functional studies performed by Tartaglia et al. disclosed that N308D increased the basal and stimulated phosphatase activity of SHP-2 [14]. Because of the possible effect of the mutant SHP-2 on GH action, investigators (including ourselves) have assessed the different response to rhGH therapy in NS patients based on their PTPN11 status. Limal et al. [17] and our group [18] have demonstrated that NS patients who harbor PTPN11 mutations have a low growth rate during rhGH therapy compared to patients with no mutations, although the difference is not statistically significant, probably due to the small sample sizes. On the other hand, Binder et al. [19] showed that changes in height SDS were significantly higher after the first year of rhGH therapy in patients with no mutations. This result is in accordance with our data showing a significant difference in height SDS gain during long-term rhGH therapy [18] in a cohort of NS patients older than those studied by Binder et al. All these data strongly suggest that PTPN11 mutations identified in NS patients cause partial GH insensitivity.

Conclusion

Long-term treatment with pharmacological doses of rhGH in this NS subject with a PTPN11 mutation, in whom a partial postreceptor GH insensitivity was expected, normalized IGF-I and IGFBP-3 levels, improved growth velocity and probably increased his final height.

de Lima Jorge

References 1 Noonan JAE: Associated noncardiac malformations in children with congenital heart disease. J Pediatr 1963;63:468. 2 Van der Burgt I, Berends E, Lommen E, Van Beersum S, Hamel B, Mariman E: Clinical and molecular studies in a large Dutch family with Noonan syndrome. Am J Med Genet 1994;53:187–191. 3 Silva EG, Slhessarenko N, Arnhold IJ, Batista MC, Estefan V, Osorio MG, Marui S, Mendonca BB: GH values after clonidine stimulation measured by immunofluorometric assay in normal prepubertal children and GH-deficient patients. Horm Res 2003; 59: 229–233. 4 Romano AA, Blethen SL, Dana K, Noto RA: Growth hormone treatment in Noonan syndrome: the national cooperative growth study experience. J Pediatr 1996;128(5 Pt 2): S18–S21. 5 Ranke MB, Lindberg A, Chatelain P, Wilton P, Cutfield W, Albertsson-Wikland K, Price DA: Prediction of long-term response to recombinant human growth hormone in Turner syndrome: development and validation of mathematical models. J Clin Endocrinol Metab 2000;85:4212–4218. 6 Jorge AA, Marchisotti FG, Montenegro LR, Carvalho LR, Mendonca BB, Arnhold IJ: Growth hormone (GH) pharmacogenetics: influence of GH receptor exon 3 retention or deletion on first-year growth response and final heights in patients with severe GH deficiency. J Clin Endocrinol Metab 2006; 91: 1076–1080. 7 Ranke MB, Lindberg A, Chatelain P, Wilton P, Cutfield W, Albertsson-Wikland K, Price DA: Derivation and validation of a mathematical model for predicting the response to exogenous recombinant human growth hormone (GH) in prepubertal children with idiopathic GH deficiency. J Clin Endocrinol Metab 1999;84:1174–1183.

Diagnosis and Long-Term rhGH Treatment in Noonan Syndrome

8 Ranke MB, Lindberg A, Cowell CT, Albertsson-Wikland K, Reiter EO, Wilton P, Price DA: Prediction of response to growth hormone treatment in short children born small for gestational age: analysis of data from KIGS (Pharmacia International Growth Database). J Clin Endocrinol Metab 2003; 88: 125–131. 9 Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H, Van der Burgt I, Crosby AH, Ion A, Jeffery S, Kalidas K, Patton MA, Kucherlapati RS, Gelb BD: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001;29:465–468. 10 Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, Van der Burgt I, Brunner HG, Bertola DR, Crosby A, Ion A, Kucherlapati RS, Jeffery S, Patton MA, Gelb BD: PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555–1563. 11 Yoshida R, Hasegawa T, Hasegawa Y, Nagai T, Kinoshita E, Tanaka Y, Kanegane H, Ohyama K, Onishi T, Hanew K, Okuyama T, Horikawa R, Tanaka T, Ogata T: Protein-tyrosine phosphatase nonreceptor type 11 mutation analysis and clinical assessment in 45 patients with Noonan syndrome. J Clin Endocrinol Metab 2004;89:3359–3364. 12 Musante L, Kehl HG, Majewski F, Meinecke P, Schweiger S, Gillessen-Kaesbach G, Wieczorek D, Hinkel GK, Tinschert S, Hoeltzenbein M, Ropers HH, Kalscheuer VM: Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 2003;11:201–206.

13 Sarkozy A, Conti E, Seripa D, Digilio MC, Grifone N, Tandoi C, Fazio VM, Di Ciommo V, Marino B, Pizzuti A, Dallapiccola B: Correlation between PTPN11 gene mutations and congenital heart defects in Noonan and LEOPARD syndromes. J Med Genet 2003; 40:704–708. 14 Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, Cordeddu V, Zampino G, van der Burgt I, Palleschi A, Petrucci TC, Sorcini M, Schoch C, Foa R, Emanuel PD, Gelb BD: Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet 2006; 78: 279–290. 15 Stofega MR, Herrington J, Billestrup N, Carter-Su C: Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phophorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:1338–1350. 16 Maile LA, Clemmons DR: Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2. J Biol Chem 2002; 277: 8955–8960. 17 Limal JM, Parfait B, Cabrol S, Bonnet D, Leheup B, Lyonnet S, Vidaud M, Le Bouc Y: Noonan syndrome: relationships between genotype, growth, and growth factors. J Clin Endocrinol Metab 2006;91:300–306. 18 Ferreira LV, Souza SA, Arnhold IJ, Mendonca BB, Jorge AA: PTPN11 (protein tyrosine phosphatase, nonreceptor type 11) mutations and response to growth hormone therapy in children with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5156–5160. 19 Binder G, Neuer K, Ranke MB, Wittekindt NR: PTPN11 mutations are associated with mild growth hormone resistance in individuals with Noonan syndrome. J Clin Endocrinol Metab 2005;90:5377–5381.

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HORMONE RESEARCH

Horm Res 2007;67(suppl 1):102–105 DOI: 10.1159/000097562

Published online: February 15, 2007

Isolated Growth Hormone Deficiency due to GH1 Gene Deletion: Central Nervous System Hypertension during Growth Hormone Treatment Sonir R. Antonini a Letícia Faleiros a Hélio R. Machado b Antonio Carlos dos Santos c Margaret de Castro c Departments of a Pediatrics, b Surgery and Anatomy and c Internal Medicine, School of Medicine of Ribeirao Preto, University of São Paulo, Brazil

Key Words Growth hormone deficiency GH1 gene deletion Intracranial hypertension Chiari malformation type I

Abstract Background: We report the case of a patient with an uncommon association of isolated growth hormone deficiency (IGHD) due to GH1 gene deletion and Chiari malformation type I. The patient presented with intracranial hypertension during recombinant human GH replacement therapy. Methods: GH deficiency (GHD) was diagnosed based on auxiological data and standard biochemical tests. Molecular analysis of the GH1 gene was performed using polymerase chain reaction amplification and SmaI enzyme restriction. The central nervous system (CNS) was evaluated by computed tomography (CT) and magnetic resonance imaging (MRI). Results: Molecular analysis showed that IGHD was due to homozygotic deletion of the 6.7-kb GH1 gene. After 28 months of GH treatment, CT and MRI scans showed enlargement of the third and lateral ventricles but a normal fourth ventricle, herniation of the cerebellar tonsils by the foramen magnum, presence of dysplastic cerebellar tonsils involving the medulla oblongata, absence of the cisterna magna and compression of the cerebellar ton-

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sils and spinal cord by the posterior arch of the atlas. The patient underwent endoscopic third ventriculostomy, which resulted in complete symptom relief. Conclusions: This case illustrates the importance of CNS evaluation, including detailed examination of the posterior fossa, in patients with GHD. Copyright © 2007 S. Karger AG, Basel

Case Report A 17-month-old Brazilian girl was referred for evaluation due to severe growth failure noted since birth. She was born by caesarian section following pelvic presentation at 34 weeks gestation, and intrauterine growth restriction (IUGR) was noted (birth length, 40 cm; birth weight, 1975 g). She is the second child of consanguineous parents (first-degree cousins); her older brother was 3 years old and had normal growth. No history of neonatal hypoglycemia or respiratory distress was reported. At presentation, she was reported to have had one episode of urinary infection at the age of 4 months and pneumonia at the age of 16 months. At the age of 17 months, her length was 55 cm (–8.2 standard deviation score [SDS]) and her weight was 4,460 g (–5.1 SDS). Her phenotype showed features typical of isolated growth hormone deficiency (IGHD): craniofacial disproportion, prominent forehead, sparse hair, small face, hypoplastic nasal bridge, high pitched voice, thin skin, and increased abdominal fat (fig. 1) and delayed walking and motor development. Her karyotype was

Sonir Roberto Antonini, MD University of São Paulo Hospital das Clinicas da Fac de Medicine 14049-900 Ribeirao Preto-SP (Brazil) Tel. +55 16 3602 2478, E-Mail [email protected]

Fig. 1. A A 17-month-old patient with typical IGHD features: craniofacial disproportion, prominent forehead, sparse hair, small face, hypoplastic nasal bridge, thin skin. B The patient had extremely short stature (–8.2 SDS) and increased abdominal fat compared with her normal older brother.

Fig. 2. Cranial CT scan showing the presence of enlarged third and lateral ventricles and no signs of cerebral mass lesions.

normal (46,XX). On the basis of biochemical evaluations, organic diseases were excluded and mild hypoglycemia was detected (2.9 mmol/l). Her thyroid function was normal (free thyroxine: 12.8 pmol/l, thyroid-stimulating hormone: 3.8 mIU/ml) as was her adrenal function (plasma cortisol: 634 nmol/l). The patient had no detectable insulin-like growth factor I in plasma and her plasma GH level was undetectable following glucagon stimulation test, confirming the diagnosis of IGHD deficiency. Ophthalmic evaluation showed a normal fundus. Magnetic resonance imaging (MRI) of the hypothalamic-pituitary region at the time of diagnosis showed a normal anterior and posterior pituitary and a normal pituitary stalk. Substitute therapy with recombinant human (rh)GH was started at the age of 2.2 years (height, –8.8 SDS) at a dose of

0.23 mg/kg/week. No side effects of the rhGH therapy were noted in the first 2 years of treatment and the patient responded very well (14 and 9 cm growth in the first and second years of treatment, respectively). At age 4.1 years her height was 81 cm (–5.1 SDS). At the age of 4.4 years she presented with severe daily headaches. Ocular fundus examination showed no abnormalities. A computed tomography (CT) scan revealed enlargement of the third and the lateral ventricles, and there were no signs of cerebral mass lesions (fig. 2). Benign intracranial hypertension (BIH) was initially suspected, and rhGH replacement therapy was stopped with complete relief of symptoms within 2 weeks. The patient remained untreated and had no symptoms over the next 3 months, but then the headaches returned and she started vomiting. An-

Growth Hormone Deficiency due to GH1 Gene Deletion

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103

Fig. 3. A Sagittal T1-weighted MRI showing enlarged third and

lateral ventricles, normal fourth ventricle, herniation of the cerebellar tonsils through the foramen magnum, dysplasic cerebellar tonsils, and the posterior arch of the atlas compressing the spinal cord (arrow). B Axial T2-weighted MRI showing dysplasic cere-

bellar tonsils involving the medulla oblongata; the cisterna magna is not visible. C Sagittal T2-weighted MRI obtained after endoscopic third ventriculostomy showing the presence of the flowvoid artifact and reduction in size of the ventricles.

Molecular Analysis 1 1900 bp

2

3

4

5

6

1472 bp

762 bp 711 bp 446 bp

Fig. 4. GH1 gene locus amplified by PCR and digested by SmaI

endonuclease. The pattern obtained using DNA from the patient indicated she was homozygous for the 6.7-kb GH1 gene deletion (lane 6).

other MRI of the central nervous system (CNS) showed enlarged third and lateral ventricles and a normal fourth ventricle. In addition, MRI showed herniation of the cerebellar tonsils by the foramen magnum. The cerebellar tonsils were dysplastic and involved the medulla oblongata. The cisterna magna was not visible, and the posterior arch of the atlas was compressing the cerebellar tonsils and the spinal cord (fig. 3A, B). Based on these findings, Chiari malformation type I (CMI) was diagnosed. The patient underwent endoscopic third ventriculostomy, which resulted in complete symptom relief. A postsurgery MRI showed reduction of the hydrocephalus and the presence of a flow-void artifact (fig. 3C). Four months after surgery, rhGH replacement was restarted.

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Molecular analyses using polymerase chain reaction and SmaI enzyme restriction were performed on the parents and both children in this family to screen for deletion within the GH1 gene cluster [1]. Informed consent was obtained from the parents for the analyses, which were approved by the local Ethics Committee. The pattern obtained using DNA from the patient indicated that she was homozygous for the 6.7-kb GH1 gene deletion (fig. 4). Both parents and the older healthy brother were heterozygous for this deletion.

Discussion

This report describes a patient with an unusual combination of IGHD due to GH1 gene deletion and CMI. The patient developed intracranial hypertension after 2 years on rhGH replacement therapy, initially presenting with signs of headache and vomiting. The patient’s CT scan showing enlarged third and lateral ventricles but no space-occupying lesion suggested BIH. BIH, or pseudotumor cerebrii related to rhGH therapy, was first reported in 1993 and is defined by the occurrence of intracranial hypertension in the absence of a space-occupying lesion. There is increased cerebrospinal fluid (CSF) pressure but CSF composition is normal. Main clinical signs are headaches, nausea, vomiting and visual changes. The prevaAntonini /Faleiros /Machado / Carlos dos Santos /de Castro

lence of BIH in GHD patients on rhGH treatment is low (approximately 1.6 per 1,000 patients) [2–5]. Ocular examination of this patient revealed no abnormalities. Although papilledema is common in this condition, its absence does not preclude the diagnosis [2]. Withdrawal of rhGH resulted in complete relief of the patient’s symptoms, reinforcing the diagnosis of BIH. When the patient’s symptoms subsequently returned after 3 months without rhGH replacement, a control MRI revealed the unexpected findings of CMI. The diagnosis of CMI in this patient raises some important questions. First, is there an association between CMI and GH1 gene deletion? There is one report in the literature of a family with IGHD in which one of the affected members has CMI, but no abnormality in the GH1 locus was found in this family [6]. Patients with GH1 gene deletion present either with normal CNS development or with a hypoplastic anterior pituitary. No other abnormalities have been reported [7]. However, abnormalities of the cerebellum resembling CMI can be found in patients with hypopituitarism due to Lhx4 gene mutation [8]. This case is the first report of a patient with both GH1 gene deletion and CMI. A second intriguing question is: did rhGH replacement contribute to development of the intracranial hypertension associated with CMI? The pathogenesis of intracranial hypertension induced by rhGH is uncertain; however, some studies have shown that – directly or indirectly – GH augmentation of insulin-like growth fac-

tor-I at the choroid plexus stimulates CSF production [9, 10]. Fagan et al. suggested that tonsillar herniation in CMI is a result of mechanical factors [11]. Thus, it is possible that increased CSF pressure induced by rhGH therapy could be involved in the appearance of clinical findings of CMI. Another question in this particular case is whether the diagnosis of CMI could have been established before the occurrence of intracranial hypertension. It is probable that enlargement of the third and lateral ventricles and herniation of the cerebellar tonsils occurred at the time of the symptom’s appearance. It is clear that the presence of congenital malformations (e.g., dysplastic cerebellar tonsils and compression of the spinal cord by the posterior arch of the atlas) could have been noted at the time of IGHD diagnosis, before the intracranial hypertension was detected. One of the main reasons why this diagnosis was missed is the fact that abnormalities in the posterior fossa have never been described in IGHD patients with GH1 gene deletions.

Conclusions

This case report describes a patient with an unusual combination of IGHD due to GH1 gene deletion and CMI. The present case illustrates the importance of detailed CNS evaluation, including examination of the posterior fossa, in patients with IGHD.

References 1 Kamijo T, Phillips JA III: Detection of molecular heterogeneity in GH-1 gene deletions by analysis of polymerase chain reaction amplification products. J Clin Endocrinol Metab 1992;74:786–789. 2 Crock PA, McKenzie JD, Nicoll AM, Howard NJ, Cutfield W, Shield LK, Byrne G: Benign intracranial hypertension and recombinant growth hormone therapy in Australia and New Zealand. Acta Paediatr 1998; 87: 381– 386. 3 Malozowski S, Tanner LA, Wysowski DK, Fleming GA, Stadel BV: Benign intracranial hypertension in children with growth hormone deficiency treated with growth hormone. J Pediatr 1995;126:996–999. 4 Drake WM, Howell SJ, Monson JP, Shalet SM: Optimizing GH therapy in adults and children. Endocr Rev 2001;22:425–450.

Growth Hormone Deficiency due to GH1 Gene Deletion

5 Wyatt D: Lessons from the National Cooperative Growth Study. Eur J Endocrinol 2004;151(suppl 1):S55–S59. 6 Hamilton J, Chitayat D, Blaser S, Cohen LE, Phillips JA III, Daneman D: Familial growth hormone deficiency associated with MRI abnormalities. Am J Med Genet 1998; 80: 128– 132. 7 Procter AM, Phillips JA III, Cooper DN: The molecular genetics of growth hormone deficiency. Hum Genet 1998;103:255–272. 8 Machinis K, Pantel J, Netchine I, Leger J, Camand OJ, Sobrier ML, Dastot-Le Moal F, Duquesnoy P, Abitbol M, Czernichow P, Amselem S: Syndromic short stature in patients with a germline mutation in the LIM homeobox LHX4. Am J Hum Genet 2001;69: 961–968.

9 Bondy C, Werner H, Roberts CT Jr, LeRoith D: Cellular pattern of type-I insulin-like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II. Neuroscience 1992;46:909–923. 10 Johansson JO, Larson G, Andersson M, Elmgren A, Hynsjo L, Lindahl A, Lundberg PA, Isaksson OG, Lindstedt S, Bengtsson BA: Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. Neuroendocrinology 1995; 61:57–66. 11 Fagan LH, Ferguson S, Yassari R, Frim DM: The Chiari pseudotumor cerebri syndrome: symptom recurrence after decompressive surgery for Chiari malformation type I. Pediatr Neurosurg 2006;42:14–19.

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Pediatric Clinical Case Sessions

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):106–108 DOI: 10.1159/000097563

Published online: February 15, 2007

Hepatic Enzyme Abnormalities in Turner Syndrome: A Case Report P. Pervanidou P. Xekouki C. Dacou-Voutetakis First Department of Pediatrics, Division of Endocrinology, Metabolism and Diabetes, University of Athens, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece

Key Words Turner syndrome Hepatic enzymes Hormone replacement therapy Growth hormone Liver

Abstract Background: Liver dysfunction has been described in subjects with Turner syndrome. The mechanism involved is not known. Methods: Using appropriate methodology, we evaluated the hepatic enzymes serum glutamic-oxaloacetic transaminase, serum glutamic pyruvic transaminase and glutamyl transpeptidase ( -GT) in a girl with 45 XO/46 Xr mosaic karyotype. She was monitored from age 9 to 20 years while she underwent various therapeutic regimens. Results: Serum glutamic-oxaloacetic transaminase, serum glutamic pyruvic transaminase and particularly -GT concentrations ranged from normal to elevated values during human growth hormone therapy, estrogen replacement therapy (conjugated estrogens were replaced after 1 year with transdermal estrogens and medroxyprogesterone), and periods without therapy. The type of the therapeutic regimen did not seem to influence the hepatic enzyme values. A correlation was found between -GT values and body mass index (BMI). Conclusions: In our patient the waxing and waning of the liver enzymes was not influenced by the therapeutic regimen, but -GT seemed to correlate with BMI. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0106$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

Turner syndrome (TS) is one of the most frequently occurring sex chromosome abnormalities. It affects one of 2,500 live female newborns (0.04/100), while it is observed in 3/100 (3%) of female fetuses. It is estimated that worldwide there are 1.5 million women with TS. TS is caused by haplo-insufficiency of X chromosome genes, and its main characteristics are short stature, gonadal dysgenesis, dysmorphic features and cardiovascular and renal anomalies. The syndrome is usually associated with a number of other disorders, including autoimmune thyroiditis, osteoporosis and inflammatory bowel disease. Liver dysfunction has recently been described in subjects with TS, but the underlying pathophysiology has not been clarified [1].

Case Report We describe hepatic enzyme values throughout the follow-up period of a girl with TS, treated with various therapeutic regimens. The patient was first examined for short stature at the age of 8.78 years in the endocrine unit of our department. She had been born after a full-term gestation with a birth weight of 2,450 kg. At presentation, her height was 107.8 cm (–3.82 standard deviation score [SDS]), her weight was 23 kg (–1.11 SDS) and her body mass index (BMI) was 19.8 kg/m2 (1.14 SDS). She was pre-

Panagiota Pervanidou, MD University of Athens, ‘Aghia Sophia’ Children’s Hospital First Department of Pediatrics, Division of Endocrinology, Metabolism and Diabetes 26 Charavgis Str., Athens, Chalandri 152 32 (Greece) Tel. +30 210 683 3936, Fax +30 210 652 5013, E-Mail [email protected]

160 140

Progesterone

120

GH

Epo

ETTS

SGOT (IU/l)

SGOT levels SGPT levels

100

-GT levels

80 60

Normal ranges

40

during various therapies. Therapies included growth hormone (GH), oral estrogen (Epo), estradiol by a transdermal therapeutic system (ETTS) and progesterone.

0 10

pubertal. Investigations revealed a 45 XO/46 Xr (ring chromosome) mosaic karyotype. The peak growth hormone value on provocative testing was 19 ng/ml. Cardiac, kidney and liver ultrasonography findings were normal. Thyroid function, as indicated by serum thyroid hormone concentrations and the thyroid autoantibodies, was normal throughout observation. Serum cholesterol and triglycerides were always normal to slightly elevated. Fasting glucose and insulin levels were normal, although impaired glucose tolerance was detected. Liver function abnormality, as indicated by increased serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and -glutamyl transpeptidase (-GT) levels, was detected at presentation before any therapy was initiated. When the patient was 9.80 years, human growth hormone (hGH) therapy was initiated at a dose of 0.8 IU/kg/week. Hepatic enzyme values during hGH therapy were normal. At the age of 13.50 years, hGH therapy was discontinued and conjugated estrogens were started. One year later medroxyprogesterone was added. During sex steroid therapy, SGOT levels, although they remained within normal range, were higher than they were prior to therapy initiation; however, SGPT and -GT levels were elevated (fig. 1). Findings on hepatic ultrasonography, and measures of serological markers of viral hepatitis were normal. When the patient was 14.60 years, physicians replaced conjugated estrogens with transdermal estradiol (twice weekly) while medroxyprogesterone was continued. Hepatic enzyme concentrations fluctuated throughout therapy, with -GT showing the most prominent alterations (fig. 1). Clinical symptoms of liver disease were absent throughout the observation period. Types and dosages of drugs did not seem to influence hepatic enzyme elevations. A significant positive correlation was found between BMI SDS, based on Greek BMI growth charts [2], and -GT levels (r = 0.52 and p = 0.028) (fig. 2). There was no correlation between BMI SDS and SGOT or SGPT levels.

Hepatic Enzyme Abnormalities in Turner Syndrome

SGOT

-GT

20

12

14

16

18

Chronological age (years)

3.0

2.5 BMI-SDS

Fig. 1. Profiles of SGOT, SGPT, and -GT

SGPT

2.0

r = 0.52 p = 0.028

1.5

1.0

n=9

20

30

40 50 -GT (IU/l)

60

70

Fig. 2. Correlation between -glutamyl transpeptidase (-GT)

levels and body mass index SDS.

Discussion

Liver enzyme abnormalities, particularly -GT, have been reported in TS patients. The prevalence of these abnormalities ranges from 20 to 80%, depending on the patient’s age [1, 3–6] inasmuch as these abnormalities become more frequent with advancing age. Horm Res 2007;67(suppl 1):106–108

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The cause of abnormal liver function in TS is not clear, but it does not seem to be related to viral hepatitis or alcohol excess. No correlation has been found between elevated hepatic enzymes and karyotype, BMI, type or duration of hormone replacement therapy or autoimmunity [1, 5]. Estrogens are thought to influence the metabolic activity of the liver, and some investigators associate estrogen therapy in TS with abnormal liver function [3]. However, others have noticed that estrogens should not be considered an important cause of hepatic enzyme elevations in TS [4, 7–9] and the findings in the case described here are in agreement with this view. Although GH treatment may result in a mild, transient increase in serum transaminases, this condition does not require further investigations [7, 10]. No elevation of liver enzymes was noted in our patient during GH administration.

Obesity is a frequent problem in adolescents and women with TS [8], and it is associated with elevated liver enzymes in normal children. Increased BMI may be a contributory or predisposing factor, rather than a cause, of liver enzyme abnormality in TS [7]. In some cases, histopathologic findings obtained from liver biopsies have shown both cirrhosis and nodular regenerative hyperplasia. Liver cirrhosis occurs in patients with TS five times more frequently than in the general population [11]. TS has also been associated with liver fatty infiltration, liver fibrosis, portal hypertension, and vascular abnormalities [12, 13]. In conclusion, hepatic enzyme abnormalities are frequent in TS but independent of hormonal treatment, and management should include hepatic function surveillance throughout follow-up.

References 1 Elsheikh M, Dunger DB, Conway GS, Wass JAH: Turner’s syndrome in adulthood. Endocrine Rev 2002;23:120–140. 2 Chiotis D, Krikos X, Tsiftis G, Hatzisymeaon M, Maniati-Christidi M, Dacou-Voutetakis A: Body mass index and prevalence of obesity in subjects of Hellenic origin aged 0–18 years, living in the Athens area. Ann Clin Pediatr Univer Atheniensis 2004;51:139–154. 3 Wemme H, Pohlenz J, Schonberger W: Effect of oestrogen/gestagen replacement therapy on liver enzymes in patients with UlrichTurner syndrome. Eur J Pediatr 1995; 154: 807–810. 4 Larizza D, Locatelli M, Vitali L, Vigano C, Calcaterra V, Tinelli C, Sommaruga MG, Bozzini A, Campani R, Severi F: Serum liver enzymes in Turner syndrome. Eur J Pediatr 2000;159:143–148.

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5 Elsheikh M, Hodgson HJ, Wass JA, Conway GS: Hormone replacement therapy may improve hepatic function in women with Turner’s syndrome. Clin Endocrinol (Oxf) 2001; 55:227–231. 6 Sylven L, Hagenfeldt K, Brondum-Nielsen K, von Schoultz B: Middle-aged women with Turner’s syndrome: medical status, hormonal treatment and social life. Acta Endocrinol 1991;125:359–365. 7 Salerno M, Di Maio S, Gasparini N, Rizzo M, Ferri P, Vajro P: Liver abnormalities in Turner syndrome. Eur J Pediatr 1999; 158: 618– 623. 8 Hannaford PC, Kay CR, Vessey MP, Painter R, Mant J: Combined oral contraceptives and liver disease. Contraception 1997; 55: 145–151.

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9 Masters KW: Treatment of Turner syndrome – a concern. Lancet 1996;348:681–682. 10 Salerno M, Di Maio S, Ferri P, Lettiero T, Di Maria F, Vajro P: Liver abnormalities during growth hormone treatment. J Pediatr Gastroenterol Nutr 2000;31:149–151. 11 Gravholt CH, Juul S, Naeraa RW, Hansen J: Morbidity in Turner syndrome. J Clin Epidemiol 1998;51:147–158 12 Roulot D, Degott C, Chazouilleres O, Oberti F, Cales P, Carbonell N, Benferhat S, Bresson-Hadni S, Valla D: Vascular involvement of the liver in Turner’s syndrome. Hepatology 2004;39:239–247. 13 Wardi J, Knobel B, Shahmurov M, Melamud E, Avni Y, Shirin H: Chronic cholestasis associated with Turner’s syndrome: 12 years of clinical and histopathological follow-up. Digestion 2003;67:96–99.

Pervanidou/Xekouki/Dacou-Voutetakis

Pediatric Clinical Case Sessions

HORMONE RESEARCH

Published online: February 15, 2007

Horm Res 2007;67(suppl 1):109–113 DOI: 10.1159/000097564

PROP1 Gene Mutations and Pituitary Size: A Unique Case of Two Consecutive Cycles of Enlargement and Regression Paraskevi Xekouki a Amalia Sertedaki a Sarantis Livadas a Maria Argyropoulou b Antonis Voutetakis a a b

Endocrine Unit, First Department of Pediatrics, Athens University School of Medicine, Athens, and Department of Radiology, University of Ioannina School of Medicine, Ioannina, Greece

Key Words PROP1 gene mutations Pituitary Growth hormone

pituitary pathology. Further changes in pituitary morphology and size can be expected; therefore, long-term followup with pituitary MRI is advised. Copyright © 2007 S. Karger AG, Basel

Abstract Background: Pituitary enlargement, which can regress with time, has been described in a number of PROP1- deficient patients. We report a PROP1-deficient patient with a unique variation in pituitary size. Case Description: A 4-year-old boy was first examined in 1989 for short stature (–2.3 standard deviation score). Growth hormone (GH) insufficiency was confirmed, and human GH (hGH) therapy was initiated and administered up to the age of 18.2 years. Levothyroxine was added 6 months after hGH initiation. Pituitary magnetic resonance imaging (MRI) obtained when the patient was 5 years old showed an enlarged pituitary gland, which grew larger by the age of 8.5 years and then regressed to normal size by the time the patient was 9.8 years old. MRI when the patient was 19 years old disclosed pituitary reenlargement, and another 3 years later indicated regression. On DNA analysis, the patient was found to be homozygous for the mutation 301–302GA of the PROP1 gene. When the patient was 18.8 years old and asymptomatic, an impaired cortisol response to glucagon was detected. Conclusions: Regression of the pituitary enlargement in PROP1- deficient patients does not seem to constitute an end stage with respect to

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0109$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

Mutations in the transcription factor PROP1 lead to combined pituitary hormone deficiency (CPHD) in mice and humans [1, 2]. Fifteen distinct mutations have been identified in patients from approximately 20 countries [3]. Although most patients with PROP1 gene mutations have a small or normal pituitary, as evidenced on magnetic resonance imaging (MRI), some patients have an enlarged pituitary. The enlargement can regress with time and lead to pituitary hypoplasia [4–8]. Herein we describe a unique variation – repeated enlargement and regression – in pituitary size and morphology in a patient with a PROP1 gene mutation. Case Description A 4-year-old boy was initially examined in 1989 because of short stature. He was the first child of healthy parents and was born at term after an uneventful pregnancy. Both the delivery and

Paraskevi Xekouki, MD ‘Choremio’ Research Laboratory ‘Aghia Sophia’ Children’s Hospital, 10 Mesologiou Str. GR–115 27 Athens (Greece) Tel. +30 210 330 1581, Fax +30 210 779 6312, E-Mail [email protected]

Fig. 1. Contrast-enhanced T1-weighted midsagittal scan at age 8.5

years shows pituitary enlargement (pituitary height, 10 mm; normal for age, 4.5 8 0.6 mm) because of a nonenhancing mass interposed between the anteriorly displaced adenohypophysis and the neurohypophysis. (Reprinted with permission from Voutetakis et al. [8].)

perinatal period were uncomplicated, and the birth weight was 4 kg. No parental consanguinity was reported. However, both parents originated from a small village. On the initial physical examination, his height was 92 cm (–2.3 standard deviation score [SDS]) and his weight was 15 kg (–1.3 SDS). The head circumference was normal (50 cm). The testes were in scrotum (1–2 ml), and penile length was 2 cm. The results of routine biochemistry and urinalysis were normal. The total thyroxine (T4) value was 82.3 nmol/l (normal range, 64.4–160 nmol/l), and the thyroid-stimulating hormone (TSH) was 1.3 mU/l (normal range, 0.5–5.0 mU/l). The prolactin value was low (3.3 g/l). The peak growth hormone (GH) value during glucagon testing was 0.8 g/l (normal value 110 g/l), while the peak cortisol response was completely normal (604.2 nmol/l). GH insufficiency was confirmed with L-Dopa testing in which the peak GH value was 1.1 g/l. Pituitary MRI obtained when the patient was 5 years old showed an enlarged pituitary gland, with the pituitary height being 8 mm (normal pituitary height for that age is 4.5 8 0.6 mm) [9]. Although no explanation was evident at that time, surgical intervention was not considered necessary. Treatment with human GH (hGH) was initiated. Six months later, the T4 value was 57.9 nmol/l and the TSH level was 1.3 mU/l, and levothyroxine substitution therapy was initiated. At that time, the patient’s 3-year-old sister was examined for short stature, and analogous findings were detected (i.e., GH deficiency, hypothyroidism and pituitary enlargement). Pituitary MRI, obtained when the boy was 8.5 years of age, showed a small further increase in the size of the ‘pituitary adenoma’ (10 mm; fig. 1), and hGH therapy was withheld. The height velocity in the subsequent 1.5 years was very low (2.3 cm/year), and hGH was readministered, although with caution. However, an MRI of

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Fig. 2. Plain T1-weighted midsagittal scan at age 9.8 years shows disappearance of the mass and normalization of the pituitary height (pituitary height, 4 mm; normal for age, 4.5 8 0.6 mm). (Reprinted with permission from Voutetakis et al. [8].)

the hypothalamic-pituitary region at the age of 9.8 years showed regression of the pituitary mass and pituitary height of 4 mm (normal pituitary height for age, 4.5 8 0.6; fig. 2). At the age of 13 years, the boy did not show any characteristics of pubertal development. Both the baseline and peak levels of serum luteinizing hormone and follicle-stimulating hormone during a gonadatrophin-releasing hormone test indicated gonadotrophin deficiency. The serum dehydroepiandrosterone sulfate (DHEA-S) value was 0.8 mol/l, which was low for age (normal range, 2.71–13.57 mol/l), whereas adrenocorticotropic hormone (ACTH) and cortisol responded normally to corticotrophin-releasing hormone (CRH) stimulation (peak value, 8.6 pmol/l and 562.8 nmol/l, respectively). Testosterone was added to the therapeutic regimen when the patient was 14 years old. It was not until 1998 that the mystery of this ‘familial’ pituitary enlargement started to unravel when a PROP1 gene mutation was reported as a cause of CPHD in humans [2]. On DNA analysis both siblings were found to be homozygous for the mutation 301–302GA of the PROP1 gene. As expected, the parents were heterozygous for the same mutation. The hGH therapy in the boy was discontinued at the age of 18.2 years, and the final height achieved was 177.2 cm (0.4 SDS), which was higher than the target height (167 8 4.5 cm). Six months after hGH discontinuation, the peak GH value during the glucagon test was even lower (0.06 g/l), and an impaired cortisol response to glucagon (350.2 nmol/l) was detected for the first time. Repeat MRI performed when the patient was 19 years old revealed pituitary reenlargement; the pituitary height was 11 mm (normal pituitary height for age, 6.1 8 0.3; fig. 3), while the morphology was similar to that observed in figure 1. A new MRI at the age of 22 years disclosed similar morphology to that observed in figures 1 and 3 but with

Xekouki /Sertedaki /Livadas / Argyropoulou /Voutetakis

years shows morphology similar to that observed in figure 1 with increased size of the pituitary gland (pituitary height, 11 mm; normal for age, 6.1 8 0.3 mm). Reprinted with permission from Voutetakis et al. [14].)

a reduction in the pituitary height (pituitary height, 6 mm; normal pituitary height for age, 5.6 8 1 mm; fig. 4) [10]. The documented changes in the pituitary gland over the years are depicted in figure 5. Currently, our patient is on L-thyroxine and testosterone substitution therapy. Since he is completely asymptomatic, cortisol therapy was not initiated. Nevertheless, he is routinely monitored and is informed of the potential for adrenal insufficiency.

Discussion

Since the original report of PROP1 gene mutations in patients with CPHD, a number of cases have been described. In fact, PROP1 alterations are the most common genetic cause of pituitary insufficiency, accounting for up to 50% of familial cases of CPHD [11–13]. We have presented long-term imaging data of pituitary abnormalities in a patient with a PROP1 gene defect, including some images previously published [8, 14]. Our patient presented with hypopituitarism in association with an enlarged pituitary gland at the age of 4 years. The pituitary size subsequently showed a unique wave-like variation with advancing age (enlargement-regression-enlargement-regression; fig. 5). The new change (‘re-regression’) was noted in the most recent MRI obtained when the patient was 22 years of age. We must underline the fact that in both the original enlargement PROP1 Gene Mutations and Pituitary Size

Fig. 4. Contrast-enhanced T1-weighted midsagittal scan at age 22 years shows morphology similar to that observed in figures 1 and 3 with a reduction in the pituitary height (pituitary height, 6 mm; normal for age, 5.6 8 1 mm).

Size of Pituitary Gland of PROP1-deficient patient

12

10 Pituitary Height (mm)

Fig. 3. Contrast-enhanced T1-weighted midsagittal scan at age 19

8

6

4

2

5

8.5

9.8 Age (year)

19

22

Fig. 5. Changes in the pituitary of the PROP1-deficient patient at

various ages (between 5 and 22 years). The maximum vertical height of the pituitary gland was measured in a midline sagittal scan and used as a marker of size. Two consecutive cycles of enlargement-regression are revealed from data extracted from longterm MRI of the hypothalamic-pituitary region.

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as well as the ‘reenlargement’, a nonenhancing mass seems to be interposed between the anteriorly displaced adenohypophysis and the neurohypophysis (fig. 1). Changes in pituitary size over a relatively short time, while the pituitary remains enlarged but before it finally becomes subnormal in size, have also been described [12]. Nevertheless, the underlying mechanism is unknown. We previously hypothesized that the pituitary enlargement originates from the intermediate lobe and may contain immature precursor cells that failed to migrate and differentiate during pituitary organogenesis [8]. This hypothesis was based on previous findings in the prop1deficient mouse model [15] and was later supported by additional mouse data [16]. Specifically, Ward et al. [16] reported that in the absence of PROP1, pituitary progenitor cells are abnormally retained within the periluminal area of Rathke’s pouch, causing the pouch to take on an overgrown, dysmorphic appearance. Therefore, pituitary enlargement in PROP1-deficient humans could be due to trapped progenitor cells, and the subsequent degeneration could be the result of apoptosis of undifferentiated cells. The low DHEA-S values in our patient should not be attributed to a deficient CRH-ACTH axis, since no such insufficiency was present at the time of DHEA-S determination. This finding could be attributed to a possible role of the PROP1 gene in the maturation of the cells that

synthesize the presumed adrenal androgen-stimulating hormone. Alternatively, if the PROP1 gene is also expressed in the zona reticularis, then subnormal DHEA-S levels could be the result of zona reticularis dysfunction [17]. Nevertheless, no embryological expression data are available to discredit or support the latter hypothesis. Current literature supports the fact that PROP1 is not expressed in corticotrophs [15]. Therefore, the underlying mechanism of the cortisol deficiency appearing with advancing age in PROP1-deficient patients remains unknown [18–20]. Conceivably, the gradual impairment of corticotroph function is the result of general pituitary dysfunction. Nevertheless, we must underline the fact that corticotroph cells are affected in prop1/lhx4 doublemutant mice, suggesting that PROP1 and LHX4 genes are important for specification of corticotrophs and that they interact to promote or maintain this cell type [15]. Regardless of the pathogenetic mechanisms involved in pituitary enlargement or regression, the possibility of a PROP1 gene defect must always be considered in patients with CPHD and pituitary mass, since early recognition and treatment are important for outcome and quality of life. Changes in pituitary morphology and size can be expected throughout life; therefore, long-term follow-up with pituitary MRI may be advised. Furthermore, the potential of corticotroph failure should be anticipated, and appropriate studies and substitution therapy should be considered.

References 1 Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, O’Connell SM, Gukovsky I, Carriere C, Ryan AK, Miller AP, Zuo L, Gleiberman AS, Andersen B, Beamer WG, Rosenfeld MG: Pituitary lineage determination by the prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384:327–333. 2 Wu W, Cogan JD, Pfaffle RW, Dasen JS, Frisch H, O’Connell SM, Flynn SE, Brown MR, Mullis PE, Parks JS, Phillips JA 3rd, Rosenfeld MG: Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nat Genet 1998;18:147–149. 3 Mody S, Brown MR, Parks JS: The spectrum of hypopituitarism caused by PROP1 mutations. Best Pract Res Clin Endocrinol Metab 2002;16:421–431.

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4 Fofanova O, Takamura N, Kinoshita E, Vorontsov A, Vladimirova V, Devov I, Peterkova V, Yamashita S: MR imaging of the pituitary gland in children and young adults with congenital combined pituitary hormone deficiency associated with PROP1 mutations. Am J Roentgenol 2000; 174: 555– 559. 5 Mendonca BB, Osorio MG, Latronico AC, Estefan V, Lo LS, Arnhold IJ: Longitudinal hormonal and pituitary imaging changes in two females with combined pituitary hormone deficiency due to depletion of A301, G302 in the PROP1 gene. J Clin Endocrinol Metab 1999;84:942–945. 6 Teinturier C, Vallette S, Adamsbaum C, Bendaoud M, Brue T, Bougneres PF: Pseudotumor of the pituitary due to PROP-1 deletion. J Pediatr Endocrinol Metab 2002; 15: 95–101.

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7 Riepe FG, Partsch CJ, Blankenstein O, Monig H, Pfaffle RW, Sippell WG: Longitudinal imaging reveals pituitary enlargement preceding hypoplasia in two brothers with combined pituitary hormone deficiency attributable to PROP1 mutation. J Clin Endocrinol Metab 2001;86:4353–4357. 8 Voutetakis A, Argyropoulou M, Sertedaki A, Livadas S, Xekouki P, Maniati-Christidi M, Bossis I, Thalassinos N, Patronas N, DacouVoutetakis C: Pituitary magnetic resonance imaging in 15 patients with Prop1 gene mutations: pituitary enlargement may originate from the intermediate lobe. J Clin Endocrinol Metab 2004;89:2200–2206. 9 Argyropoulou M, Perignon F, Brunelle F, Brauner R, Rappaport R: Height of normal pituitary gland as a function of age evaluated by magnetic resonance imaging in children. Pediatr Radiol 1991;21:247–249.

Xekouki /Sertedaki /Livadas / Argyropoulou /Voutetakis

10 Tsunoda A, Okuda O, Sato K: MR height of the pituitary gland as a function of age and sex: especially physiological hypertrophy in adolescence and in climacterium. Am J Neuroradiol 1997; 18:551–554. 11 Deladoey J, Fluck C, Buyukgebiz A, Kuhlmann BV, Eble A, Hindmarsh PC, Wu W, Mullis PE: ‘Hot spot’ in the PROP1 gene responsible for combined pituitary hormone deficiency. J Clin Endocrinol Metab 1999;84: 1645–1650. 12 Turton JPG, Reynaud R, Mehta A, Torpiano J, Saveanu A, Woods KS, Tiulpakov A, Hamilton J, Attard-Montalto S, Parascandalo R, Vella C, Clayton PE, Shalet S, Barton J, Brue T, Dattani MT: Mutations within the transcription factor PROP1 are rare in a cohort of patients with sporadic combined pituitary hormone deficiency (CPHD). Clin Endocrinol (Oxf) 2005;63(1):10–18.

PROP1 Gene Mutations and Pituitary Size

13 Parks JS, Brown MR, Hurley DL, Phelps CJ, Wajnrajch MP: Heritable disorders of pituitary development. J Clin Endocrinol Metab 1999;84(12):4362–4370. 14 Voutetakis A, Sertedaki A, Livadas S, Xekouki P, Bossis I, Dacou-Voutetakis C, Argyropoulou MI: Pituitary size fluctuation in long-term MR studies of PROP1 deficient patients: a persistent pathophysiological mechanism? J Endocrinol Invest 2006; 29: 462–466. 15 Raetzman LT, Ward R, Camper SA: Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia. Development 2002;129(18):4229–4239. 16 Ward RD, Raetzman LT, Suh H, Stone BM, Nasonkin IO, Camper SA: Role of PROP1 in pituitary gland growth. Mol Endocrinol 2005;19(3):698–710. 17 Voutetakis A, Livadas S, Sertedaki A, Maniati-Christidi M, Dacou-Voutetakis C: Insufficient adrenarche in patients with combined pituitary hormone deficiency caused by a PROP1 gene defect. J Pediatr Endocrinol Metab 2001;14:1107–1111.

18 Agarwal G, Bhatia V, Cook S, Thomas PQ: Adrenocorticotropin deficiency in combined pituitary hormone deficiency patients homozygous for a novel PROP1 deletion. J Clin Endocrinol Metab 2000;85:4556–4561. 19 Pernasetti F, Toledo SP, Vasilyev VV, Hayashida CY, Cogan JD, Ferrari C, Lourenco DM, Mellon PL: Impaired adrenocorticotropin-adrenal axis in combined pituitary hormone deficiency caused by a two-base pair deletion (301–302delAG) in the prophet of Pit-1 gene. J Clin Endocrinol Metab 2000;85: 390–397. 20 Bottner A, Keller E, Kratzsch J, Stobbe H, Weigel JF, Keller A, Hirsch W, Kiess W, Blum WF, Pfaffle RW: PROP1 mutations cause progressive deterioration of anterior pituitary function including adrenal insufficiency: a longitudinal analysis. J Clin Endocrinol Metab 2004;89(10):5256–5265.

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113

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):114 DOI: 10.1159/000097565

Published online: February 15, 2007

Hot Topics in Pediatric Endocrinology

This session explored two intriguing topics: what has been referred to as developmental programming in the fetus, leading to increased disease risk in adult life, and a congenital form of excessive antidiuresis with hyponatremia. While unrelated, both offered new insights into disease processes. Peter Gluckman addressed the issue of how events in early life are associated with changes in the risk of disease in later life. Based on results of epidemiological observations as well as on animal experimentation and molecular analyses, Gluckman and colleagues have proposed the phenomenon of ‘developmental induction’. This concept emphasizes the adaptability of the fetus to its intrauterine environment, as well as the inherent plasticity, and thus potential reversibility, of fetal programming. Stephen Rosenthal presented a new, G protein-coupled receptor disease: a nephrogenic form of inappropriate antidiuresis; it can be thought of clinically as the opposite phenotype to nephrogenic diabetes insipidus. Dr. Rosen-

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Accessible online at: www.karger.com/hre

thal reviewed the clinical and laboratory profiles of disordered water balance in two male infants presenting with hyponatremia and inappropriately concentrated urine osmolality, caused by activating mutations of the G protein-coupled V2 vasopressin receptor (as opposed to inactivating mutations seen in nephrogenic diabetes). An important clinical clue to the diagnosis was the finding of undetectable levels of arginine vasopressin. Such detailed characterization of individual cases of this nephrogenic syndrome of inappropriate antidiuresis (NSIAD) may improve understanding of fluid homeostasis and diseases involving water balance disorders and permit development of a targeted treatment. Cheri Deal Department of Pediatrics University of Montreal Montreal, Quebec, Canada

Hot Topics in Pediatric Endocrinology

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):115–120 DOI: 10.1159/000097566

Published online: February 15, 2007

Leptin Reversal of the Metabolic Phenotype: Evidence for the Role of Developmental Plasticity in the Development of the Metabolic Syndrome Peter D. Gluckman a Alan S. Beedle a Mark A. Hanson b Mark H. Vickers a a

Liggins Institute and National Research Centre for Growth and Development, University of Auckland, Auckland, New Zealand; b Centre for Developmental Origins of Health and Disease, University of Southampton, Southampton, UK

Key Words Metabolic syndrome Developmental programming Developmental induction Match-mismatch Leptin Reversibility

Abstract Events in early life are associated with changes in the risk of disease in later life. There is increasing evidence that these associations are mediated by permanent transcriptional changes in metabolic pathways, in some cases linked to epigenetic alterations. We have proposed that this phenomenon of ‘developmental induction’ is not a manifestation of pathophysiological processes but rather represents the consequence of developmental decisions made during fetal and early postnatal life to maximize subsequent fitness. However, this fitness advantage is lost if the early and later environments are mismatched. Rats undernourished in utero by maternal underfeeding develop features of the metabolic syndrome, especially if fed on a high-fat diet, but transient neonatal treatment with leptin reverses induction of this adverse metabolic phenotype. This observation demonstrates that developmental programming is reversible and provides strong support for the match-mismatch or predictive model for the origins of developmental programming. Copyright © 2007 S. Karger AG, Basel

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Introduction

There are compelling data relating events in early life to changes in the risk of developing disease in later life. However, the relative importance of such developmental induction for the ecology of disease in the developed world remains controversial. Impetus for research in this area was provided by a host of epidemiological observations relating birth size to later disease risk. In particular, most attention has focused on the association between lower birth weight and the later risk of type 2 diabetes, cardiovascular disease and obesity [1–3]. However, the data are frequently misinterpreted, adding to the confusion [4]. For example, a key point to make from the epidemiological studies is that the relationship between disease risk and birth size (generally either birth weight or ponderal index) is continuous [5]. This relationship exists even for infants born above the mean for birth weight, emphasizing that this is not a process defined by low birth weight or confined to those at the extremes of the developmental processes. Rather, this is about normative processes in development, as reflected by variation in normal birth size, leading to altered disease risk in later life. A second important point comes from studies of cohorts in Finland [6] and India [7]. For these studies the growth patterns of those who later developed disease or

Peter D. Gluckman, MD Liggins Institute, University of Auckland and National Research Center for Growth and Development, 2–6 Park Avenue Grafton, Private Bag 92019, Auckland (New Zealand) Tel. +64 9 373 7599, Fax +64 9 373 7497, E-Mail [email protected]

insulin resistance were reconstructed from birth and compared with the patterns of those who did not develop disease. Those who developed disease had a different pattern of growth, with relative weight being slightly depressed in infancy and more rapid weight gain in childhood starting from an earlier age. Importantly, the shifts in relative mean weight compared with height were small in the affected individuals (e.g., approximately 0.1 standard deviation scores [SDS] below the cohort at birth and rising to approximately 0.2 SDS above the cohort by the age of 10 years [6]). Again, these phenomena and associations fall within the normal range and do not reflect extreme pathology. These results also suggest that the association between risk of disease and pattern of growth starts before birth and extends well into childhood. Further, as we review below, the origin of the more rapid weight gain in childhood may well involve a changed sensitivity to energy-dense food which has been established earlier in development.

