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 (<–1 SD) dependent on frequency scores for licking/grooming. Retrograde tracing of projections from the ventral tegmental area by stereotactic fluorogold injection into ventral tegmental area followed by oxytocin and fluorogold immunohistochemistry. In situ dopamine concentration was measured using electrochemical probes implanted in the nucleus accumbens. Drugs were injected directly into the ventral tegmental area. Histology was used to verify proper location of electrodes and cannulae. Results: Mothers that exhibit consistently increased pup LG (i.e. high LG mothers) by comparison with low LG mothers show increased oxytocin expression in the medial preoptic area and the paraventricular nucleus of the hypothalamus and increased projections of oxytocin-positive cells from both medial preoptic area and paraventricular nucleus of the hypothalamus to the ventral tegmental area. Direct infusion of oxytocin into the ventral tegmental area increased the dopamine signal in the nucleus accumbens. High compared with low LG mothers show greater increases in dopamine signal in the nucleus accumbens during bouts of pup LG, and this difference is abolished with infusions of an oxytocin receptor antagonist directly into the ventral tegmental area. Conclusions: These studies reveal a direct effect of oxytocin on dopamine release within the mesocorticolimbic dopamine system. This supports a role for oxytocin-dopamine interactions in the establishment and maintenance of social bonds.
Promoting social behavior with oxytocin in high-functioning autism spectrum disorders Andari E, Duhamel JR, Zalla T, Herbrecht E, Leboyer M, Sirigu A Centre de Neuroscience Cognitive, Unité Mixte de Recherche, Bron, France
[email protected] Proc Natl Acad Sci USA 2010;107:4389–4394 Background: Social adaptation requires specific cognitive and emotional competences. Individuals with high-functioning autism or with Asperger syndrome cannot understand or engage in social situations despite preserved intellectual abilities. Recently, it has been suggested that oxytocin, a hormone known to promote mother-infant bonds, may be implicated in the social deficit of autism. This study investigated the behavioral effects of oxytocin. Methods: Thirteen subjects with high-functioning autism spectrum disorder were entered in a simulated ball game where participants interacted with fictitious partners and received inhaled oxytocin. Decision behavior in the game, visual examination of faces and emotional response using a self-rated scale was assessed. Results: After oxytocin inhalation, patients exhibited stronger interactions with the most socially cooperative partner and reported enhanced feelings of trust and preference. Also, during free viewing of pictures of faces, oxytocin selectively increased patients’ gazing time on the socially informative region of the face, namely the eyes.
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Conclusions: During oxytocin treatment, patients respond more strongly to others and exhibit more appropriate social behavior and affect, suggesting a therapeutic potential of oxytocin through its action on a core dimension of autism.
The first of the papers above reports on the mechanism whereby oxytocin may affect maternal licking and grooming of pups. Within a population of normal rats, the authors were able to differentiate high and low grooming mothers by the number of oxytocin neurons in several hypothalamic nuclei projecting to the ventral tegmental area and showed that oxytocin acts directly on dopamine release in the nucleus accumbens. In addition, the authors mention submitted work that associates activity of dopamine-sensitive pathways (by functional MRI) to maternal responsivity to infant-related stimuli in humans. This work opens doors to the therapeutic use of oxytocin, especially since oxytocin has been implicated in the etiology of autism. Normal subjects that receive oxytocin are more inclined to trust other players and to even send them money in simulated investment games [14], confirming a pharmacological effect of oxytocin on human behavior. The second paper describes a study in which patients with high-functioning autism spectrum disorders (HF-ASD) received inhaled oxytocin. Patients with high-functioning autism spectrum disorders have normal language and intellectual abilities but avoid eye contact, have less spontaneous interaction with people and show impairments in understanding intentions of others. Indeed, these patients had reduced oxytocin plasma concentrations compared to normal subjects. In a simulated game, oxytocin-treated subjects scanned the eye region of the face more often and reported greater trust. These results are promising for the development of pharmacological strategies to increase social interaction and adaptation in patients with autism.
New paradigm
Functional amyloids as natural storage of peptide hormones in pituitary secretory granules Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, Rissman RA, Singru PS, Nilsson KP, Simon R, Schubert D, Eisenberg D, Rivier J, Sawchenko P, Vale W, Riek R Laboratory of Physical Chemistry, Eidgenössische Technische Hochschule, Zürich, Switzerland
[email protected] Science 2009;325:328–332 Background: Amyloid fibrils are highly organized cross--sheet-rich protein or peptide aggregates that are associated with pathological conditions including several neurodegenerative diseases such as Alzheimer’s disease and other conditions including type 2 diabetes. However, amyloids may also have a normal biological function, when they are termed ‘functional amyloids’ as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. The authors hypothesized that peptide and protein hormones in secretory granules may adopt an amyloid-like structure, and that this could explain most of their properties. Methods: 42 peptide and protein hormones from multiple species and organs and with a variety of different three-dimensional structures were selected. They were assayed for their capacity to form amyloids by the amyloid-specific dyes thioflavin T and Congo Red using luminescent conjugated polyelectrolyte probes, by the conformational transition into -sheet-rich structure measured by circular dichroism spectroscopy, and by the presence of fibrils in electron microscopy images. Furthermore, x-ray fiber diffraction was measured for a subset of hormones. Results: Ten of the 42 hormones showed considerable formation of amyloids. Given the possible involvement of glycosaminoglycans (GAGs) in the formation of both secretory granules and amyloid fibrils, the amyloid formation of all 42 peptides and proteins was monitored in the presence of low molecular weight heparin as a representative of GAGs. Most hormones (n = 31) formed amyloid fibrils after 2 weeks of incubation in the presence of heparin. Adrenocorticotropic hormone (ACTH) could not form amyloidlike aggregates on its own but did so in the presence of -endorphin, which is also processed from proopiomelanocortin and secreted together with ACTH in a regulated secretory pathway. These data were
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confirmed in the mouse pituitary tumor neuroendocrine cell line AfT20. Within the mouse pituitary, immunohistochemistry revealed the abundant presence of amyloids in the anterior and posterior pituitary. Co-localization of the amyloid-specific dye Thio S and the hormones ACTH, -endorphin, prolactin and GH in the anterior lobe, ACTH in the intermediate lobe, and oxytocin and vasopressin in the posterior lobe, were indicative that their storage in the secretory granules was extensively amyloid-like. Conclusion: This study reports that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross--sheet-rich conformation. Thus, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology encompassing processes such as granule formation including hormone selection, membrane surrounding and inert hormone storage, and subsequently the release of hormones from the granules. Secretory proteins and peptides are synthesized in the endoplasmic reticulum and Golgi and then stored in vesicles or secretory granules ready for release into the extracellular space. This paper presents a highly novel concept, and suggests the association of amyloid with hormones, which then form secretory granules. The authors suggest that, although amyloid has been associated with disease processes in the past, it may play a critical functional role in hormone storage and secretion. They propose that in the Golgi, amyloid aggregation of the prohormone is initiated spontaneously above a critical prohormone concentration and/or in the presence of helper molecules such as GAGs in parallel to a possible prohormone processing. Because the prohormone may aggregate less into an amyloid entity than its hormone counterpart, prohormone processing at critical hormone concentrations may initiate the aggregation. Amyloid aggregation thereby sorts the protein/peptide hormones into secretory granule cores, concentrates them to the highest density possible, and excludes nonaggregation prone constitutively secreted proteins. During the aggregation process, the hormone amyloids become surrounded by membrane, separate from Golgi, and form mature granules, leading to long-term storage. On signaling, secretory granules are secreted and the cross -sheet structure of the amyloid enables a controlled release of monomeric functional hormone. Whether the presence of functional amyloid impacts on disease processes such as autosomal dominant GHD and autosomal dominant diabetes insipidus, where hormone secretion is abnormal, remains to be established. The authors suggest a possible functional amyloid state of many endocrine hormones in secretory granules of the hypothalamus (e.g. CRF) and pancreas (e.g. somatostatin).
New mechanism – pituitary tumors
Use of the metallothionein promoter-human growth hormone-releasing hormone (GHRH) mouse to identify regulatory pathways that suppress pituitary somatotrope hyperplasia and adenoma formation due to GHRHreceptor hyperactivation Luque RM, Soares BS, Peng XD, Krishnan S, Cordoba-Chacon J, Frohman LA, Kineman RD Section of Endocrinology, Diabetes, and Metabolism, University of Illinois, Chicago, Ill., USA
[email protected] Endocrinology 2009;150:3177–3185 Background: Hyperactivation of the GHRH receptor or downstream signaling components is associated with hyperplasia of the pituitary somatotrope population, and may result in the formation of adenomas relatively late in life. This study aimed to assess the role of GHRH and somatostatin in pituitary tumor formation. Methods: Hyperplastic and adenomatous pituitaries of metallothionein promoter-human GHRH transgenic mice (4 and > 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 NFB, 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 NFB. 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 NFB 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 NFB 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 IB␣ and subsequent nuclear translocation of the p65 subunit of NFB 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 NFB 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 (<5.0 µg/ dl) on the newborn screening test. The average total thyroxine level in the remaining 34 subjects was 9.8 ± 3.4 µg/dl. Thyrotropin levels were within the reference range in all children, as expected but not adapted for those with low T4 levels. No differences were found in the two groups for birth history, jaundice (53% overall), hypoglycemia (36% overall), or micropenis (43% of boys). 57% of children had septo-optic dysplasia, and 98% had multiple pituitary hormone deficiencies. Children with an abnormal newborn screening results were initially examined by a pediatric endocrinologist at an average age of 4.6 ± 5.0 months, and children with normal newborn screening results were initially examined at an average age of 16.9 ± 26.7 months (p = 0.037). Conclusions: Most children with congenital central hypothyroidism have normal thyroid function at birth. Normal newborn screening results can be falsely reassuring and may contribute to a delay in diagnosis of hypopituitarism despite classic clinical features.
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A 7-year experience with low blood TSH cut-off levels for neonatal screening reveals an unsuspected frequency of congenital hypothyroidism Corbetta C, Weber G, Cortinovis F, Calebiro D, Passoni A, Vigone MC, Beck-Peccoz P, Chiumello G, Persani L Laboratory for Neonatal Screening, Buzzi Children Hospital, Milan, Italy
[email protected] Clin Endocrinol (Oxf) 2009;71:739–745 Background: The guidelines of the National Academy of Clinical Biochemistry [www.nabc.org] advocated the use of low bloodspot TSH (b-TSH) threshold for newborn screening of congenital hypothyroidism (CH) [9]. Objective: The authors wished to determine the impact on CH epidemiology and classification generated by the introduction of lower b-TSH cut-off than the on they previously used. Methods: Retrospective study of 629,042 newborns screened with b-TSH cut-offs of 12 (years 1999– 2002) or 10 mU/l (2003–2005). Results were compared with those virtually obtained with the previous cut-off (20 mU/l). Clinical re-evaluation after L-T4 withdrawal of a representative group of 140 CH children at 3–5 years was also performed. Results: Low b-TSH cut-offs allowed the detection of 435 newborns with confirmed CH (incidence 1:1,446). 45% of CH infants, including 12/141 dysgenesis, would have been missed using the 20 mU/l cut-off. In contrast to current classification, 32% CH newborns had thyroid dysgenesis and 68% had a gland in situ (GIS). Premature birth was present in 20% of cases being associated with a 3- to 5-fold increased risk of GIS CH. Re-evaluation at 3–5 years showed a permanent thyroid dysfunction in 78% of 59 CH toddlers with GIS. Conclusions: The use of low b-TSH cut-off allowed the detection of an unsuspected number of children with neonatal hypothyroidism, evolving in mild permanent thyroid dysfunction later in life. The incidence of CH in this Italian population appears to be double than previously thought with a clear-cut prevalence of functional defects over dysgenetic ones.
Difficulties in selecting an appropriate neonatal thyroid-stimulating hormone screening threshold Korada SM, Pearce M, Ward Platt MP, Avis E, Turner S, Wastell H, Cheetham T Institute of Human Genetics, Newcastle University, Department of Paediatrics, Royal Victoria Infirmary, Newcastle upon Tyne, UK
[email protected] Arch Dis Child 2010;95:169–173 Background: The UK Newborn Screening Programme Centre recommends that a blood spot thyroid-stimulating hormone (TSH) cut-off of 10 mU/l is used to detect congenital hypothyroidism (CHT). As the value used varies from 5 to 10 mU/l, we examined the implications of altering this threshold. Methods: The authors’ regional blood spot TSH cut-off is 6 mU/l. Positive or suspected cases were defined as a TSH >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 <10 mU/l on the second blood spot have been diagnosed with CHT. The survey of endocrinologists highlighted significant differences in practice. Conclusions: A reduced threshold of 6 mU/l will increase the number of false positive term infants by 126%, but abnormalities of thyroid function requiring treatment will be detected. The authors suspected but did not confirm that the additional expense involved in setting a lower threshold is justified.
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Neonatal hyperthyrotropinemia: population characteristics, diagnosis, management and outcome after cessation of therapy Zung A, Tenenbaum-Rakover Y, Barkan S, Hanukoglu A, Hershkovitz E, Pinhas-Hamiel O, Bistritzer T, Zadik Z Pediatric Endocrinology Unit, Kaplan Medical Center, Rehovot, Israel
[email protected] Clin Endocrinol (Oxf) 2010;72:264–271 Background: Neonatal hyperthyrotropinemia (HT), defined by elevated TSH and normal T4, is either transient or persistent. Objective: The authors assessed perinatal parameters and diagnostic measures that may distinguish between transient and persistent HT, compared with congenital hypothyroidism (CH). They aimed to recommend optimal treatment in these forms of neonatal thyroid impairment. Methods: A multi-centre, retrospective study was conducted in six pediatric endocrinology units. 43 HT patients and 83 CH patients were included in the study. The authors evaluated differences in birth weight (BW), gestational age (GA), modes of diagnosis, screening and confirmatory T4 and TSH levels, thyroid imaging results and optimal thyroxine doses between HT and CH and between the two forms of HT. Results: Newborns with HT had lower BW and GA than those with CH. Transient (n = 18) and persistent HT (n = 25) patients were indistinguishable by most parameters, but those with persistent HT had a higher prevalence of abnormal thyroid imaging (69 vs. 8%; p = 0.005). During treatment, 79 and 55% of transient and persistent HT patients respectively experienced elevated levels of free T4. Although most HT patients were re-evaluated after 2.5 years, 6 transient HT patients stopped therapy and showed full recovery within the first year of life. Conclusion: The authors recommended obtaining thyroid imaging to distinguish between the two forms of HT. They also advised that early cessation of therapy in transient HT can prevent iatrogenic hyperthyroidism in these patients.
These four articles targeted questions related to screening of congenital hypothyroidism and extend our current knowledge. The first paper analyzed the sensitivity of combined T4/TSH bloodspot screening for detection of neonatal central hypothyroidism. Proponents of a T4 screening claim that this would identify central hypothyroidism. Only 8 (19%) of a retrospective single-center cohort of 42 children with central hypothyroidism were detected by decreased T4 values in the newborn screening. The diagnosis of central hypothyroidism was based on classic clinical and laboratory features rather than dynamic testing. This rate of normal newborn screening results of T4 in central hypothyroidism is much higher than stated in previous reports [10, 11]. This article reminds us that the possibility of late congenital hypothyroidism diagnosis should be on our mind despite normal screening results and that the clinical skills to suspect such a diagnosis should still be taught. Two papers questioned the threshold of the bloodspot TSH levels for the neonatal screening of congenital hypothyroidism. As expected, more cases were found when lowering the bloodspot TSH cutoff value to 12, 10 or 6 mU/l. Of interest, several cases of thyroid dysgenesis were found in both cohorts. On the other hand, many patients with glands in situ were identified with a high incidence of permanent thyroid dysfunction at follow-up. The worrisome question is: What happened to the children with such intermediate levels of TSH on screening at the time when the threshold was higher: were they picked up at a later age with mild hypothyroidism, should we be worried for their cognitive development? The fourth paper raised the question of the follow-up of children with neonatal hyperthyrotropinemia (HT) based on a retrospective analysis. Patients with permanent HT had a higher prevalence of abnormal thyroid imaging but were indistinguishable by other parameters from patients with transient HT. Of interest, the authors demonstrated a significant incidence of iatrogenic hyperthyroidism due to treatment. They draw our attention to this peculiar group of patients in whom a targeted dose of thyroxine and a close follow-up is necessary.
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Pediatric thyroid cancer A new bible and long-term data
Medullary thyroid cancer: management guidelines of the American Thyroid Association American Thyroid Association Guidelines Task Force, Kloos RT, Eng C, Evans DB, Francis GL, Gagel RF, Gharib H, Moley JF, Pacini F, Ringel MD, Schlumberger M, Wells SA Jr Department of Internal Medicine, Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, The Arthur G. James Cancer Hospital, Columbus, Ohio, USA
[email protected] Thyroid 2009;19:565–612 Background: Inherited and sporadic medullary thyroid cancer (MTC) is an uncommon and challenging malignancy. The American Thyroid Association (ATA) chose to create specific MTC Clinical Guidelines that would bring together and update the diverse MTC literature and combine it with evidence-based medicine and the knowledge and experience of a panel of expert clinicians. Methods: Relevant articles were identified using a systematic PubMed search and supplemented with additional published materials. Evidence-based recommendations were created and then categorized using criteria adapted from the United States Preventive Services Task Force, Agency for Healthcare Research and Quality. Results: The authors addressed initial diagnosis and therapy of preclinical disease (including RET oncogene testing and the timing of prophylactic thyroidectomy), initial diagnosis and therapy of clinically apparent disease (including preoperative testing and imaging, extent of surgery, and handling of devascularized parathyroid glands), initial evaluation and treatment of postoperative patients (including the role of completion thyroidectomy), management of persistent or recurrent MTC (including the role of tumor marker doubling times, and treatment of patients with distant metastases and hormonally active metastases), long-term follow-up and management (including the frequency of follow-up and imaging), and directions for future research. Conclusion: 122 evidence-based recommendations were created to assist in the clinical care of MTC patients and to share what the authors believed to be current, rational, and optimal medical practice.
This paper updates the 2001 consensus guidelines for treatment of MEN2 and MTC patients based on the important advances of knowledge over the last 10 years in the application of ‘codon-specific prophylactic thyroidectomy’ which is the gold standard for kindred in MEN2 families. Recommendations are stratified according to more experience with clinical outcome of patients with specific RET mutations, especially with rare ones. The management guidelines further propose a large series of recommendations dealing with diagnosis, surgical approach and long-term follow-up for medullary thyroid carcinoma, as well as for MEN2-associated pheochromocytoma and hyperparathyroidism. The 122 recommendations give answers based on the current state of the art to all clinical situations encountered in the care of affected families, from controversies concerning counseling of prenatal testing to management of persistent or recurrent metastatic disease. While the paper shows the way for the optimal management, the application of these recommendations in clinical life, especially also by pediatric endocrinologists, will need to be evaluated in the future.
Second malignant neoplasms in survivors of pediatric Hodgkin’s lymphoma treated with low-dose radiation and chemotherapy O’Brien MM, Donaldson SS, Balise RR, Whittemore AS, Link MP Department of Pediatric Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
[email protected] J Clin Oncol 2010;28:1232–1239 Background: Survivors of childhood Hodgkin’s lymphoma (HL) are at risk for second malignant neoplasms (SMNs). It is theorized that this risk may be attenuated in patients treated with lower doses of
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radiation. We report the first long-term outcomes of a cohort of pediatric survivors of HL treated with chemotherapy and low-dose radiation. Methods: Pediatric patients with HL (n = 112) treated at Stanford from 1970 to 1990 on two combined modality treatment protocols were identified. Treatment included six cycles of chemotherapy with 15–25.5 Gy involved-field radiation with optional 10-Gy boosts to bulky sites. Follow-up through September 1, 2007, was obtained from retrospective chart review and patient questionnaires. Results: 110 children completed HL therapy; median follow-up was 20.6 years. 18 patients developed one or more SMNs, including 4 leukemias, 5 thyroid carcinomas, 6 breast carcinomas, and 4 sarcomas. Cumulative incidence of first SMN was 17% (95% CI 10.5–26.7) at 20 years after HL diagnosis. The standard incidence ratio for any SMN was 22.9 (95% CI 14.2–35) with an absolute excess risk of 93.7 cases per 10,000 person-years. All 4 secondary leukemias were fatal. For those with second solid tumors, the mean (±SE) 5-year disease-free and overall survival were 76 ± 12% and 85 ± 10% with median follow-up 5 years from SMN diagnosis. Conclusion: Despite treatment with low-dose radiation, children treated for HL remain at significant risk for SMN. Sarcomas, breast and thyroid carcinomas occurred with similar frequency and latency as found in studies of children with HL who received high-dose radiation. O’Brien et al. show that low-dose irradiation of the thyroid in the context of pediatric Hodgkin’s lymphoma results in a significant risk for thyroid carcinoma on follow-up. Although reflecting the consequence of past therapies, the authors expect a reduction of second malignant neoplasms such as breast cancer by further dose reduction and shielding procedures, but not for thyroid cancer. They advocate an ‘aggressive surveillance’ of these patients. The most accurate follow-up remains to be determined but it should most probably include yearly thyroid ultrasound. As decreasing intensity of clinical follow-up of these patients often coincides with the transition from the pediatric oncologist and endocrinologist to the adult endocrinologist or general practitioner, the risk of second malignant neoplasias needs to be well communicated to the patient and the responsible physicians.
Long-term outcome in 215 children and adolescents with papillary thyroid cancer treated during 1940 through 2008 Hay ID, Gonzalez-Losada T, Reinalda MS, Honetschlager JA, Richards ML, Thompson GB Division of Endocrinology and Internal Medicine, Mayo Clinic, Rochester, Minn., USA
[email protected] World J Surg. 2010;34:1192–1202 Background: Controversy exists regarding the aggressiveness of initial therapy in childhood papillary thyroid cancer (PTC). Few studies with long-term outcome exist and second primary malignancies have rarely been analyzed. Methods: The authors studied 215 PTC patients younger than 21 years of age who were managed during 1940 through 2008. The patients were aged 3–20 years (median 16 years); median follow-up was 29 years. Recurrence and mortality details were taken from a computerized database. Results: Median primary tumor size was 2.2 cm. Six percent had distant metastases at presentation, 5% had incomplete tumor resection, 86% had nodes removed at initial surgery, and 78% had nodal metastases. After complete surgical resection, PTC recurred in 32% by 40 years. At 20 years, the recurrence rates at local, regional, and distant sites were 7, 21, and 5%, respectively. During 1940–1969, local and regional recurrence rates after unilateral lobectomy (UL) were significantly (p < 0.001) higher than after bilateral lobar resection (BLR). During 1950–2008, radioiodine remnant ablation (RRA) was administered within 18 months to 32%; it did not diminish the 25-year regional recurrence rate of 16% seen after BLR alone (p = 0.86). Only 2 fatal events from PTC occurred at 28 and 30 years, for a causespecific mortality at 40 years of only 2%. All-causes mortality rates did not exceed expectation through 20 years, but from 30 through 50 years, the number of deaths was significantly (p < 0.001) higher than predicted. 15 of 22 deaths (68%) resulted from non-thyroid malignancy. Conclusion: Survival from childhood PTC should be expected, but later death from non-thyroid malignancy is disconcerting. 73% of those who died from non-thyroid malignancy had received postoperative therapeutic irradiation.
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In general, childhood PTC certainly has an excellent prognosis. Owing to an extensively long followup period of 40 years, Han et al. revealed an unexpectedly high mortality of patients treated for PTC below age 21 years due to second primary tumors of solid organs other than thyroid between 30 and 50 years of age. The majority (73%) of these patients received postoperative therapeutic irradiation. Further, they showed that radioiodine remnant ablation did not diminish the 25-year regional recurrence rate. This result was surprising and suggests that irradiation of this patient group should be avoided as far as possible. However, in this large, single institutional cohort, the differences between survival after surgery alone and after surgery plus RRA to the endpoints of local recurrence, neck nodal metastases, locoregional recurrence, distant metastases, and all-sites recurrence were shown to be statistically insignificant. Lacking a more thorough description of the patients treated with radioiodine and the selection process of those receiving such a procedure may have induced such results. The current use of radioiodine has been validated in others studies [12].
New genetic studies for known and unknown genes
NKX2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease in ‘brain-lung-thyroid syndrome’ Guillot La, Carré Aa, Szinnai Gb, Castanet Mb, Tron Eb, Jaubert Fb, Broutin Ib, Counil Fb, Feldmann D b, Clement Ab, Polak Ma, Epaud Ra a INSERM UMR 938 and U845, Université Paris 6 and Université Paris Descartes and b Hôpital Trousseau and Necker Enfants Malades, Paris, France
[email protected],
[email protected] Hum Mutat 2010;31:E1146–1162 Background: NKX2-1 (NK2 homeobox 1) is a critical regulator of transcription for the surfactant protein (SP)-B and -C genes (SFTPB and SFTPC, respectively). Results: The authors identified and functionally characterized two new de novo NKX2-1 mutations c.493C>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 IB␣ 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 IBs, that also include IB␣. 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 IB␣, 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 IB␣, 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 IB␣ 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 <25 µg/l at postnatal day 2 were studied. The study substance was an equimolar preparation of recombinant protein complex of rhIGF-I and rhIGFBP-3 diluted with 10% glucose solution. The individual dose of rhIGF-I ranged from 1 to 12 µg/kg and was infused over 3 h. Results: The infusion of rhIGF-I and rhIGFBP-3 complex determined a marked increase of both IGF-I and IGFBP-3 concentrations. The estimated half-lives of IGF-I and IGFBP-3 were 0.86 and 0.90 h respectively for a child of 1,000 g. All the safety measures were reassuring. Conclusions: Infusion of rhIGF-I and rhIGFBP-3 complex is able to increase both circulating peptides in extremely preterm infants and is well tolerated. This study may thus represent a basis for further investigations on efficacy and safety of IGF-I in severe prematurity to stimulate growth and prevent retinopathy and brain damage.
IGF-I is a fetal growth factor essential for the development of the central nervous system. In preterm infants, low serum levels of IGF-I have been associated with slow weight gain and slow head (brain) growth as well as with the later development of retinopathy of prematurity. IGF-I promotes proliferation, maturation, and differentiation of neural stem cells. Moreover, IGF-I has been demonstrated to have neuroprotective properties both in vivo and in vitro. Finally, IGF-I plays an important role in retinal vascular development in both experimental and clinical studies. This Swedish group previously reported that serum IGF-I levels can predict which preterm babies will develop the retinopathy of prematurity [21, 22]. This paper represents the logical step ahead towards the therapeutic use of IGF-I in combination with IGFBP-3 to prevent the development of retinopathy in preterm infants. The major weakness of the study is the small number of study subjects (only 5), nevertheless it sheds, for the first time, some light on the pharmacokinetics and safety of IGF-I administration in extreme premature infants.
New paradigms Cooperation between oncogenic viruses and IGF-I receptors
Physical and functional interaction between polyoma virus middle T antigen and insulin and IGF-I receptors is required for oncogene activation and tumor initiation Novosyadlyy R, Vijayakumar A, Lann D, Fierz Y, Kurshan N, LeRoith D Division of Endocrinology, Diabetes and Bone Diseases, The Samuel Bronfman Department of Medicine, Mount Sinai School of Medicine, New York, N.Y., USA Oncogene 2009;28:3477–3486 Background: An oncogenic role of polyoma viruses in human cancers has been repeatedly proposed. Tumorigenesis seems to be mediated by polyoma virus middle T antigen (PyVmT) oncogene. PyVmT-
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transforming activity is linked with the downstream signaling after incorporation to the cell membrane. PyVnT undergoes tyrosine phosphorylation and activates MAPK and PI3K/Akt pathways. However, the mechanisms of PyVmT activation are poorly understood. The aim of this study was to test the role played by the interactions between insulin and IGF-I receptors with PyVnT in oncogene activation and tumor initiation. Methods: Met-1 and DB-7 cells derived from two different lines of mouse mammary tumor virus (MMTV)-PyVmT transgenic mice and human mammary carcinoma MCF-7 and MDA-MB-231 cells were studied. In Met-1 cells, insulin and IGF-I receptor gene silencing was induced by transfection. Thereafter, Met-1 cells were injected into inguinal mammary fat pads of wild-type FVB/N mice. After 4 weeks the size of Met-1 orthograft tumors was evaluated. Results: Insulin and IGF-I receptor (IR and IGFIR) are markedly expressed and phosphorylated in Met-1 cells overexpressing PyVmT. Treatment of these cells with insulin and IGF-I activates both PI3K and MAPK pathways inducing mitogenesis, inhibiting apoptosis, and stimulating migration and invasion. IR and IGF-IR interact with PyVmT and both insulin and IGF-I enhance this interaction and induce PyVmT tyrosine phosphorylation. IR and IGF-IR knockdown abrogates the ability of Met-1 cells to initiate tumor formation in vivo when implanted into the inguinal mammary fat pads of recipient mice. Conclusion: Although PyVmT mimics the action of tyrosine kinase receptors, it lacks intrinsic tyrosine kinase activity and requires cellular kinases for its activation. These results indicate that the interaction between PyVmT and cellular receptor tyrosine kinases such as IR and IGF-IR, plays a key role in PyVmT activation and, consequently, cancer initiation. This mechanism may represent a novel biological paradigm for oncogene activation in mammalian cells. A number of human cancers have been shown to be caused by viral infections, including Burkitt’s lymphoma, nasopharyngeal carcinoma, hepatocellular carcinoma, cervical cancer, T-cell leukemia and Kaposi’s sarcoma. The IGF system activation is also involved in the development and maintenance of tumors. High circulating IGF-I and insulin levels are associated with increased risk of many cancers, whereas low levels of these hormones are accompanied by delayed tumor development. This study demonstrates cooperation between IGF system and a viral oncogen such as polyoma virus, in promoting cell transformation via activation of common intracellular pathways including PI3K/Akt and MAPK. In particular, the tumorigenic action of polyoma virus is mainly determined by PyVmT, one of the most powerful viral oncogenes. IGFR and IR provide PyVmT, the tyrosine kinase activity necessary to initiate intracellular signaling ultimately leading to cell transformation. The potential clinical implication is that oncogenesis might be targeted by a double strategy directed to both viral infection and IGF system elements.
New paradigms Blocking IGF-I, the elixir of life?
Systemic signals regulate ageing and rejuvenation of blood stem cell niches Mayack SR, Shadrach JL, Kim FS, Wagers AJ Department of Stem Cell and Regenerative Biology, Harvard University, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Joslin Diabetes Center, Boston, Mass., USA Nature 2010;463:495–500 Background: Aging is associated with progressive decline in cell replacement and repair processes, and alterations of hematopoietic stem and progenitor cells (HSPCs) ultimately leading to defects of immune system and increased risk of malignancy. Stem cell supportive microenvironment, or niche, plays a key role in determining the fate of HSPCs in bone marrow and the authors asked whether systemic and/or local factors may affect HSPC number and function. Methods: Heterochronic parabiotic pairs were generated by surgically joining young (2 months) with older (>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 IB␣ 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
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Wnt-4
PI3K/Akt
GSK3
-catenin
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Col10a1 Proliferation
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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.
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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 <3.7 pmol/l), whereas levels in prepubertal girls were significantly higher (median 9.6 pmol/l, p < 0.001). Among the older prepubertal children (>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. <0.10 nmol/l, p < 0.01), and 17-testosterone concentrations (0.69 vs. 0.47 nmol/l, p < 0.05) compared with similarly aged prepubertal boys. Conclusions: Using an accurate and sensitive method, significantly higher levels of estrogens as well as androgen metabolites were found in prepubertal girls compared with age-matched boys. The higher prepubertal sex steroid levels in girls may contribute to their earlier onset of puberty including pubic hair development.
The authors used ultrasensitive methodology with high accuracy to determine circulating sex steroid levels in prepubertal and pubertal children of both sexes and found subtle sexual dimorphisms for
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the levels of estrogens at the low picomolar level for the levels of androgens at the subnanomolar level. This paper may serve as an important source of normative data for several sex steroids in children. However, it remains to be determined whether at these very low levels, these sex steroids exert any biological function and whether the differences observed have any phenotypic relevance.
Concerns – earlier start of puberty
Trends in puberty timing in humans and environmental modifiers Toppari Ja, Juul Ab Departments of aPhysiology and bPaediatrics, University of Turku, Turku, Finland Mol Cell Endocrinol 2010;324:339–344 Background: Secular trends in timing of puberty appear to continue although undernutrition has not been any longer a limiting factor for pubertal development. Now obesity and other environmental reasons have been suspected to cause this trend, and endocrine disrupting chemicals have become into focus as possible contributors. Methods: Epidemiological studies on endocrine disrupters are still scarce and show only weak associations between exposures and timing of puberty. Results: Since genetic background explains 50–80% of variability in the timing of puberty, it is not surprising that the observed environmental effects are rather modest when individual exposures are assessed. Despite that, some exposures have been reported to be associated with early (e.g., polybrominated biphenyls) or delayed (e.g., lead) puberty. Conclusions: The authors review the available data on recent trends in timing of puberty and the possible role of endocrine disrupters.
Gender-specific differences in birth weight and the odds of puberty: NHANES III, 1988–1994 Olivo-Marston S, Graubard BI, Visvanathan K, Forman MR The Laboratory of Human Carcinogenesis, Division of Cancer Prevention, The National Cancer Institute, The National Institutes of Health, Bethesda, Md., USA
[email protected] Paediatr Perinat Epidemiol 2010;24:222–231 Background and Methods: The association between birth weight and the odds ratio (OR) of pubertal status in girls aged between 8 and 11 and in boys aged between 8 and 12 was examined using the 1988-94 Third National Health and Nutrition Examination Survey (NHANES III). Girls (n = 956) and boys (n = 1,199) who had data on birth weight and Tanner staging were included. Maternal-reported birth weight, smoking in pregnancy and other information were provided in a home interview, while Tanner staging to assess pubertal status was part of a medical examination. Multiple logistic regression models were computed for the endpoints of the OR of being Tanner stage 2+ vs. 1 or being 2+ vs. 1 in an asynchronous pubertal pathway after adjustment for the complex sampling design of NHANES, age, race, height and body mass index (BMI). Results: Birth weight was not associated with the OR of Tanner stage 2+ among girls, however boys who were low birth weight (<2,500 g) and boys born higher than average birth weight (3,500–3,999 g) were more likely to be Tanner stage 2+ than 1. Childhood BMI was associated with the OR of having entered puberty among girls, but not boys. In an analysis of asynchronous maturation, girls born at high birth weight (>4,000 g) were more likely to have breast development 3+ than girls of normal birth weight, OR = 3.18 (95% CI 1.39, 8.25). Conclusions: The birth weight-puberty association varies by gender and by pubertal pathway. The findings need replication in prospective longitudinal studies and research to understand the mechanisms underlying the relation of early life exposures to cancer risk.
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The concept of an earlier start of puberty at the population level during the past few decades, particularly in girls, continues to provoke discussions and scientific debate. These two studies report on and discuss the possible association of earlier onset of puberty with environmental factors and with birth weight. It is clear that there is still limited data to associate specific endocrine disrupting chemicals with an earlier onset of puberty although ongoing prospective studies may add more insights into this issue. The role of body weight, BMI and the obesity epidemic as triggers of the onset of puberty has been convincingly demonstrated for certain ethnic groups but whether such mechanisms operate at the wider population level needs further attention. Longitudinal studies with prospective cohorts employing more accurate methods to determine puberty onset will add to this. Still, reports from several continents now seem to agree that there is a recent trend to start puberty earlier, which is difficult to explain as a consequence of a continuing secular trend or genetic alterations. Thus, there is urgent need to look more closely into possible environmental factors behind this phenomenon.
Follow-up on a Yearbook 2009 paper (Ong et al. [12])
LIN28B in constitutional delay of growth and puberty Tommiska Ja,b, Wehkalampi Kb,c, Vaaralahti Ka, Laitinen EMa,b, Raivio Ta,b, Dunkel Ld Department of Physiology, Institute of Biomedicine, University of Helsinki; bChildren’s Hospital, Helsinki University Central Hospital, cNational Institute for Health and Welfare, Helsinki, and dDepartment of Pediatrics, Kuopio University Hospital and University of Kuopio, Kuopio, Finland J Clin Endocrinol Metab 2010;95:3063–3066 Background: Recently, variation in LIN28B, a human ortholog of the gene-regulating processing of microRNAs (miRNAs) controlling the timing of major developmental events in the nematode Caenorhabtidis elegans, was reported to be associated with timing of puberty in humans. In C. elegans, a gain-offunction allele of lin-28 causes a retarded phenotype. The objective of the study was to evaluate the variation in the LIN28B gene in 145 subjects with constitutional delay of growth and puberty (CDGP). Methods: For this study, 115 males and 30 females with CDGP were included. CDGP was defined by Tanner genital or breast stage II and pubertal growth spurt taking place 2 SD later than average. The four coding exons (exons 1–4) and exon-intron boundaries, as well as the fragment of 3' untranslated region containing miRNA recognition elements A and B, of LIN28B were PCR amplified from genomic DNA obtained from peripheral blood leukocytes of the subjects and bidirectionally sequenced. Results: No variation in the coding region of LIN28B in the 145 subjects with CDGP was found. However, 16 of 145 subjects carried a 2-nucleotide deletion immediately 5' from miRNA recognition element A. These patients did not differ in phenotypic features as compared with non-carriers, and this variant was present in 100 controls with the same frequency. Conclusions: These results show that mutations in the coding region or 3’ untranslated region miRNA recognition elements A and B of LIN28B do not underlie CDGP. Lack of any variation in the coding region of the gene suggests that LIN28B in developmental timing is so crucial that any changes in the conserved protein would probably be lethal.
Last year we reported on the importance of microRNAs, and as one example of LIN28B, in the timing of puberty [12]. This year, Tommiska et al. tried to find variation in the LIN28B gene in individuals with constitutional delay in growth and puberty (CDGP). No variation in the coding region of the gene was found in CDGP subject. It should be remembered that the common variant of LIN28B detected by several groups [12] only modulated the age at menarche by a few weeks. The data presented here clearly indicate that variations in LIN28B are not a common cause of CDGP but leave open several possibilities. Given the sample size, it remains possible that variations in LIN28B are rare causes of CDGP not detected in the sample studied here. Alternatively, other genes in the LIN28B pathway could be involved in CDGP, a transmitted condition with so far no mechanistic basis. The last severe alterations of LIN28B might be lethal or lead to different phenotypes than the one analyzed here.
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Important for clinical practice
Congenital idiopathic hypogonadotropic hypogonadism: evidence of defects in the hypothalamus, pituitary, and testes Sykiotis GP, Hoang XH, Avbelj M, Hayes FJ, Thambundit A, Dwyer A, Au M, Plummer L, Crowley WF, Jr, Pitteloud N Harvard Reproductive Endocrine Sciences Center and the Reproductive Endocrine Unit of the Department of Medicine, Massachusetts General Hospital, Boston, Mass., USA J Clin Endocrinol Metab 2010;95:3019–3027 Background: Idiopathic hypogonadotropic hypogonadism (IHH) with normal smell (normosmic IHH) or anosmia (Kallmann syndrome) is associated with defects in the production or action of GnRH. Accordingly, most IHH patients respond to physiological pulsatile GnRH replacement by normalizing serum LH, FSH, and testosterone (T) levels and achieving gametogenesis; some patients, however, show atypical responses. Interestingly, several IHH-associated genes are expressed in multiple compartments of the hypothalamic-pituitary-gonadal axis. The aim of the study was to investigate whether the clinical, biochemical, or genetic characteristics of IHH men with atypical responses to GnRH indicate alternative or additional defects in the hypothalamic-pituitary-gonadal axis. Methods: 90 IHH men undergoing long-term pulsatile GnRH treatment over 30 years were studied. A retrospective study of response to GnRH at a Clinical Research Center was conducted. Physiological regimens of pulsatile sc GnRH were administered for at least 12 months. Dose-response studies using iv GnRH pulses assessed the pituitary LH response. Serum T, LH, FSH, and inhibin B levels, sperm in ejaculate, and sequence of IHH-associated genes was measured. Results: 26% of subjects displayed atypical responses to GnRH: (1) 10 remained hypogonadotropic and hypogonadal, demonstrating pituitary and testicular defects; (2) 8 achieved spermatogenesis and normal T but only with hypergonadotropism, indicating impaired testicular responsiveness to gonadotropins, and (3) 5 remained azoospermic despite achieving adult testicular volumes and normal hormonal profiles, suggesting primary defects in spermatogenesis. Mutations were identified only in KAL1 across groups. Conclusions: In addition to hypothalamic GnRH deficiency, IHH men can have primary pituitary and/or testicular defects, which are unmasked by GnRH replacement.
TAC3/TACR3 mutations reveal preferential activation of gonadotropinreleasing hormone release by neurokinin B in neonatal life followed by reversal in adulthood Gianetti Ea, Tusset Cb, Noel SDc, Au MGa, Dwyer AAa, Hughes VAa, Abreu APc, Carroll Jc, Trarbach Eb, Silveira LFb, Costa EMb, de Mendonca BBb, de Castro Mc, Lofrano Ac, Hall JEa, Bolu Ef, Ozata Mf, Quinton Rg, Amory JKh, Stewart SEi, Arlt Wi, Cole TRi, Crowley WFa, Kaiser UBc, Latronico ACb, Seminara SBa a Harvard Center for Reproductive Sciences and Reproductive Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Boston, Mass., USA; bLaboratorio de Hormonios e Genetica Molecular, Servico de Endocrinologia – Divisão de Clinica Medica I, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; cHarvard Center for Reproductive Sciences and Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass., USA; cDepartamento de Clinica Medica, Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Monte Alegre, Ribeirão Preto, São Paulo, cSection of Endocrinology, University Hospital of Brasilia and Molecular Pharmacology Laboratory, Faculty of Health Sciences, University of Brasilia, Brasilia, Brazil; fDepartment of Endocrinology, Gulhane School of Medicine, Ankara, Turkey; gEndocrinology Research Group, Institute for Human Genetics, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK; hDivision of General Internal Medicine, University of Washington Medical Center, Seattle, Wash., USA; iClinical Genetics Unit, Birmingham Women’s Hospital National Health Service Foundation Trust, Edgbaston, and Centre for Endocrinology, Diabetes, and Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK J Clin Endocrinol Metab 2010;95:2857–2867 Background: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. A broad
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cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Methods: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. 345 probands, 18 family members, and 292 controls were studied. Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants (3 nonsense mutations, 6 non-synonymous, 4 synonymous (1 predicted to affect splicing)). In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and 7 females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and 5 of the 7 females were assessed after discontinuation of therapy; 6 of the 7 males and 4 of the 5 females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time. These two papers represent a large body of work to better understand the etiology and pathophysiology as well as the clinical phenotypes in central hypogonadotropic hypogonadism. This is a relatively common diagnosis in the pediatric endocrinology clinic thus adding a substantial number of patients to the records. General conclusions from these studies are that it is worth looking more closely into the molecular defects when working up these cases, and that it is not uncommon that these patients suffer from several gene defects that may result in poor functional outcome with respect to fertility despite state-of-the-art endocrine treatment. One important practical implication is the recognition that some of the patients recognized in adolescence as having hypogonadism actually have reversible phenotypes and later in life have normal gonadotropic function and can reproduce without medical assistance [13]. So far, a small proportion of such patients has been identified among the vast number of patients with hypogonadism, without any clue on the factors involved in this reversible phenotype. Although not definitive, the data on patients with TAC3/TACR3 mutations suggest that these are the patients with the reversible phenotype suggesting that molecular analysis could help predict the course of the disease.
New Concerns – safety of endocrine treatment
Cognitive effects of aromatase inhibitor therapy in peripubertal boys Hero M, Maury S, Luotoniemi E, Service E, Dunkel L Department of Pediatric Endocrinology, Hospital for Children and Adolescents, Helsinki University Hospital, Helsinki, Finland Eur J Endocrinol. 2010;163:149–155 Background: Aromatase inhibitors, blockers of estrogen biosynthesis, have emerged as a new potential treatment modality for boys with short stature. The cognitive effects of such therapy are unknown. In this study, we explored the effects of aromatase inhibition on cognitive performance in peripubertal boys. Methods: Prospective, double-blind, randomized, placebo-controlled clinical study. 28 boys, aged 9.0– 14.5 years, with idiopathic short stature were treated with the aromatase inhibitor letrozole (2.5 mg/ day), or placebo, for 2 years. During the treatment, the progression of physical signs of puberty and the concentrations of sex hormones were followed up. A selection of cognitive tests, focusing on memory function, was administered to the participants at entry, at 12 months, and at 24 months after the start of the treatment. Results: Letrozole effectively inhibited the conversion of androgen to estrogen, as indicated by high serum testosterone and low serum estradiol concentrations in letrozole-treated boys who progressed into
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puberty. In both groups there was a gain in performance during the follow-up period in tests of verbal performance, in most of the tests of visuospatial performance, and in some tests of verbal memory. No significant differences between the letrozole and placebo-treated boys in development of cognitive performance were found in any of the tests during the follow-up period. Conclusions: These results suggest that blockade of estrogen biosynthesis with an aromatase inhibitor does not influence cognitive performance in peripubertal males.
Vertebral morphology in aromatase inhibitor-treated males with idiopathic short stature or constitutional delay of puberty Hero M, Toiviainen-Salo S, Wickman S, Makitie O, Dunkel L Pediatric Endocrinology and Metabolic Bone Diseases, Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland J Bone Miner Res 2010; Epub ahead of print Background: Aromatase inhibitors (AIs), blockers of estrogen biosynthesis, delay bone maturation and are therefore increasingly used to promote growth in children and adolescents with growth disorders. The effects of treatment on skeletal health are largely unknown. Methods: As estrogen deficiency is associated with various detrimental skeletal effects, we evaluated in this cross-sectional post-treatment study vertebral body morphology, dimensions and endplates, and intervertebral discs by the use of magnetic resonance imaging (MRI) in two cohorts of males previously treated with the AI letrozole, or placebo. Males with idiopathic short stature received treatment with letrozole or placebo for 2 years during pre-puberty or early puberty; males with constitutional delay of puberty received letrozole or placebo in combination with low-dose testosterone for 1 year during early or mid-puberty. Results: In males with idiopathic short stature, mild vertebral body deformities were found in 5 of 11 (45%) letrozole-treated subjects while in the placebo group no deformities were detected (p = 0.01). In the cohort of males with constitutional delay of puberty a high prevalence of endplate and intervertebral disc abnormalities was observed in both letrozole and placebo-treated males. Conclusions: The authors conclude that AI therapy during pre-puberty or early puberty may predispose to vertebral deformities, which probably reflect impaired vertebral body growth rather than impaired bone quality and compression fractures. If AIs are used in growth indications, follow-up of vertebral morphology is indicated.
These two papers deal with important safety issues in boys treated with aromatase inhibitors in order to stimulate pubertal growth. Given the common diagnosis of boys with short stature at the pediatric endocrinology clinic and the poor arsenal of tools available to stimulate their growth and increase final height, aromatase inhibitors have been increasingly tried for such purposes. It is therefore important to address the safety concerns. It is reassuring to learn that there were no observable negative effects on the cognitive performance of the treated teenager boys. However, when it comes to bone health there were clear indications that the spinal skeleton could be adversely affected by the treatment. The group of Leo Dunkel should be commended for this long-term commitment to evaluate several aspects of anti-aromatase therapy and not only the primary outcomes. These data re-emphasize the need for formal and comprehensive evaluation of new medications in the context of clinical trials before physicians even consider using them in their daily practice. It is unfortunate that this is not the case for the use of aromatase inhibitors [14].
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New mechanisms – healing by cooperation, teamwork restores the signal
Rescue of defective G-protein-coupled receptor function in vivo by intermolecular cooperation Rivero-Muller A, Chou YY, Ji I, Lajic S, Hanyaloglu AC, Jonas K, Rahman N, Ji TH, Huhtaniemi I Department of Physiology, University of Turku, Turku, Finland Proc Natl Acad Sci USA;107:2319–2324 Background: G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one-ligand/one-receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. Methods: This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. In the present study a transgenic mouse model was used. Results: The authors demonstrate, using the mouse luteinizing hormone receptor (LHR) as a model GPCR, that transgenic mice co-expressing binding-deficient and signaling-deficient forms of LHR can re-establish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. Conclusions: These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.
This paper demonstrates that a G-protein coupled receptor (GPCR) made deficient in its hormonebinding capacity, but with a still functional signaling capability, can cooperate successfully with a GPCR moiety with a reciprocal deficiency, lacking signaling activity but retaining hormone binding, to exert a normal receptor action. This is compelling evidence for the existence of interaction and functional cooperation between protein receptors at the molecular level within the cell membrane. These findings have important implications for better understanding of hormone actions and clinical phenotypes in endocrine disorders. References 1. Wilkes S, Murdoch A: Obesity and female fertility: a primary care perspective. J Fam Plann Reprod Health Care 2009;35:181–185. 2. MacDonald AA, Herbison GP, Showell M, Farquhar CM: The impact of body mass index on semen parameters and reproductive hormones in human males: a systematic review with meta-analysis. Hum Reprod Update 2010;16:293– 311. 3. Landres IV, Milki AA, Lathi RB: Karyotype of miscarriages in relation to maternal weight. Hum Reprod 2010;25:1123– 1126. 4. Kimmins S: Expanding waistlines heighten the risk for reproductive toxicity. Biol Reprod 2010;82:1–3. 5. Ghanayem BI, Bai R, Kissling GE, Travlos G, Hoffler U: Diet-induced obesity in male mice is associated with reduced fertility and potentiation of acrylamide-induced reproductive toxicity. Biol Reprod 2010;82:96–104. 6. Bukulmez O: Does assisted reproductive technology cause birth defects? Curr Opin Obstet Gynecol 2009;21:260–264. 7. Wilkins-Haug L: Assisted reproductive technology, congenital malformations, and epigenetic disease. Clin Obstet Gynecol 2008;51:96–105. 8. Hunt PA, Susiarjo M, Rubio C, Hassold TJ: The bisphenol-A experience: a primer for the analysis of environmental effects on mammalian reproduction. Biol Reprod 2009;81:807–813. 9. Phoenix CH, Goy RW, Gerall AA, Young WC: Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 1959;65:369–382. 10. Sinclair A, Smith C: Females battle to suppress their inner male. Cell 2009;139:1051–1053. 11. Murphy BD: Revisiting reproduction: what a difference a gene makes. Nat Med 2010;16:527–529. 12. Söder O, Sahlin L: Reproductive endocrinology; in Carel JC, Hochberg Z (eds): Yearbook of Pediatric Endocrinology. Basel, Karger, 2009, pp 79–92. 13. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley WF, Jr, Aparicio SA, Colledge WH: The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349:1614–1627. 14. Shulman DI, Francis GL, Palmert MR, Eugster EA, Lawson Wilkins Pediatric Endocrine Society Drug and Therapeutics Committee: Use of aromatase inhibitors in children and adolescents with disorders of growth and adolescent development. Pediatrics 2008;121:e975–e983.
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Adrenals Erica L.T. van den Akkera and Evangelia Charmandarib a
Department of Pediatric Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands Division of Endocrinology, Metabolism and Diabetes, Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, and Department of Endocrinology, Metabolism and Diabetes, University of Athens Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece
b
For this year’s chapter on ‘Adrenals’, we have searched the PubMed for articles on ‘adrenal’ and ‘steroidogenesis’ published in English between June 1, 2009 and May 31, 2010. Our search yielded more than 5,500 citations. We have examined all citations individually and selected the following collection of basic research and clinical articles. Whenever possible, we have avoided topics that have been discussed in the Yearbook 2009, unless progress in the field has been incremental. Emerging themes for this year’s chapter include the effect of circadian transcription factors on glucocorticoid receptor action, the implications of ultradian glucocorticoid pulsatility on the expression of target genes, the role of neuropeptide hormone receptors and microRNAs in the diagnosis and treatment of adrenal cancer, and recent advances in the diagnosis and treatment of congenital adrenal hyperplasia.
Mechanism of the year CLOCK/BMAL1 is a reverse-phase negative regulator of glucocorticoid action
Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications Nader N, Chrousos GP, Kino T Section on Pediatric Endocrinology, Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA
[email protected] FASEB J 2009;23:1572–1583 Background: Glucocorticoids exert their diverse actions through the glucocorticoid receptor (GR). Circulating concentrations of glucocorticoids fluctuate naturally in a circadian fashion and regulate the transcriptional activity of the GR in target tissues. The basic helix-loop-helix protein CLOCK and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms in both the central nervous system and periphery. Methods and Results: CLOCK/BMAL1 repressed the GR-induced transcriptional activity in a histone acetyltransferase (HAT) activity-dependent fashion. In serum-shock-synchronized cells, the transactivational activity of GR fluctuated spontaneously in a circadian fashion in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically, and CLOCK suppressed the binding of GR to its DNA recognition sequences by acetylating multiple lysine residues located in its hinge region. Conclusions: CLOCK/BMAL1 functions as a reverse-phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biological actions of diurnally fluctuating circulating glucocorticoids. In addition, a peripheral target tissue circadian rhythm influences the functions of every organ and tissue indirectly, through modulation of glucocorticoid action.
Interactions of the circadian CLOCK system and the HPA axis Nader N, Chrousos GP, Kino T Unit on Molecular Hormone Action, Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA
[email protected] Trends Endocrinol Metab 2010;21:277–286
Organisms have developed concurrent behavioral and physiological adaptations to the strong influence of day/night cycles, as well as to stressful stimuli. These circadian and stress-related responses are achieved by two highly conserved and interrelated regulatory networks, the circadian CLOCK and stress systems, which respectively consist of oscillating molecular pacemakers, the CLOCK/BMAL1 transcription factors, and the hypothalamic-pituitary-adrenal (HPA) axis and its end-effector, the glucocorticoid receptor. These systems communicate with each other at different signaling levels. Uncoupling of or impaired function in either system may result in the development of pathologic conditions. This review summarizes the bidirectional interaction between the circadian CLOCK system and the HPA axis, and discusses their clinical implications. In humans, circulating cortisol concentrations are tightly regulated by the central components of the HPA axis and fluctuate naturally in a circadian fashion, reaching their zenith in the early morning and their nadir in the late evening [1]. The circadian activity of the HPA axis is generated by the hypothalamic suprachiasmatic nucleus (SCN), the master oscillator and generator of the circadian rhythm of the body. Circadian rhythms of both the central nervous system and peripheral tissues and organs are generated by the coordinated activation/inactivation of self-oscillating transcription factors. Central among them are the circadian locomotor output cycle kaput (CLOCK) and its heterodimer partner brain-muscle-arnt-like protein 1 (BMAL1), which belong to the basic helix-loop-helix (bHLH)-PERARNT-SIM (PAS) superfamily of transcription factors [2]. In this study, Kino and colleagues demonstrated that CLOCK/BMAL1 is a reverse-phase negative regulator of glucocorticoid action in target tissues, antagonizing the biological actions of diurnally fluctuating circulating glucocorticoids and providing a local target tissue counter-regulatory feedback loop to the central CLOCK on the HPA axis. The circadian CLOCK system and the HPA axis regulate the activity of one another through multilevel interactions to ultimately coordinate homeostasis against the day/night change and various unforeseen random internal and external stressors (fig. 1). Uncoupling of or dysfunction in either system alters internal homeostasis and causes pathologic changes virtually in all organs and tissues, including those responsible for intermediary metabolism and immunity. Disrupted coupling of cortisol secretion and target tissue sensitivity to glucocorticoids may explain the development of central obesity and the metabolic syndrome in chronically stressed individuals, whose HPA axis circadian rhythm is characterized by blunting of the evening decreases of circulating glucocorticoids, as a result of enhanced input of higher centers upon the hypothalamic paraventricular nucleus secretion of CRH and AVP (fig. 1). Similarly, disrupted coupling of cortisol secretion and target tissue sensitivity to glucocorticoids could explain the increased cardiometabolic risk of subjects exposed to frequent jetlag because of traveling across time zones. At pharmacologic concentrations, the transactivational activity of glucocorticoids is correlated with the side effects of these steroids, while their transrepressive activity is associated mostly with their beneficial anti-inflammatory activity. Since CLOCK may differentially regulate these two major class actions of glucocorticoids, administration of these steroids at a specific period of the circadian cycle might increase their pharmacological efficacy, while at the same time reducing their unwanted side effects.
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Overnight dexamethasone test
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Fig. 1. A heuristic scheme of (a) circadian secretion of cortisol in non-stressed and chronically stressed humans (left panel) and their responses to midnight dexamethasone administration (right panel), (b) the corresponding circadian changes of target tissue sensitivity to glucocorticoids and (c) mean target tissue sensitivity to glucocorticoids in the human population. Even mild evening cortisol elevations, as those seen in chronically stressed individuals, will exert disproportionately increased glucocorticoid effects because of the natural circadian target tissue sensitivity increase at this time of the day. SS = Chronically stressed individuals, D = midnight dexamethasone administration, HS = hypersensitivity, N = normal sensitivity, NS = non-stressed individuals, R = resistance.
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New paradigms Implications of ultradian hormone pulsatility and receptor cross-talk
Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription Stavreva DA, Wiench M, John S, Conway-Campbell BL, McKenna MA, Pooley JR, Johnson TA, Voss TC, Lightman SL, Hager GL Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Md., USA Nat Cell Biol 2009;11:1093–1102 Background: Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. However, given that glucocorticoids are secreted in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Methods and Results: Ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release. Conclusions: The GR signaling pathway has been optimized for a prompt and timely response to fluctuations in glucocorticoid concentrations. These findings indicate that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation.
Glucocorticoids are released from the adrenal glands in a daily, circadian cycle as a result of the activity of the highly dynamic HPA axis. The pattern of glucocorticoid secretion is highly pulsatile (ultradian), with a periodicity of approximately 1 h. Temporal HPA axis activity drives a pattern of GR action that leads to gene stimulation that is reflective of the HPA axis profile. Physical and psychological stressors induce a rise in plasma glucocorticoid concentrations, superimposed on the ultradian and circadian rhythm. Due to its plasticity, the HPA axis integrates many internal and external stimuli, which in turn have a direct impact on GR-regulated transcriptional programs. The GR has been shown to exchange rapidly with response elements in living cells and its residence time on regulatory elements is measured in seconds. In this article, Hager and colleagues evaluated the implications of these complex aspects of glucocorticoid action (the rapid fluctuation of serum glucocorticoid concentrations and the fast dynamics of GR interactions with chromatin) on the GR-mediated transcriptional program. They demonstrated that, in contrast to treatment with long-acting glucocorticoid formulations, the ultradian mode of hormone secretion induces cyclic GR-mediated transcriptional regulation, or gene pulsing. The dynamics of GR-template interaction, as well as RNA Pol II loading and exchange, fluctuate together with the changes in extracellular hormone concentration. As a result, nascent RNA transcripts from a number of GR-regulated genes are released in distinct quanta during ultradian treatment, demonstrating profound differences in the transcriptional program induced by pulsatile ligand stimulation compared with that induced by constant stimulation. These findings suggest that gene pulsing in the GR system is necessary for correct transcriptional programming. As a result, even low doses of synthetic glucocorticoids, such as dexamethasone, are expected to alter the transcription program set by ultradian hormone release significantly. Furthermore, the inability to simulate ultradian hormone pulsatility may account for the fatigue, a common and debilitating manifestation often reported by patients with adrenal insufficiency, despite optimal glucocorticoid substitution therapy. These data provide a basis for a more complex view of gene regulation by GR and open new approaches for the development of synthetic glucocorticoids.
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Genomic and non-genomic cross talk between the gonadotropin-releasing hormone receptor and glucocorticoid receptor signaling pathways Kotitschke A, Sadie-Van Gijsen H, Avenant C, Fernandes S, Hapgood JP Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa Mol Endocrinol 2009;23:1726–1745 Background: The GnRH receptor (GnRHR) is a central regulator of reproductive function in all vertebrates. GnRH exerts its effects by binding to the GnRHR in pituitary gonadotropes. This study investigated the mechanisms of regulation of transcription of the mGnRHR gene. Methods and Results: Reporter assays with transfected mGnRHR promoter showed that both dexamethasone and GnRH increased the transcription of the mGnRHR gene via an activating protein-1 (AP-1) site. Small interfering RNA experiments revealed a requirement for the glucocorticoid receptor (GR) for both the dexamethasone and GnRH response. Chromatin immunoprecipitation (ChIP) and immunofluorescence assays showed that both GnRH and dexamethasone up-regulate the GnRHR gene via nuclear translocation and interaction of the GR with the AP-1 region on the mGnRHR promoter. GnRH activated the unliganded GR by rapid phosphorylation of the GR at Ser-234 in a GnRHRdependent fashion. Also, a direct link between GnRH-induced protein kinase C and MAPK activation was established, leading to unliganded GR phosphorylation at Ser-234 and transactivation of the glucocorticoid response element. Finally, GnRH and dexamethasone synergistically activated the endogenous GnRHR promoter via a mechanism involving steroid receptor coactivator-1 recruitment to the GnRHR AP-1 region. Conclusions: These findings demonstrate a novel mechanism of rapid non-genomic cross-talk between the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes via GnRHRdependent phosphorylation and activation of the unliganded GR in response to GnRH.
Ligand-independent activation of steroid receptors by phosphorylation is a common mechanism of receptor activation in several different cells via several different pathways. This article provides the first evidence for a novel mechanism of rapid non-genomic cross-talk between the HPG and HPA axes via GnRHR-dependent phosphorylation and activation of the unliganded GR in response to GnRH. Given that GnRH regulates several genes in pituitary gonadotropes, this non-genomic cross-talk represents a mechanism whereby the GnRHR-activated, unliganded GR may modulate the expression of several GnRH and GR target genes [3]. Furthermore, this GnRHR-GR cross-talk may have important physiologic implications, since it participates in the interplay between reproductive, stress and immune responses. It is well established that the neuroendocrine, immune, inflammatory and stressresponse systems are integrated functionally and regulated bidirectionally [4]. For example, stress or chronic activation of the HPA axis suppresses reproduction via the elevated glucocorticoid concentrations that exert their effects at all levels of the HPG axis [5]. This study shows a direct transcriptional effect of glucocorticoids on GnRHR gene expression mediated by the GR, which represents another mechanism whereby the HPA axis could modulate the HPG axis.
New hope A big role for small molecules
Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues Ziegler CG, Brown JW, Schally AV, Erler A, Gebauer L, Treszl A, Young L, Fishman LM, Engel JB, Willenberg HS, Petersenn S, Eisenhofer G, Ehrhart-Bornstein M, Bornstein SR University Hospital Carl Gustav Carus, Department of Medicine III, Dresden, Germany Proc Natl Acad Sci USA 2009;106:15879–15884 Background: Peptide analogues targeting neuropeptide receptors have been used effectively in cancer therapy. Adrenocortical tumor formation is characterized by the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and excess hormone secretion.
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Methods: A comprehensive analysis of relevant receptors in human adrenomedullary and adrenocortical tumors was performed and the antiproliferative effects of peptide analogues targeting these receptors were tested. Specifically, the receptor expression of somatostatin-type-2 (sst2) receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor were examined at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Results: The cytotoxic derivatives of somatostatin AN-238 and AN-162 reduced the cell numbers of adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the SV-1 and the LHRH receptor were expressed in adrenocortical cancer but not in pheochromocytoma cell lines. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix significantly reduced cell growth in the adrenocortical cancer cell lines. Conclusions: The expression of receptors for somatostatin, GHRH and LHRH in the normal human adrenal and in adrenal tumors, in association with the growth-inhibitory effects of antitumor peptide analogues, may improve current treatment of adrenal tumors.
The overexpression or aberrant expression of G-protein-coupled receptors for neuropeptides in human adrenal tissue has been associated with adrenal tumor formation and excessive hormone production [6, 7]. In addition, peptide hormone receptors have been detected in adrenocortical cancers, as well as in malignant pheochromocytomas. The identification of these receptors may allow the development of pharmacologic interventions as an alternative approach to current therapy. The present study evaluated specific receptor-targeted chemotherapeutic peptide antagonists and agonists for their potential future use in the antineoplastic therapy of adrenal tumors. It demonstrated that both adrenal tumor tissue and two adrenal tumor cell lines expressed receptors for somatostatin, GHRH or the SV-1 splice variant, as well as for LHRH. The immunohistochemical staining of adrenal tissue showed strong staining for sst2 in normal adrenal cortex, adrenocortical adenoma and carcinoma, as well as benign and malignant pheochromocytomas. The cytotoxic derivatives of somatostatin AN-238 and AN-162 reduced cell numbers of adrenomedullary pheochromocytoma cells and adrenocortical cancer cells, while the GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix significantly reduced cell growth in the adrenocortical cancer cell line. These results demonstrate a promising approach for delivering therapeutic compounds selectively to tumor cells, and raise hope for improved targeted treatment strategies for adrenal diseases.
MicroRNA signature of primary pigmented nodular adrenocortical disease: clinical correlations and regulation of Wnt signaling Iliopoulos D, Bimpaki EI, Nesterova M, Stratakis CA Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Mass., USA Cancer Res 2009;69:3278–3282 Background: MicroRNAs comprise a novel group of gene regulators implicated in the development of different types of cancer. However, their role in primary pigmented nodular adrenocortical disease (PPNAD), a rare form of bilateral adrenal hyperplasia caused by protein kinase A (PKA) regulatory subunit type 1A (PRKARIA)-inactivating mutations, has not been investigated. Methods and Results: A 44-microRNA gene signature of PPNAD was identified following comparison of PPNAD with normal adrenal samples: 33 microRNAs were up-regulated and 11 were down-regulated in PPNAD compared with normal adrenal tissues. Comparison of microRNA microarray data with hormonal measurements showed a negative correlation between let-7b expression and cortisol concentrations in patients with PPNAD. Also, nine microRNA-gene target pairs were identified as playing a potential role in adrenal pathogenesis. MiR-449 was up-regulated and WNT1-inducible signaling pathway protein 2 (WISP2) was identified as its direct target. Finally, pharmacologic inhibition of PKA resulted in the up-regulation of miR-449 leading to the suppression of WISP2. Conclusions: The microRNA profile and its clinical significance in PPNAD were investigated for the first time. The above findings suggest that PKA, via microRNA regulation, affects the Wnt signaling pathway, which is thought to be a primary mediator of PRKAR1A-related tumorigenesis.
