Yearbook of Pediatric Endocrinology 2010: Endorsed by the European Society for Paediatric Endocrinology

Yearbook of Pediatric Endocrinology 2010

Yearbook of Pediatric Endocrinology 2010 Endorsed by the European Society for Paediatric Endocrinology

Editors

Associate Editors

Jean-Claude Carel Ze’ev Hochberg

Gary Butler Evangelia Charmandari Francesco Chiarelli Stefano Cianfarani Mehul Dattani Nicolas De Roux Ken Ong Orit Pinhas-Hamiel Michel Polak Lars Sävendahl Olle Söder Martin Wabitsch

Diabetes: Clinical and Experimental

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Sponsored by a grant from Pfizer Endocrine Care Disclaimer. The statements, opinions 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 book 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. 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.

© Copyright 2010 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISBN 978-3-8055-9601-5 ISSN 1662-3391

Mikael Knip

Editors

Jean-Claude Carel Pediatric Endocrinology and Diabetology, and INSERM U690, University Paris 7 Denis Diderot, Hôpital Robert Debré, 48, boulevard Sérurier, FR–75935 Paris cedex 19, France Tel. +33 1 40 03 41 05; Fax +33 1 40 03 24 29; E-Mail [email protected]

Ze‘ev Hochberg Meyer Children’s Hospital, Rambam Medical Center, Faculty of Medicine Technion-Israel Institute of Technology, POB 9602 IL–31096 Haifa, Israel Fax +972 (0) 4 854 21 57; E-Mail [email protected]

Associate Editors

Gary E. Butler Department of Paediatrics and Adolescents University College London Hospital, 250 Euston Road, London NW1 2PQ, UK Tel. +44 8451 555 000 ext. 5240, E-Mail [email protected]

Evangelia Charmandari Division of Endocrinology and Metabolism, Clinical Research Center Biomedical Research Foundation of the Academy of Athens 4 Soranou tou Efessiou Street, GR–11527 Athens, Greece Tel. +30 210 659 7196, Fax +30 210 659 7545, E-Mail [email protected]

Francesco Chiarelli Department of Pediatrics, University of Chieti, Via dei Vestini, 5 IT–66013 Chieti, Italy Tel. +39 0871 358015, Fax +39 0871 574831, E-Mail [email protected]

Stefano Cianfarani Department of Public Health and Cell Biology Tor Vergata University Room E178, Via Montpellier 1, IT–00133-Rome, Italy Tel. +39 06 6859 3074, Fax +39 06 6859 2508, E-Mail [email protected]

Mehul T. Dattani Developmental Endocrine Research Group Clinical and Molecular Genetics Unit, Institute for Child Health University College London London, WC1N 1EH, UK Tel. +44 207 905 2657, Fax +44 207 404 6191, E-Mail [email protected]

Diabetes: Clinical and Experimental

Nicolas De Roux INSERM U 690, Laboratoire d’Hormonologie, AP-HP Hôpital Robert Debré, 48 Boulevard Sérurier FR–75019 Paris, France Tel. +33 1 40 03 19 85, Fax +33 1 40 40 91 95, E-Mail [email protected]

Ken Ong Medical Research Council Epidemiology Unit Institute of Metabolic Science, Addenbrooke’s Hospital, Box 285 Cambridge CB2 0QQ, UK Tel. +44 1223 769207; Fax +44 1223 330316; E-Mail [email protected]

Orit Pinhas-Hamiel Pediatric Endocrine and Diabetes Unit, Safra Children’s Hospital Sheba Medical Center Ramat-Gan IL–52621 Ramat-Gan, Israel Tel. +972 3 5305015, Fax +972 3 5305055, E-Mail [email protected]

Michel Polak Service d’endocrinologie pédiatrique INSERM EMI 0363, Hôpital Necker-Enfants Malades 149, rue de Sèvres, FR– 75015 Paris Tel. +33 1 44 49 48 03/02, Fax +33 1 44 38 16 48, E-Mail [email protected]

Lars Sävendahl Pediatric Endocrinology Unit; Q2:08 Department of Woman and Child Health Karolinska Institutet and University Hospital, Solna SE–171 76 Stockholm, Sweden Tel. +46 8 5177 2369; Fax +46 8 5177 5128; E-Mail [email protected]

Olle Söder Pediatric Endocrinology Unit, Q2:08 Department of Woman and Child Health Karolinska Institutet and University Hospital, Solna SE–171 76 Stockholm, Sweden Tel. +46 8 517 75124, Fax +46 8 517 75128, E-Mail [email protected]

Martin Wabitsch Pediatric Endocrinology, Diabetes and Obesity Unit, Department of Pediatrics and Adolescent Medicine University of Ulm, Eythstrasse 24 DE–89075 Ulm, Germany Tel. +49 731 5002 7715; +49 731 50027789; E-Mail [email protected]

Mikael Knip

Table of Contents

IX

Preface

Ze’ev Hochberg and Jean-Claude Carel 1

Neuroendocrinology

Carine Villanueva, Lukas Huihjbregts and Nicolas de Roux 13

Pituitary

Evelien F. Gevers and Mehul T. Dattani 33

Thyroid

Michel Polak, Gabor Szinnai, Aurore Carré and Mireille Castanet 49

Growth and Growth Factors

Stefano Cianfarani 65

Bone, Growth Plate and Mineral Metabolism

Terhi Heino, Dov Tiosano, Aneta Gawlik and Lars Sävendahl 83

Reproductive Endocrinology

Olle Söder and Lena Sahlin 99

Adrenals

Erica L.T. van den Akker and Evangelia Charmandari 119

Type 1 Diabetes: Clinical and Experimental

Francesco Chiarelli and M. Loredana Marcovecchio 139

Obesity and Weight Regulation

Martin Wabitsch, Daniel Tews, Michaela Keuper, Carsten Posovszky, Christian Denzer, Anja Moss, Julia von Schnurbein and Pamela Fischer-Posovszky 155

Type 2 Diabetes Mellitus, Metabolic Syndrome, Lipids

Orit Pinhas-Hamiel 171

Population Genetics and Pharmacogenetics

Ken K. Ong and Cathy Elks 189

Evidence-Based Medicine in Pediatric Endocrinology

Gary E. Butler 207

Editor’s Choice

Jean-Claude Carel and Ze’ev Hochberg 219

Science and Medicine

Ze’ev Hochberg and Jean-Claude Carel 237

Author Index

253

Subject Index

Diabetes: Clinical and Experimental

VII

Preface The approach and fusion of many basic, translational and clinical aspects of medicine make it increasingly difficult to define the limits of subjects to be included in a short presentation of pediatric endocrine highlights of the year. The enormous advances of modern medical science, as summarized in the 2010 Yearbook of Pediatric Endocrinology, are based on new knowledge and concepts in the diverse fields of genetics and genomics, innate immunology, molecular biology, systems biology, population genetics, proteomics and metabolomics, evolutionary biology – all of which require attention in our consideration of the nature and mechanism of disease processes; modern diagnosis and therapy depend on such consideration. The task of presenting recent advances in a concise but comprehensive form seems no less important as the difficulty increases. In fulfilling this task, we have kept in view the original purpose of a compact and condensed volume. As such, the Yearbook has been, and continues to be, useful to basic scientists who wish to understand the clinical issues of modern pediatric endocrinology, to clinical scientists who wish to base their research on the most recent developments in the field, and to clinicians who must maintain familiarity with the foundation sciences of medical practice as they evolve. In the Yearbook Preface, we annually recognize the anniversary of a major scientific breakthrough. 100 years ago, in 1910, Artur Biedl, Professor of General and Experimental Pathology at the German University in Prague, published the first textbook on endocrinology that was promptly translated into English: Internal Secretion: The Basic Physiology and Significance for Pathology, Urban & Schwarzenberg, Berlin. The book listed more than 8,500 references, only about 1% of which had been published prior to 1889. This averages more than 400 papers a year during the first 20 years of the field [Wilson, 2005]. The fiction author James Morrow, who is best known for his Godhead Trilogy novels, centering on the literal bodily death of the Godhead of Judeo-Christian theology, wrote: Everybody thinks he’s being oh-so-deep when he says science doesn’t have all the answers ... Science does have all the answers.... The problem is that we don’t have all the science. This Yearbook attempts to provide the 2009–10 chapter of the pediatric endocrine science. We are grateful to our twelve Associate Editors and their coauthors, who have done an enormous work to discern this year’s advances and provide their chapters in a timely fashion, to the European Society for Paediatric Endocrinology (ESPE), who endorses the Yearbook, and we acknowledge the generous support by Pfizer that makes the Yearbook project possible for its seventh year in a row. Ze’ev Hochberg (Haifa) Jean-Claude Carel (Paris) Wilson JD: The evolution of endocrinology. Clin Endocrinol (Oxf) 2005;62:389–396.

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Neuroendocrinology Carine Villanueva, Lukas Huihjbregts and Nicolas de Roux INSERM U676, Hôpital Robert Debré and Université Paris Didérot, Paris, France

New gene The most obvious candidate gene in gonadotropic deficiency validated at last Congenital idiopathic hypogonadotropic hypogonadism (IHH) is a condition characterized by a failure to undergo puberty and normal fertility in adulthood. IHH is related to a defect of GnRH secretion or GnRH activity. Genetic defects have now been described in several genes. However, to date, no mutations had been described in the most obvious candidate gene, GnRH itself. Two papers published this year have reported loss of function mutations in few cases of IHH.

Isolated familial hypogonadotropic hypogonadism and a GNRH1 mutation Bouligand J, Ghervan C, Tello JA, Brailly-Tabard S, Salenave S, Chanson P, Lombes M, Millar RP, Guiochon-Mantel A, Young J Universite Paris-Sud, Faculté de Médecine Paris-Sud and Assistance Publique-Hôpitaux de Paris, Hôpital de Bicêtre, INSERM UMR-S693, Paris, France N Engl J Med 2009;360:2742–2748

Mutations in GnRH1 which encode for the preprohormone were screened in a population of 310 hypogonadotropic hypogonadism cases. One homozygous frameshift mutation, an insertion of an adenine at nucleotide position 18, was found in 2 siblings from consanguineous parents. Both parents and 1 unaffected sibling were heterozygous for the insertion. Both affected patients have the same phenotype with isolated gonadotropic deficiency without anosmia. In transfected cells this insertion results in an aberrant peptide lacking the conserved GnRH decapeptide. The phenotype was reversed by pulsatile GnRH administration. Conclusion: This study confirms the pivotal role of GnRH in human reproduction. Methods and Results:

GNRH1 mutations in patients with idiopathic hypogonadotropic hypogonadism Chan YM, de Guillebon A, Lang-Muritano M, Plummer L, Cerrato F, Tsiaras S, Gaspert A, Lavoie HB, Wu CH, Crowley WF, Jr, Amory JK, Pitteloud N, Seminara SB Harvard Reproductive Sciences Center and Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, Mass., USA Proc Natl Acad Sci USA 2009;106:11703–11708

GnRH1 mutations were screened in 290 hypogonadotropic hypogonadism cases. One homozygous frameshift mutation leading to the synthesis of a peptide truncated at the C-terminal end of the mature peptide. This frameshift mutation was not seen in 192 controls. The mutation was found in a male with micropenis at birth, bilateral cryptorchidism and absent puberty. Four heterozygous variants were also described in 4 patients and were not seen in controls. One non-synonymous missense mutation in the eighth amino acid of the GnRH decapeptide. One nonsense mutation that causes premature termination within the GnRH-associated peptide and 2 sequence variants that cause non-synonymous amino-acid substitutions in the signal peptide and in the GnRH-associated peptide. Conclusion: GnRH1 mutation is a new genetic cause of normomics isolated hypogonadotropic hypogonadism. Methods and Results:

Screening for GnRH1 mutation was the first hypothesis tested to explain congenital IHH without anosmia. In the early 1990s, large numbers of patients were tested without success. A polymorphism

which did not fit with the hypothesis of a mendelian genetic disease with recessive transmission was reported and GnRH1 was therefore rejected. In 2009, two papers appeared at the same time and both reported a very low frequency of GnRH1 mutation in 310 or 290 IHH cases. The phenotype reported by both groups is very similar. In all cases, there was no anosmia, the gonadotropic deficiency was isolated and other endocrine functions of the anterior pituitary were normal. Both patients had blunted response to a single GnRH administration which represents an interesting finding. It confirms that the GnRH test is not appropriate to discriminate hypothalamic or pituitary defects in IHH. This point had already been described 10 years ago when the first mutation of the GnRH receptor was described in patients with low testosterone and estradiol levels, normal LH and FSH levels and adequate response to GnRH administration [1]. After 13 days of pulsatile GnRH administration, LH pulses were detected in 1 GnRH-mutated female and correlated to the GnRH pulses. A similar response was observed in patients with partial gonadotropic deficiency due to GnRH receptor mutation. The pivotal role of GnRH in the regulation of the gonadotropic axis has been known for more than 20 years. These papers confirm that the physiological function of GnRH1 in humans is restricted to the regulation of the gonadotropic axis. The very low frequency of GnRHR1 mutations indicates that it should not be the first gene to be screened in patients with IHH.

New mechanism Role of estrogen in brain sexual dimorphism

Estrogen masculinizes neural pathways and sex-specific behaviors Wu MV, Manoli DS, Fraser EJ, Coats JK, Tollkuhn J, Honda S, Harada N, Shah NM Program in Neuroscience, University of California-San Francisco, San Francisco, Calif., USA Cell 2009;139:61–72 Background: It has been clearly demonstrated that male sexual behaviors rely on a specific neural network development that requires the sex hormones testosterone and estrogen. However, how both hormonal pathways interact to govern this sexually dimorphic neural circuit remains unknown. Testosterone acts via the androgen receptor (AR) and is also metabolized in the brain into estrogens by aromatase. The authors therefore speculated that aromatase expression could be the key link between the two hormonal pathways. Methods and Results: In order to determine whether estrogens or testosterone participate in aromatase expression regulation, they designed a knock-in mouse model in which aromatase-expressing neurons specifically express the two enzymatic reporters, ␤-galactosidase and placental alkaline phosphatase. The authors show a highly sexually dimorphic distribution of aromatase-expressing neurons in terms of number and projections in the bed nucleus of the stria terminalis and the posterodorsal component of the medial amygdale. Male mice lacking a functional AR still display this sexually dimorphic trait. However, neonatal administration of estrogen or testosterone in female mice induced a pattern of brain aromatase expression similar to wild-type males. The analysis of specific apoptosis markers expression in aromatase neurons of bed nucleus of the stria terminalis and medial amygdale shows a higher level of apoptotic cells in young females relative to their male counterparts. Female mice neonatally exposed to estrogen display an increase of territorial urine marking and aggression as well as a reduced sexual receptivity to males mounting and intromission attempts. Conclusion: This study shows that the sexually dimorphic expression of aromatase is not controlled by AR. Since estrogen is barely undetectable in the male circulation, a local conversion of testosterone into estrogen by aromatase in the brain is likely to explain how estrogen determines the male-specific differentiation of aromatase neurons, probably by promoting cell survival of aromatase-expressing neurons.

The authors have designed a knock-in mouse line expressing reporter enzymes in aromatase-expressing neurons. This allowed them to highlight a sexual dimorphism of the aromatase-expressing neural network in the bed nucleus of the stria terminalis and medial amygdale, which govern the acquisition of typical male sexual behaviors, such as aggressiveness and territorial marking. In female neo-

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nates, ovaries are inactive whereas males display a surge in testosterone that is absolutely required for proper male sexual behavior acquisition. Here the authors bring evidence that this neonatal surge allows local testosterone aromatization into estrogen, the latter protecting the aromatase-expressing neurons from entering apoptosis. The major role of estrogen, testosterone and aromatase in the acquisition of male sexual behavior has been known for over 20 years. However, the underlying mechanisms have always remained unclear, especially the balanced roles between testosterone and estrogen. This work therefore brings precious insight into these mechanisms. It also raises numerous questions on how local conversion of testosterone into estrogen promotes aromatase neurons survival in the BNST and MeA, and whether other sexually dimorphic populations of neurons are determined through such a mechanism.

Conditional inactivation of androgen receptor gene in the nervous system: effects on male behavioral and neuroendocrine responses Raskin K, de Gendt K, Duittoz A, Liere P, Verhoeven G, Tronche F, Mhaouty-Kodja S Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche, Génétique MoléculaireNeurophysiologie et Comportement, and Institut de Biologie, Collège de France, Paris, France J Neurosci 2009;29:4461–4470 Background: In the developing male, testosterone (T) plays a crucial role in the establishment of malespecific neuronal network, governing typical male sexual behavior. T signaling is mediated either directly via the androgen receptor (AR) or indirectly via the estrogen receptor (ER) after T aromatization into estradiol. Because T and AR also exert major peripheral effect on male gonadal differentiation and several other physiological functions, the balance between central and peripheral influence of T on the determinism of male behavior and neuroendocrine control of reproduction is unclear. Methods and Results: In order to distinguish between the central and peripheral roles of AR, the authors used the Cre-loxP technology to generate mutant mice specifically lacking AR expression in the nervous system only (ARNesCre), thereby maintaining functional AR signaling in the peripheral tissues. Mutant males were fertile and phenotypic analyses showed that central T has little influence on the urogenital tract development. However, T and LH secretions were twofold higher than in wild-type animals with no changes in FSH levels. Mutant mice were as receptive to female pheromones as wild-type mice in terms of neuronal activity and olfaction, but showed severely impaired masculine behaviors, such as erectile activity, mounting and aggressiveness. Finally, the authors demonstrate the role of central AR in the somatotropic axis, as mutant mice showed reduced IGF-I levels and growth retardation. Conclusion: These results show that central AR is involved in male-specific acquisition of sexual and aggressive behaviors, and participates in the regulation of the somatotropic axis.

In this study, the authors designed a powerful genetic model to explore the specific role of central AR in mice. Loss of central AR did not abrogate fertility, although the authors report a high proportion of hypofertile males. Because LH secretion is increased twofold in ARNesCre mice, the data strongly suggest the existence of a major T-dependent negative feedback on GnRH release as well as a pituitary positive feedback on LH secretion, confirming previous indirect evidence of T feedback provided by the study of ER␣ knockout male mice [2]. The authors did not detect AR expression in GnRH neurons, suggesting that interneurons such as kisspeptin neurons, which express AR [3], may convey T signal toward GnRH neurons. Analysis of Kiss1 expression in ARNesCre mice would allow to test this hypothesis. Male ARNesCre mice display strongly impaired masculine behavior, suggesting that T signaling through AR in the brain plays a crucial role in the establishment of a male-specific neuronal network. However, Juntti et al. [4] recently generated another neuron-specific AR knockout mouse line called ARNsDel. In contrast with ARNesCre mice, male ARNsDel display only moderately impaired male behaviors. Moreover, the authors show that AR expression in the brain at the time of the androgen surge is very weak and is regulated by ERs. Therefore, the authors propose that estrogen metabolized from testosterone during development is responsible for a higher expression of AR in the adult brain of males, where T signals augment the male-pattern behaviors that have been differentiated under the control of estrogen signaling.

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Role of estradiol in the dynamic control of tanycyte plasticity mediated by vascular endothelial cells in the median eminence de Seranno S, d’Anglemont de Tassigny X, Estrella C, Loyens A, Kasparov S, Leroy D, Ojeda SR, Beauvillain JC, Prevot V Institut National de la Santé et de la Recherche Médicale, Unité 837, Lille, France Endocrinology 2010;151:1760–1772 Background: During low GnRH outputs, GnRH-secreting axons are enveloped by ependymoglial cells named tanycytes, which prevent GnRH crossing the basal lamina of the brain to reach the pituitary portal circulation. Prior to the preovulatory surge of LH, tanycytes no longer create a physical barrier between GnRH neuron terminal axons, therefore allowing GnRH release into the circulation. The authors previously provided evidence that endothelial cells of the median eminence play a key role in the modulation of neuroglial remodeling through the nitric oxide (NO) pathway, but the molecular mechanism governing tanycyte retraction in response to GnRH output requirements still remain unclear. Methods and Results: The authors show that tanycytes cocultured with endothelial cells of the median eminence (ECME) present an important retraction under short estradiol exposure. When cultured alone or cocultured with ECME in the presence of a NO synthase-dominant negative form, tanycytes do not retract. Triggering of NO release by L-arginine in estradiol-free cocultures induces tanycytes retracting, suggesting that estradiol triggers tanycytes retraction by inducing a NO release by ECME. Cyclooxygenase (COX)1 and COX2 are known to be involved in NO-induced actin cytoskeleton remodeling of tanycytes. Estradiol upregulates both COX1 and COX2 expression in tanycytes, leading to an increase of prostaglandin E2 (PGE2) synthesis. PGE2 treatment induces an increase of retraction of cultured tanycytes. Ultrastructural analysis of mouse hypothalamic explants of the ME showed that PGE2 induces an increase of the distance between GnRH nerve terminals and the basal lamina, suggesting a retraction of the surrounding tanycytes. Finally, they show that in vivo inhibition of COX activity in mice impairs the estrous cycle, thereby confirming the important role of COX and PGE2 in the regulation of the estrous cycle. Conclusion: The authors show that vascular endothelial cells play a critical role in the neuroendocrine brain plasticity by conveying estradiol signaling toward tanycytes in the ME.

It is now well established that neuronal signaling at the cellular level is not solely dependent on other neuronal inputs, but strongly relies on the microenvironment formed by the surrounding glia and brain capillaries. The events occurring in the median eminence of the hypothalamus during the ovarian cycle are a striking example of the dynamic morphological changes controlling neurosecretory axons. This elegant work brings further insight into the mechanisms on how endothelial cells, tanycytes and neurons coordinate their activity in response to changing outputs, such as estradiol. Here the authors suggest that estradiol induces NO release from endothelial cells of the median eminence, resulting in tanycytes retraction. This retraction could be facilitated by an estradiol-dependent increase of cyclooxygenases COX1 and 2, which metabolize PGE2, a modulator of tanycyte plasticity. These results add a new level of complexity to the understanding of estradiol-induced rise of GnRH secretion prior to the LH preovulatory surge. The question of how the negative estradiol feedback switches to a positive feedback remains unanswered.

Postnatal development of an estradiol-kisspeptin-positive feedback mechanism implicated in puberty onset Clarkson J, Boon WC, Simpson ER, Herbison AE Centre for Neuroendocrinology, Department of Physiology, University of Otago, Dunedin, New Zealand Endocrinology 2009;150:3214–3220 Background: Over the past years, the kisspeptin neuropeptide encoded by KISS1 has been extensively described as a key part in the activation of the gonadotropic axis at puberty through the modulation of GnRH release. However, the mechanisms underlying the increase of KISS1 expression in the hypothalamus prior to the onset of puberty remain unknown. Methods and Results: Using immunocytochemistry, the authors first determined the postnatal profile of kisspeptin expression in the rostral periventricular area of the third ventricule, where kisspeptin neurons

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innervate GnRH neurons. They show that kisspeptin immunoreactivity in mouse occurs around the postnatal day (P)15 and reaches its maximal level at P30, which corresponds to the onset of puberty. Female pups were then ovariectomized, which resulted in a very strong reduction in kisspeptin immunoreactivity at P30 and P60. Treatment with exogenous estrogen restores kisspeptin wild-type profile of expression. Furthermore, mice lacking a functional aromatase display a total absence of kisspeptin immunoreactivity in the periventricular area of the third ventricule, and a decrease in the arcuate nucleus. Conclusion: These results show that ovarian estrogen is required for the postnatal increase of kisspeptin expression in periventricular area of the third ventricule neurons during the prepubertal period. In this study, the authors bring evidence for an estrogen-driven positive regulation of kisspeptin expression in the hypothalamic region periventricular area of the third ventricule, occurring around P15 in mice. As the kisspeptin/KISS1R system is critical for normal puberty onset, these results account for a positive feedback loop model where estrogen-induced increase of kisspeptin activates GnRH neurons resulting in a rise of gonadotropins, which in turn stimulate gonadal steroids and so on, ultimately leading to the activation of the gonadotropic axis at puberty. Kisspeptin expression was not detected before P15, suggesting that the early activation of GnRH neurons soon after birth does not depend on kisspeptin, but rather on other neurotransmitters signaling, such as GABA and glutamate. Once ovarian function produces sufficient estrogen level, kisspeptin in periventricular area of the third ventricule facilitates GnRH impulse generator. Whether estrogen acts directly on kisspeptin neurons remains to be defined, but it would be very interesting to compare the mechanisms of prepubertal upregulation of kisspeptin in the periventricular area of the third ventricule with the upregulation occurring in the AVPV in adult females.

Concepts revised Characterization of kisspeptin neurons

Forebrain projections of arcuate neurokinin B neurons demonstrated by anterograde tract tracing and monosodium glutamate lesions in the rat Krajewski SJ, Burke MC, Anderson MJ, McMullen NT, Rance NE Department of Pathology, University of Arizona College of Medicine, Tucson, Ariz., USA Neuroscience 2010;166:680–697 Background: Current studies indicate that neurokinin-3 (NK3) receptor and kisspeptin receptor signaling play a key role in the regulation of the gonadotropic axis activity in humans. Some neurons residing in the rat arcuate nucleus (ARC) express neurokinin B (NKB), kisspeptin, dynorphin, NK3 receptor and estrogen receptor. However, how this neuronal network regulates the gonadotropic axis is unknown. Methods and Results: The projections of these neurons in the ARC were studied using NKB immunochemistry as a marker. First, arcuate nucleus was ablated by neonatal injection of monosodium glutamate. Second, neurokinin B fibers were anterogradely labeled using biotinylated dextran amine (BDA) injection into the arcuate nucleus and NKB/BDA immunoreactive axons were labeled. Four major projections pathways are described: (1) local and bilateral projections in the arcuate nucleus, (2) projections into the median eminence, (3) projections toward the periventricular zone, and (4), projections to the lateral and ventral hypothalamic tract and the medial forebrain bundle. Conclusion: The diverse projections of arcuate NKB neurons provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavior and neuroendocrine processes. This study reveals a bilateral interconnected network of sex-steroid-responsive neurons in the arcuate nucleus of the rat that project GnRH terminals in the median eminence. This circuitry provides a mechanism to modulate the pulsatile secretion of GnRH.

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Neurokinin B and dynorphin A in kisspeptin neurons of the arcuate nucleus participate in generation of periodic oscillation of neural activity driving pulsatile gonadotropin-releasing hormone secretion in the goat Wakabayashi Y, Nakada T, Murata K, Ohkura S, Mogi K, Navarro VM, Clifton DK, Mori Y, Tsukamura H, Maeda K, Steiner RA, Okamura H Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan J Neurosci 2010;30:3124–3132 Background: The pulsatile release of GnRH secretion is essential to drive pulsatile gonadotropin secretion and normal reproduction in mammals. Kisspeptin neurons serve as the central pacemaker that drives GnRH secretion. Methods and Results: Using immunochemistry, electrophysiological techniques and central injection of dynorphin (DynA), neurokinin B (NKB) or ␬-opiate receptor (KOR) antagonist, the authors have characterized kisspeptin neurons in the goat arcuate nucleus (ARC) and evaluated their role in GnRH secretion regulation. Immunohistochemistry confirmed that kisspeptin, dynorphin and neurokinin B are coexpressed in the same population of neurons. Electrophysiological techniques showed that bursts of multiple-unit activity (MUA) occurring in the medial basal hypothalamus of ovariectomized animals are associated with discrete pulses of LH. The frequency of these bursts is reduced by gonadal steroids. Central injection of dynorphin inhibits MUA volleys and pulsatile LH secretion, whereas neurokinin B induces multiple-unit activity volleys. Conclusion: Kisspeptin neurons in ARC regulate GnRH secretion, and dynorphin and neurokinin B seem to be involved in the process leading to the rhythmic discharge of kisspeptin in goat.

Tac3 encodes neurokinin B which is the ligand of the neurokinin B receptor encoded by Tacr3. Mutations in Tac3 and Tacr3 have been recently associated with gonadotropic axis deficiency [Yearbook 2009, pp 9–10]. In the human infundibular nucleus, a group of sex steroid-responsive neurons expresses neurokinin B, kisspeptin and dynorphin, a similar group of neurons were described in monkeys. Although Tacr3-invalidated adult mice were fertiles, a group of kisspeptin neurons express neurokinin B, dynorphin, and the estrogen receptor-␣ which suggest that this group of neurons also participates in the hypothalamic regulation of estrogen-negative feedback on the mouse gonadotropic axis. It is therefore fundamental to characterize the neuronal network involved in this regulation and its physiological role in the gonadotropic axis homeostasis. The first article provides compelling evidence that the majority of the neurokinin B-immunoreactive axons within the median eminence in rat originate from the arcuate nucleus and modulate GnRH secretion through the neurokinin-3 receptor via non-synaptic transmission. The importance of the neurokinin B receptor is underscored by its expression both in arcuate neurokinin B neurons and GnRH terminals in the median eminence. The multiple projections of these neurons suggest that it could also participate in the regulation of other homeostasic, behavioral and neuroendocrine circuits. In the second article, the authors provide evidence that dynorphin and neurokinin B indeed participate in the regulation of GnRH pulsatile secretion in goat. This co-expression of the three peptides in ARC neurons is thus a conserved mechanism observed in all mammals studied to date. This study also suggests a physiological function for dynorphin and neurokinin B. Kisspeptin neurons in the ARC drive pulsatile GnRH and LH secretion. After central administration of neurokinin B, dynorphin, or KOR antagonist, electrophysiological techniques show that the multiple unit activity in close proximity of kisspeptin neurons are influenced by neurokinin B and dynorphin A. The arcuate nucleus KiSS/NKB/ Dyn neurons network probably acts as a GnRH pulse generator: NKB/NK3R signalling stimulates and the Dyn/Kor signalling inhibits the activation of KiSS/NKB/Dyn neurons. These results are consistent with a model in which the pulsatile secretion of GnRH is related to a rhythmic oscillation activity in kisspeptin neurons leading to rhythmic secretion of kisspeptin. They do not preclude a similar activating-inhibiting loop on the GnRH neurons. In conclusion, these two papers confirm the essential role of neurokinin and dynorphin in the regulation of arcuate nucleus neurons expressing kisspeptin.

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New hope Sim1 as a new target in obesity

Postnatal Sim1 deficiency causes hyperphagic obesity and reduced mc4r and oxytocin expression Tolson KP, Gemelli T, Gautron L, Elmquist JK, Zinn AR, Kublaoui BM McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Tex., USA J Neurosci 2010;30:3803–3812 Background: Mutations in single-minded 1 (Sim1), which encodes a transcription factor, are associated with a monogenic obesity in humans and mice. The physiological postnatal functions of this protein have not been well established. Methods and Results: The authors designed a mouse line with a conditional postnatal CNS deletion of Sim1 by crossing ␣-calcium/calmodulin-dependent protein kinase II-Cre mice with mice carrying a floxed Sim1 allele. This conditional postnatal deletion was sufficient to cause hyperphagic obesity in Sim1 heterozygotes. Using stereological cell counting, the authors showed no hypocellularity in the paraventricular nucleus of the mutant mice hypothalamus and normal neuronal PVN projections. However, MC4R and oxytocin mRNAs were significantly decreased in this area in both conditional Sim1 homozygotes and germ line Sim1 heterozygotes. Conclusion: The hyperphagic Sim1-deficient mouse becomes obese because of feeding dysregulation due to changes in the leptin-melanocortin-oxytocin pathway.

Single-minded 1 (Sim1) deletions seem to be one of the few causes of monogenic isolated (non-syndromic) obesity but the mechanism involved in the phenotype is unknown. Sim1 null mice die perinatally and lack anterior periventricular paraventricular and supraoptic hypothalamic nuclei. Phenotypic analyzes performed in heterozygote animals did not allow to conclude whether Sim1 is only involved in hypothalamic development or also participates in postnatal physiology of the hypothalamus. This study demonstrates a role for Sim1 in food intake regulation after birth by modulating the leptinmelanocortin-oxytocin pathway. The decrease in oxytocin PVN expression after Sim1 invalidation suggests a possible role of oxytocin deficiency in the hyperphagia observed in this mouse. To support this hypothesis, the phenotype was rescued by oxytocin treatment. However, decreased expression of mc4r could also explain hyperphagia. Additional studies are needed to further identify which of Sim1 targets are indeed regulating food intake. This study also suggests that Sim1 and its potential target genes act postnatally in paraventricular hypothalamic nucleus and could be potential therapeutic targets for appetite suppression in obese individuals.

New mechanism Molecular Interface between food intake and reproduction

Melanin-concentrating hormone directly inhibits GnRH neurons and blocks kisspeptin activation, linking energy balance to reproduction Wu M, Dumalska I, Morozova E, van den Pol A, Alreja M Department of Psychiatry, Yale University School of Medicine and the Ribicoff Research Facilities, Connecticut Mental Health Center, New Haven, Conn., USA Proc Natl Acad Sci USA 2009;106:17217–17222 Background: The link between energy balance and reproduction is critical. In negative energy balance, reproductive processes need to be aborted but molecular mechanisms mediating this link remain unclear. Fasting and food restriction up-regulate the hypothalamic melanin-concentrating hormone (MCH) system that promotes feeding and decreases energy expenditure; MCH knockout mice are lean and have a higher metabolism but remain fertile. MCH also regulates other hypothalamic related function such as

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sleep, drug abuse, behavior and mood. Kisspeptin is the major regulator of GnRH secretion and therefore the central regulator of the gonadotropic axis. Two subpopulations of GnRH neurons have been described, one of which expresses the vesicular glutamate transporter 2 (vGluT2). Methods and Results: In this study, Wu et al. have tested the hypothesis that the link between feeding regulation and reproduction is mediated via a direct postsynaptic effect on vGluT2-GnRH neurons activated by kisspeptin. Using patch-clamp recording in multiple lines of transgenic mice, they demonstrated a strong inhibitory effect of MCH on vGluT2-GnRH neurons. MCH has no effect on kisspeptin-insensitive GnRH, VGluT2, cholinergic or GABAergic neurons located in the same hypothalamic nucleus. MCH blocks the excitatory effect of kisspeptin on vGluT2-GnRH neurons. This inhibitory effect of MCH is mediated via a direct postsynaptic BA2+-sensitive K+ channel mechanism involving the MCHR1 receptor. MCH fibers are in close proximity to VGluT2-GFP and GnRH-GFP neurons. Conclusion: Considering the role of MCH in regulating energy balance and of kisspeptin in regulating fertility and pubertal onset, MCH may provide a critical link between energy balance and reproduction. It is now clear that kisspeptin is the major regulator of the GnRH secretion and therefore it is involved in the timing of the pubertal onset and in the occurrence of the LH surge leading to ovulation. Normal energy balance is critical for normal fertility which occurs only when metabolic fuel is available. Peptides conveying metabolic fuel to brain act at different levels of the gonadotropic axis. Some of them, such as insulin, act directly on GnRH neurons, others such as leptin, target kisspeptins neurons. This study reports a combined effect of melanin-concentrating hormone on these two neuroendocrine targets by showing that melanin-concentrating hormone can inhibit the excitatory effect of kisspeptin on vGlutT2-GnRH neurons. The most intriguing result reported here is the capacity of melanin-concentrating hormone to specifically target kisspeptin-activated GnRH neurons and not GnRH neurons refractory to kisspeptin activation. This result provides an additional evidence of the presence of at least two GnRH neuron populations in the normal hypothalamus. This inhibitory effect of melanin-concentrating hormone was observed in prepubertal as well as pubertal mice in both sexes. The normal fertility in melanin-concentrating hormone knockout indicates that melaninconcentrating hormone effect on the gonadotropic axis is mainly required to curtail reproductive activity in conditions such as food deprivation. The next step of this interesting model would be to demonstrate that the inhibitory post-synaptic effect of melanin-concentrating hormone revealed by patch-clamp is associated with a decrease of kisspeptin-induced GnRH secretion.

New mechanism Leptin and GnRH secretion

Leptin indirectly regulates gonadotropin-releasing hormone neuronal function Quennell JH, Mulligan AC, Tups A, Liu X, Phipps SJ, Kemp CJ, Herbison AE, Grattan DR, Anderson GM Department of Anatomy and Structural Biology, Centre for Neuroendocrinology, University of Otago School of Medical Sciences, Dunedin, New Zealand Endocrinology 2009;150:2805–1812 Background: Environmental regulation of GnRH neuronal activity determines the reproductive status of the individual. Leptin is one of the hormones communicating information about metabolic status to the hypothalamic GnRH neuronal system. Methods and Results: To determine whether leptin acts directly on GnRH neurons, the authors used three complementary approaches. First, pSTAT3 immunopositive GnRH neurons were counted after intraventricular injection of leptin in males and females rats. Fertility was assessed in males and females with conditional leptin receptor knockout mice from all forebrain neurons or GnRH neurons only. Leptin receptor deletion in forebrain neurons prevented the onset of puberty resulting in infertility and blunted estradiol-induced LH surge. Leptin receptor deletion in GnRH neurons did not impair fertility. Finally, single cell-nested PCR in living brain slices showed that GnRH neurons do not express leptin receptor

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mRNA. Intracerebroventricular leptin treatment induced pSTAT3 expression within the AVPV but not in GnRH neurons. Conclusion: GnRH neurons are not direct targets for leptin in rodents. Normal fertility was observed in GnRH neuron conditional leptin receptor deletion. Single cell-nested PCR confirmed the absence of leptin receptor mRNA in GnRH neurons and pSTAT3 immunochemistry showed no co-localization with GnRH labelling. The possibility that leptin receptor could stimulate other signalling pathways than pSTAT3 cannot be excluded; however, pSTAT3 neuronal knockout is the only component of the leptin signalling pathway that results in infertility and recapitulates the leptin receptor KO phenotype. This study definitively confirms that leptin acts on the GnRH system via afferent interneurons. Kisspeptin neurons are probably one of the candidate interneurons to be tested.

New hormone A new neuropeptide involved in the regulation of food intake

Neuropeptide W: an anorectic peptide regulated by leptin and metabolic state Date Y, Mondal MS, Kageyama H, Ghamari-Langroudi M, Takenoya F, Yamaguchi H, Shimomura Y, Mori M, Murakami N, Shioda S, Cone RD, Nakazato M Frontier Science Research Center, University of Miyazaki, Kiyotake, Miyazaki, Japan Endocrinology 2010;151:2200–2210 Background: Neuropeptide W (NPW) is an anorectic peptide produced in the brain but its interactions with other peptides that regulate feeding are unknown. Methods and Results: NPW is present in several nuclei of the hypothalamus including the paraventicular, ventromedial, lateral and arcuate nuclei. NPW expression was significantly up-regulated in leptin-deficient and leptin-receptor-deficient mice. In leptin-deficient mice, leptin replacement rescued NPW hypothalamic levels. Leptin regulation of NPW expression is controlled by phosphorylation of STAT3 which induces SOCS3 in NPW-expressing neurons. The authors also report that NPW reduces feeding potentially via the melanocortin-4-receptor signaling pathway, activates POMC and inhibits NPY neurons in mice expressing promoter-driven green fluorescent protein. Conclusion: NPW plays an important role regulating feeding under the conditions of leptin insufficiency.

Previous reports have shown that NPW is expressed in different hypothalamic nuclei known as essential areas in the regulation of energy metabolism and feeding behavior. Here, it is shown that NPW expression increases under low leptin levels, which was a surprising result. Usually, anorectic peptides are up-regulated whereas orexigenic peptides are down-regulated by leptin. This control of leptin on NPW seems to be direct via activation of the leptin receptor in NPW-expressing neurons. The exact intracellular signaling pathways governing this control remain unclear. The authors studied the effect of leptin on NPW neurons in primary cells culture from adult rats and it will be necessary to confirm this effect using an in vivo approach. These results add a new complexity in the hypothalamic network regulating feeding behavior in low leptin condition. NPW seems to be a new example of a peptide having a predominant role in response to specific circumstances. It is necessary to study whether the role of NPW under normal conditions is similar.

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New mechanism From the islet to the brain and back

Influence of insulin in the ventromedial hypothalamus on pancreatic glucagon secretion in vivo Paranjape SA, Chan O, Zhu W, Horblitt AM, McNay EC, Cresswell JA, Bogan JS, McCrimmon RJ, Sherwin RS Department of Internal Medicine, Division of Endocrinology and Metabolism, Yale University School of Medicine, New Haven, Conn., USA Diabetes 2010;59:1521–1527 Background: The hypothesis that insulin regulates pancreatic ␣-cell glucagon secretion by a central effect on the hypothalamus has not been well investigated. Methods and Results: To study whether insulin acts within the ventromedial hypothalamus (VMH) to modulate the secretion of glucoregulatory hormones, the authors compared the effects of VMH exposure to insulin, anti-insulin antibody, insulin receptor antagonist, a combination of anti-insulin antibody plus GABA receptor agonist muscimol prior to a hypoglycemic clamp or under baseline conditions. Pancreatic glucagon response to central insulin was 4- to 5-fold lower than in the absence of systemic hyperinsulinemia with phloridzin-induced hypoglycemia. VMH insulin action directly suppressed glucagon response to insulin-induced hypoglycemia independently of intra-islet insulin or input from circulating catecholamines. VMH insulin microinjection suppressed glucagon response to phloridzin-induced hypoglycemia. Local blockade of VMH insulin action increased basal glucagon levels, an effect completely abolished by a simultaneous injection of ␥-aminobutyric acid A receptor agonist, muscimol. Conclusion: Insulin inhibits ␣-cell glucagon secretion both at the level of the pancreas and of the VMH.