Animal Models of Developmental Induction of Disease

Animal studies have demonstrated that there is a link between prenatal undernutrition or glucocorticoid exposure and the later risk of developing insulin resistance, endothelial dysfunction, hypertension and obesity (reviewed in [8]). A caveat must be noted that, in general, these animal studies (including our own) have used rather extreme forms of manipulation of the fetal environment. Nevertheless, the animal studies uniformly show that prenatal factors can alter long-term physiology in the very systems that cause disease in humans. Our own studies have largely focused on using maternal undernutrition throughout pregnancy in the rat. The offspring of such pregnancies have sarcopenia and develop insulin resistance, hyperleptinaemia, obesity and fatty liver [9, 10]. Intriguingly, in addition to these peripheral manifestations of obesity and insulin resistance, these animals exhibit central nervous system components as well: they are hyperphagic and in open-field testing they exercise less [11]. Whether this last observation represents an effect on lethargy or on anxiety is yet to be resolved. Studies using other developmental paradigms also entailing restriction of prenatal nutrition have shown endothelial dysfunction and alterations in food preference and neuroendocrine control of food intake [12–14]. At the molecular level there is now increasing evidence that early nutritional factors can lead to permanent tran116

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scriptional changes in metabolic pathways, with altered expression of genes for components such as glucocorticoid receptor, peroxisome proliferator-activated receptor alpha, phosphoenolpyruvate carboxykinase and protein kinase C zeta in liver, muscle and fat [15–17]. In some cases these alterations have been directly linked to epigenetic change in that the altered expression is inversely related to methylation changes in the promoter region of the gene and can be prevented by administration of a methyl donor (folate, glycine) during the period of maternal nutritional challenge [18]. Importantly, we have noted a synergistic interaction between prenatal and postnatal environments in that rats born to undernourished mothers and placed on a high-fat diet after weaning develop greater morbidity compared with rats subjected to either prenatal or postnatal manipulations alone [9, 11].

The Match-Mismatch Model of Developmental Programming

Earlier models used to explain the association between fetal growth and later disease risk focused on either purely genomic or purely environmental considerations. The ‘thrifty genotype’ model of Neel [19] later evolved into the ‘fetal insulin’ hypothesis, whereby selection had occurred for thrifty alleles of genes involved both in fetal growth regulation and in determination of postnatal insulin sensitivity [20]. Although there are genes, such as glucokinase, that would meet this criterion, the general nature of the phenomenon across all levels of birth size, the independence of postnatal effects from birth size in some clinical and experimental studies and, importantly, the experimental studies in animals of uniform genetic background make a purely genetic explanation implausible. Conversely, Hales and Barker proposed the ‘thrifty phenotype’ model, which proposed that the fetus traded off growth in utero to survive nutritional limitations. According to this model, such a trade-off had no adverse postnatal consequences provided the infant subsequently grew up in a ‘hunter-gatherer’ type environment, but was associated with a predisposition to metabolic sequelae if the infant grew up in an energy-dense environment [21]. This initial adaptive explanation has limitations. There is increasing evidence that insulin resistance need not be present at birth but can evolve later in childhood [22]. Further, there is evidence that the prenatal environment can induce effects both in other domains [23] and in nonthrifty directions [24], which limits the applicability of this model. Gluckman /Beedle /Hanson /Vickers

We have proposed that ‘developmental induction’ or ‘programming’ is not a manifestation of pathophysiological processes [25]. Rather, it is the consequence of the normative processes of developmental plasticity by which all organisms adjust their phenotype during development to better match their environment and achieve maximal reproductive fitness [26]. Clearly a mammalian fetus can be exposed to developmentally disruptive environmental cues, such as when very high maternal glucose levels in uncontrolled diabetes lead to malformation of the fetal heart. Further, some adaptive responses such as reduced growth, accelerated maturation or altered body proportions might be appropriate fetal responses to an adverse intrauterine environment to optimize the chance of survival to birth. However, most responses of the fetus are within the normative size range and reflect developmental decisions made to maximize postnatal fitness. We have described such responses to the prenatal environment for later advantage as ‘predictive adaptive responses’ (PARs), and a number of clear examples from the comparative literature are reviewed elsewhere [27]. Our current model of developmental induction is shown diagrammatically in figure 1. In the absence of any cues to the contrary, the fetus will develop along a trajectory defined by its genome and epigenome at conception. But the fetus is not immune from its maternal environment, and thus there is continuous exposure to a set of cues which, in the presumed absence of maternal and placental disease, reflects the environment of the mother. The fetus adjusts its developmental trajectory in accordance with the expectation of being born into an environment matching the one that it perceives in utero. If the prenatal and postnatal environments match, then the risk of disease is low. So when the developmental trajectory in utero has been shifted in a more thrifty direction because of poor nutrition, if that infant actually is born into and develops in a sparse environment, then its risk of metabolic disease will be low. However, if the fetus predicted a sparse environment and the postnatal environment is enriched, it will have made the wrong predictive choices and will have an enhanced risk of metabolic disease. There are several points to make. First, we do not mean to imply that plasticity is strictly a fetal phenomenon and that a non-plastic phase starts at birth. The embryo and fetus are more plastic than the neonate, but clearly metabolic plasticity extends into the neonatal period both in rodents and in humans. There is considerable evidence that altered nutrition in the neonatal peLeptin Reversal of the Metabolic Phenotype

Programming (epigenetic change, etc.)

Inherited genotype and epigenotype

Developmental Cues environment (eg UN) Matched Later environment

Mismatched

Normal Metabolic phenotype phenotype

Fig. 1. Model of developmental induction. The developmental tra-

jectory defined by the fetal genome and epigenome is represented by a black arrow. Maternal cues such as undernutrition (UN) during development cause the fetus to shift its developmental trajectory to match the perceived environment (grey arrow). If the predicted environment matches the later environment (white background), then the risk of metabolic disease is low. If there is a mismatch between the predicted environment and the later environment (black background), then the risk of metabolic disease is enhanced.

riod can affect long-term outcomes [28, 29], although it is important to distinguish between primary induction as opposed to the consequences of release from the constrained environment of fetal life. This distinction may overcome some of the confusion that currently exists in the literature [30]. Second, predictions based on this model of developmental induction need not be 100% reliable for these processes of developmental plasticity to be selected. Even if the predictive value is low, plasticity can still have a fitness advantage if the choice to select a thrifty strategy is less risky in fitness terms than the opposite choice [31]. Indeed, that would have been the case for our ancestral hunter-gatherers with a short life span and a different diet, who would not have been at risk of metabolic disease [32]. Elsewhere we have pointed out that the phenomenon of maternal constraint markedly increases the risk of false prediction in a modern world of high energy density [33]. Third, the association between disease risk and birth weight is essentially fortuitous and arises from the coincidence of immediately adaptive responses and predictive responses to similar nutritional cues. Horm Res 2007;67(suppl 1):115–120

117

Total Body Fat

Plasma Leptin

60 100

55

(%)

75

45 50

40 35

(ng/ml)

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25

30 0 AD S

AD L

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UN L

AD S

Insulin

UN L

C-peptide

2000

2.0 1500 (ng/ml)

1.5 1000

1.0

500

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0 AD S

Experimental Evidence for the Predictive Model

Recently we conducted an experiment that provides strong evidence in support of our predictive model [34]. Given that plasticity extends after birth, we hypothesized that if rat pups that had been undernourished in utero by maternal underfeeding were ‘tricked’ into seeing themselves as fatter than they were, they might reverse their predictive adaptations while still plastic. We treated female pups of maternally undernourished rats with recombinant rat leptin (2.5 g/g/day, subcutaneously) on days 3 to 10 of life and followed the animals for 170 days. At 30 days of life, after weaning, they were placed on a high-fat diet. Appropriate controls were also studied. The offspring of normally nourished mothers had no detectable phenotypic response to neonatal leptin: their pattern of growth and their insulin and leptin levels were not different from those of saline-treated rat pups from normally nourished mothers. As expected [9–11], salinetreated rat pups from undernourished mothers developed obesity, hyperleptinaemia, hyperinsulinaemia, fatty liver, hyperphagia and reduced locomotor activity in open-field testing. In contrast, the leptin-treated female rat pups from undernourished mothers were not differ118

UN S

Horm Res 2007;67(suppl 1):115–120

(pM)

Fig. 2. Reversal of developmental induction by neonatal leptin treatment. Total body fat and fasting plasma leptin, insulin and C-peptide concentrations were measured at postnatal day 170 in rats fed a high-fat diet postnatally following treatment as neonates with either saline (S) or recombinant rat leptin (L). AD = Offspring of mothers fed ad libitum throughout pregnancy; UN = offspring from mothers undernourished throughout pregnancy.

AD L

AD L

UN S

UN L

AD S

AD L

UN S

UN L

ent from saline-treated pups from normal mothers – the developmental induction of an adverse metabolic phenotype had been totally blocked (fig. 2). Intriguingly, although the phenotype was totally blocked, maternal undernutrition and leptin exposure had independent effects on gene expression in mature adipose tissue, and leptin exposure did not reverse the molecular changes caused by maternal undernutrition (KA Lillycrop, GC Burdge, MH Vickers, MA Hanson, PD Gluckman, unpublished work). These observations mean either that the molecular changes we have observed are not central to the processes of developmental induction or that, while epigenetic changes are important in the initiation of an altered trajectory, they are not essential in sustaining it.

Discussion

The totality of reversal of the adverse metabolic phenotype in the rat model described above is intriguing. It suggests that the effects of leptin are on broadly based predictive processes. The infant rats in our study were ‘tricked’ into predicting a better rather than a poorer nutritional environment, thus reversing a prediction that Gluckman /Beedle /Hanson /Vickers

would otherwise have led to mismatch, and have become better matched to their environment. In the neonatal rodent, leptin has been shown to affect neurogenesis in the hypothalamus [35], which may be central to its effects on the later phenotype, although it is noteworthy that both peripheral and central components of the phenotype were altered. Leptin also has effects on pancreatic apoptosis [36]. Preliminary data from experiments with leptin in the male rat pup do not show such a clear-cut reversal of phenotype. However, from a life-history perspective this is not surprising as rats normally live in a polygynous manner and a male rat’s life-history strategy is based on dominance in size. Thus, while a female rat’s reproductive success depends on persistent fecundity over time and thus a better match with its environment, the male drive for larger body size may play a greater role in its life-history strategy than does avoidance of metabolic mismatch. Alternatively, the timing of a presumptive critical period for plasticity may be different for male and female rats.

Conclusions

Postnatal administration of leptin totally reverses developmental induction of an adverse metabolic phenotype in female rats. This data set is interesting at multiple levels. First, it provides some evidence to support our predictive model. Second, it suggests that developmental programming can be reversed. How such information can be applied to the human is less certain. But the general point remains – namely, that optimal metabolic health requires a good match between the fetal and postnatal environments. This can be achieved either by enriching the fetal environment through improving maternal health or by ameliorating the energy-dense, exercisepoor postnatal environment of the child. Elsewhere we have pointed out that improving maternal health is key in the developing world where the risks to the fetal environment are greater [37]. A developmental perspective on metabolic health indicates pregnancy, infancy and childhood as points of intervention, although the match-mismatch model suggests that strategies will differ at different times in the life course.

References 1 Hales CN, Barker DJ, Clark PM, Cox LJ, Fall C, Winter PD: Fetal and infant growth and impaired glucose tolerance at age 64. BMJ 1991;303:1019–1022. 2 Barker DJ, Osmond C: Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1986; 1:1077–1081. 3 Eriksson J, Forsén T, Tuomilehto J, Osmond C, Barker D: Size at birth, childhood growth and obesity in adult life. Int J Obes 2001; 25: 735–740. 4 Huxley R, Neil A, Collins R: Unravelling the fetal origins hypothesis: is there really an inverse association between birthweight and subsequent blood pressure? Lancet 2002; 360:659–665. 5 Barker DJP: In utero programming of chronic disease. Clin Sci 1998;95:115–128. 6 Eriksson JG, Forsén T, Tuomilehto J, Osmond C, Barker DJ: Early adiposity rebound in childhood and risk of type 2 diabetes in adult life. Diabetologia 2003;46:190–194. 7 Bhargava SK, Sachdev HS, Fall CHD, Osmond C, Lakshmy R, Barker DJP, Biswas SK, Ramji S, Prabhakaran D, Reddy KS: Relation of serial changes in childhood body-mass index to impaired glucose tolerance in young adulthood. N Engl J Med 2004; 350: 865– 875.

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8 Gluckman P, Hanson M, Morton S, Pinal C: Life-long echoes – a critical analysis of the developmental origins of adult disease model. Biol Neonate 2005; 87:127–139. 9 Vickers MH, Breier BH, Cutfield WS, Hofman PL, Gluckman PD: Fetal origins of hyperphagia, obesity, and hypertension and postnatal amplification by hypercaloric nutrition. Am J Physiol 2000;279:E83–E87. 10 Vickers MH, Reddy S, Ikenasio BA, Breier BH: Dysregulation of the adipoinsular axis – a mechanism for the pathogenesis of hyperleptinaemia and adipogenic diabetes induced by fetal programming. J Endocrinol 2001;170:323–332. 11 Vickers M, Breier B, McCarthy D, Gluckman P: Sedentary behavior during postnatal life is determined by the prenatal environment and exacerbated by postnatal hypercaloric nutrition. Am J Physiol 2003; 285:R271– R273. 12 Brawley L, Itoh S, Torrens C, Barker A, Bertram C, Poston L, Hanson M: Dietary protein restriction in pregnancy induces hypertension and vascular defects in rat male offspring. Pediatr Res 2003;54:83–90. 13 Bellinger L, Lilley C, Langley-Evans SC: Prenatal exposure to a maternal low-protein diet programmes a preference for high-fat foods in the young adult rat. Br J Nutr 2004; 92: 513–520.

14 El-Haddad MA, Desai M, Gayle D, Ross MG: In utero development of fetal thirst and appetite: potential for programming. J Soc Gynecol Invest 2004;11:123–130. 15 Burdge GC, Phillips ES, Dunn RL, Jackson AA, Lillycrop KA: Effect of reduced maternal protein consumption during pregnancy in the rat on plasma lipid concentrations and expression of peroxisomal proliferator-activated receptors in the liver and adipose tissue of the offspring. Nutr Res 2004;24:639–646. 16 Nyirenda MJ, Lindsay RS, Kenyon CJ, Burchell A, Seckl JR: Glucocorticoid exposure in late gestation permanently programs rat hepatic phosphoenolpyruvate carboxykinase and glucocorticoid receptor expression and causes glucose intolerance in adult offspring. J Clin Invest 1998;101:2174–2181. 17 Ozanne SE, Olsen GS, Hansen LL, Tingey KJ, Nave BT, Wang CL, Hartil K, Petry CJ, Buckley AJ, Mosthaf-Seedorf L: Early growth restriction leads to down regulation of protein kinase C zeta and insulin resistance in skeletal muscle. J Endocrinol 2003; 177:235– 241. 18 Lillycrop KA, Phillips ES, Jackson AA, Hanson MA, Burdge GC: Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring. J Nutr 2005;135:1382–1386.

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19 Neel JV: Diabetes mellitus: a ‘thrifty’ genotype rendered detrimental by ‘progress’? Bull World Health Organ 1999; 77:694–703. 20 Hattersley AT, Tooke JE: The fetal insulin hypothesis: an alternative explanation of the association of low birthweight with diabetes and vascular disease. Lancet 1999;353:1789– 1792. 21 Hales CN, Barker DJ: The thrifty phenotype hypothesis. Br Med Bull 2001;60:5–20. 22 Mericq V, Ong KK, Bazaes RA, Pena V, Avila A, Salazar T, Soto N, Iniguez G, Dunger DB: Longitudinal changes in insulin sensitivity and secretion from birth to age three years in small- and appropriate-for-gestational-age children. Diabetologia 2005;48:2609–2614. 23 Wintour E, Moritz K, Johnson K, Ricardo S, Samuel CS, Dodic M: Reduced nephron number in adult sheep, hypertensive as a result of prenatal glucocorticoid treatment. J Physiol 2003;549:929–935. 24 Cutfield W: IVF children are taller with increased IGF-I, IGF-II and IGFBP-3 levels suggesting altered genetic imprinting. Proceedings of the 3rd International Congress on Developmental Origins of Health and Disease, 2005 Nov 16–19, Toronto, Canada.

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25 Gluckman PD, Hanson MA, Spencer HG, Bateson P: Environmental influences during development and their later consequences for health and disease: implications for the interpretation of empirical studies. Proc Biol Sci 2005;272:671–677. 26 West-Eberhard MJ: Developmental Plasticity and Evolution. New York, Oxford University Press, 2003. 27 Gluckman PD, Hanson MA, Spencer HG: Predictive adaptive responses and human evolution. Trends Ecol Evol 2005; 20: 527– 533. 28 Singhal A, Fewtrel M, Cole TJ, Lucas A: Low nutrient intake and early growth for later insulin resistance in adolescents born preterm. Lancet 2003;361:1089–1097. 29 Stettler N, Stallings VA, Troxel AB, Zhao J, Schinnar R, Nelson SE, Ziegler EE, Strom BL: Weight gain in the first week of life and overweight in adulthood: a cohort study of European American subjects fed infant formula. Circulation 2005;111:1897–1903. 30 Singhal A, Lucas A: Early origins of cardiovascular disease: is there a unifying hypothesis? Lancet 2004;363:1642–1645. 31 Jablonka E, Oborny B, Molnar I, Kisdi E, Hofbauer J, Czaran T: The adaptive advantage of phenotypic memory in changing environments. Philos Trans R Soc Lond B 1995; 350:133–141.

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32 Cordain L, Eaton SB, Sebastian A, Mann N, Lindeberg S, Watkins BA, O’Keefe JH, Brand-Miller J: Origins and evolution of the Western diet: health implications for the 21st century. Am J Clin Nutr 2005;81:341–354. 33 Gluckman PD, Hanson MA: Maternal constraint of fetal growth and its consequences. Semin Fetal Neonatal Med 2004;9:419–425. 34 Vickers MH, Gluckman PD, Coveny AH, Hofman PL, Cutfield WS, Gertler A, Breier BH, Harris M: Neonatal leptin treatment reverses developmental programming. Endocrinology 2005; 146:4211–4216. 35 Bouret SG, Draper SJ, Simerly RB: Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 2004;304:108– 110. 36 Islam MS, Sjöholm Å, Emilsson V: Fetal pancreatic islets express functional leptin receptors and leptin stimulates proliferation of fetal islet cells. Int J Obes Relat Metab Disord 2000;24:1246–1253. 37 Gluckman P, Hanson M: Living with the past: evolution, development, and patterns of disease. Science 2004;305:1733–1736.

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Hot Topics in Pediatric Endocrinology

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):121–125 DOI: 10.1159/000097567

Published online: February 15, 2007

Gain-of-Function Mutations in the V2 Vasopressin Receptor Stephen M. Rosenthal a Stephen E. Gitelman a Gabriel A. Vargas b Brian J. Feldman a a

Department of Pediatrics, Division of Endocrinology, and b Department of Psychiatry, University of California at San Francisco, San Francisco, Calif., USA

Key Words Antidiuresis Hyponatremia Vasopressin receptor Inappropriate secretion of antidiuretic hormone Nephrogenic syndrome of inappropriate antidiuresis

Abstract Background: Excessive antidiuresis with resulting water overload and hyponatremia are principal features of the syndrome of inappropriate secretion of antidiuretic hormone, a condition of abnormal water balance associated with a wide variety of clinical disorders. Despite clinical and laboratory evaluations consistent with the syndrome of inappropriate secretion of antidiuretic hormone, some patients may present with undetectable arginine vasopressin levels. In such cases, genetic studies may reveal novel activating mutations of the G-protein-coupled V2 vasopressin receptor, leading to what we term nephrogenic syndrome of inappropriate antidiuresis (NSIAD). We review the clinical and laboratory features of disordered water balance associated with such mutations in two pediatric NSIAD cases. Conclusions: Further characterization of NSIAD should enhance our understanding of fluid homeostasis and of clinical disease involving disorders of water balance. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0121$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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Introduction

Excessive antidiuresis with resulting water overload and hyponatremia are principal features of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), a condition of abnormal water balance associated with a wide variety of clinical disorders [1]. Normal fluid balance requires adequate water intake, regulated by an intact thirst mechanism. It also requires appropriate free water excretion by the kidneys, mediated by appropriate secretion of arginine vasopressin (AVP), also known as antidiuretic hormone [2]. AVP exerts its antidiuretic action by binding to the V2 vasopressin receptor (V2R). Located in the basolateral membrane of renalcollecting duct epithelial cells, V2R is a G-protein-coupled receptor (GPCR) that, once activated, increases intracellular cyclic adenosine monophosphate (cAMP). This, in turn, leads to trafficking of the water channel aquaporin-2 (AQP-2) to the apical membrane of collecting duct cells, resulting in increased water permeability and antidiuresis [3]. We recently evaluated two unrelated male infants who had persistent hyponatremia and were unable to excrete maximally dilute urine. Their clinical and laboratory evaluations were consistent with SIADH, yet they had undetectable AVP levels. Genetic characterization of

Stephen M. Rosenthal, MD University of California San Francisco Division of Pediatric Endocrinology, 513 Parnassus Ave. – Rm S-672 San Francisco, CA 94143-0434 (USA) Tel. +1 415 476 2266, Fax +1 415 476 8214, E-Mail [email protected]

these patients revealed novel activating mutations of the V2R, leading to what we term nephrogenic syndrome of inappropriate antidiuresis (NSIAD) [4]. Below we describe the clinical and laboratory profiles of these two previously reported pediatric cases of NSIAD [4, 5].

Case Reports We evaluated two male infants. After unremarkable early neonatal courses, one infant presented at 2.5 months of age after two generalized seizures, and the second presented at 3 months of age with irritability. Both infants were fed exclusively by bottle and thus had relatively sodium-restricted diets (7 mEq of sodium were in each liter of formula ingested [1 mEq/l = 1 mmol/l]). Only mild, intermittent systolic hypertension was remarkable on physical examination. Laboratory evaluation indicated that both infants had persistent hyponatremia (presenting serum sodium was 118 mmol/l in one infant and 123 mmol/l in the other). Potassium and bicarbonate levels were normal. Each infant also demonstrated serum hypoosmolality associated with inappropriately concentrated urine osmolality and natriuresis: one had a presenting serum osmolality of 247 mOsm/kg H2O; simultaneous urine osmolality was 390 mOsm/kg H2O, and urine sodium was 75 mmol/l. Other laboratory data were unremarkable and confirmed euvolemia, with normal renal, thyroid and adrenal function. There was also no history of central nervous system or pulmonary disease. Plasma AVP levels remained undetectable, even though the clinical findings indicated SIADH [4].

Genetic Studies

We considered the possibility that novel activating mutations of the V2R might account for the presentation of an SIADH-like clinical picture without detectable circulating levels of AVP. Genomic sequencing of each boy’s affected V2R gene (AVPR2) revealed a single nucleotide change, resulting in a missense amino acid change at codon 137 [4]. In patient 1, cytosine was mutated to thymine at nucleotide 770, resulting in a change from arginine to cysteine at codon 137 (R137C). (This patient’s mother was heterozygous for the R137C mutation.) In patient 2, guanine was mutated to thymine at nucleotide 771, resulting in a change from arginine to leucine at codon 137 (R137L). This patient had a spontaneous mutation because his mother was homozygous for wild-type AVPR2. AVPR2 is X-linked; thus, each patient was hemizygous for this mutated allele. The V2R is a well-characterized GPCR with seven transmembrane domains. As noted above, the missense mutations found in our patients map to arginine (R) at 122

Horm Res 2007;67(suppl 1):121–125

codon 137 (fig. 1). This codon is part of a highly conserved DRY/H motif located at the junction of the third transmembrane domain and second intracellular loop. This region of the receptor is thought to be critical for V2R function. More than 180 inactivating mutations of the V2R have been described in association with nephrogenic diabetes insipidus (NDI) and are scattered throughout the coding sequence [6, 7]. A missense mutation of particular interest causing V2R inactivation and NDI occurs at the same site, codon 137 of the V2R, and results in conversion of an arginine to a histidine (R137H) in the DRY/H region of the receptor [8] (fig. 1). Thus, different missense mutations at the same codon of V2R can result in opposite disease phenotypes.

Functional Assays of V2R Mutations

A functional assay for V2R activation was used to assess the effects of these novel mutations on basal, nonagonist-stimulated V2R function [4]. COS-7 cells were transfected with either an empty expression vector or an expression vector containing either the wild-type V2R sequence or sequences that contained the R137C, R137L or R137H mutations. The cAMP accumulation was indirectly measured using a plasmid vector that contained a cAMP-responsive luciferase reporter gene. As expected, cAMP-responsive luciferase activity was low in COS-7 cells transfected either with vector alone, vector containing the wild-type V2R sequence or vector containing the R137H mutation [4]. However, basal levels of cAMP-responsive luciferase activity in cells expressing V2R with the R137C or R137L mutations were 4 (p = 0.01) or 7.5 (p ! 0.004) times, respectively, the level of activity of cells expressing wild-type V2R [4]. These findings indicate that the R137C and R137L mutations result in a constitutively active V2R and provide an explanation for the SIADH-like clinical picture in the absence of circulating levels of AVP. Studies are under way to determine the molecular mechanisms responsible for constitutive V2R activation.

GPCR Mutations and Endocrine Disease

A wide variety of endocrine disorders are associated with GPCR mutations. For example, inactivating mutations of the luteinizing hormone/human chorionic gonadotropin (hCG) receptor are associated with Leydig cell hypoplasia and male pseudohermaphroditism [9]. Rosenthal /Gitelman /Vargas /Feldman

S N S S

Q

P

L

S P

L

S

P

R D T P A L L P K D L D R T W A A R L A E Q Extracellular L P L L A V F G F S I L A L A V L A VA V D L A G L S V L C GH L A V N F IH V A L I L P Intracellular A A R W R H G R RG E

R

NSIAD

H

GPV

F

A

S

T T S AM L M –NH2

R

G

A E P W P F G D C R A A S GG P E A P R G E W L L D T V V C E C Y W D T A R N G R V A A P F T W A V AQ F Q L WV A L I Y L K L L M V L F I M F L F Q V Q F M P L A S L P VG V Cell S L L L CWA L L T L A P A S M Y N S C membrane S L T V Y V V F A A G I M I S Y N A V P C Q W I V I AW V V L M T L A Y A S I F M T L L V L C C R P S F E R R D L A I S S H R V H R V R L N T S A S G A S E L R R137 W K S R I H A V S L T C V H A A V R A P A G P P P S S G M G C S E DQ P G L L G E T P A Y R H G P S G T S T A E S R R S S L A K D PG T S S –COOH G R R R GR M T C L L R D H: Congenital RH nephrogenic A diabetes I insipidus

Fig. 1. Diagram of V2R. The inset highlights changes in R137 that result in either congenital nephrogenic diabetes insipidus (R137H) or nephrogenic syndrome of inappropriate antidiuresis (R137C or R137L) [4]. (Reproduced with permission from Feldman et al. [4].)

However, activating autosomal dominant germline mutations cause Leydig cell hyperplasia (testotoxicosis), and activating somatic mutations are associated with Leydig cell adenomas [9–11]. Inactivating mutations of the follicle-stimulating hormone receptor present in women as ovarian dysgenesis [9] and as variable spermatogenic failure in men [9, 12]. In contrast, activating mutations of this receptor are associated with familial ovarian hyperstimulation syndrome in women [13] or sustained spermatogenesis in a hypophysectomized man [14]. Inactivating mutations of the thyroid-stimulating hormone (TSH) receptor are associated with TSH unresponsiveness and hypothyroidism. Activating autosomal dominant germline mutations of the TSH receptor are associated with nonautoimmune hyperthyroidism (thyrotoxi-

cosis), while somatic activating TSH receptor mutations are associated with toxic thyroid nodules and adenomas [11]. An unusual gain-of-function mutation in the TSH receptor renders it hypersensitive to hCG, resulting in familial gestational hyperthyroidism [15]. Inactivating mutations in the extracellular Ca2+-sensing receptor result in familial autosomal dominant hypocalciuric hypercalcemia or autosomal recessive neonatal severe primary hyperparathyroidism, whereas activating mutations of this receptor result in autosomal dominant hypocalcemia [11]. Interestingly, and similar to what we have observed with the V2R [4], both inactivating and activating mutations have been described in the same codon of the Ca2+-sensing receptor [16].

Gain-of-Function Mutations in the V2 Vasopressin Receptor

Horm Res 2007;67(suppl 1):121–125

123

Extrarenal Effects of V2R Activation

In addition to effects on water balance, constitutive V2R activation may have other consequences. V2R is also found on endothelial cells, where these receptors mediate vasodilation after the administration of the vasopressin analogue desmopressin [17, 18] and increase circulating levels of von Willebrand factor and tissue plasminogen activator [19]. These responses are absent in some patients with NDI [20]. However, we found no evidence of impaired coagulation in either of the patients with NSIAD [4].

Incidence of NSIAD

Neither the prevalence nor incidence of NSIAD in the general population is known. Inasmuch as 10–20% of patients with SIADH have been reported to have AVP levels that are at or below detectable levels [21], some patients identified as having SIADH may, in fact, have NSIAD because of activating V2R mutations. In addition, other patients may be identified with the NSIAD phenotype but without activating V2R mutations. Such patients may have activating mutations in AQP-2 or other defects in the signaling cascade, resulting in a syndrome of inappropriate antidiuresis.

Treatment of NSIAD

Serum sodium levels and osmolality improved with fluid restriction in the two infants identified with NSIAD; however, as they were exclusively fed with formula, restricting fluid meant limiting calorie intake [4]. In view of potentially limiting toxicities, we did not consider using agents such as demeclocycline or lithium that act

downstream of the V2R. Though AVP antagonists have recently become commercially available for adults and others are currently under clinical development [22], NSIAD’s AVP-independent nature makes their utility uncertain. One possible approach in the absence of an agonist would be to suppress receptor activity using an inverse agonist. In vitro studies of two potential nonpeptide inverse agonists of the V2R, which also behave as competitive V2R antagonists, have been reported [23]. Oral urea is an osmotic agent known to be effective in treating adults with chronic SIADH [24] and appears to be the only treatment currently available for patients with NSIAD. Urea treatment normalized fluid and electrolyte status in the two infants with NSIAD described above [4, 5].

Conclusion

Mutations of GPCRs are associated with a wide variety of physiological disorders. We have recently described two patients with novel missense mutations of the V2R receptor, R137C and R137L, which result in constitutive V2R activation and a syndrome resembling SIADH (but with undetectable levels of AVP), which we have termed nephrogenic syndrome of inappropriate antidiuresis, or NSIAD [4]. Studies to understand the molecular mechanisms by which these mutations result in constitutive V2R activation are currently under way. Characterization of NSIAD through further study should expand our understanding of fluid homeostasis and of clinical disease involving disorders of water balance. In addition, ongoing studies should expand our understanding of GPCR signaling, particularly the role of constitutively active GPCRs in disease, and may lead to development of a targeted treatment for NSIAD.

References 1 Baylis PH: The syndrome of inappropriate antidiuretic hormone secretion. Int J Biochem Cell Biol 2003; 35:1495–1499. 2 Verbalis JG: Disorders of body water homeostasis. Best Pract Res Clin Endocrinol Metab 2003;17:471–503. 3 Schrier RW, Cadnapaphornchai MA: Renal aquaporin water channels: from molecules to human disease. Prog Biophys Mol Biol 2003;81:117–131.

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4 Feldman BJ, Rosenthal SM, Vargas GA, Fenwick RG, Huang EA, Matsuda-Abdini M, Lustig RH, Mathias RS, Portale AA, Miller WL, Gitelman SE: Nephrogenic syndrome of inappropriate antidiuresis. N Engl J Med 2005;352:1884–1890. 5 Huang EA, Feldman BJ, Schwartz ID, Geller DH, Rosenthal SM, Gitelman SE: Oral urea for the treatment of chronic syndrome of inappropriate antidiuresis in children. J Pediatr 2006;148:128–131.

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6 Morello JP, Bichet DG: Nephrogenic diabetes insipidus. Annu Rev Physiol 2001; 63: 607–630. 7 Knoers NVAM: Hyperactive vasopressin receptors and disturbed water homeostasis. N Engl J Med 2005;352:1847–1850. 8 Rosenthal W, Antaramian A, Gilbert S, Birnbaumer M: Nephrogenic diabetes insipidus. A V2 vasopressin receptor unable to stimulate adenylyl cyclase. J Biol Chem 1993; 268: 13030–13033.

Rosenthal /Gitelman /Vargas /Feldman

9 Themmen APN, Huhtaniemi IT: Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev 2000;21:551–583. 10 Shenker A, Laue L, Kosugi S, Merendino JJ Jr, Minegishi T, Cutler GB Jr: A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 1993;365:652–654. 11 Spiegel AM, Weinstein LS: Inherited diseases involving G proteins and G-protein coupled receptors. Annu Rev Med 2004; 55: 27– 39. 12 Tapanainen JS, Aittomaki K, Min J, Vaskivuo T, Huhtaniemi IT: Men homozygous for an inactivating mutation of the follicle stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 1997;15:205–206. 13 Smits G, Olatunbosun O, Delbaere A, Pierson R, Vassart G, Costagliola S: Ovarian hyperstimulation syndrome due to a mutation in the follicle-stimulating hormone receptor. N Engl J Med 2003;349:760–766.

Gain-of-Function Mutations in the V2 Vasopressin Receptor

14 Simoni M, Gromoll J, Nieschlag E: The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology and pathophysiology. Endocr Rev 1997; 18: 739–773. 15 Rodien P, Jordan N, Lefevre A, Royer J, Vasseur C, Savagner F, Bourdelot A, Rohmer V: Abnormal stimulation of the thyrotrophin receptor during gestation. Hum Reprod Update 2004;10:95–105. 16 Hu J, Spiegel AM: Naturally occurring mutations of the extracellular Ca 2+-sensing receptor: implications for its structure and function. Trends Endocrinol Metab 2003;14: 282–288. 17 Hirsch AT, Dzau VJ, Majzoub JA, Creager MA: Vasopressin-mediated forearm vasodilation in normal humans: evidence for a vascular vasopressin V2 receptor. J Clin Invest 1989;84:418–426. 18 Kaufmann JE, Iezzi M, Vischer UM: Desmopressin (DDAVP) induces NO production in human endothelial cells via V2 receptors and cAMP-mediated signaling. J Thromb Haemost 2003;1:821–828. 19 Mannucci PM, Ruggeri AM, Pareti FI, Capitanio A: 1-Deamino-8-d-arginine vasopressin: a new pharmacological approach to the management of haemophilia and von Willebrands’ diseases. Lancet 1977;1:869–872.

20 Bichet DG, Razi M, Lonergan M, Arthus MF, Papukna V, Kortas C, Barjon JN: Hemodynamic and coagulation responses to 1-desamino[8-D-arginine] vasopressin in patients with congenital nephrogenic diabetes insipidus. N Engl J Med 1988;318:881–887. 21 Zerbe R, Stropes L, Robertson G: Vasopressin function in the syndrome of inappropriate antidiuresis. Annu Rev Med 1980; 31: 315–327. 22 Greenberg A, Verbalis JG: Vasopressin receptor antagonists. Kidney Int 2006; 69: 2124–2130. 23 Morin D, Cotte N, Balestre MN, Mouillac B, Manning M, Breton C, Barberis C: The D136A mutation of the V2 vasopressin receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities. FEBS Lett 1998;441:470–475. 24 Decaux G, Prospert F, Penninckx R, Namias B, Soupart A: Five-year treatment of the chronic syndrome of inappropriate secretion of ADH with oral urea. Nephron 1993; 63: 468–470.

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HORMONE RESEARCH

Horm Res 2007;67(suppl 1):126–127 DOI: 10.1159/000097568

Published online: February 15, 2007

Clinical Practice in Adult Growth Hormone Deficiency: Learnings from KIMS

KIMS (Pfizer International Metabolic Database) has been collecting data on adult patients with growth hormone (GH) deficiency since 1994. Currently, it includes data for approximately 12,000 patients. KIMS enables investigators to monitor the long-term safety and outcome of GH therapy. Further, KIMS has evolved into a valuable resource that facilitates international collaboration and enables evidence-based medicine beyond individual clinical trials. KIMS will certainly continue to serve as a powerful tool for investigators in the future. In this plenary session, four renowned experts discussed the clinical impact already manifest as a result of KIMS. Stephen M. Shalet of Christie Hospital in Manchester, UK, and Vera Popovic of the Institute of Endocrinology, University Clinical Center in Belgrade, Serbia,

conducted an interactive session on the reality of GH deficiency for patients in their mature years. John P. Monson of the London Clinic Centre for Endocrinology in London, UK, discussed improvements in lipoprotein profile observed for hypopituitary patients on maintenance therapy with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Finally, Rolf C. Gaillard of University Hospital in Lausanne, Switzerland, presented findings from KIMS showing that GHdeficient patients treated with GH have a mortality rate similar to that of the general population. Hugo Fideleff Endocrinology Unit, Department of Endocrinology Hospital T Alvarez, Buenos Aires, Argentina

Selected Bibliography Abs R, Bengtsson BA, Hernberg-Stahl EH, Monson JP, Tauber JP, Wilton P, Wuester C: GH replacement in 1034 growth hormone deficient hypopituitary adults: demographic and clinical characteristics, dosing and safety. Clin Endocrinol 1999;50:703–714. Abs R, Feldt-Rasmussen U, Mattsson AF, Monson JP, Bengtsson B-A, Góth MI, Wilton P, Koltowska-Häggström M: Determinants of cardiovascular risk in 2589 hypopituitary GH-deficient adults – a KIMS database analysis. Eur J Endocrinol 2006;155:79–90.

Bates AS, Van’t Hoff W, Jones PJ, Clayton RN: The effect of hypopituitarism on life expectancy. J Clin Endocrinol Metab 1996; 81: 1169–1172. Bates AS, Bullivant B, Sheppard MC, Steward P: Life expectancy following surgery for pituitary tumours. Clin Endocrinol 1999; 50: 315–319.

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0126$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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Brabant G, von zur Muhlen A, Wuster C, Ranke MB, Kratzsch J, Kiess W, Ketelslegers JM, Wilhelmsen L, Hulthen L, Saller B, Mattsson A, Wilde J, Schemer R, Kann P, German KIMS Board: Serum insulin-like growth factor I reference values for an automated chemiluminescence immunoassay system: results from a multicenter study. Horm Res 2003;60:53–60. Bülow B, Hagmar L, Mikoczy Z, Nordstrom CH, Erfurth EM: Increased cerebrovascular mortality in patients with hypopituitarism. Clin Endocrinol 1997;46:75–81.

Hugo Fideleff Department of Endocrinology Endocrinology Unit, Hospital T Alvarez Buenos Aires, Argentina Tel. +54 11 4501 3066, Fax +54 11 4854 5601, E-Mail [email protected]

Florakis D, Hung V, Kaltsas G, Coyte D, Jenkins PJ, Chew SL, Grossman AB, Besser GM, Monson JP: Sustained reduction in circulating cholesterol in adult hypopituitary patients given low dose titrated growth hormone replacement therapy: a two year study. Clin Endocrinol 2000;53:453–459. Gaillard RC, Bengtsson BA, Casanueva F, FeldtRasmussen U, Fideleff H, Mattsson A, Monson JP, Popovic V, Vance ML, Wilton P: GH replacement therapy in adult GH deficiency is associated with normalized mortality. Experience from KIMS. The Endocrine Society 87th Annual Meeting, 4–7 June 2005, San Diego, USA. Abstract book: P2–543 p 468. Aetiology of growth hormone deficiency (KIMS classification list) in Abs R, Feldt-Rasmussen U (eds): Growth Hormone Deficiency in Adults: 10 Years of KIMS. Oxford PharmaGenesis, 2004, Appendix D, p 346. Life Tables for 191 countries for 2000:WHO October 2001. Lissett CA, Jönsson P, Monson JP, Shalet SM: Determinants of IGF-I status in a large cohort of growth hormone-deficient (GHD) subjects: the role of timing of onset of GHD. Clin Endocrinol (Oxf) 2003;59:773–778.

Clinical Practice in Adult Growth Hormone Deficiency

Micic D, Popovic V, Doknic M, Macut D, Dieguez C, Casanueva FF: Preserved growth hormone (GH) secretion in aged and very old subjects after testing with the combined stimulus GH-releasing hormone plus GHreleasing hexapeptide-6. J Clin Endocrinol Metab 1998;83:2569–2572. Monson JP, Abs R, Bengtsson BA, Bennmarker H, Feldt-Rasmussen U, Hernberg-Stahl E, Thoren M, Westberg B, Wilton P, Wuster C: Growth hormone deficiency and replacement in elderly hypopituitary adults. KIMS Study Group and the KIMS International Board. Pharmacia and Upjohn International Metabolic Database. Clin Endocrinol (Oxf) 2000;53:281–289. Monson JP, Jönsson P: Aspects of growth hormone (GH) replacement in elderly patients with GH deficiency: data from KIMS. Horm Res 2003;55(suppl 1):112–120. Mukherjee A, Monson JP, Jönsson PJ, Trainer PJ, Shalet SM on Behalf of the KIMS International Board: Seeking the optimal target range for insulin-like growth factor I during the treatment of adult growth hormone disorders [Comment]. J Clin Endocrinol Metabol 2003;88:5865–5870.

Murray RD, Adams JE, Shalet SM: A densitometric and morphometric analysis of the skeleton in adults with varying degrees of growth hormone deficiency. J Clin Endocrinol Metabol 2006;91:432–438. Popovic V, Pekic S, Doknic M, Micic D, Damjanovic S, Zarkovic M, Aimaretti G, Corneli G, Ghigo E, Dieguez C, Casanueva FF: The effectiveness of arginine + GHRH test compared with GHRH + GHRP-6 test in diagnosing growth hormone deficiency in adults. Clin Endocrinol 2003;59:251–257. Rosen T, Bengtsson B-Å: Premature mortality due to cardiovascular disease in hypopituitarism. Lancet 1990;336:285–288. Svensson J, Bengtsson B-A, Rosen T, Oden A, Johannsson G: Malignant disease and cardiovascular morbidity in hypopituitary adults with or without growth hormone replacement therapy. J Clin Endocrinol Metab 2004; 89:3306–3312. Tomlinson JW, Stewart PM: Cortisol metabolism and the role of 11-hydroxysteroid dehydrogenase. Best Pract Res Clin Endocrinol Metab 2001;15:61–78. Toogood AA, O’Neill PA, Shalet SM: Beyond the somatopause: growth hormone deficiency in adults over the age of 60 years. J Clin Endocrinol Metab 1996;81:460–465.

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Adult Workshop 1

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):128–131 DOI: 10.1159/000097569

Published online: February 15, 2007

T4 versus T3 and T4: Is It a Real Controversy? Anthony P. Weetman The School of Medicine, University of Sheffield, Sheffield, UK

Key Words Thyroid hormone replacement Tri-iodothyroxine T4

Abstract Background: Although thyroid hormone replacement therapy has been a mainstay of endocrinologic therapy, many patients feel unwell despite apparently optimal treatment. Methods and Results: Recently, attempts have been made to improve treatment by administering tri-iodothyroxine (T3) in addition to thyroxine. Initial results of a crossover study suggested that combination T3 and T4 therapy was associated with improvements in several measures of quality of life and increased patient preference. Results of subsequent studies have not supported these initially promising findings. Conclusions: At present, combination treatment cannot be recommended. Further clinical studies that are adequately powered and comprised of homogenous patient populations are needed to determine whether any benefits are associated with sustained-release T3 preparations. Copyright © 2007 S. Karger AG, Basel

Introduction

Treatment with thyroid hormone replacement has been one of the most impressive endocrinologic therapies since its introduction by George Murray in Newcastle-

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upon-Tyne, England, in 1891. At first glance it is difficult to understand how treatment could be improved upon, given what we now know about thyroid physiology and the ready availability of both accurate measurement of thyroid-stimulating hormone (TSH) levels and purified thyroxine. Yet the fact remains that many patients feel unwell despite apparently optimal treatment. This has led to recent attempts to improve treatment by administering tri-iodothyroxine (T3) in addition to thyroxine, which in turn has created a dilemma for endocrinologists who have increasing numbers of patients demanding such replacement therapy. This development can be set against the broader conundrum of patients who have symptoms of thyroid underactivity and who request treatment despite normal TSH levels. Before considering T3 replacement, it is worth briefly reviewing the main principles of hypothyroidism treatment.

Treatment of Hypothyroidism

Thyroxine replacement is best achieved using synthetic levothyroxine sodium, which is well absorbed and has a half-life of 7 days. The optimal dose for fully hypothyroid individuals is 1.6 g/kg/day, and the goal of treatment is to achieve a TSH level in the lower half of the reference range (around 1 mU/l) [1]. Several factors may alter thyroxine dosage requirements (table 1), but provided the effect of each of these is constant, the dosage of

Anthony P. Weetman, MD School of Medicine and Biomedical Science University of Sheffield, Beech Hill Road Sheffield S10 2RX (UK) Tel. +44 114 271 2570, +44 114 271 3960, E-Mail [email protected]

thyroxine can usually be adjusted to compensate and maintain a low-normal TSH level. Some patients express concern that physicians rely too heavily on TSH levels and do not listen to their concerns and symptoms. This frustration is compounded by the recent debate over whether the reference range for TSH levels is too broad, and the knowledge that, within an individual, TSH levels may vary due to stress and diurnal variation [2, 3]. Moreover, genetic factors play a substantial role in controlling TSH levels, indicating that individualisation of TSH levels for patients taking thyroxine could be more important than previously believed [4]. Results of a recent survey showed that even when TSH levels are within the reference range, 26% more patients reported a feeling of impaired health, as assessed by the General Health Questionnaire, compared with control subjects [5]. There are several possible explanations for this. Firstly, some patients may require treatment with T3 as well as thyroxine. It is noteworthy that in this population, the TSH level was regarded as normal even if it was just below the upper limit of the reference range, rather than around 1.0 mU/l. In addition, long-term drug therapy and concomitant diseases were more prevalent among patients in this group, who therefore may have been selected because of a more general feeling of ill health unrelated to hypothyroidism.

Combined T4 and T3: Physiology

Table 1. Factors causing alteration of TSH levels in patients taking thyroxine

Factors causing alteration of TSH levels Poor adherence to treatment Formulation errors Progressive thyroid destruction Impaired thyroxine absorption Coeliac disease and other malabsorption syndromes Concurrent use of drugs (ferrous sulphate, calcium carbonate, colestyramine, sucralfate, aluminium hydroxide) Increased thyroxine clearance Pregnancy Nephrotic syndrome Concurrent use of drugs (phenytoin, carbamazepine, phenobarbitone, rifampicin, hormone replacement treatment) Impaired gastric acid production

A further consideration is the amount of T3 that is required to mimic the normal situation in a hypothyroid patient. Based on animal and human studies, a molar ratio of 14:1 T4:T3, delivering around 100 g T4 and 6 g T3 per day, appears optimal [7]. However, liothyronine tablets are 20 g in size, making any approach to normal physiology all but impossible with standard-sized tablets, especially in those who still have some degree of residual thyroid function.

Around 80% of circulating T3 arises from the peripheral tissues by deiodination of T4 ; only 20% or so is directly secreted by the thyroid gland. Thyroxine treatment in hypothyroid individuals is predicated on the assumption that this ‘missing’ 20% of T3 can be compensated for by increased peripheral deiodination. Since the deiodinase enzymes have both polymorphisms and variable tissue distribution, theoretically it is possible that some tissues could be underexposed to T3 despite apparently normal TSH levels in patients taking thyroxine. In other words, a physiologically perfect resetting of the pituitary by thyroxine replacement may not reflect such an optimal situation in other tissues. However, T3 administered as a liothyronine tablet does not reflect physiologically relevant replacement either. Its half-life is around 1 day, and administration results in undesirable, nonphysiologic peaks of serum T3 levels. Recently, slow-release formulations have been developed that can provide sustained levels of T3, but so far they have not been used extensively enough to reliably assess any benefit [6].

Early studies using T3 and T4 combinations at rather unphysiologic ratios have been reviewed elsewhere [7]. Recent interest in the field stems from a 5-week crossover study by Bunevicius et al. of a relatively small number of patients (n = 33) in whom 12.5 g T3 was substituted for 50 g of their conventional T4 dose [8]. Several measures of quality of life improved, and patients preferred the combination therapy to treatment with T4 alone. However, no benefit was observed in a subsequent study by the same group involving 10 patients treated surgically for Graves’ disease [9]. The 1999 Bunevicius study has generated considerable interest on the part of both physicians and patients. Seven subsequent studies have found no benefit from combined T3 and T4 therapy (table 2) [10–16]. These studies have generally assessed mood, hypothyroid symptoms and

T4 versus T3 and T4

Horm Res 2007;67(suppl 1):128–131

Combined T3 and T4 : Therapeutic Trials

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Table 2. Summary of trials of T3 and T4 combination treatment

Study

T3 treatment g/day

Patients

Duration weeks

Outcome (benefit/preference)

Walsh et al. [10] Sawka et al. [11] Clyde et al. [12] Saravanan et al. [13] Siegmund et al. [14] Escobar-Morreale et al. [15] Appelhof et al. [16]

T4 f 50+T310 T4 f 50%+T350 T4 f 50+T315 T4 f 50+T310 16:1 T4:T3 15:1 or 10:1 T4:T3 10:1 or 5:1 T4:T3

102 39 44 573 23 26 130

10 15 16 52 12 8 15

No/T4 = T3+T4 No/not assessed No/not assessed No/not assessed No/not assessed No/T3+T4>T4 No/T3+T4>T4

quality of life. Two studies showed a persistent, significant placebo effect that followed hormone replacement therapy [11, 13]. In two other studies, however, patients expressed a preference for the combination of T3 and T4 [15, 16], and in a third study patients reported that the effect of the T3 and T4 combination was the same as that of T4 alone. The other studies did not assess patient preference. A patient was removed from one trial after developing atrial fibrillation associated with a suppressed TSH level [14]. This event provides a cautionary note to temper the enthusiasm of some.

Conclusion

Despite initially promising results, larger and more prolonged trials have not supported the initial findings of a beneficial effect of T3 and T4 combination treatment in

patients with hypothyroidism [15]. Although in some trials patients seemed to prefer combination treatment, there was no overall objective benefit. In addition, there may be risks such as atrial fibrillation associated with sustained or fluctuating T3 levels. At present, combination treatment cannot be recommended. Future work is needed to determine whether any benefits may be associated with sustained-release T3 preparations. Such trials need to be conducted using appropriate patient populations to ensure homogeneity of the underlying thyroid condition, adequate statistical power, and appropriate randomisation. Moreover, better markers of T3 overdosage are required beyond the single TSH measurements that have been used in studies so far – bone marker and other physiologic measurements that might test thyroid hormone overdosage should be incorporated in all future studies.