An aberrant expression signature of microRNAs (miRNAs), small non-coding RNAs, is a hallmark of several diseases, including cancer. MicroRNA expression profiling by microarray techniques has pro-
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vided a powerful tool to reveal the involvement of these molecules in tumor development and progression, showing that they are differentially expressed in tumors compared with normal tissues. Moreover, specific miRNA signatures have been associated with histopathological and clinical features, suggesting a potential role of these molecules as prognostic and predictive markers [8, 9]. Focusing then on their biological effects and role in cancer, it has been shown that miRNAs can function as potential oncogenes or oncosuppressor genes, depending on the cellular context and on the target genes they regulate. This is the first study of miRNAs in PPNAD, and indeed, in any form of adrenal hyperplasia. In this study, the authors identified a miRNA gene signature for PPNAD [10] and demonstrated that let-7b expression is highly associated with midnight cortisol concentrations, an index of clinical severity of the Cushing syndrome caused by PPNAD tumors. The let-7 miRNA family appears to play a tumor suppressor role in cancer. In addition, using a PPNAD cell line, the authors identified the inhibition of miR-449 and up-regulation of its target gene WISP2. These results suggest that PKA, through miRNA regulation, affects the Wnt signaling pathway, which is important in the regulation of PRKAR1A-related tumorigenesis and in adrenocortical oncogenesis in general. The possibility to modulate miRNA expression either in vitro or in vivo by developing synthetic pre-miRNA molecules or antisense oligonucleotides provides a powerful tool towards a better understanding of the molecular mechanisms regulated by these molecules, and suggests the intriguing and promising perspective of their possible use in therapy.
New concerns Long-term effects of early-life stress
Childhood maltreatment and telomere shortening: preliminary support for an effect of early stress on cellular aging Tyrka AR, Price LH, Kao HT, Porton B, Marsella SA, Carpenter LL Mood Disorders Research Program, Butler Hospital, Providence, R.I., USA
[email protected] Biol Psychiatry 2010;676:531–534 Background: Advanced cellular aging has been proposed as a potential mechanism for the association between psychological stress and several medical and psychiatric illnesses. Previous studies linked chronic psychosocial stress and activation of the hypothalamus-pituitary-adrenal axis to shorter telomere length. Telomeres are DNA repeats that cap the ends of chromosomes and promote stability. They shorten progressively with each cell division and their length is a marker of biological aging. This study was designed to investigate the effect of childhood adversity on telomere length. Methods: 31 adults with no current or past major axis I psychiatric disorder were recruited to participate in this cross-sectional study. Subjects reported on their history of childhood maltreatment with a retrospective self-report questionnaire (CTQ). Telomere length was measured following DNA extraction from whole blood. Results: Participants reporting a history of childhood maltreatment had significantly shorter telomeres than those who did not report a history of maltreatment. This finding was not due to the effects of age, sex, smoking, body mass index or other demographic factors. Both physical neglect and emotional neglect were significantly linked to telomere length. Conclusions: Childhood maltreatment could influence cellular aging.
Stressful life experiences have been associated with an increased risk for psychiatric disorders, as well as cardiovascular and immune diseases [11]. The results of the present study extend previous reports linking shortened leukocyte telomere length and caregiver stress to more remote stressful experiences in childhood. In this study, reported maltreatment was moderate to severe, and a variety of types of abuse and neglect were represented. Analysis of subtypes of maltreatment suggested that both emotional and physical neglect may have the most robust effects. Therefore, in addition to the psychological effects of stress, it is possible that physical stressors might have contributed to these findings. Although the mechanism underlying the above association remains to be elucidated, gluco-
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corticoid-associated oxidative stress damage is likely to play an important role. Glucocorticoids increase neuronal oxidative stress damage [12] and oxidative stress reduces telomere length in vitro [13]. Limitations of this study that should be taken into consideration are the modest sample size, the cross-sectional nature of the study, and the fact that the CTQ is a brief, retrospective self-report questionnaire that may be subject to recall and other biases. Longitudinal, prospective, larger studies are needed to elucidate the effect of psychosocial stress on telomere length over time.
Quality of life in children and adolescents one year after cure of Cushing syndrome: a prospective study Keil MF, Merke DP, Gandhi R, Wiggs EA, Obunse K, Stratakis CA Program on Developmental Endocrinology and Genetics, National Institute of Child Health and Human Development, Bethesda, Md., USA
[email protected] Clin Endocrinol (Oxf) 2009;71:326–333 Background: Cushing syndrome (CS) in children is associated with symptoms that may impair healthrelated quality of life (HRQL). Prospective studies investigating the HRQL in children with CS are lacking. Methods: 40 children (age range 5–18 years) with CS were studied prospectively prior to and 1 year posttreatment for CS. The Child Health Questionnaire (CHQ) was used to assess HRQL, the Wechsler Intelligence Scale for Children (WASI) was used to assess cognitive function, and a CS symptom checklist was used to assess patient-reported symptoms. Results: Active CS was associated with low physical and psychosocial summary scores compared to US population data. Although these scores improved 1 year post-cure, residual impairment remained in physical summary and function. Incomplete recovery of adrenal function at 1 year post-treatment was associated with impaired scores. WASI IQ scores declined and a correlation was noted between age at first evaluation and IQ score changes. Most self-reported CS symptoms improved following treatment, with the exception of forgetfulness, unclear thinking and decreased attention span. Conclusions: CS in children and adolescents is associated with impaired HRQL, with residual impairment 1 year after cure.
Chronic exposure to excess endogenous glucocorticoids in adults, children and adolescents with CS is associated with detrimental health effects including truncal obesity, hypertension, insulin resistance, hyperglycemia, impaired wound healing, hypercoagulability, osteoporosis and gonadal dysfunction. Adult studies have also demonstrated that glucocorticoid excess is associated with impaired cognition. This study showed that children and adolescents with active CS had low physical and psychosocial summary scores compared to their peers in the US population. One year after successful cure of CS, children and adolescents showed residual impairments in HRQL scores for physical summary scores, physical function, global health perception, role-physical and emotional impact on parent. These findings are consistent with previous studies demonstrating that children with CS experienced a decline in cognitive function after cure of CS, despite reversal of cerebral atrophy [14]. This is in contrast to studies of adults with CS, which reported that cognitive impairment and loss of brain volume is partially reversible after successful treatment [15]. In addition, younger children were more likely to experience negative changes in cognitive function from pre- to post-treatment, although all post-treatment IQ scores remained within a normal range. Long-term follow-up of children and adolescents with CS, with particular reference to HRQL, is essential.
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Concepts revised A new role for dehydroepiandrosterone sulfate
Dehydroepiandrosterone sulfate directly activates protein kinase C- to increase human neutrophil superoxide generation Radford DJ, Wang K, McNelis JC, Taylor AE, Hechenberger G, Hofmann J, Chahal H, Arlt W, Lord JM Medical Research Council Centre for Immune Regulation, School of Immunity & Infection, University of Birmingham, Birmingham, UK
[email protected] Mol Endocrinol 2010;24;813–821 Background: Dehydroepiandrosterone (DHEA) sulfate (DHEAS) is the most abundant steroid in human circulation, and is secreted in an age-dependent fashion. DHEAS is considered an inactive metabolite that can be activated following cleavage of the sulfate group, thus generating DHEA, an important sex steroid precursor. Methods: Human neutrophils were isolated from healthy male donors. Neutrophil superoxide production, OATP, STS and SULT2A1 mRNA expression analysis, DHEAS uptake assays, protein kinase C (PKC) activation assays and phosphorylation of p47phox were studied. Results: DHEAS, but not DHEA, increased superoxide generation in primed human neutrophils in a dose-dependent fashion. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by inhibition of PKC activity. Neutrophils were found to be unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. DHEAS directly activated recombinant PKC- in a cell-free assay. Enhanced PKC- activation by DHEAS resulted in increased phosphorylation of p47phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Conclusions: PKC- acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immune senescence, which includes reduced neutrophil superoxide generation in response to pathogens, thereby impacting on a key bactericidal mechanism.
DHEA and its sulfate ester, DHEAS, are the most abundant steroids in human circulation, representing the major products of the adrenal zona reticularis. In humans and higher primates, DHEAS secretion shows a characteristic, age-associated pattern with very high concentrations in the neonatal period, a decline to very low concentrations during the first few months of life, and a continuous increase starting between the sixth and tenth year of age, also termed ‘adrenarche’ [16]. Peak DHEAS concentrations are achieved during the third decade of life followed by a steady decline starting in the fifth decade (adrenopause) with concentrations decreasing to 10–20% of maximal levels around 70 years of age [16]. This age-related decline in DHEAS does not reflect a general loss of adrenocortical output because cortisol concentrations are maintained and are even slightly raised with age [17]. This study challenges the previously held views that DHEAS is an inactive metabolite, and provides evidence for a novel signaling mechanism of DHEAS, which is distinct from its role as a precursor to DHEA and downstream sex steroid synthesis. In human neutrophils, PKC- acts as an intracellular receptor for DHEAS, mediating its stimulatory effects on neutrophil superoxide generation via activation of the NADPH oxidase complex. This is the first report of the direct activation of a major serine/ threonine protein kinase by DHEAS. These effects of DHEAS may have important clinical implications, with particular reference to immunologic conditions and/or old age.
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Important for clinical practice Immunohistochemistry and liquid chromatography-mass spectrometry in the diagnosis of adrenal disorders
Microarray gene expression and immunohistochemistry analyses of adrenocortical tumors identify IGF2 and Ki-67 as useful in differentiating carcinomas from adenomas Soon PS, Gill AJ, Benn DE, Clarkson A, Robinson BG, McDonald KL, Sidhu SB Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, St Leonards, Sydney, N.S.W., Australia
[email protected] Endocr Relat Cancer 2009;16:573–583 Background: The Weiss score is the most widely used system for the diagnosis of adrenocortical tumors (ACTs). An ACT is scored from 0 to 9, with a higher score correlating with increased malignancy. However, ACTs with a score of 3 can be phenotypically benign or malignant. Methods: Microarray profiling of a cohort of adrenocortical carcinomas (ACCs) and adrenocortical adenomas (ACAs) was used to identify discriminatory genes that could be used as an adjunct to the Weiss score. Genes with high-discriminatory power were identified by univariate and multivariate analyses and confirmed by quantitative real-time reverse transcription PCR and immunohistochemistry (IHC). Results: Compared with ACAs, ACCs demonstrated significantly higher expression of IGF2, MAD2L1 and CCNB1 but lower expression of ABLIM1, NAV3, SEPT4 and RPRM. Several proteins, including IGF2, MAD2L1, CCNB1 and Ki-67 had high-diagnostic accuracy in differentiating ACCs from ACAs. A combination of IGF2 and Ki-67 diagnosed ACCs with 96% sensitivity and 100% specificity. Conclusions: Microarray gene expression profiling accurately differentiates ACCs from ACAs. The combination of IGF2 and Ki-67 IHC is highly accurate in distinguishing between the two groups and is particularly helpful in ACTs with a Weiss score of 3.
With the advent of improved imaging techniques, adrenal tumors have been detected with increasing frequency. Distinguishing between adrenocortical adenomas (ACAs) and adrenocortical carcinomas (ACCs) can be difficult. The Weiss score, a 9-point histopathological scoring system, is presently the most widely used system for classifying adrenocortical tumors (ACTs) as benign or malignant [18]. However, whilst the Weiss score reliably classifies ACTs with a score of 0–2 as ACAs and those with a score of 4–9 as ACCs, the biological behavior of ACTs with a score of 3 can be difficult to predict accurately. Microarray gene expression profiling is a powerful tool that is able to characterize the transcription profile of a large number of genes in a tumor sample. This study has demonstrated that microarray gene expression profiling can accurately categorize ACTs into ACCs and ACAs. Immunohistochemistry (IHC) using IGF2 and a marker of proliferation can also distinguish ACCs from ACAs. Given that microarray gene expression profiling is expensive and its use in the clinical setting is limited, for the group of ACTs with a Weiss score of 3, the authors recommend the addition of IHC with IGF2 and a marker of proliferation (Ki-67 because of its wide availability and easy interpretation), in an attempt to classify and predict the biological behavior of these tumors accurately. This would lead to expeditious treatment for patients with ‘true’ ACCs, and ultimately lead to better prognosis and improved quality of life.
Serum steroid profiling for congenital adrenal hyperplasia using liquid chromatography-tandem mass spectrometry Rossi C, Calton L, Hammond G, Brown HA, Wallace AM, Sacchetta P, Morris M Centro Studi sull’Invecchiamento, Fondazione Universitaria G. d’Annunzio, Chieti, Italy Clin Chim Acta 2010;411:222–228 Background: The diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is based on the quantification of 17-hydroxyprogesterone (17-OHP) by an immunoassay. However, during the neonatal period the specificity of screening for CAH is low, leading to high false-positive rates.
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A robust, specific and selective method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) has been developed for the measurement of serum concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstene-3,17-dione (A4) and 17-OHP. Methods: The steroids were extracted from 50 µl of serum using methyl-tert-butyl-ether and the analysis was performed on a UPLC tandem quadrupole mass spectrometer system. Results: The assay was linear over each analyte concentration range. Inter- and intra-assay CVs were ≤10% across the analytical range. Simultaneous measurement of the full range of steroids in the pathway of cortisol biosynthesis allowed confirmation of the affected steroidogenic enzyme. Conclusions: A second-tier test for the diagnosis of CAH has been developed. The method allows for detection and quantification of 5 steroids with good linearity, sensitivity and precision. Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency accounts for 95% of all cases of CAH [19]. Early diagnosis and treatment is associated with a significant reduction in morbidity and mortality, and newborn screening has been advocated in many countries. The introduction of an immunoassay for 17-hydroxyprogesterone (17-OHP) in the late 1970s has contributed substantially to the uptake of CAH newborn screening programs. However, despite the wide availability and usage of this test, its diagnostic value is often limited, owing to low analytical antibody specificity and the limited diagnostic specificity of an isolated raised 17-OHP concentration. Transient elevations of 17-OHP concentrations are often seen in premature or severely ill newborns in the screening period and contribute to a high false-positive rate [20]. Analytical methods based on mass spectrometry present the most specific quantitative methods for determination of steroid concentrations [21–23]. A new method for diagnosing CAH accurately has been developed. This method is based on LC/MS/ MS. It is fast, robust and reproducible, allows quantification of 5 steroids related to CAH in 5 min, and demonstrates excellent resolution between 21-deoxycortisol and 11-deoxycortisol, thereby indicating the enzymatic defect in the vast majority of cases. The application of steroid profiling by LC/MS/ MS to the newborn screening for CAH is expected to reduce the false-positive results through its high analytical specificity and the potential to quantify several compounds in the same analytical run.
Clinical trials – new treatments A delayed- and extended-release formulation of hydrocortisone for the treatment of congenital adrenal hyperplasia
A pharmacokinetic and pharmacodynamic study of delayed- and extendedrelease hydrocortisone (ChronocortTM) vs. conventional hydrocortisone (CortefTM) in the treatment of congenital adrenal hyperplasia Verma S, Vanryzin C, Sinaii N, Kim MS, Nieman LK, Ravindran S, Calis KA, Arlt W, Ross RJ, Merke DP Reproductive Biology and Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Md., USA Clin Endocrinol (Oxf) 2010;72:441–447 Background: Current glucocorticoid therapy for congenital adrenal hyperplasia (CAH) is suboptimal and non-physiologic. This study investigated whether a new modified-release hydrocortisone (MR-HC) formulation (ChronocortTM) is able to simulate physiologic cortisol secretion more closely compared with the conventional hydrocortisone (HC) formulation (CortefTM) in patients with CAH. Methods: A phase 2, open-label, crossover pharmacokinetic and pharmacodynamic study was conducted in 14 patients (age range 17–55 years) with classic 21-hydroxylase deficiency. One week of thrice-daily HC (10, 5 and 15 mg) was followed by 1 month of once-daily MR-HC (30 mg at 22:00 h). Cortisol, 17-hydroxyprogesterone (17-OHP), androstenedione and ACTH concentrations were determined over a 24-hour period. Results: Hydrocortisone therapy resulted in three cortisol peaks, while MR-HC resulted in a single cortisol peak at approximately 06:00 h. Treatment with MR-HC resulted in significantly lower afternoon (12:00–20:00 h) and night-time (20:00–04:00 h), but higher morning (04:00–12:00 h) cortisol concen-
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trations compared with HC. Patients on MR-HC had significantly higher afternoon (12:00–20:00 h) 17-OHP, androstenedione and ACTH, but significantly lower 08:00 h 17-OHP concentrations. No serious adverse events occurred. Conclusions: Modified-release hydrocortisone represents a promising new treatment for CAH. Overnight adrenal androgens were well controlled, but rose in the afternoon with once-daily dosing, suggesting that a morning dose of glucocorticoid is needed.
Novel strategies for hydrocortisone replacement Debono M, Price JN, Ross RJ Academic Unit of Diabetes, Endocrinology and Metabolism, School of Medicine, Royal Hallamshire Hospital, Sheffield, UK Best Pract Res Clin Endocrinol Metab 2009;23:221–232
Current therapy with immediate-release hydrocortisone is the most commonly used regimen for replacement in patients with primary and secondary adrenal insufficiency. However, conventional hydrocortisone treatment cannot simulate physiologic cortisol production. Twice- or thrice-daily doses of hydrocortisone substitution inevitably result in temporary over- or under-replacement. Therefore, patients with adrenal insufficiency have a poor quality of life and an increased mortality despite optimal doses and adherence to treatment. Recent studies have investigated circadian hydrocortisone therapy imitating the physiologic circadian cortisol rhythm. Proof-of-concept studies using hydrocortisone infusions predict an improvement in both the biochemical control and the quality of life in treated patients. Delayed- and sustained-release oral formulations of hydrocortisone are being developed, which are expected to offer a more practical and effective solution for patients with adrenal insufficiency and congenital adrenal hyperplasia. Classic CAH is characterized by a defect in cortisol and aldosterone secretion, impaired development and function of the adrenal medulla, and adrenal hyperandrogenism [19]. Current treatment aims to provide adequate glucocorticoid and, when necessary, mineralocorticoid substitution to prevent adrenal crises, and to suppress the excess secretion of androgens and steroid precursors from the adrenal cortex. However, the currently available formulations of hydrocortisone are not able to simulate the normal cortisol circadian rhythm, and patients are often at risk for developing in tandem iatrogenic Cushing’s syndrome and hyperandrogenism [24, 25]. The use of a delayed- and extendedrelease formulation of hydrocortisone represents a new treatment approach to CAH that offers the prospect of a more physiologic cortisol replacement. ChronocortTM is a novel modified-release formulation of hydrocortisone designed to approximate physiologic cortisol secretion. In a proof-of-principle study, ChronocortTM was shown to mimic the circadian rhythm of serum cortisol secretion in healthy volunteers [26]. In patients with classic CAH, ChronocortTM achieved good overnight and early morning (but not afternoon or evening) control of ACTH and adrenal androgen secretion. Physiologic cortisol replacement in patients with CAH or adrenal insufficiency is expected to offer improved biochemical control and quality of life, as well as to decrease the risk for the development of co-morbidities owing to under- and/or over-treatment with glucocorticoids in these patients. Further studies are needed to determine the optimal dosing regimen and long-term clinical outcome.
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New mechanisms
Non-coding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor Kino T, Hurt DE, Ichijo T, Nader N, Chrousos GP Unit on Molecular Hormone Action, Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA
[email protected] Sci Signal 2010;3:ra8 Background: The availability of nutrients influences cellular growth and survival by affecting gene transcription. Glucocorticoids also influence gene transcription and have diverse activities on cell growth, energy expenditure and survival. Methods and Results: The growth arrest-specific 5 (Gas5) non-coding RNA, which is abundant in cells whose growth has been arrested because of lack of nutrients or growth factors, induced apoptosis by suppressing glucocorticoid-mediated induction of several responsive genes, including the cellular inhibitor of apoptosis 2. Gas5 bound to the DNA-binding domain of the glucocorticoid receptor (GR) and competed with DNA glucocorticoid-response elements for binding to the GR. Conclusions: Gas5 modulates the transcriptional activity of the GR by acting as a ‘riborepressor’, thereby influencing cell survival and metabolic activities during starvation.
Non-coding RNAs (ncRNAs) have diverse regulatory functions and may affect every aspect of organismal biology by influencing messenger RNA (mRNA) transcription, degradation and translation, the nuclear translocation of proteins, or both protein abundance and localization [27]. One such singlestrand ncRNA is the growth arrest-specific 5 (Gas5) [28]. Gas5 is expressed in growth-arrested cells as a result of nutrient deprivation or growth factor withdrawal. The present study has demonstrated that Gas5 is a strong interactant of the DNA-binding domain (DBD) of the GR, another ubiquitous molecule with major functions in behavioral, cardiovascular, metabolic and immune homeostasis. More specifically, Gas5 ncRNA interacts with the DBD of the ligand-activated GR through a decoy RNA ‘GRE’ and suppresses the GR-induced transcriptional activity of endogenous glucocorticoidresponsive genes by inhibiting binding of the GRs to target genes’ GREs. These findings indicate that Gas5 ncRNA functions as a starvation- or growth arrest-linked riborepressor of the GR and possibly other steroid hormone receptors. Given that relative starvation produces a favorable metabolic profile and prolongs life in several organisms, while increased glucocorticoid secretion or activity is associated with an unfavorable metabolic profile and decreased life expectancy, the observed Gas5-GR interaction might be of physiologic and/or pathologic importance.
Aldo-keto reductase 1B7 and prostaglandin F2␣ are regulators of adrenal endocrine functions Lambert-Langlais S, Pointud JC, Lefrançois-Martinez AM, Volat F, Manin M, Coudoré F, Val P, Sahut-Barnola I, Ragazzon B, Louiset E, Delarue C, Lefebvre H, Urade Y, Martinez A CNRS, UMR6247-Genetic, Reproduction and Development, Clermont University, Aubière, France
[email protected] PLoS One 2009;4:e7309
Prostaglandin F2␣ (PGF2␣) represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is not known. Prostaglandin biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF2␣ synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Methods and Results: This study examined the PGF2␣ biosynthetic pathway and its functional impact on both cortical and medullary zones using in vivo approaches and murine cell culture models. Both cortical and medullary compartments were shown to produce PGF2␣ but expressed different biosynthetic isozymes. In chromaffin cells, PGF2␣ secretion appeared constitutive and correlated to continuous Background:
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expression of COX1 and AKR1B3. In steroidogenic cells, PGF2␣ secretion was stimulated by ACTH and correlated to ACTH responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF2␣ release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF2␣ receptor was only detected in chromaffin cells, making medulla the primary target of PGF2␣ action. Conclusions: PGF2␣ repressed glucocorticoid secretion by a decrease in catecholamine release, which in turn decreased adrenal steroidogenesis. PGF2␣ may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. PGF2␣ is an essential autocrine/paracrine regulator of ovarian and testicular steroidogenesis. However, little was known about its possible impact on the adrenal gland. The present study provides a new insight in the understanding of PGF2␣ biosynthesis and its role in the function of the adrenal gland. Martinez and colleagues established for the first time that PGF2␣ acts as a negative regulator of both adrenocortical and adrenomedullary functions, and that the coordinate cell-specific regulation of both cyclooxygenases (COX1 and COX2) and aldo-keto reductases of the AKR1B subfamily (AKR1B7, AKR1B1, AKR1B3) could play a pivotal role in the generation of this signal.
The role of TASK1 in aldosterone production and its expression in normal adrenal and aldosterone-producing adenomas Nogueira EF, Gerry D, Mantero F, Mariniello B, Rainey WE Department of Physiology, Medical College of Georgia, Augusta, Ga., USA Clin Endocrinol (Oxf) 2009. Epub ahead of print Background: Aldosterone production in the adrenal glomerulosa is mainly regulated by angiotensin II and K+. In mice, genetic deletion of subunits of K+-selective leak-channels (KCNK), TASK1 and/or TASK3, leads to hyperaldosteronism and histological changes in the adrenal cortex. This study investigated the expression of TASK1 in human adrenocortical cells and its role in aldosterone production in H295R cells. Methods: TASK1 expression was investigated by comparative microarray analysis of aldosterone-producing adenomas (APA) and normal adrenals (NA). The effects of TASK1 knockdown by siRNA transfection were investigated in H295R cells. Fluo-4 fluorescent measurements of intracellular Ca2+ and pharmacological inhibition of Ca2+-dependent calmodulin kinases (CaMK) were performed to better define the effects of TASK1 on Ca2+ signaling pathways. Results: The expression of TASK1 did not differ between APA and NA. However, in APA, NA and H295R cells the expression of TASK1 was predominant when compared to other KCNK family members. Knockdown of TASK1 induced the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2), and stimulated pregnenolone and aldosterone production. Cells transfected with siTASK1 had increased intracellular Ca2+, leading to activation of CaMK and increased expression of CYP11B2. Conclusions: Human adrenal cortex displays predominant expression of TASK1 over other KCNK family genes. In addition, TASK1 plays an important role in the regulation of human aldosterone production through regulation of intracellular Ca2+ and CaMK signaling pathways.
Primary hyperaldosteronism (PA) is the most common cause of secondary hypertension. Aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex is physiologically regulated by angiotensin II (Ang II), K+ and ACTH. Binding of Ang II to its type 1 receptor (AT1R) stimulates a variety of signaling cascades, leading to the release of Ca2+ from the endoplasmic reticulum and subsequent cell membrane depolarization with additional flow of extracellular Ca2+ into the cytoplasm. These events stimulate the early and the late regulatory steps in aldosterone production, StAR protein and CYP11B2 respectively, culminating with elevated aldosterone production. Adrenal glomerulosa cells are sensitive to small increases in extracellular K+, a characteristic that is associated with the expression of high levels of KCNK channels, including the TWIK-related acid sensitive K+ 1 and 3 (TASK1 and TASK3). Recent studies have demonstrated that deletion of TASK1 and TASK3 in animal models leads to PA [29, 30]. Furthermore, a loss-of-function TASK3 mutation has been described in patients with Birk Barel syndrome [31]. The present study sought to define the role of TASK1 in adrenal cell aldosterone production, as well as its potential role in PA. The authors demonstrated for the first time that
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TASK1 is the predominant KCNK family member expressed in the human adrenal cortex, and that TASK1 knockdown stimulated aldosterone production through augmentation of Ca2+ flux and activation of CaMK in human adrenocortical cells. These intracellular signaling events culminated with the activation of early (StAR) and late (CYP11B2) rate-limiting steps in aldosterone production. Further studies are necessary to evaluate functional changes in TASK1 that could potentially contribute to conditions associated with increased aldosterone production. A TASK1-related clinical syndrome has not been described yet.
New genes
Germline mutations in TMEM127 confer susceptibility to pheochromocytoma Qin Y, Yao L, King EE, Buddavarapu K, Lenci RE, Chocron ES, Lechleiter JD, Sass M, Aronin N, Schiavi F, Boaretto F, Opocher G, Toledo RA, Toledo SP, Stiles C, Aguiar RC, Dahia PL Department of Medicine, San Antonio, Tex., USA Nat Genet 2010;42:229–233 Background: Pheochromocytomas are catecholamine-secreting tumors of neural crest origin that are often hereditary. The molecular basis of the majority of these tumors is not known. Methods and Results: The transmembrane-encoding gene TMEM127 was identified on chromosome 2q11 as a new pheochromocytoma susceptibility gene. In a cohort of 103 samples, truncating germline TMEM127 mutations were detected in approximately 30% of familial tumors and 3% of sporadic pheochromocytomas without a known genetic cause. In tumor DNA, the wild-type allele was consistently deleted, indicating a classic mechanism of tumor suppressor gene inactivation. Pheochromocytomas with mutations in TMEM127 showed hyperphosphorylation of mammalian target of rapamycin (mTOR) effector proteins. Accordingly, in vitro gain-of-function and loss-of-function analyses indicated that TMEM127 is a negative regulator of mTOR. Conclusions: This study identified TMEM127 as a tumor suppressor gene and validated the power of hereditary tumors to elucidate cancer pathogenesis.
The present study identified TMEM127 as a new gene conferring susceptibility to pheochromocytoma. TMEM127 has features of a classic tumor suppressor gene and TMEM127 mutations are associated with predisposition to pheochromocytoma development. Clinically, subjects with TMEM127 mutations developed pheochromocytomas in the fourth decade of life, which is similar to the mean age at diagnosis of sporadic pheochromocytomas, but notably older than the mean age at diagnosis of syndromic cases. All tumors arose from the adrenal medulla and were bilateral in approximately half of the cases. No malignancies or recurrences were detected during follow-up of the affected subjects. TMEM127 is predicted to encode a protein with three transmembrane regions and no clearly recognizable functional domains. Its sequence is highly conserved throughout evolution and putative orthologs can be identified in many species, from mammals to fish. Human TMEM127 is broadly expressed both in normal tissue and in a diverse group of cancer cell lines, with variable transcription levels that may reflect tissue of origin. The results of this study support the notion that disruptions of TMEM127 function might underlie tumors with an aberrant mTOR pathway and validate the relevance of hereditary tumor models to shed light on cell growth-related signals.
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Isolated 17,20-lyase deficiency due to the cytochrome b5 mutation W27X Kok RC, Timmerman MA, Wolffenbuttel KP, Drop SL, de Jong FH Endocrine Laboratory, Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands
[email protected] J Clin Endocrinol Metab 2010;95:994–999 Background: Cytochrome P450c17 (P450c17) is a bifunctional enzyme necessary for the production of glucocorticoids (17-hydroxylase activity) and sex steroids (17,20-lyase activity). Isolated 17,20-lyase deficiency is a rare condition characterized by deficient production of androgens, leading to 46,XY disorders of sex development (DSD), while the production of glucocorticoids is intact. Cytochrome b5 (CytB5) is an important factor in 17,20-lyase activity, probably by acting as an allosteric factor. This study investigated the role of CytB5 in a patient with 17,20-lyase deficiency. Methods: A 46,XY DSD patient with 17,20-lyase deficiency was studied. No mutation in the CYP17A1 gene had been detected in the patient and his parents. Steroid hormone concentrations were determined and sequencing the CYB5 gene was performed. Results: A homozygous W27X mutation was detected, leading to the formation of a premature stop codon. The parents of the index case were heterozygous carriers of this mutation. This mutation results in the absence of residues E48 and E49 of CytB5, which are necessary for an intact 17,20-lyase activity. Conclusion: In addition to CYP17A1 gene mutations, CytB5 mutations can result in 17,20-lyase deficiency.
Cytochrome P450c17 (P450c17) catalyzes the 17-hydroxylase and 17,20-lyase reactions required for the production of glucocorticoids and sex steroids. To exert its activities, P450c17 requires electron donation from reduced nicotinamide adenine dinucleotide phosphate through its redox partner protein cytochrome P450 oxidoreductase (POR). POR mutations have been discussed at length in previous Yearbooks. The cofactor cytochrome b5 (CytB5) is also required for optimal 17,20-lyase activity. This protein has electron-donating capacity, can accept electrons from POR and appears to participate in electron transfer for some cytochrome P450 reactions. CytB5 is not an effective electron donor to P450c17, but rather acts as an allosteric factor that fosters the interactions of POR with P450c17, enhancing 17,20-lyase activity without influencing 17-hydroxylase activity [32]. The absence of CytB5 results in low, but not absent, 17,20-lyase activity. In the patient described in this article, the clinical manifestations (bifid scrotum, penoscrotal hypospadias, bilaterally palpable gonads), endocrine evaluation and increased methemoglobin concentrations indicated impaired CytB5 function. Sequencing of the CytB5 gene revealed a mutation that led to a premature stop codon and a truncated protein lacking the E48 and E49 residues that are necessary for intact 17,20-lyase activity [33]. A case of CytB5 deficiency has been reported previously in a patient with ambiguous genitalia and methemoglobinemia, however no steroid hormone concentrations were determined [34]. In patients with 46XY DSD and evidence of 17,20-lyase deficiency, screening for mutations in the CytB5 gene, as well as for mutations in CYP17A1 and POR genes, should be considered.
Reviews
Stress and disorders of the stress system Chrousos GP Aghia Sophia Children’s Hospital, University of Athens, Greece
[email protected] Nat Rev Endocrinol 2009;5:374–381
All living organisms maintain a complex dynamic equilibrium, or homeostasis, which is constantly challenged by internal or external adverse forces termed stressors. Stress occurs when homeostasis is threatened or perceived to be so; homeostasis is re-established by various physiologic and behavioral adaptive responses. Neuroendocrine hormones have major roles in the regulation of both basal homeostasis and responses to threats, and are involved in the pathogenesis of diseases characterized by dyshomeostasis or cacostasis. The stress response is mediated by the stress system, partly located in the central nervous system
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Homeostatic effect
Allostasis (Cacostasis)
Deficiency
Eustasis
Allostasis (Cacostasis)
Optimum
Excess
Homeostatic system activity
Fig. 2. Homeostatic systems exert their effects in an inverse, U-type dose response. Eustasis is in the middle, optimal range of the curve. Suboptimal effects may be on either side of the curve and can lead to suboptimal adaptation, termed ‘allostasis’ or, more correctly, ‘cacostasis’, which may be harmful for the organism in the short or long term.
and partly in peripheral organs. Optimal basal activity and responsiveness of the stress system is essential for a sense of well-being, successful performance of tasks, and appropriate social interactions. By contrast, excessive or inadequate basal activity and responsiveness of this system might impair development, growth and body composition, and lead to a host of behavioral and somatic pathologic conditions. Stress occurs when homeostasis is threatened or perceived to be threatened. The stressors are potentially adverse forces, which can be emotional or physical. Both the magnitude and the chronicity of stressors are important. When any stressor exceeds a certain severity or temporal threshold, the adaptive homeostatic mechanisms of the organism activate compensatory responses that functionally correspond to the stressor. The homeostatic mechanisms exert their effects in an inverted U-shaped dose-response curve. Basal, healthy homeostasis (eustasis) is achieved in the central, optimal range of the curve, whereas suboptimal effects may occur on either side of the curve and can lead to insufficient adaptation (allostasis or cacostasis) (fig. 2). The latter might be harmful for the organism, and might account for many acute or chronic disorders. Prenatal development, infancy, childhood and adolescence are times of increased vulnerability to stressors.
Narrative review: the emerging clinical implications of the role of aldosterone in the metabolic syndrome and resistant hypertension Sowers JR, Whaley-Connell A, Epstein M University of Missouri, Columbia, Mo., USA Ann Intern Med. 2009;150:776–783
The prevalence of obesity, diabetes, hypertension, cardiovascular and chronic kidney disease is increasing in developed countries. Obesity, insulin resistance and hypertension commonly cluster with other risk factors for cardiovascular and chronic kidney disease to form the metabolic syndrome. Emerging evidence suggests that excess circulating aldosterone concentrations impair insulin metabolic signaling and endothelial function, which in turn leads to insulin resistance and cardiovascular and renal structural and functional abnormalities. Furthermore, the cardiovascular and renal abnormalities associated with insulin resistance are mediated in part by aldosterone acting on the mineralocorticoid receptor. Mineralocorticoid receptor blockade improves pancreatic insulin release, insulin-mediated glucose utilization, and endothelium-dependent vasorelaxation. Aldosterone excess has detrimental metabolic effects that contribute to the metabolic syndrome and endothelial dysfunction, which in turn lead to the development of resistant hypertension, as well as cardiovascular disease and chronic kidney disease.
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Obesity, insulin resistance, and hypertension commonly cluster with other risk factors for cardiovascular or chronic kidney disease to form the metabolic syndrome, which is associated with increased cardiovascular morbidity and mortality [35]. A new paradigm indicates that elevated concentrations of plasma aldosterone mediate several maladaptive changes that contribute to the pathogenesis of the metabolic syndrome, resistant hypertension, and associated cardiovascular and renal structural and functional abnormalities [36, 37]. Many of these adverse effects are mediated through rapid, membrane-initiated non-genomic actions of aldosterone. Emerging evidence suggests that mineralocorticoid receptor blockade is useful in treating resistant hypertension and in preventing cardiovascular and chronic kidney disease in patients with the metabolic syndrome and diabetes. Future research studies should further delineate the role of mineralocorticoid receptor blockade in the management of the metabolic syndrome and resistant hypertension.
Food for thought Social disadvantage and overcommitment are associated with impaired HPA function
Higher overcommitment to work is associated with higher plasma cortisol but not ACTH responses in the combined dexamethasone/CRH test in apparently healthy men and women Wirtz PH, Siegrist J, Schuhmacher A, Hoefels S, Maier W, Zobel AW Department of Clinical Psychology and Psychotherapy, University of Zurich, Zurich, Switzerland
[email protected] Psychoneuroendocrinology 2010;35:536–543 Background: Overcommitment (OC) is a pattern of excessive striving that has been associated with alterations in the hypothalamic-pituitary-adrenal (HPA) axis. This study investigated whether OC is associated with alterations in the function of HPA axis. Methods: 92 men and 108 women of a wide range of OC scores were recruited to participate in this cross-sectional study. OC, depressive symptoms (Beck Depression Inventory, BDI) and work stress (effort-reward-imbalance, ERI) were assessed by questionnaires. Cortisol and ACTH concentrations were determined following a combined dexamethasone/CRH test. Results: Independent of age and gender, higher OC was associated with higher repeated cortisol but not ACTH secretion. Similarly, higher cortisol but not ACTH increase following CRH stimulation was predicted by higher OC. Depressive symptoms (BDI score) and work stress scores (effort-reward-ratio) did not relate to neuroendocrine responses to the dexamethasone/CRH test. OC was not associated with ACTH or cortisol pre-test concentrations. Conclusions: With increasing OC scores, a higher reactivity of the adrenal cortex together with a normal reactivity of the pituitary is observed following CRH stimulation.
Overcommitment (OC) is considered to be an enduring cognitive-motivational pattern of maladaptive coping with demands characterized by the inability to withdraw from obligations combined with a high need for control and approval [38]. OC individuals are extremely ambitious and tend to exaggerate their efforts while at the same time they overestimate their resources. Prospective studies have demonstrated that OC independently increases the risk for coronary heart disease (CHD) [39–41]. Although the underlying mechanisms have not been fully delineated, emerging evidence suggests that OC is associated with alterations in the HPA axis function. In this study, Zobel and colleagues demonstrated that OC is independently associated with higher increases in cortisol but not ACTH concentrations following a combined dexamethasone/CRH test, suggesting an association between OC and HPA axis dysregulation. From a clinical perspective, OC is associated with an increased risk of depression. OC and depression are known to increase the risk of CHD and might share a common biological mechanism of HPA axis dysregulation. Further studies are required to determine the clinical implications of these findings (including insulin resistance, obesity, hyperten-
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sion and atherosclerotic cardiovascular disease), as well as the mechanisms underlying the link between OC and CHD.
Follow-up on Yearbook 2009 Fertility in non-classic congenital adrenal hyperplasia
Fertility in women with non-classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency Bidet M, Bellanné-Chantelot C, Galand-Portier MB, Golmard JL, Tardy V, Morel Y, Clauin S, Coussieu C, Boudou P, Mowzowicz I, Bachelot A, Touraine P, Kuttenn F Department of Reproductive Endocrinology, Hôpital Pitié-Salpêtrière, Paris, France
[email protected] J Clin Endocrinol Metab 2010; 95:1182–1190 Background: Although fertility has been evaluated in women with the classic form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, little is known about fertility in women with the non-classic form of the disease (NC-CAH). Methods: 161 women with NC-CAH (age range 13–52 years), who presented with hirsutism (78%), menstrual irregularities (61%) or infertility (12%), and 29 affected female relatives were studied. The diagnosis had been confirmed by an ACTH stimulation test and sequencing of the CYP21A2 gene. Results: 95 of the 190 women (age 26.7 ± 8.9 years) wanted to become pregnant; 187 pregnancies occurred in 85 women, which resulted in 141 births in 82 of them; 83% of pregnancies were obtained within 1 year. The rate of miscarriages was 6.5% for pregnancies obtained with glucocorticoid treatment and 26.3% for pregnancies obtained without treatment. Conclusions: Subfertility is mild in NC-CAH. However, the rate of miscarriages is lower in pregnancies occurring with glucocorticoid treatment, suggesting that glucocorticoid treatment should be instituted in patients who want to conceive.
Although fertility problems are addressed in adulthood, the issue is often raised by the pediatric endocrinologists and is discussed with patients and their parents during adolescence. Last year’s Yearbook addressed fertility in classic CAH. The present study provides a follow-up, investigating fertility in a large group (n = 190) of women with non-classic CAH. The study showed that fertility problems in NC-CAH are mild compared to the classic forms of the disease [42, 43]. The cumulated pregnancy rate for the 90 women who wanted to become pregnant was 76% at 1 year, which is slightly less than in the general French population (92%). Interestingly, the group of patients not receiving glucocorticoid treatment reported a higher number of miscarriages (26%) compared with the group receiving glucocorticoid treatment (6.5%) or the general population (10–15%). These findings suggest that introducing glucocorticoid treatment in women with NC-CAH who want to conceive may improve pregnancy rates. References 1. Chrousos GP, Kino T: Intracellular glucocorticoid signaling: a formerly simple system turns stochastic. Sci STKE 2005:pe48 2. Schibler U, Sassone-Corsi P: A web of circadian pacemakers. Cell 2002;111:919–922. 3. Hapgood JP, Sadie H, van Biljon W, Ronacher K: Regulation of expression of mammalian gonadotrophin-releasing hormone receptor genes. J Neuroendocrinol 2005;17:619–638. 4. Chrousos GP, Gold PW: The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA 1992;267:1244–1252. 5. Rivier C, Rivest S: Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis: peripheral and central mechanisms. Biol Reprod 1991;45:523–532. 6. Lacroix A, Ndiaye N, Tremblay J, Hamet P: Ectopic and abnormal hormone receptors in adrenal Cushing’s syndrome. Endocr Rev 2001;22:75–110. 7. Mazzuco TL, Chabre O, Feige JJ, Thomas M: Aberrant GPCR expression is a sufficient genetic event to trigger adrenocortical tumorigenesis. Mol Cell Endocrinol 2007;265-266:23–28. 8. He L, Hannon GJ: MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 2004;5:522–531. 9. Lu J, Getz G, Miska EA, et al: MicroRNA expression profiles classify human cancers. Nature 2005;435:834–838.
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Charmandari E, Johnston A, Brook CG, Hindmarsh PC: Bioavailability of oral hydrocortisone in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Endocrinol 2001;169:65–70. 25. Charmandari E, Hindmarsh PC, Johnston A, Brook CG: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: alterations in cortisol pharmacokinetics at puberty. J Clin Endocrinol Metab 2001;86:2701–2708. 26. Newell-Price J, Whiteman M, Rostami-Hodjegan A, et al: Modified-release hydrocortisone for circadian therapy: a proof-of-principle study in dexamethasone-suppressed normal volunteers. Clin Endocrinol (Oxf) 2008;68:130–135. 27. Mattick JS: The functional genomics of noncoding RNA. Science 2005;309:1527–1528. 28. Schneider C, King RM, Philipson L: Genes specifically expressed at growth arrest of mammalian cells. Cell 1988;54:787– 793. 29. Davies LA, Hu C, Guagliardo NA, et al: TASK channel deletion in mice causes primary hyperaldosteronism. Proc Natl Acad Sci USA 2008;105:2203–2208. 30. Heitzmann D, Derand R, Jungbauer S, et al: Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis. EMBO J 2008;27:179–187. 31. Barel O, Shalev SA, Ofir R, et al: Maternally inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9. Am J Hum Genet 2008;83:193–199. 32. Auchus RJ, Lee TC, Miller WL: Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer. J Biol Chem 1998;273:3158–3165. 33. Naffin-Olivos JL, Auchus RJ: Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17. Biochemistry 2006;45:755–762. 34. Hegesh E, Hegesh J, Kaftory A: Congenital methemoglobinemia with a deficiency of cytochrome b5. N Engl J Med 1986;314:757–761. 35. 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Type 1 Diabetes: Clinical and Experimental Francesco Chiarelli and M. Loredana Marcovecchio University of Chieti, Chieti, Italy
Type 1 diabetes (T1D) is one of the most common chronic disorders of childhood and adolescence and its incidence is rising worldwide, with an annual increase over the last years of about 3–4% [1]. Based on recent estimates, between 2005 and 2010, there will be a 70% increase in the prevalence of T1D, with a doubling of new cases in children younger than 5 years [1]. These data are worrisome given that a diagnosis of T1D during childhood determines a longer exposure to the metabolic derangements of the disease when compared to adult-onset T1D, therefore increasing the burden of the disease [2]. In addition, an early onset of T1D is often associated with more acute presentations, such as diabetic ketoacidosis and admission to hospital, which increase T1D morbidity [3]. Many efforts are continuously made to better understand the pathogenesis of the disease, to identify subjects at increased risk of developing it, to improve daily management of T1D and to identify factors which could predict subjects particularly predisposed to the long-term vascular complications, and towards whom more aggressive and intensive interventions should be directed. The emergence of new and promising treatment options always creates optimism among clinicians and researchers, in particular when dealing with a chronic disease such as T1D and when the aim is to improve the future of our children. All the above-mentioned aspects are the focus of the articles chosen for the 2010 Yearbook chapter on T1D.
Mechanism of the year Another key role of the KATP channel
ATP-sensitive K+ channel mediates the zinc switch-off signal for glucagon response during glucose deprivation Slucca M, Harmon JS, Oseid EA, Bryan J, Robertson RP Pacific Northwest Diabetes Research Institute and the Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine and Department of Pharmacology, University of Washington, Seattle, Wash., USA
[email protected] Diabetes 2010;59:128–134 Background: A recent hypothesis based on in vivo data from hypoglycemic rats proposes that glucagon secretion during hypoglycemia is triggered by a decrease in zinc co-secreted with insulin from -cells, rather than the decrease in insulin itself. The aim of this study was to determine whether closure of the ␣-cell ATP-sensitive K+ channel (KATP channel) is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation. Methods: Studies were performed using perifused isolated islets. Results: The expected glucagon response to an endogenous insulin switch-off signal during glucose deprivation was observed in wild-type mouse islets. In experiments with streptozotocin-treated wild-type islets, a glucagon response to an exogenous zinc switch-off signal was observed during glucose deprivation. However, this glucagon response to the zinc switch-off signal during glucose deprivation was not seen in the presence of nifedipine, diazoxide, or tolbutamide or if KATP channel knockout mouse islets were used. All islets had intact glucagon responses to epinephrine. Conclusions: This study shows that zinc co-release with insulin during glucose deprivation is a switch-off signal triggering glucagon secretion and that this zinc action is mediated through closure of KATP channels and consequent opening of calcium channels.
Glucagon release from pancreatic ␣-cells represents one of the main compensatory mechanisms stimulated by hypoglycemia. This response is often impaired, to a variable degree, in people with diabetes.
The ‘intra-islet insulin hypothesis’ is an intriguing hypothesis suggesting that glucagon secretion in conditions of glucose deprivation is mediated by the decreased intra-islet insulin secretion [4]. More recent studies have slightly modified this hypothesis proposing that other factors released by -cells, such as ␥-aminobutyric acid and zinc, together with insulin could suppress glucagon secretion when insulin levels are high and stimulate its release during hypoglycemia. In this context, zinc co-released with insulin has been shown to play an important role in several, although not all, experimental studies. In the present study the authors mark a further step in the understanding of the intra-islet insulin hypothesis. Through a series of experiments in mice, they identified the closure of KATP channels as the key effector of the zinc switch-off signal for glucagon secretion during glucose deprivation. The first step of their studies was the confirmation that cessation of zinc co-release with insulin is a key mechanism to stimulate glucagon release during hypoglycemia. Then, through manipulation of the KATP receptor with drugs known to influence its activity, such as diazoxide, which keeps the channel opened, or tolbutamide, which keeps the channel closed, they found that the channel failed to respond to the switch-off zinc signal during glucose deprivation. In a second model, where the SUR1 subunit of the KATP channel was knocked out, the impaired function of the channel led to lack of response to the switch-off zinc mechanism. A further step in their experiments was to block calcium channels with nifedipine, which therefore interfered with the downstream mechanisms following the closure of the KATP channels. The present study is of utmost importance as it strengthens the hypothesis that a key issue for keeping a good glycemic control is the intercellular hormonal dialogue within the pancreatic islets, phenomenon which is lost in people with diabetes, for lack of -cell insulin release. The results support the hypothesis that the association of high basal glucagon in T1D could at least in part be attributed to lack of insulin-bound zinc tonically suppressing ␣-cell function.
New paradigms 1 The early decline of -cell glucose sensitivity
Progression to diabetes in relatives of type 1 diabetic patients: mechanisms and mode of onset Ferrannini E, Mari A, Nofrate V, Sosenko JM, Skyler JS Department of Medicine, University of Pisa School of Medicine, Pisa, Italy
[email protected] Diabetes 2010;59:679–685 Background: In this study the mode of onset of hyperglycemia and how insulin sensitivity and -cell function contribute to the progression to T1D in relatives of patients with diabetes were assessed. Methods: 328 islet cell autoantibody-positive, non-diabetic relatives from the observational arms of the Diabetes Prevention Trial-1 Study (median age 11 years [interquartile range (IQR): 8] underwent sequential OGTT at baseline, every 6 months, and 2.7 years later, when 115 subjects developed T1D. -Cell glucose sensitivity (slope of the insulin-secretion/plasma glucose dose-response function) and insulin sensitivity were obtained by mathematical modeling of the OGTT glucose/C-peptide responses. Results: At baseline, insulin sensitivity, fasting insulin secretion, and total post-glucose insulin output did not differ between progressors and non-progressors. In contrast, -cell glucose sensitivity was significantly reduced in progressors (median 48 pmol/min/m2/mmol/l [IQR: 36] vs. 87 pmol/min/m2/mmol/l [IQR: 67]; p < 0.0001) and predicted incident diabetes (p < 0.0001) independently of gender, age, BMI, and clinical risk. Glucose sensitivity progressively declined over time with a significant fall 1.45 years before diagnosis. In contrast, 2-hour glucose levels did not significantly change until 0.78 years before diagnosis, when they started to rise rapidly (approx. 13 mmol · l–1 · year-1). During this anticipation phase, both insulin secretion and insulin sensitivity were essentially stable. Conclusions: -Cell glucose sensitivity is the earliest parameter to be impaired in relatives of patients with T1D and represents a strong predictor of diabetes progression. The time trajectories of plasma glucose
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pattern over time is often biphasic, with a slow linear increase followed by a rapid increase, and are preceded by a further deterioration of -cell glucose sensitivity. A detailed understanding of the timeline of events characterizing the development of T1D in genetically predisposed individuals could help in defining when to intervene and with which strategy, in order to reduce the risk of progressing towards overt hyperglycemia, and hopefully prevent acute presentations of T1D. Although autoantibodies can be detected during the prodromic phase of T1D, they do not give any information on the time of onset of T1D, and up to now the natural history of pancreatic -cells incompetence has not been well characterized. The present study is unique in its design, including a large cohort of predisposed individuals with serial assessments of -cell function and insulin sensitivity, in order to identify peculiar characteristics able to early distinguish, among predisposed individuals, those who will progress to T1D from nonprogressors. This study sheds light on the role of ‘–cell glucose sensitivity’, which represents the ability of -cells to rapidly adapt to acute changes in plasma glucose. Interestingly, it was found that a decrease in this parameter is the earliest defect which characterizes progressors. A clear decline in -cell glucose sensitivity was detected 1.4 years prior to diagnosis and over time represented the strongest predictor for diabetes. In contrast, no early alterations able to characterize predictors were found in insulin sensitivity and secretion. Insulin secretion showed an initial increase, reflecting an adaptation to raising glucose levels, and then decreased by only 20% at the time of diagnosis in progressors. In the majority of progressors plasma glucose showed a biphasic pattern, with an initial slow increase followed by a sudden rise around 0.7 years before diagnosis. At this time a further decline in glucose sensitivity was associated with a decline in insulin sensitivity and insulin secretion. In conclusion, this study indicates that in vivo -cell glucose insensitivity is an early defect in T1D, which could be used as a valuable parameter to identify progressors to clinical diabetes.
New paradigms 2 Growing faster increases T1D risk
Height growth velocity, islet autoimmunity and type 1 diabetes development: the Diabetes Autoimmunity Study in the Young Lamb MM, Yin X, Zerbe GO, Klingensmith GJ, Dabelea D, Fingerlin TE, Rewers M, Norris JM University of Colorado, Denver, Aurora, Colo., USA
[email protected] Diabetologia 2009;52:2064–2071 Background: The aim of the study was to assess a potential association of childhood size and growth rate with the development of islet autoimmunity (IA) and T1D. Methods: The study population was represented by participants to the Diabetes Autoimmunity Study in the Young (DAISY), which, since 1993, has followed children at increased T1D risk, based on HLA-DR, -DQ genotype or family history, for the development of IA (defined as the presence of autoantibodies to insulin, GAD or protein tyrosine phosphatase islet antigen 2 twice in succession) and T1D. Height and weight were collected starting at age 2 years, and these parameters together with BMI and velocities of growth in height, weight and BMI were assessed in relation to the development of IA and T1D, which developed in 143 and 21, respectively, of the 1,714 DAISY children aged less than 11.5 years. Results: Greater height growth velocity was associated with IA development (HR 1.63 [95% CI 1.31– 2.05]) and even more strongly with the development of T1D (HR 3.34 [95% CI 1.73–6.42]) for a 1 SD difference in velocity. Conclusions: In prepubertal children at increased genetic risk of T1D, greater height growth velocity may be involved in the progression from genetic susceptibility to autoimmunity and then to T1D.
A popular hypothesis maintains that the current childhood obesity epidemic is driving the increasing incidence and earlier age of T1D onset seen around the world. This study shows otherwise: an increased height velocity was associated with development of islet autoimmunity and progression to
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T1D in prepubertal children genetically predisposed to T1D. In contrast, BMI and rapid weight gain were not associated with study outcomes. These results raise the question as to whether increased height rate represents a direct stress factor for the -cell, which would support the ‘overload hypothesis’ [5]. This hypothesis postulates that overload of the -cells mediated by several mechanisms, including high growth rates, physical and psychological stress, insulin resistance, increases insulin demand and makes the -cells more susceptible to autoimmune attacks and apoptosis [5]. However, it needs to be acknowledged that increased growth rate may be just an epiphenomenon or, to use the authors’ words, ‘a side effect’ of the mechanisms driving the autoimmune disease process. It is possible that the primum movens is not the high height rate but a primary increase in insulin levels, probably genetically determined, which could lead to more rapid linear growth and higher demand for insulin from the -cell. The finding of a lack of association between IA/T1D and weight gain is in contrast with previous data in children [6]. However, differences in the characteristics of the study populations, such as age, presence or not of genetic susceptibility, might explain discordant findings across different studies. Interestingly, short stature was associated with autoimmunity but not with risk of progression to T1D. This is in contrast with previous findings showing an association between greater height and T1D and might reflect an adverse intrauterine environment associated later on with a higher growth velocity. One potential explanation for the findings is that increased linear growth velocity, perhaps associated with higher levels of IGF-I, may result in greater insulin secretion and insulin resistance, which have also been shown to be associated with greater IGF-I levels. Further studies are required to clarify the link between increased growth velocity and T1D risk and clarify whether the ‘overload hypothesis’ can truly explain this association.
Important for clinical practice 1 The metabolic memory wears off!
Effect of prior intensive therapy in type 1 diabetes on 10-year progression of retinopathy in the DCCT/EDIC: comparison of adults and adolescents White NH, Sun W, Cleary PA, Tamborlane WV, Danis RP, Hainsworth DP, Davis MD Washington University, St. Louis, Mo., USA
[email protected] Diabetes 2010;59:1244–1253 Background: To assess differences between adolescents and adults in the persistence of the benefits of intensive therapy 10 years after completion of the Diabetes Control and Complications Trial (DCCT). Methods: Progression of retinopathy from DCCT closeout to year 10 of the Epidemiology of Diabetes Interventions and Complications (EDIC) was evaluated in 1,055 adults and 156 adolescents. Results: During 10 years of follow-up, HbA1c was similar between previous DCCT intensive and conventional groups and between adolescents and adults. At EDIC year 10, progression of diabetic retinopathy was still slower in the intensively than in the conventionally treated adults (adjusted hazard reduction 56%, p < 0.0001), whereas in adolescents this beneficial effect had disappeared (32%, p = 0.13). The difference in the mean DCCT HbA1c between adolescents and adults (8.9 vs. 8.1%), mainly between the two intensively treated groups (8.1 vs. 7.2%), explained 79% of the observed differences between adults and adolescents in the metabolic effect on retinopathy progression. Conclusions: Glycemic control during the DCCT study is a key player in the long-term complication risk. The waning of the metabolic memory in the adolescent cohort at year 10 of the EDIC strongly highlights the importance of establishing a strict glycemic control as early as possible and of maintaining it over time.
The DCCT and its observational follow-up study, EDIC, represent landmark studies in the field of T1D and its vascular complications. The DCCT undoubtedly showed that complication risk significantly
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decreases with strict glycemic control both in adults and in adolescents [7]. The EDIC study raised the important concept of ‘metabolic memory’; in other words, although after the end of the DCCT HbA1c levels became comparable between the intensively and conventionally treated groups, patients belonging to the first group still kept an advantage from prior better HbA1c values [8]. This recent paper from White and colleagues raises the important point as to whether the benefit of a strict glycemic control during the DCCT wears off in the long run, particularly with regard to retinopathy. The most intriguing finding of the study was the emergence of differences in the persistence of the metabolic memory between the DCCT adolescent and adult cohorts. Although, at year 10 of EDIC there was a decrease in the 3-step progression risk in the intensively treated adult group when compared with year 4, the advantage of the previous intensively treated patients still persisted and was still significant. In contrast, in the adolescent cohort, retinopathy progression at year 10 of the EDIC did not differ between the previous intensively and conventionally treated groups, thus indicating loss of the metabolic memory. Interestingly, 79% of the difference in the metabolic effect between adults and adolescents after 10 years from the end of the DCCT was due to the difference in the mean HbA1c levels during the DCCT between the two cohorts. This 1% difference, which did not seem to play a major role during the DCCT and early EDIC years with regard to the outcomes, emerges as a major player in the long run. Although at the moment these results relate only to retinopathy, this is a key message, which once again strengthens the importance of establishing a good glycemic control as soon as possible in patients with T1D. Although youths with T1D rarely present advanced stages of complications, there is evidence that their pathogenesis starts soon after diagnosis and therefore preventive and therapeutic strategies should be implemented soon after diagnosis [9].
Important for clinical practice 2 Predicting T1D risk in the general population
Predictive characteristics of diabetes-associated autoantibodies among children with HLA-conferred disease susceptibility in the general population Siljander HT, Simell S, Hekkala A, Lahde J, Simell T, Vahasalo P, Veijola R, Ilonen J, Simell O, Knip M Hospital for Children and Adolescents and Folkhalsan Research Center, University of Helsinki, Helsinki, Finland
[email protected] Diabetes 2009;58:2835–2842 Background: The aim of this study was to assess the predictive performance of islet cell autoantibodies (ICAs) in combination with autoantibodies against insulin (IAAs), autoantibodies against GAD, and/or islet antigen 2 for T1D in children with HLA-defined disease predisposition recruited from the general population. Methods: The study population was made of 7,410 children, who were observed from birth (median 9.2 years) for -cell autoimmunity and T1D. If a child developed ICA positivity or diabetes, the three other antibodies were measured in all samples available from that individual. Persistent autoantibody positivity was defined as continued positivity in at least two sequential samples including the last available sample. Results: Pre-diabetic ICA positivity was observed in 1,173 subjects (15.8%), 155 of whom developed T1D. 86% of 180 progressors (median age at diagnosis 5.0 years) were identified with ICA screening. Positivity for four antibodies was associated with the highest disease sensitivity (54.4%), negative predictive values (98.3%) and the lowest negative likelihood ratio (0.5). Combining persistent ICA and IAA positivity resulted in the highest positive predictive value (91.7%), positive likelihood ratio (441.8), cumulative disease risk (100%), and specificity (100%). Young age at seroconversion, high ICA level, multipositivity, and persistent positivity for IAA were significant risk markers for T1D. Conclusion: The combination of HLA and autoantibody screening in the general population resulted in disease risks that are likely to be as high as those reported among autoantibody-positive siblings of children with T1D.
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Diabetes-associated autoantibodies, which are detectable during the preclinical phase of diabetes, are known to predict the development of T1D in siblings [10]. In contrast, their predictive value in the general population is unknown. This study is unique as it is based on a large population of 7,410 children from a country with the highest risk of T1D in the world and who were observed from birth for -cell autoimmunity and diabetes. The main inclusion criterion was carrying a high or moderate HLD haplotype for T1D susceptibility. During follow-up visits, blood samples were collected for measurement of ICAs, which were selected as the primary screening tool for -cell autoimmunity. If these autoantibodies were found in two consecutive samples, the other autoantibodies, IA-2A, GADA and IAA, were assessed. ICAs screening allowed to identify 86% of progressors, whereas this prediction was better when IAA were also assessed (97%). The combination of all four antibodies led to the highest disease sensitivity (54.4%), negative predictive value (98.3%) and the lowest likelihood ratio (0.5). The highest positive predictive value was obtained with the combination of ICA and IAA, reaching also the highest specificity and cumulative disease risk. The addition of GADA to ICA did not appear to improve prediction. Similarly to what is already known for first-degree relatives of children with T1D, a younger age at seroconversion, high ICAs levels and multipositivity for autoantibodies were associated with a higher risk of diabetes. Taken together, the findings of the present study suggest that HLA-genotyping together with regular assessment of autoantibody will represent an important tool to identify subjects at risk for T1D in the general population, once preventive programs are developed. Similar screening strategies will be of utmost importance, particularly in countries with a high incidence of T1D.
New genes Genome-wide association studies finally approach HbA1c
A genome-wide association study identifies a novel major locus for glycemic control in type 1 diabetes, as measured by both A1C and glucose Paterson AD, Waggott D, Boright AP, Hosseini SM, Shen E, Sylvestre MP, Wong I, Bharaj B, Cleary PA, Lachin JM, Below JE, Nicolae D, Cox NJ, Canty AJ, Sun L, Bull SB Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, Ont., Canada
[email protected] Diabetes 2010;59:539–549 Background: Although glycemic control is a key risk factor for diabetic complications, there are no data on its potential genetic determinants in patients with T1D. The aim of this study was to identify genetic loci associated with glycemia using longitudinal repeated measures of HbA1c from the Diabetes Control and Complications Trial (DCCT). Methods: A genome-wide association study was performed using the mean of quarterly HbA1c values measured over 6.5 years, separately in the conventional (n = 667) and intensive (n = 637) treatment groups of the DCCT. At loci of interest, linear mixed models were used to take advantage of all the repeated measures. Association of these loci with capillary glucose and repeated measures of multiple complications of diabetes were also assessed. Results: A major locus for HbA1c was found in the conventional treatment group near SORCS1 (10q25.1, p = 7 × 10–10), and it was also associated with mean glucose (p = 2 × 10–5). This was confirmed using A1C in the intensive treatment group (p = 0.01). Other loci achieved evidence close to genome-wide significance: 14q32.13 (GSC) and 9p22 (BNC2) in the combined treatment groups and 15q21.3 (WDR72) in the intensive group. The association of these loci with complication risk was also assessed and SORCS1 was linked with hypoglycemia, whereas BNC2 with renal and retinal complications. The association with glycemic control was replicated for SORCS1 in Genetics of Diabetes in Kidneys (GoKinD) study control subjects (p = 0.01) and for BNC2 in non-diabetic individuals. Conclusion: A major locus for A1C and glucose in individuals with T1D is near SORCS1.
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Hyperglycemia is a major player in the development of vascular complications of diabetes, through the activation of several metabolic pathways [11]. Landmark studies such as the DCCT and the EDIC have highlighted the key role of glycemic control in the preventing and/or slowing the development and progression of micro- and macrovascular complications. It is intriguing to observe that reaching a good glycemic control requires variable efforts in different patients and that glycemic control is often quite consistant in a given individual with diabetes. These findings underline the potential role of a genetic background in modulating plasma glucose concentrations. The role of genetic factors is also supported by twin and family studies, which have shown the heritability of measures of glycemic control, such as HbA1c, in non-diabetic individuals [12]. The present study deals with the important issue of looking for genetic determinants of glycemic control in people with T1D, using longitudinal data collected during the DCCT. A major locus (rs1358030) for HbA1c and mean glucose levels was found near SORCS1, a gene with some previous evidence of an association with glycemic traits. This locus was also replicated in a separate population, the GoKinD control subjects, and it also showed evidence for association with hypoglycemia. Other loci achieving evidence close to genome-wide significance were GSC, BNC2, and WDR72. Among them, BNC2 was replicated in a non-diabetic population (MAGIC) and was also associated with renal and retinal complications. Characterizing genes regulating glycemic control is fascinating and represents a major step forward in the management of patients with diabetes. In fact, the identification of individuals genetically predisposed to a poor metabolic control and therefore at higher risk for complications represents an important step in the long and fascinating way towards a personalized medicine, based on the concept that each patient should be considered as ‘a single one’, requiring a specific and personalized treatment plan, defined on the basis of his/her risk profile. The clear definition of genetic determinants of glycemic control is not an easy task as it requires to clearly distinguish the effect of heritability from environmental influences including treatement itself.
New genes 2 Confirmation of T1D genes
In silico replication of the genome-wide association results of the Type 1 Diabetes Genetics Consortium Qu HQ, Bradfield JP, Li Q, Kim C, Frackelton E, Grant SF, Hakonarson H, Polychronakos C Department of Pediatrics, McGill University, Montreal, Que.,, Canada
[email protected] Hum Mol Genet 2010;19:2534–2538 Background: The Type 1 Diabetes Genetics Consortium (T1DGC) recently reported 22 novel T1Dassociated loci identified by meta-analysis of three genome-wide association studies (GWASs) with a case-control design. However, the association of 10 of these 22 reported loci was not confirmed in the T1DGC family cohort. The aim of this study was to replicate the association in three independent GWAS cohorts to exclude potential bias from population stratification. Methods: Three European-descent population samples were included: 483 cases and both parents, a casecontrol cohort of 514 cases and 2,027 controls, and an additional cohort of 1,078 cases and 341 controls from the dbGaP database. Among the 22 SNPs reported by the T1DGC, there were high-quality genotypes for 15; the remaining were imputed. Results: T1D association was replicated in seven loci after Bonferroni correction for 22 independent hypotheses. An additional eight loci had nominal significance of p < 0.1 in the same direction. The genetic susceptibility conferred by non-HLA loci in the family cohort with 1 affected offspring was higher than the T1DGC multiplex families, whereas the frequency of HLA alleles in the multiplex families was higher.
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This study replicated T1D association with at least as many of these novel loci as expected from the power of the sample size, thus supporting the validity of the new discoveries.