Glucose homeostasis is regulated by insulin and glucagon with a subtle interplay between these two hormones and it is known that insulin exerts a paracrine effect in the islet on glucagon. Here, it is shown that insulin also acts centrally to regulate pancreatic glucagon secretion under fasting and mild hypoglycemic conditions. The authors cannot exclude the possibility that VMH surrounding regions also contribute to the changes observed in glucagon secretion because the cerebral injection is difficult to realize. This study provides evidence for a role of insulin signalling in the regulation of ␣-cell function in vivo. The authors suggest that the primary site for insulin-mediated glucagon regulation is the islet but highlight a secondary role for central mechanisms. An insulin central effect appears to occur under hypoglycemia and a basal condition and might contribute to glucose homeostasis disorders. The loss of glucagon response to hypoglycemia in type 1 diabetic patients might result in part from the simultaneous increase in insulin levels both in the VMH and locally in the islet caused by exogenous insulin administration. Altogether, these data point to the central importance of the hypothalamus in the regulation of metabolic fluxes and their hormonal control.

New mechanism A central clock preventing enuresis

Central clock excites vasopressin neurons by waking osmosensory afferents during late sleep Trudel E, Bourque CW Centre for Research in Neuroscience, Research Institute of the McGill University Health Center, Montreal, Que., Canada Nat Neurosci 2010;13:467–474 Background: In addition to its major function in the endocrine axis regulation, the hypothalamus is a central regulator of many fundamental behaviors like temperature regulation, food intake and circadian rhythms. Integrated regulations between circadian rhythms and endocrine homeostasis have been

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largely described. However, the molecular mechanisms by which the suprachiasmatic nucleus (SCN) regulates hormonal secretion remain unknown. Methods and Results: Clock neurons of the SCN modulate osmosensory synapses onto vasopressin neurons to facilitate osmoregulated vasopressin release during sleep. This release is facilitated at the end of the sleep cycle to prevent dehydration and enuresis. Previous work has revealed that clock neurons have low firing rates during the late sleep period. The authors found that excitation of rat vasopressin neurons by osmosensory afferents is facilitated during the late sleep period, suggesting a possible regulation of magnocellular neurosecretory cells by SCN neurons. Conclusion: Clock neurons mediate an activity-dependent presynaptic silencing of osmosensory afferent synapses onto vasopressin neurons. Osmoregulatory gain is enhanced by removal of this effect during late sleep. Several hormones are produced with a circadian timing and vasopressin is one of them. This biological rhythm over 24 h is fundamental for a normal endocrine homeostasis. Biological rhythms are under the control of the suprachiasmatic nucleus located in the hypothalamus. Neurons of this nucleus exhibit daily rhythmic activities that are regulated by cell autonomous molecular feedback loops involving clock genes. The circadian rhythm is adjusted to 24 h by external clues like daylight. The molecular mechanisms linking suprachiasmatic nucleus circadian activity and daily hormonal rhythms are poorly understood. In this paper, a molecular mechanism to explain the circadian regulation of the homeostatic circuit involved in osmotic balance is proposed. A complex biochemical interactions between osmosensory neurons located in the organum vasculosum lamina terminalis, suprachiasmatic nucleus neurons and vasopressin neurosecretory cells located in the supraoptic nucleus is shown. The experimental approach used by the authors is interesting as they were able to stimulate organum vasculosum laminae terminalis neurons with a hyperosmotic stimulus with or without electrical stimulation of the suprachiasmatic nucleus and then recorded action potentials at distance in magnocellular neurosecretory cells. Their results indicate that the increased release of vasopressin during the end of the sleep cycle is mainly regulated by the release of an inhibitory effect of suprachiasmatic nucleus neurons on osmosensory neurons. The characterization of the biochemical mechanism of this inhibitory effect will be the next step of this study. This work opens the way to understand how suprachiasmatic nucleus neurons can regulate circadian rhythms of different endocrine axes. A better understanding of these molecular mechanisms may also be important for testing new hypothesis and new treatment for nocturnal enuresis. References 1. De Roux N, Young J, Misrahi M, Genet R, Chanson P, Schaison G, et al: A family with hypogonadotropic hypogonadism and mutations in the gonadotropin-releasing hormone receptor. N Engl J Med 1997;337:1597–1602. 2. Wersinger SR, Haisenleder DJ, Lubahn DB, Rissman EF: Steroid feedback on gonadotropin release and pituitary gonadotropin subunit mRNA in mice lacking a functional estrogen receptor-␣. Endocrine 1999;11:137–143. 3. Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, et al: Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 2005;146:2976–2984. 4. Juntti SA, Tollkuhn J, Wu MV, Fraser EJ, Soderborg T, Tan S, et al: The androgen receptor governs the execution, but not programming, of male sexual and territorial behaviors. Neuron 2010;66:260–172.

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Pituitary Evelien F. Geversa,b and Mehul T. Dattania a Developmental Endocrine Research Group, Clinical and Molecular Genetics Unit, Institute for Child Health, London b Division of Molecular Neuroendocrinology, MRC-National Institute for Medical Research, London, UK

Significant advances have been made in the field of pituitary development, pituitary tumor biology and pituitary signaling, as well as the interaction between signaling, development and tumor formation. Numerous new mutations and their functional implications for pituitary function have been identified. In addition, continuing progress is being made to uncover the functions of oxytocin, especially its role in behavior and an exciting development is the pharmacological use of oxytocin to affect behavior [1].

New mutations

Heterozygous orthodenticle homeobox 2 mutations are associated with variable pituitary phenotype Dateki S, Kosaka K, Hasegawa K, Tanaka H, Azuma N, Yokoya S, Muroya K, Adachi M, Tajima T, Motomura K, Kinoshita E, Moriuchi H, Sato N, Fukami M, Ogata T Department of Endocrinology and Metabolism, National Children’s Medical Center, Tokyo, Japan [email protected] J Clin Endocrinol Metab 2010:95:756–764 Background: Orthodenticle homeobox 2 (OTX2) is a transcription factor that appears to be critical for normal forebrain and eye development. A number of heterozygous mutations in the gene encoding this protein, OTX2, have been identified in association with ocular malformations such as anophthalmia or microphthalmia, Recent studies have suggested a positive role of OTX2 in pituitary as well as ocular development and function, but there appears to be considerable variability in the phenotype, e.g. some mutations do not appear to be associated with eye phenotypes [2]. Detailed pituitary phenotypes in patients with OTX2 mutations and OTX2 target genes for pituitary function other than HESX1 and POU1F1 remain to be determined. Methods: The authors studied 94 Japanese patients with various ocular or pituitary abnormalities, and sequenced all patients for mutations in OTX2. Multiplex ligation-dependent probe amplification (MLPA) was performed for OTX2 intragenic mutation-negative patients. Transient transfection assays were performed using luciferase reporters containing IRBP, HESX1, POU1F1 and GNRH1 promoters. Human cDNA from a variety of tissues was screened for the presence of OTX2 transcripts. Results: The authors identified the following heterozygous mutations: p.K74fsX103 in case 1 with isolated GH deficiency (IGHD), anophthalmia/microphthalmia, and anterior pituitary hypoplasia (APH) and an ectopic/undescended posterior pituitary (EPP); p.A72fsX86 in case 2 with normal pituitary function and microphthalmia; p.G188X in 2 unrelated cases (case 3 with microphthalmia, combined pituitary hormone deficiency (CPHD) and APH with an EPP, and case 4 with microphthalmia and normal pituitary function), and a 2.86-Mb microdeletion including OTX2 in case 5 with IGHD and APH associated with anophthalmia/microphthalmia. Wild-type OTX2 protein transactivated the GNRH1 promoter as well as the HESX1, POU1F1, and IRBP (interstitial retinoid-binding protein) promoters, whereas the p.K74fsX103-OTX2 and p.A72fsX86-OTX2 proteins led to loss of transactivation and the p.G188X-OTX2 protein demonstrated reduced (approx. 50%) transactivation function for the four promoters, with no dominant-negative effect. cDNA screening identified positive OTX2 expression in the hypothalamus. Two additional missense mutations, p.T178S and p.A245V, were also identified in 2 further patients; these were not shown to compromise the function of the protein and may reflect rare sequence variants.

Conclusions: The results imply that OTX2 mutations are associated with variable pituitary phenotypes, with no genotype-phenotype correlations, and that OTX2 can transactivate GNRH1 as well as HESX1, POU1F1 and IRBP promoters.

Orthodenticle homeobox 2 (OTX2) is critical for normal forebrain and eye development and heterozygous mutations in OTX2 have been associated with anophthalmia or microphthalmia. Since transcriptional regulation of the development of the hypothalamus, pituitary, eyes and optic nerves overlap, this interesting study has screened a sizeable cohort of patients with ocular and/or pituitary abnormalities for mutations in OTX2. The authors report considerable variability in the phenotypes associated with the mutations with no clear genotype-phenotype correlations, although it appears likely that GH deficiency is the commonest endocrine manifestation. This is reminiscent of mutations in HESX1, which is believed to be a target of OTX2. The exact role of OTX2 in both murine and human hypothalamo-pituitary development is unclear. Additionally, the authors state that since OTX2 transactivates the GNRH1 promoter, GNRH1 may also be a target of OTX2. However, one needs to apply caution with respect to this statement. In vivo studies are needed in animals that are conditionally deleted for Otx2 in the hypothalamo-pituitary region. Similarly, one cannot extrapolate the binding of OTX2 to the POU1F1 promoter with subsequent transactivation to a direct genetic interaction between OTX2 and POU1F1 – the co-expression of the two genes needs to be shown, as does loss of expression of Pou1f1 expression in animals that are conditionally deleted for Otx2. It is therefore clear that OTX2 is an important player in hypothalamo-pituitary development in humans, but much remains to be learnt with respect to its role in normal development.

A novel mutation in the LIM homeobox 3 gene is responsible for combined pituitary hormone deficiency, hearing impairment, and vertebral malformations Kristrom B, Zdunek AM, Rydh A, Jonsson H, Sehlin P, Escher SA Department of Molecular Biology, Umeå University, Umeå, Sweden [email protected] J Clin Endocrinol Metab 2009;94:1154–1161 Background: The LIM homeobox 3 (LHX3) LIM-homeodomain transcription factor gene, found in both human and mouse, is required for development of the pituitary and motor neurons, and is also expressed in the auditory system. Mutations in the gene are associated with combined pituitary hormone deficiency (CPHD) in association with a short stiff neck, and variable sensorineural hearing impairment. To date, homozygous recessive mutations have been identified in 9 unrelated families. Methods: The objective of this study was to determine the cause of, and further explore, the phenotype in six patients (aged 6 months to 22 years) with CPHD, restricted neck rotation, scoliosis, and congenital hearing impairment. Three of the patients also have mild autistic-like behavior. The association of previously described LHX3 mutations with CPHD and restricted neck rotation led to the adoption of a candidate gene approach, and the gene was sequenced. Neck anatomy was explored by computed tomography and magnetic resonance imaging, including three-dimensional reformatting. Results: A novel recessive splice-acceptor site mutation was found. The predicted protein encoded by the mutated gene would lack the homeodomain and carboxyl terminus of the normal, functional protein. Genealogical studies revealed a common gene source for all six families dating back to the 17th century. Anatomical abnormalities in the occipito-atlantoaxial joints in combination with a basilar impression of the dens axis were found in all patients assessed. Cervical lordosis and thoraco-lumbar hyperlordosis were observed in all children by the time the children could stand. Scoliosis was identified in 5 of the children, as was significant sensorineural hearing loss. MRI revealed severe anterior pituitary hypoplasia with a cystic structure identified in patient 5. Conclusions: This study extends both the mutations known to be responsible for LHX3-associated syndromes and their possible phenotypic consequences. Previously reported traits include CPHD, restricted neck rotation and variable hearing impairment; patients examined in the present study also show a severe hearing defect. In addition, the existence of cervical vertebral malformations are revealed, and believed to be responsible for the rigid neck and the development of scoliosis.

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Initial studies suggested that LHX3 mutations were associated with CPHD excluding ACTH deficiency in association with a short stiff neck and limited rotation [3]. However, the phenotypic spectrum has recently expanded to include variability in the presence of the short stiff neck [4], the presence of a pituitary microadenoma [5] and the presence of ACTH deficiency with sensorineural hearing impairment [6]. This study is fascinating as it reports the occurrence of a founder mutation in LHX3 in a northern Swedish population, and confirms the presence of hearing deficit in affected patients, thereby confirming a role for LHX3 in human auditory development. It is important to note that the presence of hypoglycemia led to cortisol replacement in 3 of the 6 individuals. Additionally, patient 2 had a low concentration of cortisol in the neonatal period, suggesting a diagnosis of ACTH deficiency. This paper nicely describes for the first time the occipito-atlantoaxial abnormalities observed in this cohort of patients. Additionally, it describes the presence of progressive scoliosis from 5 to 6 years of age in the majority of patients. It is important to note that skeletal abnormalities were also noted in 4 patients described by Rajab et al. [6], and must now be considered to be a component of the wider LHX3 mutant phenotype.

New genes – pituitary development

A role of the LIM-homeobox gene Lhx2 in the regulation of pituitary development Zhao Y, Mailloux CM, Hermesz E, Palkovits M, Westphal H Laboratory of Mammalian Genes and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA [email protected] Dev Biol 2010:337:313–323 Background: The mammalian pituitary gland originates from two separate germinal tissues during embryonic development. The anterior and intermediate lobes of the pituitary are derived from Rathke’s pouch, a pocket formed by an invagination of the oral ectoderm. The posterior lobe is derived from the infundibulum, which is formed by evagination of the neuroectoderm in the ventral diencephalon. Previous studies have shown that development of Rathke’s pouch and the generation of distinct populations of hormone-producing endocrine cell lineages in the anterior/intermediate pituitary lobes is regulated by a number of transcription factors expressed in the pouch and by inductive signals from the ventral diencephalon/infundibulum. However, little is known about factors that regulate the development of the posterior pituitary lobe. Methods: In this study, the authors investigated the role of the LIM homeobox gene Lhx2 in hypothalamopituitary development in mice. Lhx2 null mutants (Lhx2–/–) were generated and the phenotype analyzed. The expression of a number of genes encoding signaling molecules and transcription factors was studied in embryonic sections. BrdU assays were performed to assess cell proliferation and TUNEL assays performed to detect apoptosis. Results: Lhx2 is extensively expressed in the developing ventral diencephalon, including the infundibulum and the posterior lobe of the pituitary, in addition to other tissues such as the retina, forebrain, midbrain, hindbrain and spinal cord. Deletion of Lhx2 resulted in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. Rathke’s pouch is formed and endocrine cell lineages are generated in the anterior/intermediate pituitary lobes of the Lhx2 null mutant. However, the shape and organization of the pouch and the anterior/intermediate pituitary lobes are severely altered due to the defects in development of the infundibulum and the posterior lobe, with a large mass of cells occupying the area between the third ventricle and the anterior/ intermediate lobes of the pituitary. After E15.5, the mutant mice die in utero due to a severe defect in definitive erythropoiesis. BrdU assays revealed a dramatic increase in cell proliferation in the mutants as compared with wild-type animals at E11.5, E12.5, E13.5 and E14.5. Expression of infundibular markers such as Nkx 2.1, Sox3, Six3, and the cellular retinoic acid binding protein CRABP2 was unaffected in the mutants whereas expression of posterior lobe markers such as calbindin and vasopressin was

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absent in the region corresponding to the posterior lobe. An increased number of TUNEL-positive apoptotic cells were present in the large abnormal mass occupying the ventral diencephalon of Lhx2–/– mutants. The expression domain of Fgf8 was expanded rostrally, overlying the pouch that failed to grow dorsally in the mutants. Conclusion: This study thus reveals an essential role for Lhx2 in the regulation of posterior pituitary development and suggests a mechanism whereby development of the posterior lobe may affect the development of the anterior and intermediate lobes of the pituitary gland. The normal development of the pituitary gland is a carefully orchestrated process that is dependent on the expression of a number of signaling molecules and transcription factors in a coordinated manner [7]. Early tissue recombination studies have suggested that the neuroectoderm of the ventral diencephalon, leading to neurohypophysis generation, provides inductive signals that are required for the growth and differentiation of Rathke’s pouch. This exciting work has led to the identification of a further piece of the complex jigsaw. Deletion of Lhx2 resulted in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. The importance of Lhx2 lies in the fact that it is one of the few genes that have been identified to be critical for normal development of the infundibulum and posterior pituitary. It also reiterates the importance of the inductive interaction between the neuroectoderm and the oral ectoderm. Whether it will be implicated in the etiology of hypopituitarism in humans remains to be seen, although one would predict that the phenotype in affected patients would be extensive given the presence of severely defective erythropoiesis and embryonic death in the Lhx2 mutants.

Genetic regulation of pituitary gland development in human and mouse Kelberman D, Rizzoti K, Lovell-Badge R, Robinson IC, Dattani MT Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, University College, London Institute of Child Health, London, UK [email protected] Endocr Rev 2009;30:790–829

Normal hypothalamo-pituitary development is closely related to that of the forebrain and is dependent upon a complex genetic cascade of transcription factors and signaling molecules that may be either intrinsic or extrinsic to the developing Rathke’s pouch. These factors dictate organ commitment, cell differentiation, and cell proliferation within the anterior pituitary. Abnormalities in these processes are associated with congenital hypopituitarism, a spectrum of disorders that includes syndromic disorders such as septo-optic dysplasia, combined pituitary hormone deficiencies, and isolated hormone deficiencies, of which the commonest is GH deficiency. The highly variable clinical phenotypes can now in part be explained due to research performed over the last 20 years, based mainly on naturally occurring and transgenic animal models. Mutations in genes encoding both signaling molecules such as Sonic Hedgehog and transcription factors such as HESX1, SOX2, SOX3, LHX3, LHX4, PROP1 and POU1F1 have been implicated in the etiology of hypopituitarism, with or without other syndromic features, in mice and humans. To date, mutations in known genes account for a small proportion of cases of hypopituitarism in humans. However, these mutations have led to a greater understanding of the genetic interactions that lead to normal pituitary development. This comprehensive review attempts to describe the complexity of pituitary development in the rodent, with particular emphasis on those factors that, when mutated, are associated with hypopituitarism in humans. It is clear that normal hypothalamopituitary development is a highly complex phenomenon, and features such as phenotypic variability and variability of penetrance remain to be explained. This review describes the current state of knowledge in the field of pituitary development in mouse and human. The review describes key processes in murine pituitary development and also the known genetic causes of hypopituitarism in humans, and attempts to correlate phenotypes in both mouse and human. It describes in great detail the development of the pituitary and regulation of development by genes encoding signaling molecules such as Sonic Hedgehog and transcription factors such as HESX1, SOX2, SOX3, LHX3, LHX4, PROP1 and POU1F1. It continues to describe the consequences of mutations of these genes in murine and human development of the hypothalamic-pituitary axis and

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the implication of these mutations in the etiology of hypopituitarism, with or without other syndromic features in humans.

New mechanisms – pituitary development

The notch target gene HES1 regulates cell cycle inhibitor expression in the developing pituitary Monahan P, Rybak S, Raetzman L Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Ill., USA [email protected] Endocrinology 2009;150:4386–4394 Background: Growth of the pituitary during development and adulthood is a tightly regulated process. Hairy and enhancer of split (HES1), a transcription factor whose expression is initiated by the Notch signaling pathway, is a repressor of cell cycle inhibitors. The authors hypothesized that with the loss of Hes1, pituitary progenitors are no longer maintained in a proliferative state, but chose to exit the cell cycle instead. Methods: Expression of cell cycle regulators, proliferation and apoptosis in wild-type and Hes1-deficient pituitaries was assessed using immunohistochemistry. Results: Changes in phosphohistone H3 expression in cells in Rathke’s pouch in early pituitary development [8] indicate decreased proliferation in Hes1-deficient mutants. Furthermore, pituitaries lacking Hes1 have increased cell cycle exit, shown by significant increases in the cyclin-dependent kinase inhibitors, p27 and p57, from e10.5 to e14.5. Additionally, Hes1 mutant pituitaries have ectopic expression of p21 in Rathke’s pouch progenitors, an area coincident with increased cell death. Conclusions: These data indicate a role for HES1 in the control of cell cycle exit and in balancing proliferation and differentiation, allowing for the properly timed emergence of hormone secreting cell types.

This study is not only of importance for the understanding of pituitary development, but may also be important for the understanding of pituitary tumor formation, emphasizing the close relationship between embryonic development and tumor formation. We first became interested in HES1 when we were studying regulation of chondrocyte and adipocyte proliferation and differentiation – Pref1 (DLK1) functions as a receptor in the Notch signaling system and needs down-regulation for terminal adipocyte differentiation to occur [9] but is also present in the growth plate and pituitary. Notch signaling dictates cell fate and influences cell proliferation, differentiation, and apoptosis in multiple tissues. Factors at each step – ligands, receptors, signal transducers and effectors – play critical roles in executing the effects of Notch signaling. Ligand binding to DLK1 or other Notch receptors results in regulation of Notch effectors and HES1 is one of these Notch effectors. Recent studies postulated that Notch signaling in the pituitary maintains a proliferative zone of cells lining the Rathke’s pouch. In line with a role for Notch effectors in the regulation of proliferation and differentiation, HES1 is a repressor of cell cycle inhibitors. HES–/– mice have hypomorphic pituitaries with reductions in all hormone cell types and absence of ␣-MSH-producing cells that normally appear at a more differentiated stage of pituitary development. This study further clarifies its role in pituitary progenitors located in Rathke’s pouch, which normally express HES1. Absence of Hes1 leads to dysregulation of cyclindependent kinase inhibitors like p57, p27 and p21. When mutant Hes1 is present, pituitary progenitors show an increased cell cycle exit, increased cell death and a reduction in cell proliferation resulting in a depletion of the progenitor pool. This suggests a role for HES1 in the maintenance of a balance between proliferation and differentiation, allowing cell cycle exit for differentiation to hormone-secreting cells at an appropriate time. Inappropriate regulation of this process results in a hypomorphic pituitary gland. Further work is needed to establish whether increased activation of Notch-HES1 signaling in the pituitary may lead to tumor formation.

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Co-repressors TLE1 and TLE3 interact with HESX1 and PROP1 Carvalho LR, Brinkmeier ML, Castinetti F, Ellsworth BS, Camper SA Department of Human Genetics, University of Michigan, Ann Arbor, Mich., USA [email protected] Mol Endocrinol 2010;24:754–765 Background: Pituitary hormone deficiency causes short stature in 1 in 4,000 children born and can be caused by mutations in a number of genes encoding transcription factors such as HESX1, LHX3, LHX4, SOX2, SOX3, PROP1 and POU1F1. HESX1 interacts with a member of the groucho-related gene family, TLE1, through an engrailed homology (eh1) domain and represses PROP1 activity. Mice with Prop1 deficiency exhibit failed differentiation of the POU1F1 lineage, resulting in lack of TSH, GH, and prolactin, in addition to ACTH and gonadotrophin deficiency. In addition, these mutants exhibit profound pituitary dysmorphology and overexpress Hesx1 and Tle3. Tle3 is structurally related to Tle1, and is expressed in the developing pituitary in an overlapping yet distinct pattern. The ability of HESX1 to interact with TLE3 has not been explored previously. Methods: The authors tested the ability of TLE3 to enhance HESX1-mediated repression of PROP1 activation at the POU1F1 promoter in cell culture using 293T cells (human embryonic kidney cells). In vivo studies using transgenic mice tested the functional consequences of ectopic TLE3 and HESX1 expression by driving constitutive expression in pituitary thyrotrophs and gonadotrophs using the Cga promoter. Results: In the cell culture experiments, both TLE3 and TLE1 repressed PROP1 in conjunction with HESX1 with similar efficiencies, and the eh1 domain appears to be critical for this repression. TLE1 and TLE3 could each repress PROP1 in the absence of HESX1, probably via a protein-protein interaction. In vivo, terminal differentiation of thyrotrophs and gonadotrophs was suppressed by HESX1 alone and by TLE3 and HESX1 together but not by TLE3 alone. Interestingly, whereas HESX1 alone did not affect the expression of endogenous Cga, the Tg(Cga-Tle3), Tg(Cga-Hesx1) double transgenic embryos showed a dramatic reduction in the concentration of endogenous CGA protein, suggesting that the presence of the co-repressor TLE3 in addition to HESX1 was critical for the repression of the endogenous Cga. Conclusion: This work presents evidence that HESX1 is a strong repressor that can be augmented by the co-repressors TLE1 and TLE3. In vitro studies suggest that TLE1 and TLE3 might also play roles independent of HESX1 by interacting with other transcription factors like PROP1.

Although mutations in HESX1 were first identified in 1998 [10], its function remains largely unknown. Its role as a repressor is undisputed, yet its partners and targets have not as yet been clearly established. HESX1 interacts with a member of the groucho-related gene family, TLE1, through an engrailed homology domain and represses PROP1 activity and TLE1, and the similar gene TLE3, are expressed in a pattern overlapping that of HESX1. This paper reports that TLE3 and TLE1 are potential co-repressors, as has been previously established for TLE1 [11, 12]. It is interesting that the in vivo studies report enhancement of HESX1 function by TLE3, although HESX1 can also act as a repressor independently of TLE proteins. This suggests that other co-repressors such as NCoR1 [13] may also enhance the function of HESX1 as a repressor. Much remains to be understood about normal pituitary development and the mechanisms involved, and this is underlined by our inability to identify a genetic cause in the vast majority of children with congenital hypopituitarism.

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Important for clinical practice

Use of the desmopressin test in the differential diagnosis of pseudo-Cushing state from Cushing’s disease Tirabassi G, Faloia E, Papa R, Furlani G, Boscaro M, Arnaldi G Division of Endocrinology, Polytechnic University of Marche, Ancona, Italy [email protected] J Clin Endocrinol Metab 2010;95:1115–1122 Background: The diagnosis of Cushing disease is generally based upon a combination of urinary free cortisol (UFC), midnight serum cortisol and serum cortisol after dexamethasone suppression. However, the diagnosis can be difficult to make. The desmopressin (DDAVP) test has been proposed to discriminate Cushing’s disease (CD) from pseudo-Cushing states (PC); however, current information on its value is limited and contradictory. Methods: The authors aimed to study the ability of the DDAVP test to distinguish between CD and PC, with emphasis on subjects with mild hypercortisolism. They conducted a retrospective/prospective study that included 52 subjects with CD, 28 with PC, and 31 control subjects (CT) with simple obesity in whom Cushing syndrome had previously been excluded using standard diagnostic tests. The DDAVP test was performed and compared with standard diagnostic procedures for the diagnosis of Cushing’s syndrome. The diagnosis/exclusion of CD was measured. Results: Interpretation of the DDAVP test based on percentage and absolute increment of cortisol and ACTH did not in itself give acceptable values of both sensitivity (SE) and specificity (SP). CD diagnosis based on simultaneous positivity for basal serum cortisol >331 nmol/l and absolute ACTH increment >4 pmol/l and its exclusion in subjects negative for one or both measures yielded an SE of 90.3% and an SP of 91.5%. The approach was also highly effective in distinguishing PC from: (1) CD with moderate values of urinary free cortisol (SE 86.9%, SP 92.8%); (2) CD with moderate values of serum cortisol after dexamethasone suppression (SE 86.6%, SP 92.8%), and (3) CD with moderate values of midnight serum cortisol (SE 100%, SP 92.8%). Conclusion: Interpretation of the DDAVP test through a combination of parameters allowed effective discrimination of Cushing disease from pseudo-Cushing, even in subjects with mild hypercortisolism.

Pseudo-Cushing state is caused by conditions (e.g. depression, alcoholism, polycystic ovary syndrome, severe obesity) that can activate the hypothalamic-pituitary-adrenal axis and is characterized by clinical and biochemical signs typical of Cushing’s syndrome. The overlapping clinical features with Cushing disease and the similar values frequently determined in tests such as urinary free cortisol, serum cortisol after dexamethasone suppression, and midnight serum cortisol in the respective patients make it difficult to distinguish subjects with these two entities. This study has examined the usefulness of the DDAVP test in diagnosing Cushing disease in a large cohort of adult patients and suggests that assessment of percentage and absolute increment of cortisol and ACTH concentration in the DDAVP test allows for discrimination between Cushing disease and pseudo-Cushing. Whether the test can be a useful adjunct to the standard tests in a pediatric population remains to be proven.

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New hope?

Oxytocin-dopamine interactions mediate variations in maternal behavior in the rat Shahrokh DK, Zhang TY, Diorio J, Gratton A, Meaney MJ Sackler Program for Epigenetics and Psychobiology, Douglas Mental Health University Institute, McGill University, Montreal, Canada [email protected] Endocrinology 2010;151:2276–2286 Background: Variations in maternal behavior among lactating rats associate with differences in estrogenoxytocin interactions in the medial preoptic area and in dopamine concentrations in the nucleus accumbens. Individual differences in pup licking/grooming (LG) are abolished by oxytocin receptor blockade or treatments that equalize dopamine signal in the nucleus accumbens. In this paper, novel evidence is provided for a direct effect of oxytocin at the level of the ventral tegmental area in the regulation of accumbens dopamine levels. Methods: In vivo study of normal rats divided in high LG (>1 SD) or low LG ( 10 months, respectively) were used to identify mechanisms that may prevent or delay adenoma formation in the presence of excess GHRH. Results: In hyperplastic pituitaries, expression of the late G1/G2 marker Ki67 increased, whereas the proportion of 5-bromo-2’-deoxyuridine-labeled cells (an S-phase marker) did not differ from agematched controls indicating that cell cycle progression was blocked. Further evidence suggested that

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enhanced p27 activity may contribute to this process. Adenoma formation was associated with loss of p27 activity. Increased endogenous somatostatin (SST) tone may slow the conversion from hyperplastic to adenomatous tissue since mRNA levels for SST receptors, sst2 and sst5, were elevated in hyperplastic pituitaries, whereas adenomas were associated with a decline in sst1 and sst5 mRNA. Also, SSTknockout Tg pituitaries were larger and adenomas formed earlier compared with those of SST-intact Tg mice. Unexpectedly, these changes were independent of changes in proliferation rate within the hyperplastic tissue, suggesting that endogenous SST controls GHRH-induced adenoma formation primarily via modulation of apoptotic and/or cellular senescence pathways, consistent with the predicted function of some of the most differentially expressed genes (Casp1, MAP2K1, TNFR2) identified by membrane arrays and confirmed by quantitative real-time RT-PCR. Conclusions: A block in cell cycle progression is responsible for hyperplasia of pituitaries under continuous GHRH signaling, but loss of p27 is needed for adenomatous transformation. Reduction of somatostatin tone plays a role in adenoma formation by modulation of apoptosis and senescence rather than proliferation. This paper and a paper mentioned later in this chapter investigate mechanisms involved in tumor growth, this one assessing the role of GHRH-somatostatin balance, and the next one the role of the less well known pituitary tumor transforming gene (PTTG1). GH producing adenomas are often found to overexpress GHRH and this study therefore focused on the role of GHRH in pituitary tumor formation. These tumors however often develop late in life and do not have 100% penetrance, suggesting that other factors are needed to allow for uncontrolled growth. This study aimed to find genes that allow for or halt pituitary tumor formation despite continuous GHRH stimulation. Pituitaries of mice overexpressing GHRH were used and these mice were crossed with mice deficient in somatostatin to study the contribution of somatostatin in slowing tumor formation. It was not too unexpected that loss of somatostatin tone enhanced tumor formation induced by GHRH overexpression. GHRH however seemed to act by affecting cell cycle and cyclin-dependent kinase inhibitors (p27), whereas action of somatostatin was proliferation independent and may be through modulation of apoptotic and senescence pathways. Array analysis comparing hyperplastic and adenomatous pituitaries showed differential regulation of genes involved in, amongst others, proliferation, apoptosis and microtubule formation (mitogen-activated protein kinase kinase1, adenomatosis polyposis coli binding protein (Mapre1), tumor necrosis factor receptor 1b (TNFR2)). Subsequent studies will undoubtedly assess the role of these genes in pituitary tumor formation in more detail.

New mechanisms – signaling in the pituitary and hypothalamus

ERK signaling in the pituitary is required for female but not male fertility Bliss SP, Miller A, Navratil AM, Xie J, McDonough SP, Fisher PJ, Landreth GE, Roberson MS Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, New York, N.Y., USA [email protected] Mol Endocrinol 2009;23:1092–1101 Background: Males and females require different patterns of pituitary gonadotropin secretion for fertility. The mechanisms underlying these gender-specific profiles of pituitary hormone production are unknown but there is evidence to suggest that ERK1 and 2 are essential modulators of hypothalamic GnRHmediated regulation of pituitary gonadotropin production and fertility. Methods: Mice with a pituitary-specific depletion of ERK1 and 2 were generated and a range of physiological parameters including fertility was assessed. Results: ERK signaling was found to be required in females for ovulation and fertility, but male reproductive function was unaffected. The effects of ERK pathway ablation on LH biosynthesis underlined this gender-specific phenotype, LH␤ expression being dependent on ERK-dependent up-regulation of the transcription factor Egr1. Conclusion: These findings help to elucidate the molecular basis of gender-specific regulation of the hypothalamic-pituitary-gonadal axis and sexually dimorphic control of fertility.

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This paper aims to further our understanding of the sexually dimorphic control of reproductive function at the molecular level. The mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK-ERK) signaling system comprises a three-level phosphorylation cascade (MAPK-kinasekinase Raf1, MAPK kinases MEK1 and MEK2, and MAPKs ERK1 and ERK2) that is activated upon a range of extracellular stimuli and is activated in gonadotrophs by GnRH. However, up until now, the in vivo function of this pathway has not been assessed. Mice with P-lox elements surrounding the ERK2 locus were crossed with ␣-GSU-Cre mice resulting in deletion of ERK2 in gonadotropes and thyrotropes, and then crossed to ERK1 null mice, rendering gonadotropes and thyrotropes ERK1/2 deficient. Although thyrotropes and ovaries were also ERK1 deficient, the authors argue that this is unlikely to have affected the phenotype of the mice. Ablation of the ERK signaling pathway in pituitary gonadotropes led to infertility in female but not male mice. Female mice did not have estrous cycle activity and did not mate. Ovaries contained follicles of various maturation, but lacked luteal tissue. LH␤ synthesis was reduced in gonadotropes and ovulation occurred in response to exogenous LH␤, implicating LH deficiency as the primary cause of infertility. In males, LH␤ expression was only slightly decreased and fertility was unaffected, reflecting the lower levels of LH that are required for Leydig cell function. This work implies ERK signaling as the sexually dimorphic link between genderspecific GnRH pulsatility and LH␤ synthesis. In addition, it suggests that FSH synthesis is less dependent on ERK signaling than LH synthesis.

Hypothalamic and pituitary c-Jun N-terminal kinase 1 signaling coordinately regulates glucose metabolism Belgardt BF, Mauer J, Wunderlich FT, Ernst MB, Pal M, Spohn G, Bronneke HS, Brodesser S, Hampel B, Schauss AC, Bruning JC Department of Mouse Genetics and Metabolism, Max Planck Institute for the Biology of Aging, Cologne, Germany [email protected] Proc Natl Acad Sci USA 2010;107:6028–6033 Background: c-Jun N-terminal kinase (JNK) 1-dependent signaling plays a crucial role in the development of obesity-associated insulin resistance. Methods and Results: This work demonstrates that JNK activation not only occurs in peripheral tissues, but also in the hypothalamus and pituitary of obese mice. To resolve the importance of JNK1 signaling in the hypothalamic/pituitary circuitry, the authors generated mice with a conditional inactivation of JNK1 in nestin-expressing cells (JNK1(DeltaNES) mice). JNK1(DeltaNES) mice exhibit improved insulin sensitivity both in the CNS and in peripheral tissues, improved glucose metabolism, as well as protection from hepatic steatosis and adipose tissue dysfunction upon high-fat feeding. Moreover, JNK1(DeltaNES) mice also show reduced somatic growth in the presence of reduced circulating growth hormone and insulin-like growth factor 1 concentrations, as well as increased thyroid axis activity. Conclusion: These experiments reveal an unexpected, critical role for hypothalamic/pituitary JNK1 signaling in the coordination of metabolic/endocrine homeostasis.

Recent yearbooks discussed in length the new concept of glucose homeostasis and body weight being under control of hypothalamic circuits regulating food intake, energy expenditure, and hepatic glucose production. Obesity causes an increased production of cytokines and inflammatory and stress signaling which results in activation of c-jun terminal kinase (JNK) and thereby insulin resistance. Indeed, JNK1–/– mice are protected from insulin resistance and obesity-induced hyperglycemia and are leaner. However, obesity does not only result in a reduction of insulin sensitivity in classical peripheral tissues, but also in the central nervous system thereby causing neuronal insulin and leptin resistance. In this work, mice were generated that have an ablation of JNK1 specifically in neurons. The authors started by showing that high fat feeding not only promotes JNK1 activation in peripheral tissues and CNS but also in the pituitary. Since Nestin is expressed not only in neurons but also in pituitary stem cells, use of a Nestin-Cre mouse will allow for ablation of genes in all pituitary cell lines. The authors therefore generated mice in which JNK1 was ablated in Nestin-expressing cells and confirmed ablation of JNK in both the hypothalamus and pituitary. Central JNK1 ablation resulted in increased insulin sensitivity with an increased Akt activation in response to insulin, and protection of diet induced insulin resistance. However, the mice also had reduced growth (approx. 20%) without increased adiposity as a result of a reduction in pituitary GHRH receptors, pituitary GH and peripheral

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IGF1. Unexpectedly the thyroid axis was also affected, with an increase in TRH, TRH receptors, TSH␤, and T3, a phenotype that is reminiscent of healthy ageing (increased insulin sensitivity accompanied by reduced glucose, insulin, and GH levels). This work therefore revealed an unexpected role of hypothalamic and pituitary JNK1 in the regulation of GH secretion and growth, combined with an increase in central insulin sensitivity and increased thyroid activity.

New mechanisms – GH deficiency

Growth hormone (GH)-releasing hormone increases the expression of the dominant-negative GH isoform in cases of isolated GH deficiency due to GH splice-site mutations Petkovic V, Godi M, Lochmatter D, Eble A, Fluck CE, Robinson IC, Mullis PE Department of Pediatric Endocrinology, Diabetology, and Metabolism, University Children’s Hospital, Bern, Switzerland [email protected] Endocrinology 2010;151:2650–2658 Background: IGHD II, the autosomal dominant form of isolated GH deficiency, can be due to heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of mis-splicing varies with different mutations and the level of GH expression and/or secretion. Methods: To study the functional relation between mutations and GH secretion, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Results: As expected, upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the splice site mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. Conclusion: The severity of IGHD II depends on the position of the splice site mutation and the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to produce more 17.5kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Splice site mutations in the GH-1 gene, like GH-IVS+1, GH-IVS+2, GH-IVS+6 and GH-ISE+28, result in deletion of exon 3, resulting in production of a 17.5-kDa GH isoform. This isoform is retained in the ER, disrupts the Golgi apparatus and prevents normal production and secretion of the 22-kDa isoform, so having a dominant-negative effect. Significant variation in severity and age of onset exists in patients with IGHD type II [15], with some patients showing progressive hypopituitarism, depending on the GH-1 gene alterations, but the exact reason is unclear. This study is a good example of bedside to bench work, aiming to study this clinical phenomenon using basic research tools. Mice expressing a high-copy number of a transgene mimicking the GH-IVS+1 mutation exhibit a dwarfed phenotype, and show disruption of somatotrophs and invasion of macrophages in the pituitary, and also have deficits of other pituitary hormones, the phenotype being less severe in low-copy number mice. Since the main driver of GH production is GHRH, which is overexpressed in IGHD, this study aimed to mimic the in vivo situation in vitro by transfecting wt-GH and three different exon 3 splice site mutations (GH-IVS+2, GH-IVS+6, and GH-ISE+28) which cause variable IGHD II in human patients, in a rat pituitary cell line stably expressing GHRH receptors. The results show that the three hGH splice site mutants produce different amounts of 17.5-kDa hGH after GHRH stimulation and exert

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different degrees of a dominant negative effect on endogenous wt-GH production. There is no dominant effect at the transcriptional level, but accumulation of the 17.5-kDa protein in the proteosomal degradation pathway results in impaired cell function and storage and secretion of wt-GH and finally leads to somatotroph destruction. The GH-IVS+2 mutation, which results in the most severe clinical presentation of the three, results in the most severe dominant negative effect on endogenous wt-GH production and secretion. Up-regulation of GHRH likely results in a vicious circle of increased 17.5kDa GH production and somatotroph destruction and further up-regulation of GHRH. This study demonstrates how carefully designed in vitro experiments may not only be able to explain the mechanism of a disease, but also predict severity and progress of disease.