References 1 Roberts CGP, Ladenson PW: Hypothyroidism. Lancet 2004;363:793–803. 2 Surks MI, Goswami G, Daniels GH: The thyrotropin reference range should remain unchanged. J Clin Endocrinol Metab 2005; 90: 5489–5496. 3 Wartofsky L, Dickey RA: The evidence for a narrower thyrotropin reference range is compelling. J Clin Endocrinol Metab 2005; 90:5483–5488. 4 Hansen PS, Brix TH, Sorensen TIA, Kyvic KO, Hegedus L: Major genetic influence on the regulation of the pituitary-thyroid axis: a study of healthy Danish twins. J Clin Endocrinol Metab 2004;89:1181–1187.

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5 Saravanan P, Chau WF, Roberts N, Vedhara K, Greenwood R, Dayan CM: Psychological well-being in patients on ‘adequate’ doses of L-thyroxine: results of a large, controlled community-based questionnaire study. Clin Endocrinol 2002;57:577–585. 6 Hennemann G, Docter R, Visser TJ, Postema PT, Krenning EP: Thyroxine plus low-dose, slow-release triiodothyronine replacement in hypothyroidism: proof of principle. Thyroid 2004; 14:271–275.

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7 Scobar-Morreale HF, Botella-Carretero JI, Escobar del Rey F, Morreale de Escobar G: Treatment of hypothyroidism with combinations of levothyroxine plus liothyronine. J Clin Endocrinol Metab 2005; 90: 4946– 4954. 8 Bunevicius R, Kazanavicius G, Zalinkevicius R, Prange AJ Jr: Effects of thyroxine as compared with thyroxine plus triiodothyronine in patients with hypothyroidism. N Engl J Med 1999;340:424–429. 9 Bunevicius R, Jakubonien N, Jurkevicius R, Cernicat T, Lasas L, Prange AJ Jr: Thyroxine vs thyroxine plus triiodothyronine in treatment of hypothyroidism after thyroidectomy for Graves’ disease. Endocrine 2002; 18: 129–133.

Weetman

10 Walsh JP, Shiels L, Lim EM, Bhagat CI, Ward LC, Stuckey BGA, Dhaliwal SS, Chew GT, Bhagat MC, Cussons AJ: Combined thyroxine/liothyronine treatment does not improve well-being, quality of life, or cognitive function compared to thyroxine alone: a randomized controlled trial in patients with primary hypothyroidism. J Clin Endocrinol Metab 2003;88:4543–4550. 11 Sawka AM, Gerstein HC, Marriot MJ, MacQueen GM, Joffe RT: Does a combination regimen of thyroxine (T4) and 3,5,3-triiodothyronine improve depressive symptoms better than T4 alone in patients with hypothyroidism? Results of a double-blind, randomized, controlled trial. J Clin Endocrinol Metab 2003;88:4551–4555.

T4 versus T3 and T4

12 Clyde PW, Harari AE, Getka EJ, Shakir KM: Combined levothyroxine plus liothyronine compared with levothyroxine alone in primary hypothyroidism: a randomized controlled trial. JAMA 2003;290:2952–2958. 13 Saravanan P, Simmons DJ, Greenwood R, Peters TJ, Dayan CM: Partial substitution of thyroxine (T4) with tri-iodothyronine in patients on T4 replacement therapy: results of a large community-based randomized controlled trial. J Clin Endocrinol Metab 2005; 90:805–812. 14 Siegmund W, Spieker K, Weike AI, Giessmann T, Modess C, Dabers T, Krisch G, Sanger E, Engel G, Hamm AO, Nauck M, Meng W: Replacement therapy with levothyroxine plus triiodothyronine (bioavailable molar ratio 14:1) is not superior to thyroxine alone to improve well-being and cognitive performance in hypothyroidism. Clin Endocrinol (Oxf) 2004;60:750–757.

15 Escobar-Morreale HF, Botella-Carretero JL, Gomez-Bueno M, Galan JM, Barrios V, Sancho J: Combined therapy with levo-thyroxine and liothyronine in two ratios, compared with levothyroxine mono-therapy in primary hypothyroidism: a randomized trial comparing L-thyroxine plus liothyronine with L-thyroxine alone. Ann Intern Med 2005; 142:412–424. 16 Appelhof BC, Fliers E, Wekking EM, Schene AH, Huyser J, Tijssen JG, Endert E, van Weert HC, Wiersinga WM: Combined therapy with levothyroxine and liothyronine in two ratios, compared with levothyroxine monotherapy in primary hypothyroidism: a double-blind, randomized, controlled clinical trial. J Clin Endocrinol Metab 2005; 90: 2666–2674.

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Adult Workshop 1

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):132–142 DOI: 10.1159/000097570

Published online: February 15, 2007

Recombinant Human Thyroid-Stimulating Hormone: Use in Papillary and Follicular Thyroid Cancer Martin Schlumberger a Isabelle Borget a Gérard De Pouvourville a Furio Pacini b a

Institut Gustave Roussy, Villejuif Cédex, France; b University of Siena, Siena, Italy

Key Words Recombinant human thyroid stimulating hormone Thyroid cancer

Abstract Recombinant human thyroid-stimulating hormone (rhTSH) is used in thyroid cancer patients to prepare for postoperative administration of 3.7 GBq of radioiodine (131I) and to facilitate the determination of serum thyroglobulin during follow-up. Recombinant human TSH is also used during thyroxine treatment to avoid the hypothyroid state induced by prolonged thyroid hormone withdrawal and thereby maintains a patient’s quality of life. Copyright © 2007 S. Karger AG, Basel

Introduction

Papillary and follicular thyroid carcinomas and their metastases retain two important features of normal thyroid tissue that are useful in the postsurgical follow-up and treatment regimens of persistent and recurrent disease: production of thyroglobulin (Tg) and radioiodine uptake. In the absence of thyroid-stimulating hormone (TSH) stimulation, radioiodine uptake is absent and serum Tg is undetectable in 15% of patients with distant metastases and 20% of patients with isolated lymph node

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0132$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

metastases. Since multiple metabolic defects are present in thyroid cancer tissue, both Tg production and radioiodine uptake require intensive TSH stimulation to be effective. In the past, this was typically achieved by withdrawing L-thyroxine therapy for 3 to 5 weeks, during which time high serum TSH (130 mU/l) stimulated iodine uptake and Tg secretion [1–3]. However, this withdrawal induces a transient state of hypothyroidism, which may impact the patient’s quality of life (QoL) and lead to morbidity [4–6]. To overcome the impact of this withdrawal, several procedures have been used, such as substituting the more rapidly metabolized triiodothyronine (L-T3) for thyroxine for 3 weeks and then withdrawing it for 2 weeks. This procedure reduces, but does not avoid, signs and symptoms of hypothyroidism. Other investigators have simply reduced the L-thyroxine dose by 33 to 50% [7] or have used a shorter withdrawal period [8], but the effectiveness of these procedures as compared with prolonged withdrawal has not been well documented. In addition, once L-thyroxine treatment is resumed, it sometimes takes up to 10 weeks for serum TSH to normalize, and this prolongs the impact of hypothyroidism. This period can be shortened by administering L-T3 for a few days when thyroid hormone treatment is resumed [9]. In the past, exogenous administration of bovine TSH (bTSH) (10 U for 3 consecutive days) was employed in thyroid cancer patients to stimulate radioiodine uptake.

Martin Schlumberger, MD Institut Gustave Roussy et Université Rue Camille Desmoulins FR–94805 Villejuif Cedex (France) Tel. +33 1 42 11 60 95, Fax +33 1 42 11 63 63, E-Mail [email protected]

This supplanted the need for withdrawing thyroid hormone therapy [10]. However, bTSH was less effective than endogenous TSH and was associated with adverse reactions, including urticaria and anaphylactic shock, and it induced TSH antibodies. The potential use of human TSH (hTSH) purified from pooled pituitary glands obtained at autopsy is similarly unsuitable, due to the possible transmission of Creutzfeldt-Jakob disease. The availability of rhTSH may represent a major advance in this treatment. In this review, we will discuss the benefits and efficacy, both diagnostic and therapeutic, of using rhTSH in thyroid cancer patients and avoiding the threat of hypothyroidism.

Production of rhTSH

TSH is a pituitary heterodimeric glycoprotein composed of an -subunit common to gonadotropins and a hormone-specific -subunit. Once the -subunit of the human TSH gene was cloned, the encoded protein could be overexpressed in a cell system by transfection with the human - and -subunits of complementary or genomic DNA [11–13]. With this technique, large quantities of highly purified rhTSH can be obtained. In vitro studies have shown that rhTSH can effectively stimulate cyclic adenosine monophosphate production in the thyroid cell line (FRTL-5 cells) [14] and promote the growth of human fetal thyroid cells [15]. The biological efficacy of rhTSH has been demonstrated in monkeys, where it increased serum T4 and T3 concentrations and thyroidal radioiodine uptake [16]. In human volunteers, a single dose of 0.1 mg rhTSH was a potent stimulator of thyroid function. The serum Tg response to rhTSH occurred later than the serum T3 and T4 response, with a peak serum level 2 to 3 days after rhTSH injection [17]. Thyroid uptake increased when radioiodine was given 24 h after rhTSH administration with smaller increases observed at 48 h and none at 72 h [18]. Finally, a single intramuscular (IM) dose of 0.9 mg increases thyroid uptake by approximately 100% [19]. Early studies indicated that IM injections of 0.9 mg of rhTSH on 2 consecutive days was highly efficient in promoting 131 I uptake in thyroid cancer patients without significant toxicity [20]. Pharmacokinetic studies showed that the mean peak serum TSH level was achieved within 4 to 6 h after injection. Following the IM injection of 0.9 mg of rhTSH, the mean peak serum level was 116 8 38 mU/l; serum half-life was 22.3 8 8.5 h and serum TSH remained above 30 mU/l for approximately 2 days [20]. Recombinant Human Thyroid-Stimulating Hormone

The extent of TSH stimulation depends on both serum TSH levels since rhTSH administration and the duration of elevated serum TSH levels and is best expressed by the area under the curve (AUC) of TSH level over time. In this context, the serum TSH level at the time of 131I administration or of serum Tg determination does not reflect the magnitude of previous TSH stimulation. Both peak serum TSH level and AUC were inversely correlated with lean body mass [21]. In children and teenagers, serum TSH levels achieved after rhTSH injections are similar to values reported in adults, and, therefore, dose adjustments are not generally required [22]. Recombinant human TSH was approved for human use in Europe in 2000, upon completion of two phase III pivotal studies, which studied the effects of thyrotropin for injection (Genzyme Therapeutics, Cambridge, Mass., USA) on thyroid cancer patients [23, 24]. Tolerance The use of rhTSH in thyroid cancer patients avoids the consequences of prolonged thyroid hormone withdrawal, including the usual signs or symptoms of hypothyroidism and its consequences on organ function, in particular on the brain, heart, liver and kidneys. It also avoids the worsening of any associated disease, as well as curtails the hazards of any drug therapy necessitated by decreased renal or hepatic function. Therefore, use of rhTSH avoids the morbidity that can be associated with iatrogenic hypothyroidism. The use of rhTSH also reduces the moderate-to-severe impairment of the ability to work and to operate a motor vehicle [5]. These consequences are significant because patients are typically young or middle-aged. As a result of rhTSH administration, QoL is much better than during a period of hypothyroidism. In addition, a small minority of patients may not be able to generate a sufficient response to endogenous TSH after thyroid hormone withdrawal due to older age, long-term use of suppressive therapy, a concomitant illness or pituitary insufficiency that prevents adequate uptake of 131I in metastatic foci. Paterakis et al. observed side effects in less than 10% of patients, and these consisted mainly of mild and transient fatigue, nausea and headache, and no patient developed detectable anti-rhTSH antibodies [25]. Moreover, knowing that rhTSH is available, patients strongly request treatment with this modality to avoid prolonged episodes of hypothyroidism. Finally, the use of rhTSH improves compliance with thyroid cancer monitoring [26].

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Protocol for rhTSH Use The optimal protocol for using rhTSH in thyroid cancer patients is as follows: – The dose of rhTSH is a once-daily IM injection of 0.9 mg for 2 consecutive days. – An 131I activity of 148 MBq (4 mCi) is administered for diagnostic purposes on the day following the second injection of rhTSH. – A whole body scan (WBS) is performed 48 h after radioiodine administration using standardized procedures, including appropriate equipment (a large field of view gamma camera equipped with thick crystals and high-energy collimators); the scan lasts for a minimum of 30 min or at least 140,000 counts. For spot images, scanning lasts for a minimum of 10 min or for at least 60,000 counts. – Serum Tg is determined 3 days after the second injection of rhTSH. Sera containing interferences should be excluded from the analysis, and a sensitive immunometric assay (IMA) method should be used to measure serum Tg. – Serum TSH may be measured after rhTSH injections, but only to ensure that rhTSH has been injected correctly. – For treatment, an activity of 1.1 to 3.7 GBq is administered on the day following the second injection of rhTSH, and scanning is performed 2 to 5 days later. During this procedure, thyroxine treatment is maintained. In relation to the iodine content of L-thyroxine, urinary iodine excretion is slightly increased compared with the urinary iodine excretion that accompanies hypothyroidism, but it is always far below 200 g/l [27, 28], a level generally considered to indicate an absence of iodine excess. This increase probably does not affect the iodine uptake after the intensive stimulation obtained with rhTSH. Diagnostic Use of rhTSH The availability of large quantities of rhTSH prompted researchers to conduct clinical trials in patients with papillary and follicular thyroid cancer to investigate the safety and efficacy of rhTSH in promoting radioiodine uptake and Tg secretion. Several facts have been well documented. rhTSH Stimulates 131I Uptake and Tg Secretion Initial studies demonstrated the safety and the efficacy of rhTSH in promoting 131I uptake [20, 24]. Then, a multicenter phase III trial compared two different regimens of rhTSH given at a fixed dose of 0.9 mg (once-dai134

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ly injection for 2 consecutive days versus 3 injections, 3 days apart) and a fixed activity of radioiodine (148 MBq, 4 mCi), and analyzed serum Tg as a marker of disease [23]. Serum Tg testing results and 131I-WBSs obtained after rhTSH and thyroid hormone withdrawal were compared for 49 of 220 patients with persistent or recurrent disease: 80% had concordant scans, 4% had superior rhTSH scans and 16% had superior withdrawal scans. Serum Tg was detectable (62 ng/ml) in only 80% of patients during L-thyroxine therapy and was detectable in 100% following both rhTSH therapy and withdrawal. The serum Tg level attained following rhTSH stimulation was usually lower than that following withdrawal, which underlines why a sensitive IMA method should be used and why any detectable serum Tg level – even low levels – should be taken into account. Furthermore, radioiodine uptake was lower with rhTSH administration compared with withdrawal. This was attributed to a shorter duration of stimulation and to decreased bioavailability of radioiodine after rhTSH (the euthyroid status) administration than following withdrawal (a hypothyroid state). In fact, hypothyroidism decreases renal clearance of iodine and increases its retention in the body, thus increasing its bioavailability for thyroid cells and the body radiation dose. Also, there have been anecdotal reports of patients with negative WBSs following rhTSH administration and positive WBSs following withdrawal [29, 30]. These findings underscore the need for 131I diagnostic activity level of not less than 148 MBq and for scanning 2 days later using standardized procedures. Data generated from the combined use of serum Tg and 131I-WBS are more informative than data from 131IWBS alone. More importantly, rhTSH-stimulated Tg successfully detected all patients with abnormal 131I-WBSs. A retrospective analysis of 289 thyroid cancer patients, including 139 patients with metastatic disease, confirmed that the diagnostic accuracy of 131I-WBS and/or Tg was similar among patients who were prepared by thyroid hormone withdrawal and those who received rhTSH [31]. Serum Tg Is a Highly Sensitive Tool for Detecting Persistent or Recurrent Disease Serum Tg obtained after withdrawal of thyroid hormone treatment is a highly sensitive tool for detecting persistent or recurrent disease [32]. Ablation success was defined as no or !0.1% visible thyroid bed uptake, with success verified 6–12 months after the procedure using an 131I-WBS performed with 74–185 MBq (2–5 mCi). Low 131 I thyroid bed uptake has no prognostic significance Schlumberger /Borget /De Pouvourville / Pacini

when neck ultrasonography does not show any abnormality, and a control diagnostic 131I-WBS does not add significant information in most patients when an informative postablation WBS performed with a large activity of 131I has shown the absence of uptake outside the thyroid bed [33, 34]. Several studies performed after rhTSH administration confirmed these findings and clearly demonstrated that Tg determination should be used to screen for persistent/recurrent disease [35–40]. Thus, in these patients and in the absence of anti-Tg antibodies, a diagnostic control 131I-WBS can be omitted and the current criterion for successful ablation at 9–12 months is an undetectable (!1 ng/ml) serum Tg following rhTSH stimulation [32–35]. Neck Ultrasonography Is the Most Sensitive Tool for Detecting Lymph Node Metastases Rare false-negative rhTSH-stimulated Tg measurements are observed due to isolated, small lymph-node metastases in the neck that could be detected by ultrasonography [41, 42]. A detectable serum Tg level on L-thyroxine, or its conversion from undetectable to detectable after rhTSH stimulation and/or suspicious findings on ultrasound, allows the identification of patients who require therapeutic procedures without the need for a routine diagnostic 131I-WBS [43–45]. Thus, neck ultrasonography and serum Tg determination performed 3 days after the second rhTSH injection are advised as first-line tests. The accuracy of neck ultrasonography depends on the skill of the ultrasound operator and this requires specialized training. Patients Who Have Been Treated with 131I Can Have False-Negative Serum Tg Measurements In patients who have already been treated with 131I and who have a small amount of residual disease, serum Tg may remain undetectable but 131I uptake may still be present [46, 47], and small neoplastic foci have been found at surgery [47]. In these patients, persistent or recurrent disease has already been diagnosed, and the problem is determining the optimal treatment of these known neoplastic foci. Serum Tg Becomes Detectable following rhTSH in 10–20% of Patients Who Had No Other Evidence of Disease During the first evaluation performed 6–12 months after thyroid ablation, serum Tg following rhTSH stimulation is detectable in 10–20% of patients with no other evidence of disease. In these patients, serum Tg was evalRecombinant Human Thyroid-Stimulating Hormone

uated again after rhTSH a few months later [32, 33, 48– 50]. In about two-thirds of such cases, serum Tg decreased or became undetectable in the absence of any further treatment, and these patients did not relapse. In the other third, serum Tg level remained stable or increased, and a large proportion of these patients experienced a recurrence. Thus, the trend in rhTSH-stimulated Tg level has a better predictive value than a single serum Tg level. During long-term follow-up, serum Tg observed following rhTSH stimulation is predictive of outcome; recurrences are rare when Tg is !2 ng/ml but frequent when it is 12 ng/ml [51, 52]. In patients with a serum Tg level above an arbitrary threshold (perhaps 15 to 10 ng/ml) or with an increasing serum Tg trend at consecutive determinations, one possibility is to administer a high activity of 131I after thyroid hormone withdrawal and to perform a WBS 3 to 7 days later [53]. rhTSH May Improve the Sensitivity of Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET) Scanning FDG-PET scanning is used in selected thyroid cancer patients to detect neoplastic foci when serum Tg is elevated and other imaging results are negative. In some patients, TSH stimulation obtained following thyroid hormone withdrawal or rhTSH injections increased FDG uptake by already known foci and by previously undetected neoplastic foci, thereby facilitating their discovery [54– 57]. Conclusion Based on these considerations, it appears that 131I-WBS is unnecessary for follow-up in the majority of patients with no evidence of disease (e.g., after complete surgical resection, an informative posttherapy WBS demonstrating the absence of uptake foci outside the thyroid bed), thereby reducing the risks of exposure to 131I and the cost of the follow-up protocol. According to recent consensus reports, follow-up for patients who have undergone 131I ablation may include measurement of rhTSH-stimulated serum Tg and neck ultrasonography 6 to 12 months after initial treatment [58, 59]. If neck ultrasonography does not detect any abnormality and serum Tg remains undetectable following rhTSH, the patient can be considered cured, as the risk of recurrence is ! 0.5% during the subsequent decade [33, 34]. In such cases, the thyroxine dose is decreased to achieve a serum TSH level within the normal range [60], and these patients can be followed up annually on L-thyroxine with serum TSH and Tg determinations and neck ultrasonography. For patients with deHorm Res 2007;67(suppl 1):132–142

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tectable serum Tg levels following rhTSH and no other evidence of disease, another rhTSH-stimulated Tg may be obtained some months or years later; when serum Tg becomes undetectable, the patient is considered cured. Imaging with 131I-TBS is indicated only in patients with detectable basal or stimulated Tg levels (above a cut-off value to be determined at each institution) and when it increases with time, preferably using a larger radioiodine activity [53]. Indeed, when in doubt or even as part of routine practice, rhTSH-stimulated Tg may be obtained 2 to 5 years later to ensure complete remission, but more data are needed to validate this practice. The advantages of this protocol are obvious for subsequent follow-up: it reassures patients, avoids overtreatment with L-thyroxine and simplifies long-term followup. Indeed, while false-negative serum Tg determinations following rhTSH and neck ultrasonography cannot be excluded, they are probably rare and may be discovered only after prolonged follow-up [61]. In the future, supersensitive serum Tg determinations may detect most persistent/recurrent disease during L-thyroxine suppressive therapy, but at the present time their improved sensitivity is obtained at the expense of a profoundly decreased specificity, and for this reason their use cannot replace Tg determination obtained following rhTSH stimulation. Finally, very-low-risk patients who do not undergo ablation are followed up on L-thyroxine treatment with serum TSH and Tg determinations and neck ultrasonography. There is no need for these patients to routinely receive rhTSH stimulation.

Therapeutic Use of rhTSH

Thyroid Remnant Ablation Another scenario in which rhTSH is a promising alternative to prolonged thyroid hormone withdrawal is postsurgical administration of 131I [62]. Postsurgical administration of 131I is intended to eradicate normal thyroid remnants (ablation) and neoplastic foci (treatment), and it permits a highly sensitive 131I-WBS to be performed 2 to 5 days later [3, 62]. It is not indicated for very low-risk patients (T ! 1 cm, N0, M0). In patients with known persistent disease (distant metastases or incomplete surgical tumor resection) or at high risk of persistent disease, a large activity of 131I (63.7 GBq) is administered following prolonged thyroid hormone withdrawal to destroy both normal and neoplastic cells. In low-risk patients, postsurgical 131I administration is used to eradicate normal thyroid cells, because this will facilitate subsequent follow136

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up. Postablation WBS is used to assess the absence of persistent disease. In these patients, a low (1.1 GBq) or a high (3.7 GBq) activity may be administered following either prolonged withdrawal or rhTSH stimulation. The use of rhTSH also has proven beneficial in reducing patient morbidity by avoiding hypothyroidism. Patients with thyroid cancer already have a compromised QoL due to the effects of the recent cancer diagnosis and its surgical removal. With rhTSH, patients can start thyroid hormone therapy, facilitating a normal recovery period from surgery. In the randomized clinical trial of rhTSH and withdrawal [28], QoL for patients in the rhTSH arm of the study improved from baseline (time of surgery) to week 4 (time of remnant ablation), while QoL decreased for patients in the withdrawal arm. The QoL change from baseline to time of ablation in the rhTSH group was significantly different (p ! 0.05) from that of the withdrawal group. Several studies have compared ablation success rates for patients given rhTSH and those discontinuing thyroid hormone. Retrospective studies reported no difference, but are subject to bias and confounding factors [27, 63, 64]. Two prospective trials have been performed with limited numbers of patients. One study, which used 1.1 GBq (30 mCi) as the standard ablative activity, reported that hypothyroid patients given rhTSH had similar ablation rates compared with hypothyroid patients not given rhTSH, 78 versus 84%, respectively; the ablation rate in euthyroid patients given rhTSH was significantly lower (54%, p ! 0.01) [65]. However, this observation could have been due to the trial having a stringent definition of successful ablation as absence of visible thyroid bed uptake on a diagnostic control 131I-WBS performed following thyroid hormone withdrawal. When the criterion for successful ablation was modified to no visible thyroid bed uptake on withdrawal diagnostic 131I-WBS or undetectable serum Tg following rhTSH, the success rates were more similar (95 versus 88 versus 74%) in the three groups. The other prospective trial [28] was randomized and used an ablative activity of 3.7 GBq (100 mCi). It reported similar rates of ablation success – defined as no visible uptake on an rhTSH-aided diagnostic 131I-WBS – in patients on thyroid hormone given rhTSH and in those in whom thyroid hormone was withdrawn, 96 versus 87%, respectively. Based on the primary outcome of no or !0.1% visible uptake, the ablation rate was 100% for each treatment group. Dosimetric studies showed that the residence time of 131I in thyroid remnants was similar in the two groups (indicating similar radiation doses to the remnants), but a further advantage of using rhTSH was a Schlumberger /Borget /De Pouvourville / Pacini

one-third reduction in the radiation dose delivered to the blood [28, 66, 67]. Based on these studies, rhTSH was approved by the European Agency for the Evaluation of Medicinal Products in 2005 for postoperative administration of 3.7 GBq 131 I in low-risk patients. Further studies on larger groups of patients should confirm the efficacy of its short-term use (ablation rate) and also for its long-term use (recurrence rate). These studies should determine more precisely the minimal activity that should be administered for ablation and should also evaluate the socioeconomic benefits of rhTSH. Another potential advantage of using rhTSH is it allows the possibility of treating patients with I131 a few days after surgery, thus shortening the total duration of initial treatment. Therapeutic Use The use of rhTSH is not approved for treatment of patients with 131I uptake in metastatic lesions. These patients are typically treated with 131I following thyroid hormone withdrawal. However, withdrawal of L-thyroxine therapy in preparation for 131I therapy may expose patients with metastases in critical body structures such as vertebrae or the brain to severe, mainly neurological, complications. In addition, a small minority of patients may not be able to elicit a sufficient endogenous TSH response after thyroid hormone withdrawal (TSH 130 mU/l), and thyroid hormone withdrawal may be contraindicated for medical reasons, such as a coexistent illness [68–70]. In such situations, it has been possible to deliver radioiodine therapy following stimulation by rhTSH within the framework of a ‘compassionate use’ clinical protocol [71–75]. Several hundred patients have been treated with rhTSH in the USA and Europe. Although a detailed and comprehensive review of these results is not currently available, the few reports available indicate that it is possible to deliver high radiation doses with 131I to metastatic thyroid cancer using rhTSH [71–75]. In some patients, swelling and pain due to bone metastases were observed after rhTSH administration. However, the duration of such symptoms was much shorter than when therapy is administered in a hypothyroid state. Caution should be exerted in cases of potential neurological complications, and corticosteroids should be used to prevent such complications [76–79]. The beneficial effects of radioiodine therapy are related to the radiation dose delivered to the neoplastic foci. The radiation dose is related to the uptake and to the effective half-life of radioiodine in the neoplastic focus. Following rhTSH stimulation, uptake may be Recombinant Human Thyroid-Stimulating Hormone

lower than following withdrawal, due to less intensive and shorter stimulation and to more rapid renal clearance of 131I in euthyroidism compared with hypothyroidism. A higher 131I activity should probably be administered to achieve an equivalent radiation dose to the metastatic lesions, although results from quantitative dosimetric studies are not yet available. At this stage, it is not possible to report on the longterm benefits of rhTSH in comparison with thyroid hormone withdrawal. Thus, until such data become available, patients who have a high probability of being cured with 131I treatment after prolonged withdrawal (i.e., young patients with high 131I uptake, small metastases from papillary or well-differentiated follicular thyroid carcinoma) should still be treated following withdrawal [79–82]. Furthermore, until more is known about its long-term effects, use of rhTSH to prepare patients postsurgical administration of 131I should be restricted to lowrisk patients who have no evidence of persistent disease.

The Economics of rhTSH

The clinical benefits of rhTSH have now established it as an alternative to thyroid hormone withdrawal, but its price raises the question of its relative cost-effectiveness. A full economic evaluation is required to determine the monetary impact of rhTSH on medical and societal costs such as loss of productivity, and/or to assess its utility for patients. Utility is a nonmonetary measure (a score) of the value attached by patients to a given health status. It is often measured on a scale from 0 (death) to 1 (perfect health). Knowing the time spent in a given health status, it is possible to compute a number of quality-adjusted life years (months, days), or QALYs, related to this health status, which reflects the benefit (or the loss) incurred by a patient during that period due to the particular health status. For example, if a treatment yields a benefit of 2 years of survival in a health status valued at 0.65, the economic benefit of the treatment will be 0.65 ! 2 = 1.3 QALY. Withdrawal of hormone therapy had a significant negative impact on QoL, as measured by the Functional Assessment of Cancer Therapy General Scale questionnaire [4]. By comparison, rhTSH induced significantly less symptoms related to hypothyroidism as measured by the Billewicz scale [83], and was associated with an improved score on the Profile of Mood States. The QoL was measured by the Short Form (SF)-36 QoL questionnaire [84] administered to 225 patients [6]. When compared with Horm Res 2007;67(suppl 1):132–142

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baseline before preparation, rhTSH had no negative impact on QoL; whereas, withdrawal was associated with a deterioration of QoL on eight of the eight dimensions of the SF-36 questionnaire (p ! 0.0001 for all dimensions). A method to derive a preference-based measure of health from SF-36 was developed and showed that the utility was 0.650 after withdrawal, and 0.825 for rhTSH [85]. Based on those findings, the Medical Services Advisory Committee of the Commonwealth of Australia [86] performed a 5-year cost-utility analysis comparing withdrawal to rhTSH as preparation for diagnostic WBS. In the base case, they found an incremental cost per QALY ratio of USD 39,255 (EUR 30,925). The QALY gain over 5 years with rhTSH over withdrawal was 0.087. In an additional analysis, when worker absenteeism costs were accounted for, the results were reported as USD 26,022 (EUR 20,500). Direct medical costs were USD 5,167 (EUR 4,071) for rhTSH treatment versus USD 1,764 (EUR 1,390) with withdrawal. The main contributor to the cost increment was rhTSH, which accounted for USD 2,752 (EUR 2,168), but it is important to note that the cost of rhTSH used in the analysis was at least 30% higher than the current cost in many European countries. In addition, the study estimated an increase in the number of follow-up WBSs because of improved patient compliance with rhTSH and a larger number of secondary ablations due to the lower sensitivity of rhTSH stimulation. Even though in many countries interventions with a cost per QALY of USD !50,000 (EUR 41,239) are considered cost-effective, the investigators concluded that the cost:utility ratio of rhTSH was relatively high for a procedure that was not life-saving and should be used only for patients who do not tolerate the hypothyroidism period related to withdrawal. The SF-36 questionnaire was not designed to measure the impact of a given treatment on potential loss of productivity due to sick leave. The impact of withdrawal on working time was studied on 16 active Dutch patients [87], who declared approximately 11 days out of work, corresponding to a wage loss per active person of USD 1,383 (EUR 1,090). When these results are extrapolated to the results of the Australian study, the inclusion of loss of productivity in the total cost of withdrawal would add an extra USD 816 (EUR 643) per year and lead to a final societal cost saving of USD 677 (EUR 534) per patient with rhTSH. However, this study included a small number of patients and did not measure loss of productivity related to rhTSH. The societal cost of withdrawal versus rhTSH administration was studied in Germany, using a postal survey 138

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with patients who had undergone withdrawal before a WBS [5]. In this population there was also an average sick leave duration of 11 days. Under the previous set of assumptions, rhTSH could cost an extra USD 803 (EUR 633) or generate a savings of USD 413 (EUR 326) when compared with withdrawal. The authors concluded that rhTSH avoids the morbidity, safety risks and productivity impairment of hypothyroidism at a roughly equivalent societal cost to that of withdrawal. Although there are no published studies of the socioeconomic impact of rhTSH in the context of its use in remnant ablation, it is important to note the potential economic implications of the previously described 33% reduction in radiation dose that rhTSH affords patients relative to patients prepared with withdrawal. With faster clearance of radioiodine, rhTSH use may allow for an earlier discharge from a hospital’s radioprotective ward and may potentially reduce the risk of side effects and secondary malignancies caused by radiation exposure [28, 63, 67]. The valuation of loss of productivity is a subject of controversies amongst health economists. The first debated issue is the valuation of a day out of work. The use of the average hourly or daily wage measures only the loss of income to patients, but does not capture the impact of sick leave on the production of wealth at a societal level, which comprises the value added to wages by the productive process. This would lead to higher estimations of indirect costs than the use of average daily wages. The second issue is that, in principle, utility measures will include the perceived disutility for the patient of a loss of income related to sick leave. Therefore, it is not justified to include wage loss in the numerator of the costutility ratio, since this would lead to double counting. Thus, when using either a cost-utility approach or a societal cost approach, economic analysis may lead to different conclusions for rhTSH. If the SF-36 does not capture correctly disutility related to sick leave, then it is legitimate to include a valuation of its cost in the incremental cost-utility ratio. Existing studies suggest that this may substantially lower the incremental cost per QALY of rhTSH and eventually yield a societal cost-saving. If some disutility is captured by the SF-36 questionnaire, then there is double counting and there might be an additional cost per QALY in the use of rhTSH. Indeed, future economic evaluations should use an adequate utility measure to improve the validity of results. Moreover, future studies should adopt a model similar to that of the Australian study, with a time frame of 5 years, and also include the postoperative administration of radioiodine in Schlumberger /Borget /De Pouvourville / Pacini

the analysis, since rhTSH has also demonstrated efficacy equivalent to withdrawal for this phase of the treatment process. Finally, an additional benefit of the administration of rhTSH is that it may facilitate the clearance of radioiodine relative to withdrawal, and thus allow a reduction in the length of stay for patients undergoing ablation treatment.

Conclusions

The data on rhTSH supports its safety and unequivocal ability to preserve a patient’s QoL and spare morbidity through the avoidance of hypothyroidism at the time

of Tg testing and 131I-WBS. Numerous clinical trials have demonstrated the efficacy of rhTSH in stimulating 131I uptake for 131I-WBS and Tg production. The critical advantage in defining the performance of rhTSH is its ability to identify residual or metastatic thyroid tissue. Limitations do exist in this regard when Tg testing is performed in baseline conditions (during thyroid hormone therapy). TSH stimulation by rhTSH or thyroid hormone withdrawal yields an equivalent increase in sensitivity for the detection of disease. In addition, the limitations of stimulated Tg testing and 131IWBS, such as assay variability and low iodine uptake in some metastases, do not differ for either mode of preparation.

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Adult Workshop 2

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):143–148 DOI: 10.1159/000097571

Published online: February 15, 2007

Novel Medical Approaches to the Treatment of Pituitary Tumors A.J. van der Lely Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands

Key Words Somatropin Insulin-like growth factor I Acromegaly Treatment Pituitary

Abstract Premise: Acromegaly should be treated by clinicians as not just a growth disorder, but also as a metabolic condition. That means developing a tailored approach that normalizes tissue-specific hormonal actions. All medical treatment regimens should have specific normal levels of hormones that might differ per patient. Unfortunately, the way to accomplish this has not yet been devised. Background: There have been no major breakthroughs in clinical neuroendocrinology, at least regarding pituitary tumors. Medical treatment of Cushing disease, prolactinomas, nonfunctioning pituitary tumors and thyroid-stimulating hormone-producing adenomas still relies on medications introduced 10 to 15 years ago. The only major breakthrough to impact current neuroendocrine treatment is in the field of acromegaly. This article presents a case history followed by a discussion of the mechanisms of the medical therapies for acromegaly. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0143$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Case History

The patient is a 31-year-old male with visual field disturbances and classic signs and symptoms of acromegaly. He had a high growth hormone (GH) concentration, an elevated insulin-like growth factor I (IGF-I) level and a relatively low testosterone level. His GH level did not decrease after a glucose tolerance test. Magnetic resonance imaging of the pituitary showed a large tumor impinging on the optic chiasm and surgery was the only possible treatment. A selective transsphenoidal adenomectomy was completed by an experienced neurosurgeon, the tumor was substantially debulked and the patient’s visual fields were restored. In patients with a GH concentration 630 ng/ml, the IGF-I concentration is maximally stimulated. With this kind of GH production rate, even if the tumor is surgically reduced by 50%, GH will not decrease to a level that will reduce IGF-I. The GH concentration must be decreased to !20 ng/ml before a decrease in the serum IGF-I will occur. As this case indicates, surgical approaches are often necessary for tumor debulking but their efficacy in normalising IGF-I can be quite disappointing. Although the neurosurgeon was happy with the results, the patient was unhappy that his GH and IGF-I were still elevated and that his GH levels did not decrease after glucose administration. The reason ‘successful’ surgical debulking failed to normalise IGF-I levels lies in the relationship between GH and IGF-I concentrations [1].

A.J. van der Lely, MD Department of Internal Medicine Erasmus MC, PO Box 2040 NL–3000 CA Rotterdam (The Netherlands) Tel. +31 10 463 2862, Fax +31 10 463 3639, E-Mail [email protected]

Table 1. The efficacy of long-acting somatostatin analogues in acromegaly

Authors, year

Regimen

Results

Morange et al., 1994 [4] Marek et al., 1994 [5] Stewart et al., 1995 [6] Caron et al., 1995 [7] Lancranjan et al., 1996 [8] Giusti et al., 1996 [9] Flogstad et al., 1997 [10] Caron et al., 1997 [11] Davies et al., 1998 [12] Baldelli et al., 2000 [13] Chanson et al., 2001 [14]

Lanreotide SR 30 mg/10–14 days Lanreotide SR 30 mg/14 days Octreotide LAR 20–40 mg/month Lanreotide SR 30 mg/10–14 days Octreotide LAR 20–40 mg/month Lanreotide SR 30 mg/10–14 days Octreotide LAR 20–40 mg/month Lanreotide SR 30 mg/10–14 days Octreotide LAR 20 mg/month Lanreotide SR 30 mg/10–14 days Lanreotide SR 20 mg/10–14 days Octreotide LAR 20 mg/month

GH normal: 13/19 (68%) 0.5 year IGF-I normal: 5/10 (50%) ;2 years IGF-I normal: 7/8 (88%) ;1 year IGF-I normal: 6/9 (66%) 1 year IGF-I normal: 66/101 (65%) ;2 years IGF-I normal: 19/50 (38%) 0.5 year IGF-I normal: 10/14 (71%) 1 year IGF-I normal: 14/22 (63%) 3 years IGF-I normal: 10/13 (75%) 3 years IGF-I normal: 24/34 (70%) 2 years IGF-I normal: 53/111 (48%) 0.25 year IGF-I normal: 70/107 (65%) 0.25 year

Pharmacologic Options for Controlling Acromegaly

The most important consideration in medical treatment for acromegaly is what to use, why, when and in which patients. Patients with relatively low IGF-I levels are potential candidates for treatment with the dopamine agonist cabergoline, which might normalize their IGF-I levels (fig. 1) [2]. Cabergoline is best for patients with mild acromegaly and relatively low concentrations of GH and IGF-I. This might be especially true in patients who cosecrete prolactin apart from their pathological GH hypersecretion [3]. Studies have shown that the somatostatin analogues are effective in normalizing IGF-I levels in about two thirds of patients (table 1) [4–14]. However, in most of those studies, the patients were somatostatin analoguesensitive. If somatostatin analogues were administered to treatment-naïve acromegaly patients, the efficacy rate would most likely be ^50%. Advantages of somatostatin analogues are that they shrink tumors in about 75% of patients and improve physical symptoms. Unlike cabergoline, somatostatin analogues must be injected and they have side effects, especially on the gastrointestinal tract [15]. Somatostatin analogues also inhibit insulin secretion, which is accompanied by deterioration in glucose tolerance. Furthermore, because a sizable population of acromegalic patients do not normalize with somatostatin treatment, other therapeutic options continued to be explored. In 2001, the GH-receptor antagonist (GHRA) pegvisomant was introduced. In all age-dependent groups, re144

Horm Res 2007;67(suppl 1):143–148

gardless of baseline IGF-I level, pegvisomant treatment decreases IGF-I to within normal range [15]. Pegvisomant is efficacious in all acromegalic patients, provided that enough drug is given, and it is well-tolerated. The drug does not interfere with the tumor itself; rather it interferes with GH action in peripheral tissues. Therefore, it might correct the underlying metabolic effects of acromegaly better than the somatostatin analogues. However, pegvisomant must be injected daily, it is quite expensive and its long-term safety has not been established [16– 18]. The choice of which therapy to use in everyday practice could depend on the factors discussed above. To summarize, a patient’s prolactin level might steer the clinician toward cabergoline. Tumor location and size might dictate surgery. GH and IGF-I levels might not have responded to prior therapies. Persistent symptoms on the current treatment might prompt a change in treatment. Now clinicians have the ability to control serum IGF-I concentrations in all patients with either somatostatin analogues or with GHRA, as both are very effective – and very expensive. Still, there are other factors, rather than just bringing IGF-I into a normal range, to consider. Because GH and IGF-I are considered parameters of disease activity in acromegaly, it is sometimes forgotten that these compounds are effective mediators of glycemic control. There is an interplay among IGF-I, insulin, IGFbinding protein 1 (IGFBP-1) and GH. Thus, another question to be considered in the treatment decision-making process is a patient’s glucose tolerance [15].

van der Lely

1,000 Serum IGF-I (ng/ml)

7

Pre-cabergoline On cabergoline Median glucose (mmol/l)

1,200

800 600 400 200

Reduction of fasting glucose during pegvisomant in patients with normal IGF-I on octreotide LAR

6

300

5

250

4

*

150

2

100

1

50 0 4

Fig. 1. Serum IGF-I before and during cabergoline therapy (max-

imum dose: 3.5 mg/week). (Reproduced with permission from Abs et al. [2].)

Relationship between GH and IGF-I

When a person starts fasting, the insulin level decreases, but so too will insulin-like IGF-I levels. IGF-I action is also decreased because the IGFBP-1 level is selectively increased by the absence of insulin. If IGF-I levels decrease, GH levels increase and initiate production of free fatty acids and gluconeogenesis. Changing the levels of GH, IGF-I, insulin or IGFBP-1 immediately interferes not only with the concentrations of all these factors but with their actions as well. Clinicians must look at acromegalic patients as patients in whom metabolic parameters, as well as growth factor concentrations, are changed. Patients should be treated not only to reduce IGF-I to within the normal range, but also to maintain the favorable effects on metabolism and metabolism-related mortality and morbidity. The crucial role IGF-I plays in controlling metabolism and metabolism-related changes is exemplified by results of a Russian study of patients with recently developed type 1 diabetes [19]. In this study, 36 patients were treated with either conventional insulin therapy (24 patients) or with an insulin pump that injected insulin directly into the portal vein (12 patients) to normalize insulin secretion. Hemoglobin (Hb)A1C dropped to within the normal range only in patients who had an insulin pump implanted. Moreover, when insulin was present in the portal vein, it induced a significant amount of IGF-I that ‘helped’ insulin in its hypoglycemic actions. Apparently, portal insulin can ‘call’ for IGF-I to assist in hypoglycemic effects. As the Novel Medical Approaches to the Treatment of Pituitary Tumors

200

3

0 Patients

350

IGF-I levels (ng/ml)

Serum IGF-I before and during cabergoline treatment (max dose: 3.5 mg/wk)

8

12

16 20 Weeks *Baseline vs week 32: p = 0.0005

24

28

32

Fig. 2. Reduction of fasting glucose during pegvisomant therapy in patients with normal IGF-I on octreotide LAR.

IGF-I level went up, the GH came down, which is also beneficial in patients with type 1 diabetes mellitus. In acromegalic patients, GH levels are increased and as GH increases, IGF-I concentrations increase, too. But acromegalic patients lack a proper feedback mechanism to decrease GH again. Consequently, as GH levels increase, insulin concentrations will increase also. Increased insulin will ultimately increase IGF-I levels and decrease IGFBP-1 levels. Acromegalic patients, therefore, not only have an increased IGF-I concentration but also a higher IGF-I action. Pegvisomant normalizes IGF-I action as well as its concentration. This is important because a change in IGF-I also changes a patient’s glycemic conditions. Glucose tolerance was investigated during the transition phase in a study of patients who were treated with somatostatin LAR and then switched back to pegvisomant monotherapy. In both the diabetic and nondiabetic patients, glucose concentrations decreased significantly when switched from the somatostatin LAR analogue to pegvisomant but IGF-I concentrations for the LAR and the pegvisomant groups were identical (fig. 2). However, pegvisomant was able to reduce glucose concentrations significantly, especially in the diabetic patients taking insulin who had a reduction of HbA1C of 11% [18]. IGF-I is still able to be insulin-like because it can also bind to the insulin receptor, and though IGF-I’s affinity for the insulin receptor is a hundred-fold lower, the absolute IGF-I concentration is at least a thousand times highHorm Res 2007;67(suppl 1):143–148

145

The Effect of Medical Therapy of Acromegaly on Insulin Resistance

Ezzat et al. reported that octreotide can increase IGFBP-1 levels probably by a direct effect [21]. Somatostatin analogues not only decrease GH concentrations but also decrease GH sensitivity of the liver because they also reduce portal insulin levels. In pegvisomant-treated patients, GH action drops but the GH level itself goes up. 146

Horm Res 2007;67(suppl 1):143–148

225 GH binding (% of control)

er. An individual’s IGF-I concentration is at least ten times more potent to render him or her hypoglycemic than his or her own insulin level. A person only survives this potential hypoglycemia because IGF-I is bound to the binding proteins and insulin is not; thus, regulation of these binding proteins is essential. The IGF-I actions are at least partly under the control of insulin and GH. The liver also plays a crucial role in glucose metabolism. Hepatocytes express both insulin and GH receptors. If the insulin receptors are stimulated, the hepatocyte is subjected to Janus kinase (JAK)-mediated stimulation of GH receptor production [20]. The more insulin there is, the more GH receptors. These receptors are constantly internalized by a passive method, but they are actively reshuttled to the cell surface. This active translocation can be inhibited by insulin via a phosphoinositide kinase-3 (PI3K)-dependent pathway. It becomes a matter of more insulin, more receptors but, at the same time, more insulin, less receptors at the cell surface (fig. 3). Only very high concentrations of insulin yield less receptors at the hepatocyte cell surface to which GH can bind (although the number of receptors is higher, they all stay within the cell). As noted above, if a person starts fasting, insulin levels decrease and, therefore, the number of GH receptors and the IGF-I concentration do, too. GH levels will increase, but they are no longer able to activate the liver because very few GH receptors are present at the cell surface. Thus, fasting induces a state of GH resistance in the liver, which is essential for survival. Livers of type 1 diabetes patients are also partially GH-resistant (because of low portal insulin levels and low GH receptor expression on the hepatocyte surface) and they indeed have a relatively low IGF-I level and a high GH concentration. This process is so powerful that if acromegalic patients fast, their IGF-I level can decrease in a few days to within normal range. This selective modification of the liver’s GH sensitivity is essential and it might explain the different efficacy rates of several medications.

200 175 150 125 100 75 0

0.1

1 10 100 1,000 Insulin (nmol/l)

Fig. 3. Effect of insulin on GH binding in human hepatoma cell line HuH7 model. (Reproduced with permission from Leung et al. [20].)

Insulin sensitivity as well as insulin concentrations normalize. It could be said that somatostatin analogues increase IGFBP-1 and decrease insulin action while pegvisomant normalizes IGFBP-1 and promotes insulin action. So the same absolute levels of IGF-I might have different effects over these different metabolic scenarios. The absolute concentration of IGF-I after pegvisomant treatment might not necessarily reflect the same IGF-I action as IGF-I levels during long-term somatostatin analogue treatment.

Effects of Long-Term Treatment

During long-term somatostatin analogue treatment, there is relatively weak inhibition of GH release; however, there is also a significant decrease in portal insulin secretion and therefore the liver becomes GH-resistant. These two mechanisms of somatostatin analogues normalize IGF-I in about two-thirds of patients. However, peripheral tissues still show relatively high GH concentrations, which is quite comparable to GH and IGF-I levels in type 1 diabetes. Conversely, if a patient needs high-dose pegvisomant to normalize IGF-I, there is a relatively strong inhibition of GH action. This improves insulin sensitivity compared with somatostatin analogue-treated patients partly by improving IGF-I actions. However, in high-dose pegvisomant-treated subjects, some tissues might become GH-deficient while others remain slightly acromegalic. This is the opposite glycemic situation, more similar to that for type 2 diabetes, where there is also relatively low GH action in the peripheral tissues and relatively high IGF-I action. van der Lely

IGF-I levels in 19 patients with acromegaly during high-dose long-acting monthly IM SRIF analogue therapy during same-SRIF-therapy regimen + weekly SC pegvisomant (40–80 mg)

Serum IGF-I (nmol/l)

120

80

As a proof-of-principle study, we cotreated patients with high-dose long-acting somatostatin analogues and weekly doses of pegvisomant, instead of daily injections [22]. Our rationale was that, because the somatostatin analogues reduce GH and reduce the number of GH receptors, there would be fewer receptors to block and less GH to compete with. Therefore, less pegvisomant might be needed. And indeed, of the 19 patients who completed the study, all but one showed normalization of serum IGF-I levels (fig. 4). Further studies are needed, however, before the value of combination therapy can truly be determined.

40

Conclusion

0 30

40

50 60 70 Age in years

80

Fig. 4. IGF-I levels in 19 patients with acromegaly during high-

dose long-acting monthly intramuscular SRIF analogue therapy (yellow) and during same SRIF regimen + weekly subcutaneous pegvisomant (40 to 80 mg) (blue). (Reproduced with permission from Feenstra et al. [22].)

Acromegalic patients should be treated as patients with growth disorders but also as patients with metabolic conditions. This means developing a tailored approach to normalization of tissue-specific GH and IGF-I actions. Every medical treatment should have its own normal GH and IGF-I concentrations as endpoint parameters, and these might even differ by patient. How best to accomplish such medical regimens remains to be determined.