Conclusion:
T1D is a complex disease resulting from the interaction of multiple genetic and environmental factors. Over the years many efforts have been made to understand its pathogenesis. Several genetic determinants had been identified already before the era of genome-wide association studies (GWAS). The recent development of high-throughput single nucleotide polymorphism genotyping array technologies has enabled investigators to perform high-density GWAS in search of additional T1D loci and the results of many GWAS have been reported to data. In last year chapter on T1D there were two important papers dealing with the genetics of T1D and the role of GWAS in allowing identification of a large number of loci associated with the disease [13]. The present study aimed to extend previous genetic findings in different populations. The T1DGC reported 22 novel loci associated with T1D; however, 10 of these 22 loci were not replicated in the T1DGC family cohort, raising the point as to whether heterogeneity in the populations selected in the different studies could have influenced the results [14]. Therefore, in the present study the authors attempted to validate the 22 previous identified loci, using three European descendent population samples. T1D association with seven loci was validated with a p < 4.55 × 10–3, other eight loci had a p < 0.01, whereas seven loci were not replicated. Overall this study validated the T1D association previously reported by the T1DGC and highlighted the complexity of genetics of T1D and the need of further investigations to understand the true role of the identified loci in the pathogenesis of T1D.
New concerns The lower the plasma glucose, the bigger the hippocampus
Hippocampal volumes in youths with type 1 diabetes Hershey T, Perantie DC, Wu J, Weaver PM, Black KJ, White NH Department of Psychiatry, Washington University School of Medicine, St. Louis, Mo., USA
[email protected] Diabetes 2010;59:236–241 Background: Hippocampal neurons have been suggested to be particularly vulnerable to glycemic excursions. The aim of this study was to assess the effect of hypoglycemia and hyperglycemia on hippocampal volume during brain development. Methods: The study population was represented by 95 youths with T1D and 49 sibling control subjects aged 7–17 years, who underwent magnetic brain resonance imaging. Stereologic measurements of hippocampal volumes were performed in atlas-registered space to correct for whole brain volume. T1D youths were categorized as having 0 (n = 37), 1–2 (n = 41), or 3 or more (3+; n = 17) prior severe hypoglycemic episodes. Hyperglycemia exposure was estimated from median lifetime A1C, weighted for duration of diabetes. Results: Greater exposure to severe hypoglycemia during childhood was associated with enlargement of the hippocampal volume (F [3,138] = 3.6, p = 0.016; 3+ larger than all other groups, p < 0.05). In contrast, hyperglycemia exposure was not associated with hippocampal volumes, and the 3+ severe hypoglycemia group still had larger hippocampal volumes after adjusting for age of onset and hyperglycemia exposure (main effect of hypoglycemia category, F [2,88] = 6.4, p = 0.002; 3+ larger than all other groups, p < 0.01). Conclusion: The increased hippocampal volume associated with severe hypoglycemia may be due to gliosis, reactive neurogenesis, or disruption of normal developmental pruning in the developing brain.
Diabetes can alter the function and structure of many organs, including the brain. Hyperglycemia and hypoglycemia can both alter the brain, damaging distinct areas. Data from animal studies have shown that hypoglycemia can selectively damage neurons in the medial temporal region, including
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the hippocampus. However, there are limited data in humans and in particular on the effect of hypoglycemia on the developing brain of young people with diabetes. Hershey et al. present interesting data, collected in a large group of 95 youths with T1D and 49 healthy children, on increased hippocampus volume in relation to severe hypoglycemic episodes. Hippocampal volume, measured with validated and unbiased stereologic methods, was strongly related to previous history of severe hypoglycemia, independently of potential confounders such as age, duration of diabetes, sex, and hyperglycemia. Interestingly, hyperglycemia, which is known to influence brain function, did not have any association with hippocampal volume. These data are unique as previous studies in adults with diabetes had shown no evidence of variation in hippocampal volume or, signs of neuronal death in patients with hypoglycemia. The present findings of an increased hippocampal volume might indicate a pathological response to severe hypoglycemia, expressed as gliosis, reactive neurogenesis, disruption of normal developmental pruning or a compensatory response to damage. Further studies will likely clarify the underlining mechanisms. For the moment, the available data deserve reflection on how vulnerable the developing brain to hypoglycemia can be and how the detected changes can evolve over time and influence long-term neurocognitive function.
New mechanisms 1 Epigenetic modulation of the insulin gene
Insulin gene expression is regulated by DNA methylation Kuroda A, Rauch TA, Todorov I, Ku HT, Al-Abdullah IH, Kandeel F, Mullen Y, Pfeifer GP, Ferreri K Department of Diabetes, Endocrinology, and Metabolism, Research Institute of City of Hope, Duarte, Calif., USA
[email protected] PLoS One 2009;4:e6953:1–9 Background: Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. In this study the role of DNA methylation in the regulation of mouse and human insulin gene expression was investigated. Methods: Genomic DNA samples from several tissues were bisulfite-treated and sequenced. Results: Cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in pancreatic -cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP-responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl-CpG binding protein 2 (MeCP2). In mouse embryonic stem cell cultures, the Ins2 gene is fully methylated and becomes demethylated during differentiation into insulin-expressing cells in vitro. Conclusion: Insulin promoter CpG demethylation may play a crucial role in -cell maturation and tissuespecific insulin gene expression.
Epigenetics is a central process implicated in the control of gene expression, providing the mechanism by which the environment interacts with identical genotypes to produce different phenotypes. Recently, interest has been focused on the implication of this process in the pathogenesis of T1D, through regulation of key molecules and processes involved in -cell development, survival, regeneration, function and autoimmune reactions [15]. In this paper, the emerging concept of ‘epigenetic modifications’ is assessed in relation to a key gene in the context of diabetes: the insulin gene. The authors found that methylation of this gene, as in general for other genes, is a mechanism of silencing its activity, whereas demethylation is associated with progressive expression of the gene and therefore increased insulin levels. DNA methylation patterns of the insulin gene were assessed in human and mouse -cells as well as in non--cells and in embryonic stem cells from the undifferentiated stage throughout the stages leading to insulin-expressing cells.
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The human insulin gene appears to have nine CpG sequences in its promoter. Demethylation of these sites is important for both basal and stimulated insulin gene expression. In contrast, in non--cells these sites are completely or in part methylated. Methylation of these sites induces recruitment of CpG binding protein 2 and probably of other proteins, which can interfere with binding of other factors, with the consequent suppression of gene expression. Another key finding of the study was that demethylation of the insulin promoter does not appear to be specifically involved in daily metabolic regulation, but it is more likely to be implicated in the development of insulin-producing -cells. Demethylation of the insulin gene is a late step in the differentiation of the -cell phenotype from embryonic stem cells. These findings highlight the complexity of gene regulation in humans and help to explain the difficulties met by the scientists who are trying to produce -cells in vitro, which should be then transplanted in patients with T1D. Up to now, a major problem encountered in this context has been how to obtain normal levels of insulin gene expression. The specific methylation/demethylation pattern that emerged from the present study is clearly a major aspect to consider and overcome in future studies, together with other potential epigenetic modulations of the insulin gene.
New mechanisms 2 Seeking -cell biomarkers
A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)␥a as a pancreatic -cell-specific biomarker Flamez D, Roland I, Berton A, Kutlu B, Dufrane D, Beckers MC, De Waele E, Rooman I, Bouwens L, Clark A, Lonneux M, Jamar JF, Goldman S, Marechal D, Goodman N, Gianello P, Van Huffel C, Salmon I, Eizirik DL Laboratory of Experimental Medicine, Universite Libre de Bruxelles, Brussels, Belgium
[email protected] Diabetologia 2010;53:1372–1383 Background: Non-invasive imaging of the pancreatic -cell mass requires the identification of novel and specific -cell biomarkers. In the present study a systems biology approach was used in order to identify potential -cell markers. Methods: A functional genomics strategy based on massive parallel signal sequencing and microarray data obtained in human islets, purified primary rat -cells, non--cells and INS-1E cells was applied. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays. Results: After a series of filtering steps, 12 -cell-specific membrane proteins were identified. For four of the proteins antibodies targeting specifically the human proteins and their splice variants were produced and allowed to confirm all four candidates as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na+,K+-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2␥a was restricted to pancreatic islets and selectively detected in pancreatic -cells. Analysis of human fetal pancreas samples showed the presence of FXYD2␥ at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with T1D or sections from pancreases of streptozotocin-treated M. cynomolgus monkeys indicated a close correlation between loss of FXYD2␥a and loss of insulin-positive cells. Conclusion: This study suggests human FXYD2␥a as a novel -cell-specific biomarker.
In the present study the application of a systems biology approach led to the identification of a specific new -cell biomarker, which could have important implications for assessing -cell mass with non-invasive techniques. The possibility of assessing -cell mass in humans represents an essential tool in studies aiming at better understanding the pathogenesis of the diseases as well as in evaluating the effect of emerging preventive strategies, directed at reducing -cell loss before disease onset.
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This study used a step-by-step approach consisting on functional genomics based on parallel sequencing and microarray data obtained from human islets, purified primary rat islet -cells and non--cells. Starting from 950 islet-specific genes, 114 were then identified as expressed at the membrane level and 44 of them were found to be preferentially expressed in -cells. Out of these 44 genes, 12 were further selected based on their non-responsiveness to inflammatory cytokines. The best candidate biomarker for -cells was FXYD2, with two of its three variants expressed in the human -cell and one, FXYD2␥a, exclusively expressed in these cells. This molecule represents a regulatory subunit of the ubiquitously distributed Na+,K+-ATPase [16]. Expression of this FXYD2␥a isoform emerged to be an early embryonic event and its levels decreased in parallel with -cell mass loss. These results suggest this biomarker as a potential useful one for non-invasive imaging and quantification of pancreatic -cell mass. Further studies will hopefully confirm these findings and the usefulness of this biomarker firstly in the research setting and then also in clinical practice.
New mechanisms 3 Time to focus on antigen presentation!
Targeted regulation of self peptide presentation prevents type 1 diabetes in mice without disrupting general immunocompetence Yi W, Seth NP, Martillotti T, Wucherpfennig KW, Sant’Angelo DB, Denzin LK Immunology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, N.Y., USA
[email protected] J Clin Invest 2010;120:1324–1336 Background: Peptide loading of MHC class II (MHCII) molecules is directly catalyzed by the MHCII-like molecule HLA-DM (DM). Another MHCII-like molecule, HLA-DO (DO), associates with DM, thereby modulating DM function and dampening presentation of self antigens. Methods: In order to test the idea that DO modulation of the MHCII self peptide repertoire mediates selftolerance, NOD mice (a mouse model for T1D) that constitutively overexpressed DO in DCs (referred to herein as NOD.DO mice) were generated. Results: Diabetes development was completely blocked in NOD.DO mice. NOD.DO animals selected a diabetogenic T-cell repertoire, the numbers and function of Tregs were normal, and therefore their immune system function was equivalent to that in NOD mice. However, NOD.DO DCs presented an altered MHCII-bound self peptide repertoire, thereby preventing the activation of diabetogenic T cells and subsequent diabetes development. Conclusion: DO expression can shape the overall MHCII self peptide repertoire to promote T-cell tolerance.
Up to now much of the focus in the pathogenesis of T1D has been directed towards T lymphocytes, being T1D a T-cell-mediated autoimmune disease. However, an important aspect in the development of self-reactive lymphocytes is represented by reaction with self antigens presented by specific antigen presenting cells, such as dendritic cells, in the context of the MHCII molecules [17]. The present study clearly shows that subtle changes in MHCII antigen presentation can prevent disease development, therefore proposing the manipulation of antigen presentation by dendritic cells as a new potential target toward which preventive strategies should be directed. Peptide loading of MCHII is catalyzed in late endosomal and lysosomal compartments of cells by the catalytic action of human HLA-DM. In B cells, dendritic cells and thymic epithelial cells, the peptide loading of class II molecules is modified by the expression of the non-classical class II molecule, HLA-DO [17]. The biological role of HLA-DO-mediated regulation of DM activity in vivo remains unknown; however, it has been postulated that DO expression dampens presentation of self antigens, thereby preventing inappropriate T-cell activation that ultimately leads to autoimmunity [18]. In the present study performed in a NOD.DO mice model, the development of diabetes was prevented by HLA-DO expression in dendritic cells. This protection appeared to be due to an inefficient presentation of self antigens by dendritic cells overexpressing HLA-DO, therefore maintaining periph-
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eral tolerance and confirming the above discussed hypothesis. These data suggest that when investigating the pathogenesis of T1D it is important not only to direct efforts towards autoreactive T lymphocytes, but consider the numerous players involved in the autoimmune reactions.
Clinical trial, concepts revised To treat or not to treat?
Renal and retinal effects of enalapril and losartan in type 1 diabetes Mauer M, Zinman B, Gardiner R, Suissa S, Sinaiko A, Strand T, Drummond K, Donnelly S, Goodyer P, Gubler MC, Klein R Department of Pediatrics, University of Minnesota, Minneapolis, Minn., USA
[email protected] N Engl J Med 2009;361:40–51 Background: It is unclear whether progression of nephropathy and retinopathy in people with T1D is slowed by early administration of drugs blocking the renin-angiotensin system (RAS). Methods: A multicenter, controlled trial involving 285 normotensive patients with T1D and normoalbuminuria, randomly assigned to receive losartan (100 mg daily), enalapril (20 mg daily), or placebo and followed for 5 years, was performed. The primary endpoint was a change in the fraction of glomerular volume occupied by mesangium in kidney-biopsy specimens. The retinopathy end point was a progression of two steps or more. Results: A total of 90 and 82% of patients had complete renal biopsy and retinopathy data, respectively. Change in mesangial fractional volume per glomerulus over the 5-year period did not differ between the placebo (0.016 units) and the enalapril (0.005, p = 0.38) or the losartan group (0.026, p = 0.26), nor were there significant differences in the other renal structural variables. The 5-year cumulative incidence of microalbuminuria was 6% in the placebo group; the incidence was higher with losartan (17%, p = 0.01) but not with enalapril (4%, p = 0.96). Compared with placebo, enalapril reduced the odds of retinopathy progression by 65% (odds ratio [95% confidence interval]: 0.35 [0.14–0.85]) and losartan by 70% (0.30 [0.12–0.73]), independently of blood pressure changes. There were three biopsy-related serious adverse events that completely resolved. Chronic cough occurred in 12 patients receiving enalapril, 6 receiving losartan, and 4 receiving placebo. Conclusion: Early blockade of the RAS in patients with T1D did not slow nephropathy progression but slowed the progression of retinopathy.
Several studies, manly performed in adults, have shown that treatment with angiotensin-converting enzyme inhibitors (ACEIs) reduces the rate of progression and can even promote regression of microalbuminuria [19], independently from the effect on blood pressure. ACEIs can also have a significant effect on other diabetic microvascular complications, such as retinopathy [20] and potentially cardiovascular disease [21]. The study by Mauer and colleagues does not confirm the previously observed beneficial results of ACEIs therapy in patients with diabetic nephropathy and highlights the concept that blockers of the RAS do not necessarily change renal pathology in initially normotensive normoalbuminuric subjects. The Renin-Angiotensin System Study (RASS) is up to now the largest long-term study assessing the effect of a 5-year blockage of the RAS system in people with T1D. In this study, two different strategies of inhibiting the RAS were evaluated: ACEIs and angiotensin receptor inhibitors (ARBs). In addition, a novelty of the present study was the primary study endpoint, which was represented by early renal structural alterations, assessed through renal biopsies performed at baseline and 5 years later. In contrast, the majority of previous studies had as main study endpoint changes in albumin excretion. Surprisingly, the study not only demonstrated that treatment with ACEIs or ARBs did not influence any structural renal parameter but it also showed that losartan increased the incidence of microalbuminuria when compared to placebo (17 vs. 6%), whereas enalapril did not have any effect on this secondary study endpoint.
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In contrast, inhibition of the RAS was associated with a 65–70% reduction in retinopathy progression, independently of changes in blood pressure, in patients with no sign or minimal pre-proliferative retinopathy at baseline. How can this different effect on the two vascular complications be explained? As pointed out also in the accompanying editorial of this paper, the pathogenesis of complications of diabetes is heterogeneous, with an interplay of several mechanisms, which can differ or have a different effect in the different microcirculations of the kidney and retina, particularly during the early stages of diabetic complications. With regard to differences from previous studies, the lack of an effect on renal outcomes might be related to the fact that intervention was started at an early stage of renal pathology, whereas in other investigations a beneficial effect was seen when applied at a more advanced stage of diabetic nephropathy. Again, this could implicate a different role of RAS in different stages of nephropathy.
Clinical trial, new treatments On the way towards the artificial pancreas
Manual closed-loop insulin delivery in children and adolescents with type 1 diabetes: a phase 2 randomized crossover trial Hovorka R, Allen JM, Elleri D, Chassin LJ, Harris J, Xing D, Kollman C, Hovorka T, Larsen AM, Nodale M, De Palma A, Wilinska ME, Acerini CL, Dunger DB Department of Paediatrics, University of Cambridge, Cambridge, UK
[email protected] Lancet 2010;375:743–51 Background: The effect of a closed-loop system, linking continuous glucose measurements to insulin delivery, on overnight blood glucose control was assessed in young people with T1D. Methods: Three randomized crossover studies were performed in 19 patients (age: 5–18 years; mean (SD) T1D duration: 6.4 (4.0) years). In study 1 (n = 13), a standard continuous subcutaneous insulin infusion was compared with closed-loop insulin delivery; in study 2 (n = 7) closed-loop delivery was assessed after rapidly and slowly absorbed meals; in study 3 (n = 10) a comparison was performed between closed-loop delivery and standard treatment after exercise. During closed-loop nights, glucose was assessed every 15 min and values included into a control algorithm calculating rate of insulin infusion, and a nurse adjusted the insulin pump. During control nights, patients’ standard pump settings were applied. Primary outcomes were time for which plasma glucose concentration was 3.91–8.00 or 3.90 mmol/l or lower. Results: Primary outcomes did not differ significantly between treatment groups in the three studies considered individually: study 1 (target range, median 52% closed loop vs. 39% standard treatment, p = 0.06; ≤3.90 mmol/l, 1 vs. 2%, p = 0.13), study 2 (target range, rapidly 53% vs. slowly absorbed meal 55%, p = 0.97; ≤3.90 mmol/l, 0 vs. 0%, p = 0.16), and study 3 (target range 78% closed loop vs. 43% control, p = 0.0245, not significant at corrected level; ≤3.90 mmol/l, 10 vs. 6%, p = 0.27). A secondary analysis of pooled data (study 1 + study 3) documented increased time in the target range (60 vs. 40%; p = 0.0022) and reduced time for which glucose concentrations were 3.90 mmol/l or lower (2.1 vs. 4.1%; p = 0.0304). During closed-loop delivery there were no events with plasma glucose concentration <3.0 mmol/l compared with nine events during standard treatment. Conclusions: Overnight manual closed-loop insulin delivery can improve glucose control and reduce risk of nocturnal hypoglycemia in young patients with T1D.
Hovorka et al. report the results of three randomized crossover studies performed in children and adolescents with T1D, where the effect of a manual closed-loop insulin delivery system on overnight blood glucose was assessed in comparison to a standard continuous insulin infusion. In addition, the performance of this system was investigated in relation to a variable-content evening meal and moderate-intensity evening exercise.
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Insulin therapy has been marked by unforgettable milestones starting from insulin discovery and followed, during most recent years, by the advent of insulin analogues and the implementation of pump therapy and continuous glucose monitoring. However, nowadays, treatment is still problematic and exogenous insulin administration is still far away from mimicking the endogenous insulin pattern. In addition, although intensive insulin therapy is the cornerstone of an optimal management of diabetes, aiming at reducing complication risk, the associated risk of hypoglycemia cannot be underestimated. Closed-loop insulin systems, through the combination of a continuous glucose monitor, a control algorithm and an insulin pump, aim at resembling an artificial pancreas [22]. Hovorka’s study shows that this system can achieve a safe and good overnight glucose control. By combining the data from studies 1 and 3, the authors found that closed-loop insulin delivery was associated with an increased likelihood of plasma glucose concentrations to be in the target range and reduced frequency of low glucose values, particularly after midnight when the system appears to have reached full effectiveness. Although further assessment and improvements of the system are required in the next years, the present results represent an important step forward in a still complex way towards the development and implementation of an artificial pancreas, which could not only ease daily life, but also solve the fears and consequences related to uncontrolled hyperglycemic and hypoglycemic episodes in children with T1D.
New hope 1 B-cell depletion as a novel therapy for T1D
Rituximab, B-lymphocyte depletion, and preservation of -cell function Pescovitz MD, Greenbaum CJ, Krause-Steinrauf H, Becker DJ, Gitelman SE, Goland R, Gottlieb PA, Marks JB, McGee PF, Moran AM, Raskin P, Rodriguez H, Schatz DA, Wherrett D, Wilson DM, Lachin JM, Skyler JS Indiana University School of Medicine, Indianapolis, Ind., USA
[email protected] N Engl J Med 2009;361:2143–2152 Background: There is growing evidence that B lymphocytes play a role in many T-lymphocyte-mediated diseases, such as T1D. The aim of this phase 2 study was to evaluate the role of B-lymphocyte depletion with rituximab, an anti-CD20 monoclonal antibody, in patients with T1D. Methods: In this randomized, double-blind study, 87 patients (aged 8–40 years) with newly diagnosed T1D were assigned to receive infusions of rituximab or placebo on days 1, 8, 15, and 22 of the study. The primary outcome, assessed 1 year after the first infusion, was the geometric mean area under the curve (AUC) for the serum C-peptide level during the first 2 h of a mixed-meal tolerance test. Secondary outcomes included safety and changes in HbA1c and insulin dose. Results: At 1 year, the mean AUC for the level of C peptide was significantly higher, whereas HbA1c and insulin dose were lower in the rituximab than in the placebo group. A lower rate of decline in C-peptide levels between 3 and 12 months was detected in the rituximab than in the placebo group. CD19+ B lymphocytes were depleted in patients in the rituximab group, but levels increased to 69% of baseline values at 12 months. There was no increase in infections or neutropenia with rituximab, although patients in the rituximab group were more likely to experience grade 1 or 2 adverse events after the first infusion. Conclusions: A four-dose course of rituximab partially preserved -cell function over a period of 1 year in patients with T1D, suggesting a role of B lymphocytes in the pathogenesis of T1D.
T1D is an autoimmune disease in which T lymphocytes mediate damage to pancreatic -cells. However, as for other T-cell-mediated diseases, a potential role for B lymphocytes has also been supposed and this represents the starting point for the hypothesis of the present study [23]. In this randomized double-blind study, depletion of B lymphocytes with the anti-CD20 monoclonal antibody rituximab was able to preserve -cell function in patients with newly diagnosed T1D. HbA1c and insulin dose were reduced in the rituximab group, and these differences were explained by pres-
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ervation of C-peptide levels. Although there was a higher rate of infusion reactions, there was no increased risk of neutropenia and infections, therefore supporting the safety, at least in the short term, of this treatment. Rituximab is a drug approved for treatment of B-cell lymphomas and its efficacy has also been shown in several autoimmune diseases [24]. In a previous animal study, treatment with anti-CD20 antibodies was associated with B-cell depletion, and significantly delayed and/or reduced the onset of diabetes [25]. The present study provides new hopes for preserving -cell function in people treated soon after diagnosis. This is of relevant importance as a residual insulin capacity has been associated with a better metabolic control and reduced complication risk. How can B-cell depletion be protective against diabetes? In the context of diabetes, B lymphocytes act through the production of autoantibodies, the hallmark of autoimmunity. However, it is controversial whether autoantibodies exert a main pathogenetic role in T1D. It is more likely that they enhance or facilitate T1D development without having a main role in its induction. However, B cells can also act as islet antigen-presenting cells for autoreactive T cells and B-cell depletion is associated with the generation of regulatory T and B cells, which could modulate the immune process [24, 25]. Given the demonstrated importance of both T and B cells in diabetes pathogenesis, modulation of both arms of the immune response may represent a key strategy for the development of protective immunotherapies. Further research in this area is mandatory to confirm the present findings, clarify mechanisms beyond them and to assess the long-term safety related to immunosuppressive therapies.
New hope 2 Halting glucose excursions in youths with T1D
Ramlintide lowered glucose excursions and was well tolerated in adolescents with type 1 diabetes: results from a randomized, single-blind, placebo-controlled, crossover study Chase HP, Lutz K, Pencek R, Zhang B, Porter L Barbara Davis Center for Childhood Diabetes, University of Colorado, Aurora, Colo., USA
[email protected] J Pediatr 2009;155:369–373 Background: In adults with type 1 and type 2 diabetes, pramlintide reduce gastric emptying, glucose excursions, improve glycemic control and reduced weight gain. In this study the pharmacokinetics, pharmacodynamics, safety, and tolerability of pramlintide in adolescents with T1D were assessed. Methods: 12 subjects (9 females, 3 males, age 12–17 years; A1C, 8.4%; body mass index, 25 kg/m2) were randomized to pramlintide (15 or 30 µg) or placebo administered before a standardized breakfast. Insulin lispro (50% of usual mealtime dose) was injected separately. Acetaminophen (1,000 mg) was administered orally to provide an indicator of gastric emptying rate. Results: Complete data were available for 9 participants, where plasma pramlintide concentrations increased in a dose-dependent manner. Mean peak plasma concentration (Cmax) (15-µg dose, 93±9 pg/ ml; 30-µg dose, 202±21 pg/ml) occurred approximately 0.3 h (median time to peak concentration) after administration. Pramlintide reduced incremental area under the concentration curve (AUC(0–3 h)) for glucagon and glucose versus placebo (glucagon: 15-µg dose, 4±7 pg · h/ml; 30-µg dose, 5±7 pg · h/ml; placebo, 35±9 pg · h/ml; glucose: 15-µg dose, 129±43 mg · h/dl; 30-µg dose, 132±37 mg · h/dl; placebo, 217±56 mg · h/dl). Acetaminophen C(max) decreased with pramlintide; median T(max) was delayed by approximately 2.6- to 3.8-fold. Pramlintide was well tolerated, and no treatment-related adverse events occurred. Conclusions: In adolescents with T1D, pramlintide showed a similar pharmacokinetic profile as in adults and was able to reduce postprandial glucagon and glucose excursions and slow gastric emptying. Larger and longer term studies are warranted to confirm and extend the results of this study.
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Achieving a good glycemic control is the cornerstone of treatment of diabetes but also a major challenge, particularly in adolescents with T1D. Adolescence is a period characterized by several physiological and psychological changes, which can influence glycemic control. The DCCT study clearly highlighted this issue by showing a 1% difference in HbA1c and a higher risk of hypoglycemia and weight gain between the adolescent and adult cohorts. Adjunct therapies to improve glycemic control are therefore particularly warranted in this age group. But, which therapies should be considered? In healthy subjects, glucose homeostasis is the results of an interplay of several hormones, including insulin, glucagon, glucagon-like peptide-1 and amylin [26]. Therefore, in people with diabetes, not only there is a lack of endogenous insulin production, but the secretion and/or the action of these other hormones may be impaired and contribute to the overall metabolic imbalance. In adults with diabetes, pramlintide, a synthetic analog of the pancreatic hormone amylin, has been shown to decrease HbA1c and postprandial glucose excursions as well as to reduce weight gain and gastric emptying [27]. Preliminary data in adolescents with T1D have shown that this molecule can delay gastric emptying, inhibit glucagon secretion and improve postprandial glucose excursions [28]. Chase et al. present a single-blind randomized controlled cross-over trial aiming at establishing the pharmacokinetics and safety profile of two doses of pramlintide, 15 and 30 µg, in a small group of 12 adolescents. Plasma concentrations of pramlintide were dose-dependent, whereas there was no such an effect on time for reaching peak concentration and duration of effect. Both doses of pramlintide were equally able to decrease glucagon release, reduce plasma glucose excursions and delay gastric emptying. No significant adverse effects were detected. Overall this study shows that in adolescents pramlintide has similar pharmacokinetics and safety profile than in adults and could represents a valid adjunct therapy, particularly in adolescents with fluctuating blood glucose control. Further larger and longer term studies are required and hopefully they will confirm and strengthen the findings of the present study.
Concepts revised Childhood- vs. adulthood-onset diabetes: does it matter?
Age at onset and the risk of proliferative retinopathy in type 1 diabetes Hietala K, Harjutsalo V, Forsblom C, Summanen P, Groop PH Folkhalsan Institute of Genetics, Folkhalsan Research Center, Biomedicum Helsinki, Finland
[email protected] Diabetes Care 2010; 33:1315–1319 Background: The aim of this study was to assess how age at the onset of T1D influences the long-term risk of developing proliferative retinopathy. Methods: Fundus photographs and/or ophthalmic record were available for 1117 consecutively recruited patients taking part to the FinnDiane Study. The risk of proliferative retinopathy was studied in age at onset groups 0–4, 5–14 and 15–40 years. Results: The mean durations to proliferative retinopathy were 24.3 (22.7–25.9) years in 0–4 group, 20.1 (19.2–21.1) years in 5–14 group, and 21.6 (19.8–23.3) years in 15–40 group (p < 0.001). In a Cox regression model, after adjusting for potential confounders, the highest risk of proliferative retinopathy was observed in 5–14 group (HR 1.90 [95% CI 1.45–2.48], p < 0.001). The long-term risk of proliferative retinopathy did not differ between those diagnosed aged 0–4 years compared with 5–14 years (p = 0.2). Risk of proliferative retinopathy was significantly higher in the age at onset group <15 years than in the age at onset group ≥15 years (HR 1.82 [95% CI 1.40–2.36], p < 0.001). Conclusions: An early age at onset of T1D confers a longer time free of proliferative retinopathy. However, this advantage disappears over time. The highest risk for developing proliferative retinopathy is in age at onset group 5–14, whereas the lowest risk is in the age at onset group 15–40 years.
An early onset of diabetes has been for a long time thought to be protective with respect to the development of long-term vascular complications [29]. In this recent study, Hietala et al. revise this
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concept and report that, although patients diagnosed at a young age (0–4 years) have a longer time free of proliferative retinopathy, this advantage gradually disappears over time and, after about 30-year diabetes duration, their risk of developing proliferative retinopathy is comparable to that of patients diagnosed when aged 5–14 years. These results are similar to those reported by Amin et al. [30] in 2008, for risk of microalbuminuria in a longitudinal cohort of people with childhood-onset diabetes. In Amin’s study, children with an early onset of diabetes showed a silent period, but after 15 years of diabetes duration the risk of developing microalbuminuria was similar between subjects diagnosed with diabetes before 5 years of age and those diagnosed between 5 and 11 years of age or after puberty. Taken together, the results of these two studies suggest that an early age at the onset of diabetes does not protect from complications, but it does affect the age at which complications are first detected. Hietala and colleagues offer several suggestions on the mechanisms which could explain the delayed onset of complications in their youngest age group. One hypothesis is that behavioral factors might play a role, so that younger children learn easier good self-care skills, which then lead to better metabolic control. A more stringent diabetes management might be another implicated factor. In addition, the negative effect of puberty on glycemic control needs to be considered as a potential accelerator of complications onset. Another important point raised from the Hietala’s study was the lowest risk detected in patients diagnosed at an age between 15 and 40 years. This is likely related to a less aggressive form of diabetes characterizing this age group. In particular this could be explained by a better preservation of -cell function and of C-peptide levels, factors which have been associated with a decreased incidence of complications [31].
Age at onset of type 1 diabetes in parents and recurrence risk in offspring Harjutsalo V, Lammi N, Karvonen M, Groop PH Folkhalsan Institute of Genetics, Folkhalsan Research Centre, Biomedicum Helsinki, Helsinki, Finland
[email protected] Diabetes 2010;59:210–214 Background: The aim of this study was to assess the recurrence risk of T1D in the offspring of parents with adult-onset (15–39 years) T1D and the transmission of diabetes within a continuum of parental age at onset of diabetes from childhood to adulthood. Methods: Diabetes status of all offspring (n = 9,636) in two Finnish cohorts of parents with T1D was defined until the end of year 2007. Cumulative incidences of T1D among the offspring were estimated, and several factors contributing to the risk were assessed. Results: During 137,455 person-years, a total of 413 offspring were diagnosed with type 1 diabetes. The cumulative incidence by 20 years was 4.0% (95% CI 3.1–4.8) for the offspring of parents with adultonset diabetes, with a similar risk in offspring of diabetic mothers and fathers. The cumulative incidence decreased in parallel with the increase in age at onset of diabetes in the fathers, whereas the risk did not change with age at onset in the offspring of mothers with T1D. However, the reduced risk in the maternal offspring was most pronounced in the daughters of the mothers with a diagnosis age <10 years. Conclusion: T1D transmission ratio distortion is strongly related to the sex and age at onset of diabetes in the diabetic parents.
Up to now, studies have been assessing the risk of diabetes recurrence in offspring of parents with onset of diabetes during childhood. The majority of these studies have shown a higher risk when the father is affected by T1D than when the mother is the index case. The present study represents a step forwards in the understanding of transmission of diabetes as it assesses this issue within a continuum of parental age at onset from childhood to adulthood. The study is based on population-based cohorts form Finland: 3,881 offspring in the late-onset cohort, diagnosed between 15 and 39 years of age and 5,821 in the early-onset cohort, diagnosed before the age of 15 years. T1D developed in 318 offspring in the early-onset cohort and 97 in late-onset cohort. In the late-onset cohort, where the cumulative risk was 4% by 20 years, the risk in the offspring did not differ by sex of the parents: 4.2% in the offspring of mothers vs. 3.8% in the offspring of fathers.
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Interestingly, whereas in the offspring of fathers from the late-onset cohort the risk decreased with age at diagnosis (HR = 2.9 for 15–19 years vs. 35–39), this was not the case for the offspring of mothers. This age-related trend was even more evident when both the early- and late-onset cohorts were considered together, resulting in a declining risk in the offspring of fathers with increased age at onset. For the mothers, no such trend was detected, although when considering only the offspring of mother diagnosed before 10 years of age girls showed a higher risk than boys. The findings of the present study confirm the concept that differences in the genetic, autoimmune and clinical aspects exist between childhood versus adulthood-onset diabetes. In particular, the results of this study are in line with the concept that age at onset is an indicator of genetic susceptibility. Therefore, an earlier onset underlines a stronger genetic component, and this is clearly evident from the pattern of risk in the offspring of diabetic fathers. Although in previous studies the lower risk in offspring of mothers was explained by a certain protection of the diabetic intrauterine environment, this protection cannot be claimed for offspring of mothers with adult-onset diabetes.
Food for thought Can we improve islet transplantation success?
High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice Matsuoka N, Itoh T, Watarai H, Sekine-Kondo E, Nagata N, Okamoto K, Mera T, Yamamoto H, Yamada S, Maruyama I, Taniguchi M, Yasunami Y Department of Regenerative Medicine and Transplantation, Fukuoka University, Fukuoka, Japan
[email protected] J Clin Invest 2010;120:735–743 Background: Islet transplantation for the treatment of T1D is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-␥ production by Gr-1+CD11b+ cells is essential for this loss, but the upstream events in the process remain undetermined. Methods: A mouse model of diabetes was used to assess the involvement of high-mobility group box 1 (HMGB1) in the initial events of early loss of transplanted islets. Results: A major source of HMGB1 is represented by pancreatic islets, from where this protein is released into the circulation soon after islet transplantation. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-␥ production by NKT cells and Gr-1+CD11b+ cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. HMGB1 stimulated hepatic mononuclear cells (MNCs), through an upregulation of CD40 expression and an increased production of IL-12 by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-␥ production by Gr-1+CD11b+ cells. Treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. Conclusion: HMGB1 and related activated pathways play an important role in early islet loss and represents a potential target for intervention to improve the efficiency of islet transplantation.
Islet transplantation is a promising procedure for the cure of diabetes. However, low efficiency of islet transplantation has been a major obstacle limiting its clinical applications. Early loss of transplanted islet is a key problem to be solved [32]. Animal models have shown that NKT cell-dependent IFN-␥ production by Gr-1+CD11b+ cells is an important mediator of this early loss [33]. The present study represents a step forward in understanding this process, focusing on the role of HMGB1, a protein which appears to play a key role in response to tissue damage and is released by inflammatory cells, such as dendritic cells, NK cells, and macrophages [34]. Through a series of elegant and detailed experiments in a mouse model of diabetes, the authors reached some key conclusions. They found that a major source of HMGB1 is represented by pancreatic islets, where the protein is localized mainly in nuclei and its plasma levels increase soon after transplantation, therefore repre-
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senting a potential early marker of transplant failure. HMGB1 activates the production of inflammatory cytokines including IL-12 and INF-␥. In particular, based on receptor expression pattern, the study showed that the first cell line target for HMGB is represented by dendritic cells, where they stimulate IL-12 production, which in turn could stimulate NKT-dependent INF-␥ production. These inflammatory cytokines in turn further contribute to damage of the transplanted islets. The identification and definition of this complex network represents an important discovery, which highlights potential new targets for interventions aiming at increasing the rate of success of islet transplantation.
Reviews
Mitochondria-mediated cell death in diabetes Szabadkai G, Duchen MR Department of Cell and Developmental Biology, Mitochondrial Biology Group, University College London, London, UK
[email protected] Apoptosis 2009;14:1405–1423 Overview: Mitochondrial dysfunction play a role in the pathogenesis of a wide range of diseases that involve disordered cellular fuel metabolism and survival/death pathways, including neurodegenerative diseases, cancer and diabetes. Cytokine, virus recognition and cellular stress pathways converging on mitochondria cause apoptotic and/or necrotic cell death of -cells in T1D. In addition, mitochondrial dysfunction underlies both the functional derangement of glucose-stimulated insulin secretion and stress-induced apoptotic/necrotic -cell death, which characterize type 2 diabetes. In this review a summary of the main findings supporting such a key role of the mitochondria in -cell death are summarized.
Mitochondria, the cellular aerobic bioenergy production sites, are involved in a variety of metabolic activities, and there are several lines of evidence showing a key roles of these intracellular organelles in the pathogenesis not only of diabetes but also of its complications [35]. Interestingly, mitochondrial increased oxidant generation seems to play an important role also in the context of the ‘metabolic memory’. In fact, increased mitochondrial superoxide production consequent to hyperglycemia can induce not only immediate effects, but it might also damage mitochondrial DNA and proteins, leading to synthesis of altered mitochondrial respiratory channel subunits, which could produce increased amount of superoxide, even in presence of physiological glucose levels [35]. In this review the authors report and discuss the current level of evidence supporting a key role of mitochondria in -cell death in the context of both T1D and type 2 diabetes. Although the pathogenesis of the two diseases is distinct, -cell death in both conditions appears to be mediated by a common final mechanism, represented by mitochondrial membrane permeation. In T1D, -cell death is directly linked to the autoimmune process, whereas in type 2 diabetes metabolic stress induces -cell apoptosis/necrosis. In the context of T1D, the main soluble mediator of -cell apoptosis produced by CD4+ T cells and macrophages are IL-1 , INF-␥ and TNF-␣, which stimulate NF-B activation, that in turn mediates the pro-apoptotic signal. Also, CD8+ T-cell-mediated -cell death appears to be linked to mitochondria, through the involvement of bcl-2 family. Additional mechanisms linking -cell death in T1D with mitochondria dysfunction are activation of poly(ADP-ribose) polymerase and NO competition for molecular oxygen at the level of complex IV of the mitochondria. Based on these data, mitochondria appear to be an important cellular component towards which further research should be directed to clarify aspects of the pathogenesis of diabetes.
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References 1. Patterson CC, Dahlquist GG, Gyurus E, Green A, Soltesz G: Incidence trends for childhood type 1 diabetes in europe during 1989–2003 and predicted new cases 2005–20: a multicentre prospective registration study. Lancet 2009;373:2027– 2033. 2. Narayan KM, Boyle JP, Thompson TJ, Sorensen SW, Williamson DF: Lifetime risk for diabetes mellitus in the united states. JAMA 2003;290:1884–1890. 3. Rewers A, Klingensmith G, Davis C, Petitti DB, Pihoker C, Rodriguez B, et al: Presence of diabetic ketoacidosis at diagnosis of diabetes mellitus in youth: the search for diabetes in youth study. Pediatrics 2008;121:e1258–e1266. 4. Hope KM, Tran PO, Zhou H, Oseid E, Leroy E, Robertson RP: Regulation of ␣-cell function by the -cell in isolated human and rat islets deprived of glucose: the ‘switch-off’ hypothesis. Diabetes 2004;53:1488–1495. 5. Dahlquist G: Can we slow the rising incidence of childhood-onset autoimmune diabetes? The overload hypothesis. Diabetologia 2006;49:20–24. 6. Hypponen E, Virtanen SM, Kenward MG, Knip M, Akerblom HK: Obesity, increased linear growth, and risk of type 1 diabetes in children. Diabetes Care 2000;23:1755–1760. 7. The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977–986. 8. Writing Team for the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group: Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus. JAMA 2002;287:2563–2569. 9. Marcovecchio ML, Tossavainen PH, Dunger DB: Prevention and treatment of microvascular disease in childhood type 1 diabetes. Br Med Bull 2010;94:145–164. 10. Bingley PJ, Bonifacio E, Ziegler AG, Schatz DA, Atkinson MA, Eisenbarth GS: Proposed guidelines on screening for risk of type 1 diabetes. Diabetes Care 2001;24:398. 11. Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813–820. 12. Meigs JB, Panhuysen CI, Myers RH, Wilson PW, Cupples LA: A genome-wide scan for loci linked to plasma levels of glucose and HbA1c in a community-based sample of caucasian pedigrees: The framingham Offspring Study. Diabetes 2002;51:833–840. 13. Chiarelli F, Giannini C: Type 1 diabetes: clinical and experimental; in Carel J, Hochberg Z (eds): Yearbook of Pediatric Endocrinology 2009. Basel, Karger, 2009, pp 123–124. 14. Barrett JC, Clayton DG, Concannon P, Akolkar B, Cooper JD, Erlich HA, et al: Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nat Genet 2009. 15. MacFarlane AJ, Strom A, Scott FW: Epigenetics: deciphering how environmental factors may modify autoimmune type 1 diabetes. Mamm Genome 2009;20:624–632. 16. Li C, Grosdidier A, Crambert G, Horisberger JD, Michielin O, Geering K: Structural and functional interaction sites between Na,K-ATPase and FXYD proteins. J Biol Chem 2004;279:38895–38902. 17. Hudson AW, Ploegh HL: The cell biology of antigen presentation. Exp Cell Res 2002;272:1–7. 18. Denzin LK, Fallas JL, Prendes M, Yi W: Right place, right time, right peptide: DO keeps DM focused. Immunol Rev 2005;207:279–292. 19. Lovell HG: Angiotensin-converting enzyme inhibitors in normotensive diabetic patients with microalbuminuria. Cochrane Database Syst Rev 2001:CD002183. 20. Chaturvedi N, Sjolie AK, Stephenson JM, Abrahamian H, Keipes M, Castellarin A, et al: Effect of lisinopril on progression of retinopathy in normotensive people with type 1 diabetes. The Euclid Study Group. Eurodiab controlled trial of lisinopril in insulin-dependent diabetes mellitus. Lancet 1998;351:28–31. 21. Cravedi P, Remuzzi G: Treating the kidney to cure the heart. Kidney Int 2008;74:S2–S3. 22. Kumareswaran K, Evans ML, Hovorka R: Artificial pancreas: an emerging approach to treat type 1 diabetes. Expert Rev Med Devices 2009;6:401–410. 23. Cox SL, Silveira PA: Emerging roles for B lymphocytes in type 1 diabetes. Expert Rev Clin Immunol 2009;5:311–324. 24. Bour-Jordan H, Bluestone JA: B cell depletion: a novel therapy for autoimmune diabetes? J Clin Invest 2007;117:3642– 3645. 25. Hu CY, Rodriguez-Pinto D, Du W, Ahuja A, Henegariu O, Wong FS, et al: Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice. J Clin Invest 2007;117:3857–3867. 26. Heptulla RA, Rodriguez LM, Bomgaars L, Haymond MW: The role of amylin and glucagon in the dampening of glycemic excursions in children with type 1 diabetes. Diabetes 2005;54:1100–1107. 27. Whitehouse F, Kruger DF, Fineman M, Shen L, Ruggles JA, Maggs DG, et al: A randomized study and open-label extension evaluating the long-term efficacy of pramlintide as an adjunct to insulin therapy in type 1 diabetes. Diabetes Care 2002;25:724–730. 28. Rodriguez LM, Mason KJ, Haymond MW, Heptulla RA: The role of prandial pramlintide in the treatment of adolescents with type 1 diabetes. Pediatr Res 2007;62:746–749. 29. Donaghue KC, Fairchild JM, Craig ME, Chan AK, Hing S, Cutler LR, et al: Do all prepubertal years of diabetes duration contribute equally to diabetes complications? Diabetes Care 2003;26:1224–1229. 30. Amin R, Widmer B, Prevost AT, Schwarze P, Cooper J, Edge J, et al: Risk of microalbuminuria and progression to macroalbuminuria in a cohort with childhood onset type 1 diabetes: prospective observational study. BMJ 2008;336:697– 701. 31. Steffes MW, Sibley S, Jackson M, Thomas W: Beta-cell function and the development of diabetes-related complications in the diabetes control and complications trial. Diabetes Care 2003;26:832–836. 32. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230–238. 33. Yasunami Y, Kojo S, Kitamura H, Toyofuku A, Satoh M, Nakano M, et al: V␣14 Nk T cell-triggered IFN-␥ production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets. J Exp Med 2005;202:913–918. 34. Lotze MT, Tracey KJ: High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005;5:331–342. 35. Mokini Z, Marcovecchio ML, Chiarelli F: Molecular pathology of oxidative stress in diabetic angiopathy: role of mitochondrial and cellular pathways. Diabetes Res Clin Pract 2010;87:313–321.
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Obesity and Weight Regulation Martin Wabitsch, Daniel Tews, Michaela Keuper, Carsten Posovszky, Christian Denzer, Anja Moss, Julia von Schnurbein and Pamela Fischer-Posovszky Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent Medicine, University of Ulm, Ulm, Germany
The literature on obesity and weight regulation published in the last 12 months demonstrates the activity and interest of this field of science and medicine. For the Yearbook selection this year, only articles published in journals with a high impact factor were considered, assuming that relevant findings were published in these journals. The highlights are papers about new molecules involved in the regulation of eating and physical activity behavior as well as in addiction-like reward dysfunction and compulsive eating; papers on early programming of metabolic and endocrine regulation of body weight and papers demonstrating that the search for copy number variants (CNVs) are a new strategy to discover obesity genes which might contribute to the missing heritability of obesity. In Yearbook 2009 the identification and importance of brown adipose tissue in adult humans has been demonstrated in three independent articles in the New England Journal of Medicine [1]. These findings have stimulated research in the molecular regulation of energy expenditure coming up with exciting new data presented in this year’s volume. In Yearbook 2007, two papers published in Nature showed for the first time that gut microbiota are a contributing factor to the pathophysiology of body weight regulation [2]. This story goes on as shown in an article published in Science, 2010.
Adipose tissue New molecule responsible for preadipocyte determination
Transcriptional control of preadipocytes determination by Zfp423 Gupta RK, Arany Z, Seale P, Mepani RJ, Ye L, Conroe HM, Roby YA, Kulaga H, Reed RR, Spiegelman BM Department of Cancer Biology and Division of Metabolism and Chronic Disease, Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, Mass., USA Nature 2010;464:619–623 Background: Obesity is increasing in an epidemic manner. The size of the adipose tissue mass is determined by the number of fat cells as well as their volume. It is important to understand which mechanisms control the development of fat cells. The differentiation of committed preadipocytes to adipocytes is controlled by PPAR␥ and other transcription factors. However, the molecular basis for preadipocyte determination is not understood. Methods: The authors used a new method for the quantitative analysis of transcriptional components (‘Quanttrx’) to identify new proteins involved in preadipocyte commitment. Results were confirmed using genetic approaches (ectopic expression, shRNA). Results: The zinc-finger protein Zfp423 was identified as a protein that is enriched in preadipose versus non-preadipose fibroblasts. Expression of Zfp423 in non-preadipose fibroblasts activates PPARg expression and permits cells to undergo adipocyte differentiation under permissive conditions. Knockdown of Zpf423 in preadipose 3T3-L1 cells blunts PPARg expression and diminished the ability for adipogenic differentiation. Conclusion: This study identifies Zpf423 as a transcriptional regulator of preadipocyte determination and suggests a model in which the regulation of the preadipocyte levels of PPARg by Zpf423 represents a crucial determinant of preadipocyte commitment.
There is a high turnover of fat cells in human white adipose tissue [3]. Additionally, the white adipose tissue keeps the ability to expand throughout adult life by an increase in adipocyte number [4]. For a
better understanding of the dynamics of adipose tissue and development of obesity it will be crucial to elucidate the whole adipogenic process – from stem cell precursor, via preadipose fibroblast to the mature adipocyte. The transcriptional pathways controlling adipocyte differentiation from committed preadipose fibroblasts were intensely investigated and there has been great progress. However, the transcriptional mechanisms underlying the preceding steps, the commitment of the embryonic stem cell precursor to the adipocyte lineage, remain to be characterized. The major problem seems to be that the preadipose commitment is more a quantitative than a qualitative trait [5]. Till now there is no specific marker which enables researchers to separate preadipose fibroblast from non-adipogenic fibroblast. In the present paper, for the first time, the authors were able to identify one protein that seems to be the important determinant – Zfp423. The authors showed in cell culture and in vivo (Zpf423 knockout mice) studies a genetic requirement for Zfp423 in the initial formation of both brown and white adipocytes. Taken together, this protein might help to gain more insight into the molecular basis of adipose tissue formation in physiological and pathophysiological states, thereby helping to provide new means to develop therapeutic strategies for the treatment and prevention of obesity.
New molecules involved in the regulation of eating and physical activity behavior
Regulation of adaptive behavior during fasting by hypothalamic Foxa2 Silva JP, von Meyenn F, Howell J, Thorens B, Wolfrum C, Stoffel M The Rockefeller University, Laboratory of Metabolic Diseases, New York, N.Y., USA Nature 2010;462:646–651 Background: Neurons in the lateral hypothalamus are important for feeding, behavioral arousal and addiction. These behaviors are stimulated by orexin and melanin-concentrating hormone (MCH). The aim of the study was to further understand the responsible molecular mechanisms. Methods: Mouse and metabolic studies were performed including nuclear translocation experiments, laser scanning confocal microscopy, transfection and transactivation assays, and knockout mice. Results: Forkhead box transcription factor Foxa2 is a downstream target of insulin signaling and is expressed in two distinct neuronal cell populations, namely MCH and orexin neurons. In the absence of insulin signaling, Foxa2 is recruited to the MCH and orexin promoters to activate their expression. Accordingly, during fasting resulting in low insulin levels Foxa2 binds to MCH and orexin promoters and stimulates their expression. By contrast, in the presence of insulin these two neuropeptides are down-regulated due to a change in the subcellular localization of Foxa2 which is regulated by plasma insulin concentrations. In fed and in hyperinsulinemic obese mice, Foxa2 is phosphorylated by insulin/ PI3K/Akt signaling which leads to Foxa2 inactivation and nuclear exclusion and finally to a reduced expression of MCH and orexin. Constitutive nuclear activation of Foxa2 simulating the situation in the low insulinemic state in the brain results in increased MCH and orexin expression and increased food consumption, metabolism, and also in peripheral insulin sensitivity. In addition, spontaneous physical activity is significantly increased. Conclusion: These data show that Foxa2 is a metabolic sensor in the lateral hypothalamus and integrates metabolic signals, adaptive behavior, and physiological responses.
Foxa2 in melanin-concentrating hormone- and orexin-expressing neurons is permanently inactivated in hyperinsulinemic mice with diet-induced obesity [6]. The authors show that insulin down-regulates Foxa2 expression by changing the subcellular localization of Foxa2 which is excluded from the nucleus by insulin. Hence, these studies show that there is a molecular explanation for the inverse associations of hyperinsulinemia/insulin resistance and physical activity/energy expenditure which has been observed in humans in several studies. Interestingly, the authors could show convincingly that constitutive nuclear activation of brain Foxa2 mimicking hypoinsulinemia results in increased neuronal melanin-concentrating hormone and orexin expression in obese mice and leads to increased food consumption, improved glucose homeostasis,
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decreased fat and increased lean body mass linked to an increase in physical activity in these obese animals. Pharmacological inhibition of Foxa2 phosphorylation may be a possibility to increase the levels of physical activity, overall health and longevity.
Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats Johnson PM, Kenny PJ Laboratory of Behavioral and Molecular Neuroscience, Department of Molecular Therapeutics, The Scripps Research Institute, Fla., USA
[email protected] Nature Neurosci 2010;13:635–641 Background: Food consumption is influenced by pleasure and reward. Obtaining food reward is a drive highly motivating food consumption. The hedonic mechanisms behind these observations which also might contribute to obesity remain poorly understood. The aim of the study was to investigate the effects of extended access to a palatable high-fat diet on the sensitivity of brain reward systems in rats. In addition, the role of striatal dopamine D2 receptors (D2R) in the resulting addiction-like behavioral responses was investigated. Methods: Studies were performed with Wistar rats. Methods included bipolar stimulating of the lateral hypothalamus by an electrode, training of animals in a current-threshold brain stimulation reward (BSR) procedure, and lentivirus-mediated knockdown of striatal D2R expression. Results: In the BSR procedure, rats respond vigorously to obtain rewarding electrical self-stimulation through the indwelling stimulating electrode, with the minimal stimulation intensity that maintains self-stimulation behavior termed the reward threshold. The development of obesity in rats with extended access to a cafeteria style diet consisting of palatable energy dense food was closely associated with a worsening deficit in brain reward function, reflected in progressively elevated BSR thresholds. These animals showed addiction-like reward deficits, overeating and loss of homeostatic energy balance. By contrast, restricted access to palatable food gave rise to binge-like patterns of consumption, but did not disrupt the brain reward function. Animals with extended food access took twice as much energy as compared to the restricted-access animals. Striatal expression of the D2R was lower in the extendedaccess rats than in the restricted-access or chow-only fed rats. There was also a clear inverse relationship between body weight and striatal D2R expression. There was a persistent reward dysfunction and hypophagia during abstinence in rats with extended access to a cafeteria diet. Knockdown of striatal D2R increased vulnerability to reward dysfunction in rats with extended access to a cafeteria diet. Knockdown of striatal D2 receptors markedly accelerated compulsive-like eating of palatable food. This was only observed in rats with a history of extended access to palatable food. Conclusion: These data demonstrate that overconsumption of palatable food triggers addiction-like neuroadaptive responses in brain reward circuits and raises the development of compulsive eating.
Feeding is influenced by pleasure and reward. Easy access to palatable high-fat food is considered to be an important environmental risk factor for obesity [7]. The authors show that overstimulation of brain reward systems through excessive consumption of palatable, energy-dense food induce a profound state of reward hyposensitivity and the development of compulsive-like eating. The maladaptive behavioral responses in obese rats probably arise from diet-induced deficits in striatal D2 receptor signaling. Similarly, substance abuse decreases striatal D2R density, induces a profound state of reward hypofunction and triggers the emergence of compulsive-like drug-taking behaviors. Thus, the findings of the present paper support previous work [7–22] indicating that obesity and drug addiction may arise from similar neuroadaptive responses in brain reward circuits. Conversely, individuals with anorexia nervosa have elevated striatal D2R. Weight loss after bariatric surgery is associated with elevated striatal D2R density [23, 24]. The gene polymorphism referred to as the TaqIA A1 allele results in decreased striatal D2R density, and individuals harboring this allele are over-represented in obese population [8]. So far the hedonic mechanisms contributing to obesity remain poorly understood. Interestingly in hyperphagic humans with congenital leptin deficiency, activity in the dorsal and ventral striatum, which are core components of brain reward circuits, increases markedly in response to images of food. Leptin replacement therapy attenuates both striatal activity and self-reported ‘liking’ of food
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[25]. These data already suggest that the striatum is important in hedonic aspects of feeding behavior. It also has been shown that activation of the striatum in response to highly palatable food is blunted in obese individuals when compared with lean controls [8]. Extended access to palatable high-fat food can induce addiction-like deficits in rat brain reward function. This might be an important source of motivation that may drive overeating and contribute to the development of obesity.
Regulation of energy homeostasis by bombesin receptor subtype-3: selective receptor agonists for the treatment of obesity Guan X-M, Chen H, Dobbelaar PH, Dong Y, Fong TM, Gagen K, Gorski J, He S, Howard AD, Jian T, Jiang M, Kan Y, Kelly TM, Kosinski J, Lin LS, Liu J, Marsh DJ, Metzger JM, Miller R, Nargund RP, Palyha O, Shearman L, Shen Z, Stearns R, Strack AM, Stribling S, Tang YS, Wang S-P, White A, Yu H, Reitman ML Merck Research Laboratories, Rahway, N.J., USA
[email protected],
[email protected] Cell Metabolism 2010;11:101–112 Background: Pharmacotherapy of obesity has limited success so far, due to the existence of multiple redundant and compensatory pathways in energy homeostasis. Bombesin receptor subtype-3 (BRS-3) is primarily expressed in the brain but its biological functions are not yet well investigated. Its natural ligands are unknown. The aim of the present study was to develop potent BRS-3 agonist and antagonist ligands that are suitable for exploring the role of BRS-3 in the regulation of food intake, metabolic rate, and body weight. Methods: Rat and mice studies were performed including metabolic rate measurements and binding assays. Studies were also performed using several knockdown animals. Results: A BRS-3 antagonist ligand increases food intake, body weight, and adipose tissue mass. A BRS-3 agonist reduces food intake and increases metabolic rate. BRS-3 binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was maintained in several knockdown animals (Npy–/–, Agrp –/–, Mc4r–/–, Cnr1–/– and Leprdb/db mice). BRS-3 null mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. Conclusion: This study shows that BRS-3 has an important role in energy homeostasis, complementary to several well-known pathways. BRS-3 agonists may be a potential approach for the treatment of obesity.
Bombesin receptor substrate 3 (BRS-3) is a G-protein-coupled receptor whose natural ligand is unknown. In the present study the authors developed potent, selective agonist and antagonist ligands to explore BRS-3 function. Pharmacological activation of BRS-3 presents a potential therapy for obesity. Since the data also show that the regulation of energy homeostasis by BRS-3 is complementary to the Mc4R and CB1R pathways it could be suggested that such potential therapies involving BRS-3 could also be performed in combination with the pharmacological modulation of other pathways. Food intake is under the control of both homeostatic and non-homeostatic (e.g. hedonic) mechanisms. The study supports the concept that natural BRS-3 ligands are peptides that signal BRS-3 sites at least in part in the hypothalamus. These natural ligands probably regulate energy supplies and/or hedonic drive by reaching. The precise mechanisms, including neuron types and pathways, by which BRS-3 signaling regulates energy metabolism, remain unclear. These mechanisms however seem to be complementary to the action of other factors regulating energy metabolism such as leptin, melanocortin, neuropeptide Y, and endocannabinoid.
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Signaling through Tyr985 of leptin receptor as an age/diet-dependent switch in the regulation of energy balance You J, Yu Y, Jiang L, Li W, Yu X, Gonzalez L, Yang G, Ke Z, Li W, Li C, Liu Y Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, and Graduate School of the Chinese Academy of Sciences, Shanghai, China
[email protected] Mol Cell Biol 2010;30:1650–1659 Background: Diminished leptin signaling occurs in association with aging or feeding a high-fat diet. The aim of the present study is to identify the exact physiological mechanisms linking the environmental factors to the impairment in the leptin-mediated regulation of energy metabolism. Methods: Knock-in line of mice (Y985F) expressing a mutant Ob-Rb, the long form of the leptin receptor with a phenylalanine substitution for Tyr985, one of the three intracellular tyrosines that mediate leptin’s signaling actions. Results: Surprisingly, whereas young homozygous Y985F animals were slightly leaner, they exhibit adultonset or diet-induced obesity. The observed age-dependent or diet-induced deterioration of energy balance was paralleled with pronounced leptin resistance. This leptin resistance was mainly attributable to attenuation of leptin-responsive hypothalamic STAT3 activation and elevated expression of hypothalamic SOCS3. Conclusion: The study shows that there are distinct binary roles for OBR Tyr985-mediated signaling in energy metabolism. These roles act as an age/diet-dependent regulatory switch to counteract age-associated or diet-induced obesity.
Leptin is an adipose-secreted hormone that plays a pivotal role in the regulation of energy metabolism. Acting through its receptor in distinct classes of leptin-responsive neurons, leptin activates multiple signaling pathways in the hypothalamus to regulate food intake and energy expenditure. The study shows here that the loss of Tyr985 in the long form of the leptin receptor (Ob-Rb) leads to adult onset of obesity. Adult Y985F mice exhibit impairments in both food intake and energy expenditure and a loss of Tyr985-mediated actions that exacerbates high-fat diet-induced obesity. Altogether this animal model confirms the role of the leptin receptor in the regulation of fat stores and energy metabolism and shows that mild impairment of leptin receptor signaling can produce more subtle phenotypes than those observed in human and mice so far.
Early programming of metabolic and endocrine regulation of body weight
Early overnutrition results in early-onset arcuate leptin resistance and increased sensitivity to high-fat diet Glavas MM, Kirigiti MA, Xiao XQ, Enriori PJ, Fisher SK, Evans AE, Grayson BE, Cowley MA, Smith MS, Grove KL Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oreg., USA Endocrinology 2010;151:1598–1610 Background: Childhood obesity is rising at an alarming rate, but we are far from understanding all mechanisms behind this trend. Reducing the size of a mice litter and exposing thereby mice to chronic postnatal overnutrition (CPO) is an interesting animal model to analyze mechanisms and consequences of early overfeeding. Methods: Litters from Swiss Webster dams were culled to 3 pups (CPO) or 10 pups (control). On the 23rd postnatal day all were weaned onto standard chow. At an age of 6 weeks, a subset of mice was placed on high-fat diet. In all subgroups, glucose and insulin tolerance were examined at 16–17 weeks of age. At postnatal 16 and in adulthood, leptin levels were measured and leptin sensitivity was determined by measurement of food intake and hypothalamic phosphorylated signal transducer and activator of transcription-3 (STAT3) immunoreactivity after intraperitoneal leptin injection. Results: Compared to control pups, CPO mice exhibited an increased body weight and hyperleptinemia in the pre-weaning period, but only a slightly heavier body weight and normal glucose tolerance in
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adulthood on standard chow. Throughout their whole life however, CPO mice demonstrated an attenuated clinical leptin-responsiveness and a reduced STAT3 activation in the nucleus arcuatus, despite normalized leptin levels. In response to high-fat diet, CPO mice gained weight more rapidly and in contrast to control mice displayed a pathological response to an insulin tolerance test. Conclusion: Early overfeeding leads to early and persistent leptin resistance and increases the susceptibility for overweight and insulin resistance.
Effects of maternal surgical weight loss in mothers on intergenerational transmission of obesity Smith J, Cianflone K, Biron S, Hould FS, Lebel S, Marceau S, Lescelleur O, Biertho L, Simard S, Kral JG, Marceau, P Institut Universitaire de Cardiologie et Pneumologie de Quebec, Laval University, Quebec, Canada J Clin Endocrinol Metab 2009;94:4275–4283 Background: Maternal pre-pregnancy weight, pregnancy weight gain and metabolic derangements during pregnancy influence offspring obesity rate and co-morbidities. Pre-conceptive biliopancreatic diversion bariatric surgery provides a highly specific model to study the transmission of metabolic risk factors in humans by comparing children born before (BMS) and after maternal surgery (AMS). Methods: Anthropometry and fasting blood levels were studied in 49 women who had lost 36 ± 1.8% body weight and sustained this weight-loss for 12 ± 0.8 years and in their 111 children (54 BMS and 57 AMS) aged 2.5–26 years. Results: AMS children had a lower birth weight than BMS children (2.9 ± 0.1 vs. 3.3 ± 0.1 kg, p = 0.003) and a lower prevalence of macrosomia (1.8 vs. 14.8%, p = 0.03). At the time of follow-up, AMS children showed a lower prevalence of obesity compared to BMS children (11 vs. 35%, p = 0.004). Furthermore, AMS children had a higher insulin sensitivity (HOMA index 3.4 ± 0.3 vs. 4.8 ± 0.5, p = 0.02) improved lipid profile (cholesterol/high-density lipoprotein cholesterol 2.96 ± 0.11 vs. 3.40 ± 0.18, p = 0.03; highdensity lipoprotein cholesterol 1.50 ± 0.05 vs. 1.35 ± 0.05 mmol/l, p = 0.04), lower C-reactive protein (0.88 ± 0.17 vs. 2.00 ± 0.24 µg/ml, p = 0.004), and leptin (11.5 ± 1.5 vs. 19.7 ± 2.5 ng/ml, p = 0.005) and increased ghrelin (1.28 ± 0.06 vs. 1.03 ng/ml ± 0.06) than BMS offspring. Conclusion: Improved intrauterine environment via pre-conceptive bariatric surgery not only lowers the risk of childhood/adolescence obesity but also reduces accompanying cardio-vascular risk factors.
These two studies address the interesting topic of the influence of metabolic and endocrine programming on the development of obesity and co-morbidities, albeit in two completely different approaches. The first group used an animal model of early overnutrition which leads to an increased susceptibility to overweight and hyperinsulinemia [26–28]. It has already been demonstrated that in postnatally overfed rats leptin shows a decreased inhibition of neuronal activity in the arcuate nucleus [29]. The reduced effect of leptin and a reduced induction of STAT3 expression seen here further strengthens the hypothesis that early leptin resistance might play an integral part in raising the long-term risk of obesity and metabolic derangements. Men are not mice and whereas in rodents the neuronal network responsible for food intake regulation is progressively established during early postnatal life, in humans the major part of neuronal development occurs before birth [30]. Therefore, the mechanism by which postnatal overfeeding in mice enhances the risk of adulthood obesity and insulin resistance might essentially be comparable to the consequences of human obesity during pregnancy. The study group of Kral et al. [31] is the first to examine the long-term impact of maternal weight loss through bariatric surgery on obesity rate and co-morbidity in offspring. In a previous study they were able to show an impressive reduction in childhood/adolescence obesity. Now they completed this examination by showing an associate improvement in cardiometabolic risk markers which was sustained into adolescence and young adulthood. This proves elegantly the influence of potentially modifiable epigenetic factors on childhood obesity and associated morbidity. Apart from the academic interest of this finding, it also has important practical implications. Preconceptive bariatric surgery decreases rates of gestational diabetes and neonatal macrosomia [32–35], however it increases the risk for internal hernias [32, 36, 37] and severe nutritional deficiencies [38] during pregnancy. Therefore, each decision for bariatric surgery has to be made cautiously, but this study definitely strengthens the recommendation of bariatric surgery prior to conception in severely obese women.