Follow-up on Yearbook 2009 – pituitary tumorigenesis

E2F1 induces pituitary tumor transforming gene (PTTG1) expression in human pituitary tumors Zhou C, Wawrowsky K, Bannykh S, Gutman S, Melmed S Department of Medicine, Cedars-Sinai Medical Center, UCLA, Los Angeles, Calif., USA [email protected] Mol Endocrinol 2009;23:2000–2012 Background: Rb(retinoblastoma protein)/E2F is dysregulated in murine and human pituitary tumors. Pituitary tumor transforming gene (PTTG1), a securin protein, is required for pituitary tumorigenesis, and PTTG1 deletion attenuates pituitary tumor development in Rb+/– mice. This paper examines the regulation of PTTG1 by E2F1. Methods: Immunofluorescence, transient transfection of cultured cells, chromatin immunoprecipitation, RNA down-regulation with siRNA. Results: E2F1 and PTTG1 were concordantly overexpressed in most of 46 Rb+/– murine pituitary tissues and also in over half of 80 human pituitary tumors. E2F1 specifically bound the hPTTG1 promoter, indicating that hPTTG1 may act as a direct E2F1 target. Transfection of E2F1 and its partner DP1 dose-dependently activated hPTTG1 transcription up to 3-fold in p53-devoid H1299 cells but not in p53-replete HCT116 cells. E2F1 overexpression enhanced endogenous hPTTG1 mRNA and protein levels up to 3-fold in H1299 cells. The presence of endogenous p53/p21 constrained the induction, whereas decreasing either p53 or p21 in HCT116 cells restored E2F1-induced hPTTG1 transactivation and expression. Moreover, suppressing Rb by small interfering RNA concordantly elevated E2F1 and hPTTG1 protein levels. In contrast, transfection of E2F1 small interfering RNA lowered hPTTG1 levels 24 h later in HCT116 than in H1299 cells, indicating that p53 delays E2F1 action on hPTTG1. Conclusion: These results elucidate a mechanism for abundant tumor hPTTG1 expression, whereby Rb inactivation releases E2F1 to induce hPTTG1.

Pituitary tumors account for approximately 15% of intracranial tumors in adults. As is true for tumor formation of most tissues, aberrant cell cycle regulation plays a major role in pituitary tumor formation. Retinoblastoma protein (Rb) controls G1/S phase cell phase transition and Rb+/– mice spontaneously develop pituitary tumors. PTTG is a proto-oncogene, essential for proper chromatoid separation, and facilitates cell cycle progression. Overexpression of PTTG in mice facilitates pituitary tumor development and hPTTG1 overexpression in human tumors correlates with tumor invasiveness, recurrence and prognosis. Quite a lot is known about downstream targets and action of PTTG (for example upregulation of bFGF, VEGF and c-myc) (see Yearbook 2009, Pituitary [16, 17]), but little is known about proximal regulatory mechanisms. This paper now convincingly identifies E2F1 as a direct regulator of hPTTG in several ways, including identification of potential binding sites in the hPTTG1 promoter. E2Fs are key interacting factors for Rb proteins and are universal regulators of G1/S transition and cell cycle progression [18]. This paper enhances our knowledge of Rb-E2F1-hPTTG1 signaling and the requirement of hPTTG1 for pituitary tumorigenesis.

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Concept revised

Ghrelin O-acyltransferase (GOAT) is essential for growth hormone-mediated survival of calorie-restricted mice Zhao TJ, Liang G, Li RL, Xie X, Sleeman MW, Murphy AJ, Valenzuela DM, Yancopoulos GD, Goldstein JL, Brown MS Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Tex., USA [email protected] Proc Natl Acad Sci USA 2010;107:7467–7472 Background: Ghrelin is able to enhance food intake and growth hormone secretion from pituitary cells, but its essential function is obscure since elimination of the gene encoding ghrelin or its receptor produces only mild phenotypes in mice. Ghrelin is an octanoylated peptide, and Ghrelin O-acyltransferase (GOAT) attaches octanoate to proghrelin, which is processed to ghrelin. Here, the authors eliminate the Goat gene in mice, thereby eliminating all octanoylated ghrelin from blood. Methods: Goat knockout mice (Goat–/–) were generated and used in physiological experiments. Results: Goat–/– mice grew and maintained the same weights as wild-type (WT) littermates on normal or high fat diets. When subjected to 60% calorie restriction, both WT and Goat–/– mice lost 30% of body weight and 75% of body fat within 4 days. In both lines, fasting blood glucose initially declined equally. After 4 days, glucose stabilized in WT mice at 58–76 mg/dl. In Goat–/– mice however, glucose continued to decline, reaching 12–36 mg/dl on day 7. At this point, WT mice showed normal physical activity, whereas Goat–/– mice were moribund. GH rose progressively in calorie-restricted WT mice and less in Goat–/– mice. Infusion of either ghrelin or GH normalized blood glucose in Goat–/– mice and prevented death. Conclusion: An essential function of ghrelin in mice is elevation of GH levels during severe calorie restriction, thereby preserving blood glucose and preventing death.

Ghrelin was identified more than a decade ago as the endogenous ligand for the growth hormone secretogogue receptor, located in the hypothalamus and pituitary, which on stimulation led to release of growth hormone from the pituitary. Pharmacological studies have since shown ghrelin’s potential to increase food intake, gut motility and growth hormone release. It has been much harder to identify the physiological role of ghrelin since deletion of ghrelin, its receptor or both, has no significant impact on growth or appetite, at least in mice [19]. Results of this study suggest that ghrelin is important for the maintenance of the blood-glucose concentrations needed for survival during prolonged nutrient restriction. Ghrelin is unique in that its third amino acid, serine, is modified by the attachment of a medium chain fatty acid, octanoate, and this modification is necessary for its action. In this paper, the authors take a new approach to show the physiological role of ghrelin by generating mice that lack the enzyme GOAT, which is required for the octanoylation of ghrelin. The enzyme is located in the stomach and small intestine and lacks, as far as is known, any other function. Goat–/– mice indeed did not have detectable acyl-ghrelin. Mice fed a normal ad libitum diet did not show any abnormalities, much like ghrelin or Ghs-r null mice, but experiments in this paper went one step further to expose the mice to prolonged severe nutrient restriction. Normal mice were able to cope and maintain normoglycemia in contrast to the Goat–/– mice that became hypoglycemic and died. In addition, serum GH concentration was much further increased when glucose concentration declined in WT mice, compared to Goat–/– mice, and GH treatment of Goat–/– mice prevented hypoglycemia without raising IGF1 or ghrelin. These data suggest a role for ghrelin in the release of GH during times of calorie restriction to preserve normoglycemia likely through the effect of GH on gluconeogenesis. The evidence presented reminds us also that GH is pivotal in preserving normal bloodglucose levels. This is supported by the lower concentrations of GH observed in the GOAT knockout mice and the rescue of blood glucose by infusion of GH or ghrelin.

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New mutant mouse models

Use of mutant mouse lines to investigate origin of gonadotropin-releasing hormone-1 neurons: lineage independent of the adenohypophysis Metz H, Wray S Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorder and Stroke, National Institutes of Health, Bethesda, Md., USA [email protected] Endocrinology 2010;151:766–773 Background: Mutant mouse lines have been used to study the development of specific neuronal populations and brain structures as well as behaviors. GnRH is essential for vertebrate reproduction, with either GnRH-1 or GnRH-3 controlling release of gonadotrophins from the anterior pituitary, depending on the species. It is clear that the neuroendocrine GnRH cells migrate from extra-central nervous system (CNS) locations into the forebrain. However, the embryonic origin of GnRH-1 and GnRH-3 cells is controversial and has been suggested to be nasal placode, adenohypophyseal (anterior pituitary) placode, or neural crest, again dependent on the species examined. In two zebrafish knockouts, you-too (Gli2–/–) and detour (Gli1–/–), it was reported that when the adenohypophysis was missing or reduced, so were the hypothalamic GnRH neurons, although olfactory structures were intact [20]. Methods: In this report, single [(Gli2–/–), (Gli1–/–) and (Lhx3–/–)] and double (Gli1–/–; Gli2–/–) mutant mice were used to examine the lineage of GnRH-1 cells. Standard immunohistochemistry was performed and GnRH-1 cells counted in embryos in three areas: nasal region, nasal-forebrain junction, and forebrain. For a subset of embryos, forebrain GnRH-1 cells were further characterized as being in the olfactory bulb/rostral forebrain region, the preoptic area, or the hypothalamus. Results: Mutant mice with either missing or disrupted anterior pituitaries (Gli2–/–, Gli1–/–/Gli2–/– and Lhx3–/–) exhibited a normal GnRH-1 neuronal population and these cells were still associated with the developing vomerulonasal organ. At E15.5, the proportion of the GnRH-1 population in nasal areas versus the forebrain was the same in controls (30 and 70%), single knockouts (KOs) and double knockouts (35 and 65% respectively). At E18.5, in Gli single and double KOs, GnRH-1 cells within the forebrain showed a similar distribution. There was no difference in the brain distribution among genotypes. Within the CNS, cells were detected in the olfactory bulb/rostral forebrain, preoptic area and hypothalamus. This distribution was similar to control mice. Analysis of the mutants revealed that several diencephalic regions were normal in both single- and double-Gli KOs. The vomerulonasal organ was reduced in size in Gli2–/–, Gli1+/–,Gli2–/–, and double KOs, but was structurally normal. The pituitary was normal in Gli1–/– mice but there was a missing adenohypophysis in 4 of 5 Gli2–/– and Gli1+/–,Gli2–/– mice, and all Gli1–/–, Gli2–/– mice had complete loss of the adenohypophysis. Lhx3–/– mice were characterized by the presence of an undifferentiated anterior pituitary that was still connected to the oral ectoderm; however there was no difference in GnRH-1 cell number or distribution. Conclusion: These results indicate that in mice, GnRH-1 cells develop independent of the adenohypophyseal placode and are associated early with the formation of the nasal placode.

The origin of GnRH-1 cells has been the subject of much debate. In mammals, the GnRH-1 cells are first identified in the nasal placode which also gives rise to the nonsensory respiratory epithelium, the olfactory epithelium and the vomerulonasal organ. In the mouse, it has been suggested that the GnRH-1 progenitor cells are located in an intermediate area between the anterior respiratory cells and the OE sensory cells. In the chick, GnRH-1 transcripts were identified in the primitive streak and later in the anterior neural ridge which gives rise to the anterior pituitary, with subsequent localization to the presumptive nasal cavity and olfactory placode. Ablation experiments suggested that the GnRH-1 lineage was developmentally associated with the respiratory area of the nasal placode. However, in two zebrafish KOs in the Sonic Hedgehog pathway, you-too and detour, it was reported that absence or hypoplasia of the adenohypophysis was associated with absence or reduction of GnRH neurons, yet with intact olfactory structures. The authors of this study have used these mutants in the mouse to try and establish the lineage of GnRH-1 neurons. Their data seem to confirm that the origin of the GnRH-1 cells may be independent of the adenohypophsis. However, it is important to note that the murine genetic models that they have used may not be associated with GnRH-1 cell abnormalities as the genes that have been mutated may be implicated at later stages of pituitary

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development; hence, one cannot rule out a very early common origin of GHRH-1 and pituitary progenitor cells. Additionally, whereas Gli1–/– mutants are not associated with an abnormal pituitary in the mouse, in zebrafish, the mutants are associated with a small pituitary. Hence, species differences may account for some of the discrepant results. Many unanswered questions remain, for example mutations of SOX2 in humans are associated with hypogonadotrophic hypogonadism in association with a small anterior pituitary gland, whereas the rest of the pituitary hormones are largely unaffected apart from occasional GH deficiency. However, in the mouse, there is a general reduction in all the anterior pituitary hormones [7].

New image

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function Lafont C, Desarmenien MG, Cassou M, Molino F, Lecoq J, Hodson D, Lacampagne A, Mennessier G, El Yandouzi T, Carmignac D, Fontanaud P, Christian H, Coutry N, Fernandez-Fuente M, Charpak S, Le Tissier P, Robinson IC, Mollard P Department of Endocrinology, Institute of Functional Genomics, Montpellier, France [email protected] Proc Natl Acad Sci USA 2010;107:4465–4470 Background: Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. The pulsatile release is dependent upon a carefully orchestrated pattern of GHRH and somatostatin secretion. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Methods: The authors have developed in vivo approaches using transgenic GH-eGFP mice to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. They developed optical imaging methods that can monitor directly in vivo the relationship between the blood vasculature and GH cell network function. These methods involved the modification of a fluorescent stereomicroscope with long working distance objectives to image at wide field and single cell resolution an exposed pituitary gland deep in its in vivo environment. Results: Single capillaries were identified in close proximity to structural GH cell network motifs. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. The authors also report a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Conclusion: These data suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation, with corresponding changes in oxygen supply and oxygen consumption.

In this work, in vivo techniques in transgenic GH-eGFP mice to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ, were exploited to monitor directly the relationship between the blood vasculature and GH cell network function in vivo. These elegant studies have begun to shed light on the highly complex, yet poorly understood processes leading to pulsatile GH secretion It is clear that the secretion of GH by the GH cell network is dependent upon the fine regulation of hypothalamic inputs by the pituitary microcirculation, the supply and consumption of oxygen by the vasculature and GH cells, and the dynamic uptake of secreted products by the efferent blood capillaries. The study of these processes in an in vivo setting will revolutionize our understanding of GH secretion, and will probably impact on our understanding

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of the pathophysiology associated with GH deficiency and related disorders, e.g. in children with tumors, congenital GH deficiency and those who have impaired GH secretion as a result of traumatic brain injury and radiotherapy.

Reviews

Minireview: the melanocortin 2 receptor accessory proteins Webb TR, Clark AJ Centre for Endocrinology, William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, London, UK [email protected] Mol Endocrinol 2010;24:475–484

The melanocortin 2 receptor (MC2R) accessory protein, MRAP, is one of a growing number of G-protein-coupled receptor accessory proteins that have been identified in recent years. GPCR accessory proteins modulate GPCR function, direct receptor trafficking and targeting, moderate signaling intensity, and modify receptor structure and ligand binding. MRAP interacts directly with MC2R and is essential for its trafficking from the endoplasmic reticulum to the cell surface, where it acts as the receptor for the pituitary hormone ACTH. In addition, MRAP2, a newly described homolog of MRAP, is also able to support the cell surface expression of MC2R. The mechanism of MRAP action is only beginning to be understood although it is clear that MRAP is required for MC2R function. Recent work has started to reveal which MRAP domains are involved in MC2R functional expression, and new data have shown a potential role for both MRAP and MRAP2 in the regulation of the other melanocortin receptors. This is an excellent review that highlights the importance of the various components of melanocortin receptor (MCR) signaling. MCRs act as receptors for a-, b-, and g-MSH and ACTH and have a diverse range of functions – MC1R in skin pigmentation, MC2R as the receptor for ACTH, MC3R and MC4R in energy homeostasis. MRAPs act as accessory proteins for MC2R and are required for MC2R to travel to the cell surface and for ACTH signaling, and mutations cause ACTH resistance, i.e. familial glucocorticoid deficiency. The review covers the similarities and differences of MRAP and MRAP2, their structure and function in ACTH signaling, and also discusses their MC2R independent function and ability to modulate function of other MCRs. This review facilitates the understanding of MCR signaling and its relation with disease.

Food for thought – curcumin

Growth suppression of mouse pituitary corticotroph tumor AtT20 cells by curcumin: a model for treating Cushing’s disease Bangaru ML, Woodliff J, Raff H, Kansra S Department of Endocrinology, Metabolism & Clinical Nutrition, Aurora St. Luke’s Medical Center, Milwaukee, Wisc., USA [email protected] PLoS One 2010;5:e9893 Background: Pituitary corticotroph tumors secrete excess adrenocorticotrophic hormone (ACTH) resulting in Cushing’s disease. Standard treatment includes surgery and, if not successful, radiotherapy, both of which have undesirable side effects and frequent recurrence of the tumor. Pharmacotherapy using PPAR␥ agonists, dopamine receptor agonists, retinoic acid or somatostatin analogs is still experimental. Curcumin, a commonly used food additive in South Asian cooking, has potent growth inhibitory effects on cell proliferation, possibly by inhibiting constitutively activated NF␬B, and selectively targets

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tumor cells. Increased expression of the pro-survival protein Bcl-2 is commonly observed in pituitary tumors. The pro-survival Bcl-2 family of proteins (Bcl-2, Bcl-xL, and Mcl-1) are target genes of NF␬B. The authors recently demonstrated that curcumin inhibited growth and induced apoptosis in prolactinand growth hormone-producing tumor cells. Subsequently, Schaaf et al. [21] confirmed these data and also showed the in vivo effectiveness of curcumin in suppressing pituitary tumorigenesis. However, the molecular mechanisms that mediate this effect of curcumin are still unknown. Results: Using the mouse corticotroph tumor cells, AtT20 cells, the authors report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property; significant inhibition of colony formation was observed with concentrations of 5 µM, and complete inhibition was observed with 20 µM. The curcumin-induced growth inhibition was accompanied by a dose-dependent decrease in constitutive NF␬B activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, and increased PARP cleavage, which led to increased apoptosis. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells. Conclusion: The ability of curcumin to inhibit NF␬B and induce apoptosis in pituitary corticotroph tumor cells suggests that it might be used as a novel therapeutic agent for the treatment of Cushing’s disease. The treatment of Cushing syndrome is not straightforward and often needs a combination of medical treatment, surgery and radiotherapy. Although rare in children, it can be associated with life-long complications and impact on normal growth and development and also lead to diabetes mellitus, osteoporosis and hypertension. Hence, any effective alternative therapies need to be explored. In this study, the authors have investigated the use of the potent proliferation inhibitor curcumin, which has previously been used in Indian cooking, as an anti-tumor agent. In the Indian population, it is estimated that the average daily consumption of curcumin is 60–100 mg. The results are certainly interesting, although the in vivo effects have not as yet been established in humans. Additionally, although no toxic effects are known at present, potential side effects will need to be considered, especially before use in children and adolescents; in vitro, curcumin decreased secretion of ACTH and its effects are irreversible. The proposed mechanism of action of curcumin is interesting. It suppresses the TNF␣-induced activation of IKK that leads to the inhibition of TNF-dependent phosphorylation and degradation of I␬B␣ and subsequent nuclear translocation of the p65 subunit of NF␬B to regulate expression of genes implicated in cell cycle progression (e.g. cyclin D family), apoptosis (bcl-2), and cell migration and invasion (e.g. MMP2 and MMP9). Constitutive activation of NF␬B has been reported in cell lines as well as tumor samples, and may be linked to tumor progression as well as drug resistance. Bcl-xl plays a central role in pituitary cell survival and apoptosis, and these studies suggest that Bcl-xl plays a major role in regulating cell survival in pituitary corticotrophs. Curcumin may therefore modify Bcl-xL levels and hence act as a tumor suppressor, offering new hope to the treatment of Cushing disease. References 1. Andari E, Duhamel JR, Zalla T, Herbrecht E, Leboyer M, Sirigu A: Promoting social behavior with oxytocin in highfunctioning autism spectrum disorders. Proc Natl Acad Sci USA 2010;107:4389–4394. 2. Diaczok D, Romero C, Zunich J, Marshall I, Radovick S: A novel dominant negative mutation of OTX2 associated with combined pituitary hormone deficiency. J Clin Endocrinol Metab 2008;93:4351–4439. 3. Netchine I, Sobrier ML, Krude H, Schnabel D, Maghnie M, Marcos E, et al: Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency. Nat Genet 2000;25:182–186. 4. Pfaeffle RW, Savage JJ, Hunter CS, Palme C, Ahlmann M, Kumar P, et al: Four novel mutations of the LHX3 gene cause combined pituitary hormone deficiencies with or without limited neck rotation. J Clin Endocrinol Metab 2007;92:1909– 1919. 5. Bhangoo AP, Hunter CS, Savage JJ, Anhalt H, Pavlakis S, Walvoord EC, et al: Clinical case seminar: a novel LHX3 mutation presenting as combined pituitary hormonal deficiency. J Clin Endocrinol Metab 2006;91:747–753. 6. Rajab A, Kelberman D, de Castro SC, Biebermann H, Shaikh H, Pearce K, et al: Novel mutations in LHX3 are associated with hypopituitarism and sensorineural hearing loss. Hum Mol Genet 2008;17:2150–2159. 7. Kelberman D, Rizzoti K, Lovell-Badge R, Robinson IC, Dattani MT: Genetic regulation of pituitary gland development in human and mouse. Endocr Rev 2009;30:790–829. 8. Monahan P, Rybak S, Raetzman LT: The notch target gene HES1 regulates cell cycle inhibitor expression in the developing pituitary. Endocrinology 2009;150:4386–4394. 9. Hansen LH, Madsen B, Teisner B, Nielsen JH, Billestrup N: Characterization of the inhibitory effect of growth hormone on primary preadipocyte differentiation. Mol Endocrinol 1998;12:1140–1149. 10. Dattani MT, Martinez-Barbera JP, Thomas PQ, Brickman JM, Gupta R, Martensson IL, et al: Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse. Nat Genet 1998;19:125–133.

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11. Carvalho LR, Woods KS, Mendonca BB, Marcal N, Zamparini AL, Stifani S, et al: A homozygous mutation in HESX1 is associated with evolving hypopituitarism due to impaired repressor-corepressor interaction. J Clin Invest 2003;112:1192–1201. 12. Dasen JS, Barbera JP, Herman TS, Connell SO, Olson L, Ju B, et al: Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis. Genes Dev 2001;15:3193–3207. 13. Xu L, Lavinsky RM, Dasen JS, Flynn SE, McInerney EM, Mullen TM, et al: Signal-specific co-activator domain requirements for Pit-1 activation. Nature 1998;395:301–306. 14. Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E: Oxytocin increases trust in humans. Nature 2005;435:673– 676. 15. Mullis PE, Robinson IC, Salemi S, Eble A, Besson A, Vuissoz JM, et al: Isolated autosomal dominant growth hormone deficiency: an evolving pituitary deficit? A multicenter follow-up study. J Clin Endocrinol Metab 2005;90:2089–2096. 16. Maghnie M, Secco A, Loche S: Pituitary; in Carel J-C, Hochberg Z (eds):. Yearbook of Pediatric Endocrinology 2009. Basel, Karger, 2009, pp 13–26. 17. Chesnokova V, Zonis S, Kovacs K, Ben-Shlomo A, Wawrowsky K, Bannykh S, et al: p21(Cip1) restrains pituitary tumor growth. Proc Natl Acad Sci USA 2008;105:17498–174503. 18. McClellan KA, Slack RS: Specific in vivo roles for E2Fs in differentiation and development. Cell Cycle 2007;6:2917– 2927. 19. Sun Y, Ahmed S, Smith RG: Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 2003;23:7973– 7981. 20. Whitlock KE, Wolf CD, Boyce ML: Gonadotropin-releasing hormone (GnRH) cells arise from cranial neural crest and adenohypophyseal regions of the neural plate in the zebrafish, Danio rerio. Dev Biol 2003;257:140–152. 21. Schaaf C, Shan B, Onofri C, Stalla GK, Arzt E, Schilling T, et al: Curcumin inhibits the growth, induces apoptosis and modulates the function of pituitary folliculostellate cells. Neuroendocrinology 2010;91:200–210.

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Thyroid Michel Polaka, Gabor Szinnaib, Aurore Carréa and Mireille Castanetb a

Pediatric Endocrinology and Gynecology, Hôpital Necker Enfants Malades, AP-HP, INSERM U845, Université Paris Descartes, Paris, France b Paediatric Endocrinology, University Children’s Hospital, Basel, Switzerland

The chapter aims at giving a representative choice of high-standing articles answering timely questions in the fields of thyroid physiology and disease covering areas as genetics, environmental factors, pharmacology, development and cancer. The concept of the year was the introduction of potassium channels as limiting factor of iodine accumulation in the murine thyroid by closing the gap between the sodium/iodide symporter (NIS) and the Na+,K+-ATPase by the presence of a constitutively active potassium channel. Time and future yearbooks will be able to estimate the clinical relevance of this report for understanding normal thyroid function and its disease.

Mechanism of the year in thyroidology

Kcne2 deletion uncovers its crucial role in thyroid hormone biosynthesis Roepke TK, King EC, Reyna-Neyra A, Paroder M, Purtell K, Koba W, Fine E, Lerner DJ, Carrasco N, Abbott GW Greenberg Division of Cardiology, Department of Medicine and Department of Pharmacology, Weill Medical College of Cornell University, New York, N.Y., USA [email protected] Nat Med 2009;15:1186–1194 Background: Analogous to parietal cells and colonic crypt cells in the gastrointestinal tract, thyrocytes are non-excitable, polarized epithelial cells expressing ion transporters essential for the function of the thyroid gland. The thyroid hormones (TH) triiodothyronine (T3) and tetraiodothyronine (thyroxine, or T4) are critical for normal growth and development of the fetus and newborn as well as for regulation of metabolism in virtually all tissues at all ages. I− enters thyrocytes via the basolaterally located Na+/I− symporter (NIS) and exits apically into the colloid, where it is covalently incorporated into thyroglobulin, the precursor of T3 and T4. NIS-mediated I− transport uses the downhill Na+ gradient generated by the Na+,K+-ATPase at the basolateral membrane of the thyrocyte. The role of K+ channels in the thyroid has not been described so far. Results: Targeted disruption of Kcne2, a thyrocyte K+ channel, in mice impaired thyroid iodide accumulation up to 8-fold, impaired maternal milk ejection, halved milk tetraiodothyronine (T4) content and halved litter size. Kcne2-deficient mice had hypothyroidism, dwarfism, alopecia, goiter and cardiac abnormalities including hypertrophy, fibrosis, and reduced fractional shortening. The alopecia, dwarfism and cardiac abnormalities were alleviated by triiodothyronine (T3) and T4 administration to pups, by supplementing dams with T4 before and after they gave birth or by feeding the pups exclusively from Kcne2+/+ dams; conversely, these symptoms were elicited in Kcne2+/+ pups by feeding exclusively from Kcne2–/– dams. Conclusion: The authors show that the potassium channel subunits KCNQ1 and KCNE2 form a thyroidstimulating hormone-stimulated, constitutively active, thyrocyte K+ channel required for normal thyroid hormone biosynthesis. These data provide a new potential therapeutic target for thyroid disorders and raise the possibility of an endocrine component to previously identified KCNE2- and KCNQ1linked human cardiac arrhythmias.

For us, the concept of the year is the introduction of potassium channels in the scene of thyroid hormone biosynthesis. The authors identified the presence of a heterodimeric thyrocyte potassium channel, composed of the two subunits KCNQ1 and KCNE2. They showed that both subunits were expressed in human and murine thyrocytes, where they create a TSH-stimulated constitutively active potassium current. As supposed from their co-expression with the sodium/iodide symporter (NIS) at

the basolateral membrane, deletion of Kcne2 resulted in an I– accumulation defect, the first step of thyroid hormone biosynthesis. Consequently, Kcne–/– mice were hypothyroid and developed goiter. Kcne–/– mice further had cardiac hypertrophy, and as published by the same group in a past paper, impaired ventricular repolarization. Although the concept of closing the gap between the NIS and the Na+,K+-ATPase by the presence of a constitutively active potassium channel is new and innovative, human mutations in KCNE2 have been identified in patients with long QT syndrome (LQT subtype 6), but hypothyroidism was not reported in these patients so far. As subclinical hypothyroidism is also associated with prolonged QTc, a hallmark of loss-off-function mutations in KCNE2 and KCNQ1, thorough clinical description of thyroid function in patients with mutations is warranted to further support the hypothesis of a ‘thyroid’ component to some KCNE2- or KCNQ1-associated cardiac arrhythmias. Whether further potassium channels are expressed in human thyrocytes and could be involved in human thyroid biosynthesis, remains unanswered for the moment.

Follow-up on a Yearbook 2009 paper (see Thyroid section, pp. 27–40) Protect the liver when using antithyroid drugs in children

Dissimilar hepatotoxicity profiles of propylthiouracil and methimazole in children Rivkees SA, Szarfman A Yale Pediatric Thyroid Center, Yale University School of Medicine, New Haven, Conn. and Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, Md., USA [email protected] J Clin Endocrinol Metab 2010, Epub ahead of print Background: The antithyroid drugs propylthiouracil and methimazole are estimated to be used in more than 6,000 children and adolescents per year in the USA. Over the years that these medications have been used, reports of adverse events involving hepatotoxicity have appeared. The authors have already reported in a letter format the hepatotoxic effect of propylthiouracil in children and have pleaded not to use this drug anymore in pediatric Graves’ disease [1]. However, there has not been a systematic and comparative evaluation of the adverse events associated with antithyroid drug use. Objective: The authors’ aim was to assess safety and hepatotoxicity profiles of propylthiouracil and methimazole by age in the US Food and Drug Administration’s Adverse Event Reporting System (AERS). Methods: They used a data mining algorithm to analyze more than 40 years of safety data in AERS. This algorithm allows ultimately calculating adjusted observed to expected ratios for every drug-adverse event combination in AERS, focusing on hepatotoxicity events. Results: The algorithm identified higher-than-expected reporting of severe liver injury in pediatric patients treated with propylthiouracil but not with methimazole. Propylthiouracil had a high adjusted reporting ratio for severe liver injury (17; 90% CI 11.5–24.1) in the group less than 17 years of age. The highest ratio values for methimazole were with mild liver injury in the group 61 years and older (4.8 (3.3–6.8)), which consisted of cholestasis. Vasculitis was also observed for propylthiouracil in children and adolescents, reaching higher ratio values than hepatotoxicity signals. Conclusions: Within the US Food and Drug Administration’s AERS, the authors detected higher-thanexpected reporting of severe hepatotoxicity and vasculitis in children and adolescents with propylthiouracil but not with methimazole.

This detailed paper reinforces the preliminary message of last year’s letter of the authors in the New England Journal of Medicine that propylthiouracil should not be used anymore in children with Graves’ disease [1]. By use of a data mining algorithm of more than four decades of safety data of the FDA’s AERS, they identified high reporting ratios for severe liver injury and vasculitis in the pediatric and adolescent age group for propiothiouracil vs. methimazole. The prevalence of severe liver failure was estimated to be approximately 1:2,000 children. This statement, derived from those FDA data,

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does not apply to adult patients [2]. In the treatment of pregnant women with active Graves’ disease we are faced with a dilemma: propylthiouracil may appear as the drug of choice as methimazolerelated fetal malformations were reported in some studies but not in all [3–5]. A statement was issued to use propylthiouracil during organogenesis for the first 8 weeks of development and then to switch to methimazole [2]. However, the safety of both drugs in pregnant women as well as their potential teratogenic effects should therefore be under close scrutiny from now on and further data should be gathered before formal recommendations can be issued. This paper also highlighted the utility of systematic analyse adverse events reports in large databases using methods that decrease false-positive rates due to small numbers while preserving stable signals with a small number of reports.

Clinical trials, new treatments Pro and contra for in utero treatment of congenital hypothyroidism

Experience with intra-amniotic thyroxine treatment in non-immune fetal goitrous hypothyroidism in 12 cases Ribault V, Castanet M, Bertrand AM, Guibourdenche J, Vuillard E, Luton D, Polak M, the French Fetal Goiter Study Group Pediatric Endocrinology, Université Paris Descartes, Necker Enfants Malades AP-HP, Paris, France [email protected] J Clin Endocrinol Metab 2009;94:3731–3739 Background: Non-immune fetal goitrous hypothyroidism is a rare condition that can induce obstetrical and/or neonatal complications and neurodevelopmental impairments such as those still seen in some patients with congenital hypothyroidism. Prenatal treatment to prevent these adverse outcomes is appealing, but experience is limited and the risk-to-benefit ratio controversial. Objective: The authors wished to evaluate the feasibility, safety, and effectiveness of intrauterine L-thyroxine treatment, using intra-amniotic injections in a large cohort with non-immune fetal goitrous hypothyroidism. Methods: This study was a retrospective study of 12 prenatally treated fetuses diagnosed between 1991 and 2005 in France. During pregnancy, goiter size and thyroid hormone levels were compared before and after prenatal treatment. At birth, clinical, laboratory, and ultrasound data were evaluated. Results: The practice of the prenatal treatment was found to vary widely in terms of L-thyroxine dosage (200–800 µg/injection), number of injections (1–6), and frequency (every 1–4 weeks). Fortunately, no adverse events were recorded. During pregnancy, thyroid size decreased in 8 of 9 cases and amnioticfluid TSH levels decreased in the 6 investigated cases, returning to normal in 4. However, at birth, all babies had hypothyroidism, indicating that intra-amniotic TSH levels did not reliably reflect fetal thyroid function. Conclusion: The authors confirmed the feasibility and safety of intra-amniotic L-thyroxine treatment for non-immune fetal goitrous hypothyroidism. They also could show that amniocentesis seemed inadequate for monitoring fetal thyroid function in comparison with fetal blood sampling. However, further studies are needed to determine the optimal management of this disorder.

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Non-immune goiter and hypothyroidism in a 19-week fetus: a plea for conservative treatment Stoppa-Vaucher S, Francoeur D, Grignon A, Alos N, Pohlenz J, Hermanns P, Van Vliet G, Deladoëy J Endocrinology Service and Research Center and Department of Pediatrics, CHU Sainte-Justine and Université de Montréal, Montréal, Que., Canada [email protected] J Pediatr 2010;156:1026–1029

Hypothyroidism was documented by cordocentesis at 19 weeks in a fetus with non-immune goiter. Intra-amniotic thyroxine was injected at 25 weeks when amniotic fluid volume increased. Psychomotor outcome was normal. The authors argue that intra-amniotic thyroxine should not be used to treat the hypothyroidism but only to correct the development of polyhydramnios. The diagnosis of fetal thyroid function disturbances is now possible in utero [6–8]. The feasibility of intrauterine treatment of fetuses affected by hypothyroidism has been shown. The incompletely solved question is when should we apply those new tools? While the first paper reports a multicenter retrospective experience with in utero treatment, the second paper reports a case with a review of the literature. Fetal goiter size decreased in 8 of 9 investigated cases and intra-amniotic TSH levels decreased. Some authors propose to use intra-amniotic thyroxine treatment only in case of fetal goiter that may impede vaginal delivery or result in the development of polyhydramnios, others would favor an approach using research protocols to target fetal hypothyroidism treatment itself [7, 8]. The authors rightly conclude that ‘to determine the indications and optimal modalities of the prenatal treatment of non-immune fetal goitrous hypothyroidism, larger and well-designed studies are needed and would be best conducted via international cooperation of multidisciplinary medical teams’.

Important for clinical practice Everlasting questions on congenital hypothyrodism screening

Newborn screening results in children with central hypothyroidism Nebesio TD, McKenna MP, Nabhan ZM, Eugster EA Indiana University School of Medicine, Department of Pediatrics, Riley Hospital for Children, Indianapolis, Ind., USA [email protected] J Pediatr 2010;156:990–993 Background: The authors wished to investigate newborn screening results in children with congenital hypopituitarism, due to central hypothyroidism in a screening program that uses T4 determination. They also wished whether there were differences between children who had abnormal results and children with normal newborn screening results, all with central hypothyroidism. Methods: Medical records of children with central hypothyroidism observed in their pediatric endocrinology clinics from 1990 to 2006 were reviewed. Results: 42 subjects (22 boys) were identified. 8 children (19%) had a low total thyroxine level (6 mU/l throughout the study period (1 April 2005 to 1 March 2007). All term infants (>35 weeks) whose first TSH was 6–20 mU/l had a second TSH measured. The biochemical details of infants with a TSH between 6.1 and 10.0 mU/l and then >6 mU/l on second sampling were sent to pediatric endocrinologists to determine approaches to management. Results: 148 of 65,446 infants (0.23%) had a first blood spot TSH >6.0 mU/l. 120 were term infants with 67 of these (0.1% of all infants tested) having a TSH between 6.1 and 10.0 mU/l and 53 a TSH >10.0 mU/l. Of the 67 term infants with a TSH between 6.1 and 10.0 mU/l on initial testing, 4 continued to have a TSH >6 mU/l. One with a TSH >10 mU/l and 1 infant with a TSH T (p.R165W) and c.786_787del2 (p.L263fs) in infants with closely similar severe interstitial lung disease (ILD), hypotonia, and congenital hypothyroidism. Functional analyses using A549 and HeLa cells revealed that NKX2-1-p.L263fs induced neither SFTPB nor SFTPC promoter activation and had a dominant negative effect on wild-type (WT) NKX2-1. In contrast, NKX2-1-p.R165W activated SFTPC, to a significantly greater extent than did WTNKX2-1, while SFTPB activation was only significantly reduced in HeLa cells. In accordance with their in vitro data, the authors found decreased amounts of SP-B and SP-C by Western blot in bronchoalveolar lavage fluid (patient with p.L263fs) and features of altered surfactant protein metabolism on lung histology (patient with NKX2-1-p. R165W). Conclusion: ILD in patients with NKX2-1 mutations was associated with altered surfactant protein metabolism, and both gain and loss of function of the mutated NKX2-1 genes on surfactant protein promoters were associated with ILD in ‘brain-lung-thyroid syndrome’. In contrast, in the thyroid both mutations led to loss of function.

Since the first descriptions of NKX2-1/TITF1 (NK2 homeobox 1/thyroid transcription factor 1) mutations leading to the clinical triad of congenital hypothyroidism, surfactant deficiency syndrome and benign hereditary chorea, also called ‘brain-lung-thyroid syndrome’ [13], the lung disease remained the ‘white spot’ of the disease. The data presented showed for the first time in vivo and in vitro that patients with NKX2-1 mutations with lung disease suffered from disturbed surfactant protein metabolism. This study further highlighted the variability of the transactivation capacity of mutated NKX21, as both gain and loss of function on surfactant protein promoters were associated with interstitial lung disease, probably due to altered interaction of transcriptional activators and inhibitors. The data of the paper are consistent with experimental evidence documenting the importance of NKX2-1 in pulmonary surfactant metabolism. For the neonatologists and pneumologists it is of importance to search for NKX2-1 mutations in newborns presenting neonatal surfactant deficiency syndrome with mild hyperthyrotropinemia. Whether isolated lung disease due to NKX2-1 mutations in analogy with

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isolated hereditary benign chorea exists and whether NKX2-1 mutations should be screened in such patients after exclusion of mutations of surfactant B and C remains to be shown.

Compound heterozygosity for a novel hemizygous missense mutation and a partial deletion affecting the catalytic core of the H2O2-generating enzyme DUOX2 associated with transient congenital hypothyroidism Hoste C, Rigutto S, Van Vliet G, Miot F, De Deken X IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium [email protected] Hum Mutat 2010;31:E1304–1319 Background: Dual oxidases (DUOX) 1 and 2 are components of the thyroid H2O2-generating system. H2O2 is used by thyroperoxidase to oxidize iodide for thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in transient and permanent congenital thyroid dyshormonogenesis. Results: The authors report here a novel genetic defect causing congenital hypothyroidism in a FrenchCanadian patient. At neonatal screening, the patient had high TSH and low total T4 levels. 99mTc scan showed a normally shaped orthotopic but mildly enlarged thyroid gland, suggesting dyshormonogenesis. Thyroxine treatment was given from 1 month to 17 years, after which it was stopped for re-evaluation and the patient remained euthyroid. The transient congenital hypothyroidism phenotype prompted the authors to screen for mutations in DUOX2 and DUOXA2 genes using the PCR-amplified direct sequencing method. They found complete inactivation of DUOX2 caused by a partial genomic deletion of one allele inherited from the mother associated with a paternally inherited missense mutation (c.4552G>A, p.Gly1518Ser). The deleted fragment encompasses the entire COOH-terminal end which is responsible for the NADPH-oxidase activity. The Gly1518Ser DUOX2 protein is expressed at the cell surface of transfected cells albeit at low level, but it is non-functional. Conclusion: This study provides further evidence that the permanent or transient nature of congenital hypothyroidism is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors.

The biochemical requirement of H2O2 for thyroid hormone synthesis has been known for decades. Two homologous proteins (dual oxidase 1 and 2) containing functional domains of NADPH oxidoreductases were identified in thyroid follicular cells. Mutations in the dual oxidase 2 gene (DUOX2, formerly THOX2) gene have been shown to cause transient and permanent dyshormonogenetic congenital hypothyroidism. So far, transient forms were associated with heterozygous mutations, and permanent forms with homozygous mutations in DUOX2 [14]. This interesting case, reporting a patient with transient congenital hypothyroidism and compound heterozygosity for a novel hemizygous missense mutation and a partial deletion of DUOX2, raises the question whether transiency and permanency of congenital hypothyroidism relate exclusively to monoallelic or biallelic inactivation of the gene or whether disease phenotype is dependent on residual function of the mutated DUOX2 protein. Further work on more patients affected with DUOX2 gene mutations is awaited to clarify this fundamental question.

Screening chromosomal aberrations by array comparative genomic hybridization in 80 patients with congenital hypothyroidism and thyroid dysgenesis Thorwarth A, Mueller I, Biebermann H, Ropers HH, Grueters A, Krude H, Ullmann R Institute for Experimental Pediatric Endocrinology, Charité University Medicine and Max Planck Institute for Molecular Genetics, Berlin, Germany [email protected], [email protected] J Clin Endocrinol Metab 2010, Epub ahead of print Background: A spectrum of defective thyroid morphology, termed thyroid dysgenesis (TD), represents 80% of permanent congenital hypothyroidism cases. Although several candidate genes have been implicated in thyroid development, comprehensive screens failed to detect mutation carriers in a significant number of patients with non-syndromic TD.