References 1 Barkan AL, Beitins IZ, Kelch RP: Plasma insulin-like growth factor-I/somatomedin-C in acromegaly: correlation with the degree of growth hormone hypersecretion. J Clin Endocrinol Metab 1988;67:69–73. 2 Abs R, Verhelst J, Maiter D, Van Acker K, Nobels F, Coolens JL, Mahler C, Beckers A: Cabergoline in the treatment of acromegaly: a study in 64 patients. J Clin Endocrinol Metab 1998;83:374–378. 3 Giustina A, Barkan A, Casanueva FF, Cavagnini F, Frohman L, Ho K, Veldhuis J, Wass J, Von Werder K, Melmed S: Criteria for cure of acromegaly: a consensus statement. J Clin Endocrinol Metab 2000;85:526–529. 4 Morange I, De Boisvilliers F, Chanson P, Lucas B, DeWailly D, Catus F, Thomas F, Jaquet P: Slow release lanreotide treatment in acromegalic patients previously normalized by octreotide. J Clin Endocrinol Metab 1994;79: 145–151. 5 Marek J, Hana V, Krsek M, Justova V, Catus F, Thomas F: Long-term treatment of acromegaly with the slow-release somatostatin analogue lanreotide. Eur J Endocrinol 1994; 131:20–26.

Novel Medical Approaches to the Treatment of Pituitary Tumors

6 Stewart PM, Kane KF, Stewart SE, Lancranjan I, Sheppard MC: Depot long-acting somatostatin analog (Sandostatin-LAR) is an effective treatment for acromegaly. J Clin Endocrinol Metab 1995;80:3267–3272. 7 Caron P, Cogne M, Gusthiot-Joudet B, Wakim S, Catus F, Bayard F: Intramuscular injections of slow-release lanreotide (BIM 23014) in acromegalic patients previously treated with continuous subcutaneous infusion of octreotide (SMS 201-995). Eur J Endocrinol 1995 Mar;132:320–325. 8 Lancranjan I, Bruns C, Grass P, Jaquet P, Jervell J, Kendall-Taylor P, Lamberts SW, Marbach P, Orskov H, Pagani G, Sheppard M, Simionescu L: Sandostatin LAR: a promising therapeutic tool in the management of acromegalic patients. Metabolism 1996;45(8 suppl 1):67–71. 9 Giusti M, Gussoni G, Cuttica CM, Giordano G: Effectiveness and tolerability of slow release lanreotide treatment in active acromegaly: six-month report on an Italian multicenter study. Italian Multicenter Slow Release Lanreotide Study Group. J Clin Endocrinol Metab 1996;81:2089–2097.

10 Flogstad AK, Halse J, Bakke S, Lancranjan I, Marbach P, Bruns C, Jervell J: Sandostatin LAR in acromegalic patients: long-term treatment. J Clin Endocrinol Metab 1997;82: 23–28. 11 Caron P, Buscail L, Beckers A, Esteve JP, Igout A, Hennen G, Susini C: Expression of somatostatin receptor SST4 in human placenta and absence of octreotide effect on human placental growth hormone concentration during pregnancy. J Clin Endocrinol Metab 1997;82:3771–3776. 12 Davies PH, Stewart SE, Lancranjan L, Sheppard MC, Stewart PM: Long-term therapy with long-acting octreotide (SandostatinLAR) for the management of acromegaly. Clin Endocrinol 1998;48:311–316. 13 Baldelli R, Colao A, Razzore P, Jaffrain-Rea ML, Marzullo P, Ciccarelli E, Ferretti E, Ferone D, Gaia D, Camanni F, Lombardi G, Tamburrano G: Two-year follow-up of acromegalic patients treated with slow release lanreotide (30 mg). J Clin Endocrinol Metab 2000;85:4099–4103.

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147

14 Chanson P, Daujat F, Young J, Bellucci A, Kujas M, Doyon D, Schaison G: Normal pituitary hypertrophy as a frequent cause of pituitary incidentaloma: a follow-up study. J Clin Endocrinol Metab 2001;86:3009–3015. 15 Clemmons DR, Chihara K, Freda PU, Ho KK, Klibanski A, Melmed S, Shalet SM, Strasburger CJ, Trainer PJ, Thorner MO: Optimizing control of acromegaly: integrating a growth hormone receptor antagonist into the treatment algorithm. J Clin Endocrinol Metab 2003;88:4759–4767. 16 van der Lely AJ, Hutson RK, Trainer PJ, Besser GM, Barkan AL, Katznelson L, Klibanski A, Herman-Bonert V, Melmed S, Vance ML, Freda PU, Stewart PM, Friend KE, Clemmons DR, Johannsson G, Stavrou S, Cook DM, Phillips LS, Strasburger CJ, Hackett S, Zib KA, Davis RJ, Scarlett JA, Thorner MO: Long-term treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist. Lancet 2001;358:1754–1759.

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17 Gharib H, Cook DM, Saenger PH, Bengtsson BA, Feld S, Nippoldt TB, Rodbard HW, Seibel JA, Vance ML, Zimmerman D, Palumbo PJ, Bergman DA, Garber JR, Hamilton CR Jr, Petak SM, Rettinger HI, Service FJ, Shankar TP, Stoffer SS, Tourletot JB, American Association of Clinical Endocrinologists Growth Hormone Task Force: American Association of Clinical Endocrinologists medical guidelines for clinical practice for growth hormone use in adults and children – 2003 update. Endocr Pract 2003;9:64–76. 18 Barkan AL, Burman P, Clemmons DR, Drake WM, Gagel RF, Harris PE, Trainer PJ, van der Lely AJ, Vance ML: Glucose homeostasis and safety in patients with acromegaly converted from long-acting octreotide to pegvisomant. J Clin Endocrinol Metab 2005; 90: 5684–5691.

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19 Shishko PI, Dreval AV, Abugova IA, Zajarny IU, Goncharov VC: Insulin-like growth factors and binding proteins in patients with recent-onset type 1 (insulin-dependent) diabetes mellitus: influence of diabetes control and intraportal insulin infusion. Diabetes Res Clin Pract 1994;25:1–12. 20 Leung KC, Doyle N, Ballesteros M, Waters MJ, Ho KK: Insulin regulation of human hepatic growth hormone receptors: divergent effects on biosynthesis and surface translocation. J Clin Endocrinol Metab 2000; 85: 4712–4720. 21 Ezzat S, Ren SG, Braunstein GD, Melmed S: Octreotide stimulates insulin-like growth factor binding protein-1 (IGFBP-1) levels in acromegaly. J Clin Endocrinol Metab 1991; 73:441–443. 22 Feenstra J, de Herder WW, ten Have SM, van den Beld AW, Feelders RA, Janssen JA, van der Lely AJ: Combined therapy with somatostatin analogues and weekly pegvisomant in active acromegaly. Lancet 2005;365: 1644–1646.

van der Lely

Adult Workshop 3

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):149–154 DOI: 10.1159/000097572

Published online: February 15, 2007

Genetics of Hypogonadotropic Hypogonadism Manuela Simoni Eberhard Nieschlag Institute of Reproductive Medicine, University Hospital, Münster, Germany

Key Words Hypogonadotropic hypogonadism Gonadotropinreleasing hormone receptor G-protein-coupled receptor 54 KAL1 Fibroblast growth factor receptor 1

Abstract Background: Idiopathic hypogonadotropic hypogonadism (HH) results from a defect in the normal pulsatile secretion pattern of gonadotropin-releasing hormone (GnRH) from the hypothalamus. Clinically it can be categorized as one of two types: HH associated with anosmia, known as Kallmann syndrome, and isolated HH. The anatomical explanation for Kallmann syndrome stems from incomplete or total failure of GnRH-secreting neurons to migrate from the olfactory epithelium to their final destination in the mediobasal hypothalamus. Several genes are involved in the migration of the GnRH neurons. Conclusions: Mutations of the KAL1 gene, encoding for anosmin 1, and of the FGFR1 (or KAL2) gene, encoding for fibroblast growth factor receptor 1, can be found in familial cases of Kallmann syndrome. KAL1 mutations are responsible for X-linked recessive inheritance, and FGFR1 mutations are the autosomal dominant form. Moreover, mutations of the gonadotropin-releasing hormone receptor gene and G-protein-coupled receptor 54 gene are found in over 50% of familial cases of isolated HH with autosomal recessive inheritance. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0149$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

Pubertal maturation requires pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which stimulates gonadotropin production and gonadal activation. While the neuroendocrine mechanism regulating the timing of puberty is still largely unknown, some crucial genes involved in the regulation of GnRH secretion are being discovered, and their role in idiopathic hypogonadotropic hypogonadism (HH) is being revealed. Today, we know that mutations of such genes are involved in more than 50% of the familial cases of idiopathic HH. This article summarises our current knowledge and provides some practical recommendations for genetic testing.

Background

Idiopathic HH is defined as a clinical syndrome characterised by low levels of sex steroids and gonadotropins as a result of a defect in the normal pulsatile secretion pattern of GnRH from the hypothalamus. HH has a prevalence of approximately 1 in 8,000 newborns with a 4:1 male:female ratio [1]. Clinically, it can be subdivided into HH associated with an inability to smell (anosmia), known as Kallmann syndrome, and isolated HH.

Manuela Simoni, MD Institute of Reproductive Medicine University Hospital, Domagkstrasse 11 DE–48149 Münster (Germany) Tel. +49 251 835 644, Fax +49 251 835 6093, E-Mail [email protected]

Table 1. Milestones in the pathophysiology of HH

Year

Finding

Authors

1989

Migration defect of GnRH neurons in Kallmann syndrome

Schwanzel-Fukuda et al., 1989 [2]

1991

Identification of KAL1 (anosmin-1)

Franco et al., 1991 [3]

1997

GnRHR mutations in isolated HH

de Roux et al., 1997 [4]

2003

Identification of KAL2 (FGFR1)

Dodè et al., 2003 [5]

2003

Mutations of GPR54 in isolated HH

de Roux et al., 2003 [6] Seminara et al., 2003 [7]

2006

Identification of KAL3 (PKR2)?

Matsumoto et al., 2006 [8]

The primary anatomical defect underlying Kallmann syndrome is agenesis of the bulbus olfactorius such that the GnRH neurons fail to migrate from the olfactory epithelium to the telencephalon and remain trapped in the lamina cribrosa of the ethmoid bone [2]. This defect was identified in 1989 when analysis of a 19-week-old male foetus with a deletion of the KAL1 gene revealed the abnormal location of the GnRH neurons and the lack of formation of the olfactory bulbs [2]. The gene responsible for this phenotype is KAL1, and it encodes for the anosmin-1 protein of the extracellular matrix [3]. Further milestones in the pathogenetic definition of both isolated and idiopathic HH include the first description of mutations of the GnRH receptor gene (GnRHR) [4], identification of FGFR1 as another factor involved in formation of the olfactory bulb and responsible for the autosomal dominant form of Kallmann syndrome (KAL2) [5] and discovery of the GRP54 mutations in isolated HH [6, 7]. While the role of these genes is now well established, novel putative factors continue to be identified. The most recent example is discovery of prokineticin receptor gene PKR2, elimination of which results in abnormal development of the olfactory bulb and hypogonadism in the mouse [8] (table 1).

Migration of the GnRH Neurons and Genes Involved in Kallmann Syndrome

It has long been known that there is an association between the sense of smell and gonadal function. Although a direct connection between the olfactory epithelium and the GnRH-secreting neurons has been demonstrated only recently [9, 10], complete anosmia and HH can occur as a result of hypoplasia of the olfactory bulbs and 150

Horm Res 2007;67(suppl 1):149–154

tracts as in the Kallmann syndrome, the genetic nature of which was already recognised in 1944 [11]. The anatomical explanation for Kallmann syndrome stems from incomplete or total failure of the GnRH-secreting neurons to migrate from the olfactory epithelium to their final destination in the mediobasal hypothalamus. In the sixth embryonic week of human foetal development, GnRH neurons typically start to migrate along the fibres of the terminal nerve, connecting the olfactory epithelium with the hypothalamic region. GnRH neurons enter the brain caudal to the olfactory bulb and continue their migration along the medial walls of the cerebral hemispheres. Formation of the olfactory bulbs appears to be necessary for migration of GnRH neurons, and deletions or mutations of the KAL1 gene, and possibly any genetic defect resulting in improper formation of the olfactory bulbs, may be associated with HH. The symptoms of Kallmann syndrome include complete anosmia or reduced sense of smell and HH, though a number of other symptoms also may be present. These include bimanual synkinesis, renal agenesis, cleft palate or lip, tooth agenesis, hearing loss and occasionally other anomalies. Kallmann syndrome can be familial, but most of the time it occurs as a sporadic disease. The mode of inheritance for familial forms are X-linked recessive (KAL1), autosomal dominant (KAL2) and autosomal recessive (KAL3). The KAL1 and KAL2 genes have now been identified [12], and a possible candidate for KAL3 was recently identified in an experimental model [8]. The KAL1 gene product, anosmin-1, is a protein of the extracellular matrix. Among other organs, it is localized in the olfactory bulb, which may be involved in signal transduction of fibroblast growth factor receptor 1 (FGFR1) [12]. Anosmin-1 also might be involved in the morphogenesis of the olfactory bulb. Mutations or deleSimoni/Nieschlag

Q11K

N10K M1T Y284C

T32I Q106R

A171T

C200Y

S217R

C279Y L314X

CELL MEMBRANE L266R E90K

A129D

S168R R262Q

Fig. 1. Known mutations in the GnRHR

P320L

R139H

gene in isolated HH.

tions of the KAL1 and FGFR1 (KAL2) genes result in the same phenotype, suggesting an interaction between the two factors. Hebert et al. showed that olfactory bulb formation is hampered in mutant mice in which fgfr1 expression in the telencephalon was conditionally knocked out [13]. In families with the autosomal dominant form of Kallmann syndrome, linkage analysis has identified heterozygous mutations of the FGFR1 gene [6, 7]. Homozygous loss-of-function mutations are incompatible with life. Mutations of the FGFR1 gene have been described in several families and show variable penetrance, with symptoms ranging from isolated anosmia, HH or delayed puberty even within the same family [14, 15]. Given the autosomal dominant character of the clinical manifestations, individuals with sporadic forms of Kallmann syndrome should be examined for mutations in the FGFR1 gene. GnRH or gonadotropin treatment restores normal gonadal function and fertility in affected individuals, so the genetic defect can be passed on to the next generation. Children of patients with FGFR1 mutations should be screened and monitored appropriately during puberty. Another interesting candidate gene for Kallmann syndrome is the PKR2 gene. Prokineticins are secreted proteins that regulate several functions, ranging from smooth muscle contraction in the stomach to angiogenesis in steroidogenic endocrine glands. The prokineticins PK1 and PK2 activate specific G-protein-coupled receptors, PKR1 and PKR2, which are predominantly expressed in the testis and central nervous system. A recent study in knockout mice showed that elimination of the PKR2 gene, but not the PKR1 gene, results in agenesis of the olfactory bulbs and hypogonadism due to lack of GnRH neurons in the hypothalamus [8]. PKR2 therefore represents an Genetics of Hypogonadotropic Hypogonadism

interesting experimental candidate gene for screening in individuals with the autosomal recessive form of Kallmann syndrome (KAL3), though no clinical investigations have been conducted yet.

Genes Involved in Control of GnRH Secretion and Isolated HH

GnRHR is a G-protein-coupled receptor with seven transmembrane segments and an extracellular amino terminus but no intracellular carboxyl terminus. It activates phospholipase C and mobilises intracellular calcium via G proteins. The GnRHR gene is localised on chromosome 4 at band q13.1. The coding sequence comprises three exons and spans over 20 kb. Numerous compound heterozygous and four homozygous GnRHR mutations have been described [16 and references therein], causing subtypes of isolated HH ranging from complete to partial resistance to GnRH (fig. 1). GnRHR mutations account for less than 50% of familial isolated HH cases and a small fraction of sporadic cases. The clinical manifestations of mutations of the GnRHR gene are variable, with complete or partial hypogonadism possible and without a clear relationship between type of mutation and clinical phenotype. Patient response to GnRH is usually impaired, but normal responses have been reported. Pulsatile secretion of luteinizing hormone (LH) is usually reduced [17]. In vitro testing showed that mutations resulted in reduced expression at the cell surface, decreased ligand binding or decreased or absent signal transduction [17, 18]. Common genetic variants (single nucleotide polymorphism) of the GnRHR gene are known as well, but they are not associated with either HH [16] or pubertal timing [19]. Horm Res 2007;67(suppl 1):149–154

151

L102P

R297L

CELL MEMBRANE C233R L148S X3399R

Fig. 2. Known mutations in the GPR54

G247del

1001_1002insC

R331X

gene in isolated HH.

arcuate nucleus GPR54

-

kisspeptin GnRH neuron

Fig. 3. Current model of neuroendocrine control of gonadotropin secretion. Kisspeptin, produced by the arcuate nucleus and the anteroventral paraventricular nucleus (AVPN), stimulates GnRH neurons by interacting with its receptor, GPR54, which is localised on the surface of the GnRH neurons. According to this model, the negative and positive feedback control of gonadotropin secretion by steroids and possibly by metabolic signals may occur via the kisspeptin/GPR54 system.

AVPN

+ steroids? leptin? other signals?

GnRH GnRH receptor portal pituitary circulation pituitary gonadotrope

LH/FSH

Mutations of the GPR54 gene, which is localized on the short arm of chromosome 19 (19p13), recently have been identified as a novel cause for isolated HH. GPR54 is a Gprotein-coupled receptor expressed mainly in the brain, pituitary and placenta. GPR54 is formed of five exons and encodes a 398-amino acid protein. In 2003, homozygosity mapping and linkage analysis of large consanguinous pedigrees identified loss-of-function mutations in GPR54 associated with autosomal recessive isolated HH [6, 7]. In parallel with this work, Seminara et al. independently generated and characterised a gpr54 knockout mouse model that showed a phenocopy of human isolated HH. They demonstrated that the function of GPR54 is conserved in mammals, and that GPR54 is necessary for normal functioning of the hypothalamo-pituitary-gonadal axis [7]. Since this original description, two additional cases of GPR54 mutations (one compound heterozygous 152

Horm Res 2007;67(suppl 1):149–154

mutation and one homozygous mutation) have been described in male patients with isolated HH [16, 20] (fig. 2). The discovery of GPR54 as an important factor regulating GnRH production has shed new light on neuroendocrine control of gonadotropin secretion. Shortly before the description of the first GPR54 mutations, kisspeptin (also called metastin) was identified as the natural ligand for GPR54 [21]. Kisspeptin, which is the product of the Kiss1 gene, was originally described as a 54-amino acid secreted peptide with antimetastatic properties that is highly expressed in placenta, testis, pancreas, liver, intestine and brain, especially in the hypothalamus and basal ganglia. In the forebrain, Kiss1 is expressed in the arcuate nucleus, the periventricular nucleus and the anteroventral periventricular nucleus – regions involved in the steroid-mediated feedback control of gonadotropin secreSimoni/Nieschlag

Table 2. Genes mutated in HH

Gene

Mode of inheritance

Frequency

Kallmann syndrome KAL1 (anosmin-1) FGFR1 (KAL2)

X-linked recessive Autosomal dominant

10% of familial cases 10% of familial cases Possibly frequent in sporadic cases

Isolated HH GnRHR

Autosomal recessive

GPR54

Autosomal recessive

40% of familial cases 2–3% of sporadic cases (compound heterozygosity) 15% of familial cases 2–3% of sporadic cases (compound heterozygosity)

tion [21]. Moreover, GPR54 mRNA has been found in GnRH neurons [22]. Although no direct neuroanatomical connection has been demonstrated thus far between kisspeptin-secreting neurons and GnRH neurons, injection of kisspeptin into rodents, primates and even in men rapidly stimulates secretion of LH and follicle-stimulating hormone [23], suggesting that kisspeptin can work as a true neurohormone. In addition, administration of a GnRH antagonist prevents the effect of kisspeptin, demonstrating its effect via the GnRH neurons [24]. These findings demonstrate that two novel factors, kisspeptin and its receptor GPR54, play a role in neuroendocrine control of gonadotropin secretion (fig. 3). The kisspeptin/GPR54 system might be the key mediator of steroid hormones on GnRH/gonadotropin secretion by means of negative feedback control through the arcuate nucleus and positive feedback control through the anteroventral periventricular nucleus. It may also be a major regulator of puberty [21].

Conclusion

The majority of cases of HH – with or without anosmia – are sporadic, but familial cases are not infrequent. Genetic screening should be performed in individuals with all familial forms, since gene mutations or deletions can be detected in about 20% of cases of Kallmann syndrome (KAL1 and FGFR1) and in over 50% of cases of isolated HH (GnRHR and GPR54) (table 2). Considering the phenotypic heterogeneity of the disease, genetic screening may be needed. To determine whether genetic screening is needed and, if so, which gene should be screened for, an accurate familial anamnesis is essential, asking specifically if there is consanguinity in the family, and an analysis of the patient’s pedigree and the phenotype’s mode of transmission should be conducted. Future experimental studies are needed to evaluate whether novel candidate genes such as PKR2 are part of the genetic origins of this disease.

References 1 Behre HM, Nieschlag E, Meschede D, Partsch CJ: Diseases of the hypothalamus and the pituitary gland; in Nieschlag E, Beher HM (eds): Andrology. Male Reproductive Health and Dysfunction. Berlin, Springer, 2000, ed 2, pp 125–142. 2 Schwanzel-Fukuda M, Bick D, Pfaff DW: Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Brain Res Mol Brain Res 1989;6:311–326.

Genetics of Hypogonadotropic Hypogonadism

3 Franco B, Guioli S, Pragliola A, Incerti B, Bardoni B, Tonlorenzi R, Carrozzo R, Maestrini E, Pieretti M, Taillon-Miller P, et al: A gene deleted in Kallmann’s syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 1991; 353:529–536. 4 de Roux N, Young J, Misrahi M, Genet R, Chanson P, Schaison G, Milgrom E: A family with hypogonadotropic hypogonadism and mutations in the gonadotropin-releasing hormone receptor. N Engl J Med 1997; 337:1597–1602.

5 Dodè C, Levilliers J, Dupont JM, De Paepe A, Le Du N, Soussi-Yanicostas N, Coimbra RS, Delmaghani S, Compain-Nouaille S, Baverel F, Pecheux C, Le Tessier D, Cruaud C, Delpech M, Speleman F, Vermeulen S, Amalfitano A, Bachelot Y, Bouchard P, Cabrol S, Carel JC, Delemarre-van de Waal H, GouletSalmon B, Kottler ML, Richard O, SanchezFranco F, Saura R, Young J, Petit C, Hardelin JP: Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet 2003;33:463–465.

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6 de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E: Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003; 100: 10972–10976. 7 Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS Jr, Shagoury JK, BoAbbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley WF Jr, Aparicio SA, Colledge WH: The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349:1614–1627. 8 Matsumoto S, Yamazaki C, Masumoto KH, Nagano M, Naito M, Soga T, Hiyama H, Matsumoto M, Takasaki J, Kamohara M, Matsuo A, Ishii H, Kobori M, Katoh M, Matsushime H, Furuichi K, Shigeyoshi Y: Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2. Proc Natl Acad Sci USA 2006;103:4140–4145. 9 Yoon H, Enquist LW, Dulac C: Olfactory inputs to hypothalamic neurons controlling reproduction and fertility. Cell 2005; 123: 669–682. 10 Boehm U, Zou Z, Buck LB: Feedback loops link odor and pheromone signaling with reproduction. Cell 2005;123:683–695. 11 Kalmann FJ, Schoenfeld WA, Barrera SE: The genetic aspects of primary eunuchoidism. Am J Ment Defic 1944;48:203–236. 12 Dodè C, Hardelin JP: Kallmann syndrome: fibroblast growth factor signaling insufficiency? J Mol Med 2004;82:725–734.

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13 Hebert JM, Lin M, Partanen J, Rossant J, McConnell SK: FGF signaling through FGFR1 is required for olfactory bulb morphogenesis. Development 2003;130:1101–1111. 14 de Roux N: Isolated gonadotropic deficiency with and without anosmia: a developmental defect or a neuroendocrine regulation abnormality of the gonadotropic axis. Horm Res 2005;64 (suppl 2):48–55. 15 Pitteloud N, Acierno JS Jr, Meysing AU, Dwyer AA, Hayes FJ, Crowley WF Jr: Reversible Kallmann syndrome, delayed puberty, and isolated anosmia occurring in a single family with a mutation in the fibroblast growth factor receptor 1 gene. J Clin Endocrinol Metab 2005;90:1317–1322. 16 Lanfranco F, Gromoll J, von Eckardstein S, Herding EM, Nieschlag E, Simoni M: Role of sequence variations of the GnRH receptor and G protein-coupled receptor 54 gene in male idiopathic hypogonadotropic hypogonadism. Eur J Endocrinol 2005; 153: 845– 852. 17 Karges B, Karges W, de Roux N: Clinical and molecular genetics of the human GnRH receptor. Hum Reprod Update 2003; 9: 523– 530. 18 Bedecarrats GY, Linher KD, Janovick JA, Beranova M, Kada F, Seminara SB, Michael Conn P, Kaiser UB: Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function. Mol Cell Endocrinol 2003;205:51–64.

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19 Sedlmeyer IL, Pearce CL, Trueman JA, Butler JL, Bersaglieri T, Read AP, Clayton PE, Kolonel LN, Henderson BE, Hirschhorn JN, Palmert MR: Determination of sequence variation and haplotype structure for the gonadotropin-releasing hormone (GnRH) and GnRH receptor genes: investigation of role in pubertal timing. J Clin Endocrinol Metab 2005;90:1091–1099. 20 Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, O’Rahilly S, Aparicio SA: Two novel missense mutations in G protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2005;90:1849–1855. 21 Dungan HM, Clifton DK, Steiner RA: Minireview: kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion. Endocrinology 2006;147:1154–1158. 22 Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA: Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004; 80: 264–272. 23 Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA, Bloom SR: Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005; 90:6609–6615. 24 Kaiser UB, Kuohung W: KiSS-1 and GPR54 as new players in gonadotropin regulation and puberty. Endocrine 2005;26:277–284.

Simoni/Nieschlag

Adult Workshop 3

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):155–164 DOI: 10.1159/000097573

Published online: February 15, 2007

Management of Glucocorticoid Replacement in Adult Growth Hormone Deficiency Helena Filipsson Gudmundur Johannsson Department of Endocrinology, Sahlgrenska Academy at Gothenburg’s University, Gothenburg, Sweden

Key Words Hypopituitarism Growth hormone deficiency Glucocorticoids

Abstract In multiple pituitary hormone deficiency, interactions occur among thyroxine, cortisol and growth hormone (GH). This review addresses the issues of endogenous cortisol production and exogenous cortisol replacement and discusses cortisol metabolism in the context of adult GH deficiency. In addition, we review the effect of GH on 11--hydroxysteroid dehydrogenase type 1, the current literature regarding the choice of glucocorticoid (GC) treatment and dose levels and dosing regimens. Recommendations for GC replacement therapy are also provided. Copyright © 2007 S. Karger AG, Basel

Background

Hypopituitarism acquired in adult life is often caused by pituitary or peripituitary tumours and their treatments [1, 2] (table 1). The prevalence of non-tumour origin is approximately 30% [3]. In the case of multiple pituitary hormone deficiency, various combinations of hormone replacement therapy are applied to secure the

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0155$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

patient’s well-being and to improve long-term outcome. A proper assessment of hormonal replacement is mandatory, and an understanding of interactions among pituitary axes is essential. There are interactions between the somatotropic system and the hypothalamic-pituitary-thyroid axis. Growth hormone (GH) replacement increases peripheral conversion of thyroxine (T4) to triiodothyronine (T3) and decreases conversion of T4 to reversed T3 (rT3). Many studies have reported significant changes in thyroid function, including a slight decline in serum T4, rT3 and thyroidstimulating hormone (TSH) levels and an elevation in T3 levels [4, 5]. Therefore, careful monitoring of thyroid function is also mandatory during recombinant human GH treatment [5] as it may induce hyperthyroidism, and the dose of thyroxine replacement therapy may need to be adjusted. Changes in thyroid hormone concentrations are also induced by alterations of substitution therapy in patients with adrenocortical insufficiency (ACI). Withdrawal of glucocorticoid (GC) substitution for 84 h from patients with ACI will increase serum concentrations of total T3 and decrease rT3. By contrast, subsequent oral administration of 0.5 mg dexamethasone (DX) 4 times daily for 3 days will induce a decrease in total T3 and increase rT3. In addition, serum levels of total T4 are unchanged by either withdrawal or readministration of GCs, while TSH

Gudmundur Johannsson Research Centre for Endocrinology and Metabolism Sahlgrenska University Hospital, Medicin, Gröna straket 8 SE–413 45 Göteborg (Sweden), Tel. +46 31 342 3101, Fax +46 31 821 524 E-Mail [email protected]

Table 1. Causes of secondary adrenal insufficiency

Table 2. The similar clinical features of Cushing disease and

GHD Pituitary tumours Other tumours of the hypothalamic-pituitary region Pituitary irradiation Lymphocytic hypophysitis Isolated congenital ACTH insufficiency Pro-opiomelanocortin deficiency syndrome PROP 1, HESX1 mutations Pituitary apoplexia Pituitary infiltration or granuloma Head trauma

Cushing disease

GHD

Moon face Buffalo hump Visceral adiposity Bruises Skin atrophy Insulin resistance Osteoporosis Increased vascular mortality

Dyslipidemia Increased body mass index Decreased lean body mass Visceral adiposity Insulin resistance Osteoporosis Increased vascular mortality

For a more complete review of causes, see Arlt and Allolio, Lancet 2003;361:1881–1893 [42].

concentrations are unaffected by GC withdrawal and fall during subsequent DX therapy [6]. It is important to note that thyroid hormones should never be replaced before administering GCs: euthyroidism may trigger an adrenal crisis by accelerating the metabolism of cortisol. Moreover, in the absence of GH, increased tissue exposure to cortisol occurs because of an increased conversion of inactive cortisone to active cortisol, which may contribute to the clinical features of unreplaced GH deficiency (GHD) [7], since the characteristics of GHD and excessive cortisol production are similar (table 2). Hypopituitary patients with untreated GHD have increased body mass index and fat mass, decreased lean body mass, dyslipidemia, insulin resistance and glucose intolerance [8]. Glucose intolerance is a clinical feature that may possibly be influenced by unphysiological GC replacement therapy because high doses of GC have unfavourable metabolic effects [9–11].

Normal Cortisol Physiology

In the early 1990s, Esteban et al., using isotope dilatation/mass spectrometry, found that the daily cortisol production rate from the adrenal cortex was substantially lower than previously reported [12]. This led to a reappraisal of the estimated endogenous cortisol production level to 5.7 mg/m2/day or approximately 9.9 mg/day. More recent studies, however, estimate cortisol production levels to be between 9 and 11 mg/m2/day [13]. Cortisol secretion is regulated by the paraventricular nuclei of the hypothalamus via the hypothalamic-pituitary-adrenal (HPA) axis in a circadian pattern [14]. It is well known that in humans the natural peak in cortisol level occurs early in the morning and then falls progres156

Horm Res 2007;67(suppl 1):155–164

sively during the day to a nadir around midnight [15, 16]. In addition, a pulsatile, ultradian rhythm has been found throughout the 24-hour cycle [14]. The estimated number of secretory bursts of adrenocorticotropin (ACTH) is 18/24 h [17]. It is the amplitude of each burst of ACTH, rather than the frequency, that increases and gives rise to the nocturnal surge of the 24-hour circadian pattern of cortisol secretion [14, 17]. Approximately 15 min after each burst of ACTH, a surge of cortisol is released into circulation [18]. The HPA axis is also activated by several physical and physiological stressors. Under conditions of stress such as physical activity, fever, surgery or mental stress [19], the serum cortisol concentration is increased through enhanced production and secretion from the adrenal glands via the influence of its major regulator, ACTH. Corticotropin-releasing hormone and arginine vasopressin synergistically stimulate ACTH secretion by corticotroph cells [20, 21]. Thus, HPA-axis activity is regulated not only by a negative feedback effect from circulating cortisol and ACTH secretion from the pituitary, but also by superimposition of several hypothalamic factors [14, 22]. In humans, circulating cortisol binds to a large extent with cortisol-binding globulin (CBG) and to a lesser degree with albumin. Under basal conditions, about 5–10% of the circulating cortisol is free. The free fraction mediates the GC effect in peripheral tissues [22–24]. All tissues contain and express the GC receptor, which binds cortisol and activates a number of systems. Cortisol has a similar affinity for both the mineralocorticoid receptor and the GC receptor, but the activity of 11--hydroxysteroid dehydrogenase (11--HSD) type 2 protects the mineralocorticoid receptor from overstimulation by converting active cortisol to inactive cortisone, thereby allowing aldosterone to interact with its receptor [25]. Filipsson/Johannsson

A

11--hydroxysteroid dehydrogenase (11--HSD)

Cortisone

B Cortisol

Cortisone

GHD

11--HSD type 2 in the kidney

Cortisone

Cortisol

Cortisol

Cortisone

Cortisol

GH 11--HSD type 1 in the liver and adipose tissue

Fig. 1. A In peripheral tissues, corticosteroid hormone action is determined, in part, through the activity of 11-HSD. This enzyme has two isoforms that drive the cortisone-to-cortisol shuttle in opposite directions. B The

activity of 11--HSD type 1 is increased in GHD and inhibited by GH and/or insulin-like growth factor I.

The type 2 isoform inactivates cortisol in the kidney, whereas 11--HSD type 1 principally performs the reverse action of converting cortisone to cortisol in the liver and visceral adipose tissue [26] (fig. 1A). In peripheral tissues, corticoidsteroid hormone action is determined in part through the activity of 11--HSD type 1. When expression of these 11- isoenzymes in peripheral tissues is altered, corticosteroid action is modified. Moreover, a sexual dimorphism has been found with lower 11--HSD type 1 activity in both healthy women and those with hypopituitarism [27, 28].

Diagnosis of ACTH Insufficiency

Adrenal insufficiency can be diagnosed on the basis of a morning cortisol value !100 nmol/l [29]. Subjects with a value 1500 nmol/l do not need further testing unless the purpose of the test is to exclude GHD [30]. The insulin tolerance test (ITT) has been considered the reference gold standard for assessing the adequacy of the HPA axis since hypoglycaemia (!2.2 mmol/l) is a powerful stimulus that increases all stress hormones including GH, cortisol and catecholamines [5, 31] and tests the HPA axis at all levels. An intact axis is indicated by a peak cortisol level of 1500 nmol/l. However, the ITT is contraindicated in certain patients and is labour-intensive. Sustained secondary adrenal insufficiency leads to reduced ACTH receptor expression in the adrenal gland, as Management of Glucocorticoid Replacement

ACTH upregulates its own receptor [32]. Reports in the literature have consistently found a highly significant correlation (r = 0.92) between results of the short ACTH test (SST) and the ITT [33, 34]. Various studies have evaluated the 30- and 60-min cortisol responses of the 250g SST and incremental rise in SST. The 30-min cortisol response to SST has become increasingly accepted as an alternative to the ITT [34]. Since administration of 250 g ACTH in the SST represents a massive supraphysiological challenge, a lowdose ACTH test has been proposed as a more sensitive test [35, 36]. Results of a study by Abdu et al. indicated that both SST and low-dose SST, at a cut-off in cortisol response of 600 nmol/l, are safe for the purpose of clinical decision-making with regard to steroid replacement therapy in patients with pituitary disease. As the lowdose SST produced no falsely reassuring decisions, the authors suggested that this could replace the SST and ITT for initial evaluation of the HPA axis in patients with pituitary disease [37]. Results of the SST are known to be unreliable within 2 weeks of pituitary surgery or other acute pituitary insult [29, 38]. However, a 10-year follow-up study demonstrated the safety of the SST in assessing the HPA and had a predictive value of 97% in excluding ACTH deficiency after pituitary surgery [39]. Furthermore, in a recent study of 148 patients with pituitary disease who had cortisol responses in the 2.5 to 15th percentiles (510–635 nmol/l) of normal according to the 30-min high-dose Horm Res 2007;67(suppl 1):155–164

157

Table 3. Metabolic profiles of GCs used in cortisol replacement therapy

Duration, h T½, h Mineralocorticoid effect Anti-inflammatory effect Equipotent dose, mg/day

Hydrocortisone

Cortisone acetate

Prednisolone

Dexamethasone

8–12 11/2 + + 20

8–12 2 + + 25

Intermediate 2–3 (+) ++ 5

36–72 4 – ++ 0.67

Dose equipotent schemes by Liddle [47] and Meikle and Tyler [48].

SST, only 2 patients developed adrenal insufficiency during follow-up (mean, 4.2 years) that was not otherwise explained by subsequent pituitary surgery or radiotherapy. These findings suggest that the 250-g SST is a safe test [40]. Nevertheless, in actual clinical practice, clinicians vary widely in their preferences as to which test to use and when to conduct it [41].

GC Replacement Therapy

The aims of GC replacement therapy are to mimic the circadian serum steroid profile, to respond to the increased need for cortisol during physical and physiological stimulation and to achieve normal well-being, normal metabolism and favourable long-term outcome. However, these complex systems are hard to mimic during replacement therapy, and the lack of a serum marker to assess the tissue activity of cortisol makes monitoring of replacement therapy difficult. There are several limitations to using urinary 24-hour free cortisol levels to assess replacement therapy [42]. Some researchers have advocated using serum cortisol day curves [43, 44], but these are only partially helpful. Thus, in the absence of objective variables for monitoring the adequacy of replacement therapy, the physician mainly has to rely on clinical signs and symptoms. Signs and symptoms of undertreatment include malaise, postural hypotension, poor response to stress, electrolyte disturbances and even acute adrenal crises [45]. In adults, excessive GC replacement may induce glucose intolerance, abdominal obesity, hypertension, protein catabolism and osteoporosis [9, 10]. Types of GCs Cortisol is the endogenous active steroid, and hydrocortisone (HC) is the name of synthetic cortisol. In the 1950s, empirical chemical modifications of natural ste158

Horm Res 2007;67(suppl 1):155–164

roids led to development of a number of steroids with different structural features. Cortisone acetate (CA) is a synthetic analogue that is metabolised in the liver to be converted to HC. Both HC and CA have anti-inflammatory activity and mineralocorticoid effects and are short-acting (8 to 12 h), especially HC, which has a serum half-life of 1.7 h [46]. Prednisolone is another analogue with an intermediate duration of action and a greater anti-inflammatory effect than mineralocorticoid activity. The synthetic analogue DX has mainly antiinflammatory activity with no mineralocorticoid effect at all, and it is longer-acting, with a half-life of approximately 36 to 72 h [22] (table 3). HC is probably the most commonly used GC for replacement therapy. However, some European centres also use CA mainly for practical reasons. Prednisolone and DX have been used for replacement therapy in selected patients. In terms of GC effect, equivalent doses for these steroids have been estimated to be 20 mg HC, 25 mg CA, 5 mg prednisolone and 0.65 mg DX [47, 48]. However, most estimates of bioequivalency are based on the antiinflammatory effects of GCs and thus these estimates cannot be guaranteed to serve in replacement therapy. Differences in plasma clearance rates, interindividual variations in half-life – and the facts that GC effects on GC receptors are sustained even after the GC itself has been eliminated from serum and that the effect of synthetic analogues is sustained for a longer period of time compared with cortisol – could also contribute to different metabolic profiles in GC replacement therapies. Hepatic first-pass metabolism must be considered when evaluating the optimal drug and dosage for administration of GCs [49]. Two separate reports showing that orally administered HC is almost completely bioavailable [46, 50]. The mean bioavailability of CA has been found to be about 80% of that of HC [49], but in the individual, there is a high correlation between the two Filipsson/Johannsson

steroids (r = 0.87) [49]. From the clinical point of view, if reduced bioavailability is the reason for poor clinical response to oral CA, substitution of HC in equivalent doses will not solve the problem. Instead doses may be raised. Moreover, HC does not need to be metabolised, whereas CA needs to be converted by 11--HSD type 1 to become active. Inactivation of prednisolone by 11-HSD type 2 is more effective than the enzymatic inactivation of cortisol, which explains the reduced mineralocorticoid activity of prednisolone compared with HC [45].

GC Replacement in Hypopituitarism

11--HSD in GHD The use of GH in adults has raised interest in possible interactions between GH and other hormones. A wellknown side effect of GH treatment is water retention. The antinatriuretic effect of GH reported by Ho and Weissberger in 1990 could only partly be explained by activation of the renin-angiotensin system [51]. In 1994, Weaver et al. raised the possibility that this could be a mineralocorticoid effect, but their findings did not support this hypothesis [7]. Instead, they demonstrated that GH replacement was associated with a decrease in cortisol metabolites, a possible reduction in GC sensitivity and a decrease in CBG levels, findings that were also reported by others in 1996 [7, 52]. Subsequently, it was concluded that GH therapy in hypopituitary adults is associated with an apparent reduction in the availability of administered HC as measured by urine cortisol metabolites and urine free cortisol, suggesting that GH may directly or indirectly modulate the activity of 11--HSD type 1 [7, 53, 54]. The maximum effect of GH therapy occurs at very low doses and is not mediated by changes in circulating insulin levels [53]. In patients with acromegaly, 11--HSD type 1 activity is inhibited by GH and insulin-like growth factor I [55], and blockade of GH action with pegvisomant is associated with reversal of 11--HSD type 1 inhibition and correction of cortisol metabolism [56]. By contrast, 11-HSD type 1 activity is increased in GHD [7, 54], suggesting augmented tissue exposure to GCs (fig. 1B). This could explain some of the metabolic features associated with hypopituitarism and severe GHD (table 2). Moreover, these results suggest that GH therapy in adults with GHD alters the serum cortisol profile, with a reduced concentration of total cortisol in blood after oral administration of HC. Thus, a GHD patient not on GH therapy Management of Glucocorticoid Replacement

may be adequately replaced with a given dose of HC, but that dose may become inadequate after GH therapy commences [57]. This effect is not likely to be clinically significant except possibly in patients with partial or total ACTH deficiency, in whom cortisol replacement is suboptimal, resulting in a risk of clinical manifestations of cortisol deficiency after GH therapy commences [53]. The fact that most people do not become hypoadrenal when GH therapy begins may also be indicative of previous over-replacement with HC [57]. GH replacement therapy is associated with a significant increase in mean serum dehydroepiandrostenedione sulphate in ACTH-sufficient patients only [58]. This finding reflects either GH stimulation of adrenal androgen production in the permissive presence of ACTH or an inhibitory effect of GH on 11--HSD type 1 activity, leading to enhanced cortisol clearance, subsequent activation of the HPA axis and ACTH-mediated androgen secretion. Differences between Types of GCs for Treatment of Patients with GHD Several studies have suggested that there are important differences between CA and HC. As noted previously, CA requires conversion to cortisol by 11--HSD type 1 in the liver while HC does not. In untreated GHD patients, HC – but not CA in equivalent doses – can result in a supraphysiological cortisol tissue exposure, which is attenuated by GH replacement. In addition, patients treated with CA are more vulnerable to the inhibitory effect of GH on 11--HSD type 1 with a reduction in serum cortisol levels [59]. However, it is unlikely that the bioavailability of conventional doses of CA is impaired after GH replacement [60]. We have studied whether the choice of GC was of importance for metabolic outcome in a KIMS (Pfizer International Metabolic Database) survey of GC replacement in adult ACTH-insufficient hypopituitary patients compared with ACTH-sufficient subjects before and after 1 year of GH. Patients treated with HC and prednisolone/DX had increased HbA1c and waist-hip ratio, respectively, suggesting that they are less favourable choices of GC replacement than CA [61]. Dosing Regimens DX and prednisolone are more potent GCs with longer plasma half-lives and durations of action compared with HC and CA. Clinically, they are theoretically practical as they would need to be administered only once daily. However, they are not frequently used as replacement in hypocortisolism because prednisolone and DX do not reHorm Res 2007;67(suppl 1):155–164

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sult in the most physiological serum cortisol profiles as they do not imitate the circadian rhythm. When HC is administered twice daily, two thirds of the total daily dose is typically administered in the morning and the rest in the afternoon at 16.00 h. The result is a very low cortisol level early in the morning (24.00 to 08.00 h) and at midday (15.00 to 16.00 h) [62]. This may explain some of the afternoon malaise reported by patients on GC replacement, and it may partly be prevented by administering HC on a thrice-daily regimen [63]. Moreover, based on arbitrarily defined optimal serum levels of cortisol, 60% of patients receiving HC 3 times a day (10, 5 and 5 mg) had an acceptable daytime serum cortisol profile compared with 15% of those on a twicedaily regimen [43]. In a study of 32 consecutive patients attending an outpatient clinic for evaluation of ongoing GC treatment, results of 24-hour serum cortisol sampling and 24-hour urinary free cortisol excretion showed that 28 patients (88%) required a change of treatment [44]. Twenty-four (75%) patients needed a reduction of the mean daily HC dose from 29.5 8 1.2 mg (8SEM) to 20.8 8 1.0 mg/day, 18 (56%) patients needed a change in replacement therapy regimen or drug and 14 (44%) needed both. Barbetta et al. reported that thrice-daily administration of low-dose CA (12.5 mg at 07.00 h, 6.25 mg at 12.00 h and 6.25 mg at 17.00 h) achieved more physiological cortisol levels compared with twice-daily regimens, mainly because nadir serum cortisol levels were higher [64]. Dose Levels of GCs Historically, the dose of choice for patients with adrenal insufficiency was 30 mg/day of oral HC given in divided doses [65]. By using serum cortisol day curves and 24-hour urinary free cortisol measurements to determine the total daily HC or CA dose required for replacement therapy [43, 44], the mean daily dose was reduced to 20 mg HC [44] or 25 mg CA [64]. Numerous trials have studied the effect of GC on bone and cardiovascular risk factors [24]. Zelissen et al. found an inverse correlation between bone mineral density in the lumbar spine and increasing dose of HC per kilogram body weight in men with Addison disease, but not in women [66]. Wichers et al. conducted a 6-week randomised double-blind study of patients with secondary hypocortisolism who were treated for 2 weeks with either 15, 20 or 30 mg/day of HC. They found that osteocalcin levels (a marker of osteoblast activity) fell as HC doses increased. No differences were seen 160

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in the markers of bone resorption [67]. Since long-term treatment with high-dose GC replacement therapy (30 mg/day HC) induces bone loss, they advocate substitution therapy at doses of 20 mg or even 15 mg/day HC [67]. In 1995, al-Shoumer et al. reported that hypopituitary patients on GC replacement had reduced insulin sensitivity and diminished glucose tolerance on the mornings HC was given compared with mornings when no HC was administered [68]. In contrast, Dunne and colleagues [69] found no significant difference in fasting glucose or HbA1c levels following a reduction in HC dose from 30 to 15 mg over a 3-month period. This is in keeping with the findings of McConnell et al., who studied 15 patients with secondary hypocortisolism. They found no differences in glucose metabolism between patients treated with an HC intravenous infusion to mimic the physiological cortisol day curve and those treated with 15 mg + 5 mg HC administered orally [70]. This is consistent with our findings from the KIMS survey where HbA1c was not affected by dose levels of GC. Instead, we found a clear positive dose-response relation with body mass index, total cholesterol, low-density lipoprotein cholesterol and serum triglyceride levels. However, in patients receiving HCequivalent doses !20 mg, no metabolic abnormalities were found compared to those patients with an intact HPA axis [61].

Practical Recommendations

Chronic GC Replacement Food intake prolongs HC absorption compared to the fasted state and decreases its oral clearance rate [71]. Thus, we recommend that HC and CA be ingested in a fasting state to quickly attain serum concentrations of cortisol. In addition, the cytochrome P450 CYP3A4 isozyme is involved in the hepatic metabolism of GCs. Therefore, patients who are taking drugs that may increase (phenytoin, rifampin, barbiturates) or decrease (protease inhibitors) the function of this enzyme may need higher or lower doses of GCs, respectively [72]. A thrice-daily dosing regimen seems to be the most advantageous for HC. The ideal initial dose would be 10 mg HC in the morning upon waking, with the second dose of approximately 5 mg administered at around 12.00 to 14.00 h and the third dose of 2.5 to 5 mg administered in the evening between 18.00 and 19.00 h. If CA is to be administered, we recommend a twice-daily regimen. Although there are differences between twice- and thricedaily regimens of CA in nadir serum cortisol levels, there Filipsson/Johannsson

Table 4. Replacement regimens and treatment surveillance in

chronic GC replacement in hypopituitarism Glucocorticoid replacement Substitute the HPA axis before thyroxine replacement Fasting state HC 15–20 mg or CA 25 mg/day HC is given thrice daily with most of the dose in the morning CA could be given twice daily with one-half to two-thirds of the dose at wake-up Surveillance Signs and symptoms indicative of over or underreplacement Body weight, bone mineral density, blood lipids and glucose Verification of steroid emergency card Patient reinstruction in stress management

Important information to medical staﬀ regarding patients with cortisol deﬁciency In case of fever or other greater stress, for example operations or infections, you have to give an increased dose of cortisone. • For every degree of fever over 37ºC the hydrocortisone dose has to be increased with an extra dose of 20 mg/day. • For example at 39ºC you would give 40 mg extra distributed between morning and afternoon dose. When the patient is physically unable to keep the tablets, e.g. in case of vomiting and/or diarrhoea, the patient has to be submitted to the hospital where cortisone is given by injection supplemented with intravenous ﬂuids. Produced in cooperation with CEM, Sahlgrenska University Hospital, Göteborg and Pﬁzer AB (Sweden)

are no differences in mean serum cortisol measurements (table 4). Age and gender do not influence HC kinetics, though weight, height and body surface area do [71]. In fact, Mah et al. have proposed weight-based dosing schedules for HC thrice-daily regimens [71]. In our opinion, this needs further study before it becomes part of regular clinical practice. GC replacement therapy must be individualized, and active inquiry into the symptoms prompting under- and over-replacement is mandatory. There is evidence that doses 120 mg HC could be metabolically disadvantageous, but if lower doses are used, patients must have a good understanding of the symptoms of cortisol deficiency and their management. In patients who have mild secondary adrenal insufficiency and who are asymptomatic during everyday life, HC may be required only during periods of physical stress. In cases where the clinical picture of adequate replacement therapy is doubtful (e.g., tiredness is a common symptom, but it is not always caused by the ACTH insufficiency), characterisation of full serum cortisol profiles after a single HC dose is recommended [43]. Recently, a less time-consuming method of management has been advocated based on a single measurement of serum cortisol 240 min after morning HC ingestion [71]. However, this method is based on pharmacokinetics from an open-label trial that used invalidated tools for assessing patient preference and lacked long-term morbidity data. Caution. As mentioned previously, patients with untreated GHD are at risk of supraphysiological levels of HC. Moreover, the clinical significance of modulating 11-HSD type 1 activity after GH administration is uncertain. It is possible that GH initiation could precipitate Management of Glucocorticoid Replacement

Fig. 2. A poster to increase awareness of hypocortisolism.

overt ACTH insufficiency or necessitate an increase in the dose of GC replacement, but there is no evidence such events have actually occurred. Acute GC Therapy Thorough education about hypocortisolism and GC therapy is mandatory to avoid death and disability from severe acute cortisol insufficiency. In a series of 53 patients with chronic adrenal insufficiency, representing 511 years of replacement therapy, Arlt et al. noted an overall risk of adrenal crisis requiring hospital admission of 3.3 per 100 years. Risk of crisis was much higher among patients with primary adrenal insufficiency (3.8 vs. 2.5 per 100 years) and women (4.4 vs. 2.5 per 100 years). Most crises were due to a GC dose reduction or lack of stressrelated dose adjustment by the patient or family practitioner [42]. Flemming and Kristensen found that 46% of patients were not sufficiently skilled in coping with physical stress, and this finding was more prominent among elderly patients [73]. Knowledge and awareness are mandatory and ought to be compulsory at all levels: patients, relatives and health care personnel. To this end, our institution has developed a poster to increase the awareness of hypocortisolism in the emergency room (fig. 2). Patients are advised to increase their dose of HC by 20 mg for each degree of fever. Dose increases are also needed to cover more severe illnesses, surgical procedures, heavy exercise or mental stress. In cases of serious illness, when intake of a tablet is difficult (e.g., vomiting and diarHorm Res 2007;67(suppl 1):155–164

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rhoea), 100 mg HC intravenously or intramuscularly plus intravenous saline infusion should be administered. All patients also should receive a pocket card with the same information in case of emergency and a necklace with information about their need for GC replacement. Management of acute adrenal crisis consists of immediate intravenous administration of 100 mg HC followed by 50 mg HC administered either intramuscularly or intravenously every 6 h for the first 24 h [74]. For patients who are in shock, 5% dextrose in normal saline should be given intravenously initially, with subsequent saline-anddextrose therapy depending on the results of clinical and biochemical monitoring. The GC doses commonly administered during episodes of major stress are probably excessive and are not based on any clear evidence [72, 75]. Patients with intact adrenal function typically secrete between 75 and 150 mg/day in response to major surgery [75]. Salem et al. suggest that the amount and duration of GC coverage should be determined by the patient’s preoperative dose of GC, the preoperative duration of GC administration and the nature and anticipated duration of surgery [75]. According to Omori et al., sex steroid deficiency is the greatest risk factor for adrenal crisis, based on an assessment of a subclass of 115 patients with secondary adrenal insufficiency. Patients with untreated hypogonadism had a significantly higher relative risk of 3.70 (95% CI, 1.71–7.98) compared with those without hypogonadism or with treated hypogonadism. Furthermore, among pa-

tients under the age of 50 with hypogonadism, those treated with sex hormone experienced adrenal crisis less frequently (10%) than those who went untreated (64%, p = 0.0004) [76]. The authors conclude that sex hormone replacement therapy may reduce the risk of adrenal crisis.