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Leptin resistance cannot be evaluated easily in children. However, the lower leptin levels in BMS children point to the fact that early development of leptin resistance may be a major player of increased obesity susceptibility in humans as well as in rodents.
Search for copy number variants (CNVs) a new strategy to discover obesity genes
Large, rare chromosomal deletions associated with severe early-onset obesity Bochukova EG, Huang N, Keogh J, Henning E, Purmann C, Blaszczyk K, Saeed S, Hamilton-Shield J, Clayton-Smith J, O’Rahilly S, Hurles ME, Farooqi IS University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK
[email protected],
[email protected] Nature 2010;463:666–670 Background: Genome-wide association studies have identified common single nucleotide polymorphisms (SNPs) associated with increased body mass index. However, together these account for a small percentage of inherited variations in BMI. In contrast, studies in patients with severe early-onset obesity have led to the detection of rare variants with significant impact on body weight. The aim of the present study was to explore the contribution of copy number variants (CNVs) to obesity. Methods: 300 UK Caucasian patients with severe obesity defined as a BMI standard deviation score >3 and onset of obesity before 10 years of age were selected from the Genetics of Obesity Study Cohort. Of these individuals, 143 had developmental delay. DNA samples were run on Affymetrix genomewide human SNP arrays 6.0 by Aros, Inc. Results: Large (>500 kb), rare (<1%) deletions were significantly enriched in patients compared to >7,000 controls. Several CNVs were recurrent in patients but absent or at much lower prevalence in controls. Five patients with overlapping deletions on chromosome 16p11.2 were identified. In 3 patients the deletion co-segregated with severe obesity. In an independent sample of >1,000 patients with severe obesity alone, a 16p11.2 deletion was found in an additional 2 patients. All 16p11.2 deletions encompass several genes but include SH2B1, which is known to be involved in leptin and insulin signaling. Deletion carriers exhibited hyperphagia and severe insulin resistance disproportionate for the degree of obesity. Conclusion: The CNVs contribute significantly to the genetic architecture of human obesity.
A new highly penetrant form of obesity due to deletions on chromosome 16p11.2 Walters RG, Jacquemont S, Valsesia A, de Smith AJ, Martinet D, Andersson J, Falchi M, Chen F, Andrieux J, Lobbens S, Delobel B, Stutzmann F, El-Sayed Moustafa JS, Chevre JC, Lecoeur C, Vatin V, Bouquillon S, Buxton JL, Boute O, Holder-Espinasse M, Cuisset JM, Lemaitre MP, Ambresin AE, Brioschi A, Gaillard M, Giusti V, Fellmann F, Ferrarini A, Hadjikhani N, Campion D, Guilmatre A, Goldenberg A, Calmels N, Mandel JL, Le Caignec C, David A, Isidor B, Cordier MP, Dupuis-Girod S, Labalme A, Sanlaville D, Beri-Dexheimer M, Jonveaux P, Leheup B, Ounap K, Bochukova EG, Henning E, Keogh J, Ellis RJ, Macdermot KD, van Haelst MM, Vincent-Delorme C, Plessis G, Touraine R, Philippe A, Malan V, Mathieu-Dramard M, Chiesa J, Blaumeiser B, Kooy RF, Caiazzo R, Pigeyre M, Balkau B, Sladek R, Bergmann S, Mooser V, Waterworth D, Reymond A, Vollenweider P, Waeber G, Kurg A, Palta P, Esko T, Metspalu A, Nelis M, Elliott P, Hartikainen AL, McCarthy MI, Peltonen L, Carlsson L, Jacobson P, Sjostrom L, Huang N, Hurles ME, O’Rahilly S, Farooqi IS, Mannik K, Jarvelin MR, Pattou F, Meyre D, Walley AJ, Coin LJ, Blakemore AI, Froguel P, Beckmann JS Section of Genomic Medicine, Department of Epidemiology and Public Health, Imperial College London, UK
[email protected],
[email protected] Nature 2010;463:671–677 Background: Copy number variants (CNVs) might contribute to the missing heritability of common disorders and also of obesity.
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Methods: Subjects ascertained for cognitive deficit (malformations) with or without obesity were selected from those clinically referred for genetic testing. 16p11.2 deletions were identified in these individuals by standard clinical diagnostic procedures. Algorithmic analyses of GWAS data were performed. Results: A highly penetrant form of obesity was initially observed in 31 subjects who were heterozygous for deletions at 16p11.2 and who had also cognitive deficits. Nineteen similar deletions were identified from GWAS data in >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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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 (<30 mg/g) on subsequent examination. In a subset of 2,666 youth with a median follow-up of 8.1 years, 36 non-diabetic and 30 diabetic youth with baseline ACRs of <300 mg/g developed macroalbuminuria. For a given ACR, the incidence of macroalbuminuria was 15.9-fold (95% CI 11.1–22.6) higher in the diabetic than in the non-diabetic youth. Conclusions: Elevated albuminuria is infrequent and largely transient in non-diabetic youth, but it is relatively frequent and largely persistent in those with diabetes. Microalbuminuria in youth with T2DM strongly predicts progression to macroalbuminuria, which supports annual screening for albuminuria. Objective: Methods:
The Pima Indians have been called ‘pathfinders for health’ because of their high level of volunteering to participate in research studies. The cooperative research between the Pima Indians and the NIH, which began in 1963, continues to this day. Since 1965, each member of the population, aged 5 years and older, has been invited to participate in a study investigation every other year. While the overall incidence of diabetes in the Pima Indians has not changed over the past 40 years, the incidence of diabetes among those less than 15 years of age has increased nearly 6-fold, subsequent to the increasing prevalence and degree of obesity at younger ages [5]. The current prospective study reported a 3-fold higher incidence of microalbuminuria in youth with T2DM than in those without (18.5 vs. 6.5%). Importantly, in more than half of the individuals with diabetes, microalbuminuria preceded the diagnosis of T2DM. Microalbuminuria was transient in 75% of individuals without diabetes, compared to only 7% of those with diabetes. Microalbuminuria strongly predicted progression to macroalbuminuria, with 27% of youth with T2DM progressing to macroalbuminuria. The authors concluded that the findings of this population afford examination of the impact of albuminuria in other populations. Of note, although microalbuminuria was initially attributed to diabetes among Canadian First Nation Children, biopsy results demonstrated a non-diabetic renal disease in the form of immune complex diseases or glomerulosclerosis in all cases [6].
New hopes
Metformin associated with lower cancer mortality in type 2 diabetes: ZODIAC-16 Landman GW, Kleefstra N, van Hateren KJ, Groenier KH, Gans RO, Bilo HJ Internal Medicine, Isala Clinics, Zwolle, The Netherlands
[email protected] Diabetes Care 2010;33:322–326 Background: Several studies have suggested an association between specific diabetes treatment and cancer mortality. The authors studied the association between metformin use and cancer mortality in a prospectively followed cohort.
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Methods: In 1998 and 1999, 1,353 patients with T2DM were enrolled in the Zwolle Outpatient Diabetes project Integrating Available Care (ZODIAC) study in the Netherlands. Vital status was assessed in January 2009. Cancer mortality rate was evaluated using standardized mortality ratios (SMRs). Results: Median follow-up time was 9.6 years, average age at baseline was 68 years, and average HbA1c was 7.5%. Of the patients, 570 died, of which 122 died of malignancies. The SMR for cancer mortality was 1.47 (95% CI 1.22–1.76). In patients taking metformin compared with patients not taking metformin at baseline, the adjusted hazard ratio for cancer mortality was 0.43 (95% CI 0.23–0.80), and with every increase of 1 g of metformin hazard ratio was 0.58 (95% CI 0.36–0.93). Conclusions: In general, patients with T2DM are at increased risk for cancer mortality. In this study, metformin use was associated with lower cancer mortality compared with non-use of metformin. Although the design cannot provide a conclusion about causality, data suggest a protective effect of metformin on cancer mortality.
This prospective study showed that use of metformin in patients with diabetes was associated with a lower rate of cancer-related mortality. Increasing evidence from cells, animal models and humans suggests that metformin, a mainstay of T2DM, could be a potent antitumor medicine [7]. In vitro studies with cell cultures demonstrated that metformin benefited human lung, prostate, breast, colon and ovarian cancers. In a recent study in mice, low doses of metformin combined with chemotherapy reduced tumors faster and prolonged remission to a greater degree than did chemotherapy alone. Most importantly, the mechanism involved targeting cancer stem cells, which appear to resist conventional chemotherapies. In humans, epidemiological studies reported treatment with metformin to be associated with a lower overall risk for cancer than without treatment, or than treatment with insulin or antidiabetic drugs. Patients with breast cancer who were treated with both neoadjuvant chemotherapy and metformin had a higher response rate than those treated with other diabetes medications. Finally, as presented in the current article, in patients with diabetes, metformin use was associated with lower cancer mortality. It has been suggested that metformin may influence cancer cells indirectly through insulin-mediated effects, or it may directly affect cell proliferation and apoptosis of cancer cells. Pediatricians need to follow these data closely. Recent reports suggest a relationship between childhood obesity, and an increased risk of adult pancreatic and colon cancer. Adults with T2DM are at increased risk to develop cancer. Thus, if indeed metformin treatment will be found to have antitumorgenic effect, it will be essential for children and youth with T2DM.
Screening for type 2 diabetes in obese youth Shah S, Kublaoui BM, Oden JD, White PC Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Tex., USA Pediatrics 2009;124:573–579 Objective: To assess available blood tests as potential screening tools for impaired glucose tolerance (IGT) and T2DM. Methods: 468 obese (mean BMI >34.4 kg/m2) children, including a subgroup with serum fasting insulin levels of >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 <17.0 µg/ml, with lower values for fasting BG and the HOMA-IR. In the entire study group, 9 (2%) patients had T2DM and 44 (9%) had IGT. Optimal sensitivity and specificity to detect T2DM were respectively 86 and 85% at HbA1c levels of 5.7, 88, and 93% at a fasting BG level of 104 mg/dl, and 62 and 70% at an HOMA-IR of 7.9. Conclusions: HbA1c, 1,5-anhydroglucitol, and fasting BG levels are good predictors of T2DM in obese children, whereas HOMA-IR values are not. HbA1c and 1,5-anhydroglucitol are excellent predictors of T2DM in insulin-resistant obese children.
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The escalating epidemic of childhood obesity calls for a simple diagnostic tool to identify those with impaired glucose tolerance and those with undiagnosed T2DM. Performing an oral glucose tolerance test (OGTT) on all obese children is a cumbersome task. Indeed, as the authors of the current study point out, many clinicians fail to follow the ADA guidelines for performing OGTT in obese children. Moreover, screening tests need be repeated periodically, especially when children continue to gain weight, and even when their baseline results are normal. The aim of this study was to investigate the possible utilization of fasting glucose, HbA1c, HOMA-IR and 1,5-anhydroglucitol as simpler screening tests. 1,5-Anhydroglucitol, also known as 1,5-AG, is a naturally occurring monosaccharide found in nearly all foods. Since hardly any of 1,5-AG is metabolized, its level remains relatively constant in the blood and tissues. Normally, a small amount of 1,5-AG, equal to the amount ingested, is excreted in the urine. When blood glucose exceeds 180 mg/dl, and glycosuria occurs, competitive inhibition blocks 1,5-AG from being reabsorbed, and its blood level decreases. Thus, low levels of 1,5-AG indicate hyperglycemia. Moreover, in subjects with diabetes, this assay indicates excessive glycemic variability, even in the presence of relatively low HbA1c levels. In the current study, the authors found 1,5-AG levels to only modestly predict impaired glucose tolerance among children. However, HbA1c, 1,5-AG, and fasting BG levels were found to be easily implemented screening tests for T2DM in obese children.
Food for thought
Ethnic differences in triglyceride levels and high-density lipoprotein lead to underdiagnosis of the metabolic syndrome in black children and adults Sumner AE Clinical Endocrinology Branch, NIDDK, NIH, Bethesda, Md., USA
[email protected] J Pediatr 2009;155:S7 e7–11 Background: The metabolic syndrome (MetS) was designed to identify individuals at high risk for the T2DM and cardiovascular disease. Compared with whites, blacks have higher rates of diabetes and cardiovascular disease. Paradoxically, blacks have a lower prevalence of the MetS. According to the criteria set by National Cholesterol Education Treatment Program-Adult Treatment Panel III, to diagnose the MetS, 3 of 5 characteristics must be present. These characteristics are low high-density lipoprotein levels, increased triglyceride levels, central obesity, hypertension, and fasting hyperglycemia. Examining each of these factors individually, blacks are more likely than whites to have obesity, hypertension, and diabetes. In contrast, blacks are less likely than whites to have either elevated triglyceride or low high-density lipoprotein levels. Ethnic differences in lipid levels may largely explain why blacks have a lower than expected prevalence of the MetS. Children and adults ethnic differences in the epidemiologic study of conditions associated with the MetS, as well as focus on each of the parameters of the MetS are reviewed. Conclusions: An ethnic-specific formulation of the lipid criteria in the MetS may lead to better identification of blacks at high risk for development of diabetes and cardiovascular disease.
The metabolic syndrome (MetS) comprises the clustering of risk factors that identify individuals at increased risk of CVD and diabetes. Basically, it involves the co-occurrence of at least three of the following: obesity (defined either by BMI or waist circumference), dyslipidemia including elevated TG and/or low HDL, hypertension and abnormal glucose metabolism (fasting glucose, impaired glucose tolerance). In children and in youth, the metabolic syndrome may be a particularly important tool to predict and prevent adult diseases. Between 1995 and 2000, about 10 papers regarding the metabolic syndrome in children were published, compared to around 300–400 articles per year over the last 3 years. However, the concept of the metabolic syndrome in children and adolescents has undergone long and painful labor, and the newborn still has many developmental disorders. First, several definitions were proposed by the WHO, the National Cholesterol Education Program Adult Treatment Panel III (ATP III) [5], the European Group for the Study of Insulin Resistance, and the International
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Diabetes Federation (IDF). The lack of unified criteria makes it difficult to compare prevalence rates between populations and at-risk groups. In children, the determination of cut-off points is even more complicated than in adults, as age must also be considered. Sumner’s recent article is one of several that elucidate problems in defining the metabolic syndrome. She points out that while obesity, insulin resistance, diabetes and hypertension are much more prevalent in blacks than in whites, the prevalence of the metabolic syndrome is lower in blacks. It is suggested that the factor of dyslipidemia has a disproportionate effect on metabolic syndrome diagnosis, as high triglyceride and low HDL levels occur together, due to their reciprocal clearance from blood circulation. These data may be relevant to other ethnic populations, with implications on the predictive value of the metabolic syndrome for future morbidity. Moreover, considering established risk factors that are not included in the metabolic syndrome, such as birth weight, family history, physical activity, age of puberty, and LDL cholesterol level, it seems that we might need other tools for risk prediction.
Childhood obesity, other cardiovascular risk factors, and premature death Franks PW, Hanson RL, Knowler WC, Sievers ML, Bennett PH, Looker HC Diabetes Epidemiology and Clinical Research Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Ariz., USA
[email protected] N Engl J Med 2010;362:485–493 Background: The effect of childhood risk factors for cardiovascular disease on adult mortality is poorly understood. Methods: A cohort of 4,857 American-Indian children without diabetes (mean age 11.3 years), who were born between 1945 and 1984, were studied to assess whether BMI, glucose tolerance, blood pressure and cholesterol levels predicted premature death. Risk factors were standardized according to sex and age. Results: There were 166 deaths from endogenous causes (3.4% of the cohort) during a median follow-up period of 23.9 years. Rates of death from endogenous causes among children in the highest quartile of BMI were more than double those among children in the lowest BMI quartile (incidence-rate ratio 2.30; 95% CI 1.46–3.62). Rates of death from endogenous causes among children in the highest quartile of glucose intolerance were 73% higher than those among children in the lowest quartile (incidence-rate ratio 1.73; 95% CI 1.09–2.74). No significant associations were seen between rates of death from endogenous or external causes and childhood cholesterol levels or systolic or diastolic blood-pressure levels on a continuous scale, although childhood hypertension was significantly associated with premature death from endogenous causes (incidence-rate ratio 1.57; 95% CI 1.10–2.24). Conclusions: Obesity, glucose intolerance, and hypertension in childhood were strongly associated with increased rates of premature death from endogenous causes in this population. In contrast, childhood hypercholesterolemia was not a major predictor of premature death from endogenous causes.
The opening sentence of this article is overwhelming: ‘Despite recent increases in life expectancy, the rising global prevalence of obesity may reverse this trend…’. The study assessed BMI, glucose tolerance, blood pressure and cholesterol levels in 4,857 American-Indian children and adolescents (aged 5–20 years) without diabetes. During a 24-year follow-up, 3.4% died prematurely (before age 55 years) from endogenous causes, i.e. by disease or self-inflicted injury. Of these, alcoholic liver disease accounted for the majority (59%), followed by cardiovascular disease (22%), infections (21%), cancer (12%), diabetes or diabetic nephropathy (10%) and acute alcoholic poisoning or drug overdose (9%). 33 deaths were attributed to other causes. High BMI, glucose intolerance and hypertension in childhood were strongly associated with increased rates of premature death from endogenous causes, whereas hypercholesterolemia was not. Thus childhood obesity predicted premature death. The mirror image of obesity and premature death is interesting. In rodent models, longevity has been associated with caloric restriction. Caloric restriction has been shown to extend lifespan through induction of sirtuins. Sirtuins are a family of seven proteins, SIRT1–SIRT7. Sirtuin 1 (SIRT1) is the mammalian homologue of the evolutionarily conserved silent information regulator 2 (SIR2), a deacetylase that regulates lifespan in response to caloric restriction in many organisms. In a combined in vivo and in vitro study, gene and protein expression of SIRT1 were significantly reduced in relation to insulin resistance and MetS [8]. SIRT1 was downregulated in individuals with prediabetes compared
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to those with normal glucose metabolism. Decreased SIRT1 was associated with a shorter lifespan. These data suggest that SIRT1 might be a new therapeutic target for the prevention of disease related to insulin resistance. It is therefore not surprising that in 2008, GlaxoSmithKline bought the company who developed a small molecule drug aimed at increasing SIRT1 activity as an ‘anti-aging drug’. I wait eagerly, not only to realize the promise of longevity, but to be skinny as well.
Concepts revised
Heritability of childhood weight gain from birth and risk markers for adult metabolic disease in prepubertal twins Beardsall K, Ong KK, Murphy N, Ahmed ML, Zhao JH, Peeters MW, Dunger DB Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK J Clin Endocrinol Metab 2009;94:3708–3713 Objective: Associations between size at birth, postnatal weight gain, and potential risk for adult disease have been variably explained by in utero exposures or genetic risk that could affect both outcomes. A twin model to explore these hypotheses was utilized. Methods: 100 pairs of healthy twins aged 8.9 years (range 7.2–10.9) had fasting blood samples collected, blood pressure (BP) measured, and anthropometry assessed. Results: Mean birth weights in both monozygotic and dizygotic twins were –0.90 SDS lower than the UK reference. In postnatal life, 58% of monozygotic twins and 59% of dizygotic twins showed rapid weight gain (a change of more than +0.67 in weight SDS) from birth. Postnatal weight gain was positively associated with sum of skinfolds (r = 0.51; p < 0.0005), fasting insulin levels (r = 0.35; p < 0.0005), systolic BP (r = 0.30; p < 0.0005), and diastolic BP (r = 0.15; p < 0.05) at follow-up. Heritability estimates (additive genetic components) were calculated using variance components models for: birth weight, 44%; postnatal weight gain, 80%; childhood height, 89%; body mass index, 72%; sum of skinfolds, 89%; waist circumference, 74%; fasting insulin, 65%; systolic BP, 33%, and diastolic BP, 29%. Conclusions: Postnatal weight gain from birth, rather than birth weight, was associated with childhood risk markers for adult metabolic disease. Childhood weight gain was highly heritable, and genetic factors associated with postnatal weight gain are likely to also contribute to risks for adult disease.
Timing and tempo of first-year rapid growth in relation to cardiovascular and metabolic risk profile in early adulthood Leunissen RW, Kerkhof GF, Stijnen T, Hokken-Koelega A Department of Pediatrics, Subdivision of Endocrinology, Erasmus Medical Center/Sophia Children’s Hospital, Rotterdam, The Netherlands
[email protected] JAMA 2009;301:2234–2242 Background: Growth during infancy appears to be an important determinant of cardiovascular disease and type 2 diabetes later in life. To specify which period in the first year of life is related to determinants of cardiovascular disease and T2DM in early adulthood and to investigate the association between tempo of first-year weight gain (>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 <0.05), HOMA-IR (r = –0.51; p <0.01), QUICKI (r = 0.53; p <0.01), insulin AUC (r = –0.22; NS), and sum-of-insulin (r = –0.48; p <0.05). Conclusions: In obese prepubertal children, the 13C-glucose breath test can provide a proxy estimate of IR when gold-standard techniques are either unavailable or impractical.
In healthy people, ingested glucose is metabolized in the presence of insulin, producing CO2, which is eliminated by the lungs through expiration. Since glucose can be labeled with a stable isotope tracer (13C), its metabolism to CO2 can be determined quantitatively. Among subjects with insulin resistance, glucose uptake is impaired and less CO2 is produced. Among adults, the 13C-glucose breath test was found to be highly correlated (r ≥ 0.69) with IR determined by a hyperinsulinemiceuglycemic clamp [9]. In the current study, estimates of IR generated by the 13C-glucose breath test were compared to indices derived from OGTT in prepubertal obese children. A significant, though moderate, concordance was found between 13CO2 in breath and fasting insulin, 2-hour insulin, and calculated estimates of insulin resistance such as HOMA and QUICKI. This may be useful, since clinicians often avoid testing children when repeated blood tests are necessary, as in the OGTT. Such an easy to perform, noninvasive test, applicable in both private clinical and non-clinical settings, should be investigated in different populations, including pubertal children, and children of different ethnicities. So far, it seems a promising and practical means of assessing insulin resistance in children.
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Important for clinical practice
Effect of injectable and oral contraceptives on serum lipids Berenson AB, Rahman M, Wilkinson G Department of Obstetrics and Gynecology, The Center for Interdisciplinary Research in Women’s Health, The University of Texas Medical Branch, Galveston, Tex., USA
[email protected] Obstet Gynecol 2009;114:786–794 Objective: To estimate the effects of using depot medroxyprogesterone acetate (DMPA) or oral contraceptives (OCs) containing 20 µg ethinyl estradiol and 0.15 mg desogestrel on serum lipid levels. Methods: Serum lipids were measured at baseline and every 6 months thereafter for 3 years in 703 white, African-American, and Hispanic women using DMPA, OC, or non-hormonal birth control. Those who discontinued DMPA were followed for up to 2 additional years. Participants completed questionnaires containing demographic and behavioral measures every 6 months and underwent 24-hour dietary recalls annually. Mixed-model regression analyses and general-estimating-equations procedures were used to estimate changes over time in lipids by method along with their predictors. Results: Users of OCs experienced significantly greater increases in levels of triglycerides, total cholesterol, very-low-density lipoprotein (VLDL) cholesterol, and high-density lipoprotein (HDL) cholesterol than did non-hormonal-contraceptive users (p < 0.001). However, no difference was noted in the lowdensity lipoprotein (LDL) cholesterol:HDL ratio between OC users and non-hormonal-contraceptive users. Among DMPA users, HDL levels initially decreased for 6 months but then returned to baseline. The LDL:HDL ratio rose in the first 6 months of DMPA use but then dropped back to baseline over the next 24 months. After DMPA was discontinued, triglyceride, VLDL, and HDL levels were significantly higher in women who used OCs than in those who chose non-hormonal (p < 0.05) methods. Conclusions: Use of very-low-dose OCs containing desogestrel can elevate lipid levels. Users of DMPA were at increased risk of developing an abnormally low HDL level as well as an abnormally high LDL level and an increase in the LDL:HDL cholesterol ratio, although these effects appeared to be temporary.
In the year 2010 we mark the 50th anniversary of the approval of oral contraception (OC). The enormous social impact of the pill is without question [10]. It was key to women’s gaining control over their fertility, and an ‘equalizer’ that afforded women the same sexual freedom that men had traditionally enjoyed. This revolutionary degree of autonomy, equality and freedom led to women’s modern economic role. The UN estimates that more than 100 million women worldwide take some form of hormonal contraception. The most serious risks associated with OC use include blood clots, pulmonary embolism, stroke, heart attacks and breast cancer. The current article aimed to resolve controversies on the effect of OC on lipid levels, specifically those of both depot medroxyprogesterone acetate (DMPA) and of the third-generation OC containing only 20 µg, ethinyl estradiol and 0.15 mg desogestrel. The strength of the study is the long-term follow-up: over 3 years of contraceptive use. During the study period, OC users experienced statistically significant increases in levels of triglycerides, total cholesterol, VLDL and HDL cholesterol, greater than those experienced by non-hormonal contraceptive users. In fact, OC users were 3-fold more likely to have triglycerides level >170 mg/dl, and 4-fold more likely to have LDL >160 mg/dl. The net effect of OC on lipid levels seems to depend on the balance between increased HDL cholesterol and increased triglycerides. DMPA users were 2.5 times more likely than non-hormonal contraceptive users to have a level of HDL ≤35 mg/dl, and almost 3-fold more likely to have abnormal LDL levels. These findings highlight the importance of monitoring lipid profiles in adolescents treated with OC. Of particular concern is the use of OC by adolescents with PCOS who tend to have dyslipidemia as part of their metabolic syndrome.
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Relationships between 25-hydroxyvitamin D levels and plasma glucose and lipid levels in pediatric outpatients Johnson MD, Nader NS, Weaver AL, Singh R, Kumar S Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minn., USA J Pediatr 2010;156:444–449 Objective: To study the relationships between serum vitamin D levels and plasma glucose or lipid levels in children and adolescents. Methods: This is a retrospective record review of pediatric outpatients (age 2–18 years) with simultaneous measurement of 25-hydroxyvitamin D (25[OH]D) and fasting plasma glucose (n = 302) or 25(OH) D and a lipid panel (n = 177). Pearson correlation coefficient was used to estimate the correlation between 25(OH)D and logarithmic transformed plasma glucose or lipid levels. Plasma glucose and lipid levels were compared in subjects with 25(OH)D concentrations greater or less than 30 ng/ml. Results: 25(OH)D levels were inversely correlated with fasting plasma glucose levels (r = –0.20, p < 0.001). Lower 25(OH)D levels were also associated with lower serum high-density lipoprotein cholesterol (HDL) concentrations (r = 0.41; p < or = 0.001). The relationship between 25(OH)D levels and fasting glucose and HDL levels did not vary significantly with sex, age, body mass index Z-score, or season. Children who were vitamin D insufficient (25(OH)D < or =30 ng/ml) had higher fasting plasma glucose (p = 0.002) and lower HDL levels (p < 0.001) than children who were vitamin D sufficient (25(OH)D >30 ng/ml). Conclusions: Low 25(OH)D levels in children and adolescents are associated with higher plasma glucose and lower HDL concentrations.
The discovery of the critical roles of vitamin D for overall health is a fascinating story in the history of medicine. First are its osseous effects and the association to rickets. Then came the discovery of its anti-infectious role, from the breakthrough by Niels Finsen, earning him a Nobel Prize in 1903 for the use of a form of ‘concentrated light radiation’ to treat tuberculosis skin lesions, through the sanatoriums built to treat patients with sunbathing, until the discovery of cathelicidin – an antimicrobial peptide, regulated by vitamin D, that serves a critical role in mammalian innate immune defense against invasive bacterial infection. The editorial by Chesney [11] is highly recommended. No wonder that common wisdom says, ‘Where the sun is, the doctor ain’t’. Recent studies indicate that the sunlight-generated hormone, vitamin D, with its plethora of biological effects on diverse tissues, sustains health throughout the body. It is now believed that vitamin D can protect against multiple sclerosis, T1DM and cancer. Among adults, low levels of vitamin D have been shown to be associated with increased risks of obesity, hypertension, glucose intolerance, T2DM and cardiovascular disease [12]. In the above study the authors examined the association between vitamin D, glucose and lipid levels, as features of the metabolic syndrome. Decreased levels of vitamin D were significantly associated with increasing levels of glucose. While no correlations were found between vitamin D levels and total serum cholesterol, triglyceride and non-HDL, low vitamin levels were associated with low HDL levels. These relationships persisted independent of adiposity, age and sex. Relatively few foods naturally contain vitamin D, the most abundant being oily fish like salmon, sardines, mackerel, and cod liver oil, which are not often consumed by children. In 2008 the American Academy of Pediatrics Committee on Nutrition recommended a minimum daily intake of 400 IU vitamin D for all infants and children, including adolescents. Currently, the most important question is if supplementation will prevent cardiovascular events. One systemic review found only weak evidence of the benefit from vitamin D supplementation [13], and another found no evidence [14]. Currently available data preclude definitive conclusion. We await the results of the National Institutes of Health sponsored trial, VITAL (VITamin D and OmegA-3 TriaL), which will study vitamin D (2,000 IU) and –3-fatty acid supplementation, and their effects on heart disease, stroke and cancer.
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Clinical trials, new treatments
Efficacy and safety of rosuvastatin therapy for children with familial hypercholesterolemia Avis HJ, Hutten BA, Gagne C, Langslet G, McCrindle BW, Wiegman A, Hsia J, Kastelein JJ, Stein EA Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands J Am Coll Cardiol 2010;55:1121–1126 Background: The efficacy and safety of rosuvastatin therapy for children with familial hypercholesterolemia was studied. In children, statins have been shown to be effective in reducing LDL-C, restoring flow-mediated dilation, and slowing carotid intima-media thickening. However, few children in these trials achieved current LDL-C goals. Methods: This study comprised a 12-week double-blind, randomized, placebo-controlled trial, followed by a 40-week open-label, titration-to-goal extension phase in 177 pubertal children, ages 10–17 years, with familial hypercholesterolemia. Participants were randomly assigned to placebo or rosuvastatin 5, 10, or 20 mg once daily. Results: Compared with placebo, rosuvastatin 5, 10, and 20 mg reduced LDL-C by 38, 45, and 50%, respectively (p < 0.001 for each group vs. placebo). With a maximum allowed dose of 20 mg, 40% achieved the treatment goal of <110 mg/dl during the open-label, titration-to-goal phase. Rosuvastatin was well tolerated, with no apparent adverse impact on growth or development. Conclusions: In children with familial hypercholesterolemia, rosuvastatin 20 mg daily reduced LDL-C by 50%. Nonetheless, only 40% attained the consensus LDL-C target of <110 mg/dl, reflecting these patients’ high baseline LDL-C levels (mean 232 mg/dl).
Rosuvastatin is the highest potency statin presently on the market. It is a competitive inhibitor of the enzyme HMG-CoA reductase, which is the rate-limiting enzyme of cholesterol synthesis. Inhibition of this HMG-CoA reductase in the liver decreases cholesterol synthesis, while increasing synthesis of LDL receptors, thus resulting in increased clearance of low-density lipoprotein (LDL) from the bloodstream. Rosuvastatin was first launched in 2003, however its capture of the market has followed a rough course. Though the drug was marketed as a ‘super-statin’, its introduction to clinical practice in the absence of reliable data about efficacy and safety was sharply criticized. Nonetheless, the FDA refused to withdraw Resuvastatin from the market. For a happy ending, in 2008 the JUPITER (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) trial was stopped early due to unequivocal evidence from an independent Data and Safety Monitoring Board of reduced cardiovascular morbidity and mortality in patients treated with rosuvastatin compared with placebo. As much as 50–75% of drugs used in pediatric medicine have not been studied adequately to provide appropriate labeling information. In 1997, the American Congress passed the FDA Modernization Act, which encouraged pediatric drug development by providing an incentive in the form of additional marketing exclusivity. The consequent increase in pediatric clinical studies has resulted in improved understanding of the pharmacokinetics of drugs prescribed in pediatric medicine, important dose changes, and improved safety for children taking certain drugs [15]. For rosuvastatin, as with all statins, the risk of rhabdomyolysis, and other undesired side effects, be they hepatic, skeletal or renal is a concern. This study is the first to evaluate the efficacy and safety of rosuvastatin in children aged 10–17 years. Its importance therefore lies in testing doses, compliance and safety profile.
Colesevelam hydrochloride: efficacy and safety in pediatric subjects with heterozygous familial hypercholesterolemia Stein EA, Marais AD, Szamosi T, Raal FJ, Schurr D, Urbina EM, Hopkins PN, Karki S, Xu J, Misir S, Melino M Metabolic and Atherosclerosis Research Center, Cincinnati, Ohio, USA
[email protected] J Pediatr 2010;156:231–236 e1–3 Objective: Efficacy and safety of colesevelam hydrochloride were evaluated in children with heterozygous familial hypercholesterolemia (heFH).
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Methods: This was a randomized, double-blind, multicenter site study in 194 boys and postmenarchal girls aged 10–17 years with heFH (statin-naive or on a stable statin regimen). After a 4-week stabilization period (period I), subjects were randomized to placebo (n = 65), colesevelam 1.875 g/day (n = 65), or colesevelam 3.75 g/day (n = 64) for 8 weeks (period II). All patients then received open-label colesevelam 3.75 g/day for 18 weeks (period III). Results: At week 8, a significant difference from baseline in LDL cholesterol was reported with colesevelam 1.875 g/day (–6.3%; p = 0.031) and colesevelam 3.75 g/day (–12.5%; p < 0.001) compared with placebo. Significant treatment effects were also reported for total cholesterol (–7.4%), non-HDL cholesterol (–10.9%), HDL cholesterol (+6.1%), apolipoprotein A-I (+6.9%), and apolipoprotein B (–8.3%) and a non-significant effect for triglycerides (+5.1%) with colesevelam 3.75 g/day compared with placebo at week 8. These treatment effects were maintained during period III. Conclusions: Colesevelam significantly lowered LDL cholesterol levels in children with heFH.
In October 2009 the FDA approved colesevelam HCl tablets and a new powder for oral suspension to reduce LDL-C levels in boys and postmenarchal girls aged 10–17 years with heterozygous familial hypercholesterolemia. Colesevelam belongs to the bile acid sequestrant class of drugs. The drug is a non-absorbed, lipid-lowering polymer that was engineered specifically to enhance the binding capacity for bile acids in the intestine, impeding their reabsorption. As the bile acid pool becomes depleted, the hepatic enzyme, cholesterol 7␣-hydroxylase, is upregulated, thus increasing the conversion of cholesterol to bile acids. This increases demand for cholesterol in the liver cells, resulting in the dual effect of increasing transcription and activity of the cholesterol biosynthetic enzyme, HMG-CoA reductase, and increasing the number of hepatic LDL-C receptors. Two important implications from this study are the reduction of LDL-C by colesevelam, and possible side effects in children. Among adults, colesevelam has been shown to reduce LDL-C by up to 15%. In the current study, it significantly lowered LDL-C by 12.5%. As the drug is approved only for children with LDL-C levels above 190, colesevelam use will not reach target levels. Therefore, the use of colesevelam is of particular value in patients who do not reach their LDL-C target levels with maximal tolerable dosages of statins, and for statin-intolerant patients who may benefit from colesevelam monotherapy. Bile acid sequestrants are not systematically absorbed, and indeed there were no safety or laboratory issues in this study. The drug was well tolerated with relatively good compliance. Gastrointestinal disorders were the most common adverse effects. No significant effects on growth, sexual maturation, fat-soluble vitamin levels, or clotting factors were observed.
Santa Claus: a public health pariah? Grills NJa, Halyday Bb Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, and b Department of Health, Collingwood, Vic., Australia
[email protected] BMJ 2009;339:b5261
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It appears that the awareness of Santa Claus, the childhood icon, is near universal. Given Santa’s fame, he has considerable potential to influence individual and societal behavior. However, according to the authors, this is not necessarily for the good. Epidemiological studies show there is a correlation between countries that venerate Santa Claus and those that have high levels of childhood obesity. Although the causality has not been proven, it appears that Santa promotes a message that obesity is synonymous with cheerfulness and joviality. The authors therefore offer suggestions to create a supportive environment for Santa’s dieting: ceasing the tradition of leaving him cookies, mince pies, milk, brandy or sherry, encouraging him to share the carrots and celery sticks commonly left for Rudolf, and encouraging him to adopt a more active means of delivering toys, such as swapping his reindeer for a bike or simply walking or jogging. From a public health point of view, we now know the causes and have a solution for preventing obesity, the metabolic syndrome, hyperlipidemia and T2DM!
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References 1. Steinberger J, et al: Progress and challenges in metabolic syndrome in children and adolescents: a scientific statement from the American Heart Association Atherosclerosis, Hypertension, and Obesity in the Young Committee of the Council on Cardiovascular Disease in the Young; Council on Cardiovascular Nursing; and Council on Nutrition, Physical Activity, and Metabolism. Circulation 2009;119:628–647. 2. Simmons RK, et al: The metabolic syndrome: useful concept or clinical tool? Report of a WHO Expert Consultation. Diabetologia 2010;53:600–605. 3. Gidding SS: Familial hypercholesterolemia: a decade of progress. J Pediatr 2010;156:176–177. 4. Meier JJ, Nauck MA: Is the diminished incretin effect in type 2 diabetes just an epi-phenomenon of impaired -cell function? Diabetes 2010;59:1117–1125. 5. Pavkov ME, et al: Changing patterns of type 2 diabetes incidence among Pima Indians. Diabetes Care 2007;30:1758– 1763. 6. Sellers EA, et al: Macroalbuminuria and renal pathology in First Nation youth with type 2 diabetes. Diabetes Care 2009;32:786–790. 7. Chong CR, Chabner BA: Mysterious metformin. Oncologist 2009;14:1178–1181. 8. De Kreutzenberg SV, et al: Downregulation of the longevity-associated protein sirtuin-1 in insulin resistance and metabolic syndrome: potential biochemical mechanisms. Diabetes 2010;59:1006–1015. 9. Lewanczuk RZ, Paty BW, Toth EL: Comparison of the [13C]glucose breath test to the hyperinsulinemic-euglycemic clamp when determining insulin resistance. Diabetes Care 2004;27:441–447. 10. Friedrichs E: On the pill. Nat Med 2010;16:506–508. 11. Chesney RW: Vitamin D and The Magic Mountain: the anti-infectious role of the vitamin. J Pediatr 2010;156:698–703. 12. Baz-Hecht M, Goldfine AB: The impact of vitamin D deficiency on diabetes and cardiovascular risk. Curr Opin Endocrinol Diabetes Obes 2010;17:113–119. 13. Wang L, et al: Systematic review: vitamin D and calcium supplementation in prevention of cardiovascular events. Ann Intern Med 2010;152:315–323. 14. Pittas AG, et al: Systematic review: vitamin D and cardiometabolic outcomes. Ann Intern Med 2010;152:307–314. 15. Rodriguez W, et al: Improving pediatric dosing through pediatric initiatives: what we have learned? Pediatrics 2008;121:530–539.
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Population Genetics and Pharmacogenetics Ken K. Onga,b and Cathy Elksa a
Medical Research Council Epidemiology Unit, Cambridge, and Department of Paediatrics, University of Cambridge, UK
b
Rapid technological advances continue to pave the way for exciting genetic discoveries. Just a handful of years since the appearance of genome-wide association studies that have rapidly expanded the number of common variants robustly linked to multigenic diseases and traits, whole-genome and whole-exome sequencing studies are now demonstrating their power to shed novel insights into human genetic architecture and disease etiology. This year’s review starts with four such studies all published during the first few months of 2010. Genome-wide association studies continue to generate dozens of new loci for common diseases and continuous traits, and examples described below include fasting glucose, insulin resistance and birth weight. Novel analytical approaches to genome-wide association data have described variants with parent-of-origin associations with both type 1 and type 2 diabetes. Extension of genetic studies to African-derived populations with greater power for fine mapping has finally pinpointed the first genome-wide obesity locus to the FTO gene. However, while the limits of the genome are now clearly definable and we have the tools to see it in its entirety, the percentage of disease susceptibility that can be explained remains surprisingly small. Whether you see this position as a future opportunity or as an indictment against the current efforts, it is clear that we will need new analytical strategies and likely new large familybased studies to decipher the remaining genetic basis of human phenotypic variation and disease risks.
Mechanism of the year The clinical utility of genome sequencing
Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy Lupski JR, Reid JG, Gonzaga-Jauregui C, Rio DD, Chen DC, Nazareth L, Bainbridge M, Dinh H, Jing C, Wheeler DA, McGuire AL, Zhang F, Stankiewicz P, Halperin JJ, Yang C, Gehman C, Guo D, Irikat RK, Tom W, Fantin NJ, Muzny DM, Gibbs RA Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex., USA N Engl J Med 2010;362:1181–1191 Background: Whole-genome sequencing may revolutionize medical diagnostics through rapid identification of alleles that cause disease. However, even in cases with simple patterns of inheritance and unambiguous diagnoses, the relationship between disease phenotypes and their corresponding genetic changes can be complicated. This study aimed to assess the usefulness of human whole-genome sequencing for genetic diagnosis in a patient with Charcot-Marie-Tooth disease. Methods: A family was identified with a recessive form of Charcot-Marie-Tooth disease of yet unknown genetic basis. The whole genome of the proband was sequenced and potential functional variants were genotyped in other family members. Results: Two compound, heterozygous, causative mutations in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene) were identified and validated. Separate subclinical phenotypes segregated independently with each of the two mutations; heterozygous mutations confer susceptibility to neuropathy, including the carpal tunnel syndrome. Conclusion: This study of a family with Charcot-Marie-Tooth disease shows that whole-genome sequencing can identify etiological variants and provide clinically relevant diagnostic information.
Personalised medicine is used to include a patient’s lifestyle, family history, and environment. This and the following articles illustrate the potential that rapid-sequencing technology could have for personalised medicine. While several whole genomes of healthy individuals have been published, this study is one of the first to apply whole-genome sequencing in a clinical context. Beyond the acquisition of an accurate genome sequence, two major difficulties in interpreting the data may quickly become apparent. Firstly is the realisation of non-classical mendelian inheritance patterns, involving more than one mutation in the same or even different genes; these are compound (as illustrated in this study), digenic, and trigenic inheritance patterns, and the presence of modifying genes. Secondly, many seemingly deleterious mutations will be found in each individual, and most of these will be silent or ‘non-penetrant’. In this patient, whole-genome sequencing unearthed over 3 million variants compared to the human genome reference sequence, of which 1 million were within gene regions. 9,069 were predicted to be amino-acid changing, and 121 were non-sense mutations, i.e. predicted to lead to truncation of the protein. 21 have been described to cause mendelian disease. For example the (male) proband had a mutation in ABCD1 which causes the X-linked disorder adrenoleukodystrophy (which he did not have!). The authors therefore focussed only on the 54 coding SNPs in the 40 genes with previous links to neurological conditions. Two SNPs were in SH3TC2 and one showed complete segregation with disease status in other family members. Sequencing the whole genome to find one or two causative mutations may seem like a sledgehammer to crack a small nut, particularly if most of the data is uninterpretable. However this sledgehammer is rapidly becoming lighter due to technological advances. Even within the 6-month duration of this study, the sequence yield increased threefold with no appreciable increase in cost. The estimated cost per genome in April 2009 was USD 50,000, in contrast to around USD 15,000 for a diagnostic panel for Charcot-Marie-Tooth covering only 15 genes. Current costs for whole-exome sequencing are only about USD 4,000, and this approach could have led to the same conclusion – see article below. Other studies described below illustrate further ways to interpret the data.
Exome sequencing identifies the cause of a mendelian disorder Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA, Shendure J, Bamshad MJ Department of Genome Sciences, University of Washington, Seattle, Wash., USA Nat Genet 2010;42:30–35 Background: To date, allelic variants underlying fewer than half of all monogenic disorders have been discovered. This study demonstrates the first successful application of exome sequencing to discover the gene for a rare mendelian disorder of unknown cause, Miller syndrome. Methods: For 4 affected individuals in three independent kindreds, gene-coding regions were captured and sequenced to a mean coverage of 40× and sufficient depth to call variants at approximately 97% of each targeted exome. Filtering against public SNP databases and eight HapMap exomes for genes with two previously unknown variants in each of the 4 individuals. Results: A single candidate gene, DHODH, was identified which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway. Sanger sequencing confirmed the presence of DHODH mutations in three additional families with Miller syndrome. Conclusion: Exome sequencing of a small number of unrelated affected individuals is a powerful, efficient strategy for identifying the genes underlying rare mendelian disorders and will likely transform the genetic analysis of monogenic traits.
The justification for the smaller ‘whole-exome’, rather than ‘whole-genome’, sequencing approach is that the majority of known variants for monogenic disorders disrupt the protein-coding sequence. By focusing on exons, plus splice acceptor and donor sites, only ~5% of the whole-genome sequencing load is required. Yet, this is still vast, and still results in a huge number of potential causal variants to sift through. The authors found non-synonymous, splice site or insertion/deletion variants in ~4,600 genes! Several factors increased the chances of successfully choosing the causal gene in this landmark study. Firstly, the authors chose a recessive disorder, which meant they could focus on those genes with at least two variants. Secondly, as Miller syndrome is so rare, with only ~30 cases to date, the authors argued that they could safely filter out all the variants found in existing databases. Thirdly, they
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sequenced 2 affected brothers, which further narrowed the search to those variants that were present in both exomes. The authors found further mutations in the dihydroorotate dehydrogenase gene (DHODH) in several other probands. The characteristic features of Miller syndrome include severe micrognathia, cleft lip and/or palate, hypoplasia or aplasia of the posterior elements of the limbs, and coloboma of the eyelids. It was therefore a surprise to locate gene mutations to DHODH, which encodes an enzyme that catalyzes the conversion of dihydroorotate to orotic acid, as the authors point out that inborn errors of metabolism rarely cause birth defects. The paper includes a thought-provoking discussion linking inhibitors of pyrimidine biosynthesis (such as methotrexate) to birth defects via TNF-␣, NF-B and Shh (the sonic hedgehog homolog). Intriguingly, the brothers were also compound heterozygotes for variants in DNAH5, which encodes a ciliary dynein heavy chain, and they were consequently diagnosed with primary ciliary dyskinesia. While this explained their non-Miller syndrome features of recurrent lung infections and bronchiectasis, it illustrates the potential for surprise genetic findings with clinical, and even ethical, consequences. We used to consider multiple genetic defects unique to consanguineous families. It may be more common that we expect.
Analysis of genetic inheritance in a family quartet by whole-genome sequencing Roach JC, Glusman G, Smit AF, Huff CD, Hubley R, Shannon PT, Rowen L, Pant KP, Goodman N, Bamshad M, Shendure J, Drmanac R, Jorde LB, Hood L, Galas DJ Institute for Systems Biology, Seattle, Wash., USA Science 2010;328:636–639 Background: Whole-genome sequences from 4 members of a family represent a qualitatively different type of genetic data than whole-genome sequences from individual or sets of unrelated genomes. Methods: Whole-genome sequences of a family of 4, consisting of 2 siblings and their parents, were analyzed. Family-based sequencing allowed the precise delineation of recombination sites, the identification of 70% of the sequencing errors (resulting in >99.999% accuracy), and the identification of very rare single-nucleotide polymorphisms. The directly estimated human intergeneration mutation rate was approximately 1.1 × 10–8 per position per haploid genome. Both offspring in this family have two recessive disorders: Miller syndrome, for which the gene was concurrently identified, and primary ciliary dyskinesia, for which causative genes have been previously identified. Family-based genome analysis narrowed the candidate genes for both of these mendelian disorders to only four. Conclusions: These results demonstrate the value of complete genome sequencing in families. Such data, along with relevant environmental and medical information, will characterize the integrated medical records of the future.
This paper, from the same group who reported the genetic etiology of Miller syndrome, is based on that same family and now includes their parents’ genomes. They show for the first time how whole genomes from the same family permit powerful inheritance analyses to greatly improve the detection and understanding of sequencing errors, and allow precise estimates of human mutation rates and the precise locations of recombination events. The 4 family members showed different genotypes at 3.6 million known variants, and these formed the basis for inheritance analyses. The authors describe how in a nuclear family of 4, each variant position can be allocated to one of four inheritance states with the 2 children receiving (i) the same allele from both the mother and the father (identical), (ii) the same allele from the mother but opposites from the father, (iii) the same allele from the father, but opposites from the mother, or (iv) opposites from both parents. Adjacent variants with the same inheritance states delineate inheritance blocks, and recombination must have occurred between these blocks. In contrast, blocks of variants that do not conform to these inheritance patterns (mendelian inheritance errors) must be either sequencing errors or result from hemizygous deletions. Furthermore, while the authors identified the gene for Miller syndrome in their earlier paper, the current study illustrates how family-based data would have markedly narrowed the search for the disease-causing variant. They conclude that ‘When the cost of recruiting additional families is expensive relative to sequencing costs, sequencing genomes of families will be an economical strategy for the identification of many disease-causing genes.’
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Clinical assessment incorporating a personal genome Ashley EA, Butte AJ, Wheeler MT, Chen R, Klein TE, Dewey FE, Dudley JT, Ormond KE, Pavlovic A, Morgan AA, Pushkarev D, Neff NF, Hudgins L, Gong L, Hodges LM, Berlin DS, Thorn CF, Sangkuhl K, Hebert JM, Woon M, Sagreiya H, Whaley R, Knowles JW, Chou MF, Thakuria JV, Rosenbaum AM, Zaranek AW, Church GM, Greely HT, Quake SR, Altman RB Center for Inherited Cardiovascular Disease, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, Calif., USA
[email protected] Lancet 2010;375:1525–1535 Background: The cost of genomic information has fallen steeply, but the clinical translation of genetic risk estimates remains unclear. This study aimed to undertake an integrated analysis of a complete human genome in a clinical context. Methods: A 40-year-old man with a family history of vascular disease and early sudden death underwent a routine clinical assessment plus analysis of his full genome sequence. In addition to identifying risk variants associated with mendelian disease, post-test probabilities of disease were estimated by applying likelihood ratios derived from integration of multiple common variants to age-appropriate and sexappropriate pre-test probabilities. Results: Analysis of 2.6 million single nucleotide polymorphisms and 752 copy number variations showed increased genetic risk for myocardial infarction, type 2 diabetes, and some cancers. Rare variants were found in three genes that are clinically associated with sudden cardiac death – TMEM43, DSP, and MYBPC3. A variant in LPA was consistent with a family history of coronary artery disease. The patient had a heterozygous null mutation in CYP2C19 suggesting probable clopidogrel resistance, several variants associated with a positive response to lipid-lowering therapy, and variants in CYP4F2 and VKORC1 that suggest he might have a low initial dosing requirement for warfarin. Conclusions: Although challenges remain, these results suggest that whole-genome sequencing can yield useful and clinically relevant information for individual patients.
While other studies have applied genome sequencing to rare mendelian diseases, this is the first to explore its utility in risk prediction for more complex multigenic disorders. Other than his family history, the patient was healthy and was found to have increased risks for coronary artery disease and sudden cardiac death, and his genome was informative for future drug choices and dosing. Remarkably, this assessment was performed on just 2 ml of whole blood. The authors relied on disease-specific mutation databases and pharmacogenomic databases to identify genes and mutations with known associations with disease and drug response. There were compelling findings related to drug responses, including 63 known pharmacogenomic variants that could affect the patient’s response to commonly used drugs. For example, he is likely to respond well to statins and to be at lower risk of statin-induced myopathy. Were he to need warfarin, his initial doses are likely to be low; by contrast, clopidogrel might be less effective. This approach assumes that all the identified variants are expressed and this contrasts with the findings of other studies, which showed that we each carry many mutations for mendelian disease that are somehow silenced. Nevertheless, with increasing knowledge gained from genome-wide association studies and wholegenome and whole-exome sequencing studies, the clinical value of sequence data will rapidly increase. However, clinical and ethical dilemmas need to be tackled. The patient was found to have potentially damaging variants associated with haemochromatosis and also parathyroid tumors. He had no evidence of either, but it is uncertain whether surveillance and testing for these conditions should continue. Secondly, duty of care to other family members, by informing them of their disease risks, needs to be balanced against confidentiality. Furthermore, it will be increasingly important to defend the principle that health insurers cannot use our genetic information to calculate our premiums.
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New genetic associations T2DM, insulin resistance and birth weight
New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk Dupuis J, Langenberg C, Prokopenko I, Saxena R, Soranzo N, Jackson AU, Wheeler E, Glazer NL, Bouatia-Naji N, Gloyn AL, Lindgren CM, Magi R, Morris AP, Randall J, Johnson T, Elliott P, Rybin D, Thorleifsson G, Steinthorsdottir V, Henneman P, Grallert H, Dehghan A, Hottenga JJ, Franklin CS, Navarro P, Song K, Goel A, Perry JR, Egan JM, Lajunen T, Grarup N, Sparso T, Doney A, Voight BF, Stringham HM, Li M, Kanoni S, Shrader P, Cavalcanti-Proenca C, Kumari M, Qi L, Timpson NJ, Gieger C, Zabena C, Rocheleau G, Ingelsson E, An P, O’Connell J, Luan J, Elliott A, McCarroll SA, Payne F, Roccasecca RM, Pattou F, Sethupathy P, Ardlie K, Ariyurek Y, Balkau B, Barter P, Beilby JP, Ben-Shlomo Y, Benediktsson R, Bennett AJ, Bergmann S, Bochud M, Boerwinkle E, Bonnefond A, Bonnycastle LL, Borch-Johnsen K, Bottcher Y, Brunner E, Bumpstead SJ, Charpentier G, Chen YD, Chines P, Clarke R, Coin LJ, Cooper MN, Cornelis M, Crawford G, Crisponi L, Day IN, de Geus EJ, Delplanque J, Dina C, Erdos MR, Fedson AC, Fischer-Rosinsky A, Forouhi NG, Fox CS, Frants R, Franzosi MG, Galan P, Goodarzi MO, Graessler J, Groves CJ, Grundy S, Gwilliam R, Gyllensten U, Hadjadj S, Hallmans G, Hammond N, Han X, Hartikainen AL, Hassanali N, Hayward C, Heath SC, Hercberg S, Herder C, Hicks AA, Hillman DR, Hingorani AD, Hofman A, Hui J, Hung J, Isomaa B, Johnson PR, Jorgensen T, Jula A, Kaakinen M, Kaprio J, Kesaniemi YA, Kivimaki M, Knight B, Koskinen S, Kovacs P, Kyvik KO, Lathrop GM, Lawlor DA, Le BO, Lecoeur C, Li Y, Lyssenko V, Mahley R, Mangino M, Manning AK, Martinez-Larrad MT, McAteer JB, McCulloch LJ, McPherson R, Meisinger C, Melzer D, Meyre D, Mitchell BD, Morken MA, Mukherjee S, Naitza S, Narisu N, Neville MJ, Oostra BA, Orru M, Pakyz R, Palmer CN, Paolisso G, Pattaro C, Pearson D, Peden JF, Pedersen NL, Perola M, Pfeiffer AF, Pichler I, Polasek O, Posthuma D, Potter SC, Pouta A, Province MA, Psaty BM, Rathmann W, Rayner NW, Rice K, Ripatti S, Rivadeneira F, Roden M, Rolandsson O, Sandbaek A, Sandhu M, Sanna S, Sayer AA, Scheet P, Scott LJ, Seedorf U, Sharp SJ, Shields B, Sigurethsson G, Sijbrands EJ, Silveira A, Simpson L, Singleton A, Smith NL, Sovio U, Swift A, Syddall H, Syvanen AC, Tanaka T, Thorand B, Tichet J, Tonjes A, Tuomi T, Uitterlinden AG, van Dijk KW, van HM, Varma D, Visvikis-Siest S, Vitart V, Vogelzangs N, Waeber G, Wagner PJ, Walley A, Walters GB, Ward KL, Watkins H, Weedon MN, Wild SH, Willemsen G, Witteman JC, Yarnell JW, Zeggini E, Zelenika D, Zethelius B, Zhai G, Zhao JH, Zillikens MC, Borecki IB, Loos RJ, Meneton P, Magnusson PK, Nathan DM, Williams GH, Hattersley AT, Silander K, Salomaa V, Smith GD, Bornstein SR, Schwarz P, Spranger J, Karpe F, Shuldiner AR, Cooper C, Dedoussis GV, Serrano-Rios M, Morris AD, Lind L, Palmer LJ, Hu FB, Franks PW, Ebrahim S, Marmot M, Kao WH, Pankow JS, Sampson MJ, Kuusisto J, Laakso M, Hansen T, Pedersen O, Pramstaller PP Department of Biostatistics, Boston University School of Public Health, Boston, Mass., USA Nat Genet 2010;42:105–116 Background: Levels of circulating glucose are tightly regulated. The aim of this study was to identify new genetic loci associated with various glycemic traits. Methods: A meta-analyses of 21 genome-wide association studies was performed for fasting glucose, fasting insulin and indices of -cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 non-diabetic participants. The top 25 loci were followed up in a further 76,558 participants. Results: Altogether 16 loci were identified associated with fasting glucose and HOMA-B and 2 loci associated with fasting insulin and HOMA-IR. These include 9 loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF-1). In further analyses, ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 were also demonstrated to be associated with risk of type 2 diabetes. The likely biological candidate genes within these loci influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Conclusions: These findings demonstrate that genetic studies of continuous glycemic traits can identify novel type 2 diabetes risk loci. However, other loci are associated with a modest elevation in glucose levels but not overt diabetes.
In addition to large GWA studies, distinct diseases, such as type 2 diabetes (T2DM), studying the underlying continuous traits, such as glucose levels, may uncover further genetic loci that are relevant for disease risks. Indeed, studies for BMI have been more powerful and fruitful that studies of obesity. However, there are caveats in this approach. Firstly, some variants that affect glucose metabolism may raise glucose levels only marginally and not sufficiently to contribute to risks for T2DM, or its complications. This is akin to the rare glucokinase mutations that cause the benign condition
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MODY type 2. Secondly, a recent genetic variant for HbA1c levels was surprisingly shown subsequently to influence hemoglobin levels rather than glycemia, despite being located in the hexokinase gene [1]. It is therefore reassuring to see that several of the new loci described in this study were confirmed to influence T2DM risk per se. The size of the combined effects of the 16 loci associated with fasting glucose was substantial. There was a mean differences of ~0.4 mmol/l (5.93 vs. 5.51 mmol/l) in fasting glucose when comparing the highest genetic risk group (5.6% of the sample) to the lowest (2.9% of the sample). This represents a shift of approximately 25 centile points in the distribution of fasting glucose, and corresponded to a relative risk of 1.54–1.73 for future T2DM. The one locus for insulin resistance (HOMA-IR) was near to the strongly plausible gene IGF-1, however this variant failed to reach significance in follow-up studies of over 50,000 individuals. The lack of further loci for insulin resistance is surprising as the estimates of heritability are similar for insulin resistance and insulin secretion (HOMA-B) in family studies. The authors argue that different traits could have distinct genetic architectures. For example, some such as fasting glucose could be influenced by several common variants each with modest effects. Others, such as HOMA-IR, could be putatively influenced by fewer loci, rarer variants, or by stronger environmental modifications on their genetic effects.
Genetic variant near IRS1 is associated with type 2 diabetes, insulin resistance and hyperinsulinemia Rung J, Cauchi S, Albrechtsen A, Shen L, Rocheleau G, Cavalcanti-Proenca C, Bacot F, Balkau B, Belisle A, Borch-Johnsen K, Charpentier G, Dina C, Durand E, Elliott P, Hadjadj S, Jarvelin MR, Laitinen J, Lauritzen T, Marre M, Mazur A, Meyre D, Montpetit A, Pisinger C, Posner B, Poulsen P, Pouta A, Prentki M, Ribel-Madsen R, Ruokonen A, Sandbaek A, Serre D, Tichet J, Vaxillaire M, Wojtaszewski JF, Vaag A, Hansen T, Polychronakos C, Pedersen O, Froguel P, Sladek R McGill University and Genome Quebec Innovation Centre, Montreal, Que., Canada Nat Genet 2009;41:1110–1115 Background: Previous genome-wide association studies have identified common variants that only partially explain the genetic risk for type 2 diabetes (T2D). Methods: 392,365 SNPs were tested for association with T2D in 1,376 French cases and controls. The 5% of variants most significantly associated with T2D (16,360 SNPs) were followed up in an independent sample of 4,977 French individuals using a custom designed array. The best 28 hits from this stage were subsequently selected for replication in 7,698 Danish subjects. Finally, the most significant novel locus was followed up in 14,358 French, Danish and Finnish participants from population-based cohorts with detailed T2D-related phenotypes. Results: Four SNPs were identified which showed genome-wide significant association with T2D (p < 5.0 × 10–8), one of which (rs2943641, p = 9.3 × 10–12, OR = 1.19) was located adjacent to the insulin receptor substrate 1 gene (IRS1). The C allele of rs2943641 was associated with insulin resistance and hyperinsulinemia; this allele was also associated with reduced basal levels of IRS1 protein and decreased insulin induction of IRS1-associated phosphatidylinositol-3-OH kinase activity in human skeletal muscle biopsies. Conclusions: Genetic variation near to IRS1 is associated with T2D, insulin resistance and hyperinsulinemia. Unlike previous T2D risk loci, which predominantly associate with impaired -cell function, this highlights the role of insulin resistance in T2D risk.
In GWA studies of complex traits, efforts to uncover further ‘missing heritability’ have been largely focussed on increasing statistical power by combining studies in large-scale meta-analyses. Here, the authors adopted an alternative multistage strategy whereby a GWA study was first used to test genetic association with T2D in a large well-phenotyped cohort before selecting a fraction of SNPs showing the strongest association to study in well-powered subsequent stages. The advantage of this is that statistical power is maximised cost-effectively since each stage contains a smaller number of SNPs and an increased number of individuals. The authors were rewarded by the discovery of a novel T2D locus in a possible regulatory region of the insulin receptor substrate gene (IRS1). In addition to being associated with T2D risk (OR = 1.19), the C allele at rs2943641 was also associated with insulin resistance and hyperinsulinemia, independently of BMI. In a subset of French individuals,
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the variant was associated with a 35% increased risk of T2D and a 14% increased incidence of the development of hyperglycaemia after 9 years of follow-up. In functional studies, the authors described likely regulatory effects of rs2943641 on IRS1 and insulin signaling. Interestingly, this SNP is in the same LD block as a previously identified coronary artery disease (CAD) risk locus (rs2943634, r2 = 0.8), suggesting that genetic variation near IRS1 may predispose to CAD through increased susceptibility to T2D, a known cardiovascular risk factor. Alternatively this gene region may have pleiotrophic effects on both phenotypes. In contrast to previously identified T2D loci, this variant is the first to be associated with insulin resistance and hyperinsulinemia, rather than impaired insulin secretion. This will give us new insight into the contribution of insulin sensitivity in establishing T2D risk.
Variants in ADCY5 and near CCNL1 are associated with fetal growth and birth weight Freathy RM, Mook-Kanamori DO, Sovio U, Prokopenko I, Timpson NJ, Berry DJ, Warrington NM, Widen E, Hottenga JJ, Kaakinen M, Lange LA, Bradfield JP, Kerkhof M, Marsh JA, Magi R, Chen CM, Lyon HN, Kirin M, Adair LS, Aulchenko YS, Bennett AJ, Borja JB, Bouatia-Naji N, Charoen P, Coin LJ, Cousminer DL, de Geus EJ, Deloukas P, Elliott P, Evans DM, Froguel P, Glaser B, Groves CJ, Hartikainen AL, Hassanali N, Hirschhorn JN, Hofman A, Holly JM, Hypponen E, Kanoni S, Knight BA, Laitinen J, Lindgren CM, McArdle WL, O’Reilly PF, Pennell CE, Postma DS, Pouta A, Ramasamy A, Rayner NW, Ring SM, Rivadeneira F, Shields BM, Strachan DP, Surakka I, Taanila A, Tiesler C, Uitterlinden AG, van Duijn CM, Wijga AH, Willemsen G, Zhang H, Zhao J, Wilson JF, Steegers EA, Hattersley AT, Eriksson JG, Peltonen L, Mohlke KL, Grant SF, Hakonarson H, Koppelman GH, Dedoussis GV, Heinrich J, Gillman MW, Palmer LJ, Frayling TM, Boomsma DI, Davey SG, Power C, Jaddoe VW, Jarvelin MR, McCarthy MI Genetics of Complex Traits, Peninsula College of Medicine and Dentistry, University of Exeter, Exeter, UK Nat Genet 2010;42:430–435 Background: Birth weight is a complex multifactorial trait. This study aimed to identify genetic variants associated with birth weight. Methods: The results of six genome-wide association (GWA) studies (n = 10,623 Europeans from pregnancy/birth cohorts) were meta-analyzed. Two lead signals were followed up in 13 replication studies (n = 27,591). Results: rs900400 near LEKR1 and CCNL1 (p = 2 × 10–35) and rs9883204 in ADCY5 (p = 7 × 10–15) were robustly associated with birth weight. Correlated SNPs in ADCY5 were recently implicated in regulation of glucose levels and susceptibility to type 2 diabetes, providing evidence that the welldescribed association between lower birth weight and subsequent type 2 diabetes has a genetic component, distinct from the proposed role of programming by maternal nutrition. Using data from both SNPs, the 9% of Europeans carrying four birth weight-lowering alleles were, on average, 113 g (95% CI 89–137 g) lighter at birth than the 24% with zero or one alleles (Ptrend = 7 × 10–30). Conclusion: The impact on birth weight is similar to that of a mother smoking 4–5 cigarettes per day in the third trimester of pregnancy.
The birth weight-lowering C allele of rs9883204 is in linkage disequilibrium (LD) (r2 = 0.75) with the A allele of rs11708067, which was independently shown to be associated with increased risk of type 2 diabetes, higher fasting glucose, and reduced insulin secretion. Fetal insulin is a key fetal growth factor, and these metabolic associations suggest that one mechanism explaining the ADCY5 (cyclic AMP-generating adenylate cyclase) association with birth weight might be a direct effect of the fetal risk allele on fetal growth via reduced insulin secretion, consistent with the fetal insulin hypothesis. The association with ponderal index, relative to those with birth length and head circumference, was particularly strong for the variant near the cyclin gene CCNL1, which encodes a kinase known to phosphorylate histone H1, suggesting that it might have a greater association with fat mass than with skeletal growth. However, for ADCY5, the birth measures showed more proportionate associations. Birth weight represents a complex phenotype. Beyond the usual gene-environment interactions, the fetal environment may be governed by maternal genes. It is therefore important to clarify whether apparent effects of fetal genes might be proxy markers for maternal genes acting on maternal
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metabolism. In the current study, meta-analysis of associations between birth weight and fetal genotype, conditional on maternal genotype, yielded very similar results to the original associations at both loci, showing that these are direct fetal effects. Secondly, fetal growth seems to be one of the main targets of imprinted genes. Future studies need to explore whether these fetal genes demonstrate parent of origin effects.