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Objective: Due to the sporadic occurrence of TD, the authors aimed at assessing de novo chromosomal rearrangements which conceivably represents one of the molecular mechanisms participating in its etiology. Methods: The introduction of array comparative genomic hybridization (CGH) has provided the ability to map DNA copy number variations (CNVs) genome-wide with high resolution. The authors performed an array CGH screen of 80 TD patients to determine the role of CNVs in the etiology of the disease. Results: They identified novel CNVs that have not been described as frequent variations in the healthy population in 8.75% of all patients. These CNVs exclusively affected patients with athyreosis or thyroid hypoplasia and were non-recurrent, and the regions flanking the CNVs were not enriched for segmental duplications. Conclusions: The high rate of chromosomal changes in TD argues for an involvement of CNVs in the etiology of this disease. Yet the lack of recurrent aberrations suggests that the genetic causes of TD are heterogenous and not restricted to specific genomic hot spots. Thus, future studies may have to shift the focus from singling out specific genes to the identification of deregulated pathways as the underlying cause of the disease. As in others types of endocrine disorders as well as in others fields of medicine, the study of DNA CNVs may turned out to be productive to find the etiologies of TD [15].

New methods for microarray-based genotyping of single nucleotide polymorphism (SNP) markers as well as parallel methods assessing rare and common DNA deletions or duplications across the genome as copy number variants (CNVs) permitted breakthrough identification of susceptibility genes for nonmendelian diseases [16, 17]. A recent large-scale analysis of CNVs in eight common human diseases puts the contribution of CNVs in comparison with SNP-based studies for detection of new genetic hot spots into perspective [18; also see Science and Medicine section by Z. Hochberg and J.-C. Carel, pp. 219–236]. The study of Thorwarth et al. searched for the first time for common chromosomal changes in a representative cohort of patients with thyroid dysgenesis. Their results add to current knowledge the fact that a high rate of chromosomal changes was found in these patients. However, the absence of recurrent CNVs further supports the notion that thyroid dysgenesis is rather a heterogenous disease than a monogenetic one. Future approaches aiming to decipher the genetic basis of thyroid dysgenesis should consider this new piece of knowledge.

Environment

Iodine supplementation improves cognition in mildly iodine-deficient children Gordon RC, Rose MC, Skeaff SA, Gray AR, Morgan KM, Ruffman T Department of Human Nutrition, University of Otago, Dunedin, New Zealand [email protected] Am J Clin Nutr 2009;90:1264–1271 Background: The effects of severe iodine deficiency during critical periods of brain development are well documented. Little is known about the consequences of milder forms of iodine deficiency on neurodevelopment. Objective: The authors aimed to determine whether supplementing mildly iodine-deficient children with iodine improves cognition. Methods: A randomized, placebo-controlled, double-blind trial was conducted in 184 children aged 10–13 years in Dunedin, New Zealand. Children were randomly assigned to receive a daily tablet containing either 150 µg iodine or placebo for 28 weeks. Biochemical, anthropometric, and dietary data were collected from each child at baseline and after 28 weeks. Cognitive performance was assessed through 4 subtests from the Wechsler Intelligence Scale for Children. Results: At baseline, children were mildly iodine-deficient (median urinary iodine concentration (UIC): 63 µg/l; thyroglobulin concentration: 16.4 µg/l). After 28 weeks, iodine status improved in the supplemented group (UIC: 145 µg/l; thyroglobulin: 8.5 µg/l), whereas the placebo group remained iodine-

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deficient (UIC: 81 µg/l; thyroglobulin: 11.6 µg/l). Iodine supplementation significantly improved scores for 2 of the 4 cognitive subtests (picture concepts (p = 0.023) and matrix reasoning (p = 0.040)) but not for letter-number sequencing (p = 0.480) or symbol search (p = 0.608). The overall cognitive score of the iodine-supplemented group was 0.19 SD higher than that of the placebo group (p = 0.011). Conclusion: Iodine supplementation improved perceptual reasoning in mildly iodine-deficient children and suggests that mild iodine deficiency could prevent children from attaining their full intellectual potential. The elimination of iodine deficiency by the year 2005 was a World Fit for Children target, yet a large proportion of children worldwide still have inadequate iodine intakes. Mild iodine deficiency remains the most common thyroid disease worldwide, and an important health issue also in industrialized countries. The impact of iodine supplementation on moderately iodine-deficient children was first shown in an elegant randomized controlled double-blind study by Zimmermann et al. [19]. Iodine treatment significantly improved performance in 4 of 7 cognitive and motor tests. The re-emergence of iodine deficiency in New Zealand is believed to be a consequence of lower concentrations of iodine in milk because of the discontinuation of iodine-containing sanitizers in the dairy industry, declining use of iodized salt, and an increased consumption of processed foods not made with iodized salt. Gordon et al. present us with convincing data on the impact of iodine supplementation of children in a mildly iodine-deficient area. They used a similar study design (duration of supplementation, cognitive testing) at a lower supplementation dose in analogy to the previous paper. The authors show significant association between iodine supplementation and improvement in 2 of 4 cognitive tests relative to placebo. The strength of this paper is to highlight the fact that not only in moderately but even in mildly iodine-deficient areas, iodine supplementation is beneficial for cognitive function in schoolchildren.

Thyroid hormone resistance revisited New pathophysiological insights

Resistance to thyroid hormone is associated with raised energy expenditure, muscle mitochondrial uncoupling, and hyperphagia Mitchell CS, Savage DB, Dufour S, Schoenmakers N, Murgatroyd P, Befroy D, Halsall D, Northcott S, Raymond-Barker P, Curran S, Henning E, Keogh J, Owen P, Lazarus J, Rothman DL, Farooqi IS, Shulman GI, Chatterjee K, Petersen KF University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK [email protected], [email protected] J Clin Invest 2010;120:1345–1354 Background: Resistance to thyroid hormone (RTH), a dominantly inherited disorder usually associated with mutations in thyroid hormone receptor-␤ (THRB), is characterized by elevated levels of circulating thyroid hormones (including thyroxine), failure of feedback suppression of thyrotropin, and variable tissue refractoriness to thyroid hormone action. Raised energy expenditure and hyperphagia are recognized features of hyperthyroidism, but the effects of comparable hyperthyroxinemia in RTH patients are unknown. Results: The authors showed that resting energy expenditure (REE) was substantially increased in adults and children with THRB mutations. Energy intake in RTH subjects was increased by 40%, with marked hyperphagia particularly evident in children. Rates of muscle TCA cycle flux were increased by 75% in adults with RTH, whereas rates of ATP synthesis were unchanged, as determined by 13C/31P magnetic resonance spectroscopy. Mitochondrial coupling index between ATP synthesis and mitochondrial rates of oxidation (as estimated by the ratio of ATP synthesis to TCA cycle flux) was significantly decreased in RTH patients. Conclusion: These data demonstrate that basal mitochondrial substrate oxidation is increased and energy production in the form of ATP synthesis is decreased in the muscle of RTH patients and that resting

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oxidative phosphorylation is uncoupled in this disorder. Furthermore, these observations suggest that mitochondrial uncoupling in skeletal muscle is a major contributor to increased REE in patients with RTH, due to tissue selective retention of THRB sensitivity to elevated thyroid hormone levels. This paper reports on extensive studies of energy balance in patients with thyroid hormone resistance (RTH), providing general insight into thyroid hormone-dependent energy metabolism in humans. RTH is characterized by a variable degree of tissue hyposensitivity to thyroid hormones usually associated with mutations in the thyroid hormone receptor-␤ (THRB) [20]. The authors studied an unselected group of 54 adults and 13 children from 35 unrelated families harboring 25 different THRB gene mutations. First, the authors documented significantly increased resting energy expenditure and basal metabolic rate in patients with RHT, using both ventilated hood and chamber calorimetry, respectively. The raised energy expenditure was positively correlated with heart rate and thyroid hormone levels being intermediate in RTH patients compared to euthyroid control and thyrotoxic subjects. Second, they analyzed food intake, measured by an ad libitum test meal and found a 75% increase in RTH children compared to controls, reaching levels present in hyperphagic patients with monogenetic obesity syndromes. Third, by investigating the role of skeletal muscle mitochondrial energy metabolism, they revealed a 75% increase in substrate oxidation but an identical level of ATP synthesis in the muscle of RHT patients compared to controls, suggesting that resting mitochondrial uncoupling in skeletal muscle is a major contributor to increased resting energy expenditure in patients with RTH. In conclusion, the authors suggest that thyroid hormone receptor-␣-rich organs such as skeletal muscle and myocardium are the main determinants of thyroid hormone-mediated changes in the whole body expenditure in humans.

New hope?

A thyroid hormone analog with reduced dependence on the monocarboxylate transporter 8 for tissue transport Di Cosmo C, Liao XH, Dumitrescu AM, Weiss RE, Refetoff S Department of Medicine, University of Chicago, Chicago, Ill., USA [email protected] Endocrinology 2009;150:4450–4458 Background: Mutations of the thyroid hormone (TH) cell membrane transporter MCT8, on chromosomeX, produce severe mental and neurological impairment in men. Methods: The authors generated a Mct8-deficient mouse (Mct8KO) manifesting the human thyroid phenotype. Although these mice have no neurological manifestations, they have decreased brain T3 content and high deiodinase 2 (D2) activity, reflecting TH deprivation. In contrast and as in serum, liver T3 content is high, resulting in increased deiodinase 1 (D1), suggesting that in this tissue TH entry is Mct8 independent. They tested the effect of 3,5-diiodothyropropionic acid (DITPA), a TH receptor agonist, for its dependence on Mct8 in Mct8KO and wild-type (Wt) mice tissues. After depletion of endogenous TH, mice were given three different doses of DITPA. Effects were compared with treatment with two doses of L-T4. Results: As expected, physiological doses of L-T4 normalized serum TSH, brain D2, and liver D1 in Wt mice but not the Mct8KO mice. The higher dose of T4 suppressed TSH in the Wt mice, normalized TSH and brain D2 in Mct8KO mice, but produced a thyrotoxic effect on liver D1 in both genotypes. In contrast, DITPA produced similar effects on TSH, D2, and D1 in both Wt and Mct8KO mice. The higher dose fully normalized all measurements and other parameters of TH action. Conclusions: DITPA is relatively MCT8 independent for entry into the brain and corrects the TH deficit in Mct8KO mice without causing thyrotoxic effect in liver. The potential clinical utility of this analog to patients with MCT8 mutations requires further studies.

The intracellular hypothyroidism in the brain due to the abolished activity of the thyroid hormone transporter MCT8 cannot be corrected by physiological or supraphysiological substitutive doses of

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thyroid hormones in patients with MTC8 mutations. In this context, the question, whether thyroid hormone analogues, such as the 3,5-diiodothyropropionic acid (DITPA), could be less dependent on the active transport by MTC8 for their passage from the blood to the central neurons, would be of great therapeutic importance. Di Cosmo et al. show that the transport of DITPA into the brain and liver is less dependent on MTC8. Further, they provide evidence that DITPA acts in brain without producing thyrotoxic effects in peripheral tissues, such as the liver. Although, these results are an important step forward to a clinical study of this pharmacological compound in patients with MTC8 mutations, relevant species-specific differences have to be considered. As the MTC8 knockout mouse model does not display a neurological phenotype like MTC8-deficient humans, the potential effect on the severe human neurological phenotype remains to be proven.

New concerns?

Glucagon-like peptide-1 receptor agonists activate rodent thyroid C-cells causing calcitonin release and C-cell proliferation Bjerre Knudsen L, Madsen LW, Andersen S, Almholt K, de Boer AS, Drucker DJ, Gotfredsen C, Egerod FL, Hegelund AC, Jacobsen H, Jacobsen SD, Moses AC, Mølck AM, Nielsen HS, Nowak J, Solberg H, Thi TD, Zdravkovic M Department of Biology and Pharmacology Mgt, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark [email protected] Endocrinology 2010;151:1473–1486 Background: Liraglutide is a glucagon-like peptide-1 (GLP-1) analog developed for type 2 diabetes. Longterm liraglutide exposure in rodents was associated with thyroid C-cell hyperplasia and tumors. Results: The GLP-1 receptor was localized to rodent C-cells. GLP-1 receptor agonists stimulated calcitonin release, up-regulation of calcitonin gene expression, and subsequently C-cell hyperplasia in rats and, to a lesser extent, in mice. In contrast, humans and/or cynomolgus monkeys had low GLP-1 receptor expression in thyroid C-cells, and GLP-1 receptor agonists did not activate adenylate cyclase or generate calcitonin release in primates. Moreover, 20 months of liraglutide treatment (at >60 times human exposure levels) did not lead to C-cell hyperplasia in monkeys. Mean calcitonin levels in patients exposed to liraglutide for 2 years remained at the lower end of the normal range, and there was no difference in the proportion of patients with calcitonin levels increasing above the clinically relevant cutoff level of 20 pg/ml. Conclusion: The findings delineate important species-specific differences in GLP-1 receptor expression and action in the thyroid. Indeed the data support a GLP-1 receptor-mediated mechanism for these changes in rodents. Nevertheless, the long-term consequences of sustained GLP-1 receptor activation in the human thyroid remain unknown and merit further investigation.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that promotes glucose-dependent stimulation of insulin secretion. GLP-1 receptor agonists with prolonged half-life have been developed for the treatment of type 2 diabetes. The FDA approved liraglutide for treatment of type 2 diabetes in January 2010. One major safety concern was the potential deleterious effect of liraglutide on thyroid C-cells, as the long-term exposure was associated with thyroid C-cell hyperplasia and tumors in rodents. In this context, the authors combined short- and long-term animal and human pharmacological studies to show relevant species-dependent differences of GLP-1 receptor expression as well as calcitonin release to liraglutide between rodents (rat and mouse) and humans and primates. Two-year highdose exposure studies confirmed C-cell hyperplasia and development of C-cell adenoma in rodents. In contrast, no C-cell hyperplasia was observed in primates during and after 87 weeks of treatment and basal plasma calcitonin levels were not increased in patients with type 2 diabetes receiving liraglutide in three different doses compared to placebo at repeated measurements during 2 years. In conclusion, thyroid C-cells in rats and mice differ markedly from primate and human C-cells in their response

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to GLP-1 receptor activation supporting the arguments of the FDA for approval of the drug. Longterm safety monitoring is nevertheless required by the FDA by monitoring the annual incidence of medullary thyroid cancer of over the next 15 years. For the complete discussion of the safety issue of liraglutide by the FDA, the interested reader is referred to the article by Parks and Rosebraugh [21]. References 1. Rivkees SA, Mattison DR: Ending propylthiouracil-induced liver failure in children. N Engl J Med 2009;360:1574– 1575. 2. Bahn RS, Burch HS, Cooper DS, et al: The role of propylthiouracil in the management of Graves’ disease in adults: report of a meeting jointly sponsored by the American Thyroid Association and the Food and Drug Administration. Thyroid 2009;19:673–674. 3. Clementi M, Di Gianantonio E, Pelo E, Mammi I, Basile RT, Tenconi R: Methimazole embryopathy: delineation of the phenotype. Am J Med Genet 1999;83:43–46. 4. Karlsson FA, Axelsson O, Melhus H: Severe embryopathy and exposure to methimazole in early pregnancy. J Clin Endocrinol Metab 2002;87:947–949. 5. Valdez RM, Barbero PM, Liascovich RC, De Rosa LF, Aguirre MA, Alba LG: Methimazole embryopathy: a contribution to defining the phenotype. Reprod Toxicol 2007;23:253–255. 6. Luton D, Le Gac I, Vuillard E, et al: Management of Graves’ disease during pregnancy: the key role of fetal thyroid gland monitoring. J Clin Endocrinol Metab 2005;90:6093–6098. 7. Van Vliet G, Polak M, Ritzen EM: Treating fetal thyroid and adrenal disorders through the mother. Nat Clin Pract Endocrinol Metab 2008;4:675–682. 8. Polak M, Van Vliet G: Therapeutic approach of fetal thyroid disorders. Horm Res Paediatr 2010;74:1–5. 9. Baloch Z, Carayon P, Conte-Devolx B, et al: Laboratory medicine practice guidelines. Laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid 2003;13:3–126. 10. Hanna CE, Krainz PL, Skeels MR, Miyahira RS, Sesser DE, LaFranchi SH: Detection of congenital hypopituitary hypothyroidism: ten-year experience in the Northwest Regional Screening Program. J Pediatr 1986;109:959–964. 11. Asakura Y, Tachibana K, Adachi M, Suwa S, Yamagami Y: Hypothalamo-pituitary hypothyroidism detected by neonatal screening for congenital hypothyroidism using measurement of thyroid-stimulating hormone and thyroxine. Acta Paediatr 2002;91:172–177. 12. Schlumberger M, Ricard M, De Pouvourville G, Pacini F: How the availability of recombinant human TSH has changed the management of patients who have thyroid cancer. Nat Clin Pract Endocrinol Metab 2007;3:641–650. 13. Krude H, Schutz B, Biebermann H, et al: Choreoathetosis, hypothyroidism, and pulmonary alterations due to human NKX2-1 haploinsufficiency. J Clin Invest 2002;109:475–480. 14. Moreno JC, Bikker H, Kempers MJ, et al: Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002;347:95–102. 15. Ku CS, Loy EY, Salim A, Pawitan Y, Chia KS: The discovery of human genetic variations and their use as disease markers: past, present and future. J Hum Genet 2010 (in press). 16. Manolio TA, Brooks LD, Collins FS: A HapMap harvest of insights into the genetics of common disease. J Clin Invest 2008;118:1590–1605. 17. Zhang F, Gu W, Hurles ME, Lupski JR: Copy number variation in human health, disease, and evolution. Annu Rev Genomics Hum Genet 2009;10:451–481. 18. Craddock N, Hurles ME, Cardin N, et al: Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. Nature 2010;464:713–720. 19. Zimmermann MB, Connolly K, Bozo M, Bridson J, Rohner F, Grimci L: Iodine supplementation improves cognition in iodine-deficient schoolchildren in Albania: a randomized, controlled, double-blind study. Am J Clin Nutr 2006;83:108– 114. 20. Weiss RE, Refetoff S: Resistance to thyroid hormone. Rev Endocr Metab Disord 2000;1:97–108. 21. Parks M, Rosebraugh C: Weighing risks and benefits of liraglutide – the FDA’s review of a new antidiabetic therapy. N Engl J Med 2010;362:774–777.

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Growth and Growth Factors Stefano Cianfarani Molecular Endocrinology Unit – DPUO Bambino Gesù Children’s Hospital – ‘Rina Balducci’ Center of Pediatric Endocrinology, Tor Vergata University, Rome, Italy

This collection of articles aims to provide a wide spectrum of studies on the subject of growth and growth factors, particularly focusing on those with direct or potential clinical implications. Of course, given the limited number of papers to be selected, the choice of articles has been fully arbitrary and, inevitably, other equally valuable studies have been neglected. Among experimental studies, I favored those with major clinical implications. In the last 12 months, the research in the field of growth factors has provided important achievements. Molecular biology, applied to single clinical cases or large cohorts of patients, has elucidated the mechanisms underlying some conditions characterized by severe short stature. New therapeutic tools for treating growth impairment have been tested in phase 1 and 2 clinical trials. New relationships between the IGF system and predisposition to cardiovascular disease have been described. Finally, pharmacological modulation of IGF system has been attempted to inhibit cancer growth. Although most of these papers have not yet yielded conclusive results, they nonetheless provide the basis for further clinical and experimental research. Wishing you a good reading, I would like to wrap up this short introduction quoting a sentence written on the office door of James Muorilyan Tanner, the founder of modern auxology: ‘the appetite comes reading’.

Important for clinical practice

Efficacy and safety of long-term continuous growth hormone treatment in children with Prader-Willi syndrome de Lind van Wijngaarden RF, Siemensma EP, Festen DA, Otten BJ, van Mil EG, Rotteveel J, Odink RJ, Bindels-de Heus GC, van Leeuwen M, Haring DA, Bocca G, Houdijk EC, Hoorweg-Nijman JJ, Vreuls RC, Jira PE, van Trotsenburg AS, Bakker B, Schroor EJ, Pilon JW, Wit JM, Drop SL, Hokken-Koelega AC Dutch Growth Research Foundation, Erasmus University Medical Center/Sophia Children’s Hospital, Rotterdam, The Netherlands [email protected] J Clin Endocrinol Metab 2009;94:4205–4215 Background: Prader-Willi syndrome (PWS) is a rare, complex disorder characterized by failure to thrive, early obesity starting during the second year of life, hypotonia, short stature, other endocrine dysfunctions, learning disabilities, abnormal behavior, and psychiatric problems. Hypothalamic dysfunction may account for many features of the syndrome. GH insufficiency, reduced IGF-I levels and good response to GH therapy have been described in children with PWS. The long-term efficacy of GH treatment on adult height and body composition of patients with PWS has not yet been established. The purpose of this multicenter prospective study was to test efficacy and safety of GH therapy during a 4-year follow-up. Methods: 55 prepubertal children (with a mean ± SD age of 5.9 ± 3.2 years) with PWS were recruited in the trial with GH therapy and followed for 4 consecutive years. All children had a genetically confirmed diagnosis of PWS by positive methylation test and were naive to GH treatment at the time of inclusion. Children were treated with rhGH 1 mg/m2 daily. Body composition was evaluated by dualenergy x-ray absorptiometry. Results: Fat body mass decreased only during the first year of therapy, and remained higher than +2 SDS after 4 years of therapy. Lean body mass transiently increased during therapy, remaining lower than –2 SDS after 4 years. Height and head circumference significantly increased during the first 3 years of therapy achieving a value not significantly different from 0 SDS. Body proportions expressed by the

sitting height to height ratio improved during treatment and did not significantly differ from 0 SDS after 4 years. Hand and foot length as well as arm span did not normalize during treatment. GH therapy had no significant effect on bone maturation. GH significantly increased IGF-I up to more than +2 SDS with peak after the first year of therapy. IGFBP-3 also increased but to a less extent ultimately leading to a marked increase of the IGF-I/IGFBP-3 molar ratio, which might indicate that more free IGF-I was present in the systemic circulation. Glucose and insulin levels remained unchanged during GH therapy whereas LDL cholesterol decreased significantly. Conclusions: GH long-term therapy proved to be safe and effective in improving height, body composition, head circumference and lipid profile in children with PWS. This study substantially confirms previous reports showing the effects of GH therapy on a series of features typically associated with PWS, such as short stature, alterations of body composition and microcephaly. In addition, no major side effect was observed during the 4-year follow-up in this large cohort of patients. However, since 2002 [1], a significant number of deaths in children with PWS treated with GH have been reported [2], especially during the first 6–12 months of therapy. The major cause of death in PWS children is respiratory failure [3]. Although there is no direct evidence for a causative role of GH therapy, the safety of GH treatment in this high-risk population has been questioned and warnings have been added for the use of rhGH. Although there is no clear evidence of a connection between GH treatment and risk of death, and the results of this study look reassuring, the finding of the sharp increase of IGF-I even above the upper normal range raises concern. IGF-I is a potent stimulator of lymphoid tissue growth and the excessive and rapid increase in IGF-I at the start of GH treatment could induce tonsillar and adenoid hypertrophy which may concur in increasing the risk of sleep-obstructive apnea. Therefore, further research on longer-term effects of high IGF-I levels is warranted.

Serum insulin-like growth factor-binding protein-2 levels and metabolic and cardiovascular risk factors in young adults and children born small for gestational age de Kort SW, van Doorn J, van de Sande AG, Leunissen RW, Hokken-Koelega AC Erasmus MC Sophia, Rotterdam, The Netherlands [email protected] J Clin Endocrinol Metab 2010;95:864–871 Background: Subjects born small for gestational age (SGA) are at risk of developing insulin resistance, type 2 diabetes, metabolic syndrome and cardiovascular disease in adulthood. IGF-binding protein 2 (IGFBP2) belongs to the family of 6 IGFBPs which bind IGF-I and IGF-II. IGFBP-2 has been reported to modulate intracellular insulin signaling, and IGFBP-2 levels are inversely related to insulin concentrations and insulin resistance. The aim of this study was to assess IGFBP-2 levels in a cohort of young adults and children born SGA. Methods: 151 young adults and 147 children born SGA were studied. Subjects underwent anthropometry, blood lipid profile assessment and blood pressure evaluation. Body composition was assessed by dual-energy x-ray absorptiometry. A modified, frequently sampled intravenous glucose tolerance test with tolbutamide was performed in a subgroup of study subjects. Results: SGA young adults showed reduced concentrations of IGFBP-2 independently of catch-up growth in height. Whereas IGFBP-2 did not correlate with birth size, it was inversely related to fat mass. Furthermore, in this group, IGFBP-2 correlated negatively with BMI, fat mass, blood pressure, fasting insulin, HOMA-IR, insulin secretion, cholesterol and triglycerides. On the other hand, IGFBP-2 correlated positively with insulin sensitivity. After adjustment for fat mass, the relationships with metabolic markers disappeared, thus suggesting that IGFBP-2 levels associate with FM and that a lower FM is associated with lower insulin levels and less insulin resistance. No correlation between IGFBP-2 and cardiovascular risk markers was seen in SGA children. Conclusion: In SGA young adults, IGFBP-2 may represent an indicator of the cardiovascular risk.

Concentrations of IGFBP-2, binding both IGF-I and IGF-II, increase after a prolonged period of fasting, indicating that IGFBP-2 concentrations are metabolically regulated. Evidence suggests that IGFBP-2 may alter the activity of intracellular kinases that regulate insulin signaling by both IGF-

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dependent and IGF-independent mechanisms, thereby modulating insulin sensitivity. Furthermore, studies in adult patients have shown a relationship between IGFBP-2 and cardiovascular risk factors. This study represents the first report on the association between IGFBP-2 levels and markers of cardiovascular risk in normal height and short SGA adults, and only in short SGA children. Although the results are in adults, and based on correlation analyses, the findings are suggestive for a potential use of IGFBP-2 in quantifying the metabolic risk in this population. However, it has to be pointed out that the whole set of independent variables (including IGFBP-2) only explained 20% of variance. It is noteworthy that such relationships were not present in SGA children. The search for a reliable marker of metabolic risk in children and adolescents with low birth weight looks worthwhile, but, unfortunately, no indicator has so far been identified. On the other hand, it may be argued that insulin sensitivity is mildly impaired in children born small for gestational age [4]. Efforts to evaluate metabolic risk are more meaningful in young adulthood but early predictors would be welcome.

Impact of growth hormone therapy on adult height of children born small for gestational age Maiorana A, Cianfarani S ‘Rina Balducci’ Center of Pediatric Endocrinology, Department of Public Health and Cell Biology, Tor Vergata University, Rome, Italy Pediatrics 2009;3:E519–E531 Background: The indication for growth hormone (GH) therapy in children born small for gestational age (SGA) has been approved worldwide. Although the primary outcome measure for evaluating the efficacy of such therapy is adult height, most trials have reported short-term results only. In addition, the quality of most studies is affected by the recruitment of small study cohorts, and, often, the use of historical controls. The aim of this systematic review was to examine the evidence that long-term randomized controlled trials (RCTs) of GH treatment in children born SGA may improve adult height. Methods: A meta-analysis of all RCTs conducted up to the achievement of adult height and published until November 2008 was performed. Children with birth weight and/or length below –2 SD score (SDS) and pre-therapy height less than –2 SDS treated with two dose regimens (33–67 µg/kg per day) met the inclusion criteria. Adult height and total height gain expressed in SDS were considered as the primary outcome measures. The quality of trials and strength of recommendation were assessed using the Endocrine Society grade recommendations [5]. Results: Four RCTs were identified, including a total of 270 treated and 155 untreated children (controls). The mean differences in final height and overall height again between treated and untreated subjects were 0.85 SDS (approx. 6 cm) and 1.25 SDS (approx. 8 cm) respectively. No significant difference in adult height between the two dose regimens was observed. The positive predictive factors for GH efficacy were younger age and number of prepubertal years of therapy. Moderate-quality evidence was the score assigned to all four RCTs, whereas strong recommendation was assigned to 2/4 trials. Conclusions: Despite the approval of GH therapy for improving adult height of short children born SGA, no single, long-term, randomized, controlled (in parallel), well-powered study conducted up to the achievement of adult height has been published so far. On average, GH therapy has been shown to be effective in reducing the adult height deficit, although the meta-analysis shows a wide individual variability in the response. Finally, there is no evidence for supporting the use of doses higher than 33 µg/kg per day.

This systematic review has important clinical practice implications representing a comprehensive meta-analysis appraising the effect of long-term GH therapy on adult height of short children born SGA. Although international and national drug agencies have approved the use of GH in this condition, this report re-opens the debate on the cost/benefit ratio and, more importantly, provides the evidence-based achievements that can be reasonably expected by children, parents and physicians. SGA children can expect to gain between 6 and 8 cm from years’ long daily injections of GH. Although GH seems effective in improving adult height, the magnitude of the growth-promoting effect is relatively low. In addition, the individual variability in the response to GH therapy should prompt further investigations aimed at identifying those who can substantially benefit from a long-term treatment.

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New paradigms Multiple forms of IGF-1 receptor haploinsufficiency

Two short children born small for gestational age with insulin-like growth factor 1 receptor haploinsufficiency illustrate the heterogeneity of its phenotype Ester WA, van Duyvenvoorde HA, de Wit CC, Broekman AJ, Ruivenkamp CA, Govaerts LC, Wit JM, Hokken-Koelega AC, Losekoot M Department of Pediatrics, Subdivision of Endocrinology, Erasmus Medical Center Sophia Children’s Hospital, Rotterdam, The Netherlands [email protected] J Clin Endocrinol Metab 2009;94:4717–4727 Background: Children born small for gestational age (SGA) represent a widely heterogeneous population. Genetic factors certainly play a role in determining birth size and postnatal growth, to date, however, only a few genetic alterations have been associated with intrauterine growth retardation. This study investigated copy number variations in 18 growth-related genes in short children born SGA. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to test whether copy number variations in growth-related genes (SHOX, GH1, GHR, IGF1, IGF1R, IGF2, IGFBP1–6, NSD1, GRB10, STAT5B, ALS, SOCS2, and SOCS3) were present in a cohort of 100 children born SGA with persistent short stature. In 2 subjects a deletion of the IGF1R gene was identified. The extent of the two deletions was determined with single-nucleotide polymorphism (SNP) array analysis. Finally, functional studies on dermal fibroblasts were performed to investigate the IGF1R signal transduction pathway. Results: Two patients with heterozygous de novo deletions of the insulin-like growth factor 1 receptor (IGF1R) gene were identified. Both subjects showed reduced birth length, dysmorphic features including proximal implanted thumbs, hearing problems and good response to growth hormone (GH) therapy. Patient A also showed delayed psychomotor development, whereas patient B had attention-deficit hyperactivity disorder. Unexpectedly, IGF-I circulating levels were low in patient A, probably due to partial GH deficiency. Conclusion: IGF1R haploinsufficiency may be suspected in children born SGA with short stature, dysmorphic features and developmental delay, independently of GH responses to provocative tests and IGF-I levels. Interestingly, these patients seem to respond to GH therapy.

Familial short stature caused by haploinsufficiency of the insulin-like growth factor I receptor due to nonsense-mediated messenger ribonucleic acid decay Fang P, Schwartz ID, Johnson BD, Derr MA, Roberts CT, Jr, Hwa V, Rosenfeld RG Department of Pediatrics, Oregon Health and Science University, Portland, Oreg., USA J Clin Endocrinol Metab 2009;94:1740–1747 Background: To date, only few cases of IGF-I receptor heterozygous mutations have been described in humans. These patients are characterized by severe pre- and postnatal growth retardation, microcephaly and mental retardation, a phenotype closely resembling that of subjects with IGF-I gene defects [6–9]. To ensure the accuracy of gene expression, eukaryotes have evolved several surveillance mechanisms. One of the best studied quality control mechanisms is nonsense-mediated mRNA decay (NMD), which recognizes and degrades transcripts harboring a premature translation-termination codon (PTC), thereby preventing the production of faulty proteins. NMD regulates approximately 10% of human mRNAs [10]. In this study, IGF-I receptor gene (IGF1R) was investigated in a family with severe short stature. Case Description and Methods: The proband was a boy born small for gestational age (SGA), referred for short stature (height: –3.6 SDS) and microcephaly. No mental retardation was reported. He was on treatment with methylphenidate for a diagnosis of attention-deficit hyperactivity disorder. Family history revealed severe short stature on the maternal side of the family. GH responses to stimulation tests, IGF-I, IGFBP-3 and GHBP levels were normal. GH therapy did not induce catch-up growth. Primary fibroblast cultures

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were established from skin biopsies taken from the patient and his siblings and parents. IGF1R was sequenced and both mRNA and protein expression was investigated. Results: IGF1R sequencing showed a heterozygous duplication in exon 18 in the proband and other family members with growth failure. This duplication comprised nucleotides encoding part of the tyrosine kinase domain located within the ␤-subunit. This 19Dup mutation in the mutant IGF1R allele led to degradation of the mutant mRNA through the NMD pathway, resulting in haploinsufficiency of the wild-type IGF1R protein. Conclusions: This study describes a novel heterozygous mutation in the IGF1R and indicates, for the first time, that the NMD pathway may play a key role in determining IGF1R haploinsufficiency eventually leading to the development of IGF-I resistance and human growth failure. The study of Ester et al. found IGF1R haploinsufficiency in 2 of 100 short SGA children and confirms that alterations in IGF1R expression may cause, although in a proportion of children born SGA, intrauterine growth retardation, postnatal growth impairment, peculiar phenotype and developmental delay. These findings are consistent with the description by Abuzzahab et al. [6] of 2 children with fetal and postnatal growth failure caused by defects in the IGF-IR gene. Moreover, the 2 patients with IGF1R haploinsufficiency described in this paper showed a good response to GH therapy similarly to another child previously reported by the same authors [11]. This unexpected GH effectiveness in promoting growth despite the partial absence of IGF1R was explained by both a direct effect of GH on the epiphyseal chondrocytes independent of the biological actions of IGFs, and the increased serum IGF-I levels, which may overcome the reduced peripheral sensitivity. However, it has to be pointed out that the study children did not achieve adult height yet, making the final outcome of GH therapy uncertain. Another and probably stronger merit of Ester et al.’s paper is to offer an overview of the phenotypes associated with mutations and deletions of IGF1R gene, thus providing clinical indicators to drive the investigator toward the assessment of this gene. The results open many interesting questions. The expression of the IGF1R was minimally lower in the patient’s cells, and the authors speculate that haploinsufficiency may be cell type-dependent, with possibly a relatively strong effect in growth plate chondrocytes. Low serum IGF1 and short-term response to GH therapy remain to be explained. The second paper by Fang et al. describes a novel heterozygous mutation in the tyrosine kinase domain of the IGF1R in multiple subjects of the same family apparently characterized by ‘familial short stature’. However, the severity of short stature together with intrauterine growth retardation in the proband suggested a condition different from a simple normal variant of growth. In addition, this study has the merit to provide evidence, for the first time, that a mechanism involving nonsense-mediated mRNA decay may cause IGFIR haploinsufficiency and, eventually, pre- and postnatal growth retardation.

New paradigms Is there a relationship between IGF-I and cardiovascular risk?

A significant decline in IGF-I may predispose young Africans to subsequent cardiometabolic vulnerability Schutte AE, Huisman HW, van Rooyen JM, Malan L, Malan NT, Fourie CM, Louw R, van der Westhuizen FH, Schutte R Hypertension in Africa Research Team, School for Physiology, Nutrition, and Consumer Sciences, North-West University (Potchefstroom Campus), South Africa J Clin Endocrinol Metab 2010;95:2503–2507 Background: The age-related decline of serum IGF-I has been associated with the risk of developing diabetes and cardiovascular disease. Although infectious diseases represent the major cause of death in black South Africans, the prevalence of cardiovascular disease is nevertheless high in urban areas. The aims of the study were to measure IGF-I concentrations in African and Caucasian subjects, and to correlate the IGF-I levels with risk parameters for cardiovascular disease.

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Methods: This was a cross-sectional study involving 211 African and 316 Caucasian subjects (aged 20–70 years). IGF-I, parameters of glucose homeostasis, blood pressure, and pulse wave velocity were assessed. Results: In African participants, blood pressure was significantly higher and serum IGF-I concentration significantly lower than in Caucasian counterparts. The decline of IGF-I with age was significantly faster in African subjects than in Caucasians. Finally, a significant negative correlation of IGF-I with blood pressure, pulse wave velocity, and high-density lipoprotein cholesterol was shown in young Africans. Conclusions: Africans show an accelerated decline in IGF-I levels around the age of 40 years. This finding, together with the observed relationship between IGF-I levels and cardiovascular risk factors, suggests that earlier and greater reduction of IGF-I could be associated with cardiometabolic vulnerability.

The age-related decline in IGF-I is associated with the increased incidence of cardiovascular diseases. Diabetes and cardiovascular disease are common in African urban areas. In this paper, Schutte et al. show, for the first time, an accelerated age-related decline in IGF-I circulating levels in African people. There is increasing evidence that IGF-I plays a key protective role in endothelial function, regulating nitric oxide production, improving insulin sensitivity, and exerting anti-inflammatory actions [12–14]. This study provides further indirect evidence on such a protective role of IGF-I against the development of cardiovascular disease. Although this study presents limitations such as the cross-sectional design and the lack of information on other important factors such as GH and IGF-binding proteins, and though correlation does not necessarily mean causation, the findings in African subjects are strongly consistent with the previous observations. This potential role of IGF-I warrants further research even envisaging a therapeutic implication involving perhaps low-dose IGF-I or GH treatment to prevent the early development of cardiometabolic diseases. It remains to be established whether the faster decline in IGF-I levels in Africans is due to genetic predisposition or environmental factors (such as alcohol abuse or poor nutrition) or both. Finally, it has to be pointed out that elevated levels of IGF-I have been implicated in the development and maintenance of many different cancers.

IGF-I bioactivity in an elderly population: relation to insulin sensitivity, insulin levels, and the metabolic syndrome Brugts MP, van Duijn CM, Hofland LJ, Witteman JC, Lamberts SW, Janssen JA Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Diabetes 2010;59:505–508 Background: The GH-IGF-I axis has been implicated in the development of metabolic syndrome. The aim of this study to relate the IGF-I bioactivity, measured the IGF-I kinase receptor activation assay (KIRA), with insulin sensitivity and metabolic syndrome in an elderly population-based cohort. Methods: The subjects were selected within the Rotterdam Study population, a prospective large-scale cohort study aimed at investigating incidence and risk factors of cardiovascular diseases in elderly people. 1,036 elderly subjects were recruited. The American Diabetes Association 2003 criteria were used to classify glucose tolerance. IGF-I bioactivity was determined by the IGF-I KIRA. This bioassay determines IGF-I bioactivity by measuring intracellular receptor autophosphorylation upon IGF-I binding [15]. Results: 697 subjects (69.7%) had normal fasting glucose (NFG), 165 subjects (16.3%) had impaired fasting glucose (IFG), and 153 subjects (15.1%) had diabetes. IGF-I bioactivity resulted positively related to insulin resistance markers. In non-diabetic subjects, after stratification according to deciles of HOMA-IR value, IGF-I bioactivity progressively increased up to and including the 9th decile. Thereafter, IGF-I significantly dropped in the 10th decile. IGF-I bioactivity was also directly related to the condition of metabolic syndrome, peaking when three components were present. However, a decline of IGF-I bioactivity was observed when five criteria of the metabolic syndrome were present. Conclusions: IGF-I bioactivity is closely related with insulin resistance up to a maximum threshold being significantly lower in subjects with diabetes than in subjects with NFG and IFG.

There is a complex relationship between the GH-IGF-I axis and glucose metabolism. IGF-I influences blood glucose concentrations directly by stimulating glucose uptake in target cells and indirectly by increasing the sensitivity of tissues to insulin. Unlike insulin, circulating concentrations of IGF-I do not

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fluctuate substantially with time. Instead, dynamic changes in IGF-I bioactivity are attributed to interactions with IGF-binding proteins (IGFBPs). Among the six IGFBPs, IGFBP-1 production is inhibited by insulin. Insulin is also essential for GH stimulation of hepatic IGF-I production either by regulating GH receptor expression or by modulating GH signaling. The main finding of this study is that circulating IGF-I bioactivity progressively increases with increasing severity of insulin resistance and hyperinsulinemia, reaching a plateau. Furthermore, this study shows that IGF-I bioactivity declines during progression of the metabolic syndrome. As subjects with the metabolic syndrome are chronically exposed to high insulin levels, the observed increase in IGF-I bioactivity may be secondary to an insulin-mediated suppression of IGFBP-1 levels. In subjects with more than three components of the metabolic syndrome, IGF-I bioactivity significantly declined, suggesting the development of hepatic insulin resistance (manifested by a relative increase of IGFBP-1) and hyperinsulinemia-induced GH resistance. This study suggests a close relationship between IGF-I, insulin sensitivity and, ultimately, cardiovascular risk and stimulates further studies aimed at elucidating whether IGF-I may prevent or, in specific conditions, predispose to metabolic and cardiovascular disease.