Conclusion

Increasingly, the SST is being used for assessing the HPA axis instead of the ITT, but there is still considerable variation in how to interpret the results of this test [41]. Moreover, there is uncertainty as to when to initiate lifelong GC replacement in patients with partial ACTH insufficiency. Today we have a better understanding of and ability to interpret the effects of GH on other pituitary axes in patients with multiple pituitary hormone insufficiency. Still, replacement regimens have not changed much over the years. In general, we recommend ^20 mg/ day HC or ^25 mg/day CA as the drugs of choice, but with individual consideration for each patient. Future needs are to develop new preparations of GC replacement to normalise cortisol levels and to develop markers for monitoring GC replacement to improve care for hypopituitary patients who need replacement therapy. We also must continue to thoroughly educate patients, their relatives and health care personnel about how to avoid potential adrenal crises.

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Adult Workshop 4

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):165–172 DOI: 10.1159/000097574

Published online: February 15, 2007

Ten Essential Points about Body Water Homeostasis Joseph G. Verbalis Department of Medicine, Georgetown University Medical Center, Washington, D.C., USA

Key Words Arginine vasopressin Diabetes insipidus Hypernatremia Hyponatremia Syndrome of inappropriate antidiuretic hormone secretion

Abstract Background: Disorders of body fluids are among the most commonly encountered problems in the practice of clinical medicine. This is in large part because many different disease states can potentially disrupt the finely balanced mechanisms that control the intake and output of water and solute. It therefore behooves all clinicians treating such patients to have a good understanding of the pathophysiology, the differential diagnosis and the management of these disorders. Since body water is the primary determinant of the osmolality of extracellular fluid, disorders of body water homeostasis can be divided into hypoosmolar disorders, in which there is an excess of body water relative to body solute, and hyperosmolar disorders, in which there is a deficiency of body water relative to body solute. The classic hyperosmolar disorder is diabetes insipidus, and the classic hypoosmolar disorder is the syndrome of inappropriate antidiuretic hormone secretion. Conclusions: Despite the complexity of this regulatory system, most disorders of water homeostasis can be understood by applying knowledge of the physiology and pathophysiology of arginine vaso-

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pressin (AVP) secretion and effects, as summarized in the ten essential points of this review. Understanding therapy for disorders of water homeostasis, including appropriate use of the new AVP receptor antagonists, can similarly be best understood by appreciating these same essential points. Copyright © 2007 S. Karger AG, Basel

Introduction

Disorders of body fluids are among the most commonly encountered problems in the practice of clinical medicine. This is in large part because many different disease states can potentially disrupt the finely balanced mechanisms that control the intake and output of water and solute. Since body water is the primary determinant of the osmolality of extracellular fluid (ECF), disorders of body water homeostasis can be broadly divided into hypoosmolar disorders, in which there is an excess of body water relative to body solute, and hyperosmolar disorders, in which there is a deficiency of body water relative to body solute. The classic hyperosmolar disorder is diabetes insipidus (DI), and the classic hypoosmolar disorder is the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Because sodium is the main constituent of plasma osmolality, these disorders

Joseph G. Verbalis, MD Department of Medicine, Georgetown University Medical Center 3800 Reservoir Rd, NW, 5th Floor, PHC Bldg Washington, DC 20007 (USA) Tel. +1 202 444 7520, Fax +1 202 444 7797, E-Mail [email protected]

are typically characterized by hyponatremia and hypernatremia, respectively. Rather than attempt to summarize the entire field of disorders of body water homeostasis, this article will instead briefly review our understanding of ten essential points that the author considers the most important areas that clinicians should know when treating patients with endocrine disorders associated with alterations of body water homeostasis. The practical clinical importance of each point is explained after its presentation. Interested readers are referred to several excellent comprehensive reviews that cover all aspects of body water homeostasis for additional details [1–5]. Point #1: The entire AVP-stimulated signal transduction pathway is necessary for AVP-mediated antidiuresis. The prime determinant of free water excretion in animals and man is the regulation of urine flow by circulating levels of AVP in plasma, also known as antidiuretic hormone (ADH). AVP is a nine-amino acid peptide that is synthesized in specialized (magnocellular) neural cells located in the supraoptic and paraventricular nuclei of the hypothalamus. The synthesized peptide is enzymatically cleaved from its prohormone as it is transported to the posterior pituitary gland, where it is stored within neurosecretory granules until specific stimuli cause secretion of AVP into the bloodstream [3]. Antidiuresis then occurs via interaction of the hormone with its V2type receptors (V2R) in the kidney. Binding of AVP to V2R activates the Gs adenylyl cyclase system, increasing intracellular levels of cyclic adenosine monophosphate (cAMP). The cAMP activates protein kinase A, which in turn phosphorylates preformed aquaporin-2 (AQP2) water channels localized in intracellular vesicles of collecting duct epithelial cells. Phosphorylation promotes trafficking of the vesicles to the apical membrane, which is followed by insertion of AQP2 into the apical cell membranes. This is the rate-limiting step that renders the collecting duct permeable to water. AQP2 membrane insertion, and transcription, are reduced when AVP is either absent or chronically suppressed [6]. The clinical importance of this point is that any defect in the renal AVP-signaling system can lead to pathological inability to conserve body water. Thus, mutations of the V2R gene account for 90% of cases of congenital nephrogenic DI, and most of the remaining cases are caused by mutations of the AQP2 gene [7]. Consequently, antagonism of V2R represents a logical strategy for treatment of hyponatremia caused by SIADH, since blocking the initial activation of the AVP signal transduction system will prevent inappropriate antidiuresis. 166

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Fig. 1. Schematic representation of normal physiological relationships among plasma osmolality, plasma AVP concentrations, urine osmolality, and urine volume in man. Note particularly the inverse nature of the relation between urine osmolality and urine volume, resulting in disproportionate effects of small changes in plasma AVP concentrations on urine volume at lower AVP levels.

Point #2: At physiological concentrations, AVP acts primarily as an ADH; its pressor effects require higher concentrations stimulated by substantial volume depletion or hypotension. The primary renal response to AVP is an increase in water permeability of the kidney collecting tubules [6]. With refinement of radioimmunoassays for AVP, the unique sensitivity of this hormone to small changes in osmolality, as well as the corresponding sensitivity of the kidney to small changes in plasma AVP levels, have become apparent [8]. Most studies to date have supported the concept of a discrete osmotic threshold for AVP secretion, above which a linear relationship between plasma osmolality and AVP levels occurs (fig. 1). The slope of the regression line relating AVP to plasma osmolality can vary significantly across indiVerbalis

vidual human subjects, largely because of genetic factors [9]. In general, each 1 mOsm/kg H2O increase in plasma osmolality causes an increase in plasma AVP level from 0.4 to 0.8 pg/ml. The renal response to circulating AVP is similarly linear, with urine concentrations that are directly proportionate to AVP levels from 0.5 to 4 or 5 pg/ml, after which urine osmolality is maximal and cannot increase further despite additional increases in AVP levels (fig. 1). Thus, changes of 1% or less in plasma osmolality are sufficient to cause significant increases in plasma AVP levels with proportionate increases in urine concentration, and maximal antidiuresis is achieved after increases in plasma osmolality of only 5–10 mOsm/kg H2O (2–4%) above the osmotic threshold for AVP secretion. However, this analysis underestimates this system’s sensitivity to regulate solute-free water excretion, because urine volume is inversely related to urine osmolality. Thus, an increase in plasma AVP concentration from 0.5 to 2 pg/ml has a much greater relative effect on decreasing urine flow than does a subsequent increase in AVP concentration from 2–5 pg/ml, thereby further magnifying the physiological effects of small changes in plasma AVP levels (fig. 1) [3]. The net result of these relations is a finely tuned regulatory system that adjusts the rate of solute-free water excretion very accurately to the ambient plasma osmolality via quite small changes in pituitary AVP secretion. The rapid response of pituitary AVP secretion to changes in plasma osmolality, coupled with the short half-life (10–20 min) of AVP in human plasma, enables this regulatory system to adjust renal water excretion to changes in plasma osmolality on a minute-tominute basis. Hypovolemia also is a stimulus for AVP secretion, since an appropriate physiological response to volume depletion should include urinary concentration and renal water conservation. But AVP secretion is much less sensitive to small changes in blood volume and blood pressure than to changes in osmolality [9]. Such marked differences in AVP responses represent evidence that osmolality exhibits a more sensitive regulatory system for water balance than does blood or ECF volume. Nonetheless, modest changes in blood volume and pressure influence AVP secretion indirectly, even though they are weak stimuli by themselves. This occurs via shifting the sensitivity of AVP secretion to osmotic stimuli so that a given increase in osmolality will cause a greater secretion of AVP under hypovolemic conditions than in euvolemic states [9]. Furthermore, whereas maximal antidiuresis is achieved with relatively low osmotically stimulated levels

of AVP (5–10 pg/ml), the well-known vasoconstrictive effects of AVP are not activated until much higher hypovolemia-stimulated plasma levels are achieved (i.e., 115–20 pg/ml) [10]. The clinical importance of this point is that antagonism of the AVP V1a-type receptors, which are responsible for AVP-mediated vasoconstriction of vascular smooth muscle cells, would not be predicted to affect baseline blood pressure, since ambient baseline plasma AVP levels controlled by osmotic stimulation are too low to exert significant vasopressor effects. Point #3: Plasma AVP levels do not need to be high to cause inappropriate antidiuresis and hypoosmolality. Although measurable plasma AVP levels are found in most patients with SIADH, they are rarely elevated into pathological ranges in the vast majority of cases, even those associated with ectopic AVP production from tumors [11]. Rather, in the majority of cases of SIADH, plasma AVP levels remain in ‘normal’ physiological ranges, which only become abnormal under hypoosmolar conditions when plasma AVP levels should be suppressed into unmeasurable ranges [2, 3]. Recent studies of patients with SIADH and hypopituitarism found high levels of urine AQP2 excretion, supporting persistent activation of AVP V2R as the cause of the water retention in these disorders [12]. The clinical importance of this point is that ‘normal’ plasma AVP levels, or only mildly elevated urine osmolalities, cannot be used as arguments against SIADH as an etiology for hyponatremia. Low but nonsuppressible levels of AVP can clearly cause sufficient impairment of solute-free water excretion to produce hypoosmolality when exogenous fluid intakes are high, as in psychiatric patients with polydipsia [13]. Point #4: Hyponatremia is the most common electrolyte disorder of hospitalized adult patients around the world, and is associated with poor outcomes in multiple disease states. The incidence and prevalence of hypoosmolar disorders depends on both the nature of the patient population being studied and the laboratory methods and diagnostic criteria used to ascertain hyponatremia. When hyponatremia is defined as a serum [Na+] of less than 135 mEq/l, incidences as high as 15–30% have been observed in studies of both acutely [14] and chronically [15] hospitalized patients. Incidences decrease to the range of 1 to 4% when only patients with a serum [Na+] under 130 or 131 mEq/l are included [16, 17], and this is a more appropriate level to define the occurrence of clinically significant cases of this disorder. Even using these more stringent criteria to define hypoosmolality, inci-

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Fig. 2. Schematic diagram of brain volume adaptation to hypona-

tremia. Under normal conditions brain osmolality and ECF osmolality are in equilibrium (top panel; for simplicity the predominant intracellular solutes are depicted as K+ and organic osmolytes, and the extracellular solute as Na+). Following the induction of ECF hypoosmolality, water moves into the brain in response to osmotic gradients producing brain edema (middle panel, #1). However, in response to induced swelling, the brain rapidly loses both extracellular and intracellular solutes (middle panel, #2). As water losses accompany brain solute losses, the expanded brain volume then decreases back toward normal (middle panel, #3). If hypoosmolality is sustained, brain volume eventually normalizes completely and the brain becomes fully adapted to ECF hyponatremia (bottom panel).

dences from 7 to 53% have been reported in institutionalized geriatric patients [18, 19]. Although most cases are mild, hyponatremia has been associated with poor clinical outcomes across a wide variety of disease states including heart failure, acute myocardial infarction, hepatic cirrhosis, tuberculosis and 168

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childhood diarrhea, and for many of these diseases hyponatremia has been shown to be an independent predictor of morbidity and mortality [20]. In addition, recent studies have suggested that even patients with ‘asymptomatic’ hyponatremia may have significant cognitive disturbances and gait disorders that improve following correction of hyponatremia [21]. The clinical importance of this point is that a thorough evaluation of all hyponatremic patients is indicated, regardless of the clinical setting in which they present, both to detect underlying diseases and to potentially improve clinical outcomes. Point #5: The neurological symptomatology of hyponatremia is due to cerebral edema, the severity of which is modulated by the process of brain volume regulation. Whole brain volume regulation via electrolyte losses was first described by Yannet in 1940 and has long been recognized as the mechanism by which the brain is able to adapt to hyponatremia and limit brain edema to sublethal levels [22–24]. Following the recognition that low-molecular-weight organic compounds, called organic osmolytes, also constituted a significant osmotic component in a wide variety of cell types, multiple groups have shown that the brain loses organic osmolytes in addition to electrolytes during the process of volume regulation to hypoosmolar conditions in experimental animals and patients. These losses occur relatively quickly (within 24 to 48 h in rats) and can account for as much as one third of brain solute losses during hyponatremia [25]. Such coordinate losses of both electrolytes and organic osmolytes from brain cells enable very effective regulation of brain volume during chronic hyponatremia (fig. 2). Significant neurological symptoms generally do not occur until serum [Na+] falls below 125 mEq/l, and the severity of symptoms can be roughly correlated with the degree of hypoosmolality [26, 27]. However, individual variability is marked, and for any single patient, the level of serum [Na+] at which neurological symptoms will appear cannot be predicted with great accuracy. The clinical importance of this point is that much of the variability in the presentation of hyponatremic patients can be understood within the framework provided by the process of brain volume regulation (fig. 2). Most of the neurological symptoms associated with acute hyponatremia are caused by brain edema as a result of osmotic water movement into the central nervous system. Once the brain has volume-adapted via solute losses, thereby reducing brain edema, neurological symptoms are not as prominent and may even be virtually absent. This accounts for the fairly common finding of relatively asympVerbalis

tomatic patients despite severe levels of hyponatremia [27, 28]. It is also well known from animal studies that the rate of serum [Na+] decrease is often more strongly correlated with morbidity and mortality than is the actual magnitude of the decrease [26]. This is due to the fact that the volume-adaptation process takes a finite period of time to complete; the more rapid the fall in serum [Na+], the more brain edema will be accumulated before the brain is able to lose solute and along with it part of the increased water content. These effects are responsible for the much higher incidence of neurological symptoms, as well as the higher mortality rates, in patients with acute hyponatremia than in those with chronic hyponatremia [26, 29]. This phenomenon also explains the observation that the most dramatic cases of death due to hyponatremic encephalopathy have been reported in postoperative patients in whom hyponatremia often develops rapidly as a result of intravenous infusion of hypotonic fluids [30], or with hyponatremia during endurance exercise as a result of excess water ingestion [31]. Point #6: Osmotic demyelination is an immunologically mediated process that is initiated by osmotic disruption of the blood-brain barrier. Despite the obvious survival advantages afforded by brain volume regulation in response to hyponatremia, every adaptation made by the body in response to a perturbation of homeostasis bears with it the potential to create a new set of problems. This is true for brain volume regulation as well. Over the last decade it has become apparent that the demyelinating disease of central pontine myelinolysis occurs with a significantly higher incidence in patients with hyponatremia [32]. In both animal and human studies, brain demyelination has clearly been shown to be associated with the correction of existing hyponatremia rather than simply with the presence of severe hyponatremia itself [33]. Although the mechanism(s) by which correction of hyponatremia leads to brain demyelination remain under investigation, this pathological disorder likely is precipitated by brain dehydration that occurs following correction of serum [Na+] toward normal ranges in animal models of chronic hyponatremia. Because the degree of osmotic brain shrinkage is greater in animals that are maintained chronically hyponatremic than in normonatremic animals undergoing similar increases in plasma osmolality, by analogy the brains of human patients adapted to hyponatremia are likely to be particularly susceptible to dehydration following subsequent increases in osmolality, which in turn leads to pathological demyelination in some.

Recent magnetic resonance studies have shown that chronic hypoosmolality predisposes rats to opening of the blood-brain barrier following rapid correction of hyponatremia [34] and that the disruption of the bloodbrain barrier is highly correlated with subsequent demyelination [35]. A potential mechanism by which blood-brain barrier disruption might lead to subsequent myelinolysis is via influx into the brain of complement, which is toxic to the oligodendrocytes that manufacture and maintain myelin sheaths of neurons [36]. The clinical importance of this point is that maintaining correction rates of hyponatremia below levels associated with osmotic disruption of the blood-brain barrier will prevent the development of osmotic demyelination following correction of hyponatremia, regardless of the method used to correct the serum sodium level. Point #7: Antagonism of the vasopressin V2 receptor represents the best method for treating AVP-mediated hyponatremias. Initial development of AVP receptor antagonists during the 1970s focused on peptide analogues derived from the selective V2R agonist desmopressin. In the late 1980s, it seemed likely that such agents would be successfully developed for use in humans. However, some of these agents proved to have partial V2R agonist properties in humans, and further development of peptide antagonists was abandoned. Using a functional screening strategy, several nonpeptide small-molecule AVP receptor antagonists were subsequently identified, and the first successful use of an orally active, nonpeptide V2R antagonist in humans was reported in 1993 [37]. Molecular modeling of AVP binding sites suggests that the nonpeptide antagonists penetrate deeper into the transmembrane region of the V2R than does native AVP, thereby preventing binding of native hormone without the nonpeptide antagonists themselves interacting with the H1 helix site that is critical for V2R-mediated G-protein activation [38]. Consequently, binding of the antagonists to the V2R blocks activation of the receptor by endogenous AVP. The increased urine output produced by V2R antagonists is quantitatively equivalent to loop diuretics such as furosemide, but qualitatively it is much different in that only water excretion is produced without significant increases in urine solute excretion, including sodium and potassium [37]. Thus, AVP V2R antagonists produce a solute-free water excretion in contrast to classic diuretic agents that cause both water and electrolyte excretion by virtue of their effects on inhibiting tubular sodium transporters.

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The clinical importance of this point is that the renal effects produced by AVP V2R antagonists must be differentiated from the renal effects produced by classic diuretic agents. Both increase urination, but AVP V2R antagonists increase solute-free water excretion alone, or ‘aquaresis’, whereas diuretic agents increase not only water excretion but stimulate natriuresis and kaliuresis as well. Point #8: Transsphenoidal hypophysectomy-induced hyponatremia is a variant of the triphasic response (‘isolated second phase’). In some cases, transient postoperative DI is part of a ‘triphasic’ pattern that has been well described following pituitary stalk transection [39]. The initial DI (first phase) is due to axon severance with lack of function of the damaged neurons. This phase lasts from several hours to several days, and then is followed by an antidiuresis second phase due to uncontrolled release of AVP from the disconnected and degenerating posterior pituitary. Overly aggressive administration of fluids during the second phase does not suppress AVP secretion and can lead to hyponatremia. The antidiuresis can last from 2 to 14 days, after which DI recurs (third phase) following depletion of the AVP stores from the degenerating posterior pituitary. More recently, transient hyponatremia without preceding or subsequent DI has been reported following transsphenoidal surgery for pituitary microadenomas [40]. This generally occurs 5–10 days postoperatively, and the incidence may be as high as 30% when such patients are carefully followed, although the majority of cases are mild and self-limited [41]. It is due to inappropriate AVP secretion via the same mechanism as the triphasic response, except that in these cases only the second phase occurs (‘isolated second phase’) because the neural lobe/ pituitary stalk damage is not severe enough to impair AVP secretion sufficiently to produce the clinical manifestations of DI [42]. The clinical importance of this point is that posttranssphenoidal hyponatremia can be best understood as a variant of the triphasic response, in which sufficient neurohypophyseal cells remain functional to avoid DI, but enough stalk damage occurs to denervate a critical number of AVP axon terminals in the posterior pituitary to cause transient SIADH as the damaged terminals later degenerate and release AVP stores. Point #9: Exercise-associated hyponatremia (EAH) is a variant of SIADH produced by low AVP levels in combination with excess drinking. Over the past two decades, EAH has emerged as an important complication of endurance physical activities. EAH is defined as the occur170

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rence of hyponatremia in individuals engaged in prolonged physical activity who develop a serum sodium concentration below the normal reference range of the laboratory performing the test, generally !135 mmol/l [43]. EAH has typically been classified as a form of volume depletion-related hyponatremia as a result of loss of sodium and chloride in sweat during exercise. However, recent evidence has indicated that excessive water ingestion is principally responsible for EAH. Detailed balance studies performed during recovery from ultramarathon races show that, compared with normonatremic finishers, those with EAH actually lose less sodium although they develop a greater plasma volume deficit. During recovery, runners with EAH excrete a large volume of dilute urine, whereas normonatremic finishers excrete a small volume of highly concentrated urine [44]. The change in serum sodium concentration after endurance exercise is inversely proportional to the change in body weight, and the athletes with EAH tend to gain weight during exercise [45]. In marathon runners, low body mass index, race time exceeding 4 h, consumption of fluids every mile, following advice to ‘drink as much as possible’ during the race and greater frequency of urination during the race have all been associated with EAH; in some but not all studies, female sex and the use of nonsteroidal anti-inflammatory drugs were also risk factors [46]. Thus, most runners with EAH are overhydrated as a result of excessive water ingestion over an extended race time [43, 47] during which water excretion is impaired by low levels of nonosmotically stimulated AVP secretion [48]. The clinical importance of this point is that individuals participating in endurance activities should never be told to overdrink as a means of avoiding dehydration; instead, they should drink according to their thirst, or follow recently recommended guidelines for assessing fluid intake during endurance exercise [43]. Point #10: Osmoreceptor dysfunction represents a variant of DI in which osmotic regulation of AVP secretion is absent, but baroreceptor stimulation remains intact. Extensive animal literature indicates that the primary osmoreceptors that control AVP secretion and thirst are located in the anterior hypothalamus. Lesions in this region in animals cause hyperosmolality through a combination of impaired thirst and osmotically stimulated AVP secretion [49]. Initial reports in humans described this syndrome as ‘essential hypernatremia’, and subsequent studies used the term ‘adipsic hypernatremia’ in recognition of the profound thirst deficits found in Verbalis

most. However, all of these syndromes represent disorders of osmoreceptor function. Four major patterns of osmoreceptor dysfunction have been described as characterized by defects in thirst and/or AVP secretory responses [50]. In contrast to lesions causing central DI, these lesions usually occur more anteriorly in the hypothalamus, consistent with the location of the primary osmoreceptor cells [9]. The clinical importance of this point is that in patients with osmoreceptor dysfunction, afferent neural pathways from the brainstem to the hypothalamus remain intact. Consequently, these patients will usually have normal AVP and renal-concentrating responses to baroreceptor-mediated stimuli such as hypovolemia and hypotension [50]. This can lead to confusion regarding the diagnosis, since at times these patients appear to have DI and at other times they appear to concentrate urine quite normally.

Conclusion

Body water homeostasis is accomplished by complex processes that regulate both the osmolality and volume of body fluids within very narrow tolerances. Disorders of body water homeostasis can result from a variety of derangements that impair normal pituitary secretion of AVP or normal end-organ activation of AVP receptors in the kidneys. Despite the complexity of this regulatory system, most disorders of water homeostasis can be understood by applying knowledge of the physiology and pathophysiology of AVP secretion and effects, as summarized in the essential points of this review. Understanding therapy of disorders of water homeostasis, including appropriate use of the new AVP receptor antagonists, can similarly be understood by appreciating these essential points.

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38 Macion-Dazard R, Callahan N, Xu Z, Wu N, Thibonnier M, Shoham M: Mapping the binding site of six nonpeptide antagonists to the human V2-renal vasopressin receptor. J Pharmacol Exp Ther 2006;316:564–571. 39 Verbalis JG, Robinson AG, Moses AM: Postoperative and post-traumatic diabetes insipidus; in Czernichow P, Robinson AG (eds): Diabetes Insipidus in Man. Basel, Karger, 2006, vol 13, pp 247–265. 40 Cusick JF, Hagen TC, Findling JW: Inappropriate secretion of antidiuretic hormone after transsphenoidal surgery for pituitary tumors. N Engl J Med 1984;311:36–38. 41 Olson BR, Rubino D, Gumowski J, Oldfield EH: Isolated hyponatremia after transsphenoidal pituitary surgery. J Clin Endocrinol Metab 1995;80:85–91. 42 Ultmann MC, Hoffman GE, Nelson PB, Robinson AG: Transient hyponatremia after damage to the neurohypophyseal tracts. Neuroendocrinology 1992;56:803–811. 43 Hew-Butler T, Almond C, Ayus JC, Dugas J, Meeuwisse W, Noakes T, Reid S, Siegel A, Speedy D, Stuempfle K, Verbalis J, Weschler L: Consensus statement of the 1st international exercise-associated hyponatremia consensus development conference, Cape Town, South Africa. Clin J Sport Med 2005; 15:208–213.

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44 Irving RA, Noakes TD, Buck R, van Zyl Smit R, Raine E, Godlonton J, Norman RJ: Evaluation of renal function and fluid homeostasis during recovery from exercise-induced hyponatremia. J Appl Physiol 1991; 70: 342– 348. 45 Speedy DB, Noakes TD, Rogers IR, Thompson JM, Campbell RG, Kuttner JA, Boswell DR, Wright S, Hamlin M: Hyponatremia in ultradistance triathletes. Med Sci Sports Exerc 1999;31:809–815. 46 Almond CS, Shin AY, Fortescue EB, Mannix RC, Wypij D, Binstadt BA, Duncan CN, Olson DP, Salerno AE, Newburger JW, Greenes DS: Hyponatremia among runners in the Boston marathon. N Engl J Med 2005; 352: 1550–1556. 47 Noakes T: Fluid replacement during marathon running. Clin J Sport Med 2003; 13: 309–318. 48 Siegel AJ: Exercise-associated hyponatremia: role of cytokines. Am J Med 2006; 119: S74–S78. 49 Johnson AK, Buggy J: Periventricular preoptic-hypothalamus is vital for thirst and normal water economy. Am J Physiol 1978;234: R122–R129. 50 Baylis PH, Thompson CJ: Diabetes insipidus and hyperosmolar syndromes; in Becker KL (ed): Principles and Practice of Endocrinology and Metabolism. Philadelphia, Lippincott Williams & Wilkins, 2001, pp 285–293.

Verbalis

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):173 DOI: 10.1159/000097575

Published online: February 15, 2007

Adult Clinical Case Sessions

There are intricate metabolic balances that must be addressed in any pituitary disorder or following trauma. As we learn more about these balances, medical therapies, whether as a single medication or in combination, are easier to tailor for individual patients. The adult clinical sessions address some of these metabolic issues. The first case, ‘Positive Metabolic Impact of Treatment with Pegvisomant in an Acromegalic Patient’, examined two complications of acromegaly: glucose intolerance and diabetes mellitus. This diabetic subject with acromegaly proved to be resistant to treatment with somatostatin analogues (SSAs), but treatment with pegvisomant resulted in a normalisation of insulin-like growth factor I (IGF-I) and glucose levels, with insulin and HbA1C levels gradually decreasing as well. Also, during treatment with SSA, the subject required an increased dosage of hypoglycemic agents, while with pegvisomant the amount of hypoglycemic agents needed to stabilise serum glucose levels was reduced. The occurrence of depression after traumatic brain injury (TBI) is discussed in ‘Depression following Traumatic Brain Injury Associated with Isolated Growth Hormone Deficiency: Two Case Reports’. The relationship between depression and brain injury has only recently been recognised. As many as 15% of adults with a TBI have severe and isolated growth hormone deficiency (GHD). Adult GHD is associated with body composition changes, quality-of-life impairment and depression. Two adults who suffered a TBI several years ago were evaluated and found to have isolated GHD and depression. Prior to the head injury, neither subject had any psychiatric or endocrine morbidity.

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0173$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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‘Hypodipsic Hypernatremia after Hypothalamic Infarct’ focuses on the relatively rare condition of hypothalamic hypodipsic syndrome. Hypothalamic hypodipsic syndrome is frequently associated with defective osmoregulated vasopressin secretion and diabetes insipidus, and this combination results in a greater risk of hypernatremia. Management of this hypernatremia consists of regulating vasopressin and water intake in relation to changes in daily body weight. The case presented concerns a young woman who experienced a complicated course of recovery and rehabilitation after suffering a postoperative cerebral infarct. Complications included an extensive hypothalamic syndrome, including hypodipsic hypernatremia, diabetes insipidus, hyperthermia, profound short-term memory loss and initial severe anorexia, followed by hyperphagia with an inability to maintain body weight. Fluid management was very difficult in this amnesic patient. The clinical case presentation ‘Drug-Induced Hepatitis During Combined Treatment with Pegvisomant and Octreotide LAR’ presented by Hans Feenstra was published elsewhere (Feenstra et al., Eur J Endocrinol 2006; 154:805–806). Felipe F. Casanueva Santiago de Compostela University Santiago de Compostela, Spain Ezio Ghigo Division of Endocrinology and Metabolism Department of Internal Medicine University of Turin, Turin, Italy

Adult Clinical Case Sessions

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):174–176 DOI: 10.1159/000097576

Published online: February 15, 2007

Positive Metabolic Impact of Treatment with Pegvisomant in an Acromegalic Patient Silvia Grottoli Valentina Gasco Alessandra Mainolfi Damiano De Giorgio Ezio Ghigo Division of Endocrinology and Metabolism, University of Turin, Turin, Italy

Key Words Acromegaly Diabetes mellitus Medical treatment Somatostatin analogues Pegvisomant

Abstract Background: Glucose intolerance and diabetes mellitus are important complications of acromegaly. Somatostatin analogues (SSAs) exert a dual effect on carbohydrate metabolism. Normalisation of the growth hormone/insulin-like growth factor I (GH/IGF-I) axis could improve metabolic balance, but inhibition of insulin secretion could worsen glucose tolerance. Case Description: We report a case of an acromegalic patient with diabetes mellitus that was resistant to SSA therapy. Results: Treatment with SSAs typically induces mild reductions in GH and IGF-I levels, but rarely results in normalisation of these parameters. In addition, glucose levels as well as glycosylated haemoglobin levels (HbA1c) show marked deterioration. In contrast, pegvisomant (PEG) treatment normalises IGF-I levels, and glucose, insulin and HbA1c levels gradually decrease. During SSA therapy, this patient required an increase in the dose and type of hypoglycaemic agents. During PEG treatment, however, hypoglycaemic drugs are often reduced. Conclusion: Patients with acromegaly whose disease is inadequately controlled by SSA therapy can achieve normalisation of IGF-I levels and full control of the disease, including strong improvement of glucose homeostasis despite marked reduction of antidiabetic therapy, when treated with the GH antagonist, PEG. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0174$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

Acromegaly, a chronic and systemic disease caused by growth hormone (GH) and insulin-like growth factor I (IGF-I) hypersecretion, is characterised by disabling symptoms and relevant comorbidities, namely, diabetes mellitus and cardiovascular complications, which are the primary causes of premature death [1]. The control of symptoms and comorbidities and likely survival can be achieved by normalising IGF-I concentrations. Pegvisomant (PEG), a GH receptor antagonist, is able to normalise IGF-I levels in more than 90% of patients [2]. Thus, PEG is the treatment of choice for many patients with acromegaly in whom the disease is inadequately controlled with other treatments. Dose titration of PEG is important to achieve its maximum influence on glucose metabolism in patients with acromegaly.

Case Report A 53-year-old male with acromegaly due to a pituitary adenoma was referred to our clinic for evaluation of resistant disease. The diagnosis was made in 1990 based on typical somatic modifications. Hormonal evaluation revealed a very high GH level (200 g/l), no suppression during an oral glucose tolerance test and an increased age-matched IGF-I (982 g/l) level. A magnetic resonance imaging scan (MRI) showed an extrasellar pituitary adenoma (2.5 cm). In 1991, the patient underwent transsphenoidal surgery followed by conventional radiotherapy for persistent active disease and residual adenomatous tissue. SSA therapy was started in 1992 and resulted in complete shrinkage of the adeno-

Silvia Grottoli, MD Division of Endocrinology and Metabolism Department of Internal Medicine, University of Turin IT–10126 Turin (Italy) Tel. +39 011 633 4317, Fax +39 011 664 7421, E-Mail [email protected]

Table 1. Hormonal and metabolic parameters during SSA treatment

SSA Lanreotide 30 mg

GH, g/l IGF-I, g/l Glucose, mmol/l HbA1c, % Diabetic therapy

7.6 810 8.3 7.5 Diet

Table 2. Hormonal and metabolic

7.1 2.6 827 650 12.5 9.5 8.4 8.5 Tolbutamide Glimepiride

3.9 723 10.1 9.3 Glyburide/metformin

Lanreotide 120 mg PEG during SSA treatment 10 mg

parameters during PEG treatment GH, g/l IGF-I, g/l Glucose, mg/dl HbA1c, % Glyburide/metformin, tabs/day

ma. During a routine follow-up visit in 1999, an MRI showed a secondary empty sella. GH and IGF-I levels still indicated active disease. Relevant medical history included anterior hypopituitarism, effectively compensated; obesity with obstructive sleep apnoea syndrome, and diabetes mellitus. Diabetes mellitus had been diagnosed in 1994; however, a progressive worsening of glycemic control was observed despite dose titration of hypoglycaemic agents (a sulfonylurea agent alone or with metformin) (table 1). In 2003, the patient was enrolled in a trial of PEG (from 10 up to 20 mg/day). This treatment was associated with a progressive reduction in IGF-I levels that eventually normalised following treatment with PEG 20 mg/d. Blood glucose levels (nadir vs. basal: 5.1 vs. 8.3 mmol/l) and HbA1c (6.4 vs. 9.3%) also decreased. This effect was clearly dose-dependent and so remarkable that a progressive reduction in the dose of hypoglycaemic drugs was necessary (table 2). As expected, IGF-I normalisation was associated with a clear-cut improvement in the signs and symptoms of acromegaly, including quality-of-life scores. The patient reported a reduction of soft tissue swelling that was confirmed by ring size score. After 12 weeks of PEG, the patient complained of headaches and fatigue; an MRI demonstrated the persistence of an empty sella while the headache and worsening of a well-being score were shown to be associated with hypoglycaemia.

Discussion

This case report illustrates that patients with acromegaly inadequately controlled by SSA therapy can achieve normalisation of IGF-I levels and full control of the disease, including strong improvement in glucose homeostaMetabolic Impact of Treatment with Pegvisomant

Octreotide 30 mg Lanreotide 120 mg

3.9 723 10.1 9.3 3

5.3 626 6.4 7.3 3

15 mg

20 mg

13 540 5.4 6.7 1

14 335 5.1 6.4 0.5

sis, despite a marked reduction in the use of antidiabetic therapy. GH is an important regulator of glucose metabolism and insulin sensitivity. In fact, GH overexpression increases fasting glucose levels and insulin concentrations and reduces insulin sensitivity in transgenic animals [3]. The reduction of insulin sensitivity following exposure to elevated GH levels likely reflects inadequate suppression of hepatic glucose output and peripheral utilisation [4]. In mice, reduction in the number of insulin receptors at the level of the liver has also been demonstrated [5]. Moreover, GH receptor knockout mice show enhancement of insulin sensitivity [6]. In fact, 45% of patients with acromegaly have glucose intolerance or diabetes mellitus and most are insulin resistant [7, 8]. Native somatostatin greatly affects the ability of the endocrine pancreas to inhibit insulin or glucagon secretion; somatostatin is also able to delay glucose absorption at the gastrointestinal level [9]. In patients with acromegalic GH hypersecretion, treatment with SSA inhibits GH secretion and IGF-I levels, but may also have an opposite impact on glucose metabolism [10]. Improvement of insulin sensitivity sometimes occurs during SSA treatment and is likely to reflect a reduction in GH levels [11]. However, it has been reported that long-term treatment with SSA is associated with glucose tolerance deterioration in 30% of patients [10, 12]. Metabolic balance is especially important in acromegalic patients. Prospective epidemiological studies have demonHorm Res 2007;67(suppl 1):174–176

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strated a 2-fold increase in cardiovascular risk in nonacromegalic patients with impaired glucose tolerance [13]. Moreover, the United Kingdom Prospective Diabetes Study has shown that a reduction of just 1% in HbA1c in diabetic patients is enough to significantly reduce cardiovascular events [14]. Again, fasting plasma glucose predicts cardiovascular disease-related death, independent of other traditional risk factors in diabetic patients [13]. Previous studies demonstrated that treatment with PEG is associated with a clear improvement in glucose homeostasis in patients with acromegaly [15]. PEG improves insulin sensitivity and glucose tolerance in patients on stable SSA therapy [16]. Moreover, a recent multicentre study demonstrated that PEG improves glucose homeostasis in acromegalic patients with and without diabetes regardless of the patient’s baseline IGF-I status [17].

Conclusion

Our case report agrees with evidence that PEG provides a particular benefit to acromegalic patients in terms of glucose homeostasis. Blockade of the GH receptor by PEG in patients with acromegaly is very effective in achieving normalisation of IGF-I levels and also improving glucose homeostasis. This treatment should be considered not only in patients whose disease is inadequately controlled with SSA therapy, but more generally in patients with acromegaly and associated diabetes mellitus.

References 1 Bates AS, Van’t Hoff W, Jones JM, Clayton RN: An audit of outcome of treatment in acromegaly. Q J Med 1993;86:293–299. 2 Muller AF, Kopchick JJ, Flyvbjerg A, van der Lely AJ: Growth hormone receptor antagonists. J Clin Endocrinol Metab 2004; 89: 1503–1511. 3 Costa C, Solanes G, Visa J, Bosch F: Transgenic rabbits overexpressing growth hormone develop acromegaly and diabetes mellitus. FASEB 1998;12:1455–1460. 4 Rizza RA, Mandarino LJ, Gerich JE: Effects of growth hormone on insulin action in man. Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired stimulation of glucose utilization. Diabetes 1982;31:663–669. 5 Balbis A, Bartke A, Turyn D: Overexpression of bovine growth hormone in transgenic mice is associated with changes in hepatic insulin receptors and in their kinase activity. Life Sci 1996;59:1363–1371. 6 Yakar S, Setser J, Zhao H, Stannard B, Haluzik M, Glatt V, Bouxsein ML, Kopchick JJ, LeRoith D: Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1-deficient mice. J Clin Invest 2004;113: 96–105.

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7 Kasayama S, Otsuki M, Takagi M, Saito H, Sumaitani S, Kouhara H, Koga M, Saitoh Y, Ohnishi T, Arita N: Impaired -cell function in the presence of reduced insulin sensitivity determines glucose tolerance status in acromegalic patients. Clin Endocrinol 2000; 52: 549–555. 8 Hansen I, Taslikian E, Beaufrere B, Gerich J, Haymond M, Rizza R: Insulin resistance in acromegaly: defects in both hepatic and extrahepatic insulin action. Am J Physiol 1986; 250:269–273. 9 Krejs GJ, Brown R, Raskin P: Effect of intravenous somatostatin on jejunal absorption of glucose, amino acids, water and electrolytes. Gastroenterology 1980; 78:26–31. 10 Koop BL, Harris AG, Ezzat S: Effect of octreotide on glucose tolerance in acromegaly. Eur J Endocrinol 1994;130:581–586. 11 Pereira AM, Biermasz NR, Roelfsema F, Romijn JA: Pharmacologic therapies for acromegaly: a review of their effects on glucose metabolism and insulin resistance. Treat Endocrinol 2005; 4:43–53. 12 Ronchi C, Epaminonda P, Cappiello V, BeckPeccoz P, Arosio M: Effects of two different somatostatin analogs on glucose tolerance in acromegaly. J Endocrinol Invest 2002; 25: 502–507. 13 DECODE Study Group: Glucose tolerance and mortality: comparison of WHO and American Diabetes Association diagnostic criteria. Lancet 1999;354:617–621.

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14 UKPDS Group: Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998; 352: 837– 853. 15 van der Lely A, Hutson R, Trainer P, Besser G, Barkan A, Katznelson L, Klibanski A, Herman-Bonert V, Melmed S, Vance ML, Freda PU, Stewart PM, Friend KE, Clemmons DR, Johannsson G, Stavrou S, Cook DM, Phillips LS, Strasburger CJ, Hacker S, Zib KA, Davis RJ, Scarlett JA, Thorner MO: Long-term treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist. Lancet 2001;358:1754–1759. 16 Drake WM, Rowles SV, Roberts ME, Fode FK, Besser GM, Monson JP, Trainer PJ: Insulin sensitivity and glucose tolerance improve in patients with acromegaly converted from depot octreotide to pegvisomant. Eur J Endocrinol 2003;149:521–527. 17 Barkan AL, Burman P, Clemmons DR, Drake WM, Gagel RF, Harris PE, Trainer PJ, van der Lely AJ, Vance ML: Glucose homeostasis and safety in patients with acromegaly converted from long-acting octreotide to pegvisomant. J Clin Endocrinol Metab 2005; 90: 5684–5691.

Grottoli/Gasco/Mainolfi/De Giorgio/ Ghigo

Adult Clinical Case Sessions

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):177–179 DOI: 10.1159/000097577

Published online: February 15, 2007

Depression following Traumatic Brain Injury Associated with Isolated Growth Hormone Deficiency: Two Case Reports Nadja Maric a Sandra Pekic b Mira Doknic b Miroslava Jasovic-Gasic a Vlada Zivkovic b Marko Stojanovic b Branko Djurovic c Vera Popovic b Institutes of a Psychiatry, b Endocrinology and c Neurosurgery, University Clinical Center, Belgrade, Serbia

Key Words Depression Traumatic brain injury Growth hormone Therapy Case report

Abstract Background: Depression can be a sequela of a medical condition like traumatic brain injury (TBI), but the relationship between brain trauma and depression has not been recognized until recently. Eight to 15% of adults who sustain a TBI have severe and isolated growth hormone deficiency (GHD). It has been shown that GHD in adults is associated with changes in body composition, impairment of quality of life, and depression. Methods and Results: At the time of evaluation, two adult subjects who suffered TBI several years ago presented with isolated GHD together with depression. Prior to trauma, neither subject had psychiatric or endocrine morbidity. These case reports focus on opportunities and difficulties in deciding how best to treat them. Conclusions: Both depression and TBI are highly ranked in the list of burden of disease. The mechanism of depression in GH-deficient adults who have sustained a TBI should be clarified and the efficacy and tolerability of GH replacement therapy versus placebo versus antidepressant drugs should be assessed. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0177$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Introduction

Depression can occur as a result of a medical condition like traumatic brain injury (TBI) [1], but the underlying mechanisms are not understood. Recent studies have shown that 8–15% of adult subjects with TBI have severe and isolated growth hormone deficiency (GHD) [2–4]. GHD is associated not only with changes in body composition (i.e., decrease in lean body mass, increase in percent body fat, reduced volume of extracellular water) but also with impaired quality of life and depression [5]. Moreover, symptoms of depression in adults with GHD might improve with GH replacement treatment [6]. It is reasonable to assume that GHD might be the neuroendocrinological link in depression following TBI, particularly because a decrease in the synthesis of new neurons in the adult hippocampus might be linked to major depressive disorder [7] as well as to GHD [8]. Reduced insulin-like growth factor (IGF)-I levels may be a part of the biochemical cascade occurring in association with depression [9]; however, supporting literature is limited. Case Reports The cases of two subjects who suffered TBI several years ago are presented. Subjects were identified from a study of anterior pituitary dysfunction in survivors of TBI conducted by a multidisciplinary team at the University Clinical Center in Belgrade,

Nadja Maric, MD Institute of Psychiatry University Clinic Center of Serbia, Pasterova 2 YU–11000 Belgrade (Serbia) Tel. +381 11 306 5638, E-Mail [email protected]

which included comprehensive evaluations in several domains: endocrinological, neurological, neuropsychological and psychiatric [4]. Both subjects needed psychiatric help, which typically would have involved treatment with psychotropic drugs. Given the fact that they were both survivors of TBI, the cases illustrate the dilemma of whether the typical course of therapy would be the best approach, or if other therapeutic strategies could prove to be equal or superior in terms of effectiveness. Mr. A, a 56-year-old male, sustained head trauma 3 years ago (Glasgow Coma Score, 12). He had no contact with mental health services before the trauma, but had many psychiatric complaints after the injury. He underwent a detailed psychiatric evaluation using the structured screening Mini International Neuropsychiatric Interview (MINI) [10]. Results yielded a diagnosis of severe major depression with suicidality, with no comorbid psychiatric diagnoses. Additional psychometric evaluation using the Zung Depression Inventory [11] revealed severe depression. The Symptom Checklist-90-R (SCL-90-R) is an instrument from Pearson Assessments that evaluates a broad range of psychological problems and symptoms of psychopathology [12]. The SCL-90-R scale scores for phobia, anxiety, depression, and somatization were found to be above the normal range for the general adult male population. As required by the study protocol [4], an endocrine evaluation was performed and isolated GHD was discovered (GH peak after stimulation testing with GH-releasing hormone plus GH-releasing peptide-6 [GHRH + GHRP-6] was 4.5 g/l (A peak of GH concentration !10 g/l defined as severe GHD [13]). IGF-I was 69.9 ng/ml (range 81–225 ng/ml). Other pituitary hormone levels were within normal ranges: T4 was 139 nmol/l; TSH was 0.8 mU/l; testosterone was 19.0 nmol/l; prolactin was 337 mU/l, and cortisol (sample was taken after overnight fast, at 8.00 a.m.) was 251 nmol/l. Mr. A was obese with a body mass index (BMI) of 31.0 kg/m2. An oral glucose tolerance test (OGTT) showed glucose intolerance and the subject also had dyslipidemia (total cholesterol was 6.3 mmol/l and triglycerides were 3.3 mmol/l). Mr. A was diagnosed with major depressive disorder with suicidality and severe isolated GHD. It was difficult to determine the most appropriate course of treatment: (1) should antidepressants be prescribed first; (2) should GH replacement therapy be given first, or (3) should both therapies be given concomitantly? Mr. B, a 37-year-old male, sustained head trauma 9 years ago (Glasgow Coma Score, 6) with no psychiatric history at the time of evaluation. His mental status as assessed by MINI yielded a diagnosis of major depression with atypical features, with no comorbid psychiatric diagnoses. The Zung Depression Inventory [11] evaluation revealed mild-to-moderate depression while the SCL-90-R scale scores for phobia, depression, and somatization were above the normal range for general adult male population [12]. Endocrine evaluation revealed an abnormal GH peak after stimulation testing (GHRH + GHRP-6): 7.7 g/l with normal IGF-I (236.4 ng/ml, range 109–284 ng/ml). Other pituitary hormones were within the reference ranges: T4 was 94.8 nmol/l; TSH was 1.7 mU/l; testosterone was 32.4 nmol/l; prolactin was 319 mU/ l, and cortisol (sample was taken after overnight fast, at 8.00 a.m.) was 472 nmol/l. Mr. B was of normal weight (BMI was 24.7 kg/ m2). OGTT showed glucose intolerance and the subject also had hypercholesterolemia (total cholesterol, 6.8 mmol/l).

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The criteria for major depression with atypical features were fulfilled, together with isolated GHD. Again, the condition followed TBI without any prior psychiatric or endocrinological morbidity.