New paradigms The genetics of epigenetics
Heritable individual-specific and allele-specific chromatin signatures in humans McDaniell R, Lee BK, Song L, Liu Z, Boyle AP, Erdos MR, Scott LJ, Morken MA, Kucera KS, Battenhouse A, Keefe D, Collins FS, Willard HF, Lieb JD, Furey TS, Crawford GE, Iyer VR, Birney E Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas, Austin, Tex., USA Science 2010;328:235–239 Background: The extent to which variation in chromatin structure and transcription factor binding may influence gene expression, and thus underlie or contribute to variation in phenotype, is currently unknown. Methods: Individual-to-individual variation and differences between homologous chromosomes within the same individual (allele-specific variation) in chromatin structure and transcription factor binding in lymphoblastoid cells derived from individuals of geographically diverse ancestry were catalogued. Differences between chromatin structure were compared between parents and children in order to identify whether chromatin signatures are heritable in humans. Results: Ten percent of active chromatin sites were individual-specific; a similar proportion were allelespecific. Both individual-specific and allele-specific sites were commonly transmitted from parent to child, which suggests that they are heritable features of the human genome. Conclusion: This study shows that heritable chromatin status and transcription factor binding differ as a result of genetic variation and may underlie phenotypic variation in humans.
Variation in chromatin structure can facilitate or restrict the binding of transcription factors to regulatory regions and is therefore a major component in the regulation of gene transcription. While there is much interest in such epigenetic changes as the possible mechanism to explain ‘long-term programming’ responses to specific early exposures, alterations of chromatin structure are in turn also influenced by genetic variation. In order to increase our understanding of this allele-specific gene regulation, the authors catalogued variation in transcription factor binding and chromatin structure in a European and an African family (both parents and one daughter from each) from the ‘1000 Genomes Project’ using deoxyribonuclease I hypersensitive (DNase I) site mapping and chromatin immunoprecipitation (ChIP). This allowed them to identify regulatory DNA elements such as promoters, enhancers, silencers and insulators. The study design enabled the investigators to compare related and unrelated individuals and determine whether chromatin structure is heritable. Comparison of the children’s chromatin signals to that of the parents revealed that chromatin sites were much more similar to their own parents’ than those of the unrelated family, demonstrating its strong heritability. Remarkably, only 10% of active chromatin sites were individual-specific and this indicates that one of the major types of epigenetic variation (i.e. chromatin structure) is itself largely influenced by genetic variation. Future studies should explore the specific genetic regulation of epigenetic marks.
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Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis Baranzini SE, Mudge J, van Velkinburgh JC, Khankhanian P, Khrebtukova I, Miller NA, Zhang L, Farmer AD, Bell CJ, Kim RW, May GD, Woodward JE, Caillier SJ, McElroy JP, Gomez R, Pando MJ, Clendenen LE, Ganusova EE, Schilkey FD, Ramaraj T, Khan OA, Huntley JJ, Luo S, Kwok PY, Wu TD, Schroth GP, Oksenberg JR, Hauser SL, Kingsmore SF Department of Neurology, University of California at San Francisco, San Francisco, Calif., USA
[email protected] Nature 2010;464:1351–1356 Background: Monozygotic twins are often studied to quantify the relative contributions of genetics and environment in human diseases. In multiple sclerosis (MS), disease discordance between monozygotic twin pairs has been interpreted to indicate its important environmental pathogenesis. However, both genetic and epigenetic differences between monozygotic twins have been described, challenging the accepted experimental model in disambiguating the effects of nature and nurture. Methods: Genomes of one MS-discordant monozygotic twin pair were sequenced and compared. Messenger RNA transcriptome and epigenome sequences of CD4+ lymphocytes from three MS-discordant monozygotic twin pairs were also determined and evaluated. Results: No reproducible differences were detected between co-twins among approximately 3.6 million single nucleotide polymorphisms (SNPs) or approximately 0.2 million insertion-deletion polymorphisms. Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS susceptibility SNPs, copy number variations, mRNA and genomic SNP and insertion-deletion genotypes, or the expression of approximately 19,000 genes in CD4+ T cells. Only 2–176 methylation differences out of approximately 2 million CpG dinucleotides were detected between siblings of the three twin pairs, in contrast to approximately 800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or between normal and cancerous tissues. Conclusion: In the first systematic effort to estimate sequence variation among monozygotic co-twins, there was no evidence for genetic, epigenetic or transcriptome differences that explained MS discordance. These are the first female, twin and autoimmune disease individual genome sequences reported.
Genetically identical twins discordant for a particular disease are considered a useful tool for studying a possible contribution of epigenetics to disease pathogenesis. In this study, the authors compared genetic and epigenetic differences in monozygotic twins discordant for multiple sclerosis (MS), using CD4+ lymphocytes due to their known involvement in the autoimmune pathophysiology of MS. This study of unprecedented resolution is the first to systematically characterise both genetic and epigenetic contributions to disease discordance in monozygotic twins using an impressive range of sophisticated sequencing technologies. Surprisingly, no reproducible genetic, epigenetic or transcriptome differences were identified to explain the twins discordance in MS. So what lessons can be learnt? Firstly, the lack of genetic differences between monozygotic twins at least reassures us of the validity of the monozygotic-dizygotic twins model to study the heritability of disease risk. Secondly, the further lack of epigenetic or transcriptome differences between monozygotic twins is consistent with the recent findings of McDaniell et al. (described above) in illustrating the predominant ‘genetic regulation of epigenetic marks’. And what could explain the disease discordance? The authors describe that their novel comprehensive approach still had limited coverage by exclusion of low coverage regions and repetitive sequences, by only moderate sensitivity for detection of structural variants of size 50–1,500 nucleotides, and (the most tractable explanation in our opinion) by limited feasibility to detect possible somatic mosaicism in tissues other than the CD4+ lymphocyte.
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New paradigms Parent of origin effects in genome-wide association studies
The imprinted DLK1-MEG3 gene region on chromosome 14q32.2 alters susceptibility to type 1 diabetes Wallace C, Smyth DJ, Maisuria-Armer M, Walker NM, Todd JA, Clayton DG Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Nat Genet 2010;42:68–71 Background: Genome-wide association (GWA) studies successfully mapped common disease susceptibility loci. To date, over 300 reproducibly associated loci have been reported. However, there is yet no convincing evidence for any susceptibility locus that displays parent-of-origin effects. Methods: Data were combined from three existing GWA studies comprising a total of 7,514 cases and 9,405 controls of European ancestry. Potential novel T1DM loci were tested in additional case-control and family studies. Results: There was robust evidence at rs941576 for paternally inherited risk of T1DM; ratio of allelic effects for paternal versus maternal transmissions = 0.75; 95% CI = 0.71–0.79). This marker is in the imprinted region of chromosome 14q32.2, which contains the functional candidate gene DLK1. Our meta-analysis also provided evidence for a further T1DM locus at chromosome 19p13.2. The highest association was at marker rs2304256 (odds ratio (OR) = 0.86; 95% CI = 0.82–0.90) in the TYK2 gene, which has previously been associated with systemic lupus erythematosus and multiple sclerosis. Conclusion: This study reports two novel T1DM loci, one of which is located in an imprinted gene region and conferred protection against T1DM only when paternally transmitted.
Previous analysis of these data had identified over 40 loci for T1DM [2]. This study took forward three further loci which had not quite reached the p-value thresholds for genome-wide significance (p < 10–8), but had strong ‘suggestive evidence’ for association (p < 10–7) with T1DM in additional case-control and family samples. In an overall test of GWA stage and replication data, two of these loci then reached overall genome-wide significance (p = 4 × 10–9 and p = 1.6 × 10–10). The authors noticed that one of these signals was located in a region characterised by several imprinted genes. They therefore tested the hypothesis that this association could show parent of origin effects, which they indeed showed by analysis of informative parent-offspring trios from family-based studies. As is typical of imprinted regions, the imprinted region of chromosome 14q32.2 contains several imprinted genes, some of which are only maternally expressed (BEGAIN, MEG3, MEG8 and DIO3OS), and others only paternally expressed (DLK1, RTL1 and DIO3). Notably, they found that the overall protective effect was paternally (and not maternally) inherited, and this then narrowed the list of potentially causative genes. The best candidate, DLK1, is paternally expressed, with high levels seen in human heart, pancreatic islet cells, pituitary tissue, ovaries, placenta and testes. DLK1 encodes a membrane-bound protein that is cleaved to form fetal antigen-1, which is involved in the differentiation of many cell types, including pancreatic  cells and B lymphocytes, and promotes B-cell proliferation in human peripheral blood. Thus, there are a number of ways in which variation in DLK1 expression could alter susceptibility to T1DM.
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Parental origin of sequence variants associated with complex diseases Kong A, Steinthorsdottir V, Masson G, Thorleifsson G, Sulem P, Besenbacher S, Jonasdottir A, Sigurdsson A, Kristinsson KT, Jonasdottir A, Frigge ML, Gylfason A, Olason PI, Gudjonsson SA, Sverrisson S, Stacey SN, Sigurgeirsson B, Benediktsdottir KR, Sigurdsson H, Jonsson T, Benediktsson R, Olafsson JH, Johannsson OT, Hreidarsson AB, Sigurdsson G, Ferguson-Smith AC, Gudbjartsson DF, Thorsteinsdottir U, Stefansson K deCODE genetics, Reykjavik, Iceland
[email protected] Nature 2009;462:868–874 Background: Effects of susceptibility variants may depend on from which parent they are inherited. However, in genome-wide association studies to date, the impact of parental origin has largely been ignored. Methods: From a public database of all disease-associated SNPs identified by GWA studies, those independent SNPs (n = 7) located within 500 kilobases of a known imprinted gene were selected. Their disease associations were re-examined in 38,167 Icelanders with existing GWA data. Parental origin of the alleles was determined using a combination of genealogy and long-range phasing. Results: Of the 7 SNPs analysed, 5 showed parent-of-origin specific associations (1 with breast cancer, 1 with basal-cell carcinoma, and 3 with type 2 diabetes). These variants are located in two genomic regions, 11p15 and 7q32, each harbouring a cluster of imprinted genes. SNP rs2334499 at 11p15 was identified as a novel locus for type 2 diabetes; here the allele that conferred increased risk when paternally inherited was protective when maternally transmitted. rs2334499 demonstrated correlation with methylation levels at a novel differentially methylated CTCF-binding site at 11p15. Conclusions: Genome-wide association studies have so far yielded variants that explain only a small fraction of the heritability of the traits studied. Some of the remainder may be hidden in more complex relations between variants and disease risk.
This paper, published only 1 month prior to Wallace et al. (above), took a unique systematic approach to identify SNP-disease associations that differ according to whether the allele is inherited from your mother or father. The strategy was remarkably successful in that 5 of the 7 SNPs analysed did indeed show evidence of parental origin effects. Novel mathematical approaches were developed to take advantage of this uniquely characterised population, whereby in the absence of direct genotype information on parents, the national genealogy database was used to identify even distant family relationships with other study participants (their examples even included third cousins, twice removed). This inference of parent of origin was tested in a subset of direct parent-offspring trios and was found to be 99.8% accurate. They identified a novel T2DM variant at KCNQ1, which is one of the known loci for methylation defects in Beckwith-Wiedemann syndrome. Intriguingly, this variant showed little traditional, or overall, genotype association with disease risk because its influence on increased risk if T2DM when paternally inherited was balanced by a protective effect when maternally transmitted. In the debate over the yet ‘missing heritability’ of common diseases, this study claims to open new avenues for the discovery of further disease loci. However, it is a path that will require a new generation of large and well-characterised family-based studies.
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Food for thought ‘Missing heritability’
Finding the missing heritability of complex diseases Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA, Hunter DJ, McCarthy MI, Ramos EM, Cardon LR, Chakravarti A, Cho JH, Guttmacher AE, Kong A, Kruglyak L, Mardis E, Rotimi CN, Slatkin M, Valle D, Whittemore AS, Boehnke M, Clark AG, Eichler EE, Gibson G, Haines JL, Mackay TF, McCarroll SA, Visscher PM National Human Genome Research Institute, Bethesda, Md., USA
[email protected] Nature 2009;461:747–753 Background: Genome-wide association studies have identified hundreds of genetic variants associated with complex human diseases and traits, and have provided valuable insights into their genetic architecture. Most variants confer relatively small increments in risk, and explain only a small proportion of familial clustering, leaving the question how the remaining, ‘missing’ heritability will be explained. Methods: These authors examined potential sources of missing heritability and propose research strategies, including and extending beyond current genome-wide association approaches, to illuminate the genetics of complex diseases and enhance its potential to enable effective disease prevention or treatment. Results: Strategies were identified to explore the contribution of rare, low frequency and structural variants, to investigate the potential contributions of gene-gene and gene-environment interactions and to make the most of existing and future genome-wide association studies. Conclusion: The search for missing heritability provides a potentially valuable path towards future discoveries.
The debate over the nature of the genetic contribution to individual susceptibility to multigenic diseases goes on. Genetic variants with appreciable frequency in the general population but relatively weak effects, and rare variants with relatively large effects are the likely major contributors to genetic susceptibility. The hype over the high profile results of genome-wide association studies has been countered by criticisms about the weak effect sizes and poor individual predictive ability of common variants, and the low proportion of heritability that they explain. For example, the current 40 loci for adult height together explain only around 5% of its heritability, and the corresponding figure for the 18 loci for type 2 diabetes is only 6%. The authors argue that ‘few investigators expected GWA studies immediately to find all of the variants associated with common diseases, or even most of them; the hope was that they would find at least some’. In that regard, GWA studies have been enormously successful, and the remaining or ‘missing’ heritability should be seen as the opportunity for future genetic discoveries! The authors present several suggestions. Most imminently, ‘mega’ or greatly expanded GWA studies for several traits are in progress and the results of these more powerful GWA analyses will likely be published in the coming months. Much attention is also currently focused on the role of low frequency variants with potentially relatively large effect sizes. Currently, GWA chips, and the HapMap database on which they are based, poorly represent variants with minor allele frequencies <5%. Distinct from rare deleterious mutations that cause mendelian disorders, low-frequency variants have alleles in the 0.5–5% frequency range. Systematic discovery of these variants is rapidly progressing through the ‘1000 Genomes Project’ and this information is already being applied in new genotyping study designs for obesity, type 2 diabetes and cardiovascular disease.
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Associations revisited in non-European populations
Fine mapping of the association with obesity at the FTO locus in African-derived populations Hassanein MT, Lyon HN, Nguyen TT, Akylbekova EL, Waters K, Lettre G, Tayo B, Forrester T, Sarpong DF, Stram DO, Butler JL, Wilks R, Liu J, Le ML, Kolonel LN, Zhu X, Henderson B, Cooper R, McKenzie C, Taylor HA, Jr, Haiman CA, Hirschhorn JN Department of Preventive Medicine, Keck School of Medicine and Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, Calif., USA Hum Mol Genet 2010;19:2907–2916 Background: Genome-wide association studies have typically been performed in individuals of recent European ancestry. In these populations, many common variants are tightly correlated, which considerably limits the resolution of fine mapping. Populations with recent African ancestry often have less extensive and/or different patterns of linkage disequilibrium (LD), and have been proposed to be useful in fine-mapping studies. Methods: In populations of predominantly African ancestry, variation at the FTO locus and body mass index (BMI) was explored. Single nucleotide polymorphisms that are correlated with the signal of association in individuals of European ancestry but that have varying degrees of correlation in Africanderived individuals were genotyped. Results: Most of the variants, including one previously proposed as being functionally important, showed no significant association with BMI. However, two FTO variants, rs3751812 and rs9941349, showed strong evidence of association (p = 2.58 × 10–6 and 3.61 × 10–6) in a meta-analysis of 9,881 Africanancestry individuals. Conclusions: This study both strongly replicated the FTO-BMI association in African-ancestry populations and narrowed the list of potentially causal variants to those that are correlated with rs3751812 and rs9941349 in African-derived populations. This study illustrates the potential of using populations with different LD patterns to fine map genetic associations and supports the rationale for genetically guided functional studies at the FTO locus.
The vast majority of GWA studies have been performed in studies of White-European populations. These authors argue for the study of African-ancestry populations, firstly to test whether the loci identified in European studies are also relevant in individuals of African ancestry. Second, studies of African-ancestry populations may be more efficient for ‘fine-mapping’ studies, i.e. the more precise location of the underlying causal gene or causal variant. This is because African-ancestry populations show less linkage disequilibrium, or greater genetic heterogeneity, than European populations. They found that the European obesity-associated intronic variant rs9939609 in FTO showed no association at all with obesity risk in African-derived populations, ruling this out as the causal variant. However, two further FTO variants, rs3751812 and rs9941349, did show strong associations in African-derived populations. While in Europeans the ‘FTO-association signal’ arguably extended to covered neighbouring genes, such as RPGRIP1L, the current study clearly localises FTO as the underlying gene in this locus in African populations. In addition to simple confirmation of ‘European’ GWA hits in African populations, the setting of ‘discovery’ phase studies in non-Europeans may identify new genes or loci for common disease. For example, a recent GWA study in 19,633 Japanese individuals identified IGF-1 as a major locus for adult height [3]. The 40-plus adult height loci identified in previous GWA studies contained surprisingly few genes implicated in traditional hormone pathways [4]. The minor allele frequency for the height lowering at IGF-1 was 26% in Japanese subjects compared to only 1.7% in Caucasians, which explains why those European studies had insufficient power to detect its effects.
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New mechanisms: Long-range DNA interactions
Comprehensive mapping of long-range interactions reveals folding principles of the human genome Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Mass., USA Science 2009;326:289–293 Background: Understanding how chromosomes fold can provide insight into the complex relationships between chromatin structure, gene activity, and the functional state of the cell. Previously, methods have explored interactions between target loci. Methods: Hi-C is a novel method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. Spatial proximity maps of the human genome were constructed at a resolution of 1 megabase. Results: Spatial proximity maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. An additional level of genome organization was identified, characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. Conclusions: This study demonstrates the power of the novel Hi-C method to map the dynamic conformations of whole genomes.
In its stretched out form, the human genome is roughly 2 m long. Important questions are ‘How does the linear order of basepairs relate to their spatial arrangement?’ and ‘How are genes regulated by distant elements?’ A surprising, but commonly re-occurring feature of GWA studies is that variants that are highly robustly associated with disease risks can be located very far away from genes or gene regions. For example, the obesity-susceptibility SNP rs17782313 is located 188 kb downstream of its nearest gene MC4R [5]. Furthermore, the 9p21 locus is incontrovertibly linked to risk of cardiovascular disease, yet is in an ‘intergenic’ region devoid of protein-coding genes [6]. Such loci are beyond the traditional range for binding sites of activators or regulators of gene expression, although it is possible that such regions might encode functional non-coding RNAs or copy-number variants. This study demonstrates a further possible mechanism of long-range DNA interaction due to genome folding, which could bring into close approximation distant genes and regulatory sites. In general, the ‘average intra-chromosomal contact probability’ for pairs of loci decreased monotonically with increasing genomic distance between the loci. However, even beyond distances of 200 Mb, intra-chromosomal contact probabilities were higher than the contact probabilities between different chromosomes, and blocks could be identified with either enriched or deleted interactions. Furthermore, inter-chromosomal interactions were detected, particularly between small gene-rich chromosomes (16, 17, 18, 19, 20, 21, and 22), which paves the way for exploration of gene-gene interactions. Although this global arrangement allows insights into chromatin folding principles, at this point it is not yet clear whether spatial proximity of two loci necessarily means that there is a functional relationship.
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New concepts Reciprocal imprinting in humans
Reciprocal imprinting of human GRB10 in placental trophoblast and brain: evolutionary conservation of reversed allelic expression Monk D, Arnaud P, Frost J, Hills FA, Stanier P, Feil R, Moore GE Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, London, UK
[email protected] Hum Mol Genet 2009;18:3066–3074 Background: Genomic imprinting may have evolved not only to regulate fetal growth and development, but also behaviour. The murine growth factor receptor-binding protein 10, Grb10 is a potent growth inhibitor. Its gene displays reciprocal imprinting in mice. Methods: To assess the biological relevance of this reciprocal pattern of imprinting, we explored its conservation in humans. Results: As in mice, human GRB10 gene was paternally expressed in brain. Maternal allele-specific expression was seen only in placental villous trophoblasts, an essential part of the placenta involved in nutrient transfer. All other fetal tissues tested showed equal expression from both alleles. As in the mouse, the maternal transcripts originate from several kilobases upstream of the imprinting control region (ICR) of the domain, from a promoter region at which we find no allelic chromatin differences. The brain-specific paternal expression from the ICR also shows mechanistic similarities with the mouse. This conserved CpG island is DNA-methylated on the maternal allele and is marked on the paternal allele by developmentally regulated bivalent chromatin, with the presence of both H3 lysine-4 and H3 lysine-27 methylation. Conclusions: The strong conservation of the opposite allelic expression in placenta versus brain supports the hypothesis that GRB10 imprinting evolved to mediate diverse roles in mammalian growth and behaviour.
In addition to methylation defects in the imprinted H19-IGF2 region, GRB10 has long been a candidate to explain further cases of the Silver-Russell syndrome (SRS) of low birth weight and postnatal growth failure. Indeed, as the authors point out maternal uniparental disomy (mUPD7) and small duplications of 7p12 encompassing GRB10 have been reported in ~10% of cases of SRS. However, contrary to the conflict model (that fathers’ genes favour large offspring size, and mothers’ genes favour small size), GRB10 shows biallelic expression in most tissues except brain, where this potent growth inhibitor is paternally, rather than maternally, expressed. This study now finds that GRB10 is indeed exclusively maternally expressed, and specifically in the placental cells responsible for nutrient transfer. Remarkably, as in the mouse model, in human brain GRB10 shows the opposite pattern of exclusive paternal expression. It has been suggested that imprinting evolved to allow the expression of parent-specific considerations regarding the balance of fetal survival to cost or risk to the parent. Hence promoters of fetal growth tend to be paternally expressed, while growth suppressors are maternally expressed [7]. The authors suggest that this more complex model of imprinting of GRB10 might have evolved to allow multiple actions of GRB10 to be manifested. Hence, overexpression of maternally derived GRB10 in trophoblast would limit fetal growth, resulting in IUGR, whereas a lack of paternally derived growthenhancing GRB10 in brain could explain the weaning phenotype associated with poor suckling. They further suggest that chromatin defects at the GRB10 DMR leading to lack of paternal GRB10 expression in brain could potentially result in other cases of non-syndromic postnatal growth restriction.
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Food for thought Genetic adaptation to altitude
Genetic evidence for high-altitude adaptation in Tibet Simonson TS, Yang Y, Huff CD, Yun H, Qin G, Witherspoon DJ, Bai Z, Lorenzo FR, Xing J, Jorde LB, Prchal JT, Ge R Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA Science 2010; Epub ahead of print Background: Tibetans have lived at very high altitudes for thousands of years, and they have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. These phenotypes are clearly the result of adaptation to this environment, but their genetic basis remains unknown. Methods: 31 unrelated Tibetans were genotyped for 1 million single nucleotide polymorphisms (SNPs) using the Affymetrix Genome-Wide Human SNP 6.0 Array. To pinpoint loci under positive selection, the cross-population extended haplotype homozygosity (XP-EHH) statistic was used to compare highland Tibetans to the combined HapMap Chinese (CHB) and Japanese (JPT) lowland populations. Results: There was evidence for positive selection in several regions that contain genes whose products are likely involved in high-altitude adaptation. Positively selected haplotypes of EGLN1 and PPARA were significantly associated with the decreased hemoglobin phenotype that is unique to this highland population. Conclusions: Identification of these genes provides support for previously hypothesized mechanisms of high-altitude adaptation and illuminates the complexity of hypoxia response pathways in humans.
There are yet few specific examples of genetic adaptation. Genetic variants that confer lactase persistence are thought to have evolved with the expansion of agricultural societies, while G6PD deficiency, ␣-thalassaemia, and hemoglobin C persist by protecting against malaria mortality. Since the beginning of the Himalayan climbing era, the anecdotal extraordinary physical performance at high altitude of Sherpas has intrigued climbers. These authors describe that the Tibetan highlands are one of the most extreme environments inhabited by humans. In contrast to other nonadapted populations who increase haemoglobin levels and maintain oxygen saturation in response to hypoxia (with often adverse consequences), high-altitude Tibetans show profound arterial hypoxia yet they maintain normal aerobic metabolism. This is purportedly due to more efficient oxygen transport systems, however the specific mechanisms and the genetic basis have been elusive. By comparison of genome-wide SNP frequencies in Tibetans to those in neighbouring lowland populations, this study identified evidence for evolutionary selection of several genes with a priori functional roles in hypoxia response. Remarkably, each additional copy of an advantageous haplotype at either EGLN1 (a suppressor of HIF target genes and recently implicated in growth plate processes) or PPARA (known to play a role in lipid metabolism) was associated with a massive 1.7 g/dl reduction in haemoglobin concentration, which is consistent with the previously suggested mechanism of adaptation. Further studies of these genes may lead to new approaches for the treatment of hypoxia-related diseases.
New paradigms The other genomes we carry
The cancer genome Stratton MR, Campbell PJ, Futreal PA Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
[email protected] Nature 2009;458:719–724 Background: All cancers arise as a result of changes that have occurred in the DNA sequence of the genomes of cancer cells. Over the past quarter of a century much has been learnt about these mutations
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and the abnormal genes that operate in human cancers. We are now, however, moving into an era in which it will be possible to obtain the complete DNA sequence of large numbers of cancer genomes. Methods: This paper reviews the history and recent rapid progress in this field and gives comments on its future directions. Results: Approximately 100,000 somatic mutations from cancer genomes have been reported in the quarter of a century since the first somatic mutation was found in HRAS. At least 350 (1.6%) of the 22,000 protein-coding genes in the human genome have been reported to show recurrent somatic mutations in cancer with strong evidence that these contribute to cancer development. Over the next few years several hundred million more will be revealed by large-scale, complete sequencing of cancer genomes. Conclusions: These studies will provide us with a detailed and comprehensive perspective on how individual cancers have developed and will provide new directions for the treatment of cancer. In addition to our ‘own’ germ-line genome (i.e. that inherited from our parents), there is increasing evidence of the potential disease impacts of the ‘other genomes’ that we carry, ranging from the microbiomes in body cavities to somatic genomes in our cells. Intricate DNA repair mechanisms usually reduce the impact of somatic mutations, and germ-line mutations in these housekeeping genes explain the heritability of cancer susceptibility. One of the most famous somatic genetic disruptions (i.e. acquired post-meiosis) in cancer etiology is the ‘Philadelphia translocation’ between chromosomes 9 and 22 in chronic myeloid leukaemia. These authors now describe that with the recent advent of whole-genome sequencing of DNA from cancer cells it is now recognised that cancer genomes may carry between 1,000 to 100,000 mutations. These somatic mutations range from single basepair substitutions; insertions or deletions of small or large segments of DNA; DNA rearrangements, and copy number increases and reductions. Furthermore, cancer genomes may have incorporated exogenous viral DNA, and will also have acquired epigenetic changes. Somatic mutations may be classified into two groups: ‘driver’ mutations that confer a growth advantage on the cancer cell, and ‘passenger’ mutations without any growth advantage. Recent studies indicate that there may be as many as 20 driver mutations in individual cancers, considerably more than the 5–7 previously predicted. The authors describe that these studies will generate new insights into specific disease phenotypes, detection and monitoring, prognosis, drug response and chemotherapy resistance. References 1. Bonnefond A, Vaxillaire M, Labrune Y, Lecoeur C, Chevre JC, Bouatia-Naji N, Cauchi S, Balkau B, Marre M, Tichet J, Riveline JP, Hadjadj S, Gallois Y, Czernichow S, Hercberg S, Kaakinen M, Wiesner S, Charpentier G, Levy-Marchal C, Elliott P, Jarvelin MR, Horber F, Dina C, Pedersen O, Sladek R, Meyre D, Froguel P: Genetic variant in HK1 is associated with a proanemic state and A1C but not other glycemic control-related traits. Diabetes 2009;58:2687–2697. 2. Barrett JC, Clayton DG, Concannon P, Akolkar B, Cooper JD, Erlich HA, Julier C, Morahan G, Nerup J, Nierras C, Plagnol V, Pociot F, Schuilenburg H, Smyth DJ, Stevens H, Todd JA, Walker NM, Rich SS: Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nat Gen 2009;41:703–707. 3. Okada Y, Kamatani Y, Takahashi A, Matsuda K, Hosono N, Ohmiya H, Daigo Y, Yamamoto K, Kubo M, Nakamura Y, Kamatani N: A genome-wide association study in 19 633 Japanese subjects identified LHX3-QSOX2 and IGF-1 as adult height loci. Hum Mol Genet 2010;19:2303–2312. 4. Weedon MN, Lango H, Lindgren CM, Wallace C, Evans DM, Mangino M, Freathy RM, Perry JR, Stevens S, Hall AS, Samani NJ, Shields B, Prokopenko I, Farrall M, Dominiczak A, Johnson T, Bergmann S, Beckmann JS, Vollenweider P, Waterworth DM, Mooser V, Palmer CN, Morris AD, Ouwehand WH, Zhao JH, Li S, Loos RJ, Barroso I, Deloukas P, Sandhu MS, Wheeler E, Soranzo N, Inouye M, Wareham NJ, Caulfield M, Munroe PB, Hattersley AT, McCarthy MI, Frayling TM: Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet 2008;40:575– 583. 5. Loos RJ, Lindgren CM, Li S, Wheeler E, Zhao JH, Prokopenko I, Inouye M, Freathy RM, Attwood AP, Beckmann JS, Berndt SI, Jacobs KB, Chanock SJ, Hayes RB, Bergmann S, Bennett AJ, Bingham SA, Bochud M, Brown M, Cauchi S, Connell JM, Cooper C, Smith GD, Day I, Dina C, De S, Dermitzakis ET, Doney AS, Elliott KS, Elliott P, Evans DM, Sadaf Farooqi I, Froguel P, Ghori J, Groves CJ, Gwilliam R, Hadley D, Hall AS, Hattersley AT, Hebebrand J, Heid IM, Lamina C, Gieger C, Illig T, Meitinger T, Wichmann HE, Herrera B, Hinney A, Hunt SE, Jarvelin MR, Johnson T, Jolley JD, Karpe F, Keniry A, Khaw KT, Luben RN, Mangino M, Marchini J, McArdle WL, McGinnis R, Meyre D, Munroe PB, Morris AD, Ness AR, Neville MJ, Nica AC, Ong KK, O’Rahilly S, Owen KR, Palmer CN, Papadakis K, Potter S, Pouta A, Qi L, Kraft P, Hankinson SE, Hunter DJ, Hu FB, Randall JC, Rayner NW, Ring SM, Sandhu MS, Scherag A, Sims MA, Song K, Soranzo N, Speliotes EK, Lyon HN, Voight BF, Ridderstrale M, Groop L, Syddall HE, Teichmann SA, Timpson NJ, Tobias JH, Uda M, Scheet P, Sanna S, Abecasis GR, Albai G, Nagaraja R, Schlessinger D, Ganz Vogel CI, Wallace C, Waterworth DM, Weedon MN, Willer CJ, Jackson AU, Tuomilehto J, Collins FS, Boehnke M, Mohlke KL, Wraight VL, Yuan X, Zeggini E, Hirschhorn JN, Strachan DP, Ouwehand WH, Caulfield MJ, Samani NJ, Frayling TM,
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Vollenweider P, Waeber G, Mooser V, Deloukas P, McCarthy MI, Wareham NJ, Barroso I: Common variants near MC4R are associated with fat mass, weight and risk of obesity. Nat Genet 2008;40:768–775 6. McPherson R: Chromosome 9p21 and coronary artery disease. N Engl J Med 2010;362:1736–1737 7. Haig D: Altercation of generations: genetic conflicts of pregnancy. Am J Reprod Immunol 1996;35:226–232
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Evidence-Based Medicine in Pediatric Endocrinology Gary E. Butler University College London Hospital and UCL Institute of Child Health, UK
This year’s evidence-based medicine chapter is different yet again in flavor from last year [1]. Together with large population studies and more RCTs of long-term GH treatment reporting at final height, we have important new evidence in the treatment of preterm infants with thyroxine, definite evidence for the efficacy of insulin pumps and school-based obesity interventions, and clear evidencebased guidance for our endocrine investigation protocols. We see the benefits of GH on the cardiovascular system in some groups of children, yet warnings about GH use in others. The selection was based on searching under the terms pediatric endocrinology and again under clinical trials. Over 2,000 journal articles were identified from which this selection has been made with the needs of you the clinician in mind.
Mechanism of the year Treatment of hypothyroidism in preterm infants
Phase 1 trial of four thyroid hormone regimens for transient hypothyroxinemia in neonates of <28 weeks’ gestation La Gamma EF, van Wassenaer AG, Ares S, Golombek SG, Kok JH, Quero J, Hong T, Rahbar MH, de Escobar GM, Fisher DA, Paneth N Department of Neonatal-Perinatal Medicine, Regional Neonatal Center, Maria Fareri Children’s Hospital at Westchester Medical Center, New York Medical College, Valhalla, N.Y., USA
[email protected] Pediatrics 2009;124:e258–268 Background: Transiently low levels of thyroid hormones have been shown to be associated with higher rates of cerebral palsy and cognitive impairment in approximately 50% of neonates born 24–28 weeks’ gestation. This study aimed to identify whether any of four thyroid hormone supplementation regimens could raise TT4 and FT4 levels and improve neurodevelopmental outcome without suppressing TSH (biochemical euthyroidism). Methods: Eligible subjects had gestational ages between 24 and 28 weeks, and were entered into a masked randomized trial <24 h of birth to one of six study arms (n = 20–27 per arm): placebo (vehicle: 5% dextrose), potassium iodide (30 µg/kg/day) and continuous or bolus daily infusions of either 4 or 8 µg/ kg/day of T4 for 42 days. 1 µg/kg/day of T3 was added during the first 14 postnatal days to the T4 and was infused with 1 mg/ml albumin to prevent adherence to plastic tubing. Results: FT4 levels were high in the first 7 days in all hormone-treated subjects, however only the treatment study limb which showed a significant elevation in all treatment periods was continuously 8 µg/kg/day (p < 0.002 vs. all other groups). TT4 remained elevated in the first 7 days in all hormone-treated subjects (p < 0.05 vs. placebo or iodine arms). After 14 days, both 8 µg/kg/day arms as well as the continuous 4 µg/kg/day arm produced a sustained elevation of the mean and median TT4, >7 µg/dl (90 nM/l; p < 0.002 vs. placebo). The lowest suppression of TSH occurred during the continuous infusion of 4 µg/kg/day T4 arm. Unexpectedly the duration of mechanical ventilation was significantly lower in the continuous 4 µg/kg/day T4 arm and in the bolus 8 µg/kg/day T4 arm (p < 0.05 vs. remaining arms). Retinopathy of prematurity was significantly lower in all the four thyroid hormone treatment arms compared with the combined placebo and iodine arms (p < 0.04). The incidence of necrotizing enterocolitis was higher in the combined 8 µg/kg/day arms (p < 0.05 vs. other arms).
Continuous supplement of low-dose thyroid hormone (4 µg/kg/day) for 42 days caused elevation of TT4 with only a modest suppression of TSH. This was associated with several improved clinical parameters suggesting that T4 was of benefit. Future trials will be needed to evaluate the long-term neurodevelopmental effects of such supplementation. Conclusion:
Because extremely preterm low-birthweight infants frequently suffer severe systemic illness and experience changing biological requirements during a period of critical brain development, using hormone values based either healthy fetal levels or levels observed in term neonates to determine thyroid status is probably inappropriate, and in any case does not give any guidance in the need for T4 replacement in the transient hypothyroidism of prematurity. The authors’ view is that biochemical euthyroidism is the minimum thyroid hormone threshold associated with a reduced risk of neonatal illness or better long-term neurodevelopment. This study was an elegantly designed masked randomized trial investigating two T4 dosage schedules in different delivery modes (bolus or continuous), with or without initial T3 co-supplementation and designed to avoid overtreatment which has also been reported as harmful. The goal for a minimum threshold for FT4 concentration was identified as 19 pmol/l, for TT4 concentration as 77 nmol/l, and for TT3 concentration as 0.8 nmol/l. Supplementation of the prohormone T4 with the active hormone T3 for the first 14 postnatal days raised the TT3 blood level but was depressed by TSH values to or below the assay’s level of detection, indicating overtreatment and suggesting that T3 supplementation is unnecessary. In addition, the 8% mortality rate in the two 4 µg/kg/day arms was half that of the placebo or iodine arms (16%) and also less than half that of higher dosage arms (22%). Furthermore, a statistically significant reduction in retinopathy of prematurity was found compared with the iodine/placebo arms. The authors describe this as intriguing, meriting future investigation, as thyroid hormone effects on vascular and retinal development in both animals and humans have been described. This trial accomplished the goal of elevating thyroid hormone blood levels in extremely premature neonates to exceed a predefined target threshold without completely suppressing TSH. This was most successfully achieved by using continuous 4 µg/kg/day T4 as replacement therapy.
New paradigms Patches versus pills
Conjugated oral versus transdermal estrogen replacement in girls with Turner syndrome: a pilot comparative study Nabhan ZM, Dimeglio LA, Qi R, Perkins SM, Eugster EA Section of Pediatric Endocrinology/Diabetology, Indiana University School of Medicine, Riley Hospital for Children, Indianapolis, Ind., USA
[email protected] J Clin Endocrinol Metab 2009;94:2009–2014 Background: This study aimed to compare conjugated oral versus transdermal estrogen (TD E2) on bone accrual, uterine growth, pubertal development, IGF-1, and lipids in Turner syndrome (TS) as the optimal route of estrogen delivery is unknown. Methods: Twelve prepubertal GH-treated girls aged 14.0 ± 1.7 years were randomized to conjugated oral estrogen or TD E2. The study protocol evaluated changes in dual-emission x-ray absorptiometry, pelvic ultrasound, Tanner staging, growth velocity, IGF-1, and lipid profile at 6 monthly intervals over 1 year. Results: TD E2 therapy resulted in a significantly greater change in spine bone accrual parameters at 12 months compared with conjugated oral estrogen (bone mineral content 9.0 ± 0.9 vs. 5.8 ± 0.9 g, p = 0.04; bone mineral density 0.12 ± 0.01 vs. 0.06 ± 0.01 g/cm2, p = 0.004; Z-score 0.7 ± 0.1 vs. 0.3 ± 0.1, p = 0.03). TD E2 also produced better uterine growth than the oral group at 1 year (length 4.13 ± 0.39 vs. 1.98 ± 0.39 cm, p = 0.003; volume 22.2 ± 4.4 vs. 4.0 ± 4.4 ml, p = 0.02). After 12 months, 66% of subjects in the TD group had a mature uterus compared with 0% in the oral group. No other significant differences were seen.
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In girls with TS, this pilot study showed that TD E2 resulted in faster bone accrual in the spine and increased uterine growth compared with conjugated oral estrogen. This preliminary information for optimizing estrogen replacement in this population may inform further large-scale, long-term studies.
Conclusion:
Still much controversy surrounds the choice of estrogen preparation to induce puberty in girls with TS and by inference in chromosomally normal hypogonadal girls. The authors acknowledge that this is a small-scale pilot study, but the reason for inclusion here is that it is the first prospective randomized controlled trial comparing conjugated oral versus TD E2 for pubertal induction to be performed. Conjugated oral estrogen was administered at a dose of 0.3 mg every day for the first 6 months followed by 0.3 mg alternating with 0.625 mg every day for the second 6 months, whereas the TD E2 group was treated with a 0.025-mg patch twice a week for 6 months followed by a 0.0375-mg patch twice a week for the second 6 months. These doses were chosen based on published equivalences. The significant apparent benefit of TD E2 on more rapid uterine maturation and bone accrual could be as a result of incorrect assumptions as to the bioequivalent of each estrogen preparation, but no differences in height velocity, IGF-1, lipid parameters or degree of breast development attained between the two groups were reported. It is recognized that synthetic estrogens (ethinyl estradiol) are also frequently used for the induction of puberty in TS, the authors discuss publications which cite a lack of evidence for a difference on bone mineralization between the different oral preparations, but that TD E2 had improved bone mineral density in young adult patients. This pilot study provides a good evidence base and platform for larger scale research programs to confirm that TD E2 appears to be a significantly better modality for estrogen replacement in TS and potentially other hypogonadal girls.
New hope Do we now have the evidence base for insulin pumps?
Continuous subcutaneous insulin infusion (CSII) versus multiple insulin injections for type 1 diabetes mellitus Misso ML, Egberts KJ, Page M, O’Connor D, Shaw J Australasian Cochrane Centre, Monash Institute of Health Services Research, Monash University, Clayton, Vic., Australia Cochrane Database Syst Rev 2010;CD005103 Background: This review aimed to assess the effects of continuous subcutaneous insulin infusion (CSII) compared to multiple insulin injections (MI) in people with type 1 diabetes mellitus. The onset of type 1 diabetes mellitus may occur at any age and it is one of the commonest chronic diseases of childhood and adolescence. Since there are currently no known interventions to prevent onset, it is vital that effective treatment regimes are available. Methods: Electronic searches were made of The Cochrane Library, MEDLINE, EMBASE and CINAHL. Studies were included if they were randomized controlled trials comparing CSII with three or more insulin injections per day (MI) in people with type 1 diabetes mellitus. They were subsequently assessed by two authors who independently evaluated the risk of bias and extracted characteristics of selected studies. Study investigators were contacted to obtain missing information. Generic inverse variance meta-analyses using a random-effects model were performed. Results: 23 studies randomized 976 participants with type 1 diabetes to either intervention. There was a statistically significant difference in glycosylated hemoglobin A1c (HbA1c) favoring CSII (weighted mean difference –0.3% (95% CI interval –0.1 to –0.4). There were no obvious differences between the interventions for non-severe hypoglycemia, but severe hypoglycemia appeared to be reduced in those using CSII. Quality of life measures suggest that CSII is preferred over MI. No significant difference was found for weight. Adverse events were not well reported, no information is available on mortality, morbidity and costs.
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There is some evidence to suggest that CSII may be better than MI for glycemic control in people with type 1 diabetes. Non-severe hypoglycemic events do not appear to be reduced with CSII. There is insufficient evidence regarding adverse events, mortality, morbidity and costs.
Conclusion:
The Health Technology Assessment report and the National Institute for Clinical Excellence (NICE) guidelines on the use of CSII are methodologically rigorous, however the most up-to-date search strategies were conducted in 2007 and therefore do not include recent randomized controlled trials [2]. Given the limitations of these previous systematic reviews, this review has considered these factors and used specific methodology and criteria outlined by the Cochrane Collaboration in their intent to present a comprehensive systematic review to assess the effects of CSII compared to MI. Meta-analyses were performed where possible and feasible. There may be benefit in using CSII over MI for improving glycaemic control and improving health-related quality of life in people with type 1 diabetes. Non-severe hypoglycemic events do not appear to be different between either treatment methodology. It is important to note that there is insufficient evidence regarding adverse events, reductions of diabetes late complications, mortality and cost which are all vital in deciding which treatment regimen to choose. Until evidence is available for these outcomes, firm recommendations as to whether CSII is superior in the management of people with type 1 diabetes are not possible. Long-term, large-scale and methodologically rigorous studies are needed to determine the effect of CSII and MI on outcomes such as hypoglycemia, mortality, diabetes late complications and other adverse effects, using validated scales. Cost-effectiveness data are also needed to support choice and for healthcare budgeting purposes.
New concerns
Increased risk of thyroid pathology in patients with thyroid hemiagenesis: results of a large cohort case-control study Ruchala M, Szczepanek E, Szaflarski W, Moczko J, Czarnywojtek A, Pietz L, Nowicki M, Niedziela M, Zabel M, Kohrle J, Sowinski J Department of Endocrinology, Metabolism and Internal Medicine, University of Medical Sciences, Poznan, Poland
[email protected] Eur J Endocrinol 2010;162:153–160 Background: Thyroid hemiagenesis (THA) is an anomaly resulting from the developmental failure of one lobe of the thyroid. The etiology, clinical significance, and management of patients in whom THA is identified are still unclear. The aim of the study is to provide the first controlled systematic analysis of 40 patients with THA in comparison with 80 subjects with a fully developed thyroid gland out of 2,159 participants from a cross-sectional population-based thyroid screening program. Methods: Serum TSH, free thyroxine (FT4), free triiodothyronine (FT3), and thyroid autoantibodies were measured in all subjects. In 37 of the THA patients, thyroid ultrasonography and a 99mTc thyroid scintiscan were performed, followed by fine-needle aspiration biopsy if indicated. The remaining archival 3 cases were diagnosed with the use of 131I scintiscan pre- and post-TSH stimulation. Results: In this study, a considerable prevalence of women with THA was noted, with 7:1 female-to-male ratio. Of the 40 patients, 35 had left-sided agenesis. Patients with THA were usually clinically euthyroid but levels of TSH and FT3 were significantly higher, with a higher FT3/FT4 ratio in comparison with the controls. Furthermore, a higher incidence of functional, morphological, and autoimmune thyroid disorders was seen in THA patients compared controls (p < 0.05). Conclusion: This study showed that individuals with THA are more likely to develop thyroid pathology. This may be as a result of the long-lasting TSH overstimulation. The diagnosis of THA should be followed by systematic observation and adequate levothyroxine treatment in patients with elevated TSH level.
This paper provides a long-term outlook in patients with a rare abnormality of the thyroid gland. The prevalence of THA is estimated to be 0.2–0.025%. Although conducted in a moderately iodine-
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deficient area of Poland, this study was of a prospective design with an age- and sex-matched control group. In 17/40 the anomaly was detected by screening or incidentally, all controls being recruited from the screening program. The study looked at patients between 12 and 79 years of age, so, from the pediatric perspective, is able to give us an idea of long-term prognosis if THA is identified, as in this study via a population screening program. However, the younger patients (<25 years) were less likely to have compensatory hypertrophy of the residual thyroid lobe and a lower incidence of autoimmune disease compatible with the congenital etiology. Older patients were more likely to have autoimmune disease and compensatory hypertrophy. THA was more frequently associated with hyperthyroidism and higher TSH levels and thus indicating levothyroxine replacement. In the 22 patients who had fine-needle aspiration biopsy of suspicious lesions on ultrasound scan, no malignancies were found, even though 6 proceeded to surgery due to uncertainties on the initial biopsy or goitre size. In the absence of a clear genetic cause for THA, even in familial cases ongoing follow-up is indicated in these patients. The debate also continues as to the upper normal level for plasma TSH and whether earlier levothyroxine substitution may alter outcome in some patients with this condition.
Concepts revised The age of adolescence
Recent decline in age at breast development: the Copenhagen Puberty Study Aksglaede L, Sorensen K, Petersen JH, Skakkebaek NE, Juul A Rigshospitalet, Department of Growth and Reproduction, Copenhagen, Denmark
[email protected] Pediatrics 2009;123:e932–939 Background: Data published showing an earlier onset of breast development in American girls, NHANES III, has been controversial. However, secular trend analyses are often limited by poor comparability between studies over different time periods and counties. This study presents new European data systematically collected from the same region and by the same research group at the beginning over a 15-year period up to the present. Methods: 2,095 girls aged 5.6–20.0 years were studied in 1991–1993 (1991 cohort; n = 1,100) and 2006–2008 (2006 cohort; n = 995). Puberty was staged by palpation of glandular breast tissue, and measurement of height and weight, and blood sampling for estradiol, luteinizing hormone, and folliclestimulating hormone was done. Age at entering pubertal breast stages 2–5, pubic hair stages 2–5, and menarche was documented for the two cohorts. Results: The onset of puberty (Tanner breast stage 2+) occurred significantly earlier in the 2006 cohort (estimated mean age 9.86 years) compared with the 1991 cohort (estimated mean age 10.88 years). This difference remained significant after adjustment for BMI, thus excluding obesity as a variable. Estimated ages at menarche were 13.42 and 13.13 years in the 1991 and 2006 cohorts, respectively. Serum FSH and LH did not differ between the two cohorts at any age interval. However, estradiol levels were significantly lower in 8- to 10-year-old girls from the 2006 cohort in comparison with girls of the same age from the 1991 cohort. Conclusion: This study demonstrated significantly earlier breast development but less of a reduction in the age at menarche among a recent population of girls compared with a similar one 15 years earlier. Variation in reproductive hormones and in BMI did not explain these marked changes. Other factors yet to be identified may be involved.
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Recent changes in pubertal timing in healthy Danish boys: associations with body mass index Sorensen K, Aksglaede L, Petersen JH, Juul A Department of Growth and Reproduction, Copenhagen University Hospital, Copenhagen, Denmark
[email protected] J Clin Endocrinol Metab 2010;95:263–270 Background: The American population-based study, NHANES III, identified possible secular trends in male puberty in the 1990s. However, no conclusions could be made about the age of the onset of puberty due to the lack of comparable data. This study aimed to evaluate secular trends in pubertal onset during the recent 15 years and their relation to body mass index (BMI) in boys. Methods: This was a cross-sectional study in 1991–1993 and a combined cross-sectional and longitudinal study in 2006–2008 (The Copenhagen Puberty Study). 1,528 boys aged 5.8–19.9 years were studied in 1991–1993 (1991 cohort; n = 824) and 2006–2008 (2006 cohort; n = 704) at a tertiary centre for pediatric endocrinology. Genital and pubic hair stages as well as testicular volume using an orchidometer were evaluated. Blood samples were analyzed for LH, FSH, testosterone, and SHBG. Results: The mean age of the onset of puberty (attainment of testicular volume >3 ml) of 11.66 years (95% CO 11.49–11.82) occurred significantly earlier in 2006–2008 than in the 1991–1993 cohort, 11.92 years (11.76–12.08); p = 0.025. Significantly higher LH levels were found in the 11- to 16-yearold boys from 2006–2008 compared with 1991–1993 (p = 0.020), but this was not found with testosterone. The BMI Z-score increased significantly from 0.044 (–0.016 to 0.104) in 1991–1993 to 0.290 (0.219–0.361) in 2006–2008; p < 0.001. Interestingly, pubertal onset and LH levels did not differ significantly between study periods after adjustment for BMI. Conclusion: Estimated mean age at the onset of puberty has declined significantly over the past 15 years. This decline was associated with the coincident increase in BMI.
Knowledge about the timing of sexual development in children and adolescents and the causes and consequences is fundamentally important not only for the practice of medicine but in society as well. Although a series of publications from the USA and other European countries had suggested a lowering of the age of the onset of puberty, there is a consistent finding that the tempo or transit time through the pubertal process is not necessarily accelerating at the same pace [3]. This is evident in this study at least in girls with a reduction in the age at menarche by only 0.3 years whereas age at B2 is 1 year earlier. There are different tempo patterns emerging between boys and girls too. The age of B2 is shifted to the left across the whole age spectrum, whereas earlier pubertal development in boys is limited to those beginning before age 12.5 years. Thus the latter 20% of boys entering puberty do not seem to show this secular trend. Contrary to popular myth, obesity does not appear to be implicated in these trends, the BMI effect cancels itself out in girls when controlled for, and the early developers in boys are not the ones showing a rise in BMI; paradoxically, boys going into puberty later are the ones where BMI rises, and there is no secular trend here as discussed above. The relationship between reproductive hormones and clinical pubertal changes is important to mention as the earlier physical changes were not matched by rises in gonadotrophins or sex hormones. Paradoxically, testosterone and estradiol levels were lower at maturity in the recent compared with the historical cohorts. Clearly there is much need for further exploration and research, but the value of this study is its carefully designed methodology and attempt to find the evidence base upon which we can improve our knowledge of childhood and develop our clinical practice. The picture is clearly more complicated than was expected.
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Important for clinical practice Growth in our knowledge of tests
The glucagon test in the diagnosis of growth hormone deficiency in children with short stature younger than 6 years Secco A, di Iorgi N, Napoli F, Calandra E, Ghezzi M, Frassinetti C, Parodi S, Casini MR, Lorini R, Loche S, Maghnie M Department of Pediatrics, Istituto di Ricovero e Cura a Carattere Scientifico G Gaslini Institute, University of Genova, Genova, Italy J Clin Endocrinol Metab 2009;94:4251–4257 Background: The aim of this study, conducted in two pediatric endocrinology centers, was to investigate the diagnostic value of the glucagon test as an alternative to insulin tolerance test (ITT) and arginine test in children younger than 6 years with GHD as few studies have addressed the diagnostic role of the glucagon test in children when GH deficiency (GHD) is suspected. Methods: 48 children (median age 4.2 years and median height –3.0 SD score) with GHD confirmed by a peak GH to ITT and arginine <10 and (median 4.7 and 3.4 µg/l respectively) subsequently underwent a glucagon stimulation test. Magnetic resonance imaging had showed normal hypothalamic-pituitary anatomy in 24 children, isolated anterior pituitary hypoplasia in 7, and structural hypothalamic-pituitary abnormalities in 17. Results: The median GH peak response to glucagon (13.5 µg/l) was significantly higher than that observed after ITT and arginine (p < 0.0001). 20 subjects (group 1) had a GH peak after glucagon which was <10 µg/l and 28 subjects (group 2) had peak GH levels of >10 µg/l without significant clinical or biochemical differences between the two groups. Median GH peak after glucagon was similar between patients with multiple pituitary hormone deficiency and those with isolated GHD and between subjects with and without structural hypothalamic-pituitary abnormalities. There was a negative correlation between age at diagnosis and the magnitude of the GH peak after glucagon (r = –0.636, p < 0.0001). Conclusion: Glucagon can effectively release GH and can be used to evaluate somatotroph function in young children with short stature. However, the differing results between GH levels and those following ITT and arginine in this study suggest that normative data for this test in young children need to be established.
Reassessment of the growth hormone status in young adults with childhood-onset growth hormone deficiency: reappraisal of insulin tolerance testing Secco A, di Iorgi N, Napoli F, Calandra E, Calcagno A, Ghezzi M, Frassinetti C, Fratangeli N, Parodi S, Benassai M, Leitner Y, Gastaldi R, Lorini R, Maghnie M, Radetti G Department of Pediatrics, Instituto di Ricovero e Cura a Carattere Scientifico, Giannina Gaslini Institute, University of Genova, Genova, Italy J Clin Endocrinol Metab 2009;94:4195–4204 Background: The aim of this study, performed in two pediatric endocrinology centers, was to evaluate the accuracy of the insulin tolerance test (ITT), mean 12-h spontaneous nocturnal GH (SNGH), and IGF-1 in the diagnosis of permanent GH deficiency (GHD) in young adults with childhood-onset GHD. The 2007 Consensus Statement had suggested a peak GH to the ITT of <6 µg/l as being the diagnostic cutoff for permanent GHD, although further validation was recommended. Methods: ITT, 12-h SNGH, and IGF-1 were evaluated as single or combined tests in 79 subjects with childhood-onset GHD (median age, 18.0 years). 48 of the subjects had isolated GHD or one additional pituitary defect and normal MRI or anterior pituitary hypoplasia (low likelihood GHD group), and 31 subjects had structural hypothalamic-pituitary abnormalities or multiple pituitary hormone deficiencies (high likelihood GHD). Diagnostic criteria based on the cut-offs identified by ROC analysis were combined, both in series (i.e. considering as positive a subject who was positive on each test) and in parallel (i.e. considering as positive a subject who was positive on at least one test). Results: Receiver operating characteristic curve analysis showed the most accurate parameters for the diagnosis of GHD was a peak GH in the ITT of 5.6 µg/l or lower (sensitivity 77%; specificity 94%; area under the curve (AUC) = 0.92), mean 12-h SNGH of 1.20 µg/l or lower (sensitivity 90%; specificity
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90%; AUC = 0.93), and IGF-1 of –2.8 SD score or lower (sensitivity 81%; specificity 96%; AUC = 0.93). Seven patients in the high likelihood GHD group showed a peak GH to ITT >5.6 µg/l, but a median IGF-1 that was significantly lower than that of group low likelihood GHD (–3.3 vs. –0.7 SD score; p = 0.0001). Peak GH to ITT of 3.6 µg/l or lower and to arginine of 3.1 µg/l or lower at childhood diagnosis can predict a future permanent GHD condition. Conclusion: Using a cut-off point of a peak GH to ITT <5.6 µg/l performs well as an accurate diagnostic strategy for determining persistent GHD in young adults with childhood-onset GHD. In addition, IGF-1 is a reliable marker providing information about the severity of GHD. Careful follow-up is needed for subjects who have discordant ITT and IGF-I results. Despite decades of reliance on GH stimulation tests, their use is highly problematic as discussed in the past and pointed out in this pair of papers [4]. Among the various GH stimulation tests, the glucagon test has been poorly studied in the pediatric population. In a systematic study investigating patients already confirmed as GHD on two other stimulation tests, there was a surprise finding of the GH peak after glucagon being >10 µg/l in 28 of 48 patients (58.3%), 15 (35.4%) of whom had abnormal anatomy on MRI scanning and/or MPHD. Therefore, by standard criteria, 28 of the patients would have been diagnosed as normal. In this study, the mean GH response to glucagon was higher than that observed after ITT or arginine. This could suggest that the GH-releasing effect of glucagon in young children with congenital GHD is greater than that of ITT and arginine, and thus, the same cut-off levels cannot be adopted. There was also an association between a very low IGF-1 concentration with a GH peak of <10 µg/l after both ITT and arginine. The authors caution that GH responses to glucagon may give false-negative results when the threshold of normal of 10 or 20 µg/l is used. In the absence of normative data for GH responses to pharmacological stimuli in this age group, this reinforces the caution to exert in the interpretation of GH stimulation tests. In contrast, data on re-evaluation of young adults with childhood-onset GHD provides the evidence for a good diagnostic performance for a peak GH cut-off level of <5.6 µg/l in the diagnosis of GHD. The presence of anatomical abnormalities on MRI, together with two or more pituitary hormone deficiencies, was taken as the basis for the prediction of diagnostic accuracy. ROC curve analysis indicated the best diagnostic accuracy for a peak GH to ITT of 5.6 µg/l with an AUC of 0.92, demonstrating that ITT is a reliable test for evaluation of GH secretion and confirming that the GH peak adopted by the 2007 Consensus is adequate for the definition of permanent GHD in young adults [5]. Measurement of IGF-I provides useful information in direct parallel to the degree of GHD. ROC analysis of IGF-1 showed the best diagnostic accuracy for an IGF-1 cut-off of –2.8 SDS with 96% specificity. Specificity of GH peak and IGF-1 levels are similar, suggesting that a single IGF-1 evaluation may have a role in the definition of GH status, but that normal IGF-1 concentrations do not exclude GHD in approximately 20% of young adult patients. Overnight measurement of spontaneous GH secretion was not determined to be cost-effective, either as a diagnostic test or for economic reasons. The authors did not find that clinical parameters at the time of initial diagnosis during childhood played any role in the prediction of GH status after adult height attainment, whereas ROC analysis applied to GH peaks obtained during childhood showed that a peak GH of about 3 µg/l after at least one stimulation test offered good accuracy for the discrimination of patients with a high likelihood of GHD in adult life from those who might subsequently normalize GH secretion. However, this predictive accuracy could be overestimated because it was based on observations of a cut-off point derived from retrospective data. One limitation of these analyses is that GH assay used in many previous studies is now obsolete.
Precocious pubarche: distinguishing late-onset congenital adrenal hyperplasia from premature adrenarche Armengaud JB, Charkaluk ML, Trivin C, Tardy V, Breart G, Brauner R, Chalumeau M Université Paris Descartes, Paris, France J Clin Endocrinol Metab 2009;94:2835–2840 Background: In 5–20% of children presenting with precocious pubarche (PP), late-onset congenital adrenal hyperplasia (LO-CAH) is found and an adrenal stimulation test is recommended in the diagnostic schedule, but the results are normal in more than 80% of cases and it is stressful and expensive, so the aim was to identify clinical and plasma predictors of LO-CAH among patients presenting with PP.
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Methods: This was a retrospective cohort study that included all patients seen for PP between 1999 and 2006 (n = 238). All underwent an ACTH test. LO-CAH was diagnosed by a post-ACTH 17-hydroxyprogesterone (17-OHP) plasma level >10 ng/ml (30 nmol/l) and confirmed by mutational analysis of the CYP21 gene. Results: No statistically significant difference was observed between the distributions of the age at pubic hair onset, weight, height, and pubic hair Tanner stage at the time of the first medical examination in patient with LO-CAH and PA. Basal levels of 17-OHP, ⌬4-androstenedione, and testosterone plasma levels were significantly higher in the 10 of 238 (4%) patients who had LO-CAH. A 2 ng/ml (6 nmol/l) threshold for basal 17-OHP plasma levels offered 100% (95% CI 69–100) sensitivity for the diagnosis of LO-CAH and 99% (95% CI 96–100) specificity. Conclusion: Although the three above factors were predictive of LO-CAH, 17-OHP was the most efficient. Applying a selective approach using a 2-ng/ml (6 nmol/l) basal 17-OHP plasma level cut-off would have safely avoided 99% of the unnecessary ACTH tests in this patient group.
Although a retrospective audit rather than primary research, this is the largest and most detailed study addressing this common diagnostic conundrum. The study examined clinical, basal and stimulated hormonal measures for their performance in their ability to predict LO-CAH. Presentation of clinical features such as age, sex, degree of pubic hair development did not show significant differences, although LO-CAH patients tended to have a higher BMI than those with PP. Basal 17-OHP was superior in its discriminating power (area under the ROC curve of 0.99), performing significantly better than androstenedione (0.90), testosterone (0.83) and the most discriminatory clinical parameter BMI (0.66) for comparison. Stimulated 17-OHP levels actually did not add further clarification of the diagnosis which was confirmed in all cases with mutations in the CYP21 gene. In this time of a global recession, we are increasingly being asked to look at efficiency measures in our daily clinical practice. This paper provides a good evidence base for a re-evaluation of practice. The diagnosis of LO-CAH can be made confidently with basal 17-OHP level, without the need for a stimulation test or additional hormonal analyses. If the study is confirmed on a higher number of affected individuals (only 10 here), this will allow savings in laboratory and drug costs and be better for the patient as hospital admission and a stimulation test can be avoided. One important point not discussed in the paper is the timing of basal 17-OHP measurement due to the circadian rhythm of 17-OHP secretion and risk of false negative result if blood is drawn later in the day.
Association of thyroid gland volume, serum insulin-like growth factor-I, and anthropometric variables in euthyroid prepubertal children Boas M, Hegedus L, Feldt-Rasmussen U, Skakkebaek NE, Hilsted L, Main KM University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark
[email protected] J Clin Endocrinol Metab 2009;94:4031–4035 Background: This study investigated thyroid gland volume and several hormonal and anthropometric variables in prepubertal children as previous studies had focused on the interrelation between thyroid size, anthropometric variables, and IGF-I in adults, but data such as this was lacking in children. Methods: 859 prepubertal euthyroid Danish children aged 4–9 years underwent a thorough clinical investigation, including anthropometrical measurements. Additionally, determination of TSH, thyroid hormones, autoantibodies, urinary iodine excretion was estimated, and thyroid volume (TV) measured by ultrasound. Longitudinal growth data from birth were available. Results: TV increased significantly with age (r = 0.487; p < 0.001). Mean TV ± SD for different age groups were as follows: 4 years, 2.2 ± 1.4 ml; 5 years, 2.5 ± 1.3 ml; 6 years, 2.8 ± 1.3 ml; 7 years, 3.2 ± 1.3 ml; 8 years, 3.5 ± 1.3 ml; 9 years, 3.7 ± 1.3 ml. We found a significant positive association between IGF-1 and TV (p < 0.001). Moreover, in multiple regression analyses, TV correlated significantly with TSH (p < 0.013), free T4 (p < 0.001), lean body mass (p < 0.001), and body surface area (p < 0.001) as well as other anthropometrical measurements. TV also showed a positive correlation with family history of thyroid disease and presence of incidental abnormal ultrasound findings (p = 0.025 and 0.022, respectively). Conclusion: In this study of prepubertal Danish children, the GH/IGF-1-axis was positively correlated with thyroid size, suggesting that it may have a role in the regulation of thyroid growth. Additionally, anthropometric measurements, especially body surface area, were the best predictors of TV.
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This large-scale population cohort study was able to define factors associated with thyroid gland growth, mainly by inference and comparison with other anthropometric variables and serum markers. Thyroid volume highly significantly correlated with age and anthropometric parameters: height, weight, BSA, BMI, and estimated lean body mass. Body size was strongly associated with age, but even when controlling for age by using SDS, all anthropometrical variables were positively associated with thyroid volume SDS. These positive correlations are reassuring and reassuring that in normal children the thyroid gland grows as the children themselves grow which predicates the maintenance of a euthyroid state. Of particular interest was that children with BMI >2 SD had significantly higher thyroid volume SDS, a similar finding in children exhibiting catch-up growth. This large treatise principally on normality and its definitions is a welcome addition to our knowledge and the literature.
Clinical trials of GH At the heart of the matter
Subtle alterations of cardiac performance in children with growth hormone deficiency: results of a two-year prospective, case-control study Capalbo D, Lo Vecchio A, Farina V, Spinelli L, Palladino A, Tiano C, Lettiero T, Lombardi G, Colao A, Salerno M Department of Pediatrics, Federico II University of Naples, Naples, Italy J Clin Endocrinol Metab 2009;94:3347–3355 Background: Children with GH deficiency (GHD) may have reduced left ventricular (LV) mass, this has not been documented as impairing cardiac function. The aim of this study was to evaluate effects of GHD and GH therapy on cardiac function using load-dependent and load-independent indices of myocardial contractility. Methods: Echocardiography was performed in 24 GHD children at baseline and at 1 and 2 years after GH therapy and in 24 aged-matched controls with familial short stature. Results: Compared with controls, GHD children had a had lower left ventricular mass at baseline (left ventricular mass/body surface area 50.6 ± 1.8 vs. 60.5 ± 2.4 g/m2; p < 0.002, and left ventricular mass/ height2 28.7 ± 1.2 vs. 33.6 ± 1.3 g/m2; p < 0.009). General systolic function was normal, with fractional shortening showing only a slight trend toward impairment (34.9 ± 1.5 vs. 37.6 ± 1.1%). However, subtle left ventricular dysfunction was revealed by load-dependent and load-independent indices of myocardial contractility. There was a lower rate-corrected mean velocity of circumferential fiber shortening in GHD patients compared with controls (1.0 ± 0.03 vs. 1.18 ± 0.03 circ/s; p = 0.0001) and stress shortening index (0.10 ± 0.02 vs. 0.18 ± 0.02; p < 0.007) and higher end-systolic stress (49.2 ± 1.4 vs. 45.7 ± 1.0 g/cm2; p < 0.05). There was a significant improvement of cardiac size after 1 year of GH treatment (left ventricular mass/body surface area 67.8 ± 2.9 g/m2; left ventricular mass/height2 38.2 ± 2.0 g/m2; p < 0.0001 and p = 0.0003, respectively) and in myocardial contractility (mean velocity of circumferential fiber shortening 1.2 ± 0.04 circ/s; p < 0.0002; stress shortening index 0.19 ± 0.02; p < 0.003) and reduced afterload (end-systolic stress 43.9 ± 1.4 g/cm2; p < 0.03). Conclusion: This study has demonstrated that GH deficiency is associated with abnormalities in morphology and function in not only adults but also in children and further supports the beneficial effect of GH on the heart.