Concepts revised

Partial primary deficiency of insulin-like growth factor (IGF)-I activity associated with IGF1 mutation demonstrates its critical role in growth and brain development Netchine I, Azzi S, Houang M, Seurin D, Perin L, Ricort JM, Daubas C, Legay C, Mester J, Herich R, Godeau F, Le Bouc Y Assistance Publique-Hôpitaux de Paris, Hôpital Armand-Trousseau, Explorations Fonctionnelles Endocriniennes, Paris, France [email protected] J Clin Endocrinol Metab 2009;94:3913–3921 Background: Insulin-like growth factors-I and -II (IGF-I and IGF-II) play a key role in fetal growth and development. Only few specific IGF-I gene (IGF1) defects have been described so far, all were characterized by severe pre- and postnatal growth retardation, sensorineural deafness and severe mental retardation associated with microcephaly. This study describes a child with IGF1 mutation associated with severe intrauterine growth retardation, postnatal growth impairment and brain alterations. Case Description: The patient was born at 40 weeks, to consanguineous parents, with birth weight 2,350 g (–2.4 SDS), birth length 44 cm (–3.7 SDS) and head circumference 32 cm (–2.5 SDS). At referral, the child showed severe short stature (–3.7 SDS), low weight (–5 SDS) and microcephaly (–2.5 SDS). No dysmorphic features were present. The child had anorexia and scarce adipose tissue. Hearing test was normal. Mild developmental delay was present. Conventional work-up for growth retardation was normal. GH stimulation test and spontaneous GH secretion assessment revealed normal GH concentrations. IGF-I levels were almost undetectable if measured with a highly specific monoclonal assay but elevated in a polyclonal assay. In contrast, IGFBP-3 and ALS levels were in the upper normal range or above. GHBP levels were within the normal range. The response to IGF-I generation test was subnormal. He was given GH therapy at standard dose for children born small for gestational age (SGA) with no significant improvement of his growth curve. However, when a higher dose was used, partial catchup growth was observed. Brain MRI scan was normal. Results: Due to the discrepancy between the extremely low IGF-I concentrations and normal/elevated IGFBP-3 and ALS levels, a potential IGF1 defect was investigated. A previously unidentified homozygous missense mutation in exon 4 leading to replacement of an arginine in position 36 of the C domain by a glutamine was identified. This substitution was shown to affect protein function leading to a partial loss of protein affinity for the receptor and significant reduction of mitogenic activity. Conclusions: Partial loss of IGF-I activity may cause a milder phenotype than complete IGF1 deficiency and allow a partial response to high-dose GH therapy. It is plausible that such IGF1 mutations may be not uncommon in children born SGA with microcephaly and poor response to conventional GH therapy.

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To date, 3 patients have been described with an IGF1 molecular defect leading to complete or very severe IGF-I deficiency. This paper describes the fourth case with alterations in the IGF-I gene [16–18]. The hallmark of the diagnosis is the discrepancy between the reduced IGF-I levels and IGFBP-3 and ALS serum levels in the upper normal range. Unlike the previous 3 children, this case showed a milder phenotype and only a partial loss of IGF-I binding and activity. Once again this study shows the heterogeneity of the phenotype and raises the question whether this kind of missense mutations may be commoner than expected in children born SGA. Despite the mild IGF-I deficiency, this child showed severe pre- and postnatal growth failure and mental retardation, thus confirming the pivotal role of IGF-I in growth and neurodevelopment. Therefore, this report demonstrates that even partial IGF-I deficiency has marked effects on brain development and cognitive functions. The clinical implication is that these patients are able respond to doses of GH higher than those conventionally used. Moreover, these children may also benefit from IGF-I treatment. Finally, the article reports that low IGF-I serum concentrations using a monoclonal antibody assay were found to be normal in a polyclonal antibody assay – an issue worth remembering.

Growth hormone and insulin-like growth factor I insensitivity of fibroblasts isolated from a patient with an I␬B␣ mutation Wu S, Walenkamp MJ, Lankester A, Bidlingmaier M, Wit JM, De Luca F St. Christopher’s Hospital for Children, Philadelphia, Pa., USA J Clin Endocrinol Metab 2010;95:1220–1228 Background: Nuclear factor (NF)-␬B represents a family of transcription factors including five members which in resting cells are bound to specific inhibitory proteins, the I␬Bs, that also include I␬B␣. When the cell is stimulated, NF-␬B is released from the complexes and translocates to the nucleus where it modulates the expression of target genes. In rodents, NF-␬B regulates bone growth and chondrogenesis. In a patient with heterozygous mutation of I␬B␣, partial GH insensitivity was noted [19] and the effects of GH and IGF-I on NF-␬B DNA-binding activity, cell proliferation, and target gene expression were investigated. Methods: Skin fibroblasts from the patient, his father and controls were cultured. Fibroblasts were tested for NF-␬B DNA-binding activity, cell proliferation assessed by 3H-thymidine incorporation, phosphatidylinositol 3-kinase (PI3K) assay, STAT5 phosphorylation and expression of specific genes such as GH receptor (GHR), IGF-I receptor (IGF-IR) and TDAG51, a target gene of IGF-I. Results: GH and IGF-I dose-dependent effect on NF-␬B DNA-binding activity and fibroblast incorporation of 3H-thymidine was observed in the controls and patient’s father, whereas no effect was elicited in the patient’s fibroblasts. GHR and IGF-IR expression was normal in the patient’s cells. Whilst GH addition to culture medium induced STAT5 phosphorylation and IGF-I mRNA expression in controls’ and father’s fibroblasts, no effect was elicited in the patient’s cells. Finally, IGF-I failed to stimulate PI3K activity and TDAG51 expression. Conclusions: GH and IGF-I do not stimulate cell proliferation and gene expression in fibroblasts isolated from this patient harboring a mutation of I␬B␣, thus suggesting that NF-␬B activity is necessary for the growth-promoting actions of both hormones.

This study shows for the first time the key role of NF-␬B activity in signal transduction, reporting a child with severe postnatal growth retardation associated with GH insensitivity, immunological defects, ectodermal dysplasia and delay of psychomotor development. GH and IGF-I intracellular signaling takes place through a complex network of factors whose function is finely tuned to allow the proper cell response. Alterations of this intracellular cascade induce the blockade of signal transmission ultimately leading to growth arrest. Mutations/deletions of GH and IGF receptors, and STAT5b have been described. It was previously demonstrated in rats that NF-␬B signaling facilitates longitudinal bone growth and growth plate chondrogenesis and that NF-␬B p65 in rats mediates the growthpromoting effects of IGF-I. The results of this study indicate that both GH and IGF-I independently stimulate NF-␬B DNA-binding activity and cell proliferation in human fibroblasts. The phenotype of this patient with impairment of NF-␬B activity secondary to a mutation of I␬B␣ was complex, growth retardation representing only a tiny portion of a constellation of severe symptoms and signs. The patient unfortunately died shortly after stem cell transplantation performed for the immune disorder. This report demonstrates that any alteration in the steps of the post-receptor signals may affect

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the biological action of both GH and IGF-I, often including dysregulation of other pathways converging on the same cellular junction.

Involvement of pregnancy-associated plasma protein-A2 in insulin-like growth factor (IGF)-binding protein-5 proteolysis during pregnancy: a potential mechanism for increasing IGF bioavailability Yan X, Baxter RC, Firth SM Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, N.S.W, Australia [email protected] J Clin Endocrinol Metab 2010;95:1412–1420 Background: The majority of IGFs circulate in blood in high molecular mass ternary complexes containing either IGFBP-3 or IGFBP-5 and the acid-labile subunit. IGFBP proteases are proteolytic enzymes that fragment IGFBPs and reduce their affinity for IGFs, ultimately leading to increased free IGF concentrations. Such proteolytic activity has been reported to be augmented in pregnancy, GH deficiency and catabolic conditions, coincident with an increased demand for bioavailable IGFs. During pregnancy, IGFBP-5 undergoes substantial size redistribution, the majority of the protein being in either free or IGF-IGFBP-5 binary complexed forms. The formation of binary complexes may facilitate the transfer of IGFs from circulation to tissues, eventually increasing local IGF bioactivity. The aim of the study was to characterize the IGFBP-5 proteolytic activity and determine its physiological function. Methods: Plasma from healthy non-pregnant and pregnant women was fractioned by gel-filtration chromatography. IGFBP-5 circulating forms were identified by immunoblotting. Plasma proteolytic activity against IGFBP-5 was determined. IGFBP-5 fragments were purified from pregnancy samples and analyzed by mass spectrometry. Results: Whilst the intact form of IGFBP-5 was present in non-pregnancy samples, it was absent in specimens at all stages of pregnancy which showed the presence of proteolyzed fragments in the 18to 25-kDa range, particularly in the third trimester. The level of IGFBP-5 proteolytic activity increased progressively during pregnancy. Size exclusion chromatography revealed two major sources of proteolytic activity, one in the >150-kDa fraction and the second one eluting in the approximately 40-kDa fraction. The former was present only in pregnancy plasma whereas the latter was present in both pregnant and non-pregnant samples. The protease inhibitor profile and mass spectrometry analyses showed that the >150-kDa fraction contained PAPP-A2 (pregnancy-associated plasma protein-A) or a PAPP-A2-like protease responsible for the proteolysis of IGFBP-5 during pregnancy. Finally, pregnancy plasma was able to induce proteolysis of IGF-I-IGFBP-5 complexes, and to increase IGF-I receptor phosphorylation, thus suggesting that IGFBP-5 proteolysis in pregnancy leads to increased IGF-I bioactivity. Conclusions: Circulating IGFBP-5 is fully proteolyzed by PAPP-A2 during pregnancy. This proteolysis leads to increased IGF bioavailability, which may play a key role in growth and development of fetus as well as maternal well-being.

Dissociation of IGFs from IGFBP-containing complexes is thought to be mediated by limited proteolysis of IGFBPs, resulting in fragments with reduced IGF-binding affinity. Such proteolytic activity has been reported in pregnancy and other catabolic conditions, coincident with an increased demand for bioavailable IGFs. During pregnancy, multiple endocrine adaptations occur to guarantee the best growth and development of the fetus, and the well-being of the mother. These physiological changes also involve the IGF system, resulting in increased proteolysis of IGFBP-3 finalized to augment the fraction of free IGFs. The higher proportion of IGFBP-5 in binary complexes during pregnancy may potentially facilitate the transport of IGFs to tissues and thus play a significant role in regulating IGF bioavailability. Although pregnancy plasma is reported to have proteolytic activity against IGFBP-5, there has been no direct demonstration of its proteolysis. In this study, for the first time, increased proteolysis of IGFBP-5 has been described in pregnant plasma associated with increased capacity of IGF-I to stimulate IGF-I receptor phosphorylation. In addition, PAPP-A2 was identified to be the specific protease for IGFBP-5 during pregnancy. Taken together, these data seem to be consistent with each other, suggesting a physiological adaptation to the need of a greater IGF bioactivity for fetal and maternal demands during pregnancy. Accordingly, it has recently been reported that PAPP-A2 is increased in the serum of pre-eclamptic patients [20]. This

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suggests that PAPP-A2 up-regulation reflects a compensatory mechanism finalized to preserve fetal growth and development under unfavorable conditions such as eclampsia.

Clinical trials New treatments

Inhaled growth hormone (GH) compared with subcutaneous GH in children with GH deficiency: pharmacokinetics, pharmacodynamics, and safety Walvoord EC, de la Pena A, Park S, Silverman B, Cuttler L, Rose SR, Cutler G, Drop S, Chipman JJ Department of Pediatrics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Ind., USA [email protected] J Clin Endocrinol Metab 2009;94:2052–2059 Background: Since the description of the first patient treated with growth hormone (GH) in 1958, GH has been administered initially by intramuscular and subsequently by subcutaneous injections. This annoying method of administration affects long-term compliance leading to dose missing and therapy discontinuation, sometimes even to the refusal of treatment. Therefore, a less invasive method looks desirable to guarantee a better adherence to treatment and better final results. Aerosol technology has recently allowed the development of an innovative formulation of GH, termed somatropin inhalation powder (SIP). Preliminary pharmacokinetic (PK) and pharmacodynamic (PD) data in primates were promising. This study aimed at determining bioavailability and biopotency of SIP in comparison with GH given subcutaneously. In addition, PK, PD and safety of SIP treatment in children were tested. Methods: The design was a multicenter, randomized, double-blind, placebo-controlled, crossover trial. 22 GH-deficient children were recruited. Patients underwent two 7-day treatment phases with either inhaled GH or subcutaneous GH + placebo separated by 6–13 days of washout. Results: Although the absorption of SIP was faster, the overall PK profile was similar to that of subcutaneous GH. The mean relative bioavailability for SIP administration compared with subcutaneous GH was 3.5%. The mean relative biopotency, based on IGF-I response, was 5.5%. The two routes induced similar increases in mean serum GH area under the curve and in IGF-I levels in a dose-dependent fashion. The short-term administration of SIP resulted in being safe, without any major side effects, though vomiting, headache and cough were observed in some patients. No change in pulmonary function was recorded. The questionnaires provided to patients and parents showed a clear preference for the inhalation route. Conclusions: This is the first study in children showing that inhaled GH administered to GHD children for 7 days was effective in inducing a dose-dependent raise in GH and IGF-I levels and was well tolerated. However, the bioavailability of GH administered by this route is low relatively to GH administered subcutaneously.

The search for an alternative way for administering GH in children is certainly worthwhile. Despite the use of ad-hoc devices to alleviate the burden of daily injections, long-term GH therapy remains poorly acceptable for parents and children. Recent advances in aerosol technology, by increasing particle size and lowering their density and tendency to agglomerate, have increased efficiency of deep lung delivery and improved systemic absorption This preliminary short-term investigation on inhaled GH demonstrates proof of principle and looks promising in terms of tolerability and efficacy in increasing both GH and IGF-I. Although this study demonstrates the potential feasibility of this alternative route of administration in children, the relatively low bioavailability and biopotency of inhaled GH require further extensive studies to refine this aerosol technology.

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New indications of IGF-I therapy

A pharmacokinetic and dosing study of intravenous insulin-like growth factor-I and IGF-binding protein-3 complex to preterm infants Lofqvist C, Niklasson A, Engstrom E, Friberg LE, Camacho-Hubner C, Ley D, Borg J, Smith LE, Hellstrom A Department of Ophthalmology, Sahlgrenska Academy at Gothenburg University, Goteborg Sweden Pediatr Res 2009;65:574–579 Background: Preterm babies show almost undetectable circulating levels of insulin-like growth factor-I (IGF-I) and previous reports from the same authors demonstrated an association between low IGF-I concentrations and risk of developing the retinopathy of prematurity. Moreover, IGF-I plays a key role in proliferation, maturation, differentiation and migration of neural cells during embryo-fetal brain development, and the low IGF-I levels of premature neonates have been associated with brain vulnerability. The aim of this study was to test tolerability and pharmacokinetics of IGF-I and IGFBP-3 complex administered intravenously to a small group of premature newborns. Methods: Five infants born at gestational age 26–29 weeks with IGF-I levels 21 months) mice. These animals were compared with isochronic pairs (young-young or agedaged) joined at identical ages. This parabiotic procedure provides a biological model in which the ani-

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mals share a common blood circulation, thus enabling to test whether physiological levels of circulating cells or factors can significantly alter tissue function. The possible role of niche and systemic factors in HSPC ageing was investigated using direct isolation of hematopoietic stem cell (HSC)-regulatory niche cells. Finally, osteoblastic niche cells were isolated using fluorescence-activated cell sorting (FACS) from collagenase-treated bones. Results: The exposure to young circulation of aged-heterochronic partners showed significant recovery of HSC number and function which approached normal ‘youthful’ levels. In particular, bone marrow cells from aged mice exposed to young systemic factors showed recovery of engraftment potential, manifested by increased reconstitution of peripheral blood leukocytes, as well as restoration of youthful ratios of B lymphoid to myeloid. The interaction of HSC with aged osteoblastic niche cells was sufficient to induce HSPC accumulation similarly to that observed in aged marrow. The age-dependent alterations in osteoblastic niche cell number and function were restored to youthful levels when aged animals were exposed to young circulation. Locally, the inhibition of IGF-1 signaling in aged osteoblastic niche cells promoted youthful HSC-regulatory function, indicating that IGF-1 impairs the osteoblastic niche cells appropriate regulation of HSCs, thus contributing to the age-associated hematopoietic dysfunction. Tissue, but not systemic, IGF-1 seems to induce ageing of HSC-regulatory niche cells, and neutralization of IGF-1 signaling in the bone marrow microenvironment reverts age-related changes in osteoblastic niche cells. Conclusions: The results suggest that the age-associated changes in bone marrow niche cells are both systemically and locally regulated. They can be reversed by exposure to a young circulation or by inhibition of IGF-1 in the marrow microenvironment. The finding that IGF-1 neutralization restores youthful function to aged osteoblastic niche cells highlights a new and important activity for this growth factor in controlling the fate of stem cells. In the hematopoietic system, ageing is associated with deficient immune function and increased incidence of malignancy. Age-associated blood diseases are thought to arise in part owing to discrete changes in aged hematopoietic stem and progenitor cells. Local tissue environment regulates the number and function of cells in all tissues, particularly in bone marrow. The hematopoietic stem cells (HSCs) have the potential to self-renew to maintain the HSC pool. HSCs need to be localized in a particular location (termed the HSC niche) within the bone marrow to retain their multipotency and if the HSCs are located elsewhere, they would probably commit to differentiation rather than selfrenew. Studies in genetically modified mice supported roles for cells of the osteoblast lineage in the retention and regulation of HSCs in the bone marrow. The study of Mayack et al. indicates for the first time that the regulatory role of osteoblast niche cells in hematopoietic stem cell is closely dependent on IGF-1 in mice. Switching off osteoblast IGF-1 signaling restores the cell youthful function, thus suggesting that IGF-1 impairs the osteoblast niche cell regulation of HSCs thereby contributing to ageassociated hematopoietic dysfunction. Such observation has potential major clinical implications envisaging the development of new therapeutic strategies aimed at reversing the age-related immune dysfunction and cancer risk, specifically targeting IGF-1 in the marrow microenvironment.

New anti-cancer treatments

Anti-insulin-like growth factor I receptor immunoliposomes: a single formulation combining two anticancer treatments with enhanced therapeutic efficiency Hantel C, Lewrick F, Schneider S, Zwermann O, Perren A, Reincke M, Suss R, Beuschlein F Endocrine Research Unit, Medizinische Klinik-Innenstadt, Ludwig Maximilians University, Munich, Germany J Clin Endocrinol Metab 2010;95:943–952 Background: Insulin-like growth factors (IGFs) I and II are overexpressed in the vast majority of tumors and play a pivotal role in transformation, growth and survival of cancer cells. A number of strategies have been proposed to silence the IGF system in various types of tumor cells, particularly targeting the IGF-I receptor (IGFI-R) which mediates the biological action of both IGF-I and IGF-II. The aim of this

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study was to investigate the antitumor action of a monoclonal anti-IGFI-R antibody coupled to liposomes loaded with doxorubicin (DXR) in neuroendocrine (NETs) tumors of the gastroenteropancreatic (GEP) system. Methods: Samples of gastrointestinal NETs from 59 patients and samples from normal tissues were analyzed. The anti-IGFI-R Ab was coupled to the liposomal surface via sterol-based postinsertion technique using a succinimide activated sterol-PEG1300-anchor. Tumor cell lines were used to test antibody association and internalization in vitro. For in vivo experiments commercially available sterically stabilized liposomes loaded with DXR were used. Female athymic NMRI v/v mice were inoculated with BON (GEP-NET) cells for inducing tumor development and when longest tumor diameters ranged between 0.5 and 0.7 cm, therapeutic treatments were given intravenously as single bolus. Results: All tumor tissues showed overexpression of IGFI-R. Anti-IGFI-R immunoliposomes significantly associated to cells, were internalized and resulted effective in inhibiting proliferation and inducing apoptosis of cancer cells. Treatment of human neuroendocrine BON cell xenografts increased the survival time of animals. The anti-IGFI-R immunoliposomes were also tested in other human tumor cell lines different from GEP-NET such as neuroblastoma, breast and prostate cancer cells, showing a similar capacity of binding to the cell surface and being internalized. Conclusions: In vitro and in vivo treatment with these novel anti-IGFI-R immunoliposomes resulted effective in reducing cell proliferation, inducing apoptosis, and increasing lifespan in animals bearing GEPNET tumors. Preliminary in vitro experiments indicated that this agent could also represent a promising therapeutic tool for different cancer types. This report describes the development of DXR-encapsulated immunoliposomes coupled with an IGFI-R blocking Ab to both target IGFI-R-overexpressing tumor tissues and inhibit IGF-dependent pathways. The IGFI-R is important for cancer development and progression. This role was elegantly discovered by Sell et al. who described the resistance of fibroblasts harboring a null mutation of IGFI-R to transformation induced by various viral and cellular oncogenes [23]. As IGFI-R is overexpressed in the majority of tumor cells, a number of inhibition strategies for IGFI-R signaling have been developed [24]. These include: (1) the use of antisense molecules to reduce IGF-I-R translation; (2) the use of blocking antibodies directed to the extracellular part of the receptor; (3) the use of peptides mimicking IGF-I to block the ligand/receptor interaction; (4) the use of specific inhibitors of the receptor catalytic activity; (5) the use of dominant negative gene variants, and (6) the use of peptide aptamers, a class of molecules genetically selected for specific binding to the receptor. Unfortunately the in vitro promising results obtained with all these methods were not replicable in vivo. The novelty of this study was the combined approach with a cytostatic molecule such as doxorubicin coupled to anti-IGFI-R antibodies in the same carrier. The preliminary in vitro and in vivo results are encouraging but the therapeutic efficacy and safety of such an approach in humans remain to be established.

New mechanisms

Distinct alterations in chromatin organization of the two IGF-I promoters precede growth hormone-induced activation of IGF-I gene transcription Chia DJ, Young JJ, Mertens AR, Rotwein P Department of Pediatrics, Oregon Health and Science University, Portland, Oreg., USA Mol Endocrinol 2010;24:779–789 Background: Most growth hormone (GH) biological actions are mediated by IGF-I whose expression and secretion is directly stimulated by GH in liver and other tissues. IGF-I gene contains two promoters differently active in the different tissues with the only exception of liver where both promoters are functionally active. The mechanisms by which IGF-I promoters are regulated by GH are still largely unknown. The aim of this study was to investigate the effect of GH on IGF-I promoter function. Methods: Liver from hypophysectomized male Sprague-Dawley rats was studied. IGF-I mRNA expression was assessed by RT-PCR. Quantitative Stat5 chromatin immunoprecipitation assay (ChIP) was

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performed to determine whether GH causes recruitment of Stat5b to the IGF-I promoters. Histone acetylation and methylation were determined. A series of quantitative ChIP experiments were performed. Results: Whereas in absence of GH, IGF-I gene transcription was negligible, a single systemic GH administration induced a significant increase in transcription from both liver IGF-I promoters. Stat5 ChIP experiments failed to detect a substantial association of Stat5b with either IGF-I promoter. The activation of IGF-I promoters associated with a rise in acetylation of histones H3 and H4 in promoter-associated chromatin. GH acutely modified histone lysine methylation at the IGF-I promoters. Finally, GH treatment was able to induce recruitment of polymerase II (Pol II) to promoter 2. Conclusion: The authors conclude that GH induces rapid and dramatic changes in hepatic chromatin at the IGF-I locus and activates IGF-I gene transcription in the liver by distinct promoter-specific mechanisms. Whereas GH treatment does not influence recruitment of Pol II to promoter 1 which in absence of GH shows Pol II already present in a preinitiation complex, at promoter 2, GH facilitates recruitment and then activation of RNA Pol II to initiate transcription. This exquisite molecular study sheds light on the mechanisms involved in GH-dependent activation of IGF-I gene promoters showing how GH-mediated signaling causes acute alterations in hepatic chromatin architecture at the IGF-I locus, and that GH activates IGF-I gene transcription in the liver via distinct promoter-specific mechanisms. A single GH systemic administration induces instantaneous acetylation and methylation of core histones, as well as recruitment of polymerase II. Recruitment and modification of transcriptional coregulators may represent fundamental and physiologically relevant dynamic genomic effects of GH. As Stat5b represents the connecting link between the activation of the cell membrane GH receptor and the final biological action of GH on chromatin reorganization, which leads to IGF-I gene transcription, next investigations will be targeted to elucidate the pathways downstream Stat5b activation which eventually orchestrate chromatin structure and function. The knowledge of these mechanisms could unravel processes involved in peripheral altered responses to GH and devise interventions based on the fine tuning of these pathways. References 1. Eiholzer U, Nordmann Y, L’Allemand D: Fatal outcome of sleep apnoea in PWS during the initial phase of growth hormone treatment. A case report. Horm Res 2002;58:24–26. 2. Eiholzer U: Deaths in children with Prader-Willi syndrome. A contribution to the debate about the safety of growth hormone treatment in children with PWS. Horm Res 2005;63:33–39. 3. Tauber M, Diene G, Molinas C, Hebert M: Review of 64 cases of death in children with Prader-Willi syndrome. Am J Med Genet A 2008;146A:881–887. 4. Geremia C, Cianfarani S: Insulin sensitivity in children born small for gestational age. Rev Diabet Stud 2004;1:58–65. 5. Swiglo BA, Murad MH, Schunemann HJ, et al: A case for clarity, consistency, and helpfulness: state-of-the-art clinical practice guidelines in endocrinology using the grading of recommendations, assessment, development, and evaluation system. J Clin Endocrinol Metab 2001;93:666–673. 6. Abuzzahab MJ, Schneider A, Goddard A, et al: IGF-I receptor mutations resulting in intrauterine and postnatal growth retardation. N Engl J Med 2003;349:2211–2222. 7. Inagaki K, Tiulpakov A, Rubtsov P, et al: A familial insulin-like growth factor-I receptor mutant leads to short stature: clinical and biochemical characterization. J Clin Endocrinol Metab 2007;92:1542–1548. 8. Walenkamp MJ, van der Kamp HJ, Pereira AM, et al: A variable degree of intrauterine and postnatal growth retardation in a family with a missense mutation in the insulin-like growth factor I receptor. J Clin Endocrinol Metab 2006;91:3062– 3070. 9. Kawashima Y, Kanzaki S, Yang F, et al: Mutation at cleavage site of insulin-like growth factor receptor in a short stature child born with intrauterine growth retardation. J Clin Endocrinol Metab 2005;90:4679–4687. 10. Isken O, Maquat LE: Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev 2007;21:1833–1856. 11. Walenkamp MJ, de Muinck Keizer-Schrama SM, de Mos M, Kalf ME, van Duyvenvoorde HA, Boot AM, et al: Successful long-term growth hormone therapy in a girl with haploinsufficiency of the insulin-like growth factor-I receptor due to a terminal 15q26.2-qter deletion detected by multiplex ligation probe amplification. J Clin Endocrinol Metab 2008;93:2421–2425. 12. Spies M, Nesic O, Barrow RE, Perez-Polo JR, Herndon DN: Liposomal IGF-I gene transfer modulates pro- and antiinflammatory cytokine mRNA expression in the burn wound. Gene Ther 2001;8:1409–1415. 13. Conti E, Carrozza C, Capoluongo E, Volpe M, Crea F, Zuppi C, Andreotti F: Insulin-like growth factor-1 as a vascular protective factor. Circulation 2004;110:2260–2265. 14. Sukhanov S, Higashi Y, Shai SY, Vaughn C, Mohler J, Li Y, Song YH, Titterington J, Delafontaine P: IGF-I reduces inflammatory responses, suppresses oxidative stress, and decreases atherosclerosis progression in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2007;27:2684–2690. 15. Chen JW, Ledet T, Orskov H, Jessen N, Lund S, Whittaker J, De Meyts P, Larsen MB, Christiansen JS, Frystyk J: A highly sensitive and specific assay for determination of IGF-I bioactivity in human serum. Am J Physiol 2003;284:E1149–E1155.

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16. Woods KA, Camacho-Hubner C, Savage MO, Clark AJ: Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med 1996;335:1363–1367. 17. Bonapace G, Concolino D, Formicola S, Strisciuglio P: A novel mutation in a patient with insulin-like growth factor 1 deficiency. J Med Genet 2003;40:913–917 18. Walenkamp MJ, Karperien M, Pereira AM, et al: Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab 2005;90:2855–2864. 19. Janssen R, van Wengen A, Hoeve MA, ten Dam M, van der Burg M, van Dongen J, van de Vosse E, van Tol M, Bredius R, Ottenhoff TH, Weemaes C, van Dissel JT, Lankester A: The same I␬B␣ mutation in two related individuals leads to completely different clinical syndromes. J Exp Med 2004;200:559–568. 20. Nishizawa H, Pryor-Koishi K, Suzuki M, Kato T, Kogo H, Sekiya T, Kurahashi H, Udagawa Y: Increased levels of pregnancy associated plasma protein-A2 in the serum of pre-eclamptic patients. Mol Hum Reprod 2008;14:595–602. 21. Hellstrom A, Perruzzi C, Meihua J, Engstrom E, Hard A-L, Liu J-L et al: Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity. Proc Natl Acad Sci USA 2001;98:5804–5808. 22. Hellstrom A, Engstrom E, Hard A-L, Albertsson-Wikland KA, Carlsson B, Niklasson A et al: Postnatal serum insulin-like growth factor I deficiency is associated with retinopathy of prematurity and other complications of premature birth. Pediatrics 2003;112:1016–1020. 23. Sell C, Dumenil G, Deveaud C, Miura M, Coppola D, DeAngelis T et al: Effect of a null mutation of the type I IGF receptor gene on growth and transformation of mouse embryo fibroblasts. Mol Cell Biol 1994;14:3604–3612. 24. Bahr C, Groner B: The insulin-like growth factor-1 receptor as a drug target: novel approaches to cancer therapy. Growth Horm IGF Res 2004;14:287-295.

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Bone, Growth Plate and Mineral Metabolism Terhi Heinoa, Dov Tiosanob, Aneta Gawlikc and Lars Sävendahld a

Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Finland Pediatric Endocrinology, Rambam Medical Center, Technion – Israel Institute of Technology, Haifa, Israel c Pediatric Endocrinology and Diabetes, Department of Pediatrics, Medical University of Silesia, Katowice, Poland d Pediatric Endocrinology Unit, Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden b

It has been an outstanding year of publications in the field of pediatric endocrinology. We have made a subjective selection of 19 papers and divided the chapter into ’bone’, ’growth plate’ and ’mineral metabolism’ where each paper is discussed from a pediatric endocrine point of view. A clear breakthrough and mechanism of the year is the discovery of RANKL/RANK as key thermoregulators creating a link between bone physiology and central control of body temperature and fever. A new paradigm is represented by two papers discovering that serotonin regulates bone mass accrual in opposite directions depending on site of synthesis. A paper describing a new mechanism of IGF-1 to modulate thyroid hormone effects in the growth plate is also highligted. We selected a few papers identifying new genes linked to to important clinical conditions if mutated. These include cyclophilin B – linked to severe osteogenesis imperfecta, and ENPP1 – linked to hypophosphatemic rickets. As food for thought, we included a paper disclosing how hibernating bears prevent bone loss.

New paradigms OPG autoantibodies linked to bone loss in celiac disease

Osteoporosis associated with neutralizing autoantibodies against osteoprotegerin Riches PL, McRorie E, Fraser WD, Determann C, van’t Hof R, Ralston SH Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK [email protected] N Engl J Med 2009;361:1459–1465 Background: Osteoporosis is a recognized complication of celiac disease and it is generally considered to be secondary to malabsorption and calcium and vitamin D deficiencies, rather than a consequence of an autoimmune process. Methods: Serum samples were obtained from a 40-year-old male patient with celiac disease and autoimmune hypothyroidism, who developed high-turnover osteoporosis, as well as from 10 age-matched healthy male controls, 15 patients with celiac disease, and 14 patients with autoimmune hypothyroidism. Osteoprotegerin (OPG) immunoprecipitation assays and RANK signaling assays were performed. Results: In Western blotting, OPG immunoprecipitated with patient’s serum, while serum specimens from 10 healthy controls, 12 patients with celiac disease, and 14 patients with primary hypothyroidism were negative for the autoantibody. Three samples of 15 patients with celiac disease were positive, though at a lower intensity. RANK signaling assay demonstrated that NF-B activation was inhibited by the addition of human recombinant OPG in the presence of the immunoglobulin fraction from control serum, but the inhibitory effect of OPG was lost in the presence of immunoglobulins purified from the patient’s serum. Conclusion: A high turnover osteoporosis in a patient with celiac disease was associated with the spontaneous development of autoantibodies against OPG.

This case report identifies a young man with autoimmune hypothyroidism, celiac disease and high bone turnover osteoporosis. A new pathophysiological mechanism of rapid bone loss was discovered: neutralizing autoantibodies against osteoprotegerin (OPG). The high-turnover osteoporosis, elevated alkaline phosphatase levels, and fragility fractures are consistent with the phenotype seen both in targeted OPG–/– mice and humans with juvenile Paget’s disease and OPG-inactivating mutations. His bone disease was very severe and despite the gluten-free diet, calcium and vitamin D supplementation, his osteoporosis worsened. However, a remarkable response to zoledronic acid treatment, which is a potent inhibitor of osteoclastic bone resorption, indicated that elevated bone turnover was the primary cause of this patient’s osteoporosis. This strongly suggests that his acquired illness was due to the development of neutralizing autoantibodies against OPG. It is tempting to speculate that in this case, denosumab, i.e. RANKL antagonist, would have been an even more specifically targeted therapy. The mechanism by which OPG autoantibodies developed remains unclear, but presumably, endogenous OPG had become the target of an autoimmune response in this patient. They also demonstrated the presence of autoantibodies against osteoprotegerin in 3 of 15 patients with celiac disease. The 3 patients with the autoantibodies had lower bone mineral density values than those without the autoantibodies. Future studies should determine whether OPG autoantibodies are associated with the development or severity of osteoporosis in other patients with celiac disease or other autoimmune diseases.

New genes Mutations in osteogenesis imperfecta go beyond collagen type I

Severe osteogenesis imperfecta in cyclophilin B-deficient mice Choi JW, Sutor SL, Lindquist L, Evans GL, Madden BJ, Bergen HR, 3rd, Hefferan TE, Yaszemski MJ, Bram RJ Department of Immunology, Mayo Clinic College of Medicine, Rochester, Minn., USA [email protected] PLoS Genet 2009;5:e1000750 Background: Osteogenesis imperfecta (OI) is an inherited disorder of collagen and majority of cases are caused by point mutations in the type I collagen genes COL1A1 and COL1A2. The syndrome is characterized by exquisitely fragile bones due to osteoporosis. OI also occurs in humans with homozygous mutations in prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene). P3H1 exists in a complex with CRTAP and cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been unknown. Methods: To study the role of mammalian CypB, the authors generated mice lacking this protein. Results: In early life, Ppib–/– mice developed kyphosis and severe osteoporosis. The morphology of collagen fibrils in Ppib–/– mice was abnormal and in vitro studies revealed that procollagen did not localize properly to the Golgi in CypB-deficient fibroblasts. The authors found that the levels of P3H1 were substantially reduced in Ppib–/– cells, while CRTAP was unaffected. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1 and the post-translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB-deficient cells and tissues from CypB-knockout mice. Conclusion: The data presented in this paper provide a significant new mechanistic insight into the pathophysiology of OI and reveal interactions of members of the P3H1/CRTAP/CypB complex in directing proper collagen formation.

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Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding Barnes AM, Carter EM, Cabral WA, Weis M, Chang W, Makareeva E, Leikin S, Rotimi CN, Eyre DR, Raggio CL, Marini JC National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA [email protected] N Engl J Med 2010;362:521–528 Background: OI is a heritable disorder causing bone fragility. Mutations in type I collagen result in autosomal dominant OI, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (CRTAP and P3H1) cause autosomal recessive OI with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. Methods: The authors identified two siblings in a consanguinous Senegalese family. The siblings had recessive osteogenesis imperfecta without rhizomelia and their genomic DNA was screened. Results: No mutations in COL1A1, COL1A2, CRTAP or LEPRE1 were identified, but the siblings had a homozygous start-codon mutation in the Ppib gene resulting in a lack of CypB, the third component of the complex. The proband’s collagen had normal collagen folding and normal prolyl 3-hydroxylation. Conclusions: The data demonstrates that recessive OI can be caused by a homozygous start-codon mutation in the Ppib gene. The normal collagen folding and prolyl 3-hydroxylation however indicates that CypB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought.

It is now clear that osteogenesis imperfecta (OI) is not caused only by mutations in collagen 1 and 2 chain genes. Novel mutations associated with this disease have been found in the enzymatic complex which is needed to process collagen into mature fibrils. For example, CRTAP, reviewed in Yearbook 2007 [1], and LEPRE1 [2] mutations have previously been shown to be linked to OI. The current study by Choi et al. demonstrates that mice lacking cyclophilin B, a third partner of the collagen hydroxylation enzyme complex, also show phenotypic features of OI. Interestingly, soon after the publication of this paper, Barnes et al. reported two OI siblings from a consanguineous family due to a homozygous start codon mutation in the peptidyl-prolyl isomerase B (PPIB) gene which results in a lack of cyclophilin B. They however observed a normal collagen folding and prolyl 3-hydroxylation in these patients, which is in contrast to the findings of Choi et al. showing that translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in cells and tissues from CypB-deficient mice. Thus, the fundamental questions concerning the function of the 3-hydroxylase modifications, the role of CypB in the complex and the identity of the major collagen isomerase still need to be resolved. However, these data are useful in explaining the clinical and phenotypical variety of OI. Identifying novel mutations will furthermore improve the molecular diagnosis of OI in the future.

New hope A novel role for TGF-␤ in bone remodeling

TGF-␤1-induced migration of bone mesenchymal stem cells couples bone resorption with formation Tang Y, Wu X, Lei W, Pang L, Wan C, Shi Z, Zhao L, Nagy TR, Peng X, Hu J, Feng X, Van Hul W, Wan M, Cao X Department of Pathology, University of Alabama at Birmingham, Birmingham, Ala., USA [email protected] Nat Med 2009;15:757–765 Background: Bone remodeling is an important biological process that depends on the precise coordination of bone resorption and subsequent bone formation. Disturbances in the balance of bone remodeling are associated with skeletal diseases, such as Camurati-Engelmann disease (CED).

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Methods: This study used multiple methods, including in vitro cultures of osteoclasts and bone marrow stromal cells (BMSCs), as well as in vivo studies on transforming growth factor (TGF)-1 knockout mice and CED transgenic mice. Results: Bone resorption released active TGF-1, which induced the migration of BMSCs to the bone resorptive sites. The process was mediated through a SMAD signaling pathway. Analyzing mice carrying a CED-derived mutant TGFB1 (encoding TGF-1), which showed the typical progressive diaphyseal dysplasia seen in the human disease, high levels of active TGF-1 were found in the bone marrow. Treatment with a TGF- type I receptor inhibitor partially rescued the uncoupled bone remodeling and prevented the fractures. Conclusion: Since TGF-1 functions to couple bone resorption and formation, modulation of TGF-1 activity could be an effective treatment for diseases of bone remodeling.

Transforming growth factors- (TGF-) play an important role in bone metabolism and it has been hypothesized to function as a coupling factor that links bone resorption to bone formation. However, the molecular mechanisms have not been elucidated so far. This recent study by Tang et al. demonstrates that TGF-1 is the key chemoattractant that tightly couples bone degradation and formation in space and time. It induced the migration of bone marrow stromal cells to the bone remodeling area. In addition, the authors demonstrate that mice carrying a mutation in the TGF-1 gene develop Camurati-Engelmann disease, a disease characterized by progressive diaphyseal dysplasia, and express high levels of TGF-1 in the bone marrow. Interestingly, TGF- receptor inhibition was shown to partially rescue uncoupled bone remodeling. This intruiguing study provides evidence for the critical role of TGF- in bone metabolism, but a few important points remain to be investigated. First, it should be clarified whether another important human TGF- isotype (TGF-2) has a similar activity in bone marrow stromal cells as TGF-1. Second, the potential impact of TGF- inhibitors and stimulators in osteoblast differentiation should be investigated using in vivo systems. Even though this work reveals a novel role for TGF-1 in bone remodeling and suggests TGF-1 as a potential therapeutic target for bone diseases, we do not know how molecules affecting TGF- could be delivered specifically and efficiently to target cells. These questions should be answered before considering potential clinical applications of TGF- in the treatment of bone disease.