Discussion

The typical treatment regimen for both subjects consists of several predictable steps early in the treatment phase, but because the incidence of depression in these subjects is coupled with GHD, the decision of how best to alleviate their symptoms is debatable. In the first step, both subjects would seek help from their general practitioners (if they decided to seek any medical treatment at all). In the second step, the subjects would likely be referred to a psychiatrist who would typically prescribe an antidepressant with or without cognitive-behavioral psychotherapy. Follow-up over the next few weeks (or months) would assess the effects of therapy. It is at this point in the treatment regimen that the most effective course of action is difficult to predict. Recent studies have shown that major depressive disorder often requires more than one step of treatment to elicit a remission of symptoms [14, 15]. Therefore, introduction of one or more additional drugs or recommendation to begin electroconvulsive therapy may be expected. Unfortunately, it is highly unlikely that an endocrine evaluation focused on pituitary function would be conducted; thus depression as a consequence of GHD after TBI would not be recognized, nor would GH replacement therapy be recommended. It is unknown whether TBI subjects with GHD and depression will improve with GH replacement therapy, how long the effects would last, and what side-effects might be expected. A study conducted in 25 adult subjects with GHD due to pituitary tumors showed that 2 months of GH replacement therapy resulted in significant improvements in the depression rating scale score when compared to subjects treated with placebo [6]. In a brief case study, Sonksen et al. reported that GH replacement therapy has been beneficial in reducing mental disability in a subject whose suicidality and depression appeared after surgery and radiotherapy for a pituitary tumor (and who had received replacement therapy for all pituitary hormones except for GH when the depression started) [16]. Indirectly, when depressive syndrome in GH-deficient adults was evaluated as a part of a qualityof-life assessment, symptom reduction after several months of GH replacement was confirmed [17]. InterestMaric /Pekic /Doknic /Jasovic-Gasic / Zivkovic /Stojanovic /Djurovic /Popovic

ingly, withdrawal of GH treatment from adults with severe GHD has been shown to have detrimental psychological effects: decreased energy and increased tiredness, pain, irritability, and depression [18]. It is important to add that both depression and TBI are conditions listed as causes of morbidity with very high incidence [19, 20]. Moreover, their incidence shows a

trend toward further increase. Therefore, we strongly recommend further research to (1) clarify the mechanism of depression in GH-deficient adults who sustain TBI and (2) analyze the efficacy and tolerability of GH replacement therapy compared to treatment with placebo and/or antidepressant drugs.

References 1 Fann JR, Katon WJ, Uomoto JM, Esselman PC: Psychiatric disorders and functional disability in outpatients with traumatic brain injuries. Am J Psychiatry 1995; 152: 1493–1499. 2 Agha A, Rogers B, Sherlock M, O’Kelly P, Tormey W, Phillips J, Thompson CJ: Anterior pituitary dysfunction in survivors of traumatic brain injury J Clin Endocrinol Metab 2004;89:4929–4936. 3 Aimaretti G, Ambrosio M, Di Somma C, Fusco A, Cannavo S, Gasperi M, Scaroni C, De Marinis L, Benvenga S, Degli Uberti E, Lombardi G, Mantero F, Martino E, Girodano G, Ghigo E: Traumatic brain injury and subarachnoid haemorrhage are conditions at high risk for hypopituitarism: screening study at 3 months after the brain injury. Clin Endocrinol 2004;61:320–326. 4 Popovic V, Pekic S, Pavlovic D, Maric N, Jasovic-Gasic M, Djurovic B, Medic Stojanoska M, Zivkovic V, Stojanovic M, Doknic M, Milic N, Djurovic M, Dieguez C, Casanueva FF: Hypopituitarism as a consequence of traumatic brain injury (TBI) and its possible relation with cognitive disabilities and mental distress. J Endocrinol Invest 2004; 11: 1048–1054. 5 Carroll PV, Christ ER, Bengtsson BA, Carlsson L, Christiansen JS, Clemmons D, Hintz R, Ho K, Laron Z, Sizonenko P, Sonksen PH, Tanaka T, Thorner M: Growth hormone deficiency in adulthood and the effects of growth hormone replacement: a review. J Clin Endocrinol Metab 1998;83:382–395. 6 Mahajan T, Crown A, Checkley S, Farmer A, Lightman S: Atypical depression in growth hormone deficient adults, and the beneficial effects of growth hormone treatment on depression and quality of life. Eur J Endocrinol 2004;3:325–332.

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7 Campbell S, Macqueen G: The role of the hippocampus in the pathophysiology of major depression. J Psychiatry Neurosci 2004; 29:417–426. 8 Mahmoud GS, Grover LM: Growth hormone enhances excitatory synaptic transmission in area CA1 of rat hippocampus. J Neuropsychol 2006;95:2962–2974. 9 Aberg ND, Brywe KG, Isgaard J: Aspects of growth hormone and insulin-like growth factor-I related to neuroprotection, regeneration, and functional plasticity in the adult brain. Scientific World Journal 2006; 6: 53– 80. 10 Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, Hergueta T, Baker R, Dunbar GC: The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry 1998;59(suppl 20):22–33. 11 Zung WWK: A self-rating depression scale. Arch Gen Psychiatry 1965;12:63–70. 12 Derogatis LR, Rickels K, Rock AF: The SCL90 and the MMPI: A step in the validation of a new self-report scale. Br J Psychiatry 1976; 128:280–289. 13 Popovic V, Leal A, Micic D, Koppeschaar HP, Torres E, Paramo C, Obradovic S, Dieguez C, Casanueva FF. GH-releasing hormone and GH-releasing peptide-6 for diagnostic testing in GH-deficient adults. Lancet 2000; 356(9236):1137–1142.

14 Trivedi MH, Fava M, Wisniewski SR, Thase ME, Quitkin F, Warden D, Ritz L, Nierenberg AA, Lebowitz BD, Biggs MM, Luther JF, Shores-Wilson K, Rush AJ, the STAR*D Study Team: Medication augmentation after the failure of SSRIs for depression. N Engl J Med 2006;354:1243–1252. 15 Rush AJ, Trivedi MH, Wisniewski SR, Stewart JW, Nierenberg AA, Thase ME, Ritz L, Biggs MM, Warden D, Luther JF, ShoresWilson K, Niederehe G, Fava M; STAR*D Study Team: Bupropion-SR, sertraline, or venlafaxine-XR after failure of SSRIs for depression. N Engl J Med 2006; 354: 1231– 1242. 16 Sonksen PH, McGauley G: Lies, damn lies and statistics. Growth Horm IGF Res 2005; 15:173–176. 17 Mukherjee A, Tolhurst-Cleaver S, Ryder WD, Smethurst L, Shalet SM: The characteristics of quality of life impairment in adult growth hormone (GH)-deficient survivors of cancer and their response to GH replacement therapy. J Clin Endocrinol Metab 2005; 90:1542–1549. 18 McMillan CV, Bradley C, Gibney J, Healy ML, Russell-Jones DL, Sonksen PH: Psychological effects of withdrawal of growth hormone therapy from adults with growth hormone deficiency. Clin Endocrinol (Oxf). 2003;59:467–475. 19 Anderson RN, Smith BL: Deaths: leading causes for 2002. Natl Vital Stat Rep 2005; 53: 1–89. 20 Vos T, Haby MM, Barendregt JJ, Kruijshaar M, Corry J, Andrews G: The burden of major depression avoidable by longer-term treatment strategies. Arch Gen Psychiatry 2004; 61:1097–1103.

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Adult Clinical Case Sessions

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):180–183 DOI: 10.1159/000097578

Published online: February 15, 2007

Hypodipsic Hypernatremia after Hypothalamic Infarct Mónica Marazuela a Gema López-Gallardo a María López-Iglesias a Rafael Manzanares b Departments of a Endocrinology and b Radiology, Hospital de la Princesa, Universidad Autónoma, Madrid, Spain

Key Words Hypodipsic hypernatremia Hypothalamic infarct Diabetes insipidus

Abstract Background: Hypothalamic hypodipsic syndrome is a rare condition, secondary to a defect in hypothalamic osmoreceptors that leads to impairment of water homeostasis and chronic hyperosmolality. It is frequently associated with defective osmoregulated vasopressin secretion and diabetes insipidus, and the combination results in a greater risk of hypernatremia. The principles of management consist of regulating vasopressin and water intake in relation to changes in daily body weight. Methods and Results: This case report concerns a patient who, after a postoperative cerebral infarction, developed an extensive hypothalamic syndrome including hypodipsic hypernatremia, diabetes insipidus, hyperthermia, profound short-term memory loss and initial severe anorexia, followed by hyperphagia with an inability to maintain body weight. Conclusion: The management of fluid balance proved extremely difficult in this amnesic patient, where weight alone was not sufficient to monitor water intake. Copyright © 2007 S. Karger AG, Basel

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Introduction

In a normal healthy person, plasma osmolality is maintained within a narrow physiological range by the thirst instinct and arginine vasopressin (AVP) secretion [1]. These homeostatic mechanisms are controlled by osmoreceptors located in the anterior hypothalamus, receptors that are extremely sensitive to minimal alterations in osmolality. The system is so efficient that osmolality does not vary by more than 1–2% under normal conditions, despite fluctuations in fluid intake. Hypodipsic hypernatremia is the result of a defect in the thirst mechanism, and because of the proximity between osmoreceptors for thirst and vasopressin secretion, it is usually associated with defective osmoregulation of AVP secretion and, therefore, defects in antidiuretic function [2]. Hypodipsic hypernatremia has been associated with a variety of intracranial lesions, with vascular aetiology in 15% of patients. Nevertheless, very few cases of adipsic hypernatremia secondary to vascular lesions have been reported [3]. The following case report concerns a patient with severe hypothalamic dysfunction secondary to a cerebral infarct that included defects in osmoregulation of thirst and AVP secretion, as well as cognitive, thermoregulatory and eating disorders.

Mónica Marazuela Department of Endocrinology, Hospital de la Princesa Universidad Autónoma de Madrid, c/Diego de León 62 ES–28006 Madrid (Spain) Tel. +34 91 520 2382, Fax +34 91 307 1565, E-Mail [email protected]

Fig. 1. Cerebral MRI before and after cranial surgery. A Suprasellar 4-cm mass before surgery. B Extensive anterior and midline infarct 1 week after surgery. C In-

farct in the hypothalamic region 1 month after surgery (arrow). D Persistent hypothalamic infarct 1 year after surgery (arrow).

Case Report A 19-year-old female presented with a 2-year history of headache without any other neurological symptoms. Radiological investigation showed a 4-cm suprasellar mass (fig. 1A). Her visual fields and the hypothalamic-hypophyseal axis were preserved. She underwent a left frontal craniotomy with complete tumour resection. Histological diagnosis of the tumour was a dermoid cyst. Four days postoperatively, the patient developed right hemiparesis and bilateral midriasis and was in a deep coma (5/15 on the Glasgow scale). She required assisted ventilation and was transferred to the intensive care unit. A magnetic resonance image (MRI) of the brain confirmed an extensive anterior and midline infarction, extending from the posterior arm of the right internal capsule into the hypothalamic region, in the territory of the penetrating branches of the anterior and medial cerebral arteries (fig. 1B, C). The patient recovered from the coma, but maintained residual left-sided 3/5 plegia and hypothalamic disturbances including impaired thirst perception, difficulties with body temperature regulation with persistent fever, decreased spontaneous eating and profound short-term memory loss. With conventional fluid replacement and on a fixed dose of 1-deamino-8-d-arginine vasopressin (DDAVP), the patient’s plasma osmolality and sodium fluctuated within the normal range. She was transferred to a rehabilitation unit on postoperative day 21, where she continued to have no sensation of thirst. She complained of severe anorexia, and, after a 10-kg weight loss, was started on enteral nutrition through a nasogastric tube. She also had free access to food and water.

Hypodipsic Hypernatremia after Hypothalamic Infarct

On postoperative day 27, the patient developed lethargy, irritability and nausea and vomiting secondary to potentially lifethreatening hypernatremia of 186 mmol/l, during what was considered to be a trivial infection. Physical examination showed that she was conscious, with a 39.5 ° C fever, signs of dehydration and a sore throat. Laboratory investigation revealed normal anterior pituitary function. Despite the severity of hypernatremia, an adequate level of consciousness and no restrictions of water intake, the patient was markedly hypodipsic, consistent with a significantly altered sense of thirst. Treatment with enteral and intravenous fluids, coupled with close monitoring of diuresis, water intake and electrolytes, brought about a gradual correction of hypernatremia (fig. 2). To evaluate osmoreceptor function, the patient underwent a water deprivation test to assess AVP secretion and thirst perception. During the 2.5-hour water deprivation test, plasma osmolality did not change, but urine osmolality rose from 361 to 672 mOsm/kg after DDAVP. Basal AVP level was low and did not increase after dehydration. As shown in figure 3, there was an inappropriately blunted response to a plasma osmolality of 307 mOsm/kg. Plasma AVP was measured by a sensitive and specific radioimmunoassay. Even at this high osmolality, she experienced no sensation of thirst. These data confirmed defective osmoregulated AVP release with a lack of response to hypertonic stimuli and a profound defect in osmoregulated thirst. Upon stabilization of her electrolytes and after an 8-kg weight gain with enteral nutrition, the patient was discharged home in the care of her mother. Her right hemiparesis had resolved, but

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200

150 125 100

she had persistent adipsia, low-grade fever and short-term memory loss. In addition, she developed an increased appetite and had a 12-kg weight gain in a 6-month period. Management of fluid intake was challenging because of the adipsia, amnesia and continuous weight changes. Her fluid balance was managed by obligate fluid intake based on estimated requirements, insensible losses and a fixed intranasal dose of DDAVP. Her home-monitored sodium level ranged from 133 to 160 mmol/l. Six months later, after a total 20-kg weight gain, the patient was prescribed sibutramine, which stabilized her weight. As deviations from the target weight were managed by reciprocal changes in obligate fluid intake, her fluid intake was assessed on a strict litre-per-kilogram basis. Plasma sodium was also checked regularly. Audiobased memory aides were used to manage fluid intake because of the patient’s short-term memory deficit. MRIs of the brain performed after 12 and 24 months showed no residual tumour, but there was a persistent infarct in the frontal and hypothalamic regions (fig. 1D).

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Discussion

Thirst osmoreceptors are located in the anterior hypothalamus and modulate both the sensation of thirst and AVP secretion [1]. Damage in this region causes hyperosmolality through a combination of impaired osmotic thirst and/or osmotically stimulated AVP secretion [2]. Due to the close proximity of the osmoreceptors regulating thirst and AVP release, a loss of thirst is usually associated with defective osmoregulated vasopressin secretion, as was the case with this patient. Patients with hypodipsia usually present with asymptomatic chronic hypernatremia. However, in extreme circumstances these patients can develop neurological symptoms ranging from irritability to coma [2, 3]. Hypodipsia can be diagnosed by the presence of sustained hypernatremia (plasma sodium 1150 mmol/l) in a patient who denies thirst 182

12

Plasma ADH pmol/l

Fig. 2. Variation in plasma sodium concentration with time and response to intravenous and oral fluid therapy and DDAVP (arrow). Normal plasma sodium range is represented between the dotted lines.

Na+ mmol/l

175

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LD T hirs t 280

290 300 310 Plasma osmolality (mOsm/kg)

Fig. 3. Correlation between plasma vasopressin (pg/ml) and plasma osmolality (mOsm/kg). After water deprivation test, at a plasma osmolality of 307 mOsm/kg, plasma vasopressin concentration was 2.1 pg/ml and the patient had no thirst (cross). Shaded area represents normal range.

Marazuela /López-Gallardo / López-Iglesias /Manzanares

and/or does not drink spontaneously, and in the absence of coma or other impediments to water ingestion [3, 4]. In addition, impairment of osmotically stimulated AVP secretion can be precisely diagnosed by sensitive assays to circulating AVP during hyperosmolality [3]. Hypodipsic hypernatremia is associated with a variety of intracranial pathologies including neoplasm, vascular abnormalities, trauma and surgery [5]. In this patient, injury was secondary to a postoperative infarct in the hypothalamic region. Blood supply to the AVP and thirst osmoreceptors is provided by small penetrating branches of the anterior and medial cerebral arteries. Lesions affecting these regions result in damage to the thirst osmoreceptors [6]. Very few cases of hypothalamic infarction have been reported in the literature [7–11]. Hypothalamic injury after cerebral infarction can produce paroxysmal hyperthermia. Acute damage to the preoptic anterior hypothalamus can lead to profound impairment of heat loss mechanisms, and the resulting hyperthermia can be lethal [12]. In this case, our patient developed hyperthermia that resolved after months of follow-up. The hypothalamus also receives input from the periphery that either stimulates or inhibits food intake. Lesions in the ‘feeding centre’ of the lateral hypothalamus can lead to anorexia and weight loss, whereas lesions in the ventromedial hypothalamus can cause hyperphagia and obesity. Occasionally, as was the case for our patient, hypothalamic lesions initially can produce anorexia and then, during the recovery phase, cause increased food intake with occasional hyperphagia [12, 13].

Hypothalamic injury can also produce marked, relatively focal memory deficits with disturbances of short-term memory and sparing of immediate recall and long-term memory [7]. These deficits can complicate the management of adipsic patients. Management of adipsic syndromes with associated defective osmoregulated vasopressin secretion can be difficult because of the potential danger of over- or underhydration [2, 14, 15]. Recommendations for managing water balance include using an obligate daily intake of 1.5 l and adjusting water intake to changes in daily body weight, and taking a fixed amount of an exogenous AVP analogue [2]. In addition, a small portable analyzer is useful in helping these patients control serum sodium levels at home [16]. In our patient, additional difficulties in water balance management included continuous changes in body weight and short-term memory loss associated with the hypothalamic injury. These difficulties were overcome by utilizing a combination of patient and family education, monitoring fluid intake and urine output, and home monitoring of plasma sodium when needed.

Conclusion

Management of these complex patients can be usually achieved by adjusting water intake to changes in daily body weight, providing a fixed amount of an exogenous AVP analogue and, in special cases, monitoring of plasma sodium at home.

References 1 Baylis PH, Thompson CJ: Osmoregulation of vasopressin secretion and thirst in health and disease. Clin Endocrinol 1988; 29: 549– 576. 2 Ball SG, Vaidja B, Baylis PH: Hypothalamic adipsic syndrome: diagnosis and management. Clin Endocrinol 1997; 47:405–409. 3 Verbalis JG: Disorders of water homeostasis. Best Pract Res Clin Endocrinol Metab 2003; 17:471–503. 4 Vokes TJ, Robertson GL: Disorders of antidiuretic hormone. Endocrinol Metab Clin North Am 1988;17:281–299. 5 Robertson GL, Aycinena P, Zerbe RL: Neurogenic disorders of osmoregulation. Am J Med 1982;72:339–353. 6 Daniel PM: The blood supply of the hypothalamus and pituitary gland. Br Med Bull 1966;22:202–208. 7 Nguyen BN, Yablon SA, Chen CY: Hypodipsic hypernatremia and diabetes insipidus fol-

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lowing anterior communicating artery aneurysm clipping: diagnostic and therapeutic challenges in the amnesic rehabilitation patient. Brain Inj 2001;15:975–980. Landolt AM, Yasargil MG, Krayenbuhl H: Disturbances of the serum electrolytes after surgery of intracranial arterial aneurysms. J Neurosurg 1972;37:210–218. McIver B, Connacher A, Whittle I, Baylis P, Thompson C: Adipsic hypothalamic diabetes insipidus after clipping of anterior communicating artery aneurysm. Br Med J 1991; 303:1465–1467. McMahon AJ: Diabetes insipidus developing after subarachnoid haemorrhage from an anterior communicating artery aneurysm. Scott Med J 1988;33:208–210. Takaku A, Shindo K, Tanaka S, Mori T, Suzuki J: Fluid and electrolyte disturbances in patients with intracranial aneurysms. Surg Neurol 1979;11:349–356.

12 Toni R, Malaguti A, Benfenati F, Martini L: The human hypothalamus: a morpho-functional perspective. J Endocrinol Invest 2004; 27:73–94. 13 Druce M, Bloom SR: The regulation of appetite. Arch Dis Child 2006;91:183–187. 14 Lopez-Capape M, Golmayo L, Lorenzo G, Gallego N, Barrio R: Hypothalamic adipic hypernatraemia syndrome with normal osmoregulation of vasopressin. Eur J Pediatr 2004;163:580–583. 15 Pearce SH, Argent NB, Baylis PH: Chronic hypernatremia due to impaired osmoregulated thirst and vasopressin secretion. Acta Endocr 1991; 125:234–239. 16 Green RP, Landt M: Home sodium monitoring in patients with diabetes insipidus. J Pediatr 2002;141:618–624.

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Horm Res 2007;67(suppl 1):184–185 DOI: 10.1159/000097579

Published online: February 15, 2007

Hot Topics in Adult Endocrinology

This session deals with two very important aspects of human health: aging and obesity. Statistics from the World Health Organization clearly demonstrate that inhabitants of the developed world are living longer, which has increased the burden on the health care system. A similar trend is also occurring in Asia, India and Africa, which have much larger populations. Thus, the number of individuals over 70 years of age is growing more rapidly in these areas than in western societies. Basic and clinical research aimed at improving health among older individuals is therefore of major importance. The endocrine contribution to the study of aging has focused on sex steroids and growth hormone (GH) because of the reduction in sex steroids during menopause and andropause and the reduction of GH during somatopause associated with aging. Studies with dehydroepiandrosterone (DHEA) treatment have yielded mixed results. An intervention trial found that the GH receptor knockout mouse lives longer than its littermates. By contrast, results of a few well-performed clinical trials have found that GH treatment in older, otherwise healthy, people does not convincingly demonstrate functional benefit. Still, there remains great enthusiasm around the use of endocrine intervention to influence the aging process. The finding that defects in the Klotho gene accelerate the aging process and that overexpression of the gene extends the life span in mice is an exciting discovery. More

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importantly, it has been demonstrated that the gene product is a circulating hormone that attenuates signaling of insulin-like growth factor I and insulin [1] and increases resistance to oxidative stress at the cellular level in mice. It remains to be seen whether this new discovery has applications for humans and can contribute to improving health in the aging population. Another phenomenon of major interest for the health care system is the rapidly increasing number of obese individuals in the Western world, particularly among the young. This will increase the number of subjects with type 2 diabetes mellitus and hypertension and its vascular complications. The search for new drug targets that can enhance weight loss has been intense. The endocannabinoid system is an orexigenic system with well-defined central regulation of food intake and animal behaviour [2]. Cannabinoid (CB) receptor type 1 blockers were believed to act primarily on food intake and therefore would be useful as adjuvant drug therapy for weight loss. However, the recent CB1 knockout mice have demonstrated that the endocannabinoid system has both central and peripheral actions. Therefore, drugs blocking the CB1 receptor may work through both central and peripheral receptors. Recent data show that the CB1 receptors are present in liver, adipose tissue and muscle and are regulators of fat and glucose metabolism at the peripheral tissue level. The key finding in recent animal data

suggests that the central action (anorexigenic) of CB1 blockers may be transient, but that the peripheral action may have a more sustained action and an improved metabolic profile. Results of a large clinical study showed that increased serum adiponectin levels were independent of the weight-loss promoted by treatment [3], suggesting that peripheral actions are important for the long-term metabolic effect of treatment. More drugs targeting this system will emerge in the near future and they may assist in the treatment of obesity and its metabolic consequences.

References

1 Kurosu H, Yamamoto M, Clark JD, et al: Suppression of aging in mice by the hormone. Klotho Science 2005;309:1829–1833. 2 Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R: The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocr Rev 2006;27:73– 100. 3 Despres JP, Golay A, Sjostrom L: Effects of rimonabant on metabolic risk factors in overweight patients with dyslipidemia. N Engl J Med 2005;353:2121–2134.

Gudmundur Johannsson Department of Endocrinology Sahlgrenska University Hospital Gothenburg, Sweden

Hot Topics in Adult Endocrinology

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Hot Topics in Adult Endocrinology

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):186–190 DOI: 10.1159/000097580

Published online: February 15, 2007

The Endocannabinoid System in the Physiopathology of Metabolic Disorders U. Pagotto V. Vicennati R. Pasquali Endocrinology Unit and C.R.B.A., Department of Internal Medicine and Gastroenterology, S. Orsola-Malpighi Hospital, Alma Mater Studiorum, University of Bologna, Bologna, Italy

Key Words Endocannabinoids Cannabinoid receptor type 1 (CB1) receptor Rimonabant Obesity

Abstract Background: Since the purification of 9-tetrahydrocannabinol (THC) 40 years ago, many studies have concluded that the endocannabinoid system is one of the most important orexigenic systems in the body. Endocannabinoids are endogenous lipids capable of activating the two cannabinoid receptors, CB type 1 (CB1) and CB type 2. These receptors belong to the G-protein-coupled family receptors and they were discovered while investigating the molecular mode of action of THC, to which they bind with high affinity. Endogenous cannabinoids stimulate hunger and promote appetite through activation of the CB1 receptors. The CB1 receptor is expressed in several organs that are involved at both the central and peripheral level in the control of food intake and energy metabolism. These organs include the mesolimbic system, hypothalamus, gastrointestinal tract, adipose tissue, skeletal muscles, hepatocytes and endocrine cells of the pancreas. The endocannabinoid system is believed to play a crucial role in controlling energy balance through the possible targeting of a large variety of peripheral organs while modulating metabolic processes. Conclusions: To better understand the effects of the endocannabinoid system, future studies will require detailed characterization of each individual contribution and the reciprocal

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interactions among the organs. Because the endocannabinoid system is likely overactivated in conditions such as obesity, pharmacologic therapy with a CB1 receptor antagonist like rimonabant might normalize the imbalance induced by this overactivation and produce a viable option in the fight against obesity and its associated comorbid conditions. Copyright © 2007 S. Karger AG, Basel

Introduction

Cannabinoids are historically known to stimulate hunger. In fact, due to their orexigenic properties, ancient traditions utilized marijuana (Cannabis sativa) as an ideal pharmacologic therapy in patients for whom it was necessary to promote feeding. An active component of Cannabis, 9-tetrahydrocannabinol (THC), was purified approximately 40 years ago. Since then, numerous studies have defined the endocannabinoid system as one of the most important orexigenic systems in the body [1]. Endocannabinoids, particularly anandamide and 2arachidonoylglycerol, are endogenous lipids capable of activating the two cannabinoid receptors, type 1 (CB1) and type 2 (CB2). These receptors belong to the G-protein-coupled family receptors and they were discovered during investigations of the molecular mode of action of THC, to which they bind with high affinity [2].

Uberto Pagotto, MD S. Orsola-Malpighi General Hospital Via Massarenti 9 IT–40138 Bologna (Italy) Tel. +39 051 636 3009, Fax +39 051 636 3080, E-Mail [email protected]

Endogenous cannabinoids, through activation of the CB1 receptors, stimulate hunger and promote appetite during fasting conditions [1, 3]. Moreover, the endocannabinoid system is believed to be involved in several other physiological functions, many of which are related to maintenance of homeostatic balance. Among other functions, endocannabinoids are neuroprotective modulators of nociception and motor activity, able to control certain phases of memory processing [4], and involved in the modulation of the immune and inflammatory responses; they also influence the cardiovascular and the respiratory systems by controlling heart rate, blood pressure and bronchial functions [2]. The CB1 receptor is expressed in several organs that are involved at both the central and peripheral level in the control of food intake and energy metabolism. These organs include the mesolimbic system, hypothalamus, gastrointestinal tract, adipose tissue, skeletal muscles, hepatocytes and endocrine cells of the pancreas. Notably, some of these areas are also sites of endocannabinoid production [1].

in starving conditions [7]. Therefore, the endocannabinoids are important sensors of acute changes of nutritional state by sending a strong impulse to the orexigenic pathway through CB1 receptor activation. In fact, endocannabinoids interact with many hypothalamic peptidergic circuits involved in food intake, including corticotropin-releasing hormone, cocaine-amphetamineregulated transcript, melanin-concentrating hormone, orexins, ghrelin and leptin [1]. The gastrointestinal tract is also a key target organ of endocannabinoid action. Similar to what has been described at the hypothalamic level, endocannabinoid production by the gastrointestinal tract is also regulated by the feeding state. In fact, starvation induces a 7-fold increase in anandamide levels in the small intestine, an effect that is reversed by food intake [8]. Moreover, the CB1 receptor is expressed in vagal afferent neurons projecting to the stomach and duodenum; this expression is increased by food deprivation and decreased by food intake. Therefore, the activation of CB1 receptors in these sites may be involved in mediating satiety signals originating in the gut.

Endocannabinoids and Food Intake Role of the CB1 Receptor in Metabolism

Preliminary results obtained at the end of the 1990s supported the belief that the endocannabinoid system may have a key role in regulation of the ‘reward/reinforcement’ circuitry, which is represented by an interconnection of brain nuclei associated with the medial forebrain bundle, linking the ventral tegmental area, the nucleus accumbens and the ventral pallidum. The most relevant pathway of this circuitry is the mesolimbic dopaminergic system since the relationship between the limbic endocannabinoid and dopaminergic systems in stimulating food cravings is well recognized [5]. With the advanced understanding of the crucial role of the hypothalamic neuronal network in appetite regulation, several studies have also directed attention to the putative role of the endocannabinoid system in modulation of the centers controlling satiety and famine [1]. It was recently shown that endocannabinoids induce significant hyperphagia when administered into the ventromedial hypothalamus; however, pretreatment with the CB1 receptor antagonist rimonabant attenuated the endocannabinoid-induced hyperphagia, confirming the pivotal role of CB1 receptor in mediating an orexigenic signal at the level of the hypothalamus [6]. Notably, at this site, endocannabinoid production has been shown to be closely associated with the feeding state, being very high The Endocannabinoid System in Metabolic Disorders

The most significant advancement in understanding the effects of the endocannabinoid system on peripheral metabolic functions has been provided by the recent investigation of mice lacking the CB1 receptor (CB1–/–). With a standard diet, CB1–/– mice are leaner and lighter than control wild-type littermates with a significant decrease in fat mass. ‘Pair-feeding’ experiments in these animals showed that the leaner phenotype is not only attributed to a reduction of food intake, but also due to a food intake-independent mechanism, likely related to altered metabolic processes [9]. Interestingly, the metabolic effects of the endocannabinoid system were recently confirmed in rodents treated for 40 days with rimonabant, the first CB1 receptor antagonist to be synthesized. In fact, in diet-induced obese mice, rimonabant induced a transient anorectic effect followed by a significant and more sustained reduction in body weight, due to a decrease in body fat mass [10]. These findings have greatly widened our perspectives in understanding the antiobesity mechanisms of action of CB1 receptor antagonists. Indeed, rimonabant is likely able to decrease fat accumulation initially by targeting neuronal sites and thereafter by modulating peripheral metabolic regulation.

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At the white adipocyte tissue level, CB1 receptors stimulate lipogenesis and enhance lipoprotein lipase activity; this effect can be specifically blocked by a CB1 receptor antagonist [9]. It is of interest that mature adipocytes express CB1 receptors in a predominant way in comparison with preadipocytes in both humans and rodents, suggesting that the endocannabinoid system is likely related to metabolic functions rather than to differentiation processes [11, 12]. In support of this finding is the observation of increased adiponectin release from adipocytes in vitro after treatment with a CB1 receptor antagonist [11]. Adipokine secreted by adipose tissue is known to play a crucial role in fat and glucose metabolism. In fact, adiponectin is considered to have antiatherogenic and antidiabetic properties. Moreover, CB1 receptor antagonism counteracts the alterations in gene expression induced by a highfat diet and reduces fat mass by enhancing lipolysis through induction of the -oxidation and tricarboxylic acid cycle; increasing energy expenditure through futile cycle stimulation; and stimulating glucose-transporter 4, a key player in glucose metabolism [13]. Endocannabinoids also influence metabolism directly at the hepatic level, promoting de novo lipogenesis by directly acting on hepatocytes. Accordingly, a prolonged high-fat diet in mice is associated with augmented levels of intrahepatic anandamide, inducing an up-regulation of CB1 receptor expression that may, in turn, promote the development of steatosis. In contrast, CB1–/– mice do not develop hepatic steatosis [14]. It was recently demonstrated that the endocrine pancreas expresses both CB1 and CB2 receptors. Glucagoncontaining cells mainly express the CB1 receptor while the CB2 receptor was found in both and cells [15]. At this level, endocannabinoids seem to decrease insulin secretion regulating calcium signalling [15]. Skeletal muscle also expresses CB1 receptors and treatment with rimonabant stimulates glucose uptake [16]. Endocannabinoids play a role in the development and maintenance of obesity. Recent hypotheses suggest that chronic pathologic states such as obesity can lead to longlasting and even permanent overstimulation of endocannabinoid synthesis (or hypostimulation of their degradation) (fig. 1). This overactivation then, in turn, continues to contribute to the symptoms of the disease. In fact, a series of studies on obese animals demonstrated that the CB1 receptor is overexpressed in tissues controlling energy metabolism, such as liver, skeletal muscles and adipose tissue. Finally, in humans, recent studies have shown that increased levels of circulating endocannabinoids are present in visceral obese patients when compared to sub188

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cutaneous obese patients and lean controls [3]. In particular, it has been found that abdominal fat accumulation is a critical correlate of the dysregulation of the peripheral endocannabinoid system in human obesity because circulating 2-arachidonoylglycerol levels have been found to significantly correlate with body fat, visceral fat mass and fasting plasma insulin concentrations, and negatively correlated to glucose infusion rate during clamp [3].

Clinical Trials with CB1 Antagonists

Results of several recent clinical trials have indeed shown that rimonabant, the first CB1 antagonist to have been synthesized, has a therapeutic role in the treatment of obesity [17–20]. A worldwide Phase III trial named RIO (Rimonabant In Obesity), consisting of four different clinical trials and involving more than 6,600 obese or overweight patients with or without concomitant comorbidities, has recently been concluded. The 2-year studies, RIO-Europe and RIO-North America, recruited obese or overweight patients with or without comorbidities. The 1-year studies, RIO-Lipids and RIO-Diabetes, were set up to investigate possible improvement after rimonabant treatment of the comorbid factors of hyperlipidemia and diabetes that are associated with obesity. As a general rule, treatment with 5-mg rimonabant often did not provide statistically significant changes compared with placebo, so for simplicity only the data concerning the 20mg rimonabant treatment are reviewed. Note that for all four trials a hypocaloric diet was recommended to all subjects, and at each visit subjects received dietary counselling and were encouraged to increase their level of physical activity. Analysis of the intent-to-treat population of the RIOEurope study (using the last observation carried forward) indicated a weight reduction of –6.6 kg for the 20-mg rimonabant group compared with a loss of –1.8 kg for the placebo-treated group. Similar data were obtained for the RIO-Lipids (–6.9 vs. –1.5 kg, respectively) and the RIONorth America studies (–6.3 vs. –1.6 kg, respectively). Concomitant reductions in waist circumference of –6.5, –7.1 and –6.1 cm, respectively, were observed for patients treated with rimonabant 20 mg in the three studies; whereas, the reductions seen in placebo-treated patients were –2.4, –2.4 and –2.2 cm, respectively. In the RIONorth America study, patients who received rimonabant were re-randomized at the end of the first year to receive either the same dose of rimonabant or placebo and then Pagotto/Vicennati/Pasquali

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Fig. 1. The endocannabinoid system is overactivated in obesity.

were followed up for an additional year. Patients who were switched from the 20-mg rimonabant group to the placebo group re-gained weight, while those who continued to receive 20 mg rimonabant maintained their weight loss. In all three studies significant increases in high-density lipoprotein (HDL) cholesterol and decreases of triglyceride concentrations were detected in patients treated with 20 mg rimonabant. Interestingly, logistic regression models and analysis of covariance models using weight loss as a covariate in the RIO-Europe study found that nearly half the changes in HDL-cholesterol and triglycerides were independent of weight loss, as reflected by the last weight measurement. Similarly, 57% of the increase in adiponectin levels observed in the RIO-Lipids group receiving 20 mg of rimonabant could not be attributable to weight loss. Plasma glucose and insulin levels were measured before the 75 g oral glucose tolerance test and 30, 60 and 120 min after the test, both at the beginning and end of the 1-year treatment in three out of four studies. In the RIO-

Europe study, statistically significant reductions in plasma glucose and insulin levels were achieved after treatment with 20 mg rimonabant. In both the RIO-Europe and RIO-Lipids studies there was a significant reduction from baseline in 120-min insulin levels for patients receiving 20 mg rimonabant compared with placebo-treated patients. In the RIO-Lipids study the prevalence of the metabolic syndrome was analyzed before and after 1 year of treatment. At baseline, 54% of patients met the criteria for metabolic syndrome. One year later the prevalence fell to 25.8% for the 20-mg rimonabant group compared with 41% for the placebo group. A slightly higher number of adverse or serious adverse events were reported following rimonabant treatment when compared with placebo in RIO-Europe, RIONorth-America and RIO-Lipids studies. The most commonly reported adverse events were nausea, vomiting, diarrhoea and dizziness. In all three trials a small but significant percentage of patients developed depression or

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anxiety and this led to discontinuation after rimonabant treatment. In the future, monitoring for on-treatment anxiety and depression will be necessary to ensure the safe use of rimonabant or any other CB1 receptor antagonist.

Conclusion

The endocannabinoid system plays a crucial role in controlling energy balance through the possible targeting of a large variety of peripheral organs while modulating metabolic processes. On the other hand, the endocannabinoids modulate the reward properties of food by acting at specific mesolimbic areas in the brain. In the

hypothalamus, the CB1 receptor and endocannabinoids are integrated components of the networks controlling appetite and food intake. Detailed characterizations of each individual contribution and the reciprocal interactions among the organs are critical areas of future studies. The endocannabinoid system is likely overactivated in conditions such as obesity, and in particular, in humans haematic circulating endocannabinoids seem to be directly associated with some indexes of metabolic syndrome. In the light of this evidence, drugs that interfere with the endocannabinoid system, and in particular CB1 receptor antagonists, should be considered as useful adjuncts to lifestyle and behaviour modifications in the treatment of obesity and obesity-related comorbidities.

References 1 Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R: The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocr Rev 2006;27:73– 100. 2 Piomelli D: The molecular logic of endocannabinoid signalling. Nat Rev Neurosci 2003; 4:873–884. 3 Di Marzo V, Matias I: Endocannabinoid control of food intake and energy balance. Nat Neurosci 2005;8:585–589. 4 Marsicano G, Lutz B: Neuromodulatory functions of the endocannabinoid system. J Endocrinol Invest 2006; 29(suppl 3):27–46. 5 Cota D, Tschoep MH, Horvath TL, Levine AS: Cannabinoids, opioids and eating behavior: the molecular face of hedonism? Brain Res Brain Res Rev 2006;51:85–107. 6 Jamshidi N, Taylor DA: Anandamide administration into the ventromedial hypothalamus stimulates appetite in rats. Br J Pharmacol 2001;134:1151–1154. 7 Kirkham TC, Williams CM, Fezza F, Di Marzo V: Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. Br J Pharmacol 2002;136:550–557. 8 Gomez R, Navarro M, Ferrer B, Trigo JM, Bilbao A, Del Arco I, Cippitelli A, Nava F, Piomelli D, Rodríguez de Fonseca F: A peripheral mechanism for CB1 cannabinoid receptor-dependent modulation of feeding. J Neurosci 2002;22:9612–9617. 9 Cota D, Marsicano G, Tschop M, Grubler Y, Flachskamm C, Schubert M, Auer D, Yassouridis A, Thone-Reineke C, Ortmann S, Tomassoni F, Cervino C, Nisoli E, Linthorst AC, Pasquali R, Lutz B, Stalla GK, Pagotto U:

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The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis. J Clin Invest 2003;112:423–431. Poirier B, Bidouard JP, Cadrouvele C, Marniquet X, Staels B, O’Connor SE, Janiak P, Herbert J-M: The anti-obesity effect of rimonabant is associated with an improved serum lipid profile. Diabetes Obes Metab 2005;7:65–72. Bensaid M, Gary-Bobo M, Esclangon A, Maffrand JP, Le Fur G, Oury-Donat F, Soubrie P: The cannabinoid CB1 receptor antagonist SR141716 increases Acrp30 mRNA expression in adipose tissue of obese fa/fa rats and in cultured adipocyte cells. Mol Pharmacol 2003;63:908–914. Engeli S, Böhnke J, Feldpausch M, Gorzelniak K, Janke J, Bátkai S, Pacher P, HarveyWhite J, Luft FC, Sharma AM, Jordan J: Activation of the peripheral endocannabinoid system in human obesity. Diabetes 2005; 54: 2838–2843. Jbilo O, Ravinet-Trillou C, Arnone M, Buisson I, Bribes E, Peleraux A, Penarier G, Soubrie P, Le Fur G, Galliegue S, Casellas P: The CB1 receptor antagonist rimonabant reverses the diet-induced obesity phenotype through the regulation of lipolysis and energy balance. FASEB J 2005;19:1567–1569. Osei-Hyiaman D, DePetrillo M, Pacher P, Liu J, Radaeva S, Bátkai S, Harvey-White J, Mackie K, Offertáler L, Wang L, Kunos G: Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J Clin Invest 2005;115:1298–1305.

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15 Juan-Picò P, Fuentes E, Javier Bermudez-Silva F, Javier Diaz-Molina F, Ripoll C, Rodriguez de Fonseca F, Nadal A: Cannabinoid receptors regulate Ca(2+) signals and insulin secretion in pancreatic beta-cell. Cell Calcium 2006;39:155–162. 16 Liu YL, Connoley IP, Wilson CA, Stock MJ: Effects of the cannabinoid CB1 receptor antagonist SR141716 on oxygen consumption and soleus muscle glucose uptake in Lep(ob)/ Lep(ob) mice. Int J Obes Relat Metab Disord 2005;29:183–187. 17 Blüher M, Engeli S, Klöting N, Berndt J, Fasshauer M, Batkai S, Pacher P, Schön MR, Jordan J, Stumvoll M: Dysregulation of the peripheral and adipose tissue endocannabinoid system in human abdominal obesity. Diabetes 2006;55:3053–3060. 18 Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rossner S, for the RIO-Europe study Group: Effects of the cannabinoid CB1 receptor blocker rimonabant on weight reduction and cardiovascular risk factor in overweight patients: 1 year experience from the RIO-Europe Study. Lancet 2005; 365: 1389–1397. 19 Despres JP, Golay A, Sjostrom L, for the RIOLipids Study Group: Effects of rimonabant on metabolic risk factors in overweight patients with dyslipidemia. N Engl J Med 2005; 353:2121–2134. 20 Pi-Sunyer FX, Aronne LJ, Heshamati HM, Devin J, Rosenstock J, for the RIO-North America Study Group: Effects of rimonabant, a cannabinoid-1 receptor blocker. On weight and cardiometabolic risk factors in overweight or obese patients. JAMA 2006; 295: 761–775.

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Hot Topics in Adult Endocrinology

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):191–203 DOI: 10.1159/000097581

Published online: February 15, 2007

Klotho, an Aging-Suppressor Gene Kevin P. Rosenblatt a Makoto Kuro-o b a

Division of Translational Pathology, and Clinical Proteomics Program, b Laboratory of Molecular Pathology, and Clinical Proteomics Program, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Tex., USA

Key Words Insulin signaling oxidative stress Klotho, aging FGF signaling

Abstract Suppression of aging and its effects is a far-reaching dream of human beings. To turn this dream into a scientific reality, it is essential to understand the molecular mechanisms that underlie the aging process. A coherent understanding of these mechanisms is expected to allow us to develop rational strategies for slowing the aging process, which is the most significant risk factor for many age-related disorders including diabetes, heart attack, stroke, osteoporosis, cancer, and dementia. Therefore, any remedy against the progression of aging may reduce and/or delay premature death and disability caused by multiple age-related diseases simultaneously. This could lead to a tremendous increase in both the length and quality of human life. Copyright © 2007 S. Karger AG, Basel

Introduction

Aging is an extremely complex phenomenon influenced by various genetic and environmental factors. However, recent studies have demonstrated that single gene mutations can extend life span and delay aging processes in various experimental animals. Specifically, inhibition of insulin-like signaling is one mechanism for

© 2007 S. Karger AG, Basel 0301–0163/07/0677–0191$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

extending life span that is evolutionarily conserved from worms to mammals [1]. In Caenorhabditis elegans, lossof-function mutations in daf-2 (insulin receptor) and age1 (PI3-kinase) extend the life span of adult worms [2, 3]. In Drosophila, loss-of-function mutations in inr (insulin receptor) and chico (insulin receptor substrate) increase longevity [4, 5]. In mammals, dwarf mice that have low circulating levels of insulin-like growth factor I (IGF-I) [6-8], mice heterozygous for an IGF-I receptor knockout allele (igf1r+/–) [9], and mice that lack a functional insulin receptor in adipose tissues all manifest increased longevity [10]. Furthermore, the life span of mice can be extended by overexpression of the Klotho protein, which also results in the inhibition of insulin/IGF-I signaling [11]. Disruptions of this gene in mice accelerate the development of multiple aging-like disorders and results in early death [12]. However, the mechanism by which inhibition of insulin-like signaling suppresses aging is not fully understood. Our laboratories have focused on elucidating the function of the klotho gene. The klotho gene was originally identified as a gene mutated in a mouse strain that exhibits multiple aging-like phenotypes, including a shortened life span, hypogonadism, growth arrest, hypoactivity, skin atrophy, muscle atrophy, hearing loss, premature thymic involution, cognition impairment, motor neuron degeneration, arteriosclerosis, osteopenia, soft tissue calcification, and pulmonary emphysema among others [12–15]. Thus, the klotho gene is the first documented aging suppressor gene in mammals that can extend life span

Kevin P. Rosenblatt, MD Department of Pathology, Division of Translational Pathology UT Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75390-9072 (USA) Tel. +1 214 648 4125, E-Mail [email protected]

when overexpressed and accelerate the development of multiple aging-like phenotypes when disrupted. In addition, several single-nucleotide polymorphisms in the human Klotho gene are associated with life span [16], osteoporosis [17–19], stroke, and coronary artery diseases [20, 21], suggesting that Klotho may be involved in the regulation of human aging and age-related diseases. The klotho gene encodes a single-pass transmembrane protein of 130kD with a short intracellular domain (10 amino acids). The extracellular domain is composed of two homologous domains, each having weak homology with -glucosidase of bacteria and plants [12]. However, -glucosidase-like activity is not detected in recombinant Klotho protein. Instead, -glucuronidase activity is detected in the Klotho protein [22], though its physiological relevance remains to be determined. The klotho gene is well expressed in limited tissues, notably in the kidney and the brain [12]. Nonetheless, a defect in klotho gene expression leads to multiple aging-like phenotypes involving almost all organ systems in the mouse. The noncell-autonomous effects of the klotho gene suggest that a humoral factor(s) may mediate the Klotho protein’s function. Since the extracellular domain of this protein is shed and secreted into the blood, urine, and cerebrospinal fluid, it may function as a humoral factor that mediates its anti-aging properties.

Fibroblast Growth Factor-23 Mimics Klotho’s Physiological Actions

Fibroblast growth factor-23 (FGF23) is the newest member of the FGF ligand gene family, originally identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR) [23]. Patients with ADHR carry mutations in the FGF23 gene that confer resistance to inactivation by protease cleavage, resulting in elevated serum levels of FGF23. These findings suggested that FGF23 was involved in the regulation of phosphate homeostasis. Recent studies demonstrated that administration of FGF23 increased renal phosphate excretion and decreased circulating active vitamin D (1,25-dihydroxyvitamin D3) levels in mice [24–28]. Thus, FGF23 functions as a phosphaturic hormone [29–30]. Transgenic mice that overexpress FGF23 exhibit phosphate wasting, hypophosphatemia, reduced serum 1,25-dihydroxyvitamin D3 levels, and rickets, features that mimic those seen in ADHR patients [26]. Conversely, mice defective in FGF23 expression (Fgf23–/– mice) show increased renal phos192

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phate reabsorption and hyperphosphatemia accompanied by increased serum 1,25-dihydroxyvitamin D3 [31]. FGF23 is thought to transmit its biological activity through FGF receptors (FGFR). At least four receptor tyrosine kinases encoded by distinctive genes are designated as the high-affinity FGFR (FGFR1–4) [32, 33]. Alternative RNA splicing of each FGFR gene generates multiple FGFR isoforms. Notably, alternative splicing in one of the three immunoglobulin-like domains generates ‘b’ and ‘c’ isoforms, which show distinct binding affinity to various FGF molecules [32, 33]. Although FGF23 binds to multiple FGFR c isoforms, it has low affinity to the receptors (KD = 200–700 nM) [34]. Considering that the serum FGF23 concentration is !10 pM in normal mice, a cofactor(s) must exist that significantly enhances the affinity of FGF23 for FGFR. Heparin and glycosaminoglycan can function as cofactors that increase the ability of FGF23 to activate FGF signaling in cultured cells and to inhibit phosphate transport in isolated proximal tubules in vitro [35]. However, it remains to be determined whether heparin and glycosaminoglycan would function as endogenous cofactors of FGF23 in vivo. Further analysis of klotho–/– mice has revealed abnormal phosphate metabolism similar to that observed in Fgf23–/– mice [36]. Hyperphosphatemia becomes evident as early as 2 weeks of age prior to the development of aging-like phenotypes, which are manifested at 3–4 weeks of age or after. In addition, Klotho–/– mice show increased circulating levels of 1,25-dihydroxyvitamin D3 associated with an inappropriately increased expression of 1-hydroxylase in the kidney. Interestingly, many of the aginglike phenotypes observed in Klotho–/– mice are rescued by restricting dietary vitamin D intake, suggesting that induction of the aging-like syndrome in Klotho–/– mice is partly mediated by abnormal vitamin D metabolism. Further analysis of Fgf23–/– mice has revealed multiple aging-like phenotypes similar to those observed in Klotho–/– mice [37]. Many of the aging-like phenotypes observed in Fgf23–/– mice are rescued by disrupting the 1-hydroxylase gene. The fact that Klotho–/– mice and Fgf23–/– mice show many identical phenotypes that can be rescued by ablation of vitamin D activity has raised the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s) involving FGF and vitamin D signaling. We recently found that the Klotho protein, both the full-length transmembrane form and the extracellular domain alone, bound to multiple FGFRs [38]. The Klotho/ FGFR complex bound to FGF23 with much higher affinRosenblatt /Kuro-o

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ity than to FGFR alone. In fact, Klotho expression was necessary and sufficient for FGF23 to activate FGF signaling in various types of cultured cells. These findings indicate that Klotho potentially functions as an endogenous cofactor of FGF23. Klotho is a multi-functional protein. It can potentially function as ligand, co-receptor, and/or enzyme. Understanding of Klotho protein function is expected to provide new insights into FGF signaling, phosphate/calcium homeostasis, regulation of ion channel activity, and aging.