This is the first study to document that children with GHD have mild alterations in myocardial contractility and impairment in cardiac performance. However, these abnormalities are likely of little actual clinical significance in childhood because general systolic function is within normal limits. This was a case-controlled study, and since children with GHD may have abnormal body composition, with a reduction in lean mass and an increase in body fat, the authors compared the patients with a group of controls who were younger but height and body surface area matched to better compare cardiac parameters. The results of the present study demonstrate that GH therapy is able to reverse all these subtle abnormalities. In addition to a positive effect on cardiac size, 1 year of GH treatment caused a significant decrease in wall stress and a consistent increase in cardiac contractility parameters. The findings are also in agreement with studies of cardiac function in adults with GHD showing similar
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effects but are of shorter duration in children. When atherosclerotic or thrombotic risk factors are considered, GHD was associated with higher serum homocysteine levels compared with controls. Two years of GH treatment significantly decreased homocysteine concentrations and improved lipid profiles with a decrease of total cholesterol and total to HDL cholesterol ratio, compared with both pretreatment and control values. These results suggest that children with untreated GHD may also have subtle abnormalities of their lipid profile that might place them to an increased atherosclerotic or thrombotic risk and on which GH seems to exert a beneficial effect. This study shows that children with severe GHD have a greater impairment of systolic function in addition to a more severe reduction of left ventricular mass when compared with children with partial GHD. However, as children with partial GHD also have a significant reduction in cardiac size compared with controls, myocardial contractility was only slightly impaired and afterload was not significantly increased. GH therapy normalizes cardiac size and function in both severe and partial GHD patients, and with the improvement in atherosclerotic parameters provides yet more evidence of the beneficial effect of GH treatment on the cardiovascular system, which should signal well for long-term health.
Cardiovascular and metabolic risk profile and acylation-stimulating protein levels in children with Prader-Willi syndrome and effects of growth hormone treatment de Lind van Wijngaarden RF, Cianflone K, Gao Y, Leunissen RW, Hokken-Koelega AC Dutch Growth Research Foundation, Rotterdam, The Netherlands
[email protected] J Clin Endocrinol Metab 2010;95:1758–1766 Background: The aim of this study was to investigate the metabolic and cardiovascular risk profile and acylation-stimulating protein (ASP) levels and to investigate the effects of GH treatment in children with Prader-Willi syndrome (PWS). ASP stimulates glucose uptake and triglyceride storage in adipose tissue. Methods: This was a randomized controlled GH trial. 85 children with PWS (mean ± SD age of 4.9 ± 3.0 years) participated in the study. Infants and prepubertal children received GH (1 mg/m2/day) or served as controls for 12 and 24 months, respectively. Percentage fat was measured (fat%) with dual-energy x-ray absorptiometry, together with blood pressure, fasting insulin and glucose levels, serum lipids, and ASP levels. Results: Mean ± SD fat% was 28.4 ± 6.2 in infants and 36.9 ± 8.5 in prepubertal children. 95% of prepubertal children had a fat% SD score (SDS) >2 SDS. At least one cardiovascular risk factor, defined as hypertension or dyslipidemia, was abnormal in 63% of infants and 73% of prepubertal children, with 5% of all children having the metabolic syndrome. Mean ± SD baseline ASP was 107 ± 45 nmol/l (normal <58 nmol/l) and this correlated with fat mass and TG levels, but GH treatment had no effect on mean ASP levels in this population. GH improved fat% SDS and the HDLc/LDLc ratio (p < 0.0001 and p = 0.04). Conclusion: A high proportion of children with PWS had dyslipidemia and high ASP levels. Fat% and high-density lipoprotein cholesterol/low-density lipoprotein cholesterol was improved with GH treatment, but no effect was seen on ASP. High ASP levels may prevent complete normalization of fat% SDS during GH treatment but may this effect may help to keep glucose and insulin levels within normal ranges.
This RCT of GH treatment confirms that young children with PWS have a high fat mass and dyslipidemia, whereas blood pressure and glucose homeostasis are normal in most patients with only 5% fulfilling criteria for the diagnosis of metabolic syndrome. GH-treated children showed significant improvement of height, BMI, and IGF-1 during 12 and 24 months of treatment compared with randomized controls. In addition, GH reduced fat mass and fat% and increased the HDL/LDL ratio. Acylation-stimulating protein (ASP) is produced by adipose tissue and stimulates free fatty acid incorporation into adipose tissue by increasing triglyceride synthesis and storage and by inhibiting hormone-sensitive lipase-mediated triglyceride lipolysis. ASP correlates positively with fat mass and negatively with triglyceride levels. Thus, ASP may contribute to maintaining a high fat%. The study demonstrated twofold higher ASP levels in young children with PWS, coinciding with an increased fat mass, but normal triglyceride levels. ASP levels were not altered by GH. GH treatment does not
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result in complete normalization of fat mass in children with PWS which suggests that GH insufficiency is not the only factor involved in the mechanism leading to abnormal body composition in PWS. This study provides further evidence for the metabolic benefits of GH treatment in this condition and helps to shed further light on the mechanism of fat accumulation.
New mechanisms Teaching fat-busting
School-based interventions on childhood obesity: a meta-analysis Gonzalez-Suarez C, Worley A, Grimmer-Somers K, Dones V Centre for Allied Health Evidence, University of South Australia, Adelaide, S.A., Australia
[email protected] Am J Prev Med 2009;37:418–427 Background: Childhood obesity has been recognized as an increasing health problem worldwide and predicts adulthood obesity, which in turn is strongly linked to chronic lifestyle diseases. This meta-analysis set out to evaluate the effectiveness of school-based programs in the prevention and management of childhood obesity. Methods: A comprehensive literature search for papers to be included for the meta-analysis was undertaken for RCTs and clinical controlled trials published between 1995 and 2007 on school-based interventions that addressed childhood obesity. They were restricted to those in which odds ratios (OR) or standardized mean differences and their 95% CIs were reported or could be calculated from available data. Results: The meta-analysis showed that the odds of these school-based intervention programs being significantly protective in the short term for overweight and obese participants was OR = 0.74 (95% CI 0.60, 0.92) in comparison with the control arm. Programs that were conducted for more than 1 year had a higher OR of decreasing the prevalence of obesity. However, interventions were not effective in decreasing BMI compared with control treatments, with a weighted mean difference of –0.62 (95% CI = –1.39, 0.14). Conclusion: This meta-analysis showed that in the short term, there was convincing evidence that schoolbased interventions can be effective in reducing the prevalence of childhood obesity, but not on reducing BMI. Longer-running programs were more effective than shorter ones.
Effect of school-based physical activity program (KISS) on fitness and adiposity in primary schoolchildren: cluster randomized controlled trial Kriemler S, Zahner L, Schindler C, Meyer U, Hartmann T, Hebestreit H, Brunner-La Rocca HP, van Mechelen W, Puder JJ Institute of Exercise and Health Sciences, University of Basel, Basel, Switzerland
[email protected] BMJ 2010;340:c2968 Background: This cluster randomized controlled trial aimed to assess the effectiveness of a school-based physical activity program over one school year on physical and psychological health in young schoolchildren. Methods: 28 classes from 15 elementary schools in Switzerland (540 children, of whom 502 consented and presented at baseline) were randomly selected and assigned in a 4:3 ratio to an intervention (n = 16) or control arm (n = 12) after stratification for grade (first and fifth grade), between August 2005 and June 2006. Children in the intervention arm (n = 297) received a multicomponent physical activity program that included structuring the three existing physical education lessons each week and adding two additional lessons a week, daily short activity breaks, and physical activity homework. Children (n = 205) and parents in the control group were not informed of an intervention group. For most outcome measures, the assessors were blinded. The study measures included body fat (sum of four skinfolds),
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aerobic fitness (shuttle run test), physical activity (accelerometry), and quality of life (questionnaires). Secondary outcome measures included body mass index and cardiovascular risk score (average Z score of waist circumference, mean blood pressure, blood glucose, inverted high density lipoprotein cholesterol, and triglycerides). Results: 498 children in total (mean age 6.9 (SD 0.3) years for first grade, 11.1 (0.5) years for fifth grade), completed the baseline and follow-up assessments. After adjustment for grade, sex, baseline values, and clustering within classes, in the intervention arm children showed more negative changes in the Z score of the sum of four skinfolds (–0.12, 95% CI –0.21 to –0.03; p = 0.009) compared with controls. Z scores for aerobic fitness increased more favorably (0.17, 0.01–0.32; p = 0.04), as did those for moderate-vigorous physical activity in school (1.19, 0.78–1.60; p < 0.001), all-day moderate-vigorous physical activity (0.44, 0.05–0.82; p = 0.03), and total physical activity in school (0.92, 0.35–1.50; p = 0.003). Z scores for overall daily physical activity (0.21, –0.21 to 0.63) and physical quality of life (0.42, –1.23 to 2.06) as well as psychological quality of life (0.59, –0.85 to 2.03) did not show any significant changes. Conclusion: A school-based multicomponent physical activity intervention which included some elements that were compulsory improved physical activity and fitness and reduced adiposity in children. The meta-analysis showed that long-running school-based interventions are effective in preventing childhood overweight and obesity. It found that combined interventions of physical activity in the classroom curriculum were effective in preventing childhood overweight and obesity. The duration of the intervention however was positively associated with its effectiveness. Given these results, the authors suggest that school principals and policymakers should consider implementing school-based interventions as long-term strategies for preventing and managing childhood overweight and obesity. Kreimler et al. presented such an approach in a cluster randomized controlled trial. A multicomponent physical activity intervention delivered during one school year had beneficial effects on physical activity, aerobic fitness, and adiposity and they postulated could thus reduce cardiovascular risk. They concluded that programs with compulsory physical activity components seem to be superior to those based on educational interventions, as adherence is guaranteed. Furthermore, the inclusion of all children in a class avoided any stigmatization of overweight and unfit children and gave all children an equal chance to benefit from this type of intervention. As 90% of all children and 70% of the teachers liked the program and wished it to continue, this was very reassuring. The multicomponent and systemic physical activity approach, which included a variety of strategies to enhance physical activity, may have reached more children by broadening the levels of the intervention and the spectrum of activities by making them not only fun but also developing their motor skills too. This approach of integration of increased physical activity into the regular school curriculum going in the long term is the challenge for society.
New uses for old hormones
Efficacy and safety of oxandrolone in growth hormone-treated girls with Turner syndrome Menke LA, Sas TC, de Muinck Keizer-Schrama SM, Zandwijken GR, de Ridder MA, Odink RJ, Jansen M, Delemarre-van de Waal HA, Stokvis-Brantsma WH, Waelkens JJ, Westerlaken C, Reeser HM, van Trotsenburg AS, Gevers EF, van Buuren S, Dejonckere PH, Hokken-Koelega AC, Otten BJ, Wit JM Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands
[email protected] J Clin Endocrinol Metab 2010;95:1151–1160 Background: Although GH therapy has been shown to increase growth and adult height in Turner syndrome (TS), the benefit to risk ratio of adding the weak androgen oxandrolone (Ox) in addition to GH is unclear. Methods: A double-blind, randomized, placebo-controlled, dose-response study was performed in 10 centers in The Netherlands. 133 patients with TS were split into three age groups: 1 (2–7.99 years), 2
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(8–11.99 years), or 3 (12–15.99 years). The girls received GH (1.33 mg/m2/day) from baseline, combined with placebo (Pl) or Ox in low dosage (0.03 mg/kg/day) or conventional dosage (0.06 mg/kg/day) from the age of 8 years and estrogens were added from the age of 12 years. Outcome measures included adult height gain (adult height minus predicted adult height) and safety parameters were carefully evaluated. Results: Compared with GH+Pl, GH+Ox 0.03 increased adult height gain over GH+Pl in the intentionto-treat analysis by mean 9.5 SD 4.7 vs. 7.2 SD 4.0 cm, p = 0.02) and per-protocol analysis (9.8 SD 4.9 vs. 6.8 SD 4.4 cm, p = 0.02). On account of the accelerated bone maturation (p < 0.001), adult height gain on GH+Ox 0.06 was not significantly different from that on GH+Pl (8.3 SD 4.7 vs. 7.2 SD 4.0 cm, p = 0.3). Breast development was slower on GH+Ox (GH+Ox 0.03, p = 0.02; GH+Ox 0.06, p = 0.05), and more girls reported virilization on GH+Ox 0.06 than on GH+Pl (p < 0.001). Conclusion: In GH-treated girls with TS, the authors discourage the use of the conventional Ox dosage (0.06 mg/kg/day) because of its low benefit-to-risk ratio. The addition of Ox 0.03 mg/kg/day modestly increases adult height gain and has a fairly good safety profile, except for some deceleration of breast development. This double-blind, randomized, placebo-controlled, dose-response study of careful design aimed to assess the risk-to-benefit ratio and effectiveness of low and a conventional dose of the weak anabolic steroid in augmentation of the GH induced GH response in girls with TS. Statistical pre-analysis estimated that 15 patients per dosage and age group were needed to achieve a power of 80% to detect a difference (p = 0.05, two-sided) in first-year height velocity of 2 cm with an assumed SD of 2.6. The eventual group sizes of 39–48 clearly exceeded this. Intention-to-treat analyses were performed and differences in adult height gain were also assessed by a per-protocol analysis. In the per-protocol analysis, adult height gain was 3.1 cm greater on GH+Ox 0.03 and 2.2 cm greater on GH+Ox 0.06. When correcting for bone age at starting GH therapy, the difference in adult height gain compared with GH+placebo was +1.8 cm on GH+Ox 0.03 (p = 0.05). Given the lower cost of oxandrolone compared with GH, this result could be seen to produce a greater cost-benefit ratio on improving adult height than GH alone, and avoids the need to consider the more expensive option of higher GH doses, which may in its own right have other safety considerations. Safety evaluation was a notable feature of this study, with several participants either refusing or stopping conventional dose oxandrolone and the final conclusion advises against this, both on account of a greater virilization effect and also a less good outcome for final height due to acceleration in bone age maturation. Breast development could be slightly delayed but not adversely affected. The lessons and boundaries of the use of this additional agent are clearly spelled out by this study, which provides an excellent evidence base for clinical practice.
Reviews
Prevalence and risk factors of radiation-induced growth hormone deficiency in childhood cancer survivors: a systematic review Mulder RL, Kremer LC, van Santen HM, Ket JL, van Trotsenburg AS, Koning CC, Schouten-van Meeteren AY, Caron HN, Neggers SJ, van Dalen EC Department of Paediatric Oncology, Emma Children’s Hospital/Academic Medical Center, Amsterdam, The Netherlands
[email protected] Cancer Treat Rev 2009;35:616–632 Background: The aim of this systematic review was to evaluate the existing evidence of the prevalence and risk factors of radiation-induced GHD in childhood cancer survivors. Growth hormone deficiency (GHD) is usually the first and commonest endocrine problem occurring after cranial radiotherapy (CRT). Methods: The authors searched MEDLINE, EMBASE and CENTRAL for studies reporting on radiationinduced GHD in childhood cancer survivors. A meta-regression analysis was performed on the information about study characteristics, prevalence and risk factors abstracted and the quality of each study was assessed.
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Results: Most studies had methodological limitations. The prevalence of radiation-induced GHD was estimated in 33 studies and this varied considerably between 0 and 90.9%. Only three studies had adequate peak GH cut-off limits (<5 µg/l). In these studies the prevalence ranged from 29.0 to 39.1%, with a pooled prevalence of 35.6%. The main risk factors of GHD identified from studies included in this review were higher CRT dose and longer follow-up times. The meta-regression analysis showed that the wide variation in the prevalence of GHD could be explained by differences in maximal CRT dose. Conclusion: GHD is a frequent consequence after CRT in childhood cancer survivors. The prevalence of radiation-induced GHD ranged from 29.0 to 39.1% when selecting only studies with adequate peak GH cut-off limits. Higher CRT dose and longer time from treatment are the main risk factors. Additional well-designed studies are needed to estimate the precise prevalence of GHD and to define the exact CRT threshold dose at which GHD occurs.
This systematic review and meta-analysis of factors surrounding the diagnosis of GHD in childhood cancer survivors is helpful if not new, but recommends that future studies should prospectively evaluate all children treated with CRT. Follow-up should be long enough and complete, with precise and accurate outcome definitions and uniform methods of detection. Furthermore, the evolution of different risk factors time should be investigated. The authors also recommend that appropriate multivariate analyses are necessary taking into account the separate and joint effects of CRT and other important prognostic risk factors for GHD. Time to ensure your database is up to date.
Food for thought Swings and roundabouts
Long-term follow-up of GH-treated girls with Turner syndrome: metabolic consequences Bannink EM, van der Palen RL, Mulder PG, de Muinck Keizer-Schrama SM Department of Pediatrics, Division of Endocrinology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands Horm Res 2009;71:343–349 Background: This follow-up study of a randomized GH dose-response trial study aimed to investigate the long-term metabolic consequences of childhood GH treatment in young women with Turner syndrome (TS), several years after GH had been discontinued. Methods: The original trial had three GH dosage arms (1.3, 2.0, and 2.7 mg/m2/day). 39 TS patients (20.0 ± 2.1 years) participated 4.8 ± 1.9 years after finishing GH. The mean GH treatment duration was 8.7 ± 2.0 years. Fasting glucose, insulin, and serum lipids were measured. Results: For several years after GH discontinuation, insulin sensitivity remained lower, whilst -cell function and fasting insulin levels stayed higher than before treatment. Only BMI was proportional to -cell function. Serum total cholesterol (TC), low-density lipoprotein and high-density lipoprotein (HDL) were elevated in comparison with values measured 6 months after stopping GH, resulting in higher TC, but also higher HDL levels compared to controls. The atherogenic index remained constant, but below that of the controls. Conclusion: GH therapy in girls with TS has beneficial effects on serum lipids as well as on growth promotion. Nearly 5 years after discontinuation of GH therapy the favorable effect of GH was still noticeable. The GH-induced decrease in insulin sensitivity, however, did not return to baseline. Whether this was just the natural history of TS is unclear.
Is this good or bad news? This long-term follow-up study, evaluates the metabolic consequences after GH treatment in girls with TS in three dosage schedules, but a no-treatment or placebo arm is not included, so we need to rely on historical data for comparison [6]. Insulin sensitivity remained low, not returning to baseline levels, with, pancreatic -cell function impaired and fasting insulin levels remained high. Previously, the authors hypothesized that the higher insulin levels after GH might
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result from a higher BMI, as previous studies had shown a positive correlation between insulin levels and BMI in normal children and adults and would be in agreement with a previous report on the relationship between the effect of GH on adiposity and insulin sensitivity on and after GH treatment in TS [7]. A positive association of BMI with -cell function, and with higher fasting insulin levels several years after GH therapy, supports this possible explanation. It is also reported that untreated young TS women have a higher prevalence of insulin resistance and impaired glucose tolerance, so the authors believe the findings in this study may just be part of the natural history of TS [8]. Alternatively, the persistence of low insulin sensitivity and high -cell function may be explained by the GH therapy administered. Although no association with GH dosage or GH duration is known, such an effect cannot be ruled out. During the first 4 years of GH therapy the study found a decrease in total cholesterol, LDL cholesterol, and atherogenic index, and an increase in HDL and triglycerides. Six months after GH treatment, total cholesterol, LDL and HDL cholesterol increased, resulting in a further decrease in atherogenic index. This pattern extended at long-term follow-up with lipid profiles similar to those reported in untreated TS women being age and BMI independent [6]. This effect is similar to that seen in GHD and treated SGA individuals. So what atherogenic improvements are gained on the swings need to be contrasted with losses in insulin sensitivity on the roundabouts. This is a clear message about the long-term follow-up needed for GH treated individuals.
Follow-up on YB 2009 papers GH and ISS
High-dose GH treatment limited to the prepubertal period in young children with idiopathic short stature does not increase adult height Van Gool SA, Kamp GA, Odink RJ, de Muinck Keizer-Schrama SM, Delemarre-van de Waal HA, Oostdijk W, Wit JM Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands Eur J Endocrinol 2010;162:653–660 Background: This was a randomized-controlled study to assess the long-term effect of high-dose GH treatment on growth in children with idiopathic short stature (ISS) given in the prepubertal period only. Methods: 40 prepubertal children aged 4–8 years (girls) or 4–10 years (boys), height SDS <–2.0 SDS, and birth length >–2.0 SDS, were randomly allocated to receive GH at a dose of 2 mg/m2/day (equivalent to 75 µg/kg/day at the start and 64 µg/kg/day at the finish) until the onset of puberty for at least 2 years (preceded by two 3-month periods of treatment with low or intermediate doses of GH separated by two washout periods of 3 months) or no treatment as a control group. In 28 cases, adult height (AH) was assessed at a mean (S.D.) age of 20.4 (2.3) years. Results: The children receiving GH treatment (mean time on high-dose GH 2.3 years; range 1.2–5.0) showed an increased mean height SDS at discontinuation of the treatment of –1.3 (0.8) SDS compared with –2.6 (0.8) SDS in the controls. However, bone maturation accelerated significantly in the GH-treated group compared with the controls (1.6 (0.4) vs. 1.0 (0.2) years per year, respectively), associated with an earlier onset puberty. Treatment was not given during puberty in either group and after 3–12 years, AH was –2.1 (0.7) and –1.9 (0.6) in the GH-treated and control groups respectively. Age was a positive predictor of adult height gain. Conclusion: High-dose GH treatment given only in the prepubertal period in young ISS children augments height gain during treatment but is associated with an acceleration in bone maturation. This resulted in a similar adult height compared with the untreated controls.
Each year in the Yearbook, outcomes of carefully designed, long-term RCTs of GH treatment are reported as in 2009 there were reports of apparent success [9] and failure [10] of GH treatment in short normal children. Individually they are important, even though conclusions may be different with contrasting conclusions on height gain. Inevitably, as the authors discuss here, results of this study may be limited by small size and dropout over time. Study designs are deliberately different, this one asking separate questions, i.e. What is the effect of GH treatment in ISS children when given a boost in prepuberty only? and Does the dose make a difference? This study makes several innova-
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tions such as examining acceleration in growth of different body proportions (sitting height growth accelerates much faster in the GH-treated group) and a novel technique for expressing pubertal stage in SDS (correcting for age and gender). There was a trend but no statistically significant difference between the groups at Tanner stage 2. The authors postulate that the most likely explanation is that a high GH dose (approximately three times higher than simple replacement) administered to young children not only leads to faster growth but also results in faster bone maturation. There are few data on GH treatment of young children with ISS, as most other studies started at an average age of 11 years. It seems unlikely that the high-dose per se causes the lack of effect, because children treated with a high GH dose at 11 years achieve an AH gain of 1.3 SD, which is slightly more than that would be gained at a low dose. The authors speculate that the epiphyseal plates of young children could be more sensitive to high doses of GH and/or IGF-1 than at later ages. They state that the results also imply that there may be an inverse U-shaped relationship between GH dose and AH gain if treatment is started at a young age. Standard dose GH is positively associated with AH, but in young children higher doses may decrease AH gain due to accelerated maturation of the epiphyseal plates and possibly also of the GnRH regulatory centre with the effect on growth having reached a plateau. A disappointing result from a carefully crafted study, but the discussion (as alluded to above) fosters interesting hypotheses as to the relationships between GH and maturation. Acknowledgement I would like to thank Dr. Stephen O’Riordan of the Children’s Hospital, Leicester Royal Infirmary, for assistance with the selection of some papers for review.
References 1. O’Riordan SM, Butler G: Evidence-based medicine in pediatric endocrinology; in Carel JC, Hochberg Z (eds): Yearbook of Pediatric Endocrinology. Basel, Karger 2009, pp 181–197. 2. NICE Technology Appraisal Guidance 151: Continuous subcutaneous insulin infusion for the treatment of diabetes mellitus (review of technology appraisal guidance 57) 2008. http://guidance.nice.org.uk/TA151/Guidance/pdf/English 3. Butler GE: Assessment of growth and puberty; in Ranke MB, Price DA, Reiter EO (eds): Treatment with Recombinant Human Growth Hormone in Children and Adolescence – 20 Years of KIGS. Basel, Karger 2007, chapt 2, pp 6–15. 4. Rosenfeld RG, Albertsson-Wikland K, Cassorla F, Frasier SD, Hasegawa Y, Hintz RL, LaFranchi S, Lippe BM, Loriaux DL, Melmed S, Preece MA, Ranke MB, Reiter EO, Rogol AD, Underwood LE, Werther GA: Diagnostic controversy: the diagnosis of childhood growth hormone deficiency revisited. J Clin Endocrinol Metab 1995;80:1532–1540. 5. Ho KK: Consensus guidelines for the diagnosis and treatment of adults with GH deficiency. II. A statement of the GH Research Society in association with the European Society for Pediatric Endocrinology, Lawson Wilkins Society, European Society of Endocrinology, Japan Endocrine Society, and Endocrine Society of Australia. Eur J Endocrinol 2007;157:695– 700. 6. Caprio S, Boulware S, Diamond M, Sherwin RS, Carpenter TO, Rubin K, et al: Insulin resistance: an early metabolic defect of Turner’s syndrome. J Clin Endocrinol Metab 1991;72:832–836. 7. Wooten N, Bakalov VK, Hill S, Bondy CA: Reduced abdominal adiposity and improved glucose tolerance in growth hormone-treated girls with Turner syndrome. J Clin Endocrinol Metab 2008;93:2109–2114. 8. Sagi L, Zuckerman-Levin N, Gawlik A, Ghizzoni L, Buyukgebiz A, Rakover Y, et al: Clinical significance of the parental origin of the X chromosome in Turner syndrome. J Clin Endocrinol Metab 2007;92:846–852. 9. Albertsson-Wikland K, Aronson AS, Gustaffson J, Hagenas L, Ivarsson SA et al: Dose-dependent effect of growth hormone on final height in children with short stature without growth hormone deficiency. J Clin Endocrinol Metab 2008; 93:4342–4350. 10. Elder CJ, Barton JS, Brook CG, Preece MA, Dattani MT, Hindmarsh PC: A randomised study of the effect of two doses of biosynthetic human growth hormone on final height of children with familial short stature. Horm Res 2008;70:89– 92.
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Editor’s Choice Jean-Claude Carel and Ze’ev Hochberg
Forgetful adolescents
A critical role for 4-GABAA receptors in shaping learning deficits at puberty in mice Shen H, Sabaliauskas N, Sherpa A, Fenton AA, Stelzer A, Aoki C, Smith SS Department of Physiology and Pharmacology, State University of New York (SUNY) Downstate Medical Center, Brooklyn, N.Y., USA Science 2010;327:1515–1518 Background: The onset of puberty defines a developmental stage when some learning processes are diminished, but the mechanism for this deficit remains unknown. Results: The paper reports that, at puberty, expression of inhibitory 4-aminobutyric acid type A (GABAA) receptors (GABAR) increases perisynaptic to excitatory synapses in CA1 hippocampus. Shunting inhibition via these receptors reduced N-methyl-D-aspartate receptor activation, impairing induction of long-term potentiation. Pubertal mice also failed to learn a hippocampal, long-term potentiation-dependent spatial task that was easily acquired by –/– mice. However, the stress steroid THP (3-OH-5[]-pregnan-20-one), which reduces tonic inhibition at puberty, facilitated learning. Conclusions: The emergence of 4-GABARs at puberty impairs learning, an effect that can be reversed by a stress steroid.
Certain learning and cognitive processes decline at the onset of puberty. The pubertal process that shapes this developmental decline is unknown but is likely to involve the hippocampus, which is widely regarded as the site for learning. Shen et al. found that puberty impaired learning in mice (the time to enter a shock zone) decreased by 70%, at puberty onset. Providing the stress neurosteroid progestative allopregnanolone (THP) completely reversed the learning deficit at puberty, whereas it impaired learning before puberty via its inhibition of GABA receptors for restricting hippocampal plasticity during puberty. At puberty, but not in adults or the very young, GABA receptors were targeted perisynaptically to excitatory synapses, shunting the depolarizing current necessary for NMDA receptor activation. As a consequence, signal transmission was affected and spatial learning reduced. THP effects are distinguishable from glucocorticoids, which alters learning after a delay but has no effect acutely. Thus, the stress steroid THP provides a novel means for rapid changes in synaptic plasticity at puberty. In humans, neurosteroid depletion is consistently documented in patients with current depression and may reflect their greater chronic stress. Evidence that DHEAS is a neurosteroid, together with the fact that increases in DHEAS parallel patterns of cortical maturation from approximately age 6 years to the mid-20s, suggests that DHEAS may play an important role in extended brain maturation among humans. DHEAS has demonstrated effects on mood in humans, and acts at neuron receptor sites. I suggest three ways in which DHEAS may play a role in human brain maturation: (1) increasing activity of the amgydala; (2) increasing activity of the hippocampus, and (3) promoting synaptogenesis within the cortex.
New hope The grail is in the ␣ cell
Conversion of adult pancreatic ␣ cells to  cells after extreme -cell loss Thorel F, Nepote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL Department of Cell Physiology & Metabolism, University of Geneva Faculty of Medicine, Geneva, Switzerland Nature 2010;464:1149–1154 Background: Pancreatic insulin-producing -cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, -cell loss). It is not known whether adult mammals can differentiate (regenerate) new  cells after extreme, total -cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non--cell) source. Methods: A transgenic model of diphtheria-toxin-induced acute selective near-total -cell ablation. Results: If given insulin, the mice survived and showed -cell mass augmentation with time. Lineage tracing to label the glucagon-producing ␣ cells before -cell ablation tracked large fractions of regenerated  cells as deriving from ␣ cells, revealing a previously disregarded degree of pancreatic cell plasticity. Conclusion: Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing  cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.
Finding ways to restore the pancreatic -cell mass in patients with type 1 diabetes is a challenging problem that has been addressed by several different approaches, including culture, gene transfection, fetal islet amplification, neogenesis or differentiation from stem cells. Currently, transplantation of human islets or pancreas is the only approach to replace  cells in diabetes and suffers from a lack of high-quality islets in sufficient quantity. It has been long known in animal models of diabetes due to exposure to toxic compounds such as streptozotocin that  cells could regenerate after a few months of overt diabetes. However, the exact mechanism of regeneration was not known and one limitation of these experiments was that some  cells survived to toxic exposure and could have proliferated after the initial destructive event. Here, the authors have engineered a transgenic model for virtually total ablation of pancreatic  cells, leaving intact the remaining endocrine cells. They show that over time (10 months, i.e. half a mouse lifespan) pancreatic  cells transdifferentiate from glucagon-producing ␣ cells, after a transient step of bihormonal cells (glucagon and insulin). This results in a 44-fold increase in -cell mass that is capable of maintaining glucose homeostasis. The interplay between ␣ and  cells was also demonstrated earlier this year in a series of transgenic experiments where overexpression of the transcription factor Pax4 caused the conversion of ␣ cells into  cells [1]. Depletion of ␣ cells by this mechanism caused a neogenesis of ␣ cells then converted to  cells resulting in a massive increase of -cell mass. Altogether, these results indicate that there is a very significant potential for islet cell regeneration in adults that could convert into methods to restore -cell mass in patients with diabetes [2, 3]. Conversely, the lack of such regeneration in patients further demonstrates that -cell destruction in type 1 diabetes is not a one-time event but rather continues after diagnosis and damages new cells that might be formed by the regenerative process.
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Lesson from other societies Non-universal heightism
How universal are human mate choices? Size does not matter when Hadza foragers are choosing a mate Sear R, Marlowe FW Department of Social Policy, London School of Economics, London, UK
[email protected] Biol Lett 2009;5:606–609 Background: It has been argued that size matters on the human mate market: both stated preferences and mate choices have been found to be nonrandom with respect to height and weight. But how universal are these patterns? Most of the literature on human mating patterns is based on post-industrial societies. Much less is known about mating behavior in more traditional societies. Methods: This study investigated mate choice by analyzing whether there is any evidence for nonrandom mating with respect to size and strength in a forager community, the Hadza of Tanzania. It tested whether couples assort for height, weight, body mass index (BMI), percent fat and grip strength, and whether there is a male-taller norm. Finally, it tests for an association between anthropometric variables and number of marriages. Results: Results show no evidence for assortative mating for height, weight, BMI or percent fat; no evidence for a male-taller norm and no evidence that number of marriages is associated with our size variables. Hadza couples may assort positively for grip strength, but grip strength does not affect the number of marriages. Conclusion: In contrast to post-industrial societies, mating appears to be random with respect to size in the Hadza.
Choosing the right mate is an important component of evolutionary fitness, particularly so in a species like our own, where long-term relationships between partners are common. There is now a large body of research investigating mating patterns within evolutionary studies of human behavior. In Western societies, size appears to matter for both mate preferences and mate choices; individuals take height into consideration when weighing up potential mates. Similarly, weight is a factor in both stated preferences. This study investigated mate choice in a hunter-gatherers society, the Hadza who live in the central Rift Valley of Tanzania. The results suggest that size and strength are not greatly important in this population mating. This lack of size-related mating patterns might appear surprising, since size is usually assumed to be an indicator of health, productivity and overall quality. But health and productivity may be signaled in alternative ways in the Hadza, who are a small, relatively homogeneous population. There may be some disadvantages to large size in a food-limited society, where the costs of maintaining large size during periods of food shortage may be high. Such disadvantages will not be seen in food-abundant societies, so that large size may be a better indicator of quality in Western populations. The authors conclude wisely that ‘It is time to expand our horizon to a truly cross-cultural view and begin to sort between highly variable and truly universal mate patterns.’
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Food for thought Heightism in our societies
Patient, physician, and consumer drivers: referrals for short stature and access to specialty drugs Cuttler L, Marinova D, Mercer MB, Connors A, Meehan R, Silvers JB Department of Pediatrics, Rainbow Babies and Children’s Hospital, University Hospitals Case Medical Center, Cleveland, Ohio, USA
[email protected] Med Care 2009;47:858–865 Background: Candidates for specialty drugs, the fastest growing and costliest pharmaceuticals, typically originate with primary care referrals. However, little is known about what drives such referrals – especially for large populations such as short, otherwise normal children (idiopathic short stature). Recent expanded approval of growth hormone (GH) makes more than 585,000 US children eligible for such treatment, potentially costing over USD 11 billion/year. Methods: To quantify the relative impact of patient physiological indicators, physician characteristics, and consumer preferences on referrals to endocrinologists (and potential access to GH) for short children, a national study of 1,268 randomly selected US pediatricians was conducted, based on a full factorial experimental design in a structured survey. Results: While patient indicators (height, growth pattern) influenced referrals (p < 0.001), consumer drivers (family concern) and physician attitudes had almost as great an impact – especially for children with less severe growth impairment (p < 0.001). Physician belief that short stature impairs emotional well-being and physician characteristics (female, older, shorter, beliefs about drug company information) increased referrals (p < 0.03–0.001) – independent of growth parameters. Conclusions: Referral recommendations that create the pool of candidates for the specialty drug GH are heavily swayed by physician characteristics and consumer preferences, particularly in the absence of compelling physiological evidence. This makes most of children with short stature strikingly susceptible to nonphysiological influences on referrals that render them candidates for this specialty drug. Only 1 additional referral per US pediatrician would likely increase GH costs by over USD 100 million/year.
In the vast majority of cases, evaluation of a short child by a pediatric endocrinologist results from referral by a primary care physician. This paper analyzed the determinants of referrals to pediatric endocrinologists based on 4 typical case scenarios designed to vary in 5 factors – child’s age and gender, child’s height and growth pattern, and family concern about the child’s stature. In addition, physician’s attitudes towards height and short stature evaluation were surveyed. The decision to refer patients was not only based on the patients clinical characteristics (i.e. shorter patients, falling off the centile curves more likely to be referred than milder cases) but was also very much influenced by physician-dependent factors such as age, gender, height in SDS and physician’s general attitude regarding short stature as a problem. Age of the child was not a factor affecting referral. In addition, family concern and the child’s gender also influenced the decision to refer. These data are not fully surprising to us pediatric endocrinolgists but are important for our practice and future directions. First, there is a need for higher quality evidence in our own evaluations of children with short stature [4] since better establishment of our own evaluation scheme will help in communicating it to the wider community and help referring physicians focus on patients who need a specialized evaluation most. Second, we need better evidence-based evaluation of our interventions in order to allow us to better communicate them to the medical and nonmedical community at large.
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Multigenerational stress
The ghosts of predators past: population cycles and the role of maternal programming under fluctuating predation risk Sheriff M, Krebs C, Boonstra R University of British Columbia, Vancouver, B.C., Canada Ecology doi:10.1890/09-1108 (2010) http://www.esajournals.org/doi/abs/10.1890/09-1108.1 Background: Maternal effects may be a major factor influencing the demography of populations. In mammals, the transmission of stress hormones between mother and offspring may play an important role in these effects. Laboratory studies have shown that stressors during pregnancy and lactation result in lifelong programming of the offspring phenotype. However, the relevance of these studies to free-living mammals is unclear. The 10-year snowshoe hare cycle is intimately linked to fluctuating predation pressure and predation risk. The enigma of these cycles is the lack of population growth following the decline phase when the predators have virtually all disappeared and the food supply is ample. A predator-induced increase in maternal stress hormone levels resulted in a decline in reproduction. Methods: This study examines population and hormone changes over a 4-year period from the increase (2005) to the decline (2008). Results: (1) An index of maternal stress (fecal corticosteroid metabolite [FCM] concentrations) fluctuates in synchrony with predator density during the breeding season; (2) maternal FCM levels are echoed in their offspring and this occurs at a population wide level, and (3) higher maternal FCM levels at birth are correlated with an increased responsiveness of the HPA axis in their progeny. Conclusions: The results are the first to show an intergenerational inheritance of stress hormones in a freeranging population of mammals. They propose that the lack of recovery of reproductive rates during the early low phase of the hare cycle may be the result of the impacts of intergenerational, maternallyinherited stress hormones caused by high predation risk during the decline phase.
Adaptations to the effects of environmental stressors are essential for ensuring individual fitness in natural populations. The environment that an individual’s mother experiences affects her offspring’s fitness. These nongenetic phenotypic effects can have a profound influence on offspring and can cause a resemblance not just between a mother and her offspring but also between grandparents and grand-offspring. These nongenetic maternal effects are referred to as maternal or developmental programming. A programming effect reflects the influence of a specific environmental factor during the developmental period, before or just after birth, on the organization of target tissues and/or gene-expression patterns that affect function throughout life. Mechanisms responsible for maternal programming may vary among organisms, the nature of the stressor (e.g. undernutrition, trauma, disease, psychological stress, etc.), and timing and duration of occurrence (prenatal vs. postnatal; acute vs. chronic). Glucocorticoids and their receptors play a key organization role by which these lifelong changes are brought about. This study of snowshoe hares shows that high levels of predation result in a sharp increase in levels of maternal stress hormones. These levels remain high in the offspring of these stressed animals and persist into adulthood, depressing reproduction. This suggests that the inheritance of stress levels results in a slow recovery of a population of wild mammals. The increase in stress hormones in the offspring born to stressed mothers may trade off the decrease in reproduction by increasing their offspring’s antipredator behavior. Humans also inherit a stress level from their parents, and recent results suggest that this may be epigenetically determined at the glucocorticoid receptor level.
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Concept recentered Suckling keeps us warm
Hepatic FGF21 expression is induced at birth via PPAR␣ in response to milk intake and contributes to thermogenic activation of neonatal brown fat Hondares E, Rosell M, Gonzalez FJ, Giralt M, Iglesias R, Villarroya F Departament de Bioquimica i Biologia Molecular, Institut de Biomedicina de la Universitat de Barcelona and CIBER Fisiopatologia de la Obesidad y Nutricion, Barcelona, Catalonia, Spain Cell Metab 2010;11:206–212 Background: Plasma FGF21 levels and hepatic FGF21 gene expression increase dramatically after birth in mice. This induction is initiated by suckling, requires lipid intake, is impaired in PPAR␣ null neonates, and is mimicked by treatment with the PPAR␣ activator, Wy14,643. Neonates exhibit reduced FGF21 expression in response to fasting, in contrast to the upregulation occurring in adults. Changes in FGF21 expression due to suckling or nutritional manipulations were associated with circulating free fatty acid and ketone body levels. Results: The study mimicked the FGF21 postnatal rise by injecting FGF21 into fasting neonates, and found that this enhanced the expression of genes involved in thermogenesis within brown fat, and increased body temperature. Brown adipocytes treated with FGF21 exhibited increased expression of thermogenic genes, higher total and uncoupled respiration, and enhanced glucose oxidation. Conclusions: The induction of FGF21 production by the liver mediates direct activation of brown fat thermogenesis during the fetal-to-neonatal transition.
Hepatic FGF21 has been discussed in our Yearbook in the last 2 years as a key player in metabolic adaptation to starvation, among others by stunting growth through STAT5 signaling. This year we learn that expression and plasma FGF21 levels are dramatically induced after birth, and that hepatic FGF21 induction is caused by a PPAR␣-mediated effect of lipids in the milk. White fat development determines hepatic FGF21 expression in response to fasting and FGF21 released by the liver leads to activation of neonatal brown fat thermogenesis. The net effect is that mom’s milk activates a pathway that turns on brown fat cells – the heat-generating cells in young animals. FGF21 expression could be quashed by preventing mice from suckling, and initiated by substituting lipids for the mother’s milk. FGF21, they found, turns on genes related to activation of brown fat, and was also able to activate brown adipocytes in culture.
A new cytokine More vasculature for more fat
Chemerin, a novel adipokine in the regulation of angiogenesis Bozaoglu Ka, Curran JEb, Stocker CJc, Zaibi MSc, Segal Da, Konstantopoulos Na, Morrison Sa, Carless Mb, Dyer TDb, Cole SAb, Goring HHb, Moses EKb, Walder Ka, Cawthorne MAc, Blangero Jb, Jowett JBd a Metabolic Research Unit, Deakin University, Geelong, Australia; bDepartment of Genetics, Southwest Foundation for Research, San Antonio, Tex., cClore Laboratory, University of Buckingham, Buckingham, UK, and dBaker IDI Heart and Diabetes Institute, Melbourne, Australia J Clin Endocrinol Metab 2010;95:2476–2485 Background: Chemerin is a new adipokine associated with obesity and the metabolic syndrome. Gene expression levels of chemerin were elevated in the adipose depots of obese compared with lean animals and was markedly elevated during differentiation of fibroblasts into mature adipocytes. Objective: The objective of the study was to identify factors that affect the regulation and potential function of chemerin using a genetics approach. Design: Plasma chemerin levels were measured in subjects from the San Antonio Family Heart Study, a large family-based genetic epidemiological study including 1,354 Mexican-American individuals.
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Individuals were randomly sampled without regard to phenotype or disease status. A genome-wide association analysis using 542,944 single-nucleotide polymorphisms in a subset of 523 of the same subjects was undertaken. The effect of chemerin on angiogenesis was measured using human endothelial cells and interstitial cells in coculture in a specially formulated medium. Results: Serum chemerin levels were found to be highly heritable (h2 = 0.25; p = 1.4 × 10–9). The singlenucleotide polymorphism showing strongest evidence of association (rs347344; p = 1.4 × 10–6) was located within the gene encoding epithelial growth factor-like repeats and discoidin I-like domains 3, which has a known role in angiogenesis. Functional angiogenesis assays in human endothelial cells confirmed that chemerin significantly mediated the formation of blood vessels to a similar extent as vascular endothelial growth factor. Conclusion: Plasma chemerin levels are significantly heritable and identified a novel role for chemerin as a stimulator of angiogenesis. Chemerin was reported in 2007 as a new adipokine elevated in states of obesity and the metabolic syndrome. Chemerin gene expression was significantly elevated in the adipose depots of obese compared with lean animals and was predominantly expressed by adipocytes rather than stromal and vascular cells in adipose tissue. In vitro studies have shown that chemerin expression and secretion was markedly up-regulated during differentiation of fibroblasts to mature adipocytes and was approximately 20-fold higher in fully differentiated adipocytes compared with undifferentiated fibroblasts. Furthermore, recombinant chemerin was shown to stimulate 3T3-L1 adipocyte function such as glucose transport, and plasma chemerin levels were significantly associated with characteristics of the metabolic syndrome, including body mass index, plasma triglycerides, and blood pressure in several independent human populations. This article demonstrates for the first time that variation in plasma chemerin levels is significantly heritable, and a number of polymorphisms in candidate genes that may influence plasma chemerin levels are identified. Of particular interest is a gene that has been previously shown to play a role in angiogenesis. Because the expansion of adipose tissue is dependent on the formation of new blood vessels, and given the significant association already established between chemerin and obesity, this group explored the effects of chemerin on angiogenesis and found that recombinant chemerin promoted the formation of new blood vessels. These novel data indicate a new role for chemerin in the formation of new blood vessels, and this may be an essential component of adipose tissue expansion.
Concept revised Synchronizing the mothering hormone
Synchronized network oscillations in rat tuberoinfundibular dopamine neurons: switch to tonic discharge by thyrotropin-releasing hormone Lyons DJ, Horjales-Araujo E, Broberger C Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
[email protected] Neuron 2010;65:217–229 Background: The pituitary hormone, prolactin, triggers lactation in nursing mothers. Under nonlactating conditions, prolactin secretion is suppressed by powerful inhibition from hypothalamic tuberoinfundibular dopamine (TIDA) neurons. Although a firing pattern has been suggested as integral to neuroendocrine control, the electrical behavior of TIDA cells remains unknown. Results: This report demonstrates that rat TIDA neurons discharge rhythmically in a robust 0.05-Hz oscillation. The oscillation is phase locked between neurons, and while it persists during chemical synaptic transmission blockade, it is abolished by gap junction antagonists. Thyrotropin-releasing hormone (TRH) potently stimulates prolactin release, an effect assumed to take place in the pituitary. In TIDA cells, TRH caused a transition from phasic to tonic firing through combined pre- and postsynaptic effects.
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Conclusions: These findings suggest a model for prolactin regulation where a TIDA network switch from oscillations to sustained discharge converts dopamine from an antagonist at high concentrations to a functional agonist as dopamine output from the network decreases.
The reason that women normally do not produce prolactin is that its release is normally strongly inhibited by dopamine. This is secreted by cells known as ‘TIDA’ neurons in the hypothalamus in the brain. The study has shown that the TIDA cells normally display an extremely rhythmical activity, with discharges every 20 s, required to function as a strong inhibitor of prolactin release. Thus, it seems reasonable that the functional role of the TIDA oscillation is not to time prolactin surges but to ensure sufficient inhibitory concentrations of DA in the anterior pituitary. The study has also shown that TRH can interrupt the rhythmical signaling pattern of TIDA cells. Prolactin also plays a role in reproduction and fertility, one of which is reduction of sexual libido; it is released during an orgasm. It is believed that prolactin is significant for metabolism, since patients, and children even more, with elevated levels of prolactin become overweight. In terms of basic prolactin physiology, TRH-mediated stimulation of prolactin release occurs at the hypothalamic level by shifting TIDA oscillations to tonic discharge.
A new neurohormone Blessing blockers
Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis Ubuka T, Morgan K, Pawson AJ, Osugi T, Chowdhury VS, Minakata H, Tsutsui K, Millar RP, Bentley GE Department of Integrative Biology and Helen Wills Neuroscience Institute, University of California, Berkeley, Calif., USA
[email protected] PLoS One 2009;4:e8400 Background: The existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Results: This report identifies GnIH homologs in the human hypothalamus and characterizes their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as human RFRP-1 and RFRP-3 by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone-producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca++ mobilization. It also directly modulates GnRH neuron firing. Conclusions: The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.
A neuropeptide inhibitor of gonadotropin secretion has been postulated for some years, described in birds, but remained elusive in humans. By testing the human homologs RFRP-1 and -3 to the GnIH peptide, this paper identified the GnIH. A G-protein-coupled receptor, GPR147, has been identified as their cognate receptor. GnIH neurons were observed in the dorsomedial region of the human hypothalamus in close proximity to GnRH neurons in the preoptic area, suggesting the regulation of GnRH neurons by GnIH. GnIH may regulate gonadotropin secretion by inhibiting GnRH neurons as well as directly acting on gonadotrope cells in the pituitary as was clearly demonstrated in isolated pituitary
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cells in the sheep. GnIH has the potential of an alternative or adjunct therapeutic agent to inhibit gonadotropins and steroid hormones. Thus this endogenous inhibitor of gonadotropin secretion has therapeutic potential in the treatment of hormone-dependent diseases such as precocious puberty, and may also have potential as a novel contraceptive.
Important mechanism A needed assistant
A -arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G-protein activation Gesty-Palmer D, Flannery P, Yuan L, Corsino L, Spurney R, Lefkowitz RJ, Luttrell LM Department of Medicine, Duke University Medical Center, Durham, N.C., USA Sci Transl Med 2009;1:1ra1 Background: About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through -arrestins; so-called biased agonists can selectively activate these distinct pathways. Methods: This study investigated selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (D-Trp12,Tyr34)PTH(7–34) (PTH-arr), which activates -arrestin but not classic G-protein signaling. Results: In mice, PTH-arr induces anabolic bone formation, as does the nonselective agonist PTH(1– 34), which activates both mechanisms. In -arrestin2-null mice, the increase in bone mineral density evoked by PTH(1–34) is attenuated and that stimulated by PTH-arr is ablated. The -arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. Conclusions: These results show that a biased agonist selective for the -arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.
The physiologic actions of PTH were thought to be mediated by classic G-protein signaling. We now learn that the PTH GPCR can trigger signaling mechanisms that are independent from the classic G-protein pathways. -Arrestins, a small family of intracellular proteins initially identified for their role as GPCR desensitizers, act as signal transducers through a distinct mechanism, scaffolding with accessory effector molecules. PTH stimulation of the PTH receptor promotes translocation of -arrestins 1 and 2 to the plasma membrane, association of the receptor with -arrestins, internalization of receptor--arrestin complexes, and activation of ERK1/2. This paper shows that a selective agonist for -arrestin signaling in the absence of G-protein signaling promotes osteoblastic bone formation without stimulating bone resorption and may have clinical utility as an anabolic agent in the treatment of diseases characterized by insufficient rates of bone formation, such as osteoporosis.
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In anticipation of food
Stomach ghrelin-secreting cells as food-entrainable circadian clocks LeSauter J, Hoque N, Weintraub M, Pfaff DW, Silver R Department of Psychology, Barnard College, New York, N.Y., USA Proc Natl Acad Sci USA 2009;106:13582–13587 Background: Increases in arousal and activity in anticipation of a meal, termed ‘food anticipatory activity’ (FAA), depend on circadian food-entrainable oscillators (FEOs), whose locations and output signals have long been sought. It is known that ghrelin is secreted in anticipation of a regularly scheduled mealtime. Results: This report shows that ghrelin administration increases locomotor activity in nondeprived animals in the absence of food. In mice lacking ghrelin receptors, FAA is significantly reduced. Impressively, the cumulative rise of activity before food presentation closely approximates a gaussian function (r = 0.99) for both wild-type and ghrelin receptor knockout animals, with the latter having a smaller amplitude. For both groups, once an animal begins its daily anticipatory bout, it keeps running until the usual time of food availability, indicating that ghrelin affects response threshold. Oxyntic cells coexpress ghrelin and the circadian clock proteins PER1 and PER2. The expression of PER1, PER2, and ghrelin is rhythmic in light-dark cycles and in constant darkness with ad libitum food and after 48 h of food deprivation. In behaviorally arrhythmic-clock mutant mice, unlike control animals, there is no evidence of a premeal decrease in oxyntic cell ghrelin. Rhythmic ghrelin and PER expression are synchronized to prior feeding, and not to photic schedules. Conclusions: This article concludes that oxyntic gland cells of the stomach contain FEOs, which produce a timed ghrelin output signal that acts widely at both brain and peripheral sites. It is likely that other FEOs also produce humoral signals that modulate FAA.
The body uses an endogenous circadian timing system, termed ‘food-entrainable oscillators’ (FEOs), to predict the availability of food. These activate food-seeking behaviors and enable the synthesis and secretion of enzymes necessary for digestion before mealtime. For regularly scheduled daily meals, the behavioral manifestation of this timing mechanism is the expression of food anticipatory activity (FAA), reflected in an increase in activity several hours before the appearance of food. The present studies explore the possibility that ghrelin-secreting cells of the stomach oxyntic glands are FEOs. The results show that stomach oxyntic cells fulfill several essential criteria of an FEO, and the findings provide an avenue for understanding the previous literature on FAA. Ghrelin stimulates both the appetitive (anticipatory locomotor behavior) and the consummatory component (food intake). Administration of ghrelin in the absence of food in a nondeprived animal increases activity/ arousal and increases subsequent food intake. In the absence of ghrelin receptors, food anticipatory behavior is diminished. This suggests that ghrelin increases the drive to consume food. Both ghrelin and clock genes are expressed rhythmically within oxyntic cells. The phase of this rhythm is controlled by the time of food availability. Although mice cannot be deprived for many days, in humans, ghrelin release timed to previous mealtimes persists after 1–3 days of fasting. Discovery of the brain mechanisms modulating ghrelin effects on activity and eating will further the understanding of this system in the generation of FAA.
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New hope Preventing infertility after childhood cancer treatment
Inhibition of the c-Abl-TAp63 pathway protects mouse oocytes from chemotherapy-induced death Gonfloni S, Di Tella L, Caldarola S, Cannata SM, Klinger FG, Di Bartolomeo C, Mattei M, Candi E, De Felici M, Melino G, Cesareni G Department of Biology, University of Rome Tor Vergata, Rome, Italy
[email protected] Nat Med 2009;15:1179–1185 Background: Germ cells are sensitive to genotoxins, and ovarian failure and infertility are major side effects of chemotherapy in young patients with cancer. Results: The c-Abl-TAp63 pathway is activated by chemotherapeutic DNA-damaging drugs in model human cell lines and in mouse oocytes and plays a role in cell death. In cell lines, upon cisplatin treatment, c-Abl phosphorylates TAp63 on specific tyrosine residues. Such modifications affect p63 stability and induce a p63-dependent activation of proapoptotic promoters. Similarly, in oocytes, cisplatin rapidly promotes TAp63 accumulation and eventually cell death. Treatment with the c-Abl kinase inhibitor imatinib counteracts these cisplatin-induced effects. Conclusion: The data support a model in which signals initiated by DNA double-strand breaks are detected by c-Abl, which, through its kinase activity, modulates the p63 transcriptional output. Moreover, they suggest a new use for imatinib, aimed at preserving oocytes of the follicle reserve during chemotherapeutic treatments.
In young patients with cancer, the options to preserve fertility are still limited and cryopreservation of one ovary is now a common procedure at pediatric cancer centers [5]. However, the options to use the preserved ovarian tissue are still experimental; transplantation of pieces of the cryopreserved ovarian cortex has resulted in the resumption of endocrine function and in live births but the procedure is only possible for those with low risk of preexisting contaminating cancer cells in the ovarian tissue. For others, in vitro maturation of eggs is an experimental process that has only been successful in animals so far. The use of GnRH agonist in pubertal girls has been discussed but is unlikely to have any effect [6]. Therefore, new approaches to protect gonadal tissues against the effects of chemotherapy are needed. The authors have observed that after cisplatin administration, p63 serves to eliminate the injured germ cells and that the kinase c-Abl is activated. Imatinib, an inhibitor of c-Abl, blocked the immediate appearance of apoptotic oocytes when delivered with cisplatin. Moreover, treatment of immature mice with imatinib and cisplatin resulted in normal-appearing follicles in adult ovarian tissue and restored fertility. As nicely discussed in the associated editorial [7], this breakthrough idea may raise several issues. First imatinib treatment might rescue damaged oocytes with a high risk for miscarriage and birth defects. Second, imatinib might modulate the effects of cisplatin on the target tumor, and there are some examples where it might increase or decrease the anticancer effect. Altogether, there is a need for more experimental data (action on the testis, effect on damage induced by other drugs than cisplatin) before we embark on clinical trials, but this first step is really good news.
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Reviews
Genetics, pathogenesis and clinical interventions in type 1 diabetes Bluestone JA, Herold K, Eisenbarth G Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, Calif., USA
[email protected] Nature 2010;464:1293–1300
Type 1 diabetes is an autoimmune disorder afflicting millions of people worldwide. Once diagnosed, patients require lifelong insulin treatment and can experience numerous disease-associated complications. The last decade has seen tremendous advances in elucidating the causes and treatment of the disease based on extensive research both in rodent models of spontaneous diabetes and in humans. Integrating these advances has led to the recognition that the balance between regulatory and effector T cells determines disease risk, timing of disease activation, and disease tempo. The authors describe current progress, the challenges ahead and the new interventions that are being tested to address the unmet need for preventative or curative therapies.
Etiology of type 1 diabetes Todd JA Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
[email protected] Immunity 2010;32:457–467
Recent genetic mapping and gene-phenotype studies have revealed the genetic architecture of type 1 diabetes. At least ten genes so far can be singled out as strong causal candidates. The known functions of these genes indicate the primary etiological pathways of this disease, including HLA class II and I molecules binding to preproinsulin peptides and T-cell receptors, T- and B-cell activation, innate pathogenviral responses, chemokine and cytokine signaling, and T-regulatory and antigen-presenting cell functions. This review considers research in the field of type 1 diabetes toward identifying disease mechanisms using genetic approaches. The expression and functions of these pathways, and therefore disease susceptibility, will be influenced by epigenetic and environmental factors. Certain inherited immune phenotypes will be early precursors of type 1 diabetes and could be useful in future clinical trials. Type 1 diabetes is among the most frequent conditions managed by pediatric endocrinologists and still has a very somber long-term prognosis despite advances in insulin delivery, glucose surveillance and patient education. It is also the pediatric endocrine disease on which the highest amount of research money has been spent, leading to major advances in our understanding of the mechanisms of the disease, but with limited impact on daily management. These two reviews are very timely and come from authorities in the field of diabetes genetics and immunology. They give an updated and comprehensive view on these fields and future research directions with the hope that these advances will improve patient care during the next decade. References 1. Collombat P, Xu X, Ravassard P, Sosa-Pineda B, Dussaud S, Billestrup N, et al: The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into ␣ and subsequently  cells. Cell 2009;138:449–462. 2. Liu Z, Habener JF: Alpha cells beget  cells. Cell 2009;138:424–426. 3. Zaret KS, White MF: Diabetes forum: extreme makeover of pancreatic ␣ cells. Nature 2010;464:1132–1133. 4. Grote FK, Oostdijk W, De Muinck Keizer-Schrama SM, van Dommelen P, van Buuren S, Dekker FW, et al: The diagnostic work up of growth failure in secondary health care; an evaluation of consensus guidelines. BMC Pediatr 2008;8:21. 5. West ER, Zelinski MB, Kondapalli LA, Gracia C, Chang J, Coutifaris C, et al: Preserving female fertility following cancer treatment: current options and future possibilities. Pediatr Blood Cancer 2009;53:289–295. 6. Carel JC, Eugster EA, Rogol A, Ghizzoni L, Palmert MR, Antoniazzi F, et al: Consensus statement on the use of gonadotropin-releasing hormone analogs in children. Pediatrics 2009;123:e752–e762. 7. Woodruff TK: Preserving fertility during cancer treatment. Nat Med 2009;15:1124–1125.
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Science and Medicine Ze’ev Hochberg and Jean-Claude Carel
New hope Gene therapy for adrenoleukodystrophy
Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, Vidaud M, Abel U, Dal-Cortivo L, Caccavelli L, Mahlaoui N, Kiermer V, Mittelstaedt D, Bellesme C, Lahlou N, Lefrere F, Blanche S, Audit M, Payen E, Leboulch P, l’Homme B, Bougneres P, Von Kalle C, Fischer A, Cavazzana-Calvo M, Aubourg P INSERM UMR745, University Paris-Descartes, Paris, France Science 2009;326:818–823 Background: X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). Methods: The authors performed a gene therapy trial in 2 ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then reinfused into the patients after they had received myeloablative treatment. Results: Over a span of 24–30 months of follow-up, polyclonal reconstitution was detected, with 9–14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggested that hematopoietic stem cells were transduced in the patients. Beginning 14–16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the 2 patients stopped – a clinical outcome comparable to that achieved by allogeneic HCT. Conclusion: Lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.
Although adrenoleukodystrophy often presents as an endocrine disorder with adrenal insufficiency and to a lesser extent Leydig cell insufficiency, the major impact of this devastating disease is on white matter demyelination. Approximately 50% of affected males present in mid-childhood with progressive demyelinating disease leading to death in adolescence. The group of Patrick Aubourg has been essential along the years in delineating the disease through establishment of allogeneic bone marrow transplantation as an efficient therapy, cloning the gene and refuting undue claims of dietary manipulation [1–3]. Quite interestingly, the favorable effect of allogeneic bone marrow transplantation demonstrated that replacement of deficient bone marrow-derived microglial cells was sufficient to cure the central nervous system involvement but had no effect on the adrenal dysfunction – an interesting observation for further research. However, bone marrow transplantation is a risky procedure and is often impeded by lack of familial or unrelated donor. A large collaborative group of pediatric neurologists and endocrinologists, basic scientists and clinical hematologists concurred to construct HIV-derived lentiviral vectors and transduce CD34+ hematopoietic stem cells from 2 patients, which were reinfused after myeloablation. The results, although preliminary, are quite striking since they show long-term engraftment of transduced cells, partial correction of the biochemical alterations and favorable clinical evolution. More importantly, no preferential clonal expansion was observed as compared to previously observed integration-related mutagenesis and leukemogenesis with retroviral vectors. This study is important not only for those affected or involved in the care of adrenoleukodystrophy, but also for the field of gene therapy which has been slowed down by several setbacks [4]. It confirms that a metabolic disease affecting the brain can be improved by hematopoietic stem cell correction and suggests that similar lentiviral approaches might be ben-
eficial to other genetic diseases. More importantly, it shows that persistence in clinical and basic study of a single disease can lead to major advances in medicine.
Origins and functional impact of copy number variation in the human genome Conrad DF, Pinto D, Redon R, Feuk L, Gokcumen O, Zhang Y, Aerts J, Andrews TD, Barnes C, Campbell P, Fitzgerald T, Hu M, Ihm CH, Kristiansson K, Macarthur DG, Macdonald JR, Onyiah I, Pang AW, Robson S, Stirrups K, Valsesia A, Walter K, Wei J, Tyler-Smith C, Carter NP, Lee C, Scherer SW, Hurles ME The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK Nature 2010;464:704–712
Structural variations of DNA >1 kb in size account for most bases that vary among human genomes, but are still relatively under-ascertained. Methods: This study use tiling oligonucleotide microarrays, comprising 42 million probes, to generate a comprehensive map of 11,700 copy number variations (CNVs) >443 bp, of which most (8,599) have been validated independently. For 4,978 of these CNVs, they generated reference genotypes from 450 individuals of European, African or East-Asian ancestry. Results: The predominant mutational mechanisms differ among CNV size classes. Retrotransposition has duplicated and inserted some coding and non-coding DNA segments randomly around the genome. Furthermore, by correlation with known trait-associated single nucleotide polymorphisms (SNPs), they identified 30 loci with CNVs that are candidates for influencing disease susceptibility. Conclusions: Having assessed the completeness of this map and the patterns of linkage disequilibrium between CNVs and SNPs, this report concludes that, for complex traits, the heritability void left by genome-wide association studies will not be accounted for by common CNVs. Context:
For type 2 diabetes, for example, there are now around 30 genetic variants known to influence susceptibility to the disease, but these only account for about 10% of the known inherited risk of developing these conditions. One theory for this so-called ‘missing heritability’ was that it may have been caused by copy number variations (CNVs). Yet, several studies of diabetes and other complex diseases found that commonly occurring CNV are unlikely to play a major role in such diseases. This study analyzed common CNVs in DNA samples from 3,000 healthy volunteers and compared them to 16,000 patients – 2,000 each with bipolar disorder, breast cancer, coronary artery disease, Crohn’s disease, hypertension, rheumatoid arthritis, type 1 diabetes and type 2 diabetes. They identified and confirmed three loci with commonly occurring CNV, and all three had been identified previously by searching for SNPs, and none of the three CNV loci is believed to contribute to disease. It seems unlikely that common CNVs play a major role in the genetic basis of these or other common complex diseases, either through particular CNVs having a strong effect or through a large number of CNVs each contributing a small effect.
Endogenous non-retroviral RNA virus elements in mammalian genomes Horie M, Honda T, Suzuki Y, Kobayashi Y, Daito T, Oshida T, Ikuta K, Jern P, Gojobori T, Coffin JM, Tomonaga K Department of Virology, Research Institute for Microbial Diseases (BIKEN), Osaka University, Osaka, Japan Nature 2010;463:84–87
Retroviruses are the only group of viruses known to have left a fossil record, in the form of endogenous proviruses, and approximately 8% of the human genome is made up of these elements. Although many other viruses, including non-retroviral RNA viruses, are known to generate DNA forms of their own genomes during replication, none has been found as DNA in the germline of animals. Bornaviruses, a genus of non-segmented, negative-sense RNA virus, are unique among RNA viruses in that they establish persistent infection in the cell nucleus. Results: Here they show that elements homologous to the nucleoprotein (N) gene of bornavirus exist in the genomes of several mammalian species, including humans, non-human primates, rodents and elephants. These sequences have been designated endogenous Borna-like N (EBLN) elements. Some of the primate EBLNs contain an intact open reading frame and are expressed as mRNA. Phylogenetic analyses showed that EBLNs seem to have been generated by different insertional events in each specific Context:
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animal family. Furthermore, the EBLN of a ground squirrel was formed by a recent integration event, whereas those in primates must have been formed more than 40 million years ago. They also show that the N mRNA of a current mammalian bornavirus, Borna disease virus, can form EBLN-like elements in the genomes of persistently infected cultured cells. Conclusions: These results provide the first evidence for endogenization of non-retroviral virus-derived elements in mammalian genomes and give novel insights not only into generation of endogenous elements, but also into a role of bornavirus as a source of genetic novelty in its host. This report provides evidence of endogenous sequences derived from a non-retroviral RNA virus in mammalian species. Phylogenetic analyses demonstrate that the oldest primate gene of bornavirus must have appeared in an ancestor of primates at least 40 million years, providing a new source of genetic innovation in their hosts. It is likely that these are processed pseudogenes derived from ancient bornavirus infections. Despite replication during tens of millions of years as exogenous viruses, the amino acid sequences of current Borna disease virus proteins seem surprisingly conserved. The paper also raises the possibility that, like some endogenous retroviruses, the Bornal genes may have some function in their host species – us.