New hope Embryonal stem (ES) and induced pluripotent stem (iPS) cells – powerful tools in bone biology

Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells Grigoriadis AE, Kennedy M, Bozec A, Brunton F, Stenbeck G, Park IH, Wagner EF, Keller GM Department of Craniofacial Development and Orthodontics, Guy’s Hospital, King’s College London, London, UK [email protected] Blood 2010;115:2769–2776 Background: The differentiation of human embryonic stem (ES) cells into multiple hematopoietic lineages is well established. In addition, recent discoveries in generating induced pluripotent stem (iPS) cells has further enabled understanding of specific emryonic lineages and opened new opportunities for patientspecific stem cells, drug discovery and studying disease mechanisms. Methods: In this study, the stepwise generation of bone-resorbing osteoclasts from ES and iPS cells is reported. Human ES and iPS cells were maintained and differentiated towards monocyte-macrophage lineage and finally to functional osteoclasts. Results: A precursor population enriched for cells expressing the monocyte-macrophage lineage markers CD14, CD18, CD11b, and CD115 was obtained by generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor (VEGF) and hematopoietic cytokines in serum-free media. When cultured in the presence of essential osteoclast differentiation factors, i.e. macrophage colony-stimulating factor (M-CSF) and

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receptor activator of nuclear factor- ligand (RANKL), precursor cells formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. Molecular analyses confirmed the RANKL-dependent expression of the osteoclastic marker genes NFATc1, cathepsin K, and calcitonin receptor. Conclusion: This study reports the possibility to generate large numbers of osteoclasts from human ES and iPS cells in a consistent, reproducible, and defined manner. Osteoblasts are derived from mesenchymal stem cells, whereas osteoclasts are highly specialized, multinucleated cells that are derived from hematopoietic stem cells. Induced pluripotency as a tool for biological discovery was selected as the Method of the Year 2009 by Nature Methods [3]. Since the first reports of such a form of cellular reprogramming a few years ago, research on iPS cells has progressed on a vast speed. Although the potential of mouse ES cells to differentiate into osteoclasts has been reported previously, the current study by Grigoriadis et al. for the first time demonstrates the generation of osteoclasts from human ES and iPS cells. Since human osteoclasts are traditionally obtained by a time-consuming process of isolating and differentiating peripheral blood monocytes, the new method can be utilized to more efficiently identify novel antiresorptive compounds for osteoporosis, as well as for other conditions of accelerated bone resorption. In addition, the derivation of iPS cells from patients harboring cell-autonomous genetic mutations enables studying disease pathology. This can be done by evaluating how the individual mutations affect osteoclast differentiation and function in vitro. This study furthermore establishes a basis for genetic rescue and autologous cell-based therapies for disorders characterized by increased bone loss.

New mechanisms Oxidative stress and osteogenesis

FoxO1 is a positive regulator of bone formation by favoring protein synthesis and resistance to oxidative stress in osteoblasts Rached MT, Kode A, Xu L, Yoshikawa Y, Paik JH, Depinho RA, Kousteni S Department of Medicine, Division of Endocrinology, College of Physicians and Surgeons, Columbia University, New York, N.Y., USA [email protected] Cell Metab 2010;11:147–160 Background: Osteoporosis, a disease characterized by low bone mass, is associated with decreased number of osteoblasts and increased levels of oxidative stress within osteoblasts. Methods: Since transcription factors of the FoxO family confer stress resistance, the authors investigated their potential impact on skeletal integrity. They employed osteoblast-specific deletion of FoxO proteins in mice (FoxO1ob–/–) and performed a vast range of histological, cellular, biochemical and molecular analyses. Results: Among the three FoxO proteins, only FoxO1 was required for proliferation and redox balance in osteoblasts and thereby controlling bone formation. FoxO1 regulation of osteoblast proliferation occurred through its interaction with ATF4, a transcription factor regulating amino acid import, as well as through its regulation of a stress-dependent pathway influencing p53 signaling. Bone formation and bone mass in FoxO1ob–/– mice was normalized by decreasing oxidative stress levels or increasing protein intake. Conclusion: These results identify FoxO1 as a crucial regulator of osteoblast physiology and provide a direct mechanistic link between oxidative stress and the regulation of bone remodeling.

Forkhead box (Fox) proteins are a family of transcription factors that play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity. The O subclass of human Fox proteins (FoxO) include FoxO1, FoxO3, FoxO4 and FoxO6. Oxidative stress enhances the activation of FoxO transcription factors that defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. In this report, the authors utilized osteo-

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blast-specific deletion of FoxO proteins, and showed that only FoxO1 was required for proliferation and redox balance in osteoblasts. They also demonstrated that FoxO1 regulated osteoblast proliferation via interacting with activating transcription factor 4 (ATF4), as well as via influencing p53 signaling. Bone formation in mice lacking FoxO1 in osteoblasts returned to normal by a decrease in oxidative stress or an increase in protein intake. Interestingly, in another paper in the very same volume of Cell Metabolism, Ambrogini et al. [4] performed a simultaneous conditional deletion of all three FoxOs in mice and observed an increase in bone oxidative stress and osteoblast apoptosis, as well as decrease in osteoblast number, bone formation and bone mass. They furthermore demonstrated that overexpression of a FoxO3 transgene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis, with an increase in osteoblast number, bone formation rate, and vertebral bone mass. Despite some inconsistency in the role of individual FoxOs, these papers nevertheless identify FoxO proteins as crucial regulators of bone formation and bone mass homeostasis in mice. They provide evidence for FoxO-dependent oxidative defense as a mechanism to protect against oxidative stress in osteoblasts. An increasing amount of evidence has linked oxidative stress with aging and the development of age-related diseases. Thereby it is tempting to speculate that impaired function or deletion of FoXO proteins could be involved in bone loss observed in pediatric conditions of early aging, such as Werner syndrome or Hutchinson-Gilford progeria.

Food for thought How do hibernating bears prevent bone loss?

Grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) prevent trabecular bone loss during disuse (hibernation) McGee-Lawrence ME, Wojda SJ, Barlow LN, Drummer TD, Castillo AB, Kennedy O, Condon KW, Auger J, Black HL, Nelson OL, Robbins CT, Donahue SW Department of Biomedical Engineering, Michigan Technological University, Houghton, Mich., USA [email protected] Bone 2009;45:1186–1191 Background: Reduced skeletal loading (disuse) causes an imbalance in bone formation and bone resorption, thereby leading to loss of cortical and trabecular bone. In contrast, bears completely prevent cortical bone loss by balancing intracortical bone remodeling during disuse (hibernation). Trabecular bone, however, is more detrimentally affected than cortical bone in other animal models of disuse. Methods: The effects of hibernation on bone remodeling, bone architectural properties and bone mineral density (BMD) of grizzly bear (Ursus arctos horribilis) and black bear (Ursus americanus) trabecular bone were studied. Bones were obtained from hibernating (16–18 weeks after hibernation) and active (at least 14 weeks of physical activity following hibernation) grizzly bears. In addition, bones from black bears killed by hunters in the fall or in the spring were collected. Results: No differences were observed in bone volume fraction or BMD between hibernating and active bears or between pre- and post-hibernation bears in the ilium, distal femur, or calcaneus. Even though indicators of cellular activity (mineral apposition rate, osteoid thickness) decreased, trabecular bone resorption and formation remained balanced in hibernating grizzly bears. Conclusion: Bears appear to prevent bone loss during disuse by maintaining a balance between bone formation and bone resorption, which consequently preserves bone structure and strength. Further investigation on the mechanisms of preventing disuse-induced bone loss in bears may contribute to development of novel treatments for osteoporosis.

Bears hibernate for around 6 months of the year, and thereby experience annual periods of bone disuse and remobilization that are approximately equal in length. However, the material and structural properties of black bear (Ursus americanus) and grizzly bear (Ursus arctos) bone are not compromised with age or during hibernation. The important concept based on the data presented here and in previous reports from the same group is that hibernating bears possess a mechanism by which they do not lose bone (both trabecular and cortical) associated with disuse. No increase in cortical

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porosity and no difference in trabecular bone mass were found after hibernation compared to before. In addition, trabecular bone showed no differences in eroded surface or osteoid surface, suggesting that the bone remodeling rate was not altered during hibernation. Balanced bone formation and resorption occurs likely to preserve calcium homeostasis during hibernation while the bears recycle (instead of excrete) catabolic waste products. The possible mechanisms could involve neuroendocrine control of bone remodeling and energy regulation, e.g. via PTH and serotonin. This is a fascinating study on an exceptional material and could lead to an expanded understanding of how to reduce the negative bone balance associated with disuse or age-related bone loss.

Important for clinical practice A new disease gene for brachydactyly

Deletion and point mutations of PTHLH cause brachydactyly type E Klopocki E, Hennig BP, Dathe K, Koll R, de Ravel T, Baten E, Blom E, Gillerot Y, Weigel JF, Kruger G, Hiort O, Seemann P, Mundlos S Institut fur Medizinische Genetik, Charité Universitatsmedizin Berlin, Berlin, Germany [email protected] Am J Hum Genet 2010;86:434–439 Background: Brachydactyly type E (BDE [MIM 113300]) is characterized by a general shortening of metacarpals and metatarsals and/or phalanges. The genetic cause of the great majority of BDE cases is unknown. Methods: As submicroscopic aberrations are a known cause for congenital limb malformations and brachydactylies, the authors performed array-based comparative genomic hybridization (array CGH) via a whole-genome oligonucleotide array (244K, Agilent Technologies). Results: In a large pedigree with BDE, short stature, and learning disabilities, the authors detected a microdeletion of approximately 900 kb encompassing PTHLH, the gene coding for parathyroid hormone-related protein (PTHRP). Mutation analysis performed in these individuals identified two missense (L44P and L60P), a nonstop (X178WextX*54), and a nonsense (K120X) mutation in PTHLH. One of the missense mutations was tested in chicken micromass culture with a retroviral expression system and was shown to result in a loss of function. Conclusion: Loss-of-function mutations in PTHLH cause BDE with short stature.

Brachydactylies are a family of limb malformations characterized by short hands and/or feet due to aplastic or hypoplastic skeletal elements, including Turner syndrome, or Albright hereditary osteodystrophy. The phenotype is variable even within families, ranging from moderate shortening of individual metacarpals to a shortening of all bones in the hands and/or feet. Isolated brachydactyly type E (BDE) is characterized by a general shortening of metacarpals and metatarsals and/or phalanges. BDE has been associated in sporadic cases with microdeletions of 2q37, as well as with mutations in HOXD13 [MIM 142989] [5, 6]. However, the genetic cause of the great majority of BDE cases has remained unexplained. In a large pedigree with BDE, the authors detected a microdeletion encompassing PTHLH, the gene coding for parathyroid hormone-related protein (PTHrP). PTHrP is known to regulate the balance between chondrocyte proliferation and the onset of hypertrophic differentiation during endochondral bone development. Inactivation of PTHRP in mice results in short-limbed dwarfism due to premature differentiation of chondrocyte. The primary mediator of PTHRP/PTH receptor signaling in chondrocytes is GNAS. Haploinsufficiency of GNAS in mice results in premature differentiation of growth plate chondrocytes, thus resembling the phenotype described for PTHRP/ PTH receptor as well as for PTHLH-deficient mice [7]. The here characterized pedigree with BDE adds another condition to the PTHRP-pathway disease family. The observation that heterozygous loss-offunction mutations in PTHLH result in a growth defect that is very similar to that observed in GNAS mutations suggests a particular importance for this component of the pathway in the growth of bones of the hands and feet.

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New mechanisms IGF-1 modulates thyroid hormone effects in the growth plate

Thyroid hormone-mediated growth and differentiation of growth plate chondrocytes involves IGF-1 modulation of ␤-catenin signaling Wang L, Shao YY, Ballock RT Orthopaedic Research Center, Departments of Orthopaedic Surgery and Biomedical Engineering, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA [email protected] J Bone Miner Res 2010;25:1138–1146 Background: Terminal differentiation of growth plate chondrocytes is in part regulated by thyroid hormone through modulation of the Wnt/-catenin signaling pathway. Thyroid hormone is a known stimulator of IGF-1 receptor expression and IGF-1 has been described as a stabilizer of -catenin. Methods: Chondrocytes were isolated from the resting zone of the distal femoral growth plate of 2-dayold neonatal Sprague-Dawley rats and cultured in monolayer or 3D pellets. Results: The authors show that IGF-1 and IGF1R (adenovirus transfection) stimulate Wnt-4 expression and -catenin activation in growth plate chondrocytes. The positive effects of IGF-1/IGF1R on chondrocyte proliferation and terminal differentiation was partially inhibited by Wnt antagonists. T3 activated IGF-1/IGF1R signaling and IGF-1-dependent PI3K/Akt/GSK3 signaling in proliferative and prehypertrophic growth plate chondrocytes. T3-mediated Wnt-4 expression, -catenin activation, cell proliferation and terminal differentiation of growth plate chondrocytes were partially prevented by inhibitors of the IGF1R and PI3K/Akt signaling. Conclusion: The authors conclude that interactions between thyroid hormone and -catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF-1/ IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways.

Thyroid hormone

IGF-1/IGF1R

Wnt-4

PI3K/Akt

GSK3

-catenin

Cyclin D1

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Terminal differentiation

Fig. 1. Schematic diagram of the proposed interactions between thyroid hormone, IGF-1/IGF1R, and −catenin signaling pathways in regulating cell proliferation and terminal differentiation of growth plate chondrocytes.

Terhi Heino/Dov Tiosano/Aneta Gawlik/Lars Sävendahl

Thyroid hormone is a systemic factor that potently regulates skeletal maturation in the growth plate and thyroid hormone receptor  (TR-) and is essential for regulating the process of endochondrial ossification. IGF-1 signals via the type 1 IGF receptor (IGF1R), both are expressed in the proliferating and prehypertrophic zone chondrocytes of the growth plate [8]. TR1 deleted mice have impaired Igf1r expression and IGF-1 signaling in the growth plate, suggesting that the IGF1R is a physiologic target for thyroid hormone action in the growth plate [9]. The investigators behind this report reported previously that thyroid hormone interacts with Wnt/-catenin signaling pathway in the terminal differentiation of growth plate chondrocytes [10]. They now present data supporting that IGF-1 is actively involved in the crosstalk between the Wnt/-catenin and PI3K/Akt pathways in the regulation of growth plate chondrocyte proliferation and differentiation by thyroid hormone. Thyroid hormone initially may stimulate IGF-1/IGF1R signaling in growth plate chondrocytes, activating the -catenin signaling pathway through either the PI3K/Akt pathway or the Wnt pathway, in turn regulating the transcription of -catenin responsive genes and subsequently promoting cell proliferation in the growth plate (fig. 1). Clinicians who initiate thyroxine treatment in hypothyroid children often observe a prominent catch-up growth soon after treatment start. At least partly this can now be explained by thyroid hormone interaction with IGF-1/IGF1R signaling in the growth plate.

New genes A novel activator of Ihh that paces bone growth

Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription Wang W, Lian N, Li L, Moss HE, Perrien DS, Elefteriou F, Yang X Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, Tenn., USA [email protected] Development 2009;136:4143–4153 Background: Ablation of activating transcription factor 4 (Atf4) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. Methods: Wild-type (WT) and Atf4–/– embryos and mice were obtained by crossing Atf4+/– mice. The authors also established Atf4-overexpressing chondrocytes. Results: In Atf4–/– growth plate, the typical columnar structure of proliferative chondrocytes was found to be disturbed. The proliferative zone was found to be shortened, whereas the hypertrophic zone transiently expanded. The expression of Indian hedgehog (Ihh) was markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), was found to be normal. Furthermore, forced expression of Atf4 in chondrocytes induced endogenous Ihh mRNA and Atf4 was found to directly bind to the Ihh promoter and activate its transcription. Supporting these findings, reactivation of hedgehog signaling pharmacologically in mouse limb explants corrected the Atf4–/– chondrocyte proliferation and short limb phenotypes. Conclusion: The authors conclude that Atf4 acts as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation.

The investigators behind this paper have previously shown that inactivation of the transcription factor Atf4 in mice results in severe osteopenia, which is caused by a failure of Atf4–/– osteoblasts to achieve terminal differentiation and to synthesize type I collagen, the main constituent of bone matrix [11]. This study reveals Atf4 as a crucial regulator of chondrogenesis and identifies Ihh as a transcriptional target of Atf4 in chondrocytes. Mice lacking Atf4 exhibit dwarfism and are characterized by markedly reduced growth plates, decreased chondrocyte proliferation and an abnormally

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expanded hypertrophic zone. These phenotypic abnormalities are similar to those of Ihh–/– mice [12], which, together with the dramatic decrease in Ihh expression observed in Atf4–/– growth plates, indicate that Atf4 and Ihh lie in the same genetic pathway regulating chondrogenesis during skeletal development. This study reveals a novel mechanism by which Atf4 regulates chondrocyte proliferation and differentiation via upregulating Ihh expression. It remains unclear, however, what upstream signals regulate Atf4 expression and activity and what downstream effector molecules of hedgehog signaling are controlling chondrocyte proliferation and differentiation. Many extracellular regulators, including members of the FGF, BMP, Igf1, Wnt and PTHrP families, are reported to regulate chondrocyte proliferation and differentiation. Whether they act on chondrocytes by modifying Atf4 activity remains to be determined. It is also of great interest to study whether the same mechanisms are responsible for the bone defects seen in Atf4–/– mice. No human disease has so far been identified linked to a mutated Aft4. Based on the phenotypic similarities between Aft4 and Ihh mice, a mutated Aft4 should be considered when you see a patient with features of brachydactyly A1 (MIM #112500), a disorder where a mutated ihh is found in some, but not all patients. The features to look for are short stature associated with middle phalanges of all the digits that are rudimentary or fused with the terminal phalanges.

New concerns Loop diuretics may affect growth plate chondrocytes and bone growth

A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate Bush PG, Pritchard M, Loqman MY, Damron TA, Hall AC Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, UK [email protected] J Bone Miner Res 2010; in press. DOI 10.1002/jbmr.47 Background: The underlaying mechanisms of growth plate chondrocyte volume increase and bone lengthening are poorly understood. The Na-K-Cl co-transporter (NKCC) is well known to be activated in many cell types leading to cell volume increase. The authors hypothesised that NKCC may be responsible for the volume expansion normally occuring in hypertrophic growth plate chondrocytes. Methods: Metatarsals/metscarpals from 7-day-old rat pups (P7) were incubated in the presence/absence of the specific NKCC inhibitor bumetanide and whole bone lengths and histological analysis of growth plate measured after 24 h. Fluorescent NKCC was visualised by immunohistochemistry applying confocal laser scanning microscopy on rat (P7) tibial growth plates. Microarray analysis was performed on mRNA isolated from proliferative and hypertrophic zone cells of tibial growth plates from P49, P53, and P58 rats. Results: Bumetanide resulted in a dose-dependent suppression of bone growth. Histological analysis showed that this was linked to a reduction in hypertrophic zone height. Quantification of fluorescence immunohistochemistry demonstrated a significant transition of NKCC from the intracellular space of proliferative cells to the cytosolic membrane of hypertrophic cells. In addition, microarray analysis showed an increase in NKCC1 mRNA between proliferative and hypertrophic cells. Conclusion: The authors conclude that NKCC contributes to the volume increase normally occuring in hypertrophic growth plate chondrocytes. This statement was supported by data showing an increase of NKCC1 mRNA in hypertrophic zone cells, its cellular localisation, and reduced bone growth in the presence of the NKCC inhibitor bumetanide.

The growth plate is highly organized, with a ’reserve’ zone preceding proliferative zone (PZ) chondrocytes. After a regulated period of time a marked differentiation occurs, signified by a dramatic increase in volume, resulting in the formation of hypertrophic zone (HZ) chondrocytes. The volume of a typical cell increases 15-fold in the HZ and it has been postulated that this cell volume increase occurs through a combination of classical hypertrophy and regulated volume expansion [13]. However, the mechanisms which drive growth plate HZ chondrocyte volume expansion are unknown.

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The intracellular sodium concentration ([Na+]i) plays a key role in determining cell volume, and Na+ is moved across the cytoplasmic membrane through a range of transport proteins. One of these, the Na-K-2Cl co-transporter (NKCC), hitherto known to act in neuronal tissues, is an obvious candidate for HZ chondrocyte expansion due to its ability to increase cell volume in other cell types [14]. NKCC mediates electro-neutral ion transport, and is characterized by its sensitivity to loop diuretics (e.g. bumetanide, furosemide). Two NKCC isoforms are known, the near ubiquitous NKCC1 and KCC2. Although considered to be present in all tissues only mRNA for NKCC1 has been reported in growth plates, where it demonstrates increased expression from the PZ to HZ [15]. In the present study, the authors tested the hypothesis that NKCC1 is involved with HZ chondrocyte volume increase and longitudinal bone growth. Whole metatarsal/metacarpal rudiments were incubated with the loop diuretic bumetanide, a specific NKCC inhibitor, which inhibited bone elongation by approximately 35%. If NKCC plays a major role in HZ chondrocyte volume increase, it would be expected to be under the same hormonal control that regulates bone growth. Indeed, NKCC has been shown to be regulated by cortisol in other tissue [16] but if this is the case also in the growth plate remains to be elucidated. It is important to point out that there are limited reports of skeletal growth retardation in children exposed to loop diuretics. Reduced growth might however go unreported due to the conditions which required prescription of loop diuretics. Further investigation is warranted to determine the regulatory control of NKCC in the mammalian growth plate and to the possible detrimental effect on bone growth with chronic exposure to loop diuretics in children.

New concerns Aromatase inhibition may cause osteoporosis and polycystic ovaries; at least in female rats

Marginal growth increase, altered bone quality and polycystic ovaries in female prepubertal rats after treatment with the aromatase inhibitor exemestane van Gool SA, Wit JM, De Schutter T, De Clerck N, Postnov AA, Kremer Hovinga S, van Doorn J, Veiga SJ, GarciaSegura LM, Karperien M Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands [email protected] Horm Res Paediatr 2010;73:49–60 Background: As a potential approach for growth enhancement in children with short stature aromatase inhibition has been proposed. The authors aimed to assess the effect and potential adverse effects of aromatase inhibition on growth in female rats. Methods: Prepubertal Wistar rats were given intramuscular injections with placebo or the aromatase inhibitor exemestane (10, 30 or 100 mg/kg/week) for 3 weeks. A control group was ovariectomized (OVX). Weight and length gain, tibia and femur length, growth plate width, organ weights, and insulinlike growth factor I (IGF-I) levels were measured. In addition, the histology of ovaries, uterus and brain were analyzed and X-ray microtomography was applied to the femur. Results: At 100 mg/kg/week (E100), exemestane significantly increased weight gain and growth plate width, but less prominently than OVX. In the femur, trabecular number and thickness were decreased in the metaphysis and epiphysis in both E100 and OVX rats. Importantly, E100 significantly decreased ovarian weight and multiple cysts were seen upon histological evaluation. No significant effects were found on IGF-I levels and brain morphology. Conclusion: The authors conclude that at a high dose, exemestane marginally increases growth in female rats. However this is achieved at the expense of osteopenia and polycystic ovaries.

In the present article, the authors report the results of a study, in which sexually immature female rats were treated with the steroidal, irreversible aromatase inhibitor exemestane. The main conclusion is that exemestane partially inhibits aromatase activity, which caused only a marginal length gain and appendicular growth, but also resulted in multiple ovarian cysts and changes in bone archi-

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tecture consistent with early-stage osteopenia. Although aromatase inhibition was probably incomplete, pronounced adverse effects were found in ovarian histology and bone architecture, and it remains unclear whether these would have long-term permanent consequences for reproduction and fracture risk, respectively. Species differences in dependency on estrogen signaling probably underlie the discrepancies found in growth phenotype between these aromatase inhibitor treated rats and estrogen-deficient patients described in the literature. However, considering the potential side effects on bone physiology and morphology of the ovaries, treatment with aromatase inhibitors in girls in clinical practice should not be advised.

Follow-up on a YB 2009 paper Time for new nutritional recommendations for pregnant women?

Maternal vitamin D status determines bone variables in the newborn Viljakainen HT, Saarnio E, Hytinantti T, Miettinen M, Surcel H, Makitie O, Andersson S, Laitinen K, Lamberg-Allardt C Department of Applied Chemistry and Microbiology, Division of Nutrition, University of Helsinki, Helsinki, Finland [email protected] J Clin Endocrinol Metab 2010;95:1749–1757 Background: Maternal vitamin D status may program neonatal skeletal development. The objective of this paper was to determine the association of mothers’ vitamin D status with bone variables of their newborns. Methods: 125 pregnant women participated in a longitudinal follow-up. The mean values for age, BMI before pregnancy, pregnancy weight gain, and total daily vitamin D intake in mothers were 31 years, 23.5 kg/m2, 13.1 kg, and 14.3 µg, respectively. Blood was collected from mothers during the first trimester and 2 days postpartum, and from umbilical cords at birth for analysis of 25-hydroxyvitamin D (25-OHD), PTH, and bone remodeling markers. Bone variables were measured by pQCT on average 10 days postpartum. Bone contour was analyzed for the detection of total bone mineral density (BMD), bone mineral content (BMC), and cross-sectional area (CSA). Results: The median value of the individual means for first trimester and 2-day postpartum 25-OHD level, 42.6 nmol/l, was used as cutoff to define two equal-sized groups (below or above median). Newborns of mothers with levels below median were heavier (p = 0.05), and 60% were boys. However, tibia BMC was 0.047 g/cm higher (p = 0.01), and CSA was 12.3 mm2 larger (p = 0.02), but no difference in BMD was observed above median compared with below median group. Conclusions: The data showed that although the mean total intake of vitamin D among mothers met current Nordic recommendations, 71% of women and 15% of newborns were vitamin D deficient during the pregnancy. Moreover, the results suggest that maternal vitamin D status affects bone mineral accrual during the intrauterine period and influences bone size.

Vitamin D regulates 3% of the human genome, including effects on bone health throughout life. Maternal vitamin D status during pregnancy may program skeletal development [17] and body composition in the offspring [18], by influencing the interaction between osteoblasts and adipocytes. Moreover, low maternal 25-OHD may be responsible for prematurity and in late pregnancy is associated with reduced intrauterine long bone growth and slightly shorter gestation [19]. This study examined the effect of maternal vitamin D status on skeletal variables of newborns. It confirmed that newborns whose mothers had mean 25-OHD levels above 42.6 nmol/l had higher tibial bone mineral content and higher cross-sectional area but not bone mineral density (BMD). It is important to emphasize that the average maternal intake of vitamin D in the study group was 14.3 µg, which meets current Nordic recommendations for pregnant women (10 µg or 400 IU). However, despite this, most of the mothers were vitamin D deficient throughout pregnancy. It is obvious that these recommendations need to be updated and adapted to modern lifestyle (indoors) and desired serum levels. The fact that BMD was not affected by maternal vitamin D status implies that in utero other hormones, such as PTHrP, which is secreted from the mammary glands and the placenta, promote adequate calcium supplementation to the fetus.

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New genes New gene involved in hypophosphatemic rickets

Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal-recessive hypophosphatemic rickets Lorenz-Depiereux B, Schnabel D, Tiosano D, Hausler G, Strom TM Institute of Human Genetics, Helmholtz Zentrum Munchen, German Research Center for Environmental Health, Neuherberg, Germany [email protected] Am J Hum Genet 2010;86:267–272 Background: The analysis of rare genetic disorders affecting phosphate homeostasis has led to the identification of several proteins essential for the renal regulation of phosphate homeostasis as FGF23. The authors considered ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) as a causal factor of hypophosphatemia, since it has been reported to occur in some patients with generalized arterial calcification of infancy (GACI). Methods: The cohort comprised 60 index cases with hypophosphatemia who were found negative for PHEX, FGF23 and DMP1 mutations. Using homozygosity mapping, the authors identified a candidate region on chromosome 6q23, comprising approximately 35 genes, among them the ENPP1 gene. Results: A loss-of-function mutation in the ENPP1 gene was found in members of four families affected with hypophosphatemic rickets. Conclusion: ENPP1 was identified as the fourth gene, following PHEX, FGF23, and DMP1, to cause, if mutated, hypophosphatemic rickets resulting from elevated FGF23 levels.

Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene Levy-Litan V, Hershkovitz E, Avizov L, Leventhal N, Bercovich D, Chalifa-Caspi V, Manor E, Buriakovsky S, Hadad Y, Goding J, Parvari R Department of Developmental Genetics and Virology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel [email protected] Am J Hum Genet 2010;86:273–278 Background: Human disorders of phosphate handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. Methods: An extended Bedouin family with hypophosphatemic rickets and 236 Bedouin control individuals of the same geographic region were investigated by homozygous mapping. Results: The authors identified an inactivating mutation in the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR). Conclusion: These results suggest a different pathway involved in the generation of ARHR, and possible additional functions for ENPP1.

These two papers extend our knowledge of hypophosphatemic rickets, while evoking unresolved questions. Last year we discussed hypophosphatemia as the common denominator of all rickets [20]. The three main causes for hypophosphatemia are high PTH, elevation in FGF23 and renal P loss. Now we learn that mutations in the ENPP1 gene can cause either of two diseases – generalized arterial calcification of infancy (GACI) or hypophosphatemic rickets. Interestingly, the same mutation seems responsible for these two entities, as discussed in the article by Lorentz-Depiereux et al. ENPP1 encodes the NPPI enzyme that generates pyrophosphate from ATP in the external cellular matrix. Pyrophosphate is the major hydroxyapatite inhibitor. In the absence of pyrophosphate, hydroxyapatite production is accelerated, and is accompanied by excessive secretion of collagen type I to produce the primary bone elements. In children with GACI who survived, reductions in phosphorous levels and in Tmp/GFR were observed during the second year of life. In some cases, FGF23 was ele-

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vated. It is still not clear why in some patients the main phenotype is GACI, and in others hypophosphatemic rickets.

Mechanism of the year RANKL/RANK as key thermoregulators – link between bone physiology and central control of body temperature and fever

Central control of fever and female body temperature by RANKL/RANK Hanada R, Leibbrandt A, Hanada T, Kitaoka S, Furuyashiki T, Fujihara H, Trichereau J, Paolino M, Qadri F, Plehm R, Klaere S, Komnenovic V, Mimata H, Yoshimatsu H, Takahashi N, von Haeseler A, Bader M, Kilic SS, Ueta Y, Pifl C, Narumiya S, Penninger JM IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria [email protected] Nature 2009;462:505–509

Receptor-activator of NF- ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumor necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodeling, lymph node organogenesis and formation of a lactating mammary gland. RANKL and RANK are also expressed in the central nervous system. Methods: In this study tissue-specific Nestin-Cre and GFAP-Cre rank(floxed) deleter mice were used. Two patients with autosomal-recessive osteopetrosis were also included in the study. Results: In both mice and rats, central RANKL injections triggered severe fever. The function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank(floxed) deleter mice were resistant to lipopolysaccharide-induced fever as well as to fever in response to the key inflammatory cytokines IL-1 and TNF-. RANKL activated brain regions involved in thermoregulation and induced fever via the COX2-PGE2/EP3R pathway. Moreover, female NestinCre and GFAP-Cre rank(floxed) mice exhibited increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. In addition, 2 patients with RANK mutations were found to exhibit impaired fever response during pneumonia. Conclusion: The data identify a novel function for the important osteoclast differentiation factors RANKL/ RANK in female thermoregulation. Background:

In the last years we have learned that RANKL and RANK play a major role in the activation of osteoclasts; in the absence of RANK activity, osteopetrosis develops. Surprisingly, it was shown that the RANKL/RANK regulate formation of lactating mammary gland during pregnancy [21]. Now we learn that RANK protein is also specifically expressed in the preoptic area (POA) and the medial septal nucleus (MSn). It directly connects bone physiology to the central control of female body temperature, as well as to fever in inflammation. In male mice loss of RANK in neurons had no significant effect on basal circadian body temperatures while genetic inactivation of RANK in female mice brain resulted in altered physiological thermoregulation that is regulated also by ovarian sex hormones. In this elegant study the authors have shown that RANK is a critical mediator of fever in response to inflammation in mice. However, the clinical observation in 2 siblings with homozygous RANK mutation and severe osteopetrosis, exhibiting an impaired fever response to pneumonia, suggests that RANKL/RANK regulates fever also in humans.

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New paradigms Serotonin regulates bone mass accrual in opposite directions depending on the site of synthesis

A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure Yadav VK, Oury F, Suda N, Liu ZW, Gao XB, Confavreux C, Klemenhagen KC, Tanaka KF, Gingrich JA, Guo XE, Tecott LH, Mann JJ, Hen R, Horvath TL, Karsenty G Department of Genetics and Development, Columbia University, New York, N.Y., USA [email protected] Cell 2009;138:976–989 Background: Leptin inhibition of bone mass requires the integrity of specific hypothalamic neurons but not expression of its receptor on these neurons. The same is true for its regulation of appetite and energy expenditure. This suggests that leptin acts elsewhere in the brain to achieve these three functions. Methods: In this study, mice were generated by embryonic stem cell manipulation to obtain Tph2+/– (tryptophan hydroxylase-1), and thereafter were intercrossed. For further analyses, immunohistochemistry, in situ hybridization, axonal tracing, and microcomputed tomography as well as measurement of serotonin levels in the brain, deoxypyridinoline in the serum and urinary elimination of catecholamines were performed. Results: The authors showed that brainstem-derived serotonin (BDS) favors bone mass accrual following its binding to Htr2c receptors on ventromedial hypothalamic neurons, and appetite via Htr1a and 2b receptors on arcuate neurons. Leptin inhibits these functions and increases energy expenditure because it reduces serotonin synthesis and firing of serotonergic neurons. Conclusion: The authors conclude that their study modifies the map of leptin signaling in the brain and identifies a molecular basis for the common regulation of bone and energy metabolism.

Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis Yadav VK, Balaji S, Suresh PS, Liu XS, Lu X, Li Z, Guo XE, Mann JJ, Balapure AK, Gershon MD, Medhamurthy R, Vidal M, Karsenty G, Ducy P Department of Genetics and Development, Columbia University Medical Center, New York, N.Y., USA [email protected] Nat Med 2010;16:308–312 Background: As gut-derived serotonin (GDS) inhibits bone formation, the authors asked whether hampering its biosynthesis could treat osteoporosis through an anabolic mechanism. Methods: They synthesized an inhibitor of tryptophan hydroxylase-1, the initial enzyme in GDS biosynthesis, and this small molecule was orally administered once daily for up to 6 weeks in ovariectomized rodents. Results: Oral administration of this small molecule once daily for up to 6 weeks acted as a bone metabolic agent fully rescuing gonadectomy-induced bone loss. The rescue was due to an increase in osteoblasts number and bone formation rate, whereas the osteoclast surface per bone surface was unaffected. Conclusion: The authors concluded that these results provide a proof of principle that inhibiting gutderived serotonin biosynthesis could become a new bone anabolic treatment for osteoporosis.

These two papers demonstrate the complexity of bone mass regulation by serotonin. Serotonin is probably the first example of a molecule whose influence on bone mass accrual depends on the site of synthesis: Gut-derived serotonin inhibits bone mass while brainstem-derived serotonin (BDS) favors bone density. We emphasize that although BDS, acting as a neurotransmitter, accounts for only about 5% of the total pool of serotonin in the body, its influence on bone remodeling is dominant over gut-derived serotonin (GDS). It has been shown in the past that leptin inhibits bone mass accrual by the activation of the sympathetic activity. Just the opposite, BDS decreases sympathetic activity. In this paper Yadav et al. demonstrated that leptin inhibits serotonin synthesis in Thp2 (tryptophan

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Brainstem

Hypothalamus Serotonin Htr2c Leptin Htr1a Htr2b

Appetite

Adipocytes

Bone mass

Htr1b Osteoblast Serotonin

Enterochromaffin cell

Creb

Tph1 Bone

Decreased osteoblast proliferation

Duodenum

Fig. 2. Depending on site of synthesis, serotonin regulates bone mass in opposite directions: duodenum-derived serotonin acts on osteoblasts through the Htr1b receptor and Creb (cAMP response element binding; transcription factor) response to inhibit their proliferation while brainstem-derived serotonin favors bone mass accrual following its binding to Htr2c receptors on ventromedial hypothalamic neurons and appetite via Htr1a and 2b receptors on arcuate neurons [23].

hydroxylase 2) expression neurons. The net effect is activation of the sympathetic system to reduce bone mineral density. In the second paper, the authors show that an inhibitor of GDS synthesis acts as a bone anabolic agent, and can fully rescue gonadectomy-induced bone loss. Considering the lower rate of bone remodeling in rodents, as compared to humans, as well as the lack of haversian remodeling in the former, these results need be confirmed in humans. Rodents grow throughout their lives and do not remodel their cortical bone. This biology is now harnessed with an orally available inhibitor of gut serotonin synthesis that promotes bone formation in rodents without altering brain serotonin [22] which highlights the significance of serotonin as a modulator of bone metabolism (fig. 2). References 1. Chrysis D, Heino T, Sävendahl L: Growth Plate, Bone and Calcium; in Carel J-C, Hochberg Z (eds): Yearbook of Pediatric Endocrinology 2007. Basel, Karger, 2007, pp 53–69. 2. Baldridge D, Schwarze U, Morello R, Lennington J, Bertin TK, Pace JM, et al: CRTAP and LEPRE1 mutations in recessive osteogenesis imperfecta. Hum Mutat 2008;29:1435–1442.

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3. Editorial: Method of the Year 2009. Nat Methods 2010;7:1. 4. Ambrogini E, Almeida M, Martin-Millan M, Paik JH, Depinho RA, Han L, et al: FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice. Cell Metab 2010;11:136–146. 5. Johnson D, Kan SH, Oldridge M, Trembath RC, Roche P, Esnouf RM, et al: Missense mutations in the homeodomain of HOXD13 are associated with brachydactyly types D and E. Am J Hum Genet 2003;72:984–997. 6. Wilson LC, Leverton K, Oude Luttikhuis ME, Oley CA, Flint J, Wolstenholme J, et al: Brachydactyly and mental retardation: an Albright hereditary osteodystrophy-like syndrome localized to 2q37. Am J Hum Genet 1995;56:400–407. 7. Bastepe M, Weinstein LS, Ogata N, Kawaguchi H, Juppner H, Kronenberg HM, et al: Stimulatory G protein directly regulates hypertrophic differentiation of growth plate cartilage in vivo. Proc Natl Acad Sci USA 2004;101:14794– 14799. 8. Parker EA, Hegde A, Buckley M, Barnes KM, Baron J, Nilsson O: Spatial and temporal regulation of GH-IGF-related gene expression in growth plate cartilage. J Endocrinol 2007;194:31–40. 9. O’Shea PJ, Bassett JH, Sriskantharajah S, Ying H, Cheng SY, Williams GR: Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor 1 or . Mol Endocrinol 2005;19:3045–3059. 10. Wang L, Shao YY, Ballock RT: Thyroid hormone interacts with the Wnt/-catenin signaling pathway in the terminal differentiation of growth plate chondrocytes. J Bone Miner Res 2007;22:1988–1995. 11. Yang X, Matsuda K, Bialek P, Jacquot S, Masuoka HC, Schinke T, et al: ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry syndrome. Cell 2004;117:387–398. 12. St-Jacques B, Hammerschmidt M, McMahon AP: Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13:2072–2086. 13. Bush PG, Parisinos CA, Hall AC: The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy. J Cell Physiol 2008;214:621–629. 14. Russell JM: Sodium-potassium-chloride cotransport. Physiol Rev 2000;80:211–276. 15. Wang Y, Middleton F, Horton JA, Reichel L, Farnum CE, Damron TA: Microarray analysis of proliferative and hypertrophic growth plate zones identifies differentiation markers and signal pathways. Bone 2004;35:1273–1293. 16. Kiilerich P, Kristiansen K, Madsen SS: Cortisol regulation of ion transporter mRNA in Atlantic salmon gill and the effect of salinity on the signaling pathway. J Endocrinol 2007;194:417–427. 17. Sayers A, Tobias JH: Estimated maternal ultraviolet B exposure levels in pregnancy influence skeletal development of the child. J Clin Endocrinol Metab 2009;94:765–771. 18. Pasco JA, Wark JD, Carlin JB, Ponsonby AL, Vuillermin PJ, Morley R: Maternal vitamin D in pregnancy may influence not only offspring bone mass but other aspects of musculoskeletal health and adiposity. Med Hypotheses 2008;71:266– 269. 19. Morley R, Carlin JB, Pasco JA, Wark JD: Maternal 25-hydroxyvitamin D and parathyroid hormone concentrations and offspring birth size. J Clin Endocrinol Metab 2006;91:906–912. 20. Tiosano D, Hochberg Z: Hypophosphatemia: the common denominator of all rickets. J Bone Miner Metab 2009;27:392– 401. 21. Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, et al: The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 2000;103:41–50. 22. Richards JB, Papaioannou A, Adachi JD, Joseph L, Whitson HE, Prior JC, et al: Effect of selective serotonin reuptake inhibitors on the risk of fracture. Arch Intern Med 2007;167:188–194. 23. Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, Schutz G, et al: Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008;135:825–837.