Klotho Overexpression Extends Life Span in the Mouse

lines, EFmKL46 and EFmKL48, and wild-type mice with the same genetic background were used for this study. When mice carrying the EFmKL46 or EFmKL48 transgenic alleles were fed ad libitum on a standard diet, both lines significantly outlived wild-type controls (fig. 1). The average life spans of EFmKL46 and EFmKL48 were 20.0 and 30.8% longer than that of controls in males, and 18.8 and 19.0% longer in females, respectively. The maximum life span was also extended in both lines in both sexes. The observation of significant life span extension in two independent transgenic lines provides genetic evidence that Klotho can suppress aging.

Klotho Increases Resistance to Insulin and IGF-I

As mentioned above, knocking out the Klotho gene resulted in multiple age-related phenotypes in the mice and significantly shortened their life span [12]; however, it was possible that the early demise of the animals occurred through multiple organ failure and compromised physiological state due to disease and not necessarily through accelerated aging. We therefore performed the converse experiment: overexpress the Klotho protein and examine whether age-related phenotypes are suppressed and whether the lifespan of the organism can be increased. Recently, we generated transgenic mice that overexpress Klotho under the control of the human elongation factor 1 promoter and established cohorts to compare the life span of the transgenic mice with that of wild-type controls [11]. Two independent transgenic

Moderate inhibition of the insulin-like signaling pathway is one of the mechanisms for suppressing aging that is evolutionarily conserved from worms to mammals [1], suggesting that the klotho gene is involved in the inhibition of insulin and/or IGF-I signaling. This hypothesis is consistent with earlier observations that mice defective in klotho gene expression have reduced blood glucose and insulin levels as a result of significantly enhanced sensitivity to insulin [39]. To test this hypothesis further, we compared measures of glucose metabolism in the Klothooverexpressing transgenic mice with those in wild-type animals [11]. Basal blood glucose levels were normal in the two transgenic lines; however, male EFmKL46 and EFmKL48 mice had significantly higher blood insulin levels than wild-type males. Therefore, it appears that the

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male transgenic mice required more insulin than the wild-type mice to maintain normal blood glucose levels as a result of increased insulin resistance. To confirm this notion, we performed hyperinsulinemic euglycemic clamp experiments during which a fixed amount of insulin was continuously infused. We infused glucose at the same time to maintain normal blood glucose levels. Under these conditions, the glucose infusion rate is known to be inversely correlated with the degree of insulin resistance of the animals [40]. As expected, male EFmKL46 and EFmKL48 mice required significantly lower glucose infusion rates than wild-type males to maintain normal blood glucose levels. Furthermore, insulin and IGF-I tolerance tests revealed significant attenuation in hypoglycemic response to injected insulin and IGF-I in male transgenic mice. Although insulin resistance is not detected in female transgenic mice, they are significantly resistant to IGF-I. These studies demonstrate that Klotho overexpression induces resistance to insulin and/or IGFI. Precise mechanisms of the sexual dimorphism in the effect of Klotho on insulin/IGF-I resistance remain to be determined.

Klotho Functions as a Hormone

To test the notion that the extracellular domain of Klotho functions as an endocrine factor, we generated a soluble form of recombinant Klotho protein comprising just the extracellular domain, which is equivalent to the form that circulates in the blood, and determined whether this protein is active in promoting insulin resistance when injected into mice [11]. We administered insulin (0.5 U/kg) and purified extracellular Klotho peptide (10 g/kg) simultaneously to wild-type mice by intraperitoneal injection and observed no significant attenuation of the hypoglycemic response to injected insulin either in males or in females. The results reiterate the effect of overexpression of the klotho gene that we observe in EFmKL46 and EFmKL48 males. Furthermore, injection of Klotho protein alone significantly increases blood glucose levels in wild-type mice both in males and, to a lesser extent, in females without significant changes in blood insulin and glucagon levels. To more broadly elucidate the mechanisms by which Klotho antagonizes insulin, we investigated whether recombinant extracellular Klotho peptide could reduce glucose uptake by cells through the blocking of insulin binding to the insulin receptor [11]. To do this, we stimulated cultured myoblastic cells (L6) with 10 nM of insulin 194

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in the presence or absence of 100 pM of Klotho protein and quantified cellular glucose uptake. Klotho suppressed insulin-induced glucose uptake by 55% without reducing the binding of [125I]-insulin to the cells. These observations argue against the notion that Klotho antagonizes insulin through inhibiting insulin binding to the insulin receptor, but raise the possibility that Klotho may block insulin action by transmitting a signal(s) to perturb intracellular signaling pathways activated by insulin.

Klotho Circulates in Blood and Binds to a Putative Klotho Receptor

If Klotho protein indeed functions as a humoral factor that inhibits intracellular signaling activated by insulin, we would expect receptors for the Klotho protein to be expressed on the cell surface. To demonstrate Klotho interaction with a putative receptor, we performed quantitative analysis of [125I]-labeled Klotho binding to cultured cells and observed that binding of [125I]-Klotho to the cell surface increases in a dose-dependent manner and reaches saturation when the total Klotho concentration exceeds 600 pM. We also observed that unlabeled Klotho protein inhibits the binding of [125I]-Klotho to the cell surface in a dose-dependent manner [11]. These observations indicate that binding sites specific for Klotho, or putative Klotho receptors, exist on the cell surface. The concentration of unlabeled Klotho protein necessary to obtain half-maximal inhibition of [125I]-Klotho binding is approximately 300 pM. The average number of Klotho binding sites on the cell surface is estimated at 1 ! 105/ cell in L6 cells. In order for Klotho to function as a hormone in vivo, the protein must be present in the blood. Immunoblot analyses of plasma using anti-Klotho antiserum demonstrate that the Klotho protein can be detected as a 110kDa form in wild-type, EFmKL48, and EFmKL46 mice but not in KL–/– mice [11]. Furthermore, a radioimmunoassay to quantify plasma Klotho protein concentrations revealed that the average plasma Klotho level of wild-type mice is 100 pM. EFmKL48 and EFmKL46 mice have approximately twofold and 2.5-fold higher plasma Klotho levels, respectively, than wild-type mice [11]. No significant difference is observed between males and females in each genotype. Thus, the Klotho protein circulates in the blood at a concentration that can potentially stimulate the putative Klotho receptors, corroborating the notion that Klotho functions as a hormone in vivo under physiological conditions. Rosenblatt /Kuro-o

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Fig. 2. Klotho protein inhibits intracellular insulin/IGF-I signaling. A , B Effect of

Klotho on tyrosine-phosphorylation of insulin/IGF-I receptors and IRS-1/2, association of IRS-1/2 with the PI3-kinase regulatory subunit (p85), and phosphorylation of Akt in L6 cells stimulated with 10 nM of insulin (A) or 10 nM of IGF-I (B). Antibodies used for immuno-precipitation (ip) and immunoblotting (ib) were indicated. IR; anti-insulin receptor -chain antibody, IGF-IR; anti-IGF-I receptor chain antibody, IRS-1; anti-IRS-1 antibody, IRS-2; anti-IRS-2 antibody, p85; anti-PI3-kinase regulatory subunit antibody, pY20; anti-phosphotyrosine antibody.

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Klotho Inhibits Intracellular Insulin/IGF-I Signaling

Since Klotho does not perturb insulin signaling by blocking its interaction with the insulin receptor, we examined the effect of Klotho on intracellular signaling molecules operant in the insulin/IGF-I signaling cascade in cultured cells to demonstrate an intracellular effect of the Klotho protein on the insulin pathway [11]. L6 cells or rat hepatoma cells (H4IIE) were incubated with various concentrations of recombinant extracellular Klotho peptide and then stimulated with insulin (10 nM) or IGF-I (10 nM). The cell lysates were analyzed for tyrosine phosphorylation of insulin/IGF-I receptors and IRS-1/2 and for the association of p85 with IRS proteins. Adding the Klotho protein did not inhibit the binding of [125I]-insulin or [125I]-IGF-I to the cells under these experimental conditions. However, Klotho suppressed insulin- or IGF-I-induced tyrosine phosphorylation of insulin/IGF-I receptors in a dose-dependent manner (fig. 2A, B). In addition, Klotho reduced tyrosine-phosphorylated IRS-1/2 levels and the interaction of p85 with IRS proteins (fig. 2A, B). It remains to be determined whether the effect of Klotho on IRS proteins and PI3-kinase is dependent on decreased tyrosine-phosphorylation of insulin/IGF-I receptors.

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lecular perturbations is expected to rescue some of the aging-like phenotypes in Klotho–/– mice. We explored this possibility by interfering with insulin/IGF-I signaling in Klotho–/– mice through the introduction of a mutated IRS-1 gene [11]. Klotho–/– mice heterozygous for an IRS-1 null allele (Klotho–/– IRS-1+/–) exhibit a significantly longer life span than Klotho–/– mice without the IRS-1 gene mutation both in males and, to a lesser extent, in females. In addition, Klotho–/– IRS-1+/– mice show significant improvement in all age-related pathological changes examined, including arteriosclerosis, ectopic calcification, skin atrophy, pulmonary emphysema, and hypogonadism when compared with their Klotho–/– littermates. Although reduction in IRS-1 expression extends the life span in Klotho–/– mice, this effect is minor when compared to the complete rescue observed in Klotho–/– mice carrying the EFmKL46 or EFmKL48 transgenic allele. Nonetheless, the fact that reduction in IRS-1 expression improves life spans and major aginglike pathological changes in Klotho–/– mice provides genetic evidence that the anti-aging activity of Klotho is mediated, at least in part, through its ability to inhibit insulin/IGF-I signaling.

Klotho Activates Forkhead Transcription Factors Inhibition of Insulin/IGF-I Signaling Rescues Klotho–/– Phenotypes

If the ability of Klotho to inhibit insulin/IGF-I signaling indeed contributes to its anti-aging properties, inhibition of insulin/IGF-I signaling effected by other mo-

In addition to moderate insulin resistance, increased resistance to oxidative stress has been identified as one of the mechanisms for suppressing aging, and it also is evolutionarily conserved from worms to mammals [41]. Insulin/IGF-I signaling regulates many essential intracel-

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lular signaling molecules; among them, forkhead transcription factors may play critical roles in the regulation of aging because life span extension by insulin-like signaling inhibition in C. elegans depends on expression of the forkhead transcription factor daf-16 [2, 42–44]. The mammalian homologs of daf-16 belong to the FOXO subfamily of forkhead transcription factors (FOXO1, FOXO3a, and FOXO4). The activity of these transcription factors is negatively regulated by phosphorylation via the kinase Akt, and phosphorylated FOXO factors are exported from the nucleus and exclusively localized in the cytoplasm where they cannot mediate transcription [45]. Recent studies using mammalian cells demonstrate that activation of FOXO transcription factors induces cell cycle arrest and promotes DNA repair [46, 47]. In addition, FOXO factors upregulate expression of enzymes that detoxify reactive oxygen species such as manganese superoxide dismutase (SOD2) and catalase, thereby reducing oxidative stress [48]. All these changes induced by activation of FOXO factors can antagonize aging and may be involved in the mechanism(s) by which Klotho suppresses aging. To test such a possibility, we looked for the possible reduction of Akt and FOXO phosphorylation by soluble Klotho protein [49]. When we stimulated HeLa cells with the extracellular domain of Klotho, we observed a significant reduction in the basal level of Akt and FOXO1 phosphorylation in a dose- and time-dependent manner (fig. 3A). In addition, quantitative protein microarray analyses demonstrated that the Klotho protein suppresses both basal and insulin-induced phosphorylation of Akt and FOXO1 (fig. 3B). This anti-aging hormone also suppresses phosphorylation of FOXO3a and FOXO4 (fig. 3C). These observations confirmed that treatment of cells with the Klotho protein reduces phosphorylation of FOXOs. To uncover a comparable inhibitory effect of Klotho on Akt and FOXO phosphorylation at the organism level, we analyzed tissues from Klotho–/– mice, wild-type mice, and transgenic mice that overexpress the Klotho protein (EFmKL48) [49]. Insulin-induced phosphorylation of Akt and FOXO in the muscle is enhanced in Klotho–/– mice and attenuated in EFmKL48 mice, an inverse correlation with blood Klotho levels (fig. 3D). These observations are consistent with the notion that the circulating Klotho protein has an activity that reduces FOXO phosphorylation in vivo. To demonstrate whether the Klotho-induced decrease in FOXO phosphorylation is associated with its nuclear translocation, we performed immunocytochemical anal196

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ysis [49]. Klotho protein promoted translocation of FOXO1 from the cytoplasm to the nucleus (fig. 3E). Furthermore, immunoblot analysis confirmed that Klotho resulted in an increase of FOXO1 in the nuclear fraction and a reciprocal decrease in the cytoplasmic fraction in a dose-dependent manner. This effect was evident within 15 min after the addition of Klotho protein (fig. 3F). The dose response and the time course of nuclear translocation of FOXO1 were inversely correlated with the level of its phosphorylation (fig. 3A). These observations indicate that the protein signals nuclear translocation of FOXO.

Klotho Increases Resistance to Oxidative Stress

We next investigated whether the Klotho-induced nuclear translocation of FOXO results in the transcriptional activation of target genes [49]. HeLa cells were transfected with a luciferase reporter construct containing a three-tandem array of the forkhead responsive element derived from the Fas ligand gene promoter and then incubated with Klotho protein to activate endogenous FOXO factors. Klotho stimulated the activity of this promoter; the protein also increased the activity of a human SOD2 gene promoter, which is known to contain canonical FOXO binding sites. Furthermore, a chromatin immunoprecipitation assay revealed that Klotho protein treatment increased the amount of FOXO1 bound to the native SOD2 gene promoter, demonstrating that the Klotho-induced transcriptional activation occurs via direct binding of FOXO1 to the SOD2 promoter. The increase in promoter activity is associated with an increase in SOD2 protein levels. To quantify the Klotho-induced increase in SOD2 protein we performed quantitative protein microarray analyses. Klotho protein increased the SOD2 protein levels 1.5-, 2.3-, and 5.8-fold within 16 h in COS, HeLa, and CHO cells, respectively. SOD2 mRNA levels in muscle samples from Klotho–/–, wild-type, and Klotho-overexpressing transgenic mice (EFmKL48) determined by Northern blot analysis positively correlated with circulating Klotho levels. Accordingly, SOD2 protein levels in muscles increased as the blood Klotho level increased. Quantitative protein microarray analysis revealed that the SOD2 protein level in muscles of Klotho–/– mice and EFmKL48 mice was 77 8 16% and 234 8 6% (mean 8 SD, n = 4), respectively, when compared with that of wild-type mice. All of these findings indicate that Klotho protein increases SOD2 expression both in vitro and in vivo. Rosenblatt /Kuro-o

0

25 50 100 200 400

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Fig. 3. Klotho reduces FOXO phosphory-

+

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FOXO1

+

– –

p-FOXO1

+

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KL WT EFmKL lation and promotes its nuclear translocaInsulin – + – + – + tion. A HeLa cells transfected with the FOXO1 p-Akt FOXO1-myc expression vector were stimp-FOXO3a ulated with various concentrations of Akt FOXO3a Klotho as indicated for 30 min (left panels). Cell lysates were immunoblotted with p-FOXO p-FOXO4 anti-Akt antibody (Akt), anti-myc antiFOXO1 body (FOXO1), or antibody specific to D C FOXO4 phosphorylated Akt (p-Akt) or FOXO (pFOXO1). The cells were stimulated with Klotho (200 pM) Klotho (0 pM) 200 pM Klotho for various time periods (right panels). B Protein microarray analysis of Akt and FOXO1 phosphorylation. FOXO1 HeLa cells transfected with FOXO1-myc expression vector were incubated in the presence or absence of 200 pM Klotho for 30 min and then stimulated with 10 nM inE sulin or left untreated for 30 min. Cell lysates were arrayed in quadricate in a serial Minutes after klotho stimulation Klotho (pM) dilution format. The slides were probed FOXO1 0 25 50 100 200 400 0 1 3 5 10 15 30 60 with the antibodies indicated. RepresentaCytoplasm tive blots from four independent experiNucleus ments are shown. C Klotho reduced both F basal and insulin-induced phosphorylation of all three FOXOs. HeLa cells transfected with the FOXO1-myc, FOXO3amyc, or FOXO4-myc expression vector were treated as in B. Cell ed with or without 200 pM Klotho for 30 min and then stained lysates were analyzed in the same way as in A . D Klotho reduced with anti-myc antibody. F Klotho-induced nuclear translocation insulin-induced phosphorylation of Akt and FOXO in mice. The of FOXO1 determined by immunoblotting. HeLa cells transfected lysates of muscle from KL–/– mice (KL–/–), wild-type (WT), and with the FOXO1-myc expression vector were incubated with the EFmKL48 mice (EFmKL) administered with insulin or saline indicated concentrations of Klotho for 30 min and then fractionwere immunoblotted with the antibodies indicated. Represen- ated into cytoplasm and nuclear fractions. Each fraction was imtative blots from four independent experiments are shown. munoblotted with anti-myc antibody (left panels). To determine E Klotho-induced nuclear translocation of FOXO1. HeLa cells the time-course the cells were incubated with 200 pM Klotho for transfected with the FOXO1-myc expression vector were incubat- the indicated time (right panels).

Klotho, an Aging-Suppressor Gene

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–/–

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p = 0.0019

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0 2 4 6 8 10 Time after paraquat injection (days)

Fig. 4. Klotho increases resistance to oxidative stress. A The effect

of Klotho protein on lipid oxidation in cultured cells. HeLa cells left untreated (–) or treated (+) with 100 M paraquat in the presence (+) or absence (–) of 200 pM recombinant Klotho protein were loaded with a fluorescent lipid oxidation probe C11BODIPY581/591. Yellow represents high oxidation and red represents low oxidation. A representative result from four independent experiments is shown. B –D The effect of Klotho protein on cell death induced by oxidative stress. B Representative dot plots of CHO cells stained with annexin V-FITC and propidium iodide (PI). The cells were treated with 100 M paraquat in the presence (right panel) or absence (left panel) of 200 pM Klotho protein. The bottom right quadrant represents early apoptotic cells. The top right and left quadrants represent late apoptotic and necrotic cells,

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0 Klotho D Annexin V

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respectively. The percentage counts of each quadrant are indicated. C Representative histograms of the CHO cells based on annexin V binding in the presence (red) and absence (blue) of 200 pM Klotho protein under the paraquat-induced oxidative stress. D Mean percentages of annexin V-positive and negative cells in the presence and absence of Klotho protein. The annexin V-negative cells virtually represent live cells under these experimental conditions, because necrotic cell counts were !4% (the top left quadrant in B); * p ! 0.01 by ANOVA (n = 4). E Urinary 8-OHdG levels in wild-type mice (WT) and EFmKL48 mice (EFmKL); * p = 0.02 by ANOVA (n = 4). F Kaplan-Meier analysis of survival after the paraquat challenge between age-matched male wild-type mice (WT) and EFmKL48 mice (EFmKL); p = 0.0019 by log-rank test.

Rosenblatt /Kuro-o

Klotho Binds to Multiple FGF Receptors

Identification of a Klotho receptor or binding partners of Klotho is of critical importance for understanding the Klotho signaling pathway. As mentioned previously, klotho and FGF23 knockout mice share numerous phenotypes that resemble a syndrome of early aging. In addition, Klotho and FGF23 reveal similar physiological effects, including the actions of an expected phosphaturic hormone. This genetic data supports the supposition that the FGF23 and Klotho proteins interact in some manner. Klotho, an Aging-Suppressor Gene

FGFR i.p.

Klotho (Flag)

i.b.

Mock 1b (L) 1b (S) 1c (L) 1c (S) 2b (L) 2b (M) 2b (S) 2c (L) 2c (M) 2c (S) 3b 3c 4

We then determined whether Klotho-induced FOXO activation and the concomitant increase in SOD2 expression might have physiological significance in reducing cellular reactive oxygen species [49]. To demonstrate a protective effect of the Klotho protein, we treated HeLa cells with paraquat, an herbicide that generates superoxide, and compared the extent of lipid oxidation in living cells in the presence and absence of a soluble form of recombinant Klotho protein. A fluorescent probe (C11BODIPY581/591) was employed to measure oxidized lipid [50]. Klotho protein treatment significantly suppressed paraquat-induced increases in lipid oxidation (fig. 4A). Because we suspected that Klotho protects cells from apoptosis induced by oxidative stress, which potentially promotes the survival of irreplaceable cells and contributes to the suppression of aging, we treated Chinese hamster ovary (CHO) cells with paraquat and compared the number of apoptotic cells in the presence and absence of the Klotho protein. This treatment reduced the annexin V-positive, apoptotic cell population and preserved the annexin V-negative, non-apoptotic cell population (fig. 4B–D). These observations indicate that Klotho protein confers oxidative stress resistance on mammalian cells. Since urinary 8-hydroxydeoxyguanosine (8-OHdG) is a biological marker of in vivo oxidative DNA damage [51], we compared urinary 8-OHdG levels of long-lived Klotho-overexpressing transgenic mice (EFmKL48) with those of wild-type mice. The average urinary 8-OHdG excretion in EFmKL48 mice was approximately one half of that in wild-type mice (fig. 4E), indicating that Klotho overexpression reduces overall oxidative DNA damage in mice. Additionally, we observed that EFmKL48 mice survived a challenge with lethal doses of paraquat for significantly longer periods than did wild-type mice (fig. 4F), suggesting that Klotho confers oxidative stress resistance at the organismal level as well.

FGFR (V5) Klotho

Klotho FGFR (V5) FGFR (V5)

Fig. 5. Klotho binds to multiple FGF receptors. 293 cells were sta-

bly transfected with an expression vector for the full-length transmembrane form of Klotho with a Flag epitope tag at the C-terminus (293KL). Lysates of 293KL cells transiently transfected with expression vectors for different FGFR isoforms with a V5 epitope tag were immunoprecipitated with Klotho using anti-Flag antibody and probed either with FGFRs using anti-V5 antibody or with Klotho using anti-Klotho rat monoclonal antibody KM2119 (upper two panels). The lysates were also immunoprecipitated with FGFRs using anti-V5 antibody and probed either with Klotho using KM2119 or with FGFRs using anti-V5 antibody (lower two panels). Antibodies used for immunoprecipitation (i.p.) and immunoblotting (i.b.) were indicated.

To establish whether Klotho may be involved in FGF signaling, we investigated whether Klotho could directly bind to FGF receptors (FGFRs) [38]. By transfecting 293KL cells that stably express the full-length transmembrane form of Klotho with various FGFR expression vectors, we can examine whether any of the FGFRs can be immunoprecipitated along with Klotho. Klotho bound to almost all FGFR isoforms tested (fig. 5); however, significant differences in the ability of Klotho to co-precipitate FGFRs are observed between the isoforms: (1) The c isoforms are more efficiently co-precipitated with Klotho than the b isoforms. (2) FGFR2 is less efficiently co-precipitated with Klotho than FGFR1, FGFR3, and FGFR4. Thus, Klotho may bind to multiple FGFRs with different affinities.

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00 ,0

00 ,0 10

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Klotho

Fig. 6. FGF23 preferentially binds to the Klotho/FGFR complex.

Lysates of 293 cells or 293KL cells transfected with the FGFR expression vectors were applied to anti-V5 agarose. Serum-free conditioned medium of 293 cells or 293KLTM cells (293 cells that were stably transfected with an expression vector for the extracellular domain of Klotho with a Flag epitope tag at the C-terminus) was applied to anti-Flag agarose. After washing, the beads were incubated with conditioned medium of 293 cells transfected with the mouse FGF23 (R179Q) expression vector. The washed beads were subjected to immunoblot analysis using anti-V5 antibody, KM2119, or anti-FGF23 antibody (R&D Systems).

FGF23 Requires Klotho to Bind to FGF Receptors and Activate FGF Signaling

To test whether the binding of Klotho to FGFRs may affect interaction between FGFRs and FGF23, the ability of FGFRs to immuno-precipitate with FGF23 was examined in the presence and absence of Klotho [38]. FGF23 is pulled down with FGFR1c, 3c, and 4 only in the presence of Klotho (fig. 6). We also examined the possibility that FGF23 might directly bind to Klotho; however, the extracellular domain of Klotho alone failed to pull down FGF23 under these experimental conditions (fig. 6). These observations indicate that FGF23 exhibits stronger interaction with the Klotho/FGFR complex than with Klotho or FGFR alone. Since Klotho increases the binding of FGF23 to FGFRs, we expected it might enhance the ability of FGF23 to activate FGF signaling. Thus, we stimulated 293KL cells or 293 cells with various concentrations of recombinant human FGF23 and compared the levels of phosphorylation of FGF receptor substrate-2 (FRS2) and p44/42 MAP kinase (ERK1/2). Addition of 3 nM (100 ng/ml) FGF23 200

4,

2,

00

0

0

FGFR4

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Fig. 7. Knockdown of FGF receptors by siRNA. 293KL cells transfected with siRNA for each FGFreceptor (FGFR1–4) or random siRNA (Control) as indicated were stimulated with 0.3 nM FGF23 for 10 min. Cultures were carried out in triplicate and cell lysates were printed on FAST slides in triplicate using the printing robot. The slides were probed with anti-phospho-ERK antibody (left panel). The average relative signal intensity corrected with the actin signal was determined using MicroVigene (right panel). All siRNA for FGFRs reduced the amount of phosphorylated ERK induced by FGF23.

failed to induce phosphorylation of FRS2 and ERK1/2 in 293 cells. In contrast, 0.3 nM (10 ng/ml) FGF23 activated FRS2 and ERK1/2 in 293KL cells, indicating that Klotho enhances the cellular sensitivity to FGF23 by greater than tenfold without the help of heparin or glycosaminoglycan. Similar signaling results were obtained when using two independent 293KL clones. In addition, we observed that FGF23 induces FRS2 and ERK phosphorylation in 293KLTM cells, although the levels are diminished relative to those in 293KL cells. Consistent with these findings, endogenous FGFRs in 293KL cells can be co-precipitated with the full-length transmembrane Klotho and, to a lesser extent, with the extracellular domain of Klotho. We also confirmed that the extracellular domain of Klotho binds to exogenously expressed FGFRs in the same pattern as the full-length Klotho protein. These observations suggest that FGFRs may function as receptors for circulating extracellular Klotho peptide. We conclude that both the full-length Klotho and the extracellular domain of Klotho function as cofactors that are necessary for efficient activation of FGF signaling by FGF23. Rosenblatt /Kuro-o

Discussion

Our results reveal a signaling pathway through which the anti-aging hormone Klotho increases resistance to oxidative stress. Klotho signals inhibition of FOXO phosphorylation and promotes its nuclear translocation. The nuclear FOXO then binds to the SOD2 promoter and upregulates its expression, thereby facilitating removal of reactive oxygen species and conferring resistance to oxidative stress. Although it remains to be determined whether the ability of Klotho to confer oxidative stress resistance depends entirely on the induction of SOD2 expression, this activity may be crucial for the anti-aging properties of Klotho, because overexpression of another enzyme that removes reactive oxygen species (catalase) in mitochondria extends the life span in mice [52]. We propose that the Klotho protein suppresses aging via at least two distinct anti-aging mechanisms that are evolutionarily conserved: (1) the inhibition of insulin-like signaling and (2) an increase in resistance to oxidative stress. Our results also suggest that Klotho serves as an important link between these two anti-aging mechanisms. The fact that the extracellular domain of Klotho can increase cellular sensitivity to FGF23 has raised the possibility that it may function as a paracrine factor in the kidney. Since Klotho is expressed in the distal convoluted tubules and the extracellular domain of Klotho is shed and secreted, it may act on adjacent proximal tubules and work cooperatively with FGF23 to inhibit phosphate reabsorption. It remains to be determined whether extracellular Klotho peptide could function as a paracrine and/or an endocrine factor in the regulation of FGF23 signaling. We have demonstrated that Klotho has an activity that inhibits insulin/IGF-I signaling, activates FOXOs, induces SOD2 expression, and increases resistance to oxidative stress at cellular and organismal levels. In addition, we have shown that Klotho binds to multiple FGFRs and regulates their affinity to FGFs, notably to FGF23. These findings have raised two central questions. (1) What is the relation between the insulin/IGF-I signaling and FGF signaling regulated by Klotho? Our working hypothesis is that activation of FGF signaling by Klotho may induce protein kinase C (PKC) activation, which potentially inhibits insulin receptor activity through phosphorylation [53–55]. (2) Does SOD2 induction by Klotho contribute to its anti-aging properties? We generated transgenic mice that overexpress SOD2 and are currently crossing them to the Klotho–/– background to test whether any aging-like phenotypes can be rescued. Klotho, an Aging-Suppressor Gene

Klotho is a multi-functional protein that regulates insulin/IGF-I signaling, oxidative stress resistance, FGF signaling, vitamin D metabolism, and ion transporter activity, all of which are potentially associated with its antiaging properties. We are now focusing on investigating the effect of Klotho on FGF signaling and vitamin D metabolism, and on deciphering the molecular networks that mediate its protective effects against oxidative damage. One question is whether increased activation of the Klotho signaling pathway can confer resistance to diseases thought to be mediated, at least in part, by reactive oxygen species (ROS) and free-radical damage, such as in the neurodegenerative diseases. Interestingly, epidemiological studies have targeted aging as the predominant risk factor for the development of sporadic Parkinson’s disease [56], and oxidative damage is believed to underlie some of the pathogenetic mechanisms of this and other neurodegenerative diseases [57].

Future Directions

To more fully dissect the molecular networks driving the effects of the anti-aging hormone Klotho, our laboratories in the Pathology Department at the University of Texas Southwestern Medical Center at Dallas have been collaborating to establish high-throughput screening of siRNA libraries using reverse-phase protein microarrays to monitor effects on signaling. Our laboratories previously used this method to quantify the amount of phosphorylated Akt, phosphorylated FOXO, and SOD2 proteins in cells and tissues [49]. The siRNA library for kinases (Stealth RNAi Human Kinase Collection, Invitrogen) consists of 636 siRNAs designed to knock down any human kinases. The library is aliquoted into seven 96-well plates, each well containing an individual siRNA (a cocktail of three independent siRNAs to each target) or negative controls. After transfection reagent (Oligofectamine) is added to each well, 293 or 293KL cells are plated into the wells. After transfection, the cells are stimulated with FGF23 or Klotho. Proteins are extracted from each well by adding lysis buffer containing inhibitors for phosphatases and proteinases. All media changes, cell plating, transfections, incubations and sample manipulations are carried out using the automated liquid handling systems for increased reproducibility. The protein samples are printed onto specialized substrates for protein immobilization in triplicate using microarray printing robots. Each slide is probed simultaneously with antibodies against markers of interest and house keeping genes Horm Res 2007;67(suppl 1):191–203

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such as -actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for normalization. All probing procedures (incubation, wash, etc.) are carried out with automated equipment modified in-house, which not only allows high-throughput analysis but also exhibits excellent within-run and between-run analytical precision. We have established a method for multiplexed fluorometric detection using quantum dots with different emission wavelengths. Fluorescent spots are scanned using a hyperspectral imaging system also developed on campus. All spots are normalized to -actin signal, which are carried out using proprietary and in-house software that automatically determines the spot locations and normalizes spot intensities. Our laboratory has optimized all of these processes and established a fully automated screening method for the

siRNA library using various cell lines. In addition, we have optimized the experimental condition for 293KL cells and successfully knocked down FGFRs using siRNA and observed significant reductions in phospho-ERK in response to FGF23 stimulation by protein microarray (fig. 7). We are currently optimizing the conditions for protein microarray analysis using antibodies against several markers, including pFRS, pERK and SOD2. Now that all of the procedures have been optimized, we are screening the kinase siRNA library to generate a ‘parts list’ for kinases involved in Klotho signaling and protective effects. Using the same method, we are concurrently screening a siRNA library for nuclear receptors (Stealth RNAi Human Nuclear Receptor Collection, Invitrogen) to examine the effects of nuclear hormones, including vitamin D, on Klotho signaling. These studies are expected to identify new signaling molecules and pathways involved in the regulation of aging and longevity by Klotho and FGF23.

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22 Tohyama O, Imura A, Iwano A, Freund JN, Henrissat B, Fujimori T, Nabeshima Y: Klotho is a novel beta-glucuronidase capable of hydrolyzing steroid beta-glucuronides. J Biol Chem 2004; 279:9777–9784. 23 The ADHR Consortium: Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345–348. 24 Bai XY, Miao D, Goltzman D, Karaplis AC: The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency. J Biol Chem 2003; 278:9843–9849. 25 Bai X, Miao D, Li J, Goltzman D, Karaplis AC: Transgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders. Endocrinology 2004; 145:5269–5279. 26 Larsson T, Marsell R, Schipani E, Ohlsson C, Ljunggren O, Tenenhouse HS, Juppner H, Jonsson KB: Transgenic mice expressing fibroblast growth factor 23 under the control of the alpha1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis. Endocrinology 2004;145:3087–3094. 27 Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, Takeuchi Y, Fujita T, Nakahara K, Fukumoto S, Yamashita T: FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 2004;19:429–435. 28 Yuan B, Xing Y, Horst RL,Drezner MK: Evidence for abnormal translational regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase activity in the hyp-mouse. Endocrinology 2004; 145:3804–3812. 29 Dusso AS, Brown AJ, Slatopolsky E: Vitamin D. Am J Physiol Renal Physiol 2005;289:F8– F28. 30 Inoue Y, Segawa H, Kaneko I, Yamanaka S, Kusano K, Kawakami E, Furutani J, Ito M, Kuwahata M, Saito H, Fukushima N, Kato S, Kanayama HO, Miyamoto K: Role of the vitamin D receptor in FGF23 action on phosphate metabolism. Biochem J 2005;390:325– 331. 31 Shimada T, Kakitani M, Yamazaki Y, Hasegawa H, Takeuchi Y, Fujita T, Fukumoto S, Tomizuka K, Yamashita T: Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 2004; 113: 561– 568. 32 Powers CJ, McLeskey SW, Wellstein A: Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 2000;7:165– 197. 33 Eswarakumar VP, Lax I, Schlessinger J: Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 2005;16:139–149.

Klotho, an Aging-Suppressor Gene

34 Yu X, Ibrahimi OA, Goetz R, Zhang F, Davis SI, Garringer HJ, Linhardt RJ, Ornitz DM, Mohammadi M, White KE: Analysis of the biochemical mechanisms for the endocrine actions of fibroblast growth factor-23. Endocrinology 2005;146:4647–4656. 35 Baum M, Schiavi S, Dwarakanath V, Quigley R: Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules. Kidney Int 2005;8:1148–1153. 36 Tsujikawa H, Kurotaki Y, Fujimori T, Fukuda K, Nabeshima Y: Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system. Mol Endocrinol 2003;17:2393–2403. 37 Razzaque MS, Sitara D, Taguchi T, St-Arnaud R, Lanske B: Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. FASEB J 2006;20:720–722. 38 Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M: Regulation of fibroblast growth factor-23 signaling by Klotho. J Biol Chem 2006; 281: 6120– 6123. 39 Utsugi T, Ohno T, Ohyama Y, Uchiyama T, Saito Y, Matsumura Y, Aizawa H, Itoh H, Kurabayashi M, Kawazu S, Tomono S, Oka Y, Suga T, Kuro-o M, Nabeshima Y, Nagai R: Decreased insulin production and increased insulin sensitivity in the klotho mutant mouse, a novel animal model for human aging. Metabolism 2000;49:1118–1123. 40 Halseth AE, Bracy DP, Wasserman DH: Overexpression of hexokinase II increases insulin and exercise-stimulated muscle glucose uptake in vivo. Am J Physiol 1999; 276: E70–E77. 41 Finkel T, Holbrook NJ: Oxidants, oxidative stress and the biology of ageing. Nature 2000;408:239–247. 42 Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G: The fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 1997;389:994–999. 43 Lin K, Dorman JB, Rodan A, Kenyon C: daf16:An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 1997; 278: 1319– 1322. 44 Henderson ST, Johnson TE: daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr Biol 2001; 11:1975–1980. 45 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME: Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 1999; 96: 857–868.

46 Kops GJ, Medema RH, Glassford J, Essers MA, Dijkers PF, Coffer PJ, Lam EW, Burgering BM: Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol Cell Biol 2002; 22: 2025–2036. 47 Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ, DiStefano PS, Chiang LW, Greenberg ME: DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 2002;296:530–534. 48 Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang TT, Bos JL, Medema RH, Burgering BM: Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 2002; 419:316–321. 49 Yamamoto M, Clark JD, Pastor JV, Gurnani P, Nandi A, Kurosu H, Miyoshi M, Ogawa Y, Castrillon DH, Rosenblatt KP, Kuro-o M: Regulation of oxidative stress by the anti-aging hormone Klotho. J Biol Chem 2005; 280: 38029–38034. 50 Pap EH, Drummen GP, Winter VJ, Kooij TW, Rijken P, Wirtz KW, Op den Kamp JA, Hage WJ, Post JA: Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY(581/591). FEBS Lett 1999; 453:278–282. 51 Shigenaga MK, Gimeno CJ, Ames BN: Urinary 8-hydroxy-2-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc Natl Acad Sci USA 1989; 86: 9697–9701. 52 Schriner SE, Linford NJ, Martin GM, Treuting P, Ogburn CE, Emond M, Coskun PE, Ladiges W, Wolf N, Van Remmen H, Wallace DC, Rabinovitch PS: Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 2005; 308:1909–1911. 53 Bossenmaier B, Mosthaf L, Mischak H, Ullrich A, Haring HU: Protein kinase C isoforms b1 and b2 inhibit the tyrosine kinase activity of the insulin receptor. Diabetologia 1997;40:863–866. 54 Itani S, Zhou Q, Pories W, MacDonald K, Dohm G: Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity. Diabetes 2000;49:1353–1358. 55 Pillay TS, Xiao S, Keranen L, Olefsky JM: Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with beta isoenzymes and interaction with the catalytic domain of betaII. Cell Signal 2004;16:97–104. 56 Bowling AC, Beal MF: Bioenergetic and oxidative stress in neurodegenerative diseases. Life Sci 1995;56:1151–1171. 57 Abou-Sleiman PM, Muqit MMK, Wood NW: Expanding insights of mitochondrial dysfunction in Parkinson’s disease. Nat Rev Neurosci 2006;7:207–219.

Horm Res 2007;67(suppl 1):191–203

203

Abstract Winners

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):204–205 DOI: 10.1159/000097582

Published online: February 15, 2007

Vitamin D Stimulates Growth Hormone-InsulinLike Growth Factor (GH-IGF) Gene Axis Expression and Potentiates GH Effect to Reverse the Inhibition Produced by Glucocorticoids in Human Growth Plate Chondrocytes M. Fernández-Cancio P. Andaluz N. Torán C. Esteban A. Carrascosa L. Audí Pediatric Endocrinology Research Unit, Hospital Maternoinfantil Vall d’Hebron, Autonomous University of Barcelona, Barcelona, Spain

Key Words Growth plate Chondrocyte Vitamin D Insulin-like growth factor-I Insulin-like growth factor binding protein-3

ficiency. The interaction of VitD with GH to reverse gene expression inhibition produced by Dx was also analyzed.

Materials and Methods Introduction

Chronic treatment with high glucocorticoid (GC) doses has an inhibitor effect on skeletal growth. Growth plate and bone are target tissues for 1,25(OH)2-vitamin D (VitD), and chronic VitD deficiency, whether due to nutritional deprivation and/or lack of exposure to sunlight, results in bone diseases such as rickets. Moreover, growth retardation occurs in VitD-resistance states (human VDR mutations and VDR knockout mice).

Aims

Results

Dexamethasone (Dx) and VitD effects on proliferation and gene expression were studied in chondrocytes from human fetal epiphyseal growth plate cartilage to analyze the molecular mechanisms involved in skeletal growth inhibition produced by GC excess or by VitD de-

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Chondrocytes from human fetal epiphyseal growth plates were obtained at first passage, serum-deprived for 48 h and incubated for a further 48 h with Dx (10 –9 to 10 –6 M), VitD (10 –11 to 10 –6 M) or GH (500 ng/ml) or combinations of Dx, VitD and GH. Cell proliferation was determined as 3H-thymidine incorporation into DNA (n = 25), and gene expression of growth factors and binding proteins (IGF-I, IGF-II, IGF binding protein-3 [IGFBP3]), receptors (IGF-IR, GHR), SOX9 transcription factor and matrix proteins (aggrecan, COL2A1, COMP) were analyzed by total RNA extraction and real-time quantitative polymerase chain reaction (n = 8).

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Proliferation Low-dose Dx (10 –9 M) maintained proliferation (112 8 7%), suggesting that low GC concentrations did not interfere with basal cell proliferation whereas higher concentrations dose-dependently inhibited it (64 8 6 % at

Laura Audí, MD Pediatric Endocrinology Research Unit Hospital Maternoinfantil Vall d’Hebron, Paseo Vall d’Hebron 119 ES–8035 Barcelona (Spain) Tel./Fax +34 93 489 40 30, E-Mail [email protected]

Table 1. mRNA relative quantities of genes compared with control (n = 100) as regulated by Dx, VitD and GH (mean 8 SE)

Dx 10–6 M VitD 10–6 M GH 500 ng/ml Dx 10–6 + VitD 10–6 + GH

IGF-I

IGFBP-3

GHR

IGF-IR

SOX9

COL2A1

2086** 1,0538540 3068189 1,52081067

882** 241832* 133829 1284*

8684* 425896* 149832 158844

183828* 14289* 99821 131825

5087** 2786** 83831 6786* 116818 2108131 48816 21817

Aggrecan COMP 5687* 6088* 81815 52823

267884 162819* 123830 2308151

* p < 0.05; ** p < 0.0001.

10 –6 M). A similar effect was observed with VitD incubation (41 8 3.5 %), whereas GH had no significant effect (104 8 12%). The addition of GH to Dx (10–6 M) and VitD (10 –6 M) did not reverse the inhibitory effects of Dx and VitD alone. Gene Expression Dx dose-dependently inhibited expression of GH-IGF axis genes (IGF-I, IGFBP-3, GHR), transcription factors and matrix proteins (SOX9, COL2A1, aggrecan), whereas it stimulated expression of IGF-IR and the matrix protein COMP (table 1, at 10 –6 M). VitD had an opposite effect, which was also dose-dependent, by stimulating expression of IGF-I, IGFBP-3, IGF-IR, GHR and COMP, whereas it inhibited expression of SOX9, COL2A1 and aggrecan (table 1, at 10 –6 M). GH alone had a variable effect on gene

VitD and GH-IGF in Growth Retardation due to VitD-Resistant States

expression, which did not reach statistical significance. The combination of VitD with GH not only completely reversed inhibition by Dx of GHR and IGF-I expression, but stimulated it (table 1).

Conclusion

In the presence of pharmacological GC concentrations, VitD potentiated the GH effect, resulting in significant stimulation of GH-IGF axis gene expression (mainly IGF-I and GHR, but not IGFBP-3) in human growth plate chondrocytes. These results concur with the improvement in growth under GH therapy observed in children treated with chronic GC therapy, which requires maintenance of adequate VitD levels.

Horm Res 2007;67(suppl 1):204–205

205

Abstract Winners

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):206–207 DOI: 10.1159/000097583

Published online: February 15, 2007

Abnormalities of Pituitary Function after Traumatic Brain Injury in Children T. Niederland a H. Makovi a V. Gál a B. Andréka b C.S. Ábrahám c J. Kovács d Departments of a Pediatrics and b Isotopic Diagnostics, Petz Aladár County Teaching Hospital, Győr, c Department of Pharmaceutical Technology, University of Debrecen, Debrecen, and d Department of Pediatrics, University of Szeged, Szeged, Hungary

Key Words Children Head injury Pituitary dysfunction

Background

Traumatic brain injury (TBI) is known to be associated with pituitary lesions both in adults and children. Although the relationship between head trauma and hypopituitarism was originally described at the beginning of the last century, Benvenga et al. [1] presented the first meta-analysis in 2000 about the association of TBI with selective or combined pituitary hormone deficiency. More authors have emphasized that head trauma frequently occurs in young adults, with around 100 TBI hospitalizations per 100,000 total hospitalizations/year, and endocrine consequences may develop in about 30 to 50% of affected victims. Although brain injuries are also common in childhood, there are only a few case reports about the endocrine sequelae.

Aim

Methods Our endocrine follow-up study was performed between October 2003 and February 2004 in the Pediatric Department of Petz Aladár County Teaching Hospital, Győr, Hungary. Twenty-six children with a history of head injury (17 boys and 9 girls; mean age, 11.47 8 0.75 years; mean lapsed time after head injury, 30.6 8 8.3 months) and 21 age-matched controls were enrolled. Basal and stimulated anterior pituitary and peripheral hormone concentrations were measured by routine laboratory methods.

Statistical Analysis

All data presented are mean 8 SEM (n = 26 in head trauma group and n = 21 in the control group). Statistical analysis of different groups and at various time-points was performed using repeated measures analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test, Friedman test followed by Dunn’s multiple comparison test, unpaired Student t test, or MannWhitney test, as appropriate. The following statistical software was used for the analysis of data: GraphPad Prism, version 3.02 (GraphPad Software Inc., San Diego, Calif., USA).

Our main goal was to reveal the anterior pituitary function of children with a history of hospitalization due to mild-to-severe head trauma 30.6 8 8.3 months after head injury.

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T. Niederland, MD Department of Pediatrics Petz Aladár County Teaching Hospital HU–9024 Győr (Hungary) Tel. +36 209 495 735, Fax +36 9650 7913, E-Mail [email protected]

Table 1. Cumulative and peak GH levels during L-dopa and insulin tolerance tests (mean 8 SEM) L-dopa test

TBI Controls p

ITT

Basal GH ␮U/ml

Peak GH ␮U/ml

AUC GH

Mean GH ␮U/ml

Basal GH ␮U/ml

Peak GH ␮U/ml

AUC GH

Mean GH ␮U/ml

6.382.1 7.582.1 NS 0.1565

16.883.12 32.083.8 0.0031

5518116 1,1488155 0.0028

8.981.5 17.082.35 0.0001

9.982.5 5.882.1 NS 0.5316

20.583.74 27.183.81 NS 0.2253

7238147 9568142 NS 0.2605

11.681.5 15.181.9 NS 0.0576

Table 2. Cortisol values in insulin

tolerance test (mean 8 SEM) TBI Controls p

Basal cortisol nmol/l

Peak cortisol nmol/l

AUC cortisol nmol/l ! min

Mean cortisol nmol/l

313827 454837 0.0029

511836 712850 0.0016

24,15981,628 35,41182,064 <0.0001

388817 544828 <0.0001

Results

Pituitary dysfunction was detected in 61% of patients with a history of TBI. All growth hormone (GH) parameters measured and calculated were significantly (p ! 0.05) lower in the TBI group than in the controls after Ldopa stimulation. Similar differences were detected 60 min after insulin provocation. A total of 42% of all TBI children showed insufficient GH response after both stimulation tests, and 73% of these cases were boys. Cortisol levels of TBI patients were significantly (p ! 0.05) lower all through the insulin test than were values for subjects in the control group. The degree of pituitary dysfunction was independent of the severity of TBI. The height standard deviation score (SDS) corrected for chronological age was slightly decreased among TBI patients who were GH-deficient compared with TBI patients who were GH-sufficient (mean and SEM of height SDS was +0.1 8 0.74 vs. +0.29 8 0.38, respectively); however, the difference was not statistically significant (p = 0.59).

Conclusion

pituitary function after any kind of brain trauma requiring hospitalization in childhood. Pediatricians and endocrinologists should keep in mind the possibility of TBI as a cause of pituitary dysfunction (even in the absence of manifest clinical signs). These recommendations are in full concordance with the consensus guidelines on screening for hypopituitarism following TBI in adults [2–4].

References

1 Benvenga S, Campennı A, Ruggeri, RM, Trimarchi F: Hypopituitarism secondary to head trauma. J Clin Endocrinol Metabol 2000;85:1353–1361. 2 Ghigo E, Masel B, Aimaretti G, Léon-Carrión J, Casanueva FF, Dominguez-Morales MR, Elovic E, Perrone K, Stalla G, Thompson C, Urban R: Consensus guidelines on screening for hypopituitarism following traumatic brain injury. Brain Inj 2005; 19: 711–724. 3 Casanueva FF, Leal A, Koltowska-Haggstrom M, Jonsson P, Goth MI: Traumatic brain injury as a relevant cause of growth hormone deficiency in adults: a KIMS-based study. Arch Phys Med Rehabil 2005;86:463–468. 4 Urban RJ, Harris P, Masel B: Anterior hypopituitarism following traumatic brain injury. Brain Inj 2005;19:349–358.

Our study confirms the high risk for hypopituitarism in children with TBI despite a lack of obvious clinical symptoms. Our suggestion is to screen and follow-up Abnormalities of Pituitary Function after Traumatic Brain Injury in Children

Horm Res 2007;67(suppl 1):206–207

207

TBI Monograph

HORMONE RESEARCH

Horm Res 2007;67(suppl 1):208–221 DOI: 10.1159/000097584

Published online: February 15, 2007

Hypopituitarism in Adults and Children following Traumatic Brain Injury Felipe F. Casanueva a Ezio Ghigo b Michel Polak c Martin O. Savage d a

Division of Endocrinology, Department of Medicine and Complejo Hospitalario de Santiago, Santiago de Compostela, Spain; b Division of Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy; c Service d’Endocrinologie Pédiatrique Hôpital Necker, Paris, France; d Department of Endocrinology, St Bartholomew’s and the London School of Medicine and Dentistry, London, UK

Key Words Traumatic brain injury Hypopituitarism Growth hormone deficiency Diagnosis Cognitive function

Introduction

‘Traumatic Brain Injury and Hypopituitarism’ was an international workshop held on 9–10 April 2006 in Granada, Spain, as a satellite meeting to the 38th International GH and IGF-I Symposium organized by Pfizer Endocrine Care. Publications over the last 5 to 10 years have led to the understanding that in adults, hypopituitarism after traumatic brain injury (TBI) occurs more often than previously thought. However, there has been little formal investigation of pituitary function in children after TBI. A principal aim of this meeting was to define methods for prospective investigation of children at risk from this complication. More than 100 delegates from numerous countries attended and pediatric and adult endocrinologists, basic scientists and specialists in the fields of rehabilitation, neurology, sports medicine, military injuries and assessment of cognitive function were among the presenters.