DNA methylation and gene expression differences in children conceived in vitro or in vivo Katari S, Turan N, Bibikova M, Erinle O, Chalian R, Foster M, Gaughan JP, Coutifaris C, Sapienza C Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pa., USA Hum Mol Genet 2009;18:3769–3778
Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth-weight, major and minor birth defects, and rare disorders involving imprinted genes, suggesting that epigenetic changes may be associated with assisted reproduction. Methods: This study examined DNA methylation at more than 700 genes (1,536 CpG sites) in placenta and cord blood and measured gene expression levels of a subset of genes that differed in methylation levels between children conceived in vitro versus in vivo. Results: The results suggest that in vitro conception is associated with lower mean methylation at CpG sites in placenta and higher mean methylation at CpG sites in cord blood. It also finds that in vitro conception-associated DNA methylation differences are associated with gene expression differences at both imprinted and non-imprinted genes. The range of inter-individual variation in gene expression of the in vitro and in vivo groups overlaps substantially but some individuals from the in vitro group differ from the in vivo group mean by more than two standard deviations. Several of the genes whose expression differs between the two groups have been implicated in chronic metabolic disorders, such as obesity and type 2 diabetes. Conclusions: These findings suggest that there may be epigenetic differences in the gametes or early embryos derived from couples undergoing treatment for infertility. Alternatively, assisted reproduction technology may have an effect on global patterns of DNA methylation and gene expression. In either case, these differences or changes may affect long-term patterns of gene expression. Context:
IVF now accounts for at least 1–2% of all live births in the Western world. Although the overall rate of congenital anomalies in children conceived by IVF is low (4–6%), this rate still represents a significant increase over the background rate of major malformations (3%). When indices of pre- and postnatal development are measured, IVF children, as a group, do not differ significantly from their control counterparts, except for having an increased incidence of low birth-weight and being slightly taller. This report analyzed DNA methylation at a large number of CpG sites in placenta and cord blood, comparing children conceived by IVF with a control group. They observed an overall lower level of specific CpG site methylation in placenta and higher level in cord blood. If these differences are characteristic of differences in embryonic versus extraembryonic tissues, it may suggest differences in the way ‘outer’ and ‘inner’ blastomeres of pre-implantation embryos respond to in vitro culture. These epigenetic differences have the potential to affect embryonic development and fetal growth, as well as to influence the long-term patterns of gene expression associated with the increased risk of many human diseases. The authors suggest that there is also a possibility that these changes could pass to the children of IVF babies and spread through the human gene pool.
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Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors Goriely Aa, Hansen RMa, Taylor IBa, Olesen IAb, Jacobsen GKc, McGowan SJd, Pfeifer SPe, McVean GAe, Meyts ERb, Wilkie AOa a Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK; Departments of bGrowth & Reproduction and cPathology, Copenhagen University Hospital, Copenhagen, Denmark; d Computational Biology Research Group, Oxford, and eDepartment of Statistics, University of Oxford, Oxford, UK Nat Genet 2009;41:1247–1252
Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism’s life. Here we unify these processes to a single cellular event for mutations arising in male germ cells that show a paternal age effect. Results: Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA showed that levels of the FGFR3 mutation increase with paternal age and that the mutation spectrum at the Lys650 codon is similar to that observed in bladder cancer. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. Conclusions: This article proposes that paternal age-effect mutations activate a common ‘selfish’ pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition. Context:
This article offers a link between achondroplasia and Apert, Noonan and Costello syndromes and seminomas of older men: they all may arise from germ cell defects. FGFR3 and HRAS mutations induce gain of function, and develop as the testicle ages, and encourage the mutant cells to divide and multiply with copies of the mutation to each daughter cell. Hence, the number of sperm carrying this mutation increases as men get older, raising the risk to older fathers of having affected children. The findings link the processes of mutation in the soma (causing neoplasia) and germline (causing heritable disorders in the next generation), which normally occur in different cells, to a mutational event likely happening in the same cell. The authors call them ‘selfish’ because the mutations benefit the germ cell but are harmful to offspring. But other diseases seem to be more frequent in the offspring of older fathers, including breast cancer, autism and schizophrenia; the authors suggest that mutations might contribute to these diseases.
New mechanisms All in the Aire – cytokine antibodies, mucocutaneous candidiasis and neonatal tolerance
Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I Puel A, Doffinger R, Natividad A, Chrabieh M, Barcenas-Morales G, Picard C, Cobat A, Ouachee-Chardin M, Toulon A, Bustamante J, Al-Muhsen S, Al-Owain M, Arkwright PD, Costigan C, McConnell V, Cant AJ, Abinun M, Polak M, Bougneres PF, Kumararatne D, Marodi L, Nahum A, Roifman C, Blanche S, Fischer A, Bodemer C, Abel L, Lilic D, Casanova JL Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Institut National de la Santé et de la Recherche Médicale (INSERM), U550, Paris, France
[email protected] J Exp Med 2010;207:291–207 Background: Most patients with autoimmune polyendocrine syndrome type I (APS-I) display chronic mucocutaneous candidiasis.
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Methods: The authors hypothesized that this chronic mucocutaneous candidiasis might result from autoimmunity to interleukin (IL)-17 cytokines. Results: High titers of autoantibodies against IL-17A, IL-17F, and/or IL-22 were found in the sera of all 33 patients tested, as detected by multiplex particle-based flow cytometry. The auto-Abs against IL-17A, IL-17F, and IL-22 were specific in the 5 patients tested, as shown by Western blotting. The auto-Abs against IL-17A were neutralizing as shown by bioassays of IL-17A activity. None of the 37 healthy controls and none of the 103 patients with other autoimmune disorders tested had such autoantibodies. None of the patients with APS-I had auto-Abs against cytokines previously shown to cause other welldefined clinical syndromes in other patients (IL-6, interferon-␥, or granulocyte/macrophage colonystimulating factor) or against other cytokines (IL-1, IL-10, IL-12, IL-18, IL-21, IL-23, IL-26, interferon-, tumor necrosis factor-␣, or transforming growth factor-). Conclusion: The findings suggest that autoantibodies against IL-17A, IL-17F, and IL-22 may cause chronic mucocutaneous candidiasis in patients with APS-I.
Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity Guerau-de-Arellano M, Martinic M, Benoist C, Mathis D Section on Immunology and Immunogenetics, Joslin Diabetes Center, Harvard Medical School, Boston, Mass., USA J Exp Med 2009;206:1245–1252 Background: There has long been conceptual and experimental support for, but also challenges to, the notion that the initial period of the immune system’s development is particularly important for the establishment of tolerance to self. The display of self antigens by thymic epithelial cells is key to inducing tolerance in the T-lymphocyte compartment, a process enhanced by the Aire transcription factor. Methods: Doxycycline-regulated transgene to target Aire expression to the thymic epithelium, complementing the Aire knockout in a temporally controlled manner. Results: Aire is essential in the perinatal period to prevent the multiorgan autoimmunity that is typical of Aire deficiency. Aire could be shut down soon thereafter and remain off for long periods, with few deleterious consequences. The lymphopenic state present in neonates was a factor in this dichotomy because inducing lymphopenia during Aire turnoff in adults recreated the disease, which, conversely, could be ameliorated by supplementing neonates with adult lymphocytes. Conclusion: Aire expression during the perinatal period is both necessary and sufficient to induce longlasting tolerance and avoid autoimmunity. Aire-controlled mechanisms of central tolerance are largely dispensable in the adult, as a previously tolerized T-cell pool can buffer newly generated autoreactive T cells that might emerge.
Autoimmune polyendocrine syndrome type I (APS-I) is a rare and severe monogenic disorder due to loss of function of the Aire gene, resulting in a failure of central (thymic) tolerance mechanisms and in multiorgan autoimmunity. The list of autoimmune targets is extremely vast, including endocrine (parathyroid, adrenals, islets, thyroid, …) and non-endocrine (liver, bronchi, digestive tract, …) systems. One of the unique features of the disease is the susceptibility to chronic mucocutaneous candidiasis, which is often the presenting sign in early childhood and highly prevalent in affected individuals, as compared to other involvements. This susceptibility to candidiasis is quite unique and is not associated with risk of infection with other pathogens. The general mechanism for the disease lies in the role of the Aire gene in directing the expression of self antigens in specialized cells of the thymic medulla (medullary thymic epithelial cells) where these self antigens allow for the elimination of highly self-reactive T lymphocytes [5]. Although this general mechanism has been demonstrated in human and animal systems, it could not easily provide a basis for the unique susceptibility to candidiasis. Here it is shown that antibodies to IL-17 cytokines, produced by Th17 CD4+ T cells [6], probably explains the candidiasis observed in APS-I patients. Similar data were presented in an accompanying paper [7], showing in addition that patients with thymomas, a situation of acquired defective central T-cell tolerance similarly have antibodies to Th17 cytokines. In addition to providing a mechanistic basis for candidiasis, these results give ground to clinical evaluation of immunosuppressive therapies such as ones targeting B cells in patients with APS-I [8]. Complementing this particular aspect of APS-I, the paper by Guerau-de-Arellano et al. addresses a fundamental question regarding the immune system of whether the neonatal period is unique as a
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time when the immune system becomes tolerant to any (most?) antigens that it encounters. Increased autoimmunity with age could be viewed as an escape from these mechanisms, and failure to establish neonatal tolerance results in catastrophic autoimmune diseases such as APS-I or IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked). The discovery of Aire and the establishment of its role in central tolerance has greatly enhanced our understanding of immune tolerance. It turns out that the thymus continues to play a role throughout life, as exemplified by premature immune aging in young adults thymectomized during early childhood and autoimmune diseases arising in patients with thymomas [9]. In this study, the Mathis group used a clever system of inducible Aire transgene in a background of Aire knockout mice, allowing them to control Aire expression throughout development. Expression of Aire during fetal life only had a minimal effect in preventing autoimmunity, whereas expression of Aire during fetal and the first 3 weeks of life almost completely prevented autoimmunity. In addition, infusions of physiologically lymphopenic Aire-deficient neonates with adult T cells partially prevented the autoimmune pathology, whereas transient depletion of lymphocytes in protected adult mice worsened the pathology. Altogether, these results provide important and new understanding of the regulation of autoimmunity in the neonatal period. Study of neonatal T tolerance in subjects at risk for autoimmune diseases might result in new paradigms for the treatment or prevention of autoimmunity by targeting this specific period of life. If we ever find a way to cure APS-I, we will have to diagnose it intrauterinely or in early infancy.
Concepts revised Bugs like sweets – with a little help from GRP78
The endothelial cell receptor GRP78 is required for mucormycosis pathogenesis in diabetic mice Liu M, Spellberg B, Phan QT, Fu Y, Lee AS, Edwards JE, Jr, Filler SG, Ibrahim AS Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, Calif., USA J Clin Invest 2010;120:1914–1924 Background: Mucormycosis is a fungal infection of the sinuses, brain, or lungs that causes a mortality rate of at least 50% despite first-line therapy. Angioinvasion is a hallmark of mucormycosis infections and endothelial cell receptor(s) for fungi of the order Mucorales (the fungi that cause mucormycosis) are not determined. Patients with elevated available serum iron, including those with diabetic ketoacidosis (DKA), are uniquely susceptible to mucormycosis, suggesting a role of iron and glucose in regulating the expression of such a receptor. Methods and Results: Glucose-regulated protein 78 (GRP78) was identified as a novel host receptor that mediates invasion and damage of human endothelial cells by Rhizopus oryzae, the most common etiologic species of Mucorales, but not Candida albicans or Aspergillus fumigatus. Elevated concentrations of glucose and iron, consistent with those seen during DKA, enhanced GRP78 expression and the resulting R. oryzae invasion and damage of endothelial cells in a receptor-dependent manner. Mice with DKA, which have enhanced susceptibility to mucormycosis, exhibited increased expression of GRP78 in sinus, lungs, and brain compared with normal mice. Finally, GRP78-specific immune serum protected mice with DKA from mucormycosis. Conclusion: The results suggest a unique susceptibility of patients with DKA to mucormycosis and provide a foundation for the development of new therapeutic interventions for these deadly infections.
Infections, in particular fungal, are a hallmark of diabetes and an important cause of morbidity and mortality in diabetic patients. The most generally invoked mechanism for this increased susceptibility is the increased availability of glucose as a metabolic substrate for pathogen growth. This paper revisits this concept and shows that such simplistic views should be revised. The authors have analyzed the pathogenesis of Rhizopus oryzae, the pathogen involved in mucormycosis, a deadly fungal infection that can occur in immunocompromised patients and in diabetic ketoacidosis. They show that a normal endothelial protein with unknown physiological functions serves as receptor for the
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fungus. Moreover, high glucose, low pH and high iron concentrations, all observed during diabetic ketoacidosis, concur to increase the expression of the receptor. GRP78 is a member of the HSP70 protein family and functions as a chaperone. It has also been reported as a receptor for a variety of ligands, including an angiogenesis inhibitor, an activated proteinase inhibitor, a synthetic peptide a dengue virus and a Coxsackievirus. These data demonstrate the plasticity of pathogens which have adapted to mammalian proteins and use them as unwanted receptors or adhesion molecules. We should refrain from simplistic explanations for phenomena we believe to understand. Last and more importantly, blocking GRP78 with an antibody prevents mucormycosis and might become a useful approach to treat this deadly condition.
Food for thought Tell me who you cite, I will tell you who you are
How citation distortions create unfounded authority: analysis of a citation network Greenberg SA Children’s Hospital Informatics Program and Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass., USA
[email protected] BMJ 2009;339:b2680 Background: During the preparation and writing of manuscripts, authors must consider carefully the selection, completeness, and appropriateness of the articles referenced to ensure adequate and accurate citation as a necessity of scientifically and methodologically sound research. The aim of the paper was to understand belief in a specific scientific claim by studying the pattern of citations among papers stating it. Methods: A complete citation network was constructed from all PubMed indexed English literature papers addressing the belief that -amyloid, a protein accumulated in the brain in Alzheimer’s disease, is produced by and injures skeletal muscle of patients with inclusion body myositis. Social network theory and graph theory were used to analyze this network. Citation bias, amplification, and invention, and their effects on determining authority were studied. Results: The network contained 242 papers and 675 citations addressing the belief, with 220,553 citation paths supporting it. Unfounded authority was established by citation bias against papers that refuted or weakened the belief; amplification, the marked expansion of the belief system by papers presenting no data addressing it; and forms of invention such as the conversion of hypothesis into fact through citation alone. Extension of this network into text within grants funded by the National Institutes of Health and obtained through the Freedom of Information Act showed the same phenomena present and sometimes used to justify requests for funding. Conclusion: The author concludes that citation is an impartial scholarly method and a powerful form of social communication. Distortions in its use including bias, amplification, and invention, can be used to generate information cascades resulting in unfounded authority of claims. Construction and analysis of a claim-specific citation network may clarify the nature of a published belief system and expose distorted methods of social citation.
Citations of peer-reviewed papers constitute one of the bases of medical writing and allow authors to make statements based on ‘current knowledge’ or on previously established facts in the literature. Since most journals allow only a limited number of references, authors invariably have to make choices in the references they use. Although we have all noticed distortions in citations in specific manuscripts, this is the first systematic approach to such a phenomenon. The author used a very limited research field (the role of -amyloid in inclusion body myositis) and applied social networks techniques to address the question of how citation distortion can create what the author calls ‘unfounded authority’. 242 papers generated a network of more than 220,000 citation paths. Ten authoritative papers were all from the same group with only 4 of them containing primary data. Six
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papers with primary data that were critical of the hypothesis were seldom of never cited. Citation distortions were noted, as well as amplification over time and impact on NIH funding. Whether the same is true to other fields of science is not known and more studies like this will be needed in other research areas to establish how widespread the process is. In the field of clinical medicine, the recognition of such citation biases has led to the development of meta-analyses and systematic reviews where all primary evidence is re-evaluated and weighed appropriately. However, this has not been the case for basic science, or for mechanistic or diagnostic studies. The accompanying Editorial calls for systematic reviews included or available for all grant applications. In addition, calling the attention of authors on the fairness of the selection of papers they cite could be one step forward [10]. In the meantime, we should all pay attention to citation distortion as readers, reviewers or authors of manuscripts to avoid carrying over inaccurate information from one paper to the next as elegantly demonstrated here.
Important for clinical practice Beware of how you say it
Effect of alternative summary statistics for communicating risk reduction on decisions about taking statins: a randomized trial Carling CL, Kristoffersen DT, Montori VM, Herrin J, Schunemann HJ, Treweek S, Akl EA, Oxman AD Norwegian Knowledge Centre for the Health Services, Oslo, Norway
[email protected] PLoS Med 2009;6:e1000134 Background: Different ways of presenting treatment effects can affect healthcare decisions but little is known about which presentations best help people make decisions consistent with their own values. Methods: Six summary statistics for communicating coronary heart disease (CHD) risk reduction with statins were compared: relative risk reduction and five absolute summary measures – absolute risk reduction, number needed to treat, event rates, tablets needed to take, and natural frequencies. A randomized trial was conducted to determine which presentation would result in choices most consistent with participants’ values. Adult volunteers were recruited who participated through an interactive website. Participants rated the relative importance of outcomes using visual analog scales (VAS). They were randomized to one of the six summary statistics and asked to choose whether to take statins based on this information. A relative importance score was calculated by subtracting the VAS for the downsides of taking statins from the VAS score for coronary heart disease. Logistic regression was used to determine the association between participants’ relative importance score and their choice. 2,978 participants completed the study. Results: Relative risk reduction resulted in a 21% higher probability of choosing to take statins over all values of relative importance score compared to the absolute summary statistics. This corresponds to a number needed to treat of 5, i.e. for every 5 participants shown the relative risk reduction, 1 additional participant chose to take statins, compared to the other summary statistics. There were no significant differences among the absolute summary statistics in the association between relative importance score and participants’ decisions whether to take statins. Natural frequencies were best understood (86% reported they understood them well or very well), and participants were most satisfied with this information. Conclusion: Presenting the benefits of taking statins as a relative risk reduction increases the likelihood of people accepting treatment compared to presenting absolute summary statistics, independent of the relative importance they attach to the consequences. Natural frequencies may be the most suitable summary statistic for presenting treatment effects, based on self-reported preference, understanding of and satisfaction with the information, and confidence in the decision.
Communication in modern medical practice is essential since patients are more and more associated with difficult choices offered by several possibilities of contemporary medicine. These choices are increasingly complex for physicians in their own field, and approaches to present them both to
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healthcare professionals and to patients have been developed. However, communication is never neutral and we know from daily practice that the ways we explain treatment affect patients’ choice. This study addressed the issue in a systematic way by randomizing volunteers to 6 groups who were presented with the same therapeutic scenario (10-year coronary heart disease of risk of 6% without statins and 30% relative reduction in the risk with statins) using 6 different methods: relative risk reduction, number needed to treat, tablets needed to treat, absolute risk reduction, event rate and natural frequencies. Individuals in the group that was presented with relative risk reduction selected the treatment in 74% of cases, as compared with ≈50% in all other groups. A clear limitation of the study is that the recruitment was internet-based, and the volunteering subjects were not really involved in the decision process. However, the majority of volunteers was above 40 years of age and therefore potentially implicated in similar choices. Although subjectivity is an important part of medical decision-making, healthcare professionals increasingly share evidence-based medical information with their patients to allow them to participate in medical decisions. This study shows that this is not enough and that we should pay attention not only to the information we provide but also how we provide it.
New concerns EPO and mortality in cancer patients
Recombinant human erythropoiesis-stimulating agents and mortality in patients with cancer: a meta-analysis of randomized trials Bohlius J, Schmidlin K, Brillant C, Schwarzer G, Trelle S, Seidenfeld J, Zwahlen M, Clarke M, Weingart O, Kluge S, Piper M, Rades D, Steensma DP, Djulbegovic B, Fey MF, Ray-Coquard I, Machtay M, Moebus V, Thomas G, Untch M, Schumacher M, Egger M, Engert A Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland Lancet 2009;373:1532–1542 Background: Erythropoiesis-stimulating agents reduce anemia in patients with cancer and could improve their quality of life, but these drugs might increase mortality. Methods: Meta-analysis of randomized controlled trials in which these drugs plus red blood cell transfusions were compared with transfusion alone for prophylaxis or treatment of anemia in patients with cancer. Data for patients treated with epoetin alfa, epoetin beta, or darbepoetin alfa were obtained and analyzed by independent statisticians using fixed-effects and random-effects meta-analysis. Analyses were by intention to treat. Primary endpoints were mortality during the active study period and overall survival during the longest available follow-up, irrespective of anticancer treatment, and in patients given chemotherapy. Tests for interactions were used to identify differences in effects of erythropoiesisstimulating agents on mortality across pre-specified subgroups. Results: Data from a total of 13,933 patients with cancer in 53 trials were analyzed. 1,530 patients died during the active study period and 4,993 overall. Erythropoiesis-stimulating agents increased mortality during the active study period (combined hazard ratio 1.17, 95% CI 1.06–1.30) and worsened overall survival (1.06, 1.00–1.12), with little heterogeneity between trials. 10,441 patients on chemotherapy were enrolled in 38 trials. The combined hazard ratio for mortality during the active study period was 1.10 (0.98–1.24), and 1.04 (0.97–1.11) for overall survival. There was little evidence for a difference between trials of patients given different anticancer treatments. Conclusion: Treatment with erythropoiesis-stimulating agents in patients with cancer increased mortality during active study periods and worsened overall survival. The increased risk of death associated with treatment with these drugs should be balanced against their benefits.
The value of this paper is really to illustrate the power of high-quality meta-analyses to elucidate rare or difficult-to-detect events in a complex situation such as the treatment of cancer. Patients with cancer are often anemic and it has been debated whether erythropoiesis-stimulating agents that increase the comfort and quality of life of patients might increase mortality. 53 out of 102 eligible studies were selected and the meta-analysis on more than 14,000 patients showed a 17% increased
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risk of death during treatment and 6% overall. For pediatric endocrinologists, the value of the study is not really to discuss whether the use of EPO is warranted during cancer treatment or not, but rather to show how large dataset analyses can increase our knowledge and provide answers to pending questions. The practical implications of such answers are then, as we just learned in the previous article, a matter of how the results are communicated.
Interesting mechanism End of the chromosome and the end of life
Genetic variation in human telomerase is associated with telomere length in Ashkenazi centenarians Atzmon G, Cho M, Cawthon RM, Budagov T, Katz M, Yang X, Siegel G, Bergman A, Huffman DM, Schechter CB, Wright WE, Shay JW, Barzilai N, Govindaraju DR, Suh Y Department of Medicine and Genetics, Albert Einstein College of Medicine, Bronx, N.Y., USA
[email protected] Proc Natl Acad Sci USA 2010;107(suppl 1):1710–1717
Telomere length in humans is emerging as a biomarker of aging because its shortening is associated with aging-related diseases and early mortality. However, genetic mechanisms responsible for these associations are not known. Methods: Here, in a cohort of Ashkenazi Jewish centenarians, their offspring, and offspring-matched controls, the inheritance and maintenance of telomere length and variations in two major genes associated with telomerase enzyme activity, hTERT and hTERC, were studied. Results: The study demonstrated that centenarians and their offspring maintain longer telomeres compared with controls with advancing age and that longer telomeres are associated with protection from age-related diseases, better cognitive function, and lipid profiles of healthy aging. Sequence analysis of hTERT and hTERC showed overrepresentation of synonymous and intronic mutations among centenarians relative to controls. Moreover, they identified a common hTERT haplotype that is associated with both exceptional longevity and longer telomere length. Conclusions: Thus, variations in human telomerase gene that are associated with better maintenance of telomere length may confer healthy aging and exceptional longevity in humans. Context:
Telomeres consist of the TTAGGG tandem repeats at the ends of chromosomes and are known to protect these regions from degradation and DNA repair activities. Telomeres progressively shorten with each cell division in cultured primary human cells until a critically shortened length is achieved, upon which the cells enter replicative senescence. This study assessed the telomere length in blood leukocytes among subjects with exceptional longevity (centenarians) to investigate if centenarians survived with long telomeres, an indicator of better telomere length maintenance, or short telomeres reflecting chronologically old age. They demonstrated that not only centenarians, but their offspring too, maintain longer telomeres compared with controls with advancing age and that longer telomeres are associated with protection from age-related diseases, better cognitive function, and lipid profiles of healthy aging. They identified a common telomerase gene haplotype that is associated with both exceptional longevity and longer telomere length.
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Important to know Know your elder
Ardipithecus ramidus and the paleobiology of early hominids White TD, Asfaw B, Beyene Y, Haile-Selassie Y, Lovejoy CO, Suwa G, WoldeGabriel G Human Evolution Research Center and Department of Integrative Biology, University of California, Berkeley, Calif., USA
[email protected] Science 2009;326:75–86
Hominid fossils predating the emergence of Australopithecus have been sparse and fragmentary. The evolution of our lineage after the last common ancestor we shared with chimpanzees has therefore remained unclear. Methods: Ardipithecus ramidus, recovered in ecologically and temporally resolved contexts in Ethiopia’s Afar Rift, now illuminates earlier hominid paleobiology and aspects of extant African ape evolution. More than 110 specimens recovered from 4.4-million-year-old sediments include a partial skeleton with much of the skull, hands, feet, limbs, and pelvis. Results: This hominid combined arboreal palmigrade clambering and careful climbing with a form of terrestrial bipedality more primitive than that of Australopithecus. Ar. ramidus had a reduced canine/ premolar complex and a little-derived cranial morphology and consumed a predominantly C3 plantbased diet (plants using the C3 photosynthetic pathway). Its ecological habitat appears to have been largely woodland-focused. Ar. ramidus lacks any characters typical of suspension, vertical climbing, or knuckle-walking. Ar. ramidus indicates that despite the genetic similarities of living humans and chimpanzees, the ancestor we last shared probably differed substantially from any extant African ape. Conclusions: Hominids and extant African apes have each become highly specialized through very different evolutionary pathways. This evidence also illuminates the origins of orthogrady, bipedality, ecology, diet, and social behavior in earliest Hominidae and helps to define the basal hominid adaptation, thereby accentuating the derived nature of Australopithecus. Context:
A special issue of Science was devoted to describe the eldest hominid, Ardipithecus ramidus, who roamed what is now Ethiopia 4.4 million years ago. The most complete skeleton of a female was nicknamed ‘Ardi’, who lived more than a million years before ‘Lucy’. Ardi, who weighed about 50 kg and stood about 120 cm tall, had a mix of ‘primitive’ traits, shared with her predecessors, the primates of the Miocene epoch, and ‘derived’ traits, which it shares exclusively with later hominids. The investigators described her as a mosaic creature, i.e. neither chimpanzee nor human. Ardi lived in a woodland environment where she climbed on all fours along tree branches but walked, upright, on two legs, while on the ground.
We keep evolving The following review and the subsequent four articles deal with ongoing evolution of us - modern humans
Has human evolution stopped? Templeton AR Departments of Biology and Genetics, Washington University, St. Louis, Mo., USA; Department of Evolutionary and Environmental Biology, University of Haifa, Israel Rambam Maimonides Med J 2010;1:e0006
It has been argued that human evolution has stopped because humans now adapt to their environment via cultural evolution and not biological evolution. However, all organisms adapt to their
Context:
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environment, and humans are no exception. Culture defines much of the human environment, so cultural evolution has actually led to adaptive evolution in humans. Evidence: Examples are given to illustrate the rapid pace of adaptive evolution in response to cultural innovations. These adaptive responses have important implications for infectious diseases, mendelian genetic diseases, and systemic diseases in current human populations. Moreover, evolution proceeds by mechanisms other than natural selection. The recent growth in human population size has greatly increased the reservoir of mutational variants in the human gene pool, thereby enhancing the potential for human evolution. The increase in human population size coupled with our increased capacity to move across the globe has induced a rapid and ongoing evolutionary shift in how genetic variation is distributed within and among local human populations. In particular, genetic differences between human populations are rapidly diminishing and individual heterozygosity is increasing, with beneficial health effects. Finally, even when cultural evolution eliminates selection on a trait, the trait can still evolve due to natural selection on other traits. Conclusions: Our traits are not isolated, independent units, but rather are integrated into a functional whole, so selection on one trait can cause evolution to occur on another trait, sometimes with mildly maladaptive consequences. The distinguished paleontologist Stephen Jay Gould stated: ‘There’s been no biological change in humans in 40,000 or 50,000 years. Everything we call culture and civilization we’ve built with the same body and brain.’ Templeton argues that human evolution has not stopped, and our ongoing evolution has many medical and health implications. The rationale for the cessation of human evolution is based on the premise that cultural evolution eliminates adaptive evolution via natural selection. However, all organisms adapt to their environment, and in humans much of our environment is defined by our culture. Hence, cultural change can actually spur on adaptive evolution in humans. The only way to truly stop any biological organism from evolving is extinction. Evolution would be slowed by reducing and keeping population size to a small number of individuals. This will lead to a loss of most genetic variations through genetic drift and minimize the input of new mutations into the population. Our population size has been increasing over the last 10,000 years, and is now so large that the current human gene pool contains an immense reservoir of genetic variation; our evolutionary potential has never been higher. Our evolution is further driven by a radical change in the balance of genetic drift and gene flow that is rapidly causing a major evolutionary change in the human species in how genetic variation is distributed within and among local populations. Even when our cultural innovations do eliminate selection on a trait, that trait can still evolve as a correlated response to evolution of another trait, often in a non-adaptive fashion and sometimes in a mildly maladaptive fashion. As long as humans persist as a reproducing population, humans will evolve. This has been the lesson of the past 10,000 years, and is certainly what we can expect to continue for as long as our species persists on the Earth.
Natural selection in a contemporary human population Byars SG, Ewbank D, Govindaraju DR, Stearns SC Department of Ecology and Evolutionary Biology, Yale University, New Haven, Conn., USA Proc Natl Acad Sci USA 2010;107(suppl 1):1787–1792
The aims of this study were to demonstrate that natural selection is operating on contemporary humans, predict future evolutionary change for specific traits with medical significance, and show that for some traits we can make short-term predictions about our future evolution. Methods: To do so, they measured the strength of selection, estimated genetic variation and covariation, and predicted the response to selection for women in the Framingham Heart Study, a project of the National Heart, Lung, and Blood Institute and Boston University that began in 1948. Results: They found that natural selection is acting to cause slow, gradual evolutionary change. The descendants of these women are predicted to be on average slightly shorter and stouter, to have lower total cholesterol levels and systolic blood pressure, to have their first child earlier, and to reach menopause later than they would in the absence of evolution. Conclusions: Selection is tending to lengthen the reproductive period at both ends. To better understand and predict such changes, the design of planned large, long-term, multicohort studies should include input from evolutionary biologists. Context:
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Natural selection is acting slowly and gradually on traits of medical importance and on life history traits. The study examined the vital statistics of 2,238 postmenopausal women participating in the Framingham Heart Study, searching for correlations between women’s height, weight, blood pressure and cholesterol levels and the number of offspring they produced. Both age at first birth and age at menopause appear to be changing so as to lengthen the reproductive period, which is consistent with previous findings. Slightly overweight (but not obese) women tended to have more children, as did women with lower blood pressure and cholesterol levels. Using a sophisticated statistical analysis that controlled for any social or cultural factors that could impact childbearing, the researchers determined that these characteristics were passed on genetically from mothers to daughters and granddaughters. It was suggested that if these trends were to continue with no cultural changes for the next 10 generations, by 2409 the average Framingham woman would be 2 cm (0.8 in) shorter, 1 kg (2.2 lb) heavier, have a healthier heart, have her first child 5 months earlier and enter menopause 10 months later than a woman today.
High frequency of lactose intolerance in a prehistoric hunter-gatherer population in northern Europe Malmstrom H, Linderholm A, Liden K, Stora J, Molnar P, Holmlund G, Jakobsson M, Gotherstrom A Department of Evolutionary Biology, Uppsala University, Uppsala, Sweden BMC Evol Biol 2010;10:89
Genes and culture are believed to interact, but it has been difficult to find direct evidence for the process. One candidate example that has been put forward is lactase persistence in adulthood, i.e. the ability to continue digesting the milk sugar lactose after childhood, facilitating the consumption of raw milk. This genetic trait is believed to have evolved within a short time period and to be related with the emergence of sedentary agriculture. Results: This study investigated the frequency of an allele (−13910*T) associated with lactase persistence in a Neolithic Scandinavian population. From the 14 individuals originally examined, 10 yielded reliable results. They find that the T allele frequency was very low (5%) in this Middle Neolithic huntergatherer population, and that the frequency is dramatically different from the extant Swedish population (74%). Conclusions: They conclude that this difference in frequency could not have arisen by genetic drift and is either due to selection or, more likely, replacement of hunter-gatherer populations by sedentary agriculturalists. Context:
The ability to drink milk as an adult occurs at a high frequency in present-day Caucasians, who have been practicing dairying and cattle rearing for 10,000 years, and less so in African and Far Eastern populations who started farming only some 2,000 years ago. This capacity is closely associated with the transition from hunter-gatherer to agricultural societies. Apparently, the capacity to consume unprocessed milk into adulthood is regarded as having been of great significance for human prehistory. This has been regarded as an adaptive genetic trait exposed to positive selection induced by cultural practices. But the hunter-gatherers who inhabited Scandinavia 4,000 years ago were lactoseintolerant. They differed significantly from modern Swedes in terms of the DNA sequence that we generally associate with a capacity to digest lactose into adulthood. The paper suggests two possible explanations for the DNA differences. One possibility is that these differences are evidence of a powerful selection process, through which the Stone Age hunter-gatherers’ genes were lost due to some significant advantage associated with the capacity to digest milk. The other is that today’s Scandinavians are not descended from the Stone Age people in question, but from a group that arrived later. Most importantly, these findings describe a possible scenario where cultural practices could have had a tremendous impact on the genetic composition of human populations.
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The origins of lactase persistence in Europe Itan Y, Powell A, Beaumont MA, Burger J, Thomas MG Research Department of Genetics, Evolution and Environment, University College London, London, UK PLoS Comput Biol 2009;5:e1000491
Lactase persistence (LP) is common among people of European ancestry, but with the exception of some African, Middle Eastern and Southern Asian groups, is rare or absent elsewhere in the world. Lactase gene haplotype conservation around a polymorphism strongly associated with LP in Europeans (−13,910 C/T) indicates that the derived allele is recent in origin and has been subject to strong positive selection. Furthermore, ancient DNA work has shown that the −13,910*T (derived) allele was very rare or absent in early Neolithic Central Europeans. It is unlikely that LP would provide a selective advantage without a supply of fresh milk, and this has led to a gene-culture co-evolutionary model where LP is only favored in cultures practicing dairying, and dairying is more favored in lactase-persistent populations. Methods: The study developed a flexible demic computer simulation model to explore the spread of LP, dairying, other subsistence practices and unlinked genetic markers in Europe and Western Asia’s geographic space. Results: Using data on −13,910*T allele frequency and farming arrival dates across Europe, and approximate bayesian computation to estimate parameters of interest, they infer that the −13,910*T allele first underwent selection among dairying farmers around 7,500 years ago in a region between the Central Balkans and Central Europe, possibly in association with the dissemination of the Neolithic Linearbandkeramik culture over Central Europe. Furthermore, the results suggest that natural selection favoring a LP allele was not higher in northern latitudes through an increased requirement for dietary vitamin D. Conclusions: The results provide a coherent and spatially explicit picture of the co-evolution of LP and dairying in Europe. Context:
Before the evolution of lactase persistence, humans typically lost their ability to digest lactose around the age of 5. This is thought to have helped motivate weaning. Still today, most of the world’s population, including Asians and Africans, can only tolerate milk for the first few years of life. But, through at least four parallel evolutions starting several thousand years ago, lactase persistence spread throughout human populations. In Europe, a single genetic variant, −13,910*T, is strongly associated with lactase persistence and appears to have been favored by natural selection. Since adult consumption of fresh milk was only possible after the domestication of animals, it is likely that lactase persistence co-evolved with the cultural practice of dairying, although it is not known when lactase persistence first arose in Europe or what factors drove its rapid spread. Using a simulation model of the spread of lactase persistence, dairying, and farmers in Europe, integrating genetic and archaeological data the report shows that lactase persistence/dairying co-evolution began around 7,500 years ago in the Balkans and Central Europe, and not in the northern part of the continent, as previously thought.
Variants in neuropeptide Y receptors 1 and 5 are associated with nutrient-specific food intake and are under recent selection in Europeans Elbers CC, de Kovel CG, van der Schouw YT, Meijboom JR, Bauer F, Grobbee DE, Trynka G, van Vliet-Ostaptchouk JV, Wijmenga C, Onland-Moret NC Complex Genetics Section, Department of Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands PLoS One 2009;4:e7070
There is a large variation in caloric intake and macronutrient preference between individuals and between ethnic groups, and these food intake patterns show a strong heritability. The transition to new food sources during the agriculture revolution around 11,000 years ago probably created selective pressure and shaped the genome of modern humans. One major player in energy homeostasis is the appetite-stimulating hormone neuropeptide Y, in which the stimulatory capacity may be mediated by the neuropeptide Y receptors 1, 2 and 5 (NPY1R, NPY2R and NPY5R).
Context:
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Methods: The study assesses association between variants in the NPY1R, NPY2R and NPY5R genes, and nutrient intake in a cross-sectional, single-center study of 400 men aged 40–80 years, and examines whether genomic regions containing these genes show signatures of recent selection in 270 HapMap individuals (90 Africans, 90 Asians, and 90 Caucasians) and in 846 Dutch blood bank controls. Results: The results show that derived alleles in NPY1R and NPY5R are associated with lower carbohydrate intake, mainly because of a lower consumption of mono- and disaccharides. They also show that carriers of these derived alleles, on average, consume meals with a lower glycemic index (GI) and glycemic load (GL), and have higher alcohol consumption. One of these variants shows the hallmark of recent selection in Europe. Conclusions: The data suggest that lower carbohydrate intake, consuming meals with a low GI and GL, and/or higher alcohol consumption, gave a survival advantage in Europeans since the agricultural revolution. This advantage could lie in overall health benefits, because lower carbohydrate intake, consuming meals with a low GI and GL, and/or higher alcohol consumption, are known to be associated with a lower risk of chronic diseases.
The appetite-stimulating hormone neuropeptide Y (NPY) evokes through the NPY receptors eating behavior, inducing particularly carbohydrate intake. Injection of NPY in the brain elicits a strong feeding response even in satiated animals, eventually leading to obesity. The results of this study show that derived alleles in NPY1R and NPY5R are associated with lower relative carbohydrate intake, mainly because of a lower consumption of mono- and disaccharides. Carriers of these derived alleles on average consume meals with a lower glycemic index (GI) and glycemic load (GL). However, the same alleles are associated with increased alcohol consumption. The derived allele appears to be under recent selection in the European population, and probably originates from around 4,000 years ago. A predicted selective sweep of around 4,000 years ago fits the theory of adaptation to novel food sources during the agriculture revolution, which started in Europe around 6,000 years ago and was gradually further developed from that point on. The data suggest that a lower carbohydrate intake, consumption of meals with a low GI and GL, and/or higher alcohol consumption gave a survival advantage in Europeans during the agricultural revolution.
Homo floresiensis: a cladistic analysis Argue D, Morwood MJ, Sutikna T, Jatmiko, Saptomo EW Australian National University, Canberra, ACT, Australia
[email protected] J Hum Evol 2009;57:623–639 Context: The announcement of a new species, Homo floresiensis, a primitive hominin that survived until relatively recent times, is an enormous challenge to paradigms of human evolution. Until this announcement, the dominant paradigm stipulated that: (1) only more derived hominins had emerged from Africa, and (2) Homo sapiens was the only hominin since the demise of Homo erectus and Homo neanderthalensis. Resistance to H. floresiensis has been intense, and debate centers on two sets of competing hypotheses: (1) that it is a primitive hominin, and (2) that it is a modern human, either a pygmoid form or a pathological individual. Despite a range of analytical techniques having been applied to the question, no resolution has been reached. Methods: Here, they use cladistic analysis, a tool that has not, until now, been applied to the problem, to establish the phylogenetic position of the species. Results: The results produce two equally parsimonious phylogenetic trees. The first suggests that H. floresiensis is an early hominin that emerged after Homo rudolfensis (1.86 Ma) but before Homo habilis (1.66 Ma, or after 1.9 Ma if the earlier chronology for H. habilis is retained). The second tree indicates H. floresiensis branched after H. habilis.
In 2005, the Yearbook enthusiastically discussed the finding of a 20,000-year ancient hominin in Indonesia with a stature of 1 m and a small skull. Following several years of a debate whether this was a diseased H. sapiens or a new species, this series of articles conclude that it was indeed a new species. We were naive to assume that we all come from Africa during the 100–40,000 years ago ‘out of Africa’ migration. Body size reduction in mammals is usually associated with only moderate brain size reduction, because the brain and sensory organs complete their growth before the rest of the
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body during ontogeny. But the small skull of H. floresiensis may teach us otherwise. Indeed, this trend has been questioned in the special case of dwarfism of mammals on islands, the so-called insular dwarfism. The nature of the proportional change in brain size in insular dwarfism indicates that selective pressures on brain size are potentially independent of those on body size.
Concepts revised If injured, run in bare feet
Foot strike patterns and collision forces in habitually barefoot versus shod runners Lieberman DE, Venkadesan M, Werbel WA, Daoud AI, D’Andrea S, Davis IS, Mang’eni RO, Pitsiladis Y Department of Human Evolutionary Biology, Harvard University, Cambridge, Mass., USA
[email protected] Nature 2010;463:531–535 Background: Humans have engaged in endurance running for millions of years, but the modern running shoe was not invented until the 1970s. For most of human evolutionary history, runners were either barefoot or wore minimal footwear such as sandals or moccasins with smaller heels and little cushioning relative to modern running shoes. The authors wondered how runners coped with the impact caused by the foot colliding with the ground before the invention of the modern shoe. Methods: Kinematic studies of foot strike in 5 groups of runners habitually shod or unshod. Results: Habitually barefoot endurance runners often land on the fore-foot (fore-foot strike) before bringing down the heel, but they sometimes land with a flat foot (mid-foot strike) or, less often, on the heel (rear-foot strike). In contrast, habitually shod runners mostly rear-foot strike, facilitated by the elevated and cushioned heel of the modern running shoe. Kinematic and kinetic analyses show that even on hard surfaces, barefoot runners who fore-foot strike generate smaller collision forces than shod rear-foot strikers. This difference results primarily from a more plantar-flexed foot at landing and more ankle compliance during impact, decreasing the effective mass of the body that collides with the ground. Conclusion: Fore-foot- and mid-foot-strike gaits were probably more common when humans ran barefoot or in minimal shoes, and may protect the feet and lower limbs from some of the impact-related injuries now experienced by a high percentage of runners.
We generally tend to think that modern life utensils protect us from injuries and provide us with improved performances. This study, reminiscent of the Noble Savage theory [11], reminds us that human body evolution over millenaries cannot be ‘improved’ in 40 years by designers from Nike®, Reebok® and consorts. The authors are evolutionary biologists, bioengineers and physical medicine specialists and observed foot impact of various groups of runners while shod or unshod. ‘Modern’ usually shod runners mostly strike the heel first, while unshod runners from Kenya mostly strike the fore-foot first. The shod heel first pattern resulted in more repetitive high impact forces while unshod running was associated with a more diverse way of running, resulting in a lessened risk of injury. The findings of the study complement the endurance-running hypothesis for the transformation of the human body plan with the emergence of the genus Homo. The much earlier australopithecine version of bipedalism (as seen in ‘Lucy’, Australopithecus afarensis) received a major makeover near the Pliocene/Pleistocene boundary about 2 million years ago with longer hind limbs and shorter toes. If the endurance-running hypothesis is correct, the evolution of these features are probably linked directly to barefoot running as an integral part of an adaptive strategy for pursuit hunting [12]. Many shod modern-day runners develop injuries [13] and new footwear now use barefoot like design and might protect them from repetitive stress injury. Nowadays, every big marathon like New York, Paris or Berlin brings together 30,000–40,000 participants somehow holding an atavistic ceremony commemorating their ancient past. We might see them leaving their shoes in the locker room before long.
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Mechanism of the year MicroRNA and cholesterol regulation
MiR-33 contributes to the regulation of cholesterol homeostasis Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N, Fisher EA, Moore KJ, Fernandez-Hernando C Department of Medicine, Leon H. Charney Division of Cardiology and the Marc and Ruti Bell Vascular Biology and Disease Program, New York University School of Medicine, New York, N.Y., USA Science 2010;328:1570–1573
Cholesterol metabolism is tightly regulated at the cellular level. miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element-binding factor-2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate-binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Conclusion: miR-33 regulates both HDL biogenesis in the liver and cellular cholesterol efflux. Background: Results:
MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis Najafi-Shoushtari SH, Kristo F, Li Y, Shioda T, Cohen DE, Gerszten RE, Naar AM Massachusetts General Hospital Cancer Center, Charlestown, Mass., USA Science 2010;328:1566–1569 Background: Proper coordination of cholesterol biosynthesis and trafficking is essential to human health. The sterol regulatory element-binding proteins (SREBPs) are key transcription regulators of genes involved in cholesterol biosynthesis and uptake. Results: MicroRNAs (miR-33a/b) embedded within introns of the SREBP genes target the adenosine triphosphate-binding cassette transporter A1 (ABCA1), an important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport, for posttranscriptional repression. Antisense inhibition of miR-33 in mouse and human cell lines causes up-regulation of ABCA1 expression and increased cholesterol efflux, and injection of mice on a Western-type diet with locked nucleic acidantisense oligonucleotides results in elevated plasma HDL. Conclusion: miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis. miR-33 may represent a therapeutic target for ameliorating cardiometabolic diseases.
PubMed provides 8,186 citations to microRNA, which were discovered as late as 2001. They have emerged as essential players in gene regulation. Initially identified in the cancer field, which still constitutes three quarters of microRNA literature, they are now recognized as essential players in gene expression in all tissues. MicroRNAs are small (22-nt) endogenous double-stranded RNAs that regulate physiological processes at the posttranscriptional level [14, 15]. They bind to complementary target sites in the 3⬘ untranslated regions of mRNAs, causing translational repression and/or mRNA destabilization [3]. A single microRNA can have multiple targets, potentially regulating several genes involved in a physiological pathway. miR-122 has been previously implicated in cholesterol regulation [16]. These two papers further extend the role of microRNAs in metabolic regulation and miR-33 appears as a key regulator of HDL cholesterol metabolism. Using different approaches, the authors identified two isoforms of miR-33 that are embedded in introns of SREBP genes 1 & 2, encoding sterol regulatory element-binding proteins which in turn regulate the synthesis of fatty acids and cholesterol respectively. miR-33 targets ABCA1, a regulator of HDL synthesis, and implicated in Tangier disease, a condition of plasma HDL deficiency. The studies demonstrate an impact on macrophages and, to a lesser extent, on the liver. In vivo manipulation of miR-33 levels using lentiviral vectors increased plasma HDL levels by 25% using anti-miR-33 and decreased plasma HDL levels by 22% using miR-33. Decreased plasma HDL is a key feature of the metabolic syndrome, and miR-33 could
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play a central role downstream of the enhanced liver insulin signaling of the metabolic syndrome. Quite interestingly, the authors show that individuals with mutations in miR-33b might be protected from decreased HDL and the metabolic syndrome. Agents that antagonize miR-33 will have to be tested to evaluate their potential to increase plasma HDL levels and treat the metabolic syndrome. References 1. Aubourg P, Blanche S, Jambaque I, Rocchiccioli F, Kalifa G, Naud-Saudreau C, et al: Reversal of early neurologic and neuroradiologic manifestations of X-linked adrenoleukodystrophy by bone marrow transplantation. N Engl J Med 1990;322:1860–1866. 2. Aubourg P, Adamsbaum C, Lavallard-Rousseau MC, Rocchiccioli F, Cartier N, Jambaque I, et al: A two-year trial of oleic and erucic acids (Lorenzo’s oil) as treatment for adrenomyeloneuropathy. N Engl J Med 1993;329:745–752. 3. Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, et al: Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 1993;361:726–730. 4. Naldini L: Medicine. A comeback for gene therapy. Science 2009;326:805–806. 5. Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, et al: Projection of an immunological self-shadow within the thymus by the Aire protein. Science 2002;298:1395–1401. 6. Korn T, Bettelli E, Oukka M, Kuchroo VK: IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485–517. 7. Kisand K, Boe Wolff AS, Podkrajsek KT, Tserel L, Link M, Kisand KV, et al: Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines. J Exp Med 2010;207:299– 308. 8. Gavanescu I, Benoist C, Mathis D: B cells are required for Aire-deficient mice to develop multi-organ autoinflammation: a therapeutic approach for APECED patients. Proc Natl Acad Sci USA 2008;105:13009–13014. 9. Cheng MH, Fan U, Grewal N, Barnes M, Mehta A, Taylor S, et al: Acquired autoimmune polyglandular syndrome, thymoma, and an Aire defect. N Engl J Med 2010;362:764–766. 10. Fergusson D: Inappropriate referencing in research. BMJ 2009;339:b2049. 11. http://en.wikipedia.org/wiki/Noble_savage 12. Jungers WL: Biomechanics: barefoot running strikes back. Nature 2010;463:433–434. 13. Van Gent RN, Siem D, van Middelkoop M, van Os AG, Bierma-Zeinstra SM, Koes BW: Incidence and determinants of lower extremity running injuries in long distance runners: a systematic review. Br J Sports Med 2007;41:469–480. 14. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–297. 15. Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215–233. 16. Elmen J, Lindow M, Schutz S, Lawrence M, Petri A, Obad S, et al: LNA-mediated microRNA silencing in non-human primates. Nature 2008;452:896–899.