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Reproductive Endocrinology Olle Söder and Lena Sahlin Paediatric Endocrinology Unit, Department of Women’s and Children’s Health, Astrid Lindgren Children’s Hospital; Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden

Reproductive endocrinology is a wide field encompassing areas such as germ stem cell biology, assisted reproduction technologies, reproductive behavior, gonadal physiology, puberty, endocrine pharmacology, and many other topics of great interest for pediatric endocrinologists. This years’ chapter includes references to recent papers on reproductive concerns of parental obesity, possible adverse effect caused by use of aromatase inhibitors for short stature, guidelines on endocrine treatment of transsexual adolescents, and many more. There were of course several other excellent papers published in these areas during the past year, some which we might have missed in our search of the area and yet others which were not possible to include due to space limitation. Although the present selection of papers obviously represents our own bias, we hope you will find them enjoyable to read and some provocative and helpful for your activity in the pediatric endocrinology arena.

Important for clinical practice/new concerns in obesity

Maternal obesity, inflammation, and fetal skeletal muscle development Du M, Yan X, Tong JF, Zhao J, Zhu MJ Department of Animal Science, University of Wyoming, Laramie, Wyo., USA [email protected] Biol Reprod 2010;82:4–12 Background: Maternal obesity coupled with Western-style and high-energy diets represents a special problem that can result in poor fetal development, leading to harmful, persistent effects on offspring, including predisposition to obesity and type 2 diabetes. Methods: Since skeletal muscle is the principal site for glucose and fatty acid utilization and composes 40–50% of total body mass, changes in the properties of offspring skeletal muscle and its mass resulting from maternal obesity may be responsible for the increase in type 2 diabetes and obesity. There is no net increase in the muscle fiber number after birth, therefore the fetal stage is crucial for skeletal muscle development. Its development involves myogenesis, adipogenesis, and fibrogenesis, which are all derived from mesenchymal stem cells. Shifting commitment of mesenchymal stem cells from myogenesis to adipogenesis and fibrogenesis will result in increased intramuscular fat and connective tissue, as well as reduced numbers of muscle fiber and/or diameter, all of which have lasting negative effects on offspring muscle function and properties. Results: Maternal obesity leads to low-grade inflammation, changing the commitment of mesenchymal stem cells in fetal muscles through several possible mechanisms: (1) inflammation down-regulates wingless and int (WNT) signaling, which attenuates myogenesis; (2) inflammation inhibits AMP-activated protein kinase, which promotes adipogenesis, and (3) inflammation may induce epigenetic modification through polycomb group proteins. Conclusion: More studies are needed to further explore the underlying mechanisms associated with maternal obesity, inflammation, and the commitment of fetal mesenchymal stem cells.

Maternal metabolism and obesity: modifiable determinants of pregnancy outcome Nelson SM, Matthews P, Poston L Division of Developmental Medicine, Reproductive and Maternal Medicine, Faculty of Medicine, University of Glasgow, Glasgow, UK [email protected] Hum Reprod Update 2010;16:255–275 Background: Obesity among pregnant women is highly prevalent worldwide and is associated in a linear manner with markedly increased risk of adverse outcome for mother and infant. The role of maternal metabolism in determining these outcomes and the potential for lifestyle modification are largely unknown. Methods: Studies were identified by searching PubMed, the metaRegister of clinical trials and Google Scholar without limitations. Sensitive search strategies were combined with relevant medical subject headings and text words. Results: Maternal obesity and gestational weight gain have a significant impact on maternal metabolism and offspring development. Insulin resistance, glucose homeostasis, fat oxidation and amino acid synthesis are all disrupted by maternal obesity and contribute to adverse outcomes. Modification of lifestyle is an effective intervention strategy for improvement of maternal metabolism and the prevention of type 2 diabetes and, potentially, gestational diabetes. Conclusion: Maternal obesity requires the development of effective interventions to improve pregnancy outcome. Strategies that incorporate a detailed understanding of the maternal metabolic environment and its consequences for the health of the mother and the growth of the child are likely to identify the best approach.

Maternal and paternal obesity has a known negative impact on fertility and pregnancy outcome. The two selected papers show a need of pre-maternity advice on lifestyle factors, since a reduction in weight will reduce the risk of negative effects on the child, but also increase the likelihood of becoming pregnant [1]. The impact of obesity and the low-grade inflammation on mesenchymal stem cells with potentially negative effects on offspring muscle function and metabolism need further investigation. A meta-analysis aimed at determining if paternal factors like semen parameters and reproductive hormones are affected by obesity [2]. The authors found no relation between increased BMI and semen parameters, but strong evidence of a negative relationship for testosterone, SHBG and free testosterone with increased BMI. A significantly higher number of embryos with a normal karyotype were found in miscarriages of overweight and obese women as compared to normal weight women. The results indicate that the excess risk of miscarriages in the overweight and obese population is independent of embryonic aneuploidy [3]. A recent comment in Biology of Reproduction [4] stated that the high incidence of obesity may aggravate adverse effects of environmental pollutants. Many of these environmental toxicants are lipophilic and thus stored and accumulated in fat tissue. An increased fat mass will therefore increase the toxic dose in obese individuals. Paradoxically, such toxic effects may increase during weight loss when compounds stored in fat are released to the systemic circulation. Combined interaction of reproductive toxicants and obesity is indicated to be additive [5], which predicts an increased need for assisted reproductive technologies (ART) in the future. Of great concern with such development is that more children may be at risk to be born with birth defects and possibly imprinting disorders [6]. A likely explanation for the abnormalities in children born after ART is the link to an altered embryonic epigenome [7]. Preventive measures at the population level and with focus on risk groups, like education in the importance of lifestyle factors, are of great importance for future reproductive health and favorable pregnancy outcome.

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Endocrine treatment of transsexual persons: an Endocrine Society clinical practice guideline Hembree WC, Cohen-Kettenis P, Delemarre-van de Waal HA, Gooren LJ, Meyer WJ, 3rd, Spack NP, Tangpricha V, Montori VM The Endocrine Society, Chevy Chase, Md., USA J Clin Endocrinol Metab 2009;94:3132–3154 Background: The aim was to formulate practice guidelines for endocrine care and treatment of transsexual persons including children. Methods: An evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe the strength of recommendations and the quality of evidence, which was low or very low. Results: Committees and members of The Endocrine Society, European Society of Endocrinology, European Society for Paediatric Endocrinology, Lawson Wilkins Pediatric Endocrine Society, and World Professional Association for Transgender Health commented on preliminary drafts of these guidelines. Conclusions: Transsexual persons seeking to develop the physical characteristics of the desired gender require a safe, effective hormone regimen that will (1) suppress endogenous hormone secretion determined by the person’s genetic/biologic sex and (2) maintain sex hormone levels within the normal range for the person’s desired gender. A mental health professional must recommend endocrine treatment and participate in ongoing care throughout the endocrine transition and decision for surgical sex reassignment. Because a diagnosis of transsexualism in a prepubertal child cannot be made with certainty, the authors do not recommend endocrine treatment of prepubertal children. They recommend treating transsexual adolescents (Tanner stage 2 or later) by suppressing puberty with GnRH analogues until age 16 years, after which cross-sex hormones may be given under strict criteria. They suggest suppressing endogenous sex hormones, maintaining physiologic levels of gender-appropriate sex hormones and monitoring for known risks in adult transsexual persons.

Psychosexual identity is probably the most important part of the individual’s sex and gender complex. Still, we know very little about its underlying biology and disorders, and the diagnostic criteria of gender identity disorders (GID) are based on a weak evidence base. This guidelines paper is therefore an important tool for pediatric endocrinologists caring for children with GID. An increasingly high number of adolescents with GID are referred to many centers, probably due to greater awareness of the condition and changes in healthcare-seeking behavior. Although the diagnostic work-up of GID is mainly psychiatric, the pediatric endocrinologist has an important role as a team member not only in the pharmacological treatment but also in the initial phase to exclude a cryptic DSD. GID patients and particularly adolescents create many challenges for the physician and it is therefore especially important to have evidence- and experience-based guidelines to consult for the daily routine.

Food for fertility...

Childhood nutrition and later fertility: pathways through education and pre-pregnant nutritional status Graff M, Yount KM, Ramakrishnan U, Martorell R, Stein AD Carolina Population Center, University of North Carolina at Chapel Hill, Chapel Hill, N.C., USA [email protected] Demography 2010;47:125–144 Background: Better childhood nutrition is associated with earlier physical maturation during adolescence and increased schooling attainment. However, as earlier onset of puberty and increased schooling can have opposing effects on fertility, the net effect of improvements in childhood nutrition on a woman’s fertility are uncertain.

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Methods: Using path analysis, the strength of the pathways was estimated between childhood growth and subsequent fertility outcomes in Guatemalan women followed prospectively since birth. Results: Height for age z score at 24 months was positively related to body mass index (BMI) and height in adolescence and to schooling attainment. BMI was negatively associated and schooling was positively associated with age at first birth. Total associations with the number of children born were positive with BMI and negative with schooling. Height was not related to age at first birth or the number of children born. Conclusions: In summary, childhood nutrition, as reflected by height at 2 years, was positively associated with delayed age at first birth and fewer children born. If schooling is available for girls, increased growth during childhood will most likely result in a net decrease in fertility.

This investigation links early childhood nutritional aspects with later fertility parameters in an underprivileged society. The paper shows that a well-nourished girl (as determined by height at 2 years) spends more time at school, postpones the birth of her first child and also that she will have fewer children. If the girl has an increased BMI (is overnourished) she will give birth earlier and also have more children, and spend less time at school. Although there are obvious questions as to who is the hen and who is the egg for several associations described, the long-term prospective nature of this paper makes it quite valuable.

An old concern maintained – bisphenol-A

Neonatal bisphenol-A exposure alters rat reproductive development and ovarian morphology without impairing activation of gonadotropinreleasing hormone neurons Adewale HB, Jefferson WN, Newbold RR, Patisaul HB Department of Biology, North Carolina State University, Raleigh, N.C., USA Biol Reprod 2009;81:690–699 Background: Developmental exposure to endocrine-disrupting compounds (EDCs) is hypothesized to adversely affect female reproductive physiology by interfering with the organization of the hypothalamic-pituitary-gonadal axis. Methods: The effects of neonatal exposure to two environmentally relevant doses of bisphenol-A (BPA) was compared with the ER␣-selective agonist PPT on the development of the female rat hypothalamus and ovary. Oil vehicle and estradiol benzoate (E2) were used as negative and positive controls, respectively. Results: Exposure to E2, PPT, or the low dose of BPA advanced pubertal onset. A total of 67% of females exposed to the high BPA dose were acyclic by 15 weeks after vaginal opening compared with 14% of those exposed to the low BPA dose, all of the E2- and PPT-treated females, and none of the control animals. Ovaries from the E2-treated females were undersized and showed no evidence of folliculogenesis, whereas ovaries from the PPT-treated females were characterized by large antral-like follicles, which did not appear to support ovulation. Severity of deficits within the BPA-treated groups increased with dose and included large antral-like follicles and lower numbers of corpora lutea. Fos induction in hypothalamic gonadotropic (GnRH) neurons after hormone priming was impaired in the E2- and PPT-treated groups but neither of the BPA-treated groups. Conclusion: These data suggest that BPA disrupts ovarian development but not the ability of GnRH neurons to respond to steroid-positive feedback.

The research field of endocrine-disrupting compounds is controversial and the risks of exposure to bisphenol-A have been particularly debated. This paper adds novel hard data to the field. Previously, bisphenol-A has been shown to affect the regulation of vascular endothelial growth factor in rat uterine endothelial cells thus affecting fertility. In addition, the severity of reproductive tract deficits within neonatally bisphenol-A-treated animals increased with the dose of bisphenol-A and included large antral-like follicles and lower numbers of corpora lutea. The present results demonstrate that

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bisphenol-A disrupted ovarian development but not the ability of GnRH neurons to respond to steroid-positive feedback. Thus, this endocrine-disrupting compounds is primarily affecting fertility at the gonadal level. A recent review by Hunt et al. [8] discusses, in light of the ‘bisphenol-A saga’, the importance to analyze environmental factors to determine their potential effects on reproduction in mammals – in order to preserve reproductive health.

Food for thought Sex without reproduction – reproduction without sex

Sex and reproduction: an evolving relationship Benagiano G, Carrara S, Filippi V Department of Obstetrics and Gynaecology, Sapienza University, Rome, Italy [email protected] Hum Reprod Update 2010;16:96–107 Background: Technological advances now allow for both sex without reproduction and reproduction without sex. This review summarizes social and ethical commentaries on the new relationship between sex and reproduction. Methods: This is a literature study where a systematic search was made using PubMed, Medline, ScienceDirect, classic books, Google and/or religious websites. The search focused on publications between 1975 and 2009, but some older materials were also utilized. Results: The classic picture of sex for reproduction and bonding between mating partners is increasingly being replaced by reproduction separate from sexual activity. Although not every advance in assisted reproduction resulted in a further separation from sexual intercourse, these two fundamental human activities are today increasingly carried out independently. Thus, reproduction is possible, not only without sex, but even through the intervention of more than two partners. The possibility of reproduction with only one or even no gametes, although highly controversial and not yet feasible, is nonetheless being investigated. Conclusion: Technological advances in the field of reproductive biology have enabled couples considered infertile to conceive and have healthy babies, causing a revolution in culture and customs. The independence of sex and reproduction is now established and in the future human reproduction may move even further away from the sexual act, an option definitely unacceptable to some ethicists.

Since more and more children are conceived in a laboratory setting rather than during parental intercourse, a need for increasing awareness is needed in examination of these children for adverse effects from advanced ART. The effect on the genome, transferred though generations, is a risk that should not be ignored [6, 7]. As fantastic as the prospect to give infertile couples the possibility of parenthood, the awareness of potentially adverse, maybe subtle, effects in their children must be brought to attention and any deviations reported. As a consequence of obesity as discussed above, an increased need for ART is evolving in parallel with the increased incidence of obesity in a large part of the world. The accumulation of endocrine disrupting compounds (EDCs) in fat and their potentially detrimental effect on fertility point to the risk of the combination of obesity, EDCs and ART to set the scene for a dim scenario for future generations.

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Concepts revised – a female (hormone) behind the man

The androgen receptor governs the execution, but not programming, of male sexual and territorial behaviors Juntti SA, Tollkuhn J, Wu MV, Fraser EJ, Soderborg T, Tan S, Honda S, Harada N, Shah NM Graduate Program in Neuroscience, University of California, San Francisco, Calif., USA Neuron 2010;66:260–272 Background: Testosterone and estrogen are essential for male behaviors in vertebrates. How these two sex steroid and their signaling pathways interact to control masculinization of the brain and behavior is not known. Circulating testosterone activates the androgen receptor (AR) and also serves as a substrate for local production of estrogen in the brain. Methods: AR was specifically deleted in the mouse nervous system. This approach permitted determination of the function of AR in sexually dimorphic behaviors in males while maintaining circulating testosterone levels within the normal range. Results: The AR mutant males were found to exhibit masculine sexual and territorial displays, but to have striking deficits in specific components of these behaviors. The mutant mice were for example less likely to initiate mating and spent less time fighting to protect their home cage, as compared to the wildtype mice. Conclusions: Taken together with the very limited expression of AR in the developing brain, these results indicate that testosterone most probably acts as a precursor to estrogen to masculinize the brain and behavior. The AR mutant mice exhibited striking deficits in the pattern and extent of male sexual and territorial behaviors. AR is not essential for the masculinization of mating, aggression and urine marking, but rather serves to amplify the display of this behavioral repertoire in males.

For decades the dominating concept has been that the male fetus is masculinized by a prenatal androgen surge [9]. This new evidence using genetically modified mice shows that animals lacking AR in the brain do develop male sexual and territorial behaviors. Thus, there is always an imposing female (factor) behind every man! On the same theme, see the paper by Wu et al. analyzed page 2 in the neuroendocrinology chapter.

Stem cells are here to stay

A signaling principle for the specification of the germ cell lineage in mice Ohinata Y, Ohta H, Shigeta M, Yamanaka K, Wakayama T, Saitou M Center for Developmental Biology, RIKEN Kobe Institute, Minatojima-Minamimachi, Chuo-ku, Japan Cell 2009;137:571–584 Background: Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Methods and Results: The authors demonstrate that germ cell fate in the epiblast is a direct consequence of BMP4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). BMP8b from the ExE restricts AVE development, thereby contributing to BMP4 signaling. In addition, Wnt3 in the epiblast ensures its responsiveness to BMP4. Serum-free, defined cultures revealed that, in response to BMP4, competent epiblasts uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquire germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Conclusions: Induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, this study presents a strategy for reconstituting the mammalian germ cell lineage in vitro.

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A novel approach for the derivation of putative primordial germ cells and Sertoli cells from human embryonic stem cells Bucay N, Yebra M, Cirulli V, Afrikanova I, Kaido T, Hayek A, Montgomery AM Whittier Institute, Department of Pediatrics, University of California, San Diego, La Jolla, Calif., USA Stem Cells 2009;27:68–77 Background: In vitro gametogenesis established from stem cells without use of viral vectors and genetic manipulation is highly warranted for regenerative medicine purposes in humans, e.g. to preserve fertility in children receiving gonadotoxic chemotherapy for cancer. A number of recent studies have shown that gametes can be derived from murine embryonic stem cells (mESCs) but simple approaches to for derivation of such cells in humans are yet to be demonstrated. Methods: Using human ESCs, the authors describe a novel method for rapid derivation and enrichment of primordial germ cells (PGCs) and Sertoli cells. The methodology does not require genetic manipulation or complex three-dimensional culture. Results: The authors determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions, these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli-like cells, known to provide trophic support and immunoprotection to developing germ cells. Conclusions: The putative Sertoli-germ cell co-cultures generated in this study may ultimately be developed to establish autologous human gametogenesis as a mean to rescue male fertility in selected cases with gonadal damage.

The above two studies cited are examples of the continuous contribution and impact of the stem cell field to our understanding of the early steps of sexual differentiation and germ cell development. This also keeps open and widens the roads to future exploitation in human reproductive medicine. Results first obtained in mouse models are now being increasingly translated into human systems, with success in most cases so far. Although it is still a long journey before the efficacy and safety of these novel cell therapy methods will be ascertained for clinical use in human medicine, the relatively simple and straightforward approaches used will facilitate their clinical implementation. It is therefore important for the clinical pediatric endocrinologist to be well informed and updated on these continuous developments.

New genes – sex determination

New candidate genes identified for controlling mouse gonadal sex determination and the early stages of granulosa and Sertoli cell differentiation Bouma GJ, Hudson QJ, Washburn LL, Eicher EM The Jackson Laboratory, Bar Harbor, Me., USA [email protected] Biol Reprod 2010;82:380–389 Background: Mammalian gonadal sex-determining (GSD) genes are expressed in a unique population of somatic cells that differentiate into granulosa cells in XX gonads or Sertoli cells in XY gonads. The ability to efficiently isolate these somatic support cells (SSCs) during the earliest stages of gonad development would facilitate identifying (1) new candidate GSD genes that may be involved in cases of unexplained abnormal gonad development and (2) genes involved in the earliest stages of granulosa and Sertoli cell differentiation. Methods: A unique mouse model was developed, carrying two transgenes that allow XX and XY mice to be distinguished as early as embryonic day 11.5 (E11.5) and allow SSCs to be isolated from undifferen-

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tiated (E11.5) and early differentiated (E12.5) fetal gonads. The Mouse Genome 430v2.0 GeneChip (Affymetrix) was used to identify transcripts exhibiting a sexual dimorphic expression pattern in XX and XY isolated SSCs. Results: The analysis revealed previously unidentified sexually dimorphic transcripts, including low-level expressed genes such as Sry, a gene not identified in other microarray studies. Multi-gene real-time PCR analysis of 57 genes verified that 53 were expressed in fetal gonads in a sexually dimorphic pattern, and whole-mount in situ hybridization analysis verified 4930563E18Rik, Pld1, and Sprr2d are expressed in XX gonads, and Fbln2, Ppargc1a, and Scrn1 are expressed in XY gonads. Conclusion: The data provide a comprehensive resource for the spatial-temporal expression pattern of genes that are part of the genetic network underlying the early stages of mammalian fetal gonadal development, including the development of granulosa and Sertoli cells. In XY mammalian embryos, including humans, initial expression of Sry occurs in Sertoli cell precursors in the gonadal anlagen, which thereafter develop into testes. In the absence of functional Sry the same precursor cells develop into granulosa cells determining the ovary. However, there are still other molecular events and genes involved in sex determination yet to be discovered. Most DSD cases with gonadal dysgenesis not caused by numerical chromosome aberrations have still an unknown origin. In this study employing an elegantly created mouse model, the authors detected more than 50 genes that were expressed in early development in fetal gonadal tissue in a sexually dimorphic fashion. Some of these identified genes can be studied for a role in the pathogenesis of human DSDs.

New mechanisms

The ovary Basic biology and clinical implications Richards JS, Pangas SA Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Tex., USA [email protected] J Clin Invest 2010;120:963–972 Background: The classical view of ovarian follicle development is that it is regulated by the hypothalamicpituitary-ovarian axis, in which gonadotropin-releasing hormone (GnRH) controls the release of the gonadotropic hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and that ovarian steroids exert both negative and positive regulatory effects on GnRH secretion. More recent studies in mice and humans indicate that many other intra-ovarian signaling cascades affect follicular development and gonadotropin action in a stage- and context-specific manner. Methods: Review paper with update on current status of our understating of ovarian function and folliculogenesis and their translational implications. Results and Conclusions: Evidence from mutant mouse models and clinical observations indicate that some of the most powerful intra-ovarian regulators of follicular development include the TGF-␤/SMAD, WNT/ FZD/␤-catenin, and RAS/ERK1/2 signaling pathways and the FOXO/FOXL2 transcription factors.

Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, Treier AC, Klugmann C, Klasen C, Holter NI, Riethmacher D, Schutz G, Cooney AJ, Lovell-Badge R, Treier M Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Cell 2009;139:1130–1142 Background: In mammals, the transcription factor SRY, encoded by the Y chromosome, is normally responsible for triggering the indifferent gonads to develop as testes rather than ovaries. However, testis differentiation can occur in its absence. Methods: Inducible deletion of Foxl2 in adult ovarian follicles.

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Results: The forkhead transcriptional regulator FOXL2, as a single factor, was shown to be required to prevent transdifferentiation of an adult ovary to a testis. Deletion of Foxl2 leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9. Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY male littermates. Conclusions: Maintenance of the ovarian phenotype is an active process throughout life. They might also have important medical implications for the understanding and treatment of some disorders of sexual development in children and premature menopause in women.

These two papers on the ovary are excellent updates on important basic mechanisms and clinical implications of ovarian development and function. The original paper on FOXL2 published in Cell presents seminal work demonstrating that the ovarian-specific gene FOXL2 has a role to suppress testis transdifferentiation in the adult ovary, i.e. that females continue to fight against their inner males throughout life [10]. Thus, a single gene deletion could induce male transformation – the old dogma that the testis and ovary are terminally differentiated tissues is not any longer valid. This paper was also commented on in Nature Medicine [11], where Foxl2’s possible impact on the polycystic ovary syndrome (PCOS) is discussed. The transient knockout of FOXL2 expression results in ovaries that, as in PCOS, produce primarily androgens rather than estrogens.

New methodology – can you be too sensitive?

Assessment of circulating sex steroid levels in prepubertal and pubertal boys and girls by a novel ultrasensitive gas chromatography-tandem mass spectrometry method Courant F, Aksglaede L, Antignac JP, Monteau F, Sorensen K, Andersson AM, Skakkebaek NE, Juul A, Bizec BL Laboratoire d’Etude des Residus et Contaminants dans les Aliments, Unité Sous Contrat 2013, Institut National de la Recherche Agronomique, Ecole Nationale Vétérinaire de Nantes, Nantes, France J Clin Endocrinol Metab 2010;95:82–92 Background: Estrogens and androgens play key roles for pubertal onset and sexual maturation. Most currently used immunoassays are not sensitive enough to accurately measure the low circulating levels of sex steroids in children without any signs of puberty. However, this does not exclude that sex steroids have important biological roles in prepubertal children. The aim of the study was to accurately determine levels of sex steroid hormones and their metabolites in serum of healthy children before any physical signs of puberty and to evaluate possible sex differences. Methods: Total (unconjugated plus conjugated) serum levels of 17␤-testosterone, 17␣-testosterone, 5␣-dihydrotestosterone, 5␤-dihydrotestosterone, androsterone, etiocholanolone, estradiol, and estrone measured by an ultrasensitive method based on gas chromatography-tandem mass spectrometry in samples from 81 healthy school children (42 boys) without any signs of puberty. For comparison, 48 pubertal children were studied. Results: 17␤-Estradiol levels in prepubertal boys were undetectable or extremely low (median 8 years), girls had significantly higher androsterone (4.07 vs. 1.45 nmol/l, p < 0.05), etiocholanolone (5.45 vs. 1.95 nmol/l, p < 0.0001), 5␣-dihydrotestosterone (0.11 vs. 16,000 individuals from eight European cohorts. These deletions were absent from healthy non-obese controls and accounted for 0.7% of the morbid obesity cases (BMI ≥40 or BMI-SDS ≥4). Conclusion: These data demonstrate the potential importance of rare variants with strong effects in common diseases as it could be shown here for a CNV in 16p11.2 for obesity.

These two studies show a promising strategy for identifying missing heritability in obesity (and other complex traits). Cohorts with extreme phenotypes are likely to be enriched for rare variants, with increased chance of discovery. The loci identified by this strategy may be subsequently analyzed and reveal additional rare variants that further contribute to the missing heritability. It seems to be very productive to combine ‘the power of the extreme’ [40]: small, well-phenotyped cohorts with targeted follow-up and large population cohorts. Strategies aimed at looking for rare variants near common susceptibility loci may well prove to be fruitful in other common complex disease. The study by Bochukova et al. showed that a deletion of 16p11.2 is associated with highly penetrant familial severe early-onset obesity and severe insulin resistance. The data suggest that although the contribution of other genes or non-coding genetic material cannot be excluded, the phenotype is consistent with a role for SH2B1 in human energy homeostasis and glucose metabolism. The prevalence of the SH2B1 containing deletion in patients with severe early-onset obesity was significantly greater than in controls. SH2B1 encodes an adaptor protein involved in leptin and insulin signaling. Disruption of SH2B1 in mice results in obesity and severe insulin resistance [39].

Molecular regulation of energy expenditure

Sarcolemmal ATP-sensitive K+ channels control energy expenditure determining body weight Alekseev AE, Reyes S, Yamada S, Hodgson-Zingman DM, Sattiraju S, Zhu Z, SierraA, Gerbin M, Coetzee WA, Goldhamer DJ, Terzic A, Zingman LV Department of Medicine, Mayo Clinic, Rochester, Minn., USA Cell Metab 2010;11:58–69 Background: Energy-conserving mechanisms promote obesity in an environment of hyperalimentation and sedentary lifestyle. Comprehension of these mechanisms could help to interfere with their efficiency to advance obesity treatment and prevention. ATP-sensitive K+ channels are involved in setting muscle energy expenditure. Methods: Two mouse models of KATP channel deficiency were characterized in terms of energy metabolism. Results: The disruption of KATP channel function raised energy expenditure in both cardiac and skeletal muscle, while locomotor activity and blood substrate availability were unaltered. As a consequence of this fuel metabolism inefficiency, glycogen and body fat depots were reduced, leading to a lean phenotype. Under high-fat diet, the phenotype of a reduced body weight of KATP channel-deficient mice persisted. However, this obesity retardation was accompanied by a decreased workload endurance. Conclusions: Downregulation of sarcolemmal KATP channels could provide a novel option against obesity by interfering with muscle energy efficiency.

KATP channels are hetero-octameric proteins composed of inwardly rectifying K+ channel (Kir6.x) and sulfonylurea receptor (SUR) subunits. Different combinations of Kir6.x and SUR subunits comprise KATP channels with distinct electrophysiological and pharmacological properties. Coupling of phos-

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photransfer pathways with KATP channels permits a transduction of nucleotide fluxes into changes in membrane excitability, matching energy demands with metabolic resources. KATP channels are expressed in various tissues including heart, kidney, pancreatic ␤ cells, central neurons and skeletal and smooth muscle [41], there functioning as metabolic sensors assigned to protective responses under severe energy insult. Former studies with Kir6.2-deficient mice suggest a role for these channels in skeletal muscle glucose uptake [42]. The present study describes a novel role for sarcolemmal KATP channels under non-stressed, physiological conditions. Lack of these channels led to an increase in energy expenditure which resulted in the decrease of body glycogen and fat stores promoting a lean phenotype, even under high-fat diet. The authors propose that the protective effect of the downregulation of KATP channels may be a novel option for obesity therapy. However, the described reductions in workload endurance will limit this approach, for instance by interfering with lifestyle intervention programs.

Fyn-dependent regulation of energy expenditure and body weight is mediated by tyrosine phosphorylation of LKB1 Yamada E, Pessin JE, Kurland IJ, Schwartz GJ, Bastie CC Department of Medicine, Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, N.Y., USA [email protected] Cell Metabolism 2010;11:113–124 Background: Fyn null mice show an increased fatty acid oxidation and energy expenditure resulting in a reduced adipose tissue mass and enhanced insulin sensitivity. Fyn is a negative regulator of fatty acid oxidation through inhibition of the AMP-dependent protein kinase (AMPK). AMPK is regarded as an energy sensor. It is regulated directly by the intracellular AMP/ATP ratio. In a state of caloric excess, AMPK is inactive, resulting for instance in an increased fatty acid storage. An upstream kinase activator of AMPK in peripheral tissue is LKB1 which is inactive as long as it is localized in the nucleus. Methods: The authors inhibited Fyn kinase with SU6656 in WT mice. They determined whole-body energy expenditure via indirect calorimetry and total fat mass by magnetic resonance. Fatty acid oxidation was assessed in isolated skeletal muscles. Additionally, they treated murine muscle and adipocyte cell lines with SU6656 followed by localization studies of LKB1. Validation experiments were performed with genetic approaches. Results: Selective pharmacological inhibition of Fyn in wild-type mice resulted in a similar phenotype as Fyn-null mice (increased fatty acid oxidation, elevated energy expenditure, lean). The authors showed that Fyn-dependent phosphorylation of LKB1 is responsible for the nuclear localization of LKB1. Inhibition of Fyn kinase results in a reduced rate of nuclear import of LKB1. A higher amount of LKB1 in the cytoplasm leads to activation of AMPK, thereby increasing the fatty acid oxidation and the energy expenditure. Conclusion: The positive metabolic effects seen in Fyn-null mice (decreased adiposity, increased energy expenditure) can be reproduced by the acute pharmacological inhibition of Fyn activity. These data highlight the therapeutic potential of inhibiting Fyn kinase signaling for obesity and its related disorders.

By inhibiting the Fyn kinases, an important player in energy metabolism, the authors showed an increased energy expenditure and increased fatty acid oxidation leading to a decreased fat mass in mice. These effects are similar to those they already observed in a knockout mouse for Fyn [43]. The finding of Yamada and colleagues’ suggest that this enzyme which belongs to the Src family of nonreceptor tyrosine kinases might offer a useful target for obesity therapy. However, the inhibitor SU6656 used in the present study is not a drug candidate. Both Fyn kinase as well as the downstream target of Fyn, AMPK, are important in the brain besides their action in fat and muscle. Therefore, it will be necessary to find something acting only on adipose tissue and muscle. Another possible way might be to manipulate the Fyn kinase gently with nutrients. For instance, epigallocatechin gallate which can be found in green tea inhibits Fyn kinase [44] and mimics thereby the action of the SU6656. Green tea has already been associated with weight loss which was explained by thermogenic effects probably partly due to the caffeine in the green tea [45–47]. The role of Fyn in this context has now to be considered. Taken together, this publication shows a new possible target for obesity therapy – the Fyn kinase. But due to its important role in the brain as well, more research has to be done. It will be essential to map

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all regulatory elements of this signaling pathway to identify its regulatory actions and to elucidate how this pathway functionally integrates with other pathways to further support the potential of Fyn as a drug target for the treatment of obesity.

Gs␣ deficiency in adipose tissue leads to a lean phenotype with divergent effects on cold tolerance and diet-induced thermogenesis Chen M, Chen H, Nguyen A, Gupta D, Wang J, Lai EW, Pacak K, Gavrilova O, Quon MJ, Weinstein S Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Md., USA Cell Metabolism 2010;11:320–330

Gs, a G-protein-mediating receptor-stimulated cAMP production, is involved in adipogenesis and adipose tissue function. Mutations of this protein lead to an obesogenic phenotype in both mice and patients with Albright hereditary osteodystrophy (AHO). Methods: An adipose-specific Gs-deficient mouse model was generated and analyzed in terms of adipose function and thermogenic properties. Results: Heterozygeous animals did not show an obvious phenotype, indicating that obesity seen in AHO is not caused by an adipose-specific Gs deficiency. Homozygotes displayed a strong reduction of adipose tissue, pointing to a role of Gs in adipogenesis. This came along with impaired cold tolerance and reduced brown adipose tissue (BAT) response to sympathetic stimuli. However, diet-induced thermogenesis and fatty acid oxidation in skeletal muscle were increased. Under high-fat diet, wild-type mice displayed enhanced sympathetic nerve activity in muscle, but not in BAT. Conclusion: The results of this study indicate that cold- and diet-induced thermogenesis occurs in different tissues. According to these data, BAT is not the tissue responsible for diet-induced thermogenesis. Background:

Brown adipose tissue (BAT) is the site of active glucose uptake in humans as recently demonstrated by PET/CT imaging. This has led to a renewed interest in the function of BAT thermogenesis in the regulation of body weight. Diet-induced thermogenesis (DIT) results from increased energy expenditure secondary to changes in nutrient uptake and is partly mediated by the sympathetic nervous system (SNS). SNS induced thermogenesis in BAT is partially mediated by Gs␣, a ubiquitously expressed G protein which couples hormone and neurotransmitter receptors to the generation of intracellular ATP. In the present study, the authors investigated the participation of this protein in thermogenesis in a Gs␣-deficient mouse model. These mice were cold-intolerant due to disruption of SNS signaling to BAT indicating a defect in cold-induced thermogenesis. Under high-fat diet, DIT was paradoxically increased in knock-out mice. In the absence of BAT thermogenesis this indicates that another tissue must be responsible for DIT, suggestively the skeletal muscle. DIT is due to an increase in energy expenditure after changes in nutrient uptake. So far, it was thought to result from adrenergic stimulation of BAT and to be mediated by BAT uncoupling protein UCP-1 [48]. However, UCP-1-ablated mice do not show an obesogenic phenotype unless they are housed at physiological temperatures [49, 50]. Altogether, this model questions the involvement of BAT tissue in DIT and points to additional tissue sites involved. To further confirm these data, studies with human brown adipocytes ex vivo are needed. The mechanisms for obesity in Gs␣ deficiency in humans remain elusive.

Initiation of myoblast to brown fat switch by a PRDM16–C/EBP-␤ transcriptional complex Kajimura S, Seale P, Kubota K, Lunsford E, Frangioni JV, Gygi SP, Spiegelman BM Dana-Farber Cancer Institute, Department of Cell Biology, Harvard Medical School, Division of Hematology/ Oncology, Beth Israel Deaconess Medical Center, Mass., USA Nature 2009;460:1154–1159

Recent publications by this group identified PRDM16 (PR domain zinc finger protein 16) as a factor of brown fat determination. It stimulates brown fat-selective gene expression while suppressing the expression of genes selective for white fat cells. Brown adipocytes arise from precursors expressing the myoblast lineage marker Myf5. However, the mechanism of this determination is currently unknown.

Background:

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Methods: The authors identified putative binding partners of PRDM16 by proteomic analysis of transcriptional complexes formed with wild-type and differentiation-incompetent mutant PRDM16. Subsequently, the expressions of these candidates in white and brown adipose tissue were analyzed and their function in the process of myoblast to brown fat conversion by PRDM16 was investigated. Results: Proteomic analyses and expression profiling revealed C/EBP-␤ as a binding partner of PRDM16. Overexpression of both factors in fibroblastic cells including mouse and human skin fibroblasts was sufficient to induce a fully functional brown fat program. Transplants of fibroblasts expressing both PRDM16 and C/EBP-␤ into mice led to the development of a fat pad with morphological and biochemical characteristics of brown adipose tissue. By using PET-CT scans, this fat pad was shown to act as a sink for glucose similar to endogenous brown adipose tissue. Conclusions: The data show that the induction of brown fat is under the control of PRDM16 by formation of a transcription complex with C/EBP-␤. This might offer novel therapeutic opportunities for obesity or type 2 diabetes.

In mammals, there are two types of adipose tissue with distinct functions. While white adipose tissue is the main storage organ of the body, brown adipose tissue regulates the body temperature by dissipating energy in the form of heat. This process is driven by uncoupling mitochondrial respiratory chain from ATP production by the protein UCP-1 (uncoupling protein 1). Since recruitment of brown adipose tissue is currently discussed as a strategy to eliminate excess energy in obesity, it is important to understand the molecular mechanisms of brown fat cell determination. Recent studies suggest different origins of brown and white adipose tissue. Atit et al. [51] proposed a common lineage for muscle and brown fat on the basis of their finding that specific Myf5-expressing cells of the dermomyotome give rise to both muscle and brown fat cells but not to white adipocytes. Beneath several transcriptional regulators, PRDM16 was recently described as a switch in brown fat cell development by inducing brown adipocyte differentiation from white preadipocytes and myoblastic precursors [52]. However, disruption of DNA binding of PRDM16 does not interfere with its ability to induce a brown phenotype [53]. The authors suggested therefore that PRDM16 acts predominantly by protein-protein interactions rather than by DNA interaction. Indeed, PRDM16 coactivates the transcriptional regulators PGC-1 and PPAR␥. By proteomic analyses, the authors could show here C/EBP-␤ as a binding partner of PRDM16 which is involved in adipocyte development. This study provides a detailed insight into brown adipocyte determination and introduces a new potential target for enhancing brown adipose tissue in the context of obesity therapy.

Gut microbiota The story goes on

Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5 Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S, Sitaraman SV, Knight R, Ley RE, Gewirtz AT Department of Pathology, Emory University, Atlanta, Ga., USA [email protected] Science 2010;328:228–231 Background: Recent research has linked the mammalian host-gut microbial relationship to human obesity. There is growing evidence for a role of gut microbiota in both chronic inflammation and insulin resistance. Methods: By using mice genetically deficient in Toll-like receptor 5 (TLR5), this study supports a direct relationship between impairment of the innate immune system, altered microbiota composition and development of metabolic syndrome. Mice embryos were transplanted into standard mice to standardize the microbiota in KO and control mice. Results: Genetically deficient in TLR5, T5KO mice had a mild colitis and paradoxically, at 20 weeks of age, 20% higher body mass index than WT mice. Serum triglycerides, cholesterol and blood pressure

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were increased compared to WT mice. In addition, a higher ex vivo production of proinflammatory cytokines such as interferon-␥ and interleukin-1␤ was observed in adipose tissue of T5KO than in WT mice. T5KO mice also exhibited hyperglycemia after fasting and an impaired glucose tolerance, elevated basal serum insulin levels and insulin resistance with high lipocalin-2 levels and increased number and size of pancreatic islets. Metabolic syndrome developed in T5KO after administering an 8-week high-fat diet. In contrast to WT mice, which also showed significant increase in body mass, T5KO became diabetic with fasting blood glucose concentrations above 120 mg/dl. They developed hepatic steatosis and inflammatory infiltrates in the pancreatic islets. The metabolic syndrome depends on hyperphagia since T5KO consume about 10% more food than WT littermates. Twelve weeks of food restriction prevented many of the metabolic abnormalities in T5KO mice. The metabolic syndrome in T5KO mice is independent of the adaptive immune system and TLR2/ TLR4, but results from the gut microbiota. The gut microbiota by broad-spectrum antibiotics in 4-week-old mice lowered the bacterial load by 90% and corrected food intake, fat pad and fasting glucose. Even the gut microbiota from T5KO mice intragastrically transferred in 4-week old WT germ-free mice induced elevated food intake, weight gain and lower insulin sensitivity with increase of inflammatory cytokine production in these mice. Conclusion: Malfunctions of the innate immune system in TLR5-deficient mice alter the gut microbiota and contribute to the development of an increased fat mass and the metabolic syndrome in mice. Nutritional and genetic factors alter the intestinal microbiota and may predispose individuals to the metabolic syndrome. The influence of gut microbiota on nutrient absorption and metabolic regulation has been demonstrated in human and animal studies showing alterations of the gut microbiota [54, 55]. Obesity is associated with inflammation which might contribute to the increased risk of cardiovascular disease and type 2 diabetes. Innate immune dysfunction, such as Toll-like receptor deficiency, results in serum antibody production against commensal microbiota and disturbed host-commensal mutualism [56]. TLR5 is the main component of the innate immune system in the intestine. Surprisingly, Vijay-Kumar presented a TLR5 null mice phenotype with only mild colitis, but increased fat mass and typical signs of metabolic syndrome. This is the first link between malfunction of the innate immune system, changes in gut microbiota and the metabolic syndrome. Alterations in host-microbiota interactions may drive obesity and metabolic syndrome. Additional studies will show whether it is possible to change the microbiota in a way that ameliorates the metabolic profile in terms of food absorption or energy storage and hyperphagia.