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Background

Epidemiology, Incidence and Prevalence of TBI TBI is a critical problem affecting public health. Some sequelae of TBI, including cognition and memory impairments, are often not visible. Because awareness of TBI among the general public is rather limited, it is frequently referred to as the ‘silent epidemic’. Each year in the United States, at least 1.4 million people sustain a TBI. Of them, about 1.1 million are treated and released from an emergency department, 235,000 are hospitalized and 50,000 die [1]. Half of these deaths result directly from head injury. These figures do not include the estimated 439,000 TBIs treated by physicians in their offices or the 89,000 mild-to-moderate TBIs treated in other outpatient settings. Also not represented are data from military, federal and Veterans Administration facilities. Worldwide incidence and mortality rates are similar. Recent data from Germany show an incidence rate of serious head injury of 33.5 per 100,000 (68.4% die before hospital admission) [2]. The prevalence of TBI, roughly around 150 to 200 cases per 100,000, is generally similar in countries with a common sociopolitical framework. According to estimates, in any given industrialized city, 5% of emergency room hospitalizations are for severe TBI and 10% of these patients require intensive care admission.

Felipe F. Casanueva School of Medicine, Santiago de Compostela University Calle San Franciso SN, PO Box 563 ES–15780 Santiago de Compostela (Spain) Tel. +34 981 572 121, Fax +34 919 966 6025, E-Mail [email protected]

TBI is the leading cause of death and disability among children and young adults in the US [1]. Approximately 475,000 TBIs occur annually among children aged 14 years and younger. Emergency department visits account for 190% of the TBIs in this age group [1]. The types of accidents resulting in head trauma are diverse, but the majority of adult cases fall into three main categories: falls, motor vehicle/traffic accidents and violence; in children under the age of 19 years, a fourth category is sports injuries. Traffic accidents are responsible for 50 to 75% of all cases of TBI [3]. Legislation requiring wearing of seat belts and crash helmets, better emergency services on the accident scene and shortened hospital transportation times, as well as adopting valid hospital admission criteria, have lowered the number of admissions for head injury and have resulted in major cost savings [4]. The consequences of TBI are substantial and greatly affect the quality of life of patients and their families. Dr Brent Masel of the Transitional Learning Center in Galveston, Tex., USA, stressed that TBI is not just an event, but a lifelong disease that never completely resolves. Medical complications are manifested in numerous body systems, but approximately 50% of patients who experience a TBI have abnormalities in the neurologic, gastrointestinal and endocrine systems (table 1). In addition to the medical consequences, patients experience disturbances in motor function, difficulty in communicating with others, abnormally heightened or decreased sensations, increased occurrence of emotional and psychosocial issues and impaired cognition (particularly in the categories of attention, orientation, memory, visual spatial processing and reasoning). Recent studies of TBI patients in the chronic recovery phase indicate specific deficits in general health, vitality and mental health, with depression and anxiety being particularly common. These psychosocial consequences often become the major impediments to successful rehabilitation [5]. There are an estimated 2.5 to 6.5 million survivors of TBI [4]. Over 200,000 survivors will demonstrate persistent cognitive, physical and/or emotional deficits that will prevent functioning at preinjury levels; 70,000 to 90,000 will survive with substantial loss of function. Acute care and rehabilitation cost approximately USD 10 billion annually. Estimates of the lifetime average cost of caring for a survivor of TBI range from USD 600,000 to USD 1.9 million [4].

Hypopituitarism in Adults and Children following Traumatic Brain Injury

Table 1. Percentage of patients with TBI reporting a medical complication, by body system

Body system

Patients, %

Neurologic Endocrine Gastrointestinal Genitourinary Respiratory Cardiovascular Dermatologic Musculoskeletal

56 50 50 45 34 32 21 21

Other Sepsis Anemia

5 2

Basis for Endocrine Dysfunction following TBI The first case of pathological hypopituitarism, termed ‘hypophyseal cachexia’, was described by Simmonds in 1914. Four years later, Cyran first reported hypopituitarism related to head trauma. In 1942, Escamilla and Lisser published a study and a complete literature review of pathological hypopituitarism. They found that only four of 595 cases (0.7%) were related to head trauma. More recently, in 1986, Edwards and Clark reviewed the literature and reported on 53 patients [6], and in 2000, Benvenga and colleagues reviewed 367 cases of posttraumatic hypopituitarism [7]. The increasing numbers of individuals diagnosed with posttraumatic hypopituitarism may likely reflect increased road traffic accidents and subsequently increased numbers of survivors of TBI due to improved emergency services, neurosurgical techniques, etc. [8]. There is great variability in the manifestation of endocrine disorders after TBI. One reason is that injury may involve the hypothalamus, stalk or pituitary gland. With regard to the pituitary gland, possible causes of hypopituitarism include hemorrhage, infarction, ischemia, swelling or direct trauma. The vascular nature of the hypophyseal system makes it vulnerable to trauma. About 70–90% of the blood supply to the anterior lobe of the pituitary gland is supplied by branches from the long hypophyseal portal system. These branches originate from above the diaphragm and pass through the diaphragmatic sella. In contrast, the short hypophyseal portal veins arise below the diaphragmatic sella. They supply the anterior lobe with !30% of its blood supply, mostly in the medial portion, and they supply the posterior lobe [9]. In Horm Res 2007;67(suppl 1):208–221

209

one series of 76 brain injury patients who underwent computed tomography (CT) or magnetic resonance imaging, most injuries were vascular: 25% of patients had infarction of the anterior lobe; 26%, hemorrhage of the posterior lobe, and 29%, hemorrhage of the hypothalamus [7]. Numerous case studies and reviews of patients with TBI-induced hypopituitarism indicate a hierarchy of vulnerability in the pituitary cells during trauma, with somatotrophs and gonadotrophs apparently the most vulnerable, and corticotrophs and thyrotrophs being more resilient. The somatotrophs and gonadotrophs are located in the lateral wings of the pituitary gland, where the vascular supply comes from the hypothalamo-pituitary portal vessels. Therefore, damage to this area would impair the blood and oxygen supply, resulting in cell death. In contrast, the cells that secrete adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone are located ventrally in the more protected, medial portion of the pituitary. The vascular supply for the medial portion comes from the portal vessels and the anterior pituitary artery branch, which provide nutrients and oxygen to this area and to all the cells located in the subcapsular area [10]. There is a prevalent lag time between the time of trauma and the diagnosis of subsequent hypopituitarism. A study of 202 reported cases of posttraumatic hypopituitarism by Benvenga et al. showed that most subjects (71%) were diagnosed within the first year after the injury. However, one case was not diagnosed until more than 40 years after the accident [7]. The delay may be due to the fact that TBI survivors are often ‘lost’ within the healthcare system after discharge from rehabilitation centers, which generally occurs within that first year, suggesting the probability of underdiagnosis [8]. Because the patient’s memory of the event may be compromised, family members should be questioned about the patient’s history to avoid potential delay in diagnosis of posttraumatic hypopituitarism [8]. Subjects at highest risk appear to be those who have suffered a moderate-to-severe head trauma; however, even mild trauma may precede hypopituitarism. Pituitary dysfunction may be present in any subject regardless of the degree of injury; thus, subjects may remain undiagnosed and untreated while attempting to recover from brain injury [11]. Major studies on posttraumatic hypopituitarism published within the last few years have shown that isolated pituitary deficits are noted more frequently than total pituitary dysfunction. In one review of 344 patients who endured a TBI some time between 1 month and 23 years 210

Horm Res 2007;67(suppl 1):208–221

preceding the study, roughly 43% of patients were found to have some form of pituitary dysfunction. Gonadotropin and growth hormone deficiencies (GHDs) were the most common, although other studies noted a high occurrence of central hypothyroidism. Diabetes insipidus, well recognized as associated with TBI, is considered a rare complication; it is usually transient, occurring most often in the early, acute phase after injury [12].

Clinical Management

Identification Systematic screening of pituitary function is recommended for all patients with moderate-to-severe TBI since they are at risk of developing hormone deficiencies. Patients with TBI are typically first seen and treated by trauma surgeons and neurosurgeons with subsequent treatment provided by neurologists and rehabilitation specialists. These front-line specialists must be able to discern which patients may be at risk for posttraumatic hypopituitarism and to identify the signs and symptoms of pituitary dysfunction. Since diagnosis can be delayed for many years, physicians must continually monitor patients for signs and symptoms of hypothalamic-pituitary impairment. Endocrinologists and internists, though not usually the first to see trauma patients, must be encouraged to share expertise with rehabilitation physicians to increase their awareness of possible endocrinopathies [10]. In an effort to identify those TBI patients who could potentially benefit from hormone replacement therapy, Dr Günter Stalla from the Max Planck Institute in Munich, Germany, stressed the need for development of a network for patient identification and referral, continuing medical education and local databases to collect information about hypopituitarism after brain injuries. Patient identification could be greatly aided by establishing networks between endocrinologists and rehabilitation centers. Consultation services could include evaluation and discussion of laboratory results by telephone or electronic mail and regular on-site medical education. Because many TBI patients are unable to be transported, endocrinologists could provide on-site visits on demand. Dissemination of current data from interdisciplinary screening centers could occur at annual expert meetings and press conferences and may include literature reviews, case studies and ‘hot topic’ presentations. In addition to expert meetings, regional educational conferences could be held in specialized hospitals/treatment centers and reCasanueva /Ghigo /Polak /Savage

habilitation clinics. Dr Stalla stated that publications in interdisciplinary and specialized medical journals have increased in Germany over the last few years and, it is hoped, might also occur elsewhere. Local databases need to be established to investigate the prevalence and quality of pituitary insufficiency in patients after TBI, the influence of risk factors on the number of deficient hormone axes and the influence of the number of deficient hormone axes on patients’ quality of life. Timing and Testing Dr Chris Thompson from Beaumont Hospital, Dublin, Ireland, and Dr Randall Urban from The University of Texas Medical Branch in Galveston, Tex., USA, discussed the identification of pituitary deficits, the timeframe for initiation of hormone replacement therapy and the treatments available. Signs and symptoms associated with hypopituitarism often mimic the sequelae of TBI. Clinical manifestations of TBI vary widely depending on the type, location and severity of the injury. Pituitary gland dysfunction as a result of a TBI is particularly problematic because of the gland’s critical role in regulating essential hormones – involving many processes with profound physiological and/or psychological consequences – from the thyroid, gonads and adrenals. Consequently, pituitary hormone deficiencies could result in suboptimal rehabilitation for patients with posttraumatic hypopituitarism [10]. Basal hormonal evaluations are an essential means of demonstrating hypopituitarism, including deficits of the adrenal, thyroid and gonadal axis; in addition, they can avoid the need for provocative testing. Routine baseline hormonal testing should be performed on any patient who sustains a moderate-to-severe TBI that requires hospitalization and rehabilitation and on any patient who sustains a mild TBI and subsequently develops symptoms of fatigue, loss of energy or diminished productivity [10]. During the acute phase following TBI, the most clinically relevant evaluations include assessment of ACTH dysfunction and syndrome of inappropriate antidiuretic hormone secretion. Prospectively, all TBI patients, regardless of the severity of their injury, should undergo baseline hormonal evaluation 3 and 12 months after the primary brain insult or discharge from the hospital or intensive care unit. Patients with adrenal insufficiency, diabetes insipidus or other clinical symptoms of hypopituitarism should undergo immediate testing of the rest of the pituitary axis without waiting 3 months. Patients with clinical signs or symptoms suggestive of impaired hypothalamic-pituitary function (e.g., polyuria) should Hypopituitarism in Adults and Children following Traumatic Brain Injury

Table 2. Suggested endocrine tests for diagnosis of hypopituita-

rism Assess for

Suggested test(s)

Gonadotropin dysfunction

Total testosterone and FSH (males) Menstrual history (females) Prolactin (males and females)

Thyroid dysfunction

Free T4 and TSH

ACTH/cortisol dysfunction

Morning cortisol Glucagon stimulation Short synacten test Cosyntropin stimulation Metyrapone

GHD

Serum IGF-I GH provocation Insulin tolerance test Glucagon stimulation GHRH + arginine

be investigated whenever medically warranted (e.g., a decrease in ability that cannot be otherwise accounted for by changes in CT scan or changes in medication). Retrospectively, all patients with any signs or symptoms of hypopituitarism who experienced a moderate or severe TBI more than 12 months before their onset should undergo immediate hormonal testing. Given the 12-month passage of time, it is unlikely that any hormonal deficit would be transient, so for these patients the screening can be done in a single session [10]. A summary of the clinical tests most often used in diagnosing hypopituitarism can be found in table 2. Regarding the diagnosis of GHD, a growing body of evidence indicates that insulin-like growth factor I (IGFI) levels are the best marker of GH status as they provide an integrated measure of GH secretion [10]. Because normal IGF-I levels do not rule out the possibility of severe GHD, when GHD is suspected, further investigation by means of provocative testing is required. The insulin-tolerance test (ITT) is considered the gold standard for the diagnosis of GHD as well as for ACTH deficiency; however, the ITT is generally contraindicated in patients who have central nervous system pathologies, and hypoglycemia-induced side effects are known to be potentially hazardous. Among classic provocative tests, glucagon is considered a good alternative to ITT for investigation of either GH or ACTH and cortisol secretion. However, GHreleasing hormone (GHRH) in combination with arginine or a GH secretagogue (GHS) (e.g., synthetic GHS such as GHRP-6 or hexarelin or the natural GHS ghrelin) Horm Res 2007;67(suppl 1):208–221

211

Table 3. Why is timing of assessment of the hypothalamic-pitu-

itary-adrenal axis critical? Time after TBI

Pituitary recovery

~6 months

Many hormone deficiencies recover Some new deficiencies manifest

`6 months

No new abnormalities appear after 6 months Recovery rare after 6 months

is the best alternative test for the diagnosis of GHD. Other available provocative tests used to further investigate the existence of deficiencies of other anterior pituitary hormones include gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone, corticotropinreleasing hormone, ACTH and metyrapone. Specific provocation tests to be performed for each patient should be determined by the endocrinologist in collaboration with the rehabilitation clinicians [10, 13]. Physicians should consider retesting of patients with borderline abnormalities at 12 months. Treatment Considerable uncertainty exists within the medical community over the potential benefits of hormone replacement therapy (HRT) in TBI patients. Well-controlled studies of HRT in the treatment of patients with TBI-induced hypopituitarism are necessary to determine the degree to which patients will benefit in terms of rehabilitation and endocrinological function. This uncertainty underscores the need for physicians to be able to accurately identify which subpopulation of patients with TBI could benefit from HRT. Even then, the advantages and disadvantages of hormone therapy must be carefully weighed in the decision-making process. Present knowledge dictates that immediate HRT should be instituted for patients with isolated hypopituitarism in the case of diabetes insipidus, secondary adrenal insufficiency and secondary thyroid insufficiency (just after adrenal replacement has been started). Replacement therapy for a gonadal deficit is always recommended in the context of panhypopituitarism or multiple deficits including a gonadal deficit. However, both GH and gonadal deficiencies may be transient (table 3). Replacement of other hormonal deficits can restore normal GH response to provocative testing; thus, GHD needs to be confirmed after appropriate replacement of other hormone deficiencies. Isolated insufficiency of the gonadal axis could reflect functional stress-induced impairment. 212

Horm Res 2007;67(suppl 1):208–221

Patients should be retested before hormone replacement is initiated [10]. All patients with proven posttraumatic hypopituitarism who are receiving HRT should undergo periodic follow-up testing by an endocrinologist to monitor overall health. According to the Consensus Guidelines for Diagnosis and Treatment of GHD, for patients with total hypopituitarism or multiple deficits including severe GHD, recombinant human (rh)GH replacement therapy should be initiated for confirmed severe GHD after appropriate (optimal) replacement of other pituitary deficits for at least 3 to 6 months. For patients with an isolated pituitary deficit of severe GHD, rhGH replacement therapy should be initiated if persistent, severe GHD is evident upon retesting 12 months after TBI. For patients with isolated, secondary hypogonadism, HRT should be considered as appropriate. For instance, it may be helpful in men to take advantage of the anabolic action of testosterone. In contrast, a better approach for women with secondary amenorrhea may be to postpone HRT and monitor menses over time, since postmenopausal women do not typically receive estradiol replacement therapy. As in the case of immediate HRT, the modalities and dosages of delayed therapy should be determined by the endocrinologist in collaboration with the rehabilitation physician [10].

Other Possible Causes of TBI-Induced Hypopituitarism

The term ‘traumatic brain injury’ most likely brings to mind catastrophic injuries such as those sustained in a traffic accident or serious fall; however, approximately three fourths of TBIs that occur each year are concussions or other mild forms of brain injury [14]. Yet even these seemingly milder injuries can cause pituitary dysfunction in persons participating in combative sports such as boxing, football and ice hockey [15]. Another relevant cause of TBI is injury during military service. The incidence of TBI amongst active duty military personnel in war zones is growing [14]. Since the beginning of the Iraqi war, so many soldiers have been diagnosed with TBI that military doctors are calling it the signature injury of the conflict. At Walter Reed Army Medical Center in Washington, D.C., USA, 60% of patients wounded in Iraq have been diagnosed with TBI [16]. Participants in contact sports, including boxing, soccer, football, ice hockey and the martial arts, are at risk for acute or chronic TBI [17]. A literature search on the Casanueva /Ghigo /Polak /Savage

Hypopituitarism in Adults and Children following Traumatic Brain Injury

70

50 GH peak (µg/l)

topic of TBI and sports revealed that nearly 70% of the studies focused on boxers (who are known to be at risk for both acute and chronic neurological injury). Acute injuries range from mild concussions to cerebral hemorrhage, diffuse axonal injury and even death [18]. Snowboarders, particularly beginners, are also reported to be at high risk of head injury. In one study, more than half of snowboarders suffered closed head injuries, and most were diagnosed with concussion. The use of helmets was subsequently proposed for increased protection [19, 20]. Among children, an estimated 300,000 sports-related brain injuries occur each year in the US [14]. The relationship between sports and TBI is well documented; however, chronic trauma in sports (especially in boxing) and its pituitary consequences have not been investigated before [21]. Professor Fahrettin Kelestimur, an expert in sports medicine at Erciyes University Medical School in Kayseri, Turkey, has investigated several boxers and found significant evidence of pituitary deficiencies, principally GHD [21]. In one study, the pituitary function of 11 male boxers (actively competing or retired) was assessed. As shown in figure 1, five boxers (45%) had peak GH levels !10 g/l, indicative of severe GHD. In 4 boxers, peak GH levels were between 10 and 20 g/l, indicating borderline GHD. None of the boxers had reported any comorbidity or previous pituitary disorder and none were taking any medications. Seven boxers complained of memory impairment and four complained of fatigue. No other symptoms suggestive of a pituitary deficit were reported. One simple punch, rather than accumulated damage from multiple punches, can reportedly result in cerebral concussion and many boxers experience memory disturbance, not just after a fight, but in daily life [15]. Professor Kelestimur presented unpublished data from a study of pituitary function in 23 amateur kickboxers (17 males, 6 females; mean age of 27.4 8 7.0 years). Seventeen subjects were active kickboxers who were members of a national kickboxing team; the other six were retired. The mean body mass index of the group was 24.2 8 3.8 and the subjects had been kickboxing for a mean of 9.2 8 5.4 years. Frank GHD (GH levels !10 g/l) was evident in three male subjects (1 retired, 2 active). Another three male subjects had GH levels between 10 and 20 g/l; therefore, additional provocative testing with glucagon and GHRH + GHRP-6 was conducted. Two of these three subjects with uncertain GH levels were found to be GH deficient. Of the 23 total subjects in this study, 5 (22%) were found to be GH deficient. Rabadi and Jordan reviewed the literature and reported

30

20

10

Boxer

Control Groups

Fig. 1. Chronic, repetitive head trauma during boxing is associated with isolated GHD. Data show individual GHRH+GHRP6-stimulated GH peaks in controls and boxers. The cut-off peak GH value for GHD was ^10 g/l. Peak GH values 620 g/l were considered normal. Reproduced, with permission, from Editrice Kurtis.

certain risk factors for TBI in boxing: retirement after the age of 28 years; boxing 610 years; participation in 6150 bouts; increased sparring exposure, and previous history of a technical knockout or a knockout and poor performance [17]. Another emerging cause of TBI involves military personnel injured during active service during war. Dr Warren Lux of the Defense and Veterans Brain Injury Center (DVBIC) at Walter Reed Army Medical Center (WRAMC) in Washington, D.C., USA, presented information about TBIs in the military. Decades ago, many service personnel who were critically wounded in action died; however, improvements in protective equipment, advances in acute trauma care and heightened efficiency of triage in the field have led to decreasing case fatality rates. Thus, we are now seeing survivors with eye and ear injuries, amputations and brain injuries of all severities in persons who in prior conflicts would have succumbed to their injuries. As seen in table 4, blasts are the most frequent causes of injury leading to medical evacuation among armed forces in Operation Iraqi Freedom. This, together with Horm Res 2007;67(suppl 1):208–221

213

Table 4. Top three reasons for wounded-in-action evacuation of US Army personnel during Operation Iraqi Freedom: 19 Mar 2003–30 Sep 2005

Injury type

Total number

Total wounded-inaction evacuations, %

Explosion* Gunshot Rocket-propelled grenade

1,944 446 228

66.7 15.3 7.8

Source: AMEDD OTSG Website (http://www.armymedicine. army.mil); 14 Dec 2005. * Includes improvised explosive device, land mine, grenade, shrapnel and blast injury.

the historical data on the high frequency of head/brain injury in a blast environment, suggests that a careful examination of the relationship between blasts and brain injury is important to any review of brain injury in the military. There are four possible blast injury mechanisms: primary, secondary, tertiary and quaternary. In a primary blast injury, there is direct exposure of the body to the overpressurization wave. Secondary injury occurs when there is impact from blast-energized debris, both penetrating and nonpenetrating. Tertiary injury involves displacement of the person by the blast, and quaternary injury includes burns and/or inhalation of gases as a result of the blast. It is important to note that whether or not there is also primary blast injury, the secondary and tertiary mechanical closed brain injuries seen in a blast environment are the same kinds of injuries as those seen in ordinary motor vehicle accidents, falls and assaults. The incidence of TBIs in active-duty military personnel during Operation Iraqi Freedom has been evaluated by the DVBIC at WRAMC. The first study included patients whose injuries were severe enough to require evacuation to a tertiary-care medical facility in the US. All returning patients were screened by the TBI team based on mechanism of injury: (1) penetrating wounds of head, neck or face; (2) falls; (3) motor vehicle accident, or (4) exposure to a blast. Any loss or alteration of consciousness or presence of postconcussive symptoms was noted and cognitive impairment was assessed. Comprehensive multidisciplinary evaluations were conducted, if indicated, to further characterize patients who screened positive for brain injury. A total of 466 patients were found to have brain injuries between January 2003 when the screens be214

Horm Res 2007;67(suppl 1):208–221

gan and the end of February 2005. The data on the total number of screens done prior to December 2004 were not robust, but the rate of positive screens was approximately 60%. Screening conducted on 112 patients between December 2004 and February 2005 showed that 53% were positive for TBI during this period. In some patients, the brain injury had not been identified previously, usually due to a clinical focus on severe systemic injuries requiring immediate attention (e.g., traumatic amputation). Patients injured by blasts had positive screens for brain injuries at a rate similar to the overall rate. More recent screens conducted between August 2004 and September 2005 by the WRAMC TBI team on 405 patients injured by one of the four specific mechanisms (i.e., penetrating wounds of head/neck/face, falls, motor vehicle accidents or blasts) showed that 221 patients (approximately 55%) were positive for TBI. A larger cohort of 1,116 patients admitted to WRAMC between January 2003 and April 2005 with a variety of battle injuries was also screened for TBI: 342 (31%) were positive. As one would expect, the rate of moderate-to-severe brain injuries in the United States Army has gone up since Operation Iraqi Freedom began (fig. 2). Since trauma-related hypopituitarism tends to occur more frequently in patients experiencing moderate-to-severe injuries, further evaluation of the role of pituitary function in these patients is warranted.

Epidemiology and Pathophysiology of TBI in Childhood

Dr Roberta DePompei with the School of Speech-Language Pathology and Audiology at the University of Akron, Ohio, USA, reviewed the epidemiology of TBI in children and discussed the effects of unrecognized pituitary damage in children after TBI including the psychological and behavioral changes that often go undiagnosed and, therefore, untreated. TBI is the leading cause of death and disability among children and young adults in the US. Approximately 475,000 TBIs occur annually among children ^14 years of age. More than 90% of the TBIs in this age group result in emergency department visits [1]. Children and infants have large, heavy heads and weaker cervical ligaments and muscles than adults. Given the same deceleration of the body, head trauma is therefore more likely in younger children. Similarly, the resulting brain injury may be more severe due to the thin, pliable skull and unfused sutures. The causes of TBI differ by age groups. Infants Casanueva /Ghigo /Polak /Savage

Apparent low incidence

50

Incidence rate/100,000

45 40

Mild

35

Severe

30 25

Lack of awareness

20

Underidentiﬁcation

Lack of training

Unknown Moderate

15 10 5 0 2000

2001

2002 Fiscal year

2003

2004

Fig. 2. Incidence of TBI-related hospitalizations in the US Army

by TBI severity for the fiscal years 2000 to 2004.

Lack of research money

Lack of right services for kids who are ID

Fig. 3. Cycle of underidentification of children with TBI in the education system.

mostly suffer from falls or are assaulted; these still account for the majority of injuries in toddlers, but they are also more frequently injured in motor vehicle accidents. As children grow older, TBI is more often caused by traffic accidents and from contact sports [22]. The lack of accurate identification of children with TBI in the education system can be attributed to many factors that culminate in the lack of proper resources for those few children who are eventually identified (fig. 3). There are also many community myths that contribute to the lack of proper services for children living with TBI. Some believe that TBI is an educational problem only and that free services for management of these individuals are provided in the school systems; therefore, there is no need for other means of support and funding. However, now that TBI is a known and accepted medical condition that requires rehabilitation in adults, why would this not be the case in pediatrics? A shift in thinking is necessary to ensure that the survivors of childhood TBI and their families are provided with adequate treatment and support. This is a growing population and recent studies have shown that even such mild injuries as concussions can lead to lifelong problems that may affect language, cognition and learning. TBI often results in debilitating cognitive impairments including impaired attention, perception and/or memory; inflexibility, impulsivity and/or disorganized thinking or acting, and inefficient processing of information including the rate, amount and complexity of infor-

mation. Children with TBI experience difficulties processing abstract information as well as learning new information, rules and procedures. Their ability to retrieve old or stored information is also compromised. Deficiencies may involve not only language and cognitive skills associated with learning, but also social and behavioral skills. These children may have difficulties with problemsolving and judgment, exhibit inappropriate or unconventional social behaviors and experience impaired executive functioning, e.g., self-awareness of strengths and weaknesses, goal-setting, planning, self-initiating, monitoring and evaluating. Since children with TBI won’t just ‘grow out of it’, the responsibility of educating the community lies largely with medical leaders. Children may fail to reach developmental milestones; however, no one equates this lack of development to injury sustained years earlier. Because their brains continue to develop and change over time, children have to recover at each new stage of development. Materials and methods used in the treatment of adults with TBI are not necessarily applicable or beneficial in the pediatric population; therefore, further research into medical/pharmacological interventions and the effects of TBI on learning, behavior, cognitive capacity, social communication and physical/ emotional development in childhood survivors is necessary.

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215

Table 5. Characteristics of TBI-induced hypopituitarism in children

Study

Benvenga, 2004 [23] Eichler, 1988 [24] Paxson, 1976 [25] Girard, 1977 [26] Miller, 1980 [27] Mariani, 1996 [28] Barbeau, 1998 [29] Grossman, 1994 [30] Yamanaka, 1993 [31] Valenta, 1980 [32] Total Percentage

Patients

4 1 1 1 3 3 3 1 2 1

Sex

M, F, F, NS M F M F, M, M F, M, M M, M, F F M, M F 20 100

Age at injury years

Type of injury

7–16 12 14 3.7 0.1–0.3 2–9 0.5–3 0.8 3, 11 16

Fall (!2), MVA (!2) NA MVA MVA Abuse MVA, judo, fall NA MVA Fall (!2) Skull fracture

GH

TBI-Induced Hypopituitarism in Childhood

Because no definitive study of pituitary function following TBI in children has been published, Dr Martin Savage of St Bartholomew’s and the London School of Medicine and Dentistry, UK, presented a review of published cases of anterior pituitary deficiencies in children with TBI from 1976 through 2004. A total of 20 patients ranging in age from 0.1 to 16 years were included in the retrospective analysis; the severity of head injuries varied from mild without loss of consciousness to severe (table 5) [23–32]. As expected, most injuries were the result of falls or traffic accidents; however, there were also documented cases of hypopituitarism following abuse (e.g., direct injury, shaking) and sports-related injuries (e.g., judo). The most common symptoms prompting further medical evaluation of these patients were delayed growth and pubertal abnormalities (early-onset or lack of onset). The lag time between injury and diagnosis of endocrine abnormalities varied from 1 year to more than 40 years after TBI. In the previously noted study by Benvenga et al., four children presented with apathy and abnormal libido as teenagers and adults even though their injuries were sustained during childhood [7]. Dr Carlo Acerini of the University of Cambridge, Cambridge, UK, reported another retrospective analysis of the chronic endocrine sequelae of TBI using data extrapolated from the Pfizer International Growth Study Database (KIGS). This pharmacoepidemiological database, begun in 1987, functions as a register of surveillance and outcome for children and adolescents receiving any 216

Horm Res 2007;67(suppl 1):208–221

Deficiencies

2 1 1 1 3 2 3 1 2 1 17 85

TSH 4 – 1 1 2 2 2 1 1 1 15 75

ACTH 1 – 1 1 3 2 2 1 – – 11 55

LH/ FSH 4 – 1 – 3 2 2 1 2 1 16 80

ADH – – – – – 2 – – – – 2 10

type of GH therapy. Patient registration is voluntary, and currently, there are approximately 51,000 patients registered from more than 40 countries. The aims of this retrospective analysis were to collate and compare the clinical data of subjects registered in KIGS with etiological classifications of TBI or idiopathic GHD (IGHD). As of January 2006, baseline clinical characteristics and subsequent responses to GH treatment were assessed in approximately 24,000 patients with IGHD (68% male) and 141 patients with TBI (66% male). No data, however, were available on the epidemiology or characteristics of the brain injuries suffered by these children. As summarized in table 6, children with TBI were slightly older than those with IGHD with a slower height velocity at the time of initiation of GH replacement therapy. Multiple pituitary hormone deficiencies were noted in a significantly greater percentage of patients with TBI than with IGHD (36 vs. 9%, p ! 0.001). Both groups responded well to GH replacement therapy. These results confirm similar data presented in a KIGS analysis by Price and Jonsson in 1996 [33]. Since there is a dearth of data on pituitary function in childhood and adolescent survivors of TBI, hormonal evaluations in adult patients with TBI can provide valuable clues for the identification and treatment of these problems in the pediatric population. In collaboration with neurosurgeons, neurologists and endocrinologists, and under the auspices of the Italian Society of Endocrinology, a prospective study evaluated hormone levels in 100 adult patients with TBI at 3 and 12 months after injury. Results from this and similar studCasanueva /Ghigo /Polak /Savage

Table 6. Endocrine sequelae of patients

with IGHD vs. TBI: review of KIGS data through January 2006

Table 7. Retrospective evaluation of

pituitary function after TBI in children and adolescents in Italy

Parameter

IGHD (n = 23,722)

TBI (n = 141)

Baseline characteristics Age onset of therapy, years Height, SDS Height velocity, cm/year BMI, SDS Max GH, mU/l Prepubertal, %

10.3 –2.43 4.43 –0.26 5.95 87.7

11.0 –2.44 3.80 0.23 3.55 87.4

0.045 NS <0.001 <0.001 <0.001 NS

Pituitary hormone deficiencies, % Isolated GHD Multiple deficiencies TSH ACTH Gonadotropin ADH

87.4 8.9 7.4 3.1 3.2 0.7

58.9 36.2 30.5 23.9 16.9 9.3

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Response to GH therapy Time on GH therapy, years GH dose, mg/kg/week Height, SDS Height velocity, cm/year height velocity, cm/year BMI, SDS

3.6 0.19 –1.49 5.62 3.72 –0.11

3.8 0.18 –0.99 4.59 4.87 0.14

0.014 0.016 NS 0.007 0.016 NS

Patient

p value

Age years

Sex

Pituitary abnormality

Therapy

1

5.2

M

GH, thyroxine, hydrocortisone

2 3 4 5 6 7

14.1 14.4 14.5 10.4 13.9 13.1

M M M M M M

Multiple deficiencies (GH, LH/ FSH, ACTH, TSH) Isolated GHD Isolated GHD Isolated GHD Precocious puberty LH/FSH deficiency ACTH deficiency

* * * GnRH analogs Testosterone Hydrocortisone**

* Still under investigation. ** Only required in times of stress.

ies showed that there is a high risk for anterior pituitary dysfunction in TBI patients and that the early diagnosis of total hypopituitarism is consistently confirmed in the long term after brain injury. Pituitary function in brain-injured patients may improve over time; rarely, pituitary function that is normal shortly after brain injury may become impaired 12 months later. Some degree of pituitary impairment is present in approximately 22– 37.5% of patients. In adults, severe GHD is the most

common pituitary abnormality, followed by secondary hypogonadism. The consequences of hypopituitarism in general and of GHD in particular on growth and development are likely to be critical in children and adolescents during the so-called transition phase. To test this hypothesis, Aimaretti and colleagues evaluated a population of adolescents and young adults 3 and 12 months after brain injury [34]. A total of 23 patients (14 males, 9 females) between the

Hypopituitarism in Adults and Children following Traumatic Brain Injury

Horm Res 2007;67(suppl 1):208–221

217

% 100 Total hypopituitarism 80 Multiple hypopituitarism 60

Isolated hypopituitarism

40

Partial GHD Normal pituitary function

20

Fig. 4. Percentage of normal pituitary function and various degrees of hypopituitarism in patients with TBI in the transition phase (ages 16–25 years).

0

3

12 Months

0

Magnitude of impairment

–0.2 –0.4 –0.6 –0.8 –1.0 –1.2 –1.4 Psychomotor Mild TBI (n = 52)

Fig. 5. Magnitude of cognitive impairment

Attention

Working memory

Memory

Executive function

Memory

Executive function

Moderate-to-severe TBI (n = 37)

in TBI 1 year after injury.

0

Magnitude of impairment

–0.2 –0.4 –0.6 –0.8 –1.0 –1.2 –1.4

Psychomotor

Attention

Working memory

Moderate-to-severe TBI (n = 7) (mean age 23.5 ± 3.5)

Fig. 6. Magnitude of cognitive impairment

Moderate-to-severe TBI with GH deﬁciency (n = 5) (mean age 23.5 ± 3.5)

in TBI with GHD.

218

Horm Res 2007;67(suppl 1):208–221

Casanueva /Ghigo /Polak /Savage

ages of 16 and 25 years were included in the study. As seen in figure 4, hypopituitarism was present in 34.6% at 3 months and in 30.3% at 12 months. Total hypopituitarism was always confirmed at retesting. As in adults, GHD and secondary hypogonadism were the most common acquired pituitary deficits in this transition population. These results show that the high risk of TBI-induced hypopituitarism is also applicable in the transition age. A retrospective evaluation of pituitary function after TBI in children and adolescents was conducted by investigators at the Regina Margherita Children Hospital, Turin, and the University of Piemonte Orientale, Novara, Italy. Patients were considered for inclusion if they were under the age of 18 at the time of trauma and had a brain injury with pathological CT findings according to Marshall criteria and a Glasgow coma score 1 4. A total of 79 cases were identified (64 males; 15 females). The age at trauma ranged from 3 months to 16.5 years and time since injury from 6 months to 7 years. Seven of these patients (9%) exhibited some degree of hypopituitarism; six had isolated deficits and one patient experienced multiple hormonal deficiencies (table 7).

Clinical Endpoints for Adult and Pediatric Endocrinologists

Cognition is an accepted surrogate marker of central nervous system function and a regulatory-approved outcome in the US and Europe. Cognitive impairment is an enduring consequence of TBI. Assessment of cognitive function provides a framework for communicating the nature of the injury and benefits of treatment. However, because humans generally have poor insight into their own cognitive function, it must be assessed objectively. Dr Paul Maruff of the Centre for Neuroscience, University of Melbourne, Melbourne, Australia, discussed the use of cognitive function as a clinical endpoint following TBI. Since GHD is one of the most common pituitary abnormalities following head trauma, he also discussed how GH relates to cognitive function. The consequences of TBI can be severe, including disability in motor function, speech, cognition and psychosocial and emotional skills (fig. 5). Clinical studies of pituitary function after TBI have consistently demonstrated a 30–40% occurrence of pituitary abnormalities involving at least one anterior pituitary hormone following moderate-to-severe TBI. GHD is the most common pituitary hormone disorder, occurring in approximately 20% of patients when multiple tests of GHD are used [35]. Hypopituitarism in Adults and Children following Traumatic Brain Injury

Studies have shown that GH-deficient patients show cognitive impairment when compared to controls and that GH replacement therapy can improve cognitive function. Dr Maruff presented findings from a small study of patients with moderate-to-severe brain injuries. One group of patients had adequate GH reserves; the other group had concomitant GHD. As evident in figure 6, the TBI patients with GHD had greater magnitudes of impairment in each of the five categories of cognitive function. These findings may have significant implications for the recovery and rehabilitation of patients with TBI. Normal cognitive function requires normal GH. There is converging evidence that TBI is associated with GHD, and the concomitant existence of these abnormalities may exacerbate cognitive impairment. Studies in patients with nontrauma-related GHD have shown that GH replacement therapy results in major neurobehavioral, mental well-being and cognitive improvements. To determine whether this theory holds true in patients with GHD arising as a sequela of TBI, prospective, randomized, controlled trials are warranted. Children and adolescents who suffer a TBI are also vulnerable to complications involving skeletal growth and gonadal development, since GH is a crucial mediator of these functions. Dr Savage discussed some approaches for determining whether further testing is necessary in pediatric patients following TBI. For patients with a history of TBI, prospective investigations (biochemical, neurological, etc.) should commence. In patients referred for short stature, clinicians should take a detailed medical history including a history of brain injury of even mild severity. Physical examinations may provide some clues as to endocrine function, even before any biochemical investigations are performed. Diminished height and abnormal pubertal development are good indicators of endocrine dysfunction. Pediatricians following patients after a TBI need to have a firm understanding of normal puberty and pubertal growth so that they can easily identify children who are developing abnormally. Establishment of formal joint collaborative consultations among neurologists, rehabilitation specialists, developmental pediatricians and pediatric endocrinologists is essential to ensure optimal diagnosis, proper treatment and adequate follow-up of childhood and adolescent survivors of TBI.

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219

Conclusions

Although recent literature has focused mainly on adults with pituitary abnormalities following motor vehicle accidents, prospective studies to establish the frequency, type and natural history of endocrine deficits in survivors of pediatric TBI are urgently needed since childhood and adolescence are critical times for linear growth and sexual maturation, both of which may be notably compromised by untreated pituitary deficiencies that often arise following head trauma. In an effort to improve awareness, a model of interaction between neurosurgeons, neurologists, rehabilitation specialists and pediatric endocrinologists, with support from adult endocrinologists, should be established to optimize identification, investigation and treatment of pediatric TBI survivors. Detailed follow-up will be required to assess the clinical impact of pituitary anomalies on growth (both linear growth and brain growth), puberty and neurocog-

nitive function and to determine the role of hormone replacement therapy during this critical period of maturation. If funding permits, another satellite meeting following the 39th International GH and IGF-I Symposium – to be held in Berlin, Germany in 2007 – would allow dissemination of results from recent studies including military service-related TBIs, child abuse cases and sportsrelated injuries. To promote awareness of the growing problem of TBIs and their endocrine sequelae, a special issue dedicated to research and findings of pituitary abnormalities in adult and pediatric survivors of TBI should be published in a peer-reviewed journal.

Acknowledgment Funding for this satellite symposium was provided by Pfizer, Inc.

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Author Index Vol. 67, Suppl. 1, 2007

HORMONE RESEARCH

Ábrahám, C.S. 206 Ambrosini, L. 71 Andaluz, P. 204 Andréka, B. 206 Antonini, S.R. 102 Argyropoulou, M. 109 Audí, L. 204

Gluckman, P.D. 115 Grottoli, S. 174

Barrell, G.K. 81 Beedle, A.S. 115 Binder, G. 45 Borget, I. 132

Jasovic-Gasic, M. 177 Jessen, N. 33 Johannsson, G. 155, 184 Jørgensen, J.O.L. 33

Carlos dos Santos, A. 102 Carrascosa, A. 204 Casanueva, F.F. 173, 208 Christiansen, J.S. 33 Clayton, P.E. 10

Knutson, K. 2 Kovács, J. 206 Krag, M. 33 Kuro-o, M. 191

Hanson, M.A. 115 Hardin, D.S. 32 Holmbäck, U. 2 Hughes, I.A. 91

Dacou-Voutetakis, C. 96, 106 Darendeliler, F. 43 Darlow, B.A. 81 de Castro, M. 102 De Giorgio, D. 174 de Lima Jorge, A.A. 98 De Pouvourville, G. 132 Deal, C. 114 di Iorgi, N. 71 Djurovic, B. 177 Doknic, M. 177 Dunger, D. 37 Eastell, R. 23 Espiner, E.A. 81 Esteban, C. 204 Faleiros, L. 102 Feldman, B.J. 121 Fernández-Cancio, M. 204 Ferrández Longás, Á. 43 Fideleff, H. 126 Filipsson, H. 155 Fricke, O. 16 Gál, V. 206 Gasco, V. 174 Ghigo, E. 173, 174, 208 Gitelman, S.E. 121

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Larsen, R.L. 33 Latronico, A.C. 96 Leproult, R. 2 Livadas, S. 109 López-Gallardo, G. 180 López-Iglesias, M. 180 Machado, H.R. 102 Maghnie, M. 71 Mainolfi, A. 174 Makovi, H. 206 Manzanares, R. 180 Marazuela, M. 180 Maric, N. 177 Miller, A. 2 Møller, L. 33 Møller, N. 33 Napoli, F. 71 Nedeltcheva, A. 2 Niederland, T. 206 Nieschlag, E. 149 Nir, A. 77 Nørrelund, H. 33 Pacini, F. 132 Pagotto, U. 186 Pannain, S. 2 Pasquali, R. 186 Pekic, S. 177

Penev, P. 2 Pervanidou, P. 106 Polak, M. 208 Popovic, V. 177 Prickett, T.C. 81 Ranke, M.B. 45 Reiter, E.O. 58 Rosenblatt, K.P. 191 Rosenthal, S.M. 121 Salgin, B. 37 Savage, M.O. 208 Schlumberger, M. 132 Schoenau, E. 16 Sertedaki, A. 109 Simoni, M. 149 Spiegel, K. 2 Stojanovic, M. 177 Sullivan, M.J. 81 Tasali, E. 2 The Scientific Planning Committee 1 Thissen, J.-P. 64 Torán, N. 204 Trounson, A. 28 Van Cauter, E. 2 van der Lely, A.J. 143 Vargas, G.A. 121 Verbalis, J.G. 165 Vicennati, V. 186 Vickers, M.H. 115 Voutetakis, A. 109 Weetman, A.P. 128 Wellby, M. 81 Whatmore, A.J. 10 Wit, J.M. 50 Wittekindt, N. 45 Xekouki, P. 106, 109 Yandle, T.G. 81 Yuen, K. 37 Zivkovic, V. 177

Subject Index Vol. 67, Suppl. 1, 2007

HORMONE RESEARCH

Acromegaly 143, 174 Alendronate 23 Amino-terminal pro-CNP 81 Anterior pituitary 71 Antidiuresis 121 Arginine vasopressin 165 Bone mineral density 23 Bone density 16 Calcitonin 23 Cannabinoid receptor type 1 (CB1) receptor 186 Case report 177 Chiari malformation type I 102 Chondrocytes 81, 204 Cognitive function 208 C-type natriuretic peptide 81 Cytokines 64 Densitometry 16 Depression 177 Developmental induction 115 – programming 115 Diabetes 2, 28 – insipidus 71, 165, 180 – mellitus 174 Disorders of sex development 91 Embryonic stem cells 28 Endocannabinoids 186 Familial short stature 50 Fibroblast growth factor receptor 1 149 – signaling 191 Fracture risk 23 Functional muscle-bone unit 16 Gender dysphoria 91 – identity disorder 91 GH1 gene deletion 102 Ghrelin 2 Glucocorticoids 155 Gonadotropin-releasing hormone receptor 149 G-protein-coupled receptor 54 149

© 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/hre

Growth hormone 33, 37, 58, 106, 109, 177 – – deficiency 10, 33, 37, 102, 155, 208 – – insensitivity 98 – – receptor 10 – – replacement 33 – – resistance 64 – plate 81, 204 – retardation 64 – velocity 98 Head injury 206 Heart disease 77 Hepatic enzymes 106 Hormone replacement therapy 106 Hypernatremia 165 Hypodipsic hypernatremia 180 Hypogonadotropic hypogonadism 149 Hyponatremia 121, 165 Hypopituitarism 155, 208 Hypothalamic infarct 180 Idiopathic short stature 50 Inappropriate secretion of antidiuretic hormone 121 Insulin resistance 2, 33 – secretion 37 – sensitivity 37 – signaling oxidative stress 191 Insulin-like growth factor binding protein-3 204 – – – deficiency 58 – – – I 10, 37, 64, 143 – – factor-I 204 Intracranial hypertension 102 JAK2-STAT5 45 KAL1 149 Klotho, aging 191 Leptin 2, 115 Linear growth 81 Liver 106 Magnetic resonance imaging 71 Match-mismatch 115 Metabolic syndrome 115 Muscle force 16

223

Natriuretic peptides 77 Nephrogenic syndrome of inappropriate antidiuresis 121 Non-familial short stature 50 Noonan syndrome 45, 98 Obesity 2, 186 Osteoporosis 16, 23 Pancreatic differentiation 28 Pegvisomant 174 Pituitary 109, 143 – dysfunction 206 Posterior pituitary 71 Preimplantation embryos 28 PROP1 gene mutations 109 PTPN11 45 – mutations 98 Raloxifene 23 Recombinant human thyroid stimulating hormone 132 Rimonabant 186 Risedronate 23

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Horm Res Vol. 67, 2007

Sexual differentiation 91 Short stature 58, 98 SHP2 45 Skeletal muscle 64 Small for gestational age 10, 50 Somatostatin analogues 174 Somatropin 143 Syndrome of inappropriate antidiuretic hormone secretion 165 T4 128 Teriparatide 23 Therapy 177 Thyroid cancer 132 – hormone replacement 128 Traumatic brain injury 177, 208 Tri-iodothyroxine 128 Turner syndrome 10, 106 Vasopressin 71 – receptor 121 Vitamin D 204

Subject Index

38th International Symposium on Growth Hormone and Growth Factors in Endocrinology and Metabolism: Granada, Spain, April 7-8, 2006 (Hormone Research)

Growth Hormone

Human Growth Hormone: Research and Clinical Practice (Contemporary Endocrinology)

Human Growth Hormone: Research and Clinical Practice (Contemporary Endocrinology)

Human Growth Hormone. Research and Clinical Practice

Growth Hormone Receptor

Growth Hormone Deficiency in Adults (Frontiers of Hormone Research)

Challenges in Growth Hormone Therapy

Obesity and Metabolism (Frontiers of Hormone Research)

Growth Hormone in Adults: Physiological and Clinical Aspects, Second Edition

Growth Hormone in Adults: Physiological and Clinical Aspects, Second Edition

Hypertension and Hormone Mechanisms (Contemporary Endocrinology)

Growth Hormone Secretagogues: Basic Findings and Clinical Implications

Growth Hormone Related Diseases and Therapy: A Molecular and Physiological Perspective for the Clinician (Contemporary Endocrinology)

Hypertension and Hormone Mechanisms

Hematopoietic Growth Factors

Transforming Growth Factors

Lysophospholipid Growth Factors

Insulin-like growth factors

Hormone Deception

MRAM Market Size and Growth Factors Research and Projection 2026

Growth Factors and Psychiatric Disorders (Novartis Foundation Symposium 289)

Neuro-endocrinology of Growth and Energy Balance

Endocrinology and Metabolism

Advances in Plant Ethylene Research: Proceedings of the 7th International Symposium on the Plant Hormone Ethylene

Growth Factors, Part A (Growth Factors & Cytokines in Health & Disease) (Growth Factors & Cytokines in Health & Disease)

Growth Hormone Deficiency - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References

Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction

Growth Factors, Part A (Growth Factors & Cytokines in Health & Disease) (Growth Factors & Cytokines in Health & Disease)

Growth Theory and Growth Policy

International trade, growth, and development

38th International Symposium on Growth Hormone and Growth Factors in Endocrinology and Metabolism: Granada, Spain, April 7-8, 2006 (Hormone Research)

Growth Hormone

Human Growth Hormone: Research and Clinical Practice (Contemporary Endocrinology)

Human Growth Hormone: Research and Clinical Practice (Contemporary Endocrinology)

Human Growth Hormone. Research and Clinical Practice

Growth Hormone Receptor

Growth Hormone Deficiency in Adults (Frontiers of Hormone Research)

Challenges in Growth Hormone Therapy

Obesity and Metabolism (Frontiers of Hormone Research)

Growth Hormone in Adults: Physiological and Clinical Aspects, Second Edition

Growth Hormone in Adults: Physiological and Clinical Aspects, Second Edition

Hypertension and Hormone Mechanisms (Contemporary Endocrinology)

Growth Hormone Secretagogues: Basic Findings and Clinical Implications

Growth Hormone Related Diseases and Therapy: A Molecular and Physiological Perspective for the Clinician (Contemporary Endocrinology)

Hypertension and Hormone Mechanisms

Hematopoietic Growth Factors

Transforming Growth Factors

Lysophospholipid Growth Factors

Insulin-like growth factors

Hormone Deception

MRAM Market Size and Growth Factors Research and Projection 2026

Growth Factors and Psychiatric Disorders (Novartis Foundation Symposium 289)

Neuro-endocrinology of Growth and Energy Balance

Endocrinology and Metabolism

Advances in Plant Ethylene Research: Proceedings of the 7th International Symposium on the Plant Hormone Ethylene

Growth Factors, Part A (Growth Factors & Cytokines in Health & Disease) (Growth Factors & Cytokines in Health & Disease)

Growth Hormone Deficiency - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References

Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction

Growth Factors, Part A (Growth Factors & Cytokines in Health & Disease) (Growth Factors & Cytokines in Health & Disease)

Growth Theory and Growth Policy

International trade, growth, and development

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