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Author Index A Abbott, G.W. 33 Abel, L. 222 Abel, U. 219 Abinun, M. 222 Abreu, A.P. 94 Acerini, C.L. 131 Adachi, M. 13 Adair, L.S. 177 Adewale, H.B. 86 Aerts, J. 220 Afrikanova, I. 89 Aguiar, R.C. 113 Ahmed, M.L. 163 Aitken, J.D. 149 Akl, E.A. 226 Aksglaede, L. 91, 193, 194 Akylbekova, E.L. 183 Al-Abdullah, I.H. 127 Al-Muhsen, S. 222 Al-Owain, M. 222 Albrechtsen, A. 176 Alekseev, A.E. 146 Allen, J.M. 131 Almholt, K. 46 Alos, N. 36 Alreja, M. 7 Altman, R.B. 174 Altshuler, D. 156 Ambresin, A.E. 145 Amit, I. 184 Amory, J.K. 1, 94 An, P. 175 Andari, E. 20 Andersen, S. 46 Anderson, G.M. 8 Anderson, M. 150 Anderson, M.J. 5 Andersson, A.M. 91 Andersson, J. 145 Andersson, S. 76 Andreasen, C.H. 156 Andrews, T.D. 220 Andrieux, J. 145 Anlag, K. 90 Antignac, J.P. 91 Aoki, C. 207 Arany, Z. 139 Ardlie, K. 156, 175 Ares, S. 189 Argue, D. 233 Ariyurek, Y. 175 Arkwright, P.D. 222 Arlt, W. 94, 107, 109 Armengaud, J.B. 196 Arnaldi, G. 19
Arnaud, P. 185 Aronin, N. 113 Asfaw, B. 229 Ashley, E.A. 174 Atzmon, G. 228 Au, M. 94 Au, M.G. 94 Aubourg, P. 219 Audit, M. 219 Auger, J. 70 Aulchenko, Y.S. 156, 177 Avbelj, M. 94 Avenant, C. 103 Avis, E. 37 Avis, H.J. 168 Avizov, L. 77 Avril, I. 208 Azuma, N. 13 Azzi, S. 55
B Bachelot, A. 117 Bacot, F. 176 Bader, M. 78 Bai, Z. 186 Bainbridge, M. 171 Bakker, B. 49 Balaji, S. 79 Balapure, A.K. 79 Balise, R.R. 39 Balkau, B. 145, 156, 175, 176 Ball, G.D. 165 Ballock, R.T. 72 Bamshad, M. 173 Bamshad, M.J. 172 Bangaru, M.L. 30 Bannink, E.M. 203 Bannykh, S. 26 Baranzini, S.E. 179 Barcenas-Morales, G. 222 Barkan, S. 38 Barlow, L.N. 70 Barnes, A.M. 67 Barnes, C. 220 Barroso, I. 156 Barter, P. 175 Bartholomae, C.C. 219 Barzilai, N. 228 Bastie, C.C. 147 Baten, E. 71 Battenhouse, A. 178 Bauer, F. 232 Baxter, R.C. 57 Beardsall, K. 163
Beaumont, M.A. 232 Beauvillain, J.C. 4 Beck-Peccoz, P. 37 Becker, D.J. 132 Beckers, M.C. 128 Beckmann, J.S. 145 Befroy, D. 44 Beilby, J.P. 175 Belgardt, B.F. 24 Belisle, A. 176 Bell, C.J. 179 Bellanné-Chantelot, C. 117 Bellesme, C. 219 Below, J.E. 124 Ben-Shlomo, Y. 175 Benagiano, G. 87 Benassai, M. 195 Bender, M.A. 184 Benediktsdottir, K.R. 181 Benediktsson, R. 175, 181 Benn, D.E. 108 Bennett, A.J. 155, 175, 177 Bennett, P.H. 159, 162 Benoist, C. 223 Bentley, G.E. 214 Bercovich, D. 77 Berenson, A.B. 166 Bergen 3rd, H.R. 66 Bergman, A. 228 Bergman, R.N. 156 Bergmann, S. 145, 175 Beri-Dexheimer, M. 145 Berlin, D.S. 174 Bernstein, B. 184 Berry, D.J. 177 Berton, A. 128 Bertrand, A.M. 35 Besenbacher, S. 181 Beuschlein, F. 61 Beyene, Y. 229 Bharaj, B. 124 Bibikova, M. 221 Bidet, M. 117 Bidlingmaier, M. 56 Biebermann, H. 42 Bielinski, S.J. 156 Biertho, L. 144 Bigham, A.W. 172 Bilo, H.J. 159 Bimpaki, E.I. 104 Bindels-de Heus, G.C. 49 Birney, E. 178 Biron, S. 144 Bistritzer, T. 38 Bizec, B.L. 91 Bjerre Knudsen, L. 46
Black, H.L. 70 Black, K.J. 126 Blakemore, A.I. 145 Blanche, S. 219, 222 Blangero, J. 212 Blaszczyk, K. 145 Blaumeiser, B. 145 Bliss, S.P. 23 Blom, E. 71 Bluestone, J.A. 218 Boaretto, F. 113 Boas, M. 197 Bocca, G. 49 Bochud, M. 175 Bochukova, E.G. 145 Bodemer, C. 222 Boehnke, M. 156, 182 Boerwinkle, E. 156, 175 Boesgaard, T.W. 156 Bogan, J.S. 10 Bohlius, J. 227 Bolu, E. 94 Bonnefond, A. 156, 175 Bonnycastle, L.L. 156, 175 Boomsma, D.I. 177 Boon, W.C. 4 Boonstra, R. 211 Borch-Johnsen, K. 156, 175, 176 Borecki, I.B. 175 Borg, J. 59 Boright, A.P. 124 Borja, J.B. 177 Bornstein, S.R. 103, 156, 175 Boscaro, M. 19 Bottcher, Y. 156, 175 Bouatia-Naji, N. 156, 175, 177 Boudou, P. 117 Bougneres, P. 219 Bougneres, P.F. 222 Bouligand, J. 1 Bouma, G.J. 89 Bouquillon, S. 145 Bourque, C.W. 10 Boute, O. 145 Bouwens, L. 128 Boyle, A.P. 178 Bozaoglu, K. 212 Bozec, A. 68 Bradfield, J.P. 125, 177 Brailly-Tabard, S. 1 Bram, R.J. 66 Brauner, R. 196 Breart, G. 196 Brillant, C. 227 Brinkmeier, M.L. 18 Brioschi, A. 145 Broberger, C. 213
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Brodesser, S. 24 Broekman, A.J. 52 Bronneke, H.S. 24 Broutin, I. 41 Brown, H.A. 108 Brown, J.W. 103 Brown, M.S. 27 Brown, W.A. 150 Brugts, M.P. 54 Bruning, J.C. 24 Brunner, E. 156, 175 Brunner-La Rocca, H.P. 200 Brunton, F. 68 Bryan, J. 119 Bucay, N. 89 Buchanan, T.A. 156 Buckingham, K.J. 172 Budagov, T. 228 Buddavarapu, K. 113 Bull, S.B. 124 Bumpstead, S.J. 156, 175 Burger, J. 232 Buriakovsky, S. 77 Burke, M.C. 5 Burton, P.R. 150 Bush, P.G. 74 Bustamante, J. 222 Butler, G.E. 189 Butler, J.L. 183 Butte, A.J. 174 Buxton, J.L. 145 Byars, S.G. 230
C Cabral, W.A. 67 Caccavelli, L. 219 Caiazzo, R. 145 Caillier, S.J. 179 Calandra, E. 195 Calcagno, A. 195 Caldarola, S. 217 Calebiro, D. 37 Calis, K.A. 109 Calmels, N. 145 Calton, L. 108 Camacho-Hubner, C. 59 Campbell, P. 220 Campbell, P.J. 186 Camper, S.A. 18 Campion, D. 145 Candi, E. 217 Cannata, S.M. 217 Cant, A.J. 222 Canty, A.J. 124 Cao, X. 67 Capalbo, D. 198 Cardon, L.R. 182 Carel, J.-C. IX, 207, 219
Carless, M. 212 Carling, C.L. 226 Carlsson, L. 145 Carmignac, D. 29 Caron, H.N. 202 Carpenter, L.L. 105 Carrara, S. 87 Carrasco, N. 33 Carré, A. 33, 41 Carroll, J. 94 Carter, E.M. 67 Carter, N.P. 220 Carter, R.C. 151 Cartier, N. 219 Carvalho, F.A. 149 Carvalho, L.R. 18 Casanova, J.L. 222 Casini, M.R. 195 Cassou, M. 29 Castanet, M. 33, 35, 41 Castillo, A.B. 70 Castinetti, F. 18 Cauchi, S. 176 Cavalcanti-Proenca, C. 156, 175, 176 Cavazzana-Calvo, M. 219 Cawthon, R.M. 228 Cawthorne, M.A. 212 Cerrato, F. 1 Cesareni, G. 217 Chahal, H. 107 Chakravarti, A. 182 Chalian, R. 221 Chalifa-Caspi, V. 77 Chalumeau, M. 196 Chan, O. 10 Chan, Y.M. 1 Chang, W. 67 Chanson, P. 1 Charkaluk, M.L. 196 Charmandari, E. 99 Charoen, P. 177 Charpak, S. 29 Charpentier, G. 156, 175, 176 Chase, H.P. 133 Chassin, L.J. 131 Chatterjee, K. 44 Cheetham, T. 37 Chen, C.M. 177 Chen, D.C. 171 Chen, F. 145 Chen, H. 142, 148 Chen, M. 148 Chen, R. 174 Chen, Y.D. 156, 175 Chera, S. 208 Chevre, J.C. 145 Chia, D.J. 62
Author Index
Chiarelli, F. 119 Chiesa, J. 145 Chines, P. 175 Chines, P.S. 156 Chipman, J.J. 58 Chiumello, G. 37 Cho, J.H. 182 Cho, M. 228 Chocron, E.S. 113 Choi, J.W. 66 Chou, M.F. 174 Chou, Y.Y. 97 Chowdhury, V.S. 214 Chrabieh, M. 222 Christian, H. 29 Chrousos, G.P. 99, 100, 111, 114 Church, G.M. 174 Cianfarani, S. 49, 51 Cianflone, K. 144, 199 Cirulli, V. 89 Clark, A. 128 Clark, A.G. 182 Clark, A.J. 30 Clarke, M. 227 Clarke, R. 175 Clarkson, A. 108 Clarkson, J. 4 Clauin, S. 117 Clayton, D.G. 180 Clayton-Smith, J. 145 Cleary, P.A. 122, 124 Clement, A. 41 Clendenen, L.E. 179 Clifton, D.K. 6 Coats, J.K. 2 Cobat, A. 222 Coetzee, W.A. 146 Coffin, J.M. 220 Cohen, D.E. 235 Cohen-Kettenis, P. 85 Coin, L.J. 145, 175, 177 Colao, A. 198 Cole, S.A. 212 Cole, T.R. 94 Collins, F.S. 156, 178, 182 Condon, K.W. 70 Cone, R.D. 9 Confavreux, C. 79 Connors, A. 210 Conrad, D.F. 220 Conroe, H.M. 139 Conway-Campbell, B.L. 102 Cooney, A.J. 90 Cooper, C. 156, 175 Cooper, M.N. 175 Cooper, R. 183 Corbetta, C. 37 Cordier, M.P. 145
Author Index
Cordoba-Chacon, J. 22 Cornelis, M. 156, 175 Corsino, L. 215 Cortinovis, F. 37 Costa, E.M. 94 Costigan, C. 222 Coudoré, F. 111 Counil, F. 41 Couper, D.J. 156 Courant, F. 91 Cousminer, D.L. 177 Coussieu, C. 117 Coutifaris, C. 221 Coutry, N. 29 Cowley, M.A. 143 Cox, N.J. 124, 182 Crawford, G. 175 Crawford, G.E. 178 Crawford, G.J. 156 Cresswell, J.A. 10 Crisponi, L. 175 Crowley Jr, W.F. 1, 94 Crowley, W.F. 94 Cuisset, J.M. 145 Cullender, T.C. 149 Curran, J.E. 212 Curran, S. 44 Curtis, J.M. 159 Cutler, G. 58 Cuttler, L. 58, 210 Czarnywojtek, A. 192
D Dabelea, D. 121 Dahia, P.L. 113 Daito, T. 220 Dal-Cortivo, L. 219 Damron, T.A. 74 D’Andrea, S. 234 d’Anglemont de Tassigny, X. 4 Daniels, S.R. 157, 158 Danis, R.P. 122 Daoud, A.I. 234 Date, Y. 9 Dateki, S. 13 Dathe, K. 71 Dattani, M.T. 13, 16 Daubas, C. 55 Davalos, A. 235 Davey, S.G. 177 David, A. 145 Davis, I.S. 234 Davis, M.D. 122 Day, I.N. 175 de Boer, A.S. 46 de Castro, M. 94 De Clerck, N. 75
De Deken, X. 42 de Escobar, G.M. 189 De Felici, M. 217 de Gendt, K. 3 de Geus, E.J. 175, 177 de Guillebon, A. 1 de Jong, F.H. 114 de Kort, S.W. 50 de Kovel, C.G. 232 de la Pena, A. 58 de Lind van Wijngaarden, R.F. 49, 199 De Luca, F. 56 de Mendonca, B.B. 94 de Muinck Keizer-Schrama, S.M. 201, 203, 204 De Palma, A. 131 de Ravel, T. 71 de Ridder, M.A. 201 de Roux, N. 1 De Schutter, T. 75 de Seranno, S. 4 de Smith, A.J. 145 De Waele, E. 128 de Wit, C.C. 52 Debono, M. 110 Dedoussis, G.V. 175, 177 Dehghan, A. 175 Dejonckere, P.H. 201 Dekker, J. 184 Deladoëy, J. 36 Delarue, C. 111 Delemarre-van de Waal, H.A. 85, 201, 204 Delobel, B. 145 Deloukas, P. 177 Delplanque, J. 156, 175 Dent, K.M. 172 Denzer, C. 139 Denzin, L.K. 129 Depinho, R.A. 69 Derr, M.A. 52 Desarmenien, M.G. 29 Desgraz, R. 208 Determann, C. 65 Dewey, F.E. 174 Di Bartolomeo, C. 217 Di Cosmo, C. 45 di Iorgi, N. 195 Di Tella, L. 217 Dimeglio, L.A. 190 Dina, C. 156, 175, 176 Dinh, H. 171 Diorio, J. 20 Dixon, J.B. 150 Djulbegovic, B. 227 Dobbelaar, P.H. 142 Doffinger, R. 222 Dolan, L.M. 157, 158
239
Donahue, S.W. 70 Donaldson, S.S. 39 Dones, V. 200 Doney, A. 156, 175 Dong, Y. 142 Donnelly, S. 130 Dorschner, M.O. 184 Drmanac, R. 173 Drop, S. 58 Drop, S.L. 49, 114 Drucker, D.J. 46 Drummer, T.D. 70 Drummond, K. 130 Du, M. 83 Duchen, M.R. 137 Ducy, P. 79 Dudley, J.T. 174 Dufour, S. 44 Dufrane, D. 128 Duhamel, J.R. 20 Duittoz, A. 3 Dumalska, I. 7 Dumitrescu, A.M. 45 Dunger, D.B. 131, 163 Dunkel, L. 93, 95, 96 Dupuis-Girod, S. 145 Dupuis, J. 156, 175 Durand, E. 176 Dwyer, A. 94 Dwyer, A.A. 94 Dyer, T.D. 212
E Eble, A. 25 Ebrahim, S. 175 Edwards Jr, J.E. 224 Egan, J.M. 156, 175 Egberts, K.J. 191 Egerod, F.L. 46 Egger, M. 227 Ehrhart-Bornstein, M. 103 Eicher, E.M. 89 Eichler, E.E. 182 Eisenbarth, G. 218 Eisenberg, D. 21 Eisenberger, T. 90 Eisenhofer, G. 103 Eizirik, D.L. 128 El-Sayed Moustafa, J.S. 145 El Yandouzi, T. 29 Elbers, C.C. 232 Elefteriou, F. 73 Elks, C. 171 Elleri, D. 131 Elliott, A. 175 Elliott, P. 145, 155, 175, 176, 177 Ellis, R.J. 145
240
Ellsworth, B.S. 18 Elmquist, J.K. 7 Eng, C. 39 Engel, J.B. 103 Engert, A. 227 Engstrom, E. 59 Enriori, P.J. 143 Epaud, R. 41 Epstein, M. 115 Erdos, M.R. 156, 175, 178 Eriksson, J.G. 177 Erinle, O. 221 Erler, A. 103 Ernst, M.B. 24 Escher, S.A. 14 Esko, T. 145 Ester, W.A. 52 Estrella, C. 4 Eugster, E.A. 36, 190 Evans, A.E. 143 Evans, D.B. 39 Evans, D.M. 177 Evans, G.L. 66 Ewbank, D. 230 Eyre, D.R. 67
F Falchi, M. 145 Faloia, E. 19 Fang, P. 52 Fantin, N.J. 171 Farina, V. 198 Farmer, A.D. 179 Farooqi, I.S. 44, 145 Fedson, A.C. 175 Feil, R. 185 Feldmann, D. 41 Feldt-Rasmussen, U. 197 Fellmann, F. 145 Feng, X. 67 Fenton, A.A. 207 Ferguson-Smith, A.C. 181 Fernandes, S. 103 Fernandez-Fuente, M. 29 Fernandez-Hernando, C. 235 Ferrannini, E. 120 Ferrarini, A. 145 Ferreri, K. 127 Ferrucci, L. 156 Festen, D.A. 49 Feuk, L. 220 Fey, M.F. 227 Fierz, Y. 59 Filippi, V. 87 Filler, S.G. 224 Fine, E. 33 Fingerlin, T.E. 121 Firmann, M. 156
Firth, S.M. 57 Fischer, A. 219, 222 Fischer-Posovszky, P. 139 Fischer-Rosinsky, A. 175 Fisher, D.A. 189 Fisher, E.A. 235 Fisher, P.J. 23 Fisher, S.K. 143 Fishman, L.M. 103 Fitzgerald, M.L. 235 Fitzgerald, T. 220 Flamez, D. 128 Flannery, P. 215 Florez, J.C. 156 Fluck, C.E. 25 Fong, T.M. 142 Fontanaud, P. 29 Forman, M.R. 92 Forouhi, N.G. 156, 175 Forrester, T. 183 Forsblom, C. 134 Foster, M. 221 Fourie, C.M. 53 Fox, C.S. 156, 175 Frackelton, E. 125 Francis, G.L. 39 Francoeur, D. 36 Frangioni, J.V. 148 Franklin, C.S. 175 Franks, P.W. 162, 175 Frants, R. 175 Franzosi, M.G. 175 Fraser, E.J. 2, 88 Fraser, W.D. 65 Frassinetti, C. 195 Fratangeli, N. 195 Frayling, T.M. 155, 156, 177 Freathy, R.M. 155, 177 Friberg, L.E. 59 Frigge, M.L. 181 Froguel, P. 145, 156, 176, 177 Frohman, L.A. 22 Frost, J. 185 Fu, Y. 224 Fujihara, H. 78 Fukami, M. 13 Furey, T.S. 178 Furlani, G. 19 Furuyashiki, T. 78 Futreal, P.A. 186
G Gagel, R.F. 39 Gagen, K. 142 Gagne, C. 168 Gaillard, M. 145 Galan, P. 175
Author Index
Galand-Portier, M.B. 117 Galas, D.J. 173 Gandhi, R. 106 Gans, R.O. 159 Ganusova, E.E. 179 Gao, X.B. 79 Gao, Y. 199 Gao, Z. 158 Garcia-Segura, L.M. 75 Gardiner, R. 130 Gaspert, A. 1 Gastaldi, R. 195 Gaughan, J.P. 221 Gautron, L. 7 Gavrilova, O. 148 Gawlik, A. 65 Ge, R. 186 Gebauer, L. 103 Gehman, C. 171 Gemelli, T. 7 Gerbin, M. 146 Gerry, D. 112 Gershon, M.D. 79 Gerszten, R.E. 235 Gesty-Palmer, D. 215 Gevers, E.F. 13, 201 Gewirtz, A.T. 149 Ghamari-Langroudi, M. 9 Gharib, H. 39 Ghervan, C. 1 Ghezzi, M. 195 Gianello, P. 128 Gianetti, E. 94 Gibbs, R.A. 171 Gibson, G. 182 Gieger, C. 175 Gill, A.J. 108 Gillerot, Y. 71 Gillman, M.W. 177 Gingrich, J.A. 79 Giralt, M. 212 Gitelman, S.E. 132 Giusti, V. 145 Glaser, B. 177 Glavas, M.M. 143 Glazer, N.L. 156, 175 Gloyn, A.L. 175 Glusman, G. 173 Gnirke, A. 184 Godeau, F. 55 Godi, M. 25 Goding, J. 77 Goel, A. 175 Gojobori, T. 220 Gokcumen, O. 220 Goland, R. 132 Goldenberg, A. 145 Goldhamer, D.J. 146 Goldman, S. 128
Author Index
Goldstein, D.B. 182 Goldstein, J.L. 27 Golmard, J.L. 117 Golombek, S.G. 189 Gomez, R. 179 Gonfloni, S. 217 Gong, L. 174 Gonzaga-Jauregui, C. 171 Gonzalez, F.J. 212 Gonzalez, L. 143 Gonzalez-Losada, T. 40 Gonzalez-Suarez, C. 200 Goodarzi, M.O. 156, 175 Goodman, N. 128, 173 Goodyer, P. 130 Gooren, L.J. 85 Gordon, R.C. 43 Goriely, A. 222 Goring, H.H. 212 Gorski, J. 142 Gotfredsen, C. 46 Gotherstrom, A. 231 Gottlieb, P.A. 132 Govaerts, L.C. 52 Govindaraju, D.R. 228, 230 Graessler, J. 156, 175 Graff, M. 85 Grallert, H. 156, 175 Grant, S.F. 125, 177 Grarup, N. 156, 175 Grattan, D.R. 8 Gratton, A. 20 Graubard, B.I. 92 Gray, A.R. 43 Grayson, B.E. 143 Greely, H.T. 174 Greenbaum, C.J. 132 Greenberg, S.A. 225 Grignon, A. 36 Grigoriadis, A.E. 68 Grills, N.J. 169 Grimmer-Somers, K. 200 Grobbee, D.E. 232 Groenier, K.H. 159 Groop, L. 156 Groop, P.H. 134, 135 Groudine, M. 184 Grove, K.L. 143 Groves, C.J. 155, 175, 177 Grueters, A. 42 Grundy, S. 175 Guan, X.-M. 142 Gubler, M.C. 130 Gudbjartsson, D.F. 181 Gudjonsson, S.A. 181 Guerau-de-Arellano, M. 223 Guibourdenche, J. 35 Guillot, L. 41 Guilmatre, A. 145
Guiochon-Mantel, A. 1 Guo, D. 171 Guo, X.E. 79 Gupta, D. 148 Gupta, R.K. 139 Gutman, S. 26 Guttmacher, A.E. 182 Gwilliam, R. 175 Gygi, S.P. 148 Gylfason, A. 181 Gyllensten, U. 175
H Haby, M.M. 151 Hacein-Bey-Abina, S. 219 Hadad, Y. 77 Hadjadj, S. 175, 176 Hadjikhani, N. 145 Hager, G.L. 102 Haile-Selassie, Y. 229 Haiman, C.A. 183 Haines, J.L. 182 Hainsworth, D.P. 122 Hakonarson, H. 125, 177 Hall, A.C. 74 Hall, J.E. 94 Hallmans, G. 175 Halperin, J.J. 171 Halsall, D. 44 Halyday, B. 169 Hamilton-Shield, J. 145 Hammond, G. 108 Hammond, N. 175 Hampel, B. 24 Han, X. 175 Hanada, R. 78 Hanada, T. 78 Hansen, R.M. 222 Hansen, T. 156, 175, 176 Hanson, R.L. 159, 162 Hantel, C. 61 Hanukoglu, A. 38 Hanyaloglu, A.C. 97 Hapgood, J.P. 103 Harada, N. 2, 88 Haring, D.A. 49 Harjutsalo, V. 134, 135 Harmon, J.S. 119 Harris, J. 131 Hartikainen, A.L. 145, 175, 177 Hartmann, T. 200 Hasegawa, K. 13 Hassanali, N. 175, 177 Hassanein, M.T. 183 Hattersley, A.T. 155, 156, 175, 177 Hauser, S.L. 179
241
Hausler, G. 77 Hay, I.D. 40 Hayek, A. 89 Hayes, F.J. 94 Hayward, C. 175 He, S. 142 Heath, S.C. 175 Hebert, J.M. 174 Hebestreit, H. 200 Hechenberger, G. 107 Hefferan, T.E. 66 Hegedus, L. 197 Hegelund, A.C. 46 Heino, T. 65 Heinrich, J. 177 Hekkala, A. 123 Hellstrom, A. 59 Hembree, W.C. 85 Hen, R. 79 Henderson, B. 183 Henneman, P. 175 Hennig, B.P. 71 Henning, E. 44, 145 Herbison, A.E. 4, 8 Herbrecht, E. 20 Hercberg, S. 175 Herder, C. 175 Herich, R. 55 Hermanns, P. 36 Hermesz, E. 15 Hero, M. 95, 96 Herold, K. 218 Herrera, P.L. 208 Herrin, J. 226 Hershey, T. 126 Hershkovitz, E. 38, 77 Hicks, A.A. 175 Hietala, K. 134 Hillman, D.R. 175 Hills, F.A. 185 Hilsted, L. 197 Hindorff, L.A. 182 Hingorani, A. 156 Hingorani, A.D. 175 Hiort, O. 71 Hirschhorn, J.N. 177, 183 Hivert, M.F. 156 Hoang, X.H. 94 Hochberg, Z. IX, 207, 219 Hodges, L.M. 174 Hodgson-Zingman, D.M. 146 Hodson, D. 29 Hoefels, S. 116 Hofland, L.J. 54 Hofman, A. 175, 177 Hofmann, J. 107 Hokken-Koelega, A. 163 Hokken-Koelega, A.C. 49, 50, 52, 199, 201
242
Holder-Espinasse, M. 145 Holly, J.M. 177 Holmlund, G. 231 Holter, N.I. 90 Honda, S. 2, 88 Honda, T. 220 Hondares, E. 212 Honetschlager, J.A. 40 Hong, T. 189 Hood, L. 173 Hoorweg-Nijman, J.J. 49 Hopkins, P.N. 168 Hoque, N. 216 Horblitt, A.M. 10 Horie, M. 220 Horjales-Araujo, E. 213 Horvath, T.L. 79 Hosseini, S.M. 124 Hoste, C. 42 Hottenga, J.J. 175, 177 Houang, M. 55 Houdijk, E.C. 49 Hould, F.S. 144 Hovorka, R. 131 Hovorka, T. 131 Howard, A.D. 142 Howell, J. 140 Hreidarsson, A.B. 181 Hsia, J. 168 Hu, F.B. 156, 175 Hu, J. 67 Hu, M. 220 Huang, N. 145 Hubley, R. 173 Hudgins, L. 174 Hudson, Q.J. 89 Huff, C.D. 172, 173, 186 Huffman, D.M. 228 Hughes, V.A. 94 Huhtaniemi, I. 97 Hui, J. 175 Huihjbregts, L. 1 Huisman, H.W. 53 Hung, J. 175 Hunter, D.J. 182 Huntley, J.J. 179 Hurles, M.E. 145, 220 Hurt, D.E. 111 Hutten, B.A. 168 Hwa, V. 52 Hypponen, E. 155, 177 Hytinantti, T. 76
I Ibrahim, A.S. 224 Ichijo, T. 111 Iglesias, R. 212 Ihm, C.H. 220
Ikuta, K. 220 Iliopoulos, D. 104 Illig, T. 156 Ilonen, J. 123 Imakaev, M. 184 Ingelsson, E. 156, 175 Irikat, R.K. 171 Isidor, B. 145 Isomaa, B. 156, 175 Itan, Y. 232 Itoh, T. 136 Iyer, V.R. 178
J Jabs, E.W. 172 Jackson, A.U. 156, 175 Jacobsen, G.K. 222 Jacobsen, H. 46 Jacobsen, S.D. 46 Jacobson, P. 145 Jacquemont, S. 145 Jaddoe, V.W. 177 Jakob, S. 90 Jakobsson, M. 231 Jamar, J.F. 128 Jansen, M. 201 Janssen, J.A. 54 Jarvelin, M.R. 145, 155, 176, 177 Jatmiko 233 Jaubert, F. 41 Jefferson, W.N. 86 Jern, P. 220 Jessberger, S. 21 Jetha, M.M. 165 Ji, I. 97 Ji, T.H. 97 Jian, T. 142 Jiang, L. 143 Jiang, M. 142 Jing, C. 171 Jira, P.E. 49 Johannsson, O.T. 181 John, S. 102 Johnson, B.D. 52 Johnson, M.D. 167 Johnson, P.M. 141 Johnson, P.R. 175 Johnson, T. 156, 175 Johnson, T.A. 102 Jonas, K. 97 Jonasdottir, A. 181 Jonsson, H. 14 Jonsson, T. 181 Jonveaux, P. 145 Jorde, L.B. 173, 186 Jorgensen, T. 156, 175 Jowett, J.B. 212
Author Index
Jula, A. 175 Juntti, S.A. 88 Juul, A. 91, 92, 193, 194
K Kaakinen, M. 175, 177 Kageyama, H. 9 Kaido, T. 89 Kaiser, U.B. 94 Kajimura, S. 148 Kamp, G.A. 204 Kan, Y. 142 Kandeel, F. 127 Kanoni, S. 175, 177 Kansra, S. 30 Kao, H.T. 105 Kao, W.H. 156, 175 Kaprio, J. 175 Karki, S. 168 Karpe, F. 175 Karperien, M. 75 Karsenty, G. 79 Karvonen, M. 135 Kasparov, S. 4 Kastelein, J.J. 168 Katari, S. 221 Katz, M. 228 Ke, Z. 143 Keefe, D. 178 Keil, M.F. 106 Kelberman, D. 16 Keller, G.M. 68 Kelly, T.M. 142 Kemp, C.J. 8 Kennedy, M. 68 Kennedy, O. 70 Kenny, P.J. 141 Keogh, J. 44, 145 Kerkhof, G.F. 163 Kerkhof, M. 177 Kesaniemi, Y.A. 175 Ket, J.L. 202 Keuper, M. 139 Khan, O.A. 179 Khankhanian, P. 179 Khoury, P.R. 157, 158 Khrebtukova, I. 179 Kiermer, V. 219 Kilic, S.S. 78 Kim, C. 125 Kim, F.S. 60 Kim, M.S. 109 Kim, N.H. 159 Kim, R.W. 179 Kimball, T.R. 157, 158 Kineman, R.D. 22 King, E.C. 33 King, E.E. 113
Author Index
Kingsmore, S.F. 179 Kino, T. 99, 100, 111 Kinoshita, E. 13 Kirigiti, M.A. 143 Kirin, M. 177 Kitaoka, S. 78 Kivimaki, M. 156, 175 Klaere, S. 78 Klasen, C. 90 Kleefstra, N. 159 Klein, R. 130 Klein, T.E. 174 Klemenhagen, K.C. 79 Klingensmith, G.J. 121 Klinger, F.G. 217 Kloos, R.T. 39 Klopocki, E. 71 Kluge, S. 227 Klugmann, C. 90 Knight, B. 175 Knight, B.A. 177 Knight, R. 149 Knip, M. 123 Knowler, W.C. 159, 162 Knowles, J.W. 174 Koba, W. 33 Kobayashi, Y. 220 Kode, A. 69 Kohno, K. 208 Kohrle, J. 192 Kok, J.H. 189 Kok, R.C. 114 Koll, R. 71 Kollman, C. 131 Kolonel, L.N. 183 Komnenovic, V. 78 Kong, A. 181, 182 Koning, C.C. 202 Konstantopoulos, N. 212 Kooy, R.F. 145 Koppelman, G.H. 177 Korada, S.M. 37 Kosaka, K. 13 Kosinski, J. 142 Koskinen, S. 175 Kotitschke, A. 103 Kottgen, A. 156 Kousteni, S. 69 Kovacs, P. 156, 175 Krajewski, S.J. 5 Kral, J.G. 144 Krause-Steinrauf, H. 132 Krebs, C. 211 Kremer Hovinga, S. 75 Kremer, L.C. 202 Kress, J. 90 Kriemler, S. 200 Krishnan, S. 22 Kristiansson, K. 220
Kristinsson, K.T. 181 Kristo, F. 235 Kristoffersen, D.T. 226 Kristrom, B. 14 Krohn, K. 156 Krude, H. 42 Kruger, G. 71 Kruglyak, L. 182 Ku, H.T. 127 Kublaoui, B.M. 7, 160 Kubota, K. 148 Kucera, K.S. 178 Kulaga, H. 139 Kumar, S. 167 Kumararatne, D. 222 Kumari, M. 156, 175 Kurg, A. 145 Kurland, I.J. 147 Kuroda, A. 127 Kurshan, N. 59 Kutlu, B. 128 Kutschera, I. 219 Kuttenn, F. 117 Kuusisto, J. 156, 175 Kwok, P.Y. 179 Kyvik, K.O. 175
L La Gamma, E.F. 189 Laakso, M. 156, 175 Labalme, A. 145 Lacampagne, A. 29 Lachin, J.M. 124, 132 Lafont, C. 29 Lahde, J. 123 Lahlou, N. 219 Lai, E.W. 148 Laitinen, E.M. 93 Laitinen, J. 176, 177 Laitinen, K. 76 Lajic, S. 97 Lajoie, B.R. 184 Lajunen, T. 175 Lamb, M.M. 121 Lamberg-Allardt, C. 76 Lambert-Langlais, S. 111 Lamberts, S.W. 54 Lammi, N. 135 Lander, E.S. 184 Landman, G.W. 159 Landreth, G.E. 23 Lang-Muritano, M. 1 Lange, L.A. 177 Langenberg, C. 156, 175 Lango, H. 155 Langslet, G. 168 Lankester, A. 56 Lann, D. 59
243
Larsen, A.M. 131 Lathrop, G.M. 175 Latronico, A.C. 94 Laurie, C. 150 Lauritzen, T. 156, 176 Lavoie, H.B. 1 Lawlor, D.A. 175 Lazarus, J. 44 Le Bacquer, O. 156 Le, B.O. 175 Le Bouc, Y. 55 Le Caignec, C. 145 Le, M.L. 183 Le Tissier, P. 29 Lebel, S. 144 Leboulch, P. 219 Leboyer, M. 20 Lechleiter, J.D. 113 Lecoeur, C. 145, 156, 175 Lecoq, J. 29 Lee, A.S. 224 Lee, B.K. 178 Lee, C. 172, 220 Lefebvre, H. 111 Lefkowitz, R.J. 215 Lefrançois-Martinez, A.M. 111 Lefrere, F. 219 Legay, C. 55 Leheup, B. 145 Lei, W. 67 Leibbrandt, A. 78 Leikin, S. 67 Leitner, Y. 195 Lemaitre, M.P. 145 Lenci, R.E. 113 Lerner, D.J. 33 LeRoith, D. 59 Leroy, D. 4 LeSauter, J. 216 Lescelleur, O. 144 Lettiero, T. 198 Lettre, G. 183 Leunissen, R.W. 50, 163, 199 Leventhal, N. 77 Levy-Litan, V. 77 Levy-Marchal, C. 156 Lewanczuk, R.Z. 165 Lewrick, F. 61 Ley, D. 59 Ley, R.E. 149 l’Homme, B. 219 Li, C. 143 Li, L. 73 Li, M. 156, 175 Li, Q. 125 Li, R.L. 27 Li, W. 143 Li, Y. 175, 235
244
Li, Z. 79 Lian, N. 73 Liang, G. 27 Liao, X.H. 45 Liden, K. 231 Lieb, J.D. 178 Lieberman-Aiden, E. 184 Lieberman, D.E. 234 Liere, P. 3 Lightman, S.L. 102 Lilic, D. 222 Lin, L.S. 142 Lind, L. 175 Linderholm, A. 231 Lindgren, C.M. 155, 175, 177 Lindquist, L. 66 Link, M.P. 39 Liu, J. 142, 183 Liu, M. 224 Liu, X. 8 Liu, X.S. 79 Liu, Y. 143 Liu, Z. 178 Liu, Z.W. 79 Lo Vecchio, A. 198 Lobbens, S. 145 Loche, S. 195 Lochmatter, D. 25 Lofqvist, C. 59 Lofrano, A. 94 Lombardi, G. 198 Lombes, M. 1 Lonneux, M. 128 Looker, H.C. 162 Loos, R.J. 156, 175 Loqman, M.Y. 74 Lord, J.M. 107 Loredana Marcovecchio, M. 119 Lorenz-Depiereux, B. 77 Lorenzo, F.R. 186 Lorini, R. 195 Losekoot, M. 52 Louiset, E. 111 Louw, R. 53 Lovejoy, C.O. 229 Lovell-Badge, R. 16, 90 Loyens, A. 4 Lu, X. 79 Luan, J. 156, 175 Lunsford, E. 148 Luo, S. 179 Luotoniemi, E. 95 Lupski, J.R. 171 Luque, R.M. 22 Luton, D. 35 Luttrell, L.M. 215 Lutz, K. 133 Lyon, H.N. 177, 183
Lyons, D.J. 213 Lyssenko, V. 156, 175
M McArdle, W.L. 177 Macarthur, D.G. 220 McAteer, J.B. 156, 175 McCarroll, S.A. 175, 182 McCarthy, M.I. 145, 155, 156, 177, 182 McConnell, V. 222 McCoy, C.E. 157 McCrimmon, R.J. 10 McCrindle, B.W. 168 McCulloch, L.J. 175 McDaniell, R. 178 Macdermot, K.D. 145 Macdonald, J.R. 220 McDonald, K.L. 108 McDonough, S.P. 23 McElroy, J.P. 179 McGee-Lawrence, M.E. 70 McGee, P.F. 132 McGowan, S.J. 222 McGrice, M. 150 McGuire, A.L. 171 Machtay, M. 227 Mackay, T.F. 182 McKenna, M.A. 102 McKenna, M.P. 36 McKenzie, C. 183 McMullen, N.T. 5 McNay, E.C. 10 McNelis, J.C. 107 McPherson, R. 175 McRorie, E. 65 McVean, G.A. 222 Madden, B.J. 66 Madsen, L.W. 46 Maeda, K. 6 Maghnie, M. 195 Magi, R. 175, 177 Magnusson, P.K. 175 Mahlaoui, N. 219 Mahley, R. 175 Maier, W. 116 Mailloux, C.M. 15 Main, K.M. 197 Maiorana, A. 51 Maisuria-Armer, M. 180 Maji, S.K. 21 Makareeva, E. 67 Makitie, O. 76, 96 Malan, L. 53 Malan, N.T. 53 Malan, V. 145 Malmstrom, H. 231 Mandel, J.L. 145
Author Index
Mang’eni, R.O. 234 Mangino, M. 175 Manin, M. 111 Mann, J.J. 79 Mannik, K. 145 Manning, A.K. 156, 175 Manoli, D.S. 2 Manolio, T.A. 182 Manor, E. 77 Mantero, F. 112 Marais, A.D. 168 Marceau, P. 144 Marceau, S. 144 Mardis, E. 182 Marechal, D. 128 Mari, A. 120 Marini, J.C. 67 Mariniello, B. 112 Marinova, D. 210 Marks, J.B. 132 Marlowe, F.W. 209 Marmot, M. 156, 175 Marodi, L. 222 Marre, M. 176 Marsella, S.A. 105 Marsh, D.J. 142 Marsh, J.A. 177 Martillotti, T. 129 Martinet, D. 145 Martinez, A. 111 Martinez-Larrad, M.T. 175 Martinic, M. 223 Martorell, R. 85 Maruyama, I. 136 Masson, G. 181 Mathieu-Dramard, M. 145 Mathis, D. 223 Matsuoka, N. 136 Mattei, M. 217 Matthews, P. 84 Mauer, J. 24 Mauer, M. 130 Maury, S. 95 May, G.D. 179 Mayack, S.R. 60 Mayor, V. 156 Mazur, A. 176 Meaney, M.J. 20 Medhamurthy, R. 79 Meehan, R. 210 Meigs, J.B. 156 Meijboom, J.R. 232 Meisinger, C. 175 Melino, G. 217 Melino, M. 168 Melmed, S. 26 Melzer, D. 175 Meneton, P. 175 Menke, L.A. 201
Author Index
Mennessier, G. 29 Mepani, R.J. 139 Mera, T. 136 Mercer, M.B. 210 Merke, D.P. 106, 109 Mertens, A.R. 62 Mester, J. 55 Metspalu, A. 145 Metz, H. 28 Metzger, J.M. 142 Meyer 3rd, W.J. 85 Meyer, U. 200 Meyre, D. 145, 156, 175, 176 Meyts, E.R. 222 Mhaouty-Kodja, S. 3 Miettinen, M. 76 Millar, R.P. 1, 214 Miller, A. 23 Miller, N.A. 179 Miller, R. 142 Mimata, H. 78 Minakata, H. 214 Miot, F. 42 Mirny, L.A. 184 Misir, S. 168 Misso, M.L. 191 Mitchell, B.D. 156, 175 Mitchell, C.S. 44 Mittelstaedt, D. 219 Moczko, J. 192 Moebus, V. 227 Mogi, K. 6 Mohlke, K.L. 156, 177 Mølck, A.M. 46 Moley, J.F. 39 Molino, F. 29 Mollard, P. 29 Molnar, P. 231 Monahan, P. 17 Mondal, M.S. 9 Monk, D. 185 Monteau, F. 91 Montgomery, A.M. 89 Montori, V.M. 85, 226 Montpetit, A. 176 Moodie, M.L. 151 Mook-Kanamori, D.O. 177 Moore, G.E. 185 Moore, K.J. 235 Mooser, V. 145, 156 Moran, A.M. 132 Morel, Y. 117 Morgan, A.A. 174 Morgan, K. 214 Morgan, K.M. 43 Mori, M. 9 Mori, Y. 6 Moriuchi, H. 13 Morken, M.A. 156, 175, 178
Morozova, E. 7 Morris, A.D. 156, 175 Morris, A.P. 175 Morris, M. 108 Morrison, S. 212 Morwood, M.J. 233 Moses, A.C. 46 Moses, E.K. 212 Moss, A. 139 Moss, H.E. 73 Motomura, K. 13 Mowzowicz, I. 117 Mudge, J. 179 Mueller, I. 42 Mukherjee, S. 175 Mulder, P.G. 203 Mulder, R.L. 202 Mullen, Y. 127 Mulligan, A.C. 8 Mullis, P.E. 25 Mundlos, S. 71 Murakami, N. 9 Murata, K. 6 Murgatroyd, P. 44 Muroya, K. 13 Murphy, A.J. 27 Murphy, N. 163 Muzny, D.M. 171 Mwangi, S. 149
N Naar, A.M. 235 Nabhan, Z.M. 36, 190 Nader, N. 99, 100, 111 Nader, N.S. 167 Nagata, N. 136 Nagy, T.R. 67 Nahum, A. 222 Naitza, S. 175 Najafi-Shoushtari, S.H. 235 Nakada, T. 6 Nakazato, M. 9 Napoli, F. 195 Nargund, R.P. 142 Narisu, N. 156, 175 Narumiya, S. 78 Nathan, D.M. 156, 175 Natividad, A. 222 Navarro, P. 175 Navarro, V.M. 6 Navratil, A.M. 23 Nazareth, L. 171 Nebesio, T.D. 36 Neff, N.F. 174 Neggers, S.J. 202 Nelis, M. 145 Nelson, O.L. 70 Nelson, R.G. 159
245
Nelson, S.M. 84 Nepote, V. 208 Nesterova, M. 104 Netchine, I. 55 Neville, M.J. 175 Newbold, R.R. 86 Ng, S.B. 172 Nguyen, A. 148 Nguyen, T.T. 183 Nickerson, D.A. 172 Nicolae, D. 124 Niedziela, M. 192 Nielsen, H.S. 46 Nieman, L.K. 109 Niklasson, A. 59 Nilsson, K.P. 21 Nilsson, P. 156 Nodale, M. 131 Noel, S.D. 94 Nofrate, V. 120 Nogueira, E.F. 112 Norris, J.M. 121 Northcott, S. 44 Novosyadlyy, R. 59 Nowak, J. 46 Nowicki, M. 192 Nzekwu, U. 165
O Obici, S. 152 O’Brien, M.M. 39 O’Brien, P.E. 150 Obunse, K. 106 O’Connell, J. 156, 175 O’Connor, D. 191 Oden, J.D. 160 Odink, R.J. 49, 201, 204 Ogata, T. 13 Ohinata, Y. 88 Ohkura, S. 6 Ohta, H. 88 Ojeda, S.R. 4 Okamoto, K. 136 Okamura, H. 6 Oksenberg, J.R. 179 Olafsson, J.H. 181 Olason, P.I. 181 Olesen, I.A. 222 Olivo-Marston, S. 92 Ong, K.K. 163, 171 Onland-Moret, N.C. 232 Onyiah, I. 220 Oostdijk, W. 204 Oostra, B.A. 175 Opocher, G. 113 O’Rahilly, S. 145 O’Reilly, P.F. 177 Ormond, K.E. 174
246
Orru, M. 175 Oseid, E.A. 119 Oshida, T. 220 Osugi, T. 214 Otten, B.J. 49, 201 Ouachee-Chardin, M. 222 Ounap, K. 145 Oury, F. 79 Owen, P. 44 Oxman, A.D. 226 Ozata, M. 94
P Pacak, K. 148 Pacini, F. 39 Page, M. 191 Paik, J.H. 69 Pakyz, R. 156, 175 Pal, M. 24 Palkovits, M. 15 Palladino, A. 198 Palmer, C.N. 156, 175 Palmer, L.J. 175, 177 Palta, P. 145 Palyha, O. 142 Pando, M.J. 179 Paneth, N. 189 Pang, A.W. 220 Pang, L. 67 Pangas, S.A. 90 Pankow, J.S. 156, 175 Pant, K.P. 173 Paolino, M. 78 Paolisso, G. 175 Papa, R. 19 Paranjape, S.A. 10 Parathath, S. 235 Park, I.H. 68 Park, S. 58 Paroder, M. 33 Parodi, S. 195 Parvari, R. 77 Pascoe, L. 156 Passoni, A. 37 Paterson, A.D. 124 Patisaul, H.B. 86 Pattaro, C. 175 Pattou, F. 145, 156, 175 Paul, E. 150 Pavkov, M.E. 159 Pavlovic, A. 174 Pawson, A.J. 214 Payen, E. 219 Payne, F. 156, 175 Pearce, M. 37 Pearson, D. 156, 175 Peden, J.F. 175 Pedersen, N.L. 175
Pedersen, O. 156, 175, 176 Peeters, M.W. 163 Peltonen, L. 145, 177 Pencek, R. 133 Peng, X. 67 Peng, X.D. 22 Pennell, C.E. 177 Penninger, J.M. 78 Perantie, D.C. 126 Perin, L. 55 Perkins, S.M. 190 Perola, M. 175 Perren, A. 61 Perrien, D.S. 73 Perrin, M.H. 21 Perry, J.R. 155, 175 Persani, L. 37 Pescovitz, M.D. 132 Pessin, J.E. 147 Petersen, J.H. 193, 194 Petersen, K.F. 44 Petersenn, S. 103 Petkovic, V. 25 Pfaff, D.W. 216 Pfeifer, G.P. 127 Pfeifer, S.P. 222 Pfeiffer, A.F. 175 Phan, Q.T. 224 Philippe, A. 145 Phipps, S.J. 8 Picard, C. 222 Pichler, I. 175 Pietz, L. 192 Pifl, C. 78 Pigeyre, M. 145 Pilon, J.W. 49 Pinhas-Hamiel, O. 38, 155 Pinto, D. 220 Piper, M. 227 Pisinger, C. 176 Pitsiladis, Y. 234 Pitteloud, N. 1, 94 Plehm, R. 78 Plessis, G. 145 Plummer, L. 1, 94 Pohlenz, J. 36 Pointud, J.C. 111 Polak, M. 33, 35, 41, 222 Polasek, O. 175 Polychronakos, C. 125, 176 Pooley, J.R. 102 Porter, L. 133 Porton, B. 105 Posner, B. 176 Posovszky, C. 139 Posthuma, D. 175 Postma, D.S. 177 Postnov, A.A. 75 Poston, L. 84
Author Index
Potter, S.C. 175 Poulsen, P. 176 Pouta, A. 155, 175, 176, 177 Powell, A. 232 Power, C. 155, 177 Pramstaller, P.P. 175 Prchal, J.T. 186 Prentki, M. 176 Prevot, V. 4 Price, J.N. 110 Price, L.H. 105 Pritchard, M. 74 Prokopenko, I. 156, 175, 177 Province, M.A. 175 Psaty, B.M. 156, 175 Puder, J.J. 200 Puel, A. 222 Purmann, C. 145 Purtell, K. 33 Pushkarev, D. 174
Q Qadri, F. 78 Qi, L. 156, 175 Qi, R. 190 Qin, G. 186 Qin, Y. 113 Qu, H.Q. 125 Quake, S.R. 174 Quennell, J.H. 8 Quero, J. 189 Quinton, R. 94 Quon, M.J. 148
R Raal, F.J. 168 Rached, M.T. 69 Rades, D. 227 Radetti, G. 195 Radford, D.J. 107 Raetzman, L. 17 Raff, H. 30 Ragazzon, B. 111 Raggio, C.L. 67 Ragoczy, T. 184 Rahbar, M.H. 189 Rahman, M. 166 Rahman, N. 97 Rainey, W.E. 112 Raivio, T. 93 Ralston, S.H. 65 Ramakrishnan, U. 85 Ramaraj, T. 179 Ramasamy, A. 177 Ramos, E.M. 182 Rance, N.E. 5 Randall, J. 175
Author Index
Raskin, K. 3 Raskin, P. 132 Rathmann, W. 156, 175 Rauch, T.A. 127 Ravindran, S. 109 Ray-Coquard, I. 227 Raymond-Barker, P. 44 Rayner, K.J. 235 Rayner, N.W. 175, 177 Redon, R. 220 Reed, R.R. 139 Reeser, H.M. 201 Refetoff, S. 45 Reid, J.G. 171 Reinalda, M.S. 40 Reincke, M. 61 Reinehr, T. 152 Reitman, M.L. 142 Rewers, M. 121 Reyes, S. 146 Reymond, A. 145 Reyna-Neyra, A. 33 Ribault, V. 35 Ribel-Madsen, R. 176 Rice, K. 156, 175 Richards, J.S. 90 Richards, M.L. 40 Riches, P.L. 65 Ricort, J.M. 55 Riek, R. 21 Riethmacher, D. 90 Rigutto, S. 42 Ring, S.M. 155, 177 Ringel, M.D. 39 Rio, D.D. 171 Ripatti, S. 175 Rissman, R.A. 21 Rivadeneira, F. 175, 177 Rivero-Muller, A. 97 Rivier, J. 21 Rivkees, S.A. 34 Rizzoti, K. 16 Roach, J.C. 173 Robbins, C.T. 70 Roberson, M.S. 23 Roberts Jr, C.T. 52 Robertson, R.P. 119 Robinson, B.G. 108 Robinson, I.C. 16, 25, 29 Robson, S. 220 Roby, Y.A. 139 Roccasecca, R.M. 175 Rocheleau, G. 175, 176 Roden, M. 175 Rodriguez, H. 132 Roepke, T.K. 33 Roifman, C. 222 Roland, I. 128 Rolandsson, O. 175
Rooman, I. 128 Ropers, H.H. 42 Rose, M.C. 43 Rose, S.R. 58 Rosell, M. 212 Rosenbaum, A.M. 174 Rosenfeld, R.G. 52 Ross, R.J. 109, 110 Rossi, C. 108 Rothman, D.L. 44 Rotimi, C.N. 67, 182 Rotter, J.I. 156 Rotteveel, J. 49 Rotwein, P. 62 Rowen, L. 173 Ruchala, M. 192 Ruffman, T. 43 Ruivenkamp, C.A. 52 Rung, J. 176 Ruokonen, A. 155, 176 Rybak, S. 17 Rybin, D. 156, 175 Rydh, A. 14
S Saarnio, E. 76 Sabaliauskas, N. 207 Sabo, P.J. 184 Sacchetta, P. 108 Sadie-Van Gijsen, H. 103 Saeed, S. 145 Sagreiya, H. 174 Sahlin, L. 83 Sahut-Barnola, I. 111 Saitou, M. 88 Salenave, S. 1 Salerno, M. 198 Salmon, I. 128 Salomaa, V. 175 Sampson, M. 156 Sampson, M.J. 175 Sandbaek, A. 156, 175, 176 Sandhu, M. 175 Sandstrom, R. 184 Sangkuhl, K. 174 Sanlaville, D. 145 Sanna, S. 175 Sant’Angelo, D.B. 129 Sapienza, C. 221 Saptomo, E.W. 233 Sarpong, D.F. 183 Sas, T.C. 201 Sass, M. 113 Sato, N. 13 Sattiraju, S. 146 Savage, D.B. 44 Sävendahl, L. 65 Sawaya, M.R. 21
247
Sawchenko, P. 21 Sawyer, S.M. 150 Saxena, R. 156, 175 Sayer, A.A. 156, 175 Schally, A.V. 103 Schatz, D.A. 132 Schauss, A.C. 24 Schechter, C.B. 228 Scheet, P. 175 Scherer, S.W. 220 Schiavi, F. 113 Schilkey, F.D. 179 Schindler, C. 200 Schlumberger, M. 39 Schmidlin, K. 227 Schmidt, M. 219 Schnabel, D. 77 Schneider, S. 61 Schoenmakers, N. 44 Schouten-van Meeteren, A.Y. 202 Schroor, E.J. 49 Schroth, G.P. 179 Schubert, C.M. 164 Schubert, D. 21 Schuhmacher, A. 116 Schumacher, M. 227 Schunemann, H.J. 226 Schurr, D. 168 Schutte, A.E. 53 Schutte, R. 53 Schutz, G. 90 Schwartz, G.J. 147 Schwartz, I.D. 52 Schwarz, P. 156, 175 Schwarzer, G. 227 Scott, L.J. 156, 175, 178 Seale, P. 139, 148 Sear, R. 209 Secco, A. 195 Seedorf, U. 175 Seemann, P. 71 Segal, D. 212 Sehlin, P. 14 Seidenfeld, J. 227 Sekido, R. 90 Sekine-Kondo, E. 136 Seminara, S.B. 1, 94 Serrano-Rios, M. 175 Serre, D. 176 Service, E. 95 Seth, N.P. 129 Sethupathy, P. 175 Seurin, D. 55 Shadrach, J.L. 60 Shah, A.S. 158 Shah, N.M. 2, 88 Shah, S. 160 Shahrokh, D.K. 20
248
Shannon, P.T. 172, 173 Shao, Y.Y. 72 Sharp, S.J. 156, 175 Shaw, J. 191 Shay, J.W. 228 Shearman, L. 142 Shen, E. 124 Shen, H. 207 Shen, L. 176 Shen, Z. 142 Shendure, J. 172, 173 Sheriff, M. 211 Sherpa, A. 207 Sherwin, R.S. 10 Shi, Z. 67 Shields, B. 155, 175 Shields, B.M. 177 Shigeta, M. 88 Shimomura, Y. 9 Shioda, S. 9 Shioda, T. 235 Shrader, P. 156, 175 Shuldiner, A.R. 156, 175 Shulman, G.I. 44 Sidhu, S.B. 108 Siegel, G. 228 Siegrist, J. 116 Siemensma, E.P. 49 Sierra, A. 146 Sievers, M.L. 162 Sigurdsson, A. 181 Sigurdsson, G. 181 Sigurdsson, H. 181 Sigurethsson, G. 175 Sigurgeirsson, B. 181 Sijbrands, E. 156 Sijbrands, E.J. 175 Silander, K. 175 Siljander, H.T. 123 Silva, J.P. 140 Silveira, A. 175 Silveira, L.F. 94 Silver, R. 216 Silverman, B. 58 Silvers, J.B. 210 Simard, S. 144 Simell, O. 123 Simell, S. 123 Simell, T. 123 Simon, R. 21 Simonson, T.S. 186 Simpson, E.R. 4 Simpson, L. 175 Sinaii, N. 109 Sinaiko, A. 130 Singh, R. 167 Singleton, A. 156, 175 Singru, P.S. 21 Sirigu, A. 20
Siscovick, D.S. 156 Sitaraman, S.V. 149 Sjostrom, L. 145 Skakkebaek, N.E. 91, 193, 197 Skeaff, S.A. 43 Skinner, S. 150 Skyler, J.S. 120, 132 Sladek, R. 145, 156, 176 Slatkin, M. 182 Sleeman, M.W. 27 Slucca, M. 119 Smit, A.F. 173 Smith, G.D. 155, 175 Smith, J. 144 Smith, L.E. 59 Smith, M.S. 143 Smith, N.L. 156, 175 Smith, S.S. 207 Smyth, D.J. 180 Soares, B.S. 22 Söder, O. 83 Soderborg, T. 88 Solberg, H. 46 Song, K. 156, 175 Song, L. 178 Soon, P.S. 108 Soranzo, N. 175 Sorensen, K. 91, 193, 194 Sosenko, J.M. 120 Sovio, U. 175, 177 Sowers, J.R. 115 Sowinski, J. 192 Spack, N.P. 85 Sparso, T. 156, 175 Spellberg, B. 224 Spiegelman, B.M. 139, 148 Spinelli, L. 198 Spohn, G. 24 Spranger, J. 175 Spurney, R. 215 Srinivasan, S. 149 Stacey, S.N. 181 Stamatoyannopoulos, J. 184 Stanier, P. 185 Stankiewicz, P. 171 Stavreva, D.A. 102 Stearns, R. 142 Stearns, S.C. 230 Steegers, E.A. 177 Steensma, D.P. 227 Stefansson, K. 156, 181 Stein, A.D. 85 Stein, E.A. 168 Steiner, R.A. 6 Steinthorsdottir, V. 156, 175, 181 Stelzer, A. 207 Stenbeck, G. 68
Author Index
Stewart, S.E. 94 Stijnen, T. 163 Stiles, C. 113 Stirrups, K. 220 Stocker, C.J. 212 Stoffel, M. 140 Stokvis-Brantsma, W.H. 201 Stoppa-Vaucher, S. 36 Stora, J. 231 Strachan, D.P. 155, 177 Strack, A.M. 142 Stram, D.O. 183 Strand, T. 130 Stratakis, C.A. 104, 106 Stratton, M.R. 186 Stribling, S. 142 Stringham, H.M. 156, 175 Strom, T.M. 77 Stumvoll, M. 156 Stutzmann, F. 145 Suarez, Y. 235 Suda, N. 79 Suh, Y. 228 Suissa, S. 130 Sulem, P. 181 Summanen, P. 134 Sumner, A.E. 161 Sun, L. 124 Sun, S.S. 164 Sun, W. 122 Surakka, I. 177 Surcel, H. 76 Suresh, P.S. 79 Suss, R. 61 Sutikna, T. 233 Sutor, S.L. 66 Suwa, G. 229 Suzuki, Y. 220 Sverrisson, S. 181 Swift, A. 175 Swift, A.J. 156 Swinburn, B.A. 151 Syddall, H. 156, 175 Sykiotis, G.P. 94 Sylvestre, M.P. 124 Syvanen, A.C. 156, 175 Szabadkai, G. 137 Szaflarski, W. 192 Szamosi, T. 168 Szarfman, A. 34 Szczepanek, E. 192 Szinnai, G. 33, 41
T Taanila, A. 177 Tabor, H.K. 172 Tajima, T. 13 Takahashi, N. 78
Author Index
Takenoya, F. 9 Tamborlane, W.V. 122 Tamehiro, N. 235 Tan, S. 88 Tanaka, H. 13 Tanaka, K.F. 79 Tanaka, T. 156, 175 Taneera, J. 156 Tang, Y. 67 Tang, Y.S. 142 Tangpricha, V. 85 Taniguchi, M. 136 Tardy, V. 117, 196 Taylor, A.E. 107 Taylor, I.B. 222 Taylor Jr, H.A. 183 Tayo, B. 183 Tecott, L.H. 79 Telling, A. 184 Tello, J.A. 1 Templeton, A.R. 229 Tenenbaum-Rakover, Y. 38 Terzic, A. 146 Tews, D. 139 Thakuria, J.V. 174 Thambundit, A. 94 Thi, T.D. 46 Thomas, G. 227 Thomas, M.G. 232 Thompson, G.B. 40 Thorand, B. 175 Thorel, F. 208 Thorens, B. 140 Thorleifsson, G. 156, 175, 181 Thorn, C.F. 174 Thorsteinsdottir, U. 156, 181 Thorwarth, A. 42 Tiano, C. 198 Tichet, J. 175, 176 Tiesler, C. 177 Timmerman, M.A. 114 Timpson, N.J. 155, 175, 177 Tiosano, D. 65, 77 Tirabassi, G. 19 Todd, J.A. 180, 218 Todorov, I. 127 Toiviainen-Salo, S. 96 Toledo, R.A. 113 Toledo, S.P. 113 Tollkuhn, J. 2, 88 Tolson, K.P. 7 Tom, W. 171 Tommiska, J. 93 Tomonaga, K. 220 Tong, J.F. 83 Tonjes, A. 156, 175 Toppari, J. 92 Toulon, A. 222
Touraine, P. 117 Touraine, R. 145 Trarbach, E. 94 Treier, A.C. 90 Treier, M. 90 Trelle, S. 227 Treszl, A. 103 Treweek, S. 226 Trichereau, J. 78 Trivin, C. 196 Tron, E. 41 Tronche, F. 3 Trudel, E. 10 Trynka, G. 232 Tsiaras, S. 1 Tsukamura, H. 6 Tsutsui, K. 214 Tuomi, T. 156, 175 Tuomilehto, J. 156 Tups, A. 8 Turan, N. 221 Turner, S. 37 Tusset, C. 94 Tyler-Smith, C. 220 Tyrka, A.R. 105
U Ubuka, T. 214 Ueta, Y. 78 Uhlenhaut, N.H. 90 Uitterlinden, A.G. 175, 177 Ullmann, R. 42 Untch, M. 227 Urade, Y. 111 Urbina, E.M. 157, 158, 168
V Vaag, A. 176 Vaaralahti, K. 93 Vadodaria, K. 21 Vahasalo, P. 123 Val, P. 111 Vale, W. 21 Valenzuela, D.M. 27 Valle, D. 182 Valle, T.T. 156 Valsesia, A. 145, 220 van Berkum, N.L. 184 van Buuren, S. 201 van Dalen, E.C. 202 van de Sande, A.G. 50 van den Akker, E.L.T. 99 van den Pol, A. 7 van der Palen, R.L. 203 van der Schouw, Y.T. 232 van der Westhuizen, F.H. 53
249
van Dijk, K.W. 175 van Doorn, J. 50, 75 van Duijn, C. 156 van Duijn, C.M. 54, 177 van Duyvenvoorde, H.A. 52 van Gool, S.A. 75 Van Gool, S.A. 204 van Haelst, M.M. 145 van Hateren, K.J. 159 van, H.M. 175 van Hoek, M. 156 Van Huffel, C. 128 Van Hul, W. 67 van Leeuwen, M. 49 van Mechelen, W. 200 van Mil, E.G. 49 van Rooyen, J.M. 53 van Santen, H.M. 202 van Trotsenburg, A.S. 49, 201, 202 van Velkinburgh, J.C. 179 Van Vliet, G. 36, 42 van Vliet-Ostaptchouk, J.V. 232 van Wassenaer, A.G. 189 Vanryzin, C. 109 van’t Hof, R. 65 Varma, D. 175 Vatin, V. 145 Vaxillaire, M. 176 Veiga, S.J. 75 Veijola, R. 123 Veit, F. 150 Venkadesan, M. 234 Veres, G. 219 Verhoeven, G. 3 Verma, S. 109 Vidal, M. 79 Vidaud, M. 219 Vigone, M.C. 37 Vijay-Kumar, M. 149 Vijayakumar, A. 59 Viljakainen, H.T. 76 Villanueva, C. 1 Villarroya, F. 212 Vincent-Delorme, C. 145 Visscher, P.M. 182 Visvanathan, K. 92 Visvikis-Siest, S. 175 Vitart, V. 175 Vogelzangs, N. 175 Voight, B.F. 156, 175 Volat, F. 111 Vollenweider, P. 145, 156 von Haeseler, A. 78 Von Kalle, C. 219 von Meyenn, F. 140 von Schnurbein, J. 139 Voss, T.C. 102
250
Vreuls, R.C. 49 Vuillard, E. 35
W Wabitsch, M. 139 Waeber, G. 145, 156, 175 Waelkens, J.J. 201 Wagers, A.J. 60 Waggott, D. 124 Wagner, E.F. 68 Wagner, P.J. 175 Wakabayashi, Y. 6 Wakayama, T. 88 Walder, K. 212 Walenkamp, M.J. 56 Walker, M. 156 Walker, N.M. 180 Wallace, A.M. 108 Wallace, C. 180 Walley, A. 156, 175 Walley, A.J. 145 Walter, K. 220 Walters, G.B. 175 Walters, R.G. 145 Walvoord, E.C. 58 Wan, C. 67 Wan, M. 67 Wang, J. 148 Wang, K. 107 Wang, L. 72 Wang, S.-P. 142 Wang, W. 73 Ward, K.L. 175 Ward Platt, M.P. 37 Wareham, N.J. 156 Warrington, N.M. 177 Washburn, L.L. 89 Wastell, H. 37 Watanabe, R.M. 156 Watarai, H. 136 Waters, K. 183 Waterworth, D. 145 Waterworth, D.M. 156 Watkins, H. 175 Wawrowsky, K. 26 Weaver, A.L. 167 Weaver, P.M. 126 Webb, T.R. 30 Weber, G. 37 Weedon, M.N. 155, 156, 175 Wehkalampi, K. 93 Wei, J. 220 Weigel, J.F. 71 Weil, E.J. 159 Weingart, O. 227 Weinstein, S. 148 Weintraub, M. 216 Weis, M. 67
Weiss, R.E. 45 Wells Jr, S.A. 39 Werbel, W.A. 234 Westerlaken, C. 201 Westphal, H. 15 Whaley-Connell, A. 115 Whaley, R. 174 Wheeler, D.A. 171 Wheeler, E. 175 Wheeler, M.T. 174 Wherrett, D. 132 White, A. 142 White, N.H. 122, 126 White, P.C. 160 White, T.D. 229 Whittemore, A.S. 39, 182 Wichmann, H.E. 156 Wickman, S. 96 Widen, E. 177 Wiegman, A. 168 Wiench, M. 102 Wiggs, E.A. 106 Wijga, A.H. 177 Wijmenga, C. 232 Wild, S.H. 175 Wilinska, M.E. 131 Wilkie, A.O. 222 Wilkinson, G. 166 Wilks, R. 183 Willard, H.F. 178 Willemsen, G. 175, 177 Willenberg, H.S. 103 Williams, G.H. 156, 175 Williams, L. 184 Wilson, D.M. 132 Wilson, J.F. 156, 177 Wirtz, P.H. 116 Wit, J.M. 49, 52, 56, 75, 201, 204 Witherspoon, D.J. 186 Witteman, J.C. 54, 175 Wojda, S.J. 70 Wojtaszewski, J.F. 176 WoldeGabriel, G. 229 Wolffenbuttel, K.P. 114 Wolfrum, C. 140 Wong, I. 124 Woodliff, J. 30 Woodward, J.E. 179 Woon, M. 174 Worley, A. 200 Wray, S. 28 Wright, W.E. 228 Wu, C.H. 1 Wu, J. 126 Wu, M. 7 Wu, M.V. 2, 88 Wu, S. 56 Wu, T.D. 179
Author Index
Wu, X. 67 Wucherpfennig, K.W. 129 Wunderlich, F.T. 24
X Xiao, X.Q. 143 Xie, J. 23 Xie, X. 27 Xing, D. 131 Xing, J. 186 Xu, J. 168 Xu, L. 69
Y Yadav, V.K. 79 Yamada, E. 147 Yamada, S. 136, 146 Yamaguchi, H. 9 Yamamoto, H. 136 Yamanaka, K. 88 Yan, X. 57, 83 Yancopoulos, G.D. 27 Yang, C. 171 Yang, G. 143 Yang, X. 73, 228 Yang, Y. 186 Yao, L. 113 Yarnell, J.W. 175 Yasunami, Y. 136 Yaszemski, M.J. 66 Ye, L. 139
Author Index
Yebra, M. 89 Yi, W. 129 Yin, X. 121 Yokoya, S. 13 Yoshikawa, Y. 69 Yoshimatsu, H. 78 You, J. 143 Young, J. 1 Young, J.J. 62 Young, L. 103 Yount, K.M. 85 Yu, H. 142 Yu, X. 143 Yu, Y. 143 Yuan, L. 215 Yun, H. 186
Z Zabel, M. 192 Zabena, C. 175 Zadik, Z. 38 Zahner, L. 200 Zaibi, M.S. 212 Zalla, T. 20 Zandwijken, G.R. 201 Zaranek, A.W. 174 Zdravkovic, M. 46 Zdunek, A.M. 14 Zeggini, E. 155, 156, 175 Zelenika, D. 175 Zerbe, G.O. 121 Zethelius, B. 175
Zhai, G. 175 Zhang, B. 133 Zhang, F. 171 Zhang, H. 177 Zhang, L. 179 Zhang, T.Y. 20 Zhang, Y. 220 Zhao, J. 83, 177 Zhao, J.H. 156, 163, 175 Zhao, L. 67 Zhao, T.J. 27 Zhao, Y. 15 Zhou, C. 26 Zhu, M.J. 83 Zhu, W. 10 Zhu, X. 183 Zhu, Z. 146 Ziegler, C.G. 103 Zillikens, M.C. 175 Zingman, L.V. 146 Zinman, B. 130 Zinn, A.R. 7 Zobel, A.W. 116 Zung, A. 38 Zwahlen, M. 227 Zwermann, O. 61
251
Subject Index A ABCA1, MiR-33 and cholesterol homeostasis regulation 235 ABCD1, gene therapy with lentiviral vector for X-linked adrenoleukodystrophy 219, 220 ACE inhibitors, see Angiotensin-converting enzyme inhibitors ACTH, see Adrenocorticotropic hormone Activating transcription factor 4 (Atf4), chondrocyte proliferation and differentiation regulation 73, 74 Acylation-stimulating protein (ASP), Prader-Willi syndrome levels and growth hormone therapy effects 199 ADCY5, see Cyclic AMP-generating adenylate cyclase Adipocyte, Zfp423 transcriptional control of preadipocyte determination 139, 140 Adrenal tumor neuropeptide hormone receptors 103, 104 DNA microarray analysis of adrenocortical tumors 108 TASK1 expression 112, 113 Adrenocorticotropic hormone (ACTH), see also Cushing’s disease curcumin effects on corticotrophs 30, 31 work overcommitment studies 116, 117 Aging child abuse and stress induction 105, 106 telomerase variants in Ashkenazi centenarians 228 Aire, perinatal window for control of autoimmunity 223, 224 AKR1B7, see Aldo-keto reductase 1B7 Albuminuria, American-Indian youths 159
ALD, see X-linked adrenoleukodystrophy Aldo-keto reductase 1B7 (AKR1B7), adrenal regulation 111, 112 Aldosterone metabolic syndrome and resistant hypertension 115, 116 TASK1 role in production 112, 113 Alpha cell, beta cell conversion after extreme beta cell loss 208 Altitude, see High-altitude adaptation Amyloid fibril, pituitary secretory granules and peptide hormone storage 21, 22 Androgen receptor (AR) conditional inactivation 3 male sexual and territorial behavior role 88 Angiotensin-converting enzyme (ACE) inhibitors, enalapril and losartan effects on kidney and retina 136, 137 APS-I, see Autoimmune polyendocrine syndrome type I AR, see Androgen receptor Arcuate nucleus discovery of neurokinin B neurons 5 kisspeptin neurons in pulsatile gonadotropinreleasing hormone secretion 6 Ardipithecus ramidus, hominid paleobiology 228, 229 Aromatase estrogen aromatization and male sexual behavior 2, 3 inhibitors bone effects 75, 76 cognitive effects in prepubertal boys 95, 96 vertebral morphology in male idiopathic short stature or constitu-
tional delay of puberty 96 ASP, see Acylation-stimulating protein Atf4, see Activating transcription factor 4 ATP-sensitive potassium channel energy expenditure control and body weight determination 146, 147 glucagon response role during glucose deprivation 119, 120 Autism, oxytocin for social behavior promotion in autism 20, 21 Autoimmune polyendocrine syndrome type I (APS-I), interleukin autoantibodies 222, 223 Autosomal-recessive hypophosphatemic rickets, see Rickets
B Bariatric surgery laparoscopic gastric banding in severely obese adolescents 150, 151 maternal intergenerational transmission of obesity 144, 145 BAT, see Brown adipose tissue Beta cell alpha cell conversion after extreme beta cell loss 208 FXYD2␥a as biomarker 128, 129 HMGB1 role in early loss of transplanted islets in mice 136, 137 rituximab and preservation of function 132, 133 Bisphenol-A (BPA), neonatal exposure effects in rat reproduction 86, 87 BMPs, see Bone morphogenetic proteins Body mass index (BMI) mate selection in Hadza foragers 209 puberty timing in boys 194
Bombesin receptor, receptor subtype-3 agonists for obesity management 142 Bone morphogenetic proteins (BMPs), germ cell specification signaling 88 Bone remodeling, transforming growth factor- role 67, 68 BPA, see Bisphenol-A Brachydactyly, parathyroid hormone-related protein gene mutations in brachydactyly type E 71 Brain-lung-thyroid syndrome, NKX2-1 mutations 41, 42 Breast development, age decline 193 Brown adipose tissue (BAT) FGF21 in thermogenic activation 212 Gs␣ deficiency effects 148 transcriptional regulation of myoblast to brown fat switch 148, 149
C c-Abl, pathway inhibition in oocyte chemotherapy protection 217 CAH, see Congenital adrenal hyperplasia Calcitonin, glucagon-like peptide-1 receptor agonist activation of thyroid C-cells 46, 47 Calorie restriction, ghrelin O-acyltransferase in growth hormone mediation of calorie-restricted mouse survival 27 Cancer, see also specific cancers erythropoiesis-stimulating agents and mortality 227, 228 FGFR3 and HRAS activating mutations in congenital disorders and testicular tumors 222 genomics 186, 187 growth hormone deficiency induction by radiation in cancer survivors 202, 203 insulin-like growth factor-1 receptor immunoliposomes for treatment 61, 62
254
metformin and mortality reduction 159, 160 CCNL1, see Cyclins C/EBP-, transcriptional regulation of myoblast to brown fat switch 148, 149 CGH, see Comparative genomic hybridization Charcot-Marie-Tooth disease, whole-genome sequencing 171, 172 Chemerin, angiogenesis stimulation 211, 212 Child abuse, stress and aging induction 105, 106 Chondrocyte Atf4 regulation of proliferation and differentiation 73, 74 NKCC1 mediation of volume increase in growth plate 74, 75 thyroid hormone effects in growth plate 72, 73 Chromatin, heritable signatures 178 Citations, distortions and unfounded authority 225, 226 CLOCK/BMAL glucocorticoid receptor regulation 99 hypothalamic-pituitaryadrenal axis interactions 100, 101 CNV, see Copy number variation Colesevelam hydrochloride, familial hypercholesterolemia management 168, 169 Communications, alternative summary statistics for communicating risk reduction with statins 226, 227 Comparative genomic hybridization (CGH), findings in congenital hypothyroidism and thyroid dysgenesis 42, 43 Congenital adrenal hyperplasia (CAH) differential diagnosis of late-onset congenital adrenal hyperplasia from premature adrenarche 196, 197
fertility in women with 21-hydroxylase deficiency 117 hydrocortisone therapy pharmacology study 109, 110 replacement strategies 110 serum steroid profiling with liquid chromatographymass spectrometry 108, 109 Copy number variation (CNV), origins and functional impact 220 Cortisol fetal corticosteroid metabolite concentrations and maternal effects 211 work overcommitment studies 116, 117 Curcumin, Cushing’s disease management rationale 30, 31 Cushing’s disease curcumin management rationale 30, 31 desmopressin test for differential diagnosis from pseudo-Cushing state 19 quality of life one year after cure 106 Cyclic AMP-generating adenylate cyclase (ADCY5), variant effects on fetal growth and birth weight 177, 178 Cyclins, CCNL1 variant effects on fetal growth and birth weight 177, 178 Cyclophilin B (CypB), osteogenesis imperfecta human deficiency studies 67 knockout mice 66 CypB, see Cyclophilin B Cytochrome b5, mutation in 17,20-lyase deficiency 114
D DDAV test, see Desmopressin test Dehydroepiandrosterone sulfate (DHEAS), protein kinase C- activation and neutrophil superoxide generation 107
Subject Index
Depot medroxyprogesterone acetate (DMPA), serum lipid response 166 Desmopressin (DDAV) test, Cushing's disease differential diagnosis from pseudoCushing state 19 DHEAS, see Dehydroepiandrosterone sulfate Diabetes type 1 age of onset parent age of onset and offspring risk 135, 136 proliferative retinopathy risks 134, 135 angiotensin-converting enzyme inhibitor effects on kidney and retina 136, 137 autoantibodies and human leukocyte antigenconferred disease susceptibility 123, 124 closed-loop insulin delivery system 131, 132 continuous subcutaneous insulin infusion versus multiple injections 191, 192 etiology 218 FXYD2␥a as beta cell biomarker 128, 129 genome-wide association studies glycemic control locus 124, 125 in silico replication 125, 126 height growth velocity and risks 121, 122 hippocampal volume 126, 127 HMGB1 role in early loss of transplanted islets in mice 136, 137 imprinted DLK-MEG3 gene region and susceptibility 180 insulin gene methylation 127, 128 intensive therapy and retinopathy outcomes 122, 123 mitochondria dysfunction 137 pramlintide and glucose excursion lowering 133, 134
Subject Index
progression mechanisms and mode of onset 120, 121 rituximab and preservation of beta cell function 132, 133 self peptide presentation 129, 130 Diabetes type 2 albuminuria in AmericanIndian youths 159 alleles near insulin receptor substrate-1 gene 176, 177 atherosclerotic vascular changes in adolescents and young adults 158 genetic loci in fasting glucose homeostasis 175, 176 glucagon-like peptide-1 receptor alleles 156 low birth weight as risk factor 155, 156 metformin and cancer mortality reduction 159, 160 parental origin of sequence variants 181 screening in obesity 160, 161 3,5-Diiodothyropropionic acid (DITPA), Mct8 transport studies 49, 50 DITPA, see 3,5-Diiodothyropropionic acid DMPA, see Depot medroxyprogesterone acetate DNA methylation insulin gene methylation 127, 128 patterns for in vitro fertilization 221 DNA microarray, adrenocortical tumor analysis 108 Dopamine D2 receptor role in addiction and compulsive eating in obese rats 141, 142 oxytocin interactions in maternal behavior 20 thyrotropin-releasing hormone and tuberoinfundibular dopamine neuron effects 213, 214 Dual oxidase (DUOX), DUOX2 mutation in transient
congenital hypothyroidism 42 DUOX, see Dual oxidase Dynorphin A leptin regulation of neuron function 8, 9 neurokinin B and dynorphin A in arcuate nucleus kisspeptin neurons regulation of gonadotropin-releasing hormone pulsatile secretion 6
E E2F1, pituitary tumor transforming gene induction in pituitary tumors 26 EBLN elements, see Endogenous Borna-like N elements Ectonucleotide pyrophosphate/phosphodiesterase (ENPP1), generalized arterial calcification of infancy and rickets mutations 77 Embryonic stem (ES) cell, germ cell derivation 89 Enalapril, see Angiotensinconverting enzyme inhibitors Endogenous Borna-like N (EBLN) elements, human genome 220, 221 Endothelial cell, tanycyte plasticity role in median eminence with estrogen 4 ENPP1, see Ectonucleotide pyrophosphate/phosphodiesterase ERK, see Mitogen-activated protein kinase Erythropoietin, therapy and cancer mortality 227, 228 ES cell, see Embryonic stem cell Estrogen aromatization and male sexual behavior 2, 3 estradiol-kisspeptin positive feedback mechanism in puberty onset 4, 5 gas chromatography-mass spectrometry 91, 92 tanycyte plasticity role in median eminence 4 Turner syndrome replacement therapy 190, 191
255
Evidence-based medicine breast development age decline 193 differential diagnosis of late-onset congenital adrenal hyperplasia from premature adrenarche 196, 197 estrogen replacement in Turner syndrome 190, 191 growth hormone deficiency cardiac function 198, 199 diagnosis glucagon test 195 insulin tolerance test 195, 196 induction by radiation in cancer survivors 202, 203 Prader-Willi syndrome growth hormone therapy 199, 200 pubertal timing in boys and body mass index 194 school-based interventions in obesity, meta-analysis 200, 201 thyroid gland volume, insulin-like growth factor-1, and anthropometric variables in euthyroid prepubertal children 197, 198 thyroid hemiagenesis and thyroid pathology risks 192, 193 thyroid hormone regimens for transient hypothyroxinemia in neonates 189, 190 Turner syndrome growth hormone therapy metabolic consequences 203, 204 oxandrolone efficacy and safety 201, 202 Evolution, see Natural selection Exemestane, bone effects 75, 76 Exome sequencing, advantages in mendelian disorders 172, 173
256
F Familial hypercholesterolemia colesevelam hydrochloride therapy 168, 169 rosuvastin therapy 168 FCM, see Fetal corticosteroid metabolites FEO, see Food-entrainable oscillator Fetal corticosteroid metabolites (FCM), maternal effects on concentration 211 FGF, see Fibroblast growth factor Fibroblast growth factor (FGF) FGF21 in thermogenic activation of neonatal brown fat 212 FGFR3 activating mutations in congenital disorders and testicular tumors 222 Food-entrainable oscillator (FEO), stomach ghrelinsecreting cells 216 Foot strike patterns, barefoot versus shod runners 233, 234 Foxa2, adaptive behavior regulation during fasting 140, 141 Foxl2, somatic sex reprogramming of ovaries to testes by ablation 90, 91 FoxO1, osteoblast effects 69, 70 FTO, locus studies of obesity in African-derived populations 183 FXYD2␥a, beta cell biomarker 128, 129 Fyn, energy expenditure control and body weight determination 147, 148
G ␥-Aminobutyric acid (GABA), 4--GABAA receptor in puberty-associated learning deficits 207 GABA, see ␥-Aminobutyric acid GACI, see Generalized arterial calcification of infancy Gas5, glucocorticoid receptor repression 111
Gas chromatography-mass spectrometry, sex steroid level determination 91, 92 Gastric inhibitory peptide, see Glucagon-like peptide-1 Generalized arterial calcification of infancy (GACI), ectonucleotide pyrophosphate/phosphodiesterase mutations 77 Genome sequencing Charcot-Marie-Tooth disease 171, 172 clinical assessment incorporating personal genome 174 exome sequencing advantages 172, 173 family-based sequencing 173 Germ cell derivation from embryonic stem cells 89 signaling in specification 88 GH, see Growth hormone Ghrelin O-acyltransferase (GOAT), growth hormone mediation of