Bariatric surgery in adolescents

Laparoscopic adjustable gastric banding in severely obese adolescents: a randomized trial O’Brien PE, Sawyer SM, Laurie C, Brown WA, Skinner S, Veit F, Paul E, Burton PR, McGrice M, Anderson M, Dixon JB FRACS, Centre for Obesity Research and Education, Monash University Medical School, Alfred Hospital, Melbourne, Vic., Australia [email protected] JAMA 2010;303:519–526 Background: Adolescents with extreme obesity is a serious health challenge. Extreme obesity in adolescents is associated with both immediate and late health effects. Bariatric surgery has been shown to be the only effective treatment in obese adults leading to sizeable weight reduction. There are only a few observative studies in adolescents applying bariatric surgery. The aim of the study therefore was to compare the outcomes of gastric banding with an optimal lifestyle program in adolescent obesity. Methods: Prospective, randomized, controlled study with 50 adolescents (age 14–18 years, BMI >35), follow-up visits during 2 years. Eligibility criteria included identifiable medical complications such as hypertension, metabolic syndrome, asthma, back pain, physical limitations such as an inability to play

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a sport, difficulties with activities of daily lining, or psychosocial difficulties such as isolation or low self-esteem and evidence of attempts to lose weight by lifestyle means more than 3 years. Results: In the gastric banding group a weight loss of 34.6 kg (30.2–39.0) representing an excess weight loss of 79% and a BMI z score change from 2.4 to 1.3 was achieved. The mean weight loss in the lifestyle group was 3.0 kg (2.1–8.1) representing excess weight loss of 13% and a BMI z score change from 2.4 to 23. In the gastric banding group a more favorable improvement in metabolic factors was observed as well as in quality of life. There were no perioperative adverse events. However, 8 operations (33%) were required in 7 patients for revisional procedures either for proximal pouch dilatation or tubing injury during follow-up. Conclusion: Gastric banding in obese adolescents resulted in much greater weight losses than lifestyle intervention and was associated with more favorable benefits to health and quality of life. This study shows that gastric banding in obese adolescents proves to be an effective intervention leading to substantial and durable reduction of obesity and to better health. It should however be mentioned that the gastric banding approach to weight loss is not a quick fix. For optimal effectiveness, it requires long-term supportive follow-up by trained health professionals. The need for additional surgery for enlargement of the stomach above the band or injury to the tubing is intrinsic to the gastric banding procedure. Eating small meals slowly is central to avoiding this problem after the gastric banding procedure. For adolescents, additional education and supervision of eating are necessary to reduce the need for revisions. Severe obesity in adolescents is associated with multiple serious diseases, impaired quality of life, and an increased risk for later cardiovascular and other diseases. This study confirms that lifestyle treatments can achieve weight loss and improvement in health for some individuals. Diligent application of these approaches should remain the first option for obese adolescents. However, the majority of patients do not respond to this approach. Therefore, laparoscopic adjustable gastric banding may be a future option for these patients. It also has been argued that adolescents with severe obesity need treatment during adolescence rather than deferring until adulthood. In the context of the study it should be mentioned that the Endocrine Society has published guidelines for bariatric surgery in obese adolescents [1].

Obesity prevention in schools No clarity about cost-effectiveness

The cost-effectiveness of Australia’s active after-school communities program Moodie ML, Carter RC, Swinburn BA, Haby MM Deakin Health Economics, Public Health Research Evaluation and Policy Cluster, Deakin University, Burwood, Vic., Australia [email protected] Obesity (Silver Spring). 2009, Nov 5. Epub ahead of print

The school is an ideal place for obesity prevention programs. So-called ‘after-school programs’ are therefore reasonable because the time after school lessons is an optimal time to increase the physical activity within sport programs and simultaneous reduce the common consumption of sugar and fatty snacks at this time of day. For more than 5 years the Australian government has supported the so-called active-after-school-communities (AASC) program within a comprehensive national initiated program for obesity prevention. Cost-effectiveness analysis, conducted by health economists using simulation and modeling not described here showed that the program is not cost-effective as such. The authors suggest to improve the cost-effectiveness by increasing the duration of physical activity and raising the number of participating school children.

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Reviews

Minireview: Molecular targets for obesity therapy in the brain Obici S University of Cincinnati, Obesity Research Center, Cincinnati, Ohio, USA [email protected] Endocrinology 2009;150:2512–2517

This review summarizes recent progress in the identification of the neural pathways that respond to peripheral signals of energy availability such as leptin and macronutrients. The control of energy balance is achieved through neural pathways that receive metabolic signals. These pathways integrate these signals and activate behavioral and metabolic responses in order to maintain constant body weight and energy stores. These homeostatic mechanisms are mediated by neurons in the hypothalamus. In addition, other areas of the brain are involved in hedonic rewarding, and motivational aspects of ingestive behavior which contribute to modulate energy balance. This review summarizes recent progress in understanding the neural mechanisms by which leptin and nutrients modulate energy balance. Several molecular pathways recognized as general sensors of energy flux are emerging as pivotal sensors of energy availability in the hypothalamus. The convergence of nutrients and hormonal signals on a number of signaling pathways in the hypothalamus suggests that several nutritional cues are sensed and integrated to regulate energy balance. The disruption of the sensing and integration of these neural signals might be a crucial factor that leads to the development of obesity. Furthermore, these pathways represent a likely target for therapeutic intervention.

Obesity and thyroid function Reinehr T. Department of Paediatric Nutrition Medicine, Vestische Hospital for Children and Adolescents, University of Witten/Herdecke, Datteln, Germany [email protected] Mol Cell Endocrinol 2010;316:165–171

Advanced understanding of central mechanisms controlling energy homeostasis and data from clinical research characterizing hormonal and metabolic phenotypes of obese humans has renewed scientific interest in exploring the role of thyroid function in obesity. Elevated levels of thyrotropin (TSH) with concurrently normal thyroxine (T4) and normal to slightly elevated levels of free triiodothyronine (fT3) are a common finding in obese children and adolescents, as well as in adults. Nonetheless, cause and consequences of the obesity-associated state of ‘subclinical hypothyroidism’ have not been fully elucidated. Here, published data since the 1970s on the impact of T3 on energy expenditure and T3 metabolism in states of over- and undernutrition are reviewed. Increased TSH and fT3 levels in obesity could be interpreted as an adaptational process increasing resting energy expenditure and therefore reducing the conversion of excess energy into fat stores. As a second focus, the review presents available data on the association of leptin and TSH levels and current hypotheses on possible interactions of TSH and leptin signaling. While identifying the exact molecular mechanisms linking obesity and thyroid function and learning how to manipulate thyroid hormone pathways is a rewarding task in obesity research, clinicians should be assured that at least thyroxine supplementation never has been – and probably never will be – a viable treatment option for obesity at any age. References 1. Wabitsch M, Horenburg S, Denzer C, von Schnurbein J, Keuper M, Tews D, Moss A, Posovszky C, Fischer-Posovszky P: Obesity and weight regulation; in Carel J-C, Hochberg Z (eds): Yearbook of Pediatric Endocrinology. Basel, Karger, 1990. 2. Wabitsch M, Fuchs M, Horenburg S, Denzer C, von Puttkamer J, Moss A, Lahr G, Fischer-Posovszky P: Obesity and weight regulation; in Carel J-C, Hochberg Z (eds): Yearbook of Pediatric Endocrinology. Basel, Karger, 2007, pp 119– 132. 3. Spalding KL, Arner E, Westermark PO, Bernard S, Buchholz BA, Bergmann O, et al: Dynamics of fat cell turnover in humans. Nature 2008;453:783–787. 4. Prins JB, O’Rahilly S: Regulation of adipose cell number in man. Clin Sci (Lond) 1997;92:3–11. 5. Green H, Kehinde O: An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 1975;5:19–27.

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6. Byberg L, Zethelius B, McKeigue PM, Lithell HO: Changes in physical activity are associated with changes in metabolic cardiovascular risk factors. Diabetologia 2001;44:2134–2139. 7. Volkow ND, Wise RA: How can drug addiction help us understand obesity? Nat Neurosci 2005;8:555–260. 8. Stice E, Spoor S, Bohon C, Small DM: Relation between obesity and blunted striatal response to food is moderated by TaqIA A1 allele. Science 2008;322:449–452. 9. Noble EP: Addiction and its reward process through polymorphisms of the D2 dopamine receptor gene: a review. Eur Psychiatry 2000;15:79–89. 10. Wang GJ, Volkow ND, Fowler JS: The role of dopamine in motivation for food in humans: implications for obesity. Expert Opin Ther Targets 2002;6:601–609. 11. Booth ML, Wilkenfeld RL, Pagnini DL, Booth SL, King LA: Perceptions of adolescents on overweight and obesity: the weight of opinion study. J Paediatr Child Health 2008;44:248–252. 12. Puhl RM, Moss-Racusin CA, Schwartz MB, Brownell KD: Weight stigmatization and bias reduction: perspectives of overweight and obese adults. Health Educ Res 2008;23:347–358. 13. American Medical Association: Diagnostic and Statistical Manual of Mental Disorders, ed 4 (DSM-IV). Washington, American Psychiatric Association Publishing, 1994. 14. Markou A, Koob GF: Construct validity of a self-stimulation threshold paradigm: effects of reward and performance manipulations. Physiol Behav 1992;51:111–119. 15. Rolls BJ, Rowe EA, Turner RC: Persistent obesity in rats following a period of consumption of a mixed, high energy diet. J Physiol 1980;298:415–427. 16. Ahmed SH, Kenny PJ, Koob GF, Markou A: Neurobiological evidence for hedonic allostasis associated with escalating cocaine use. Nat Neurosci 2002;5:625–626. 17. Markou A, Koob GF: Postcocaine anhedonia. An animal model of cocaine withdrawal. Neuropsychopharmacology 1991;4:17–26. 18. Kenny PJ, Chen SA, Kitamura O, Markou A, Koob GF: Conditioned withdrawal drives heroin consumption and decreases reward sensitivity. J Neurosci 2006;26:5894–5900. 19. Cottone P, Sabino V, Steardo L, Zorrilla EP: Opioid-dependent anticipatory negative contrast and binge-like eating in rats with limited access to highly preferred food. Neuropsychopharmacology 2008;33:524–535. 20. Llado I, Estrany ME, Rodriguez E, Amengual B, Roca P, Palou A: Effects of cafeteria diet feeding on ␤3-adrenoceptor expression and lipolytic activity in white adipose tissue of male and female rats. Int J Obes Relat Metab Disord 2000;24:1396–1404. 21. Fishburn CS, Elazar Z, Fuchs S: Differential glycosylation and intracellular trafficking for the long and short isoforms of the D2 dopamine receptor. J Biol Chem 1995;270:29819–29824. 22. Vanderschuren LJ, Everitt BJ: Drug seeking becomes compulsive after prolonged cocaine self-administration. Science 2004;305:1017–1019. 23. Wang GJ, Tomasi D, Backus W, Wang R, Telang F, Geliebter A, et al: Gastric distention activates satiety circuitry in the human brain. Neuroimage 2008;39:1824–1831. 24. Frank GK, Bailer UF, Henry SE, Drevets W, Meltzer CC, Price JC, et al: Increased dopamine D2/D3 receptor binding after recovery from anorexia nervosa measured by positron emission tomography and [11C]raclopride. Biol Psychiatry 2005;58:908–912. 25. Farooqi IS, Bullmore E, Keogh J, Gillard J, O’Rahilly S, Fletcher PC: Leptin regulates striatal regions and human eating behavior. Science 2007;317:1355. 26. Plagemann A, Harder T, Rake A, Waas T, Melchior K, Ziska T, et al: Observations on the orexigenic hypothalamic neuropeptide Y-system in neonatally overfed weanling rats. J Neuroendocrinol 1999;11:541–546. 27. Plagemann A, Heidrich I, Gotz F, Rohde W, Dorner G: Obesity and enhanced diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding. Exp Clin Endocrinol 1992;99:154–158. 28. Rodrigues AL, de Moura EG, Passos MC, Trevenzoli IH, da Conceicao EP, Bonono IT, et al: Postnatal early overfeeding induces hypothalamic higher SOCS3 expression and lower STAT3 activity in adult rats. J Nutr Biochem 2010, Epub ahead of print. 29. Davidowa H, Plagemann A: Decreased inhibition by leptin of hypothalamic arcuate neurons in neonatally overfed young rats. Neuroreport 2000;11:2795–2798. 30. McMillen IC, Robinson JS: Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. Physiol Rev 2005;85:571–633. 31. Kral JG, Biron S, Simard S, Hould FS, Lebel S, Marceau S, et al: Large maternal weight loss from obesity surgery prevents transmission of obesity to children who were followed for 2 to 18 years. Pediatrics 2006;118:e1644–e1649. 32. Patel JA, Patel NA, Thomas RL, Nelms JK, Colella JJ: Pregnancy outcomes after laparoscopic Roux-en-Y gastric bypass. Surg Obes Relat Dis 2008;4:39–45. 33. Wittgrove AC, Jester L, Wittgrove P, Clark GW: Pregnancy following gastric bypass for morbid obesity. Obes Surg 1998;8:461–464. 34. Ducarme G, Revaux A, Rodrigues A, Aissaoui F, Pharisien I, Uzan M: Obstetric outcome following laparoscopic adjustable gastric banding. Int J Gynaecol Obstet 2007;98:244–147. 35. Dixon JB, Dixon ME, O‘Brien PE: Birth outcomes in obese women after laparoscopic adjustable gastric banding. Obstet Gynecol 2005;106:965–972. 36. Marceau P, Kaufman D, Biron S, Hould FS, Lebel S, Marceau S, et al: Outcome of pregnancies after biliopancreatic diversion. Obes Surg 2004;14:318–324. 37. Wax JR, Wolff R, Cobean R, Pinette MG, Blackstone J, Cartin A: Intussusception complicating pregnancy following laparoscopic Roux-en-Y gastric bypass. Obes Surg 2007;17:977–979. 38. Guelinckx I, Devlieger R, Vansant G: Reproductive outcome after bariatric surgery: a critical review. Hum Reprod Update 2009;15:189–201. 39. Ren D, Li M, Duan C, Rui L: Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice. Cell Metab 2005;2:95–104. 40. Froguel P, Blakemore AI: The power of the extreme in elucidating obesity. N Engl J Med 2008;359:891–893. 41. Ashcroft FM: Adenosine 5⬘-triphosphate-sensitive potassium channels. Annu Rev Neurosci 1988;11:97–118. 42. Miki T, Minami K, Zhang L, Morita M, Gonoi T, Shiuchi T, et al: ATP-sensitive potassium channels participate in glucose uptake in skeletal muscle and adipose tissue. Am J Physiol 2002;283:E1178–E1184.

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43. Bastie CC, Zong H, Xu J, Busa B, Judex S, Kurland IJ, et al: Integrative metabolic regulation of peripheral tissue fatty acid oxidation by the SRC kinase family member Fyn. Cell Metab 2007;5:371–381. 44. He Z, Tang F, Ermakova S, Li M, Zhao Q, Cho YY, et al: Fyn is a novel target of (–)-epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation. Mol Carcinog 2008;47:172–183. 45. Thielecke F, Boschmann M: The potential role of green tea catechins in the prevention of the metabolic syndrome – a review. Phytochemistry 2009;70:11–24. 46. Shixian Q, VanCrey B, Shi J, Kakuda Y, Jiang Y: Green tea extract thermogenesis-induced weight loss by epigallocatechin gallate inhibition of catechol-O-methyltransferase. J Med Food 2006;9:451–458. 47. Dulloo AG, Seydoux J, Girardier L, Chantre P, Vandermander J: Green tea and thermogenesis: interactions between catechin-polyphenols, caffeine and sympathetic activity. Int J Obes Relat Metab Disord 2000;24:252–258. 48. Rothwell NJ, Stock MJ: A role for brown adipose tissue in diet-induced thermogenesis. Nature 1979;281:31–35. 49. Enerback S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper ME, et al: Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 1997;387:90–94. 50. Feldmann HM, Golozoubova V, Cannon B, Nedergaard J: UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality. Cell Metab 2009;9:203–209. 51. Atit R, Sgaier SK, Mohamed OA, Taketo MM, Dufort D, Joyner AL, et al: Beta-catenin activation is necessary and sufficient to specify the dorsal dermal fate in the mouse. Dev Biol 2006;296:164–176. 52. Seale P, Bjork B, Yang W, Kajimura S, Chin S, Kuang S, et al: PRDM16 controls a brown fat/skeletal muscle switch. Nature 2008;454:961–967. 53. Seale P, Kajimura S, Yang W, Chin S, Rohas LM, Uldry M, et al: Transcriptional control of brown fat determination by PRDM16. Cell Metab 2007;6:38–54. 54. Ley RE, Peterson DA, Gordon JI: Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 2006;124:837–848. 55. Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI: The human microbiome project. Nature 2007;449:804–810. 56. Slack E, Hapfelmeier S, Stecher B, Velykoredko Y, Stoel M, Lawson MA, et al: Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science 2009;325:617–620.

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Type 2 Diabetes Mellitus, Metabolic Syndrome, Lipids Orit Pinhas-Hamiel Pediatric Endocrinology and Diabetes Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat-Gan and Maccabi Juvenile Diabetes Center, Raanana, Sackler School of Medicine, Tel-Aviv University, Israel

Identifying the genetic variants that increase the risk of type 2 diabetes mellitus (T2DM) in humans continues to be a challenge. In the search to identify diabetes susceptibility genes, genome-wide association (GWA) studies have revealed not only new loci but also new possible underlying mechanisms and hypotheses of T2DM pathogenesis. The impact of T2DM during childhood on the early development of diabetes complications, including abnormalities in carotid structure and function, as well as the development and progression of microalbuminuria in children with T2DM, has been the subject of a number of recent studies. Research on metformin treatment as protection against cancer incidence and mortality in patients with T2DM is presented. Longitudinal follow-up has demonstrated a high risk of premature mortality among subjects who had features of the metabolic syndrome (MetS) during their childhood. A number of articles published this year have highlighted dilemmas in definitions of the MetS, as well as revelations and challenges [1, 2]. Rapid weight gain in newborns with intrauterine growth retardation and precocious puberty has both been found to be associated with the development of the MetS. Hyperlipidemia among children and adolescents continues to be on the front line, as expressed in an editorial entitled ‘a decade of progress’ [3] and as manifested by the emergence of new drugs for children with familial hyperlipidemia. Hyperlipidemia has been associated to another ‘hot topic’, vitamin D. Finally, we discuss the impact of the contraception pill, now celebrating its 50th anniversary on lipids.

New paradigms

Type 2 diabetes risk alleles are associated with reduced size at birth Freathy RM, Bennett AJ, Ring SM, Shields B, Groves CJ, Timpson NJ, Weedon MN, Zeggini E, Lindgren CM, Lango H, Perry JR, Pouta A, Ruokonen A, Hypponen E, Power C, Elliott P, Strachan DP, Jarvelin MR, Smith GD, McCarthy MI, Frayling TM, Hattersley AT Genetics of Complex Traits, Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, UK Diabetes 2009;58:1428–1433 Background: Low birth weight is associated with an increased risk of T2DM. The mechanisms underlying this association are unknown and may represent intrauterine programming or two phenotypes of one genotype. The fetal insulin hypothesis proposes that common genetic variants that reduce insulin secretion or action may predispose to both T2DM and also reduce birth weight, since insulin is a key fetal growth factor. Methods: Single-nucleotide polymorphisms (SNPs) at five recently identified T2DM loci (CDKAL1, CDKN2A/B, HHEX-IDE, IGF2BP2, and SLC30A8) were genotyped in 7,986 mothers and 19,200 offspring white Europeans. The association between maternal or fetal genotype at each locus and birth weight of the offspring was tested. Results: T2M risk alleles at the CDKAL1 and HHEX-IDE loci were associated with reduced birth weight when inherited by the fetus (21 g [95% CI 11–31], p = 2 × 10–5, and 14 g [4–23], p = 0.004, lower birth weight per risk allele, respectively). The 4% of offspring carrying four risk alleles at these two loci were 80 g (95% CI 39–120) lighter at birth than the 8% carrying none (ptrend = 5 × 10–7). There were no associations between birth weight and fetal genotypes at the three other loci or maternal genotypes at any locus.

Conclusions: These findings provide robust evidence that common disease-associated variants can alter size at birth directly through the fetal genotype.

Low birth weight is an established risk factor for the development of T2DM. An explanation based on the insulin resistance model suggests that metabolic adaptation to fetal undernutrition persists into adult life. According to this theory, fetal programming, which basically expresses plasticity for shortterm survival, results later in adult life in a range of metabolic abnormalities. Alternatively, according to the fetal insulin hypothesis, the same genetic variants that reduce insulin secretion or insulin sensitivity also reduce birth weight. Two observations support this hypothesis. First, patients with monogenic neonatal diabetes have reduced birth weights. Second, offspring of fathers who develop diabetes later in life have, on average, lower birth weights than those born to fathers who do not develop diabetes. This is consistent with the fact that the fetus inherits about 50% of the father’s genetic predisposition to diabetes, as well as the genetic predisposition to reduced fetal growth. Interestingly, the authors discuss that maternal genotypes cannot help us understand such inheritance, since the effect of maternal genes is confounded by that of maternal hyperglycemia on birth weight. To test the fetal insulin hypothesis, the authors investigated the relationship between the size of the fetus at birth and five known T2DM variants identified through genomic-wide association. Two risk alleles that have been associated with reduced ␤-cell function, CDKAL1 and HHEX-IDE, were associated with reduced birth weight. The authors hypothesized that reduced fetal insulin secretion in utero results in reduced fetal size at birth, and subsequently leads to T2DM. The example presented of two phenotypes for a common genotype is compelling. Nevertheless, in the editorial accompanying this article [4], Meier discusses the idea that the pathogenesis of T2DM likely involves a combination of several mechanisms with varying contributions in different people.

Genetic variation in GIPR influences the glucose and insulin responses to an oral glucose challenge Saxena R, Hivert MF, Langenberg C, Tanaka T, Pankow JS, Vollenweider P, Lyssenko V, Bouatia-Naji N, Dupuis J, Jackson AU, Kao WH, Li M, Glazer NL, Manning AK, Luan J, Stringham HM, Prokopenko I, Johnson T, Grarup N, Boesgaard TW, Lecoeur C, Shrader P, O’Connell J, Ingelsson E, Couper DJ, Rice K, Song K, Andreasen CH, Dina C, Kottgen A, Le Bacquer O, Pattou F, Taneera J, Steinthorsdottir V, Rybin D, Ardlie K, Sampson M, Qi L, van Hoek M, Weedon MN, Aulchenko YS, Voight BF, Grallert H, Balkau B, Bergman RN, Bielinski SJ, Bonnefond A, Bonnycastle LL, Borch-Johnsen K, Bottcher Y, Brunner E, Buchanan TA, Bumpstead SJ, Cavalcanti-Proenca C, Charpentier G, Chen YD, Chines PS, Collins FS, Cornelis M, Crawford GJ, Delplanque J, Doney A, Egan JM, Erdos MR, Firmann M, Forouhi NG, Fox CS, Goodarzi MO, Graessler J, Hingorani A, Isomaa B, Jorgensen T, Kivimaki M, Kovacs P, Krohn K, Kumari M, Lauritzen T, Levy-Marchal C, Mayor V, McAteer JB, Meyre D, Mitchell BD, Mohlke KL, Morken MA, Narisu N, Palmer CN, Pakyz R, Pascoe L, Payne F, Pearson D, Rathmann W, Sandbaek A, Sayer AA, Scott LJ, Sharp SJ, Sijbrands E, Singleton A, Siscovick DS, Smith NL, Sparso T, Swift AJ, Syddall H, Thorleifsson G, Tonjes A, Tuomi T, Tuomilehto J, Valle TT, Waeber G, Walley A, Waterworth DM, Zeggini E, Zhao JH, Illig T, Wichmann HE, Wilson JF, van Duijn C, Hu FB, Morris AD, Frayling TM, Hattersley AT, Thorsteinsdottir U, Stefansson K, Nilsson P, Syvanen AC, Shuldiner AR, Walker M, Bornstein SR, Schwarz P, Williams GH, Nathan DM, Kuusisto J, Laakso M, Cooper C, Marmot M, Ferrucci L, Mooser V, Stumvoll M, Loos RJ, Altshuler D, Psaty BM, Rotter JI, Boerwinkle E, Hansen T, Pedersen O, Florez JC, McCarthy MI, Boehnke M, Barroso I, Sladek R, Froguel P, Meigs JB, Groop L, Wareham NJ, Watanabe RM Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Mass., USA Nat Genet 2010;42:142–148 Background: Glucose levels 2 h after an oral glucose challenge are a clinical measure of glucose tolerance used in the diagnosis of T2DM. Methods: A meta-analysis of 9 genome-wide association studies (n = 15,234 non-diabetic individuals) and a follow-up of 29 independent loci (n = 6,958–30,620). Results and Conclusion: Variants at the gastric inhibitory polypeptide receptor (GIPR) locus associated with 2 h glucose level (rs10423928, ␤ (SEM) = 0.09 (0.01) mmol/l per A allele, p = 2.0 × 10–15) were identified. The GIPR A-allele carriers also showed decreased insulin secretion (n = 22,492; insulinogenic index, p = 1.0 × 10–17; ratio of insulin to glucose area under the curve, p = 1.3 × 10–16) and diminished incretin effect (n = 804; p = 4.3 × 10–4). Other variants at ADCY5 (rs2877716, p = 4.2 × 10–16), VPS13C (rs17271305, p = 4.1 × 10–8), GCKR (rs1260326, p = 7.1 × 10–11) and TCF7L2 (rs7903146, p = 4.2 ×

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10–10) were also associated with 2 h glucose. Of the three newly implicated loci (GIPR, ADCY5 and VPS13C), only ADCY5 was found to be associated with T2DM in collaborating studies (n = 35,869 cases, 89,798 controls, OR = 1.12, 95% CI 1.09–1.15, p = 4.8 × 10–18). The ‘incretin effect’ denotes the phenomenon by which plasma insulin levels increase more from oral glucose intake than from the same amount of glucose administered intravenously. The incretins are hormones secreted from the gastrointestinal tract into the circulation in response to nutrient ingestion. They potentiate glucose-stimulated insulin secretion from islet ␤-cells. Glucose-dependent insulinotropic polypeptide, also known as gastric inhibitory polypeptide (GIP), and glucagon-like peptide-1 (GLP-1), act in an additive manner and are considered responsible for 50–70% of the postprandial insulin responses. In patients with T2DM, however, their contribution was found to be less than 20%. Interestingly, despite the reduced insulinotropic effect in patients with T2DM, the secretion of GIP appears to be relatively unchanged, suggesting a possible problem with its receptor GIPR. Genomic-wide association (GWAS) has contributed to the identification of many T2DM-associated loci. In the current study, GWAS revealed 3 new loci associated with 2-h glucose: GIPR (gastric inhibitory polypeptide receptor, rs10423928; VPS13C [vacuolar protein sorting 13 homolog C]) and ADCY5 (adenylate cyclase). Investigations of the latter two loci did not reveal a role in early insulin secretion. The GIPR variant, the SNP rs10423928 A allele, however, was associated with increased 2-h glucose; a lower insulinogenic index, representing reduction in the early phase of insulin secretion; a lower ratio of insulin to glucose area under the curve, which is an integrated measure of insulin response over the 2-h OGTT, and a lower 2-h insulin level. As expected, GIPR variation was not associated with insulin measures of intravenous glucose tolerance test. Interestingly, deletion of GIPR in an animal model shows mild glucose intolerance and reduced insulin secretion in response to an oral glucose challenge, but normal response to an intraperitoneal glucose challenge. Finally, the rs10423928 A allele was moderately associated with an increased risk of T2D in 19,091 individuals with diabetes, compared with 38,508 individuals without diabetes. These data support the hypothesis that a defect of the GIPR could be part of the T2D pathophysiology [4].

New concerns

Youth with obesity and obesity-related type 2 diabetes mellitus demonstrate abnormalities in carotid structure and function Urbina EM, Kimball TR, McCoy CE, Khoury PR, Daniels SR, Dolan LM Department of Pediatrics, Cincinnati Children’s Hospital and University of Cincinnati, Cincinnati, Ohio, USA [email protected] Circulation 2009;119:2913–1919 Background: Adults with obesity or T2DM are at higher risk for stroke and myocardial infarction. Increased carotid intima-media thickness (cIMT) and stiffness are associated with these adverse outcomes. The aim of this study was to determine whether similar changes exist in youth with obesity and T2DM. Methods: Carotid ultrasound for cIMT measurement was performed, as well as measures of stiffness (the Young elastic modulus and ␤ stiffness index), anthropometric and laboratory values and blood pressure were measured in 182 lean, 136 obese, and 128 T2DM youth (aged 10–24 years). Results: Cardiovascular risk factors worsened from lean to obese to T2DM groups. T2DM subjects had greater cIMT than that in lean and obese subjects for the common carotid artery and bulb. For the internal carotid artery, cIMT measurements in both obese and T2DM groups were thicker than in the lean group. The carotid arteries were stiffer in obese and T2DM groups than in the lean group. Determinants of cIMT were group, group × age interaction, sex, and systolic blood pressure for the common carotid artery (r2 = 0.17); age, race, and systolic blood pressure for the bulb (r2 = 0.16); and age, race, sex, systolic blood pressure, and total cholesterol for the internal carotid artery (r2 = 0.21). Age, systolic blood pressure, and diastolic blood pressure were determinants of all measures of carotid

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stiffness, with sex adding to the Young elastic modulus (r2 = 0.23), and body mass index Z score, group, and group × age interaction contributing to the ␤ stiffness index (r2 = 0.31; all p < 0.0001). Conclusions: Youth with obesity and T2DM have abnormalities in carotid thickness and stiffness that should alert healthcare practitioners to address cardiovascular risk factors early to prevent an increase in the incidence of stroke and myocardial infarction.

Influence of duration of diabetes, glycemic control, and traditional cardiovascular risk factors on early atherosclerotic vascular changes in adolescents and young adults with type 2 diabetes mellitus Shah AS, Dolan LM, Kimball TR, Gao Z, Khoury PR, Daniels SR, Urbina EM Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Cincinnati, Ohio, USA [email protected] J Clin Endocrinol Metab 2009;94:3740–1745 Background: Carotid intima-media thickness (IMT) provides a mechanism for detecting early atherosclerosis. Factors that may contribute to early changes in carotid IMT in youth with T2DM were studied. Moreover, predictors of increased carotid IMT were sought. Methods: Demographic, anthropometric, laboratory data and carotid imaging were obtained in 129 youth of mixed ethnicity, ages 10–23 years. Results: Carotid IMT increased with higher glycosylated hemoglobin (HbA1c) levels and longer duration of diabetes. Regression modeling showed that HbA1c and duration of diabetes in the presence of traditional cardiovascular risk factors (male sex, LDL cholesterol, and blood pressure) were independent determinants of carotid IMT. Logistic regression analysis demonstrated that each 1% increase in HbA1c or each year increase in duration of T2DM is associated with approximately 30% increased odds of a thicker carotid IMT. Conclusions: Poorer glycemic control and longer disease duration have independent adverse effects on carotid IMT in youth with T2DM. These adverse effects appear to be more prominent in males.

‘For the thing which I did fear is come upon me, and that which I was afraid of hath overtaken me’ (Job chapter 3, verse 25). One of the main concerns with the new epidemic of ‘Diabesity’ is the secondary morbidity associated with these disorders, and mainly the appearance of complications early in life, subjecting affected people to many years of morbidity. These two articles describe early atherosclerotic changes in obese and T2DM patients. Although the clinical manifestations of cardiovascular disease appear in adulthood, it is well known that atherosclerosis begins already in childhood. Hyperlipidemia, obesity, hypertension, diabetes and smoking are risk factors for the acceleration of atherosclerosis both in adults and in children. While the development of atherosclerosis in children was initially based on autopsy studies, non-invasive techniques now enable detection of early anatomical and physiological changes. Carotid intima-media thickness (IMT) measurements, which have been used as a surrogate endpoint for the progression and regression of atherosclerotic cardiovascular disease since the 1990s, enable identification of target organ damage. An association between IMT and the presence and severity of atherosclerosis has been documented in adults. In children, IMT was shown to be significantly increased in those with familial hypercholesterolemia, morbid obesity, metabolic syndrome or type 1 diabetes mellitus (T1DM). The study by Urbina et al. is the first to examine the effect of T2DM on IMT among adolescents. Early changes in both vascular structure and function are demonstrated in obese adolescents, and more severely in those with T2DM. Shah et al.’s study investigated factors that may contribute to changes in carotid IMT. Of youth with T2DM, common carotid IMT was elevated in 13.4%, bulb IMT in 16.5%, and internal carotid IMT in 18.9%. These prevalence rates are concerning, as young adults with increased IMT have increased likelihood of myocardial infarction and stroke. Elevated carotid IMT was found to be associated with higher HbA1c concentrations and longer duration of T2DM. In addition, the traditional cardiovascular risk factors, including blood pressure, LDL cholesterol and male sex were also important determinants of carotid IMT in this population. These data establish HbA1c and duration of diabetes as independent factors in the progressive thickening of carotid IMT. However, there is light in the tunnel. Evidence that improvement in glucose control at an early age may reduce the progression of atherosclerosis should encourage clinicians to aggressive interventions for risk reduction.

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Predictive value of albuminuria in American-Indian youth with or without type 2 diabetes Kim NH, Pavkov ME, Knowler WC, Hanson RL, Weil EJ, Curtis JM, Bennett PH, Nelson RG Diabetes Epidemiology and Clinical Research Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Ariz., USA Pediatrics 2010;125:e844–851

To examine the prognostic significance of elevated albuminuria in youth with T2DM. Cross-sectional and prospective studies were conducted on Pima Indian youth aged 5–19 years at baseline between 1982 and 2007. Prevalence and sequential changes in the level of microalbuminuria (30 < or = albumin-to-creatinine ratio [ACR] < 300 mg/g) and macroalbuminuria (ACR > or = 300 mg/g) and incidence of macroalbuminuria were computed according to the presence or absence of T2DM. Results: The prevalence of microalbuminuria and macroalbuminuria was 6.5 and 0.6% in the 3,856 non-diabetic youth and 18.5 and 2.9% in the 103 youth with diabetes, respectively. 141 of 187 (75.4%) non-diabetic youth, but only 1 of 14 (7.1%) diabetic youth with an elevated ACR (> or = 30 mg/g) regressed to an undetectable or normal ACR (15 µIU/ml, were studied. An oral glucose tolerance test was performed on each patient, fasting and 2-hour post-load serum glucose and insulin levels were obtained as well as HbA1c, and 1,5-anhydroglucitol (insulin-resistant subgroup only) levels. All values and calculated HOMA-IR were used as predictors for exceeding various 2-hour BG cut-offs. Results: In the insulin-resistant subgroup, 3 (2%) patients had T2DM and 23 (12%) had IGT. Optimal sensitivity and specificity to detect T2DM were respectively 99 and 96% at HbA1c > or = 6.0%, and 96 and 88% at 1,5-anhydroglucitol 0.67 SDs) and these determinants. Methods: Observational study using longitudinal data collected in the Programming Factors for Growth and Metabolism (PROGRAM) study of 217 healthy participants, aged 18–24 years, including a relatively large sample of participants born small for gestational age and participants with short stature. The association of cardiovascular disease and T2DM with tempo of weight gain was assessed in a subgroup of 87 participants. Results: Weight gain in the first 3 months of life was inversely associated with insulin sensitivity (␤ –0.223; 95% CI –0.386 to –0.060) and serum high-density lipoprotein cholesterol level (␤ –0.053; 95% CI –0.090 to –0.016) and positively associated with waist circumference (␤ 1.437; 95% CI 0.066–

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2.808), acute insulin response (␤ 0.210; 95% CI 0.024–0.395), ratio of total cholesterol to high-density lipoprotein cholesterol (␤ 0.052; 95% CI 0.010–0.094), and level of triglycerides (␤ 0.066; 95% CI 0.003–0.129) in early adulthood. Rapid weight gain during the first 3 months of life resulted in a higher percentage of body fat, more central adiposity, and reduced insulin sensitivity in early adulthood than when slower weight gain occurred during the entire first year. Conclusion: Rapid weight gain in the first 3 months of life is associated with several determinants of cardiovascular disease and T2DM in early adulthood. Since the introduction of Barker’s hypothesis regarding the developmental origins of adult diseases, many epidemiological studies have reported a strong association between growth retardation in uterus and long-term adverse effects such as T2DM, coronary heart disease, hypertension and stroke. The above two articles show the period of postnatal catch-up growth, and not just fetal life, to be an important determinant of adult disease risk. In the first article, Beardsall et al. used models of monozygotic (MZ) and dizygotic (DZ) twins to explore the contribution of postnatal weight gain to markers of adult metabolic disease states, including adiposity, arterial blood pressure and fasting insulin levels. This elegant model enables estimation of the contribution of fetal genotype, maternal environment and postnatal weight gain. Both MZ and DZ twins, who were growth restricted at birth, showed catch-up growth in both weight and height by ages 7–11 years. Neither birth weight nor discordance between twin pairs was associated with markers of metabolic disease. Outcomes were strongly related to postnatal weight gain. The heritability of postnatal weight gain and fasting insulin were assessed at 80 and 65% respectively. While Beardsall et al.’s study did not investigate the exact timing of catch-up growth, Leunissen et al.’s study found that in the first 3 months of life, an increased gain in weight, relative to height, was associated with all the important determinants of the metabolic syndrome, specifically: increased waist circumference, low insulin sensitivity, low HDL and increased triglyceride levels in early adulthood. Moreover, the tempo of weight gain was also found important. Children with rapid weight gain within the first 3 months had higher percentages of body fat, central adiposity and reduced insulin sensitivity later. Although nutritional data was not available, the use of nutrient-enriched formulas may be one explanation for rapid weight gain. Anxious to see their ‘malnourished baby’ achieving normal weight percentiles as soon as possible, parents often implement aggressive dietary management with considerable caloric intake supplementation. In light of these findings, we should be cautious in our aims for rapid catch-up.

Prolonged juvenile states and delay of cardiovascular and metabolic risk factors: the Fels Longitudinal Study Sun SS, Schubert CM Department of Biostatistics, School of Medicine, Virginia Commonwealth University, Richmond, Va., USA [email protected] J Pediatr 2009;155:S7 e1–6 Background: The influence of a prolonged juvenile state on the onset of the metabolic syndrome, cardiovascular disease, and T2DM later in life was studied. Methods: Prolongation of a juvenile state as a retarded tempo of growth was determined by the timing of peak height velocity in each subject and relate the retarded tempo of growth to metabolic syndrome, cardiovascular disease, and T2DM later in life by use of serial data of 237 study participants (119 men and 118 women) enrolled in the Fels Longitudinal Study. Results: Children who matured early tended to have greater body mass index, waist circumference, and percent of body fat and were more likely to have adverse cardiovascular risk profiles than children who matured late. The differences in these risk factors between early and late maturers were significant for percent body fat, fasting plasma triglycerides, and fasting plasma insulin. Conclusions: The analyses disclosed a clear separation between early and late maturers in the appearance of these risk factors in young adulthood.

This study demonstrates the value and power of longitudinal studies in revealing predictors of certain conditions. The Fels Longitudinal Study began in 1929, after the president of Antioch College in Yellow Springs, Ohio, posed the question, ‘What makes people different?’ He believed that a longi-

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tudinal study from birth to adulthood would be required to answer this question. Samuel Fels, a Philadelphia businessman and philanthropist, established the Fels Research Institute. Data measured by the Fels Longitudinal Study has since become the core of numerous works. The current study is the first to elucidate the effects of maturation rate on metabolic variables and risk factors for cardiovascular disease over 40 years of lifespan. It is known that overweight children, particularly those of certain minority groups, those born small for gestational age, females with PCOS, and children with family histories of T2DM, are at risk for developing the MetS. Early maturation is now shown to be another risk factor for developing the MetS. Children who matured early tended to have greater BMI and waist circumference, a higher percentage of body fat, and elevated levels of fasting plasma triglycerides and fasting plasma insulin in adulthood compared with late maturers. As such, a new risk group for development of the MetS has been identified, for which careful follow-up is needed.

A novel, non-invasive 13C-glucose breath test to estimate insulin resistance in obese prepubertal children Jetha MM, Nzekwu U, Lewanczuk RZ, Ball GD Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alta., Canada J Pediatr Endocrinol Metab 2009;22:1051–1059 Background: Insulin resistance (IR) is an important risk factor for the development of T2DM in obese boys and girls. The objective of this cross-sectional study of obese prepubertal children (n = 39) was to compare estimates of IR using a novel, non-invasive technique (13C-glucose breath test) with common indices of IR derived from an oral glucose tolerance test (OGTT). Methods: For the 13C-glucose breath test, samples were collected before and 90 min after ingestion of 25 mg 13C-labeled glucose. For the OGTT, glucose and insulin samples were collected at 0, 15, 30, 45, 60, 90 and 120 min. The homeostatic model assessment of insulin resistance (HOMA-IR), quantitative insulin sensitivity check index (QUICKI), insulin area-under-the-curve (AUC), and sum-of-insulin were calculated as indices of IR. Results: A significant, but moderate, associations between the 13C-glucose breath test and fasting insulin (r = –0.50; p < 0.01), 2-hour insulin (r = –0.40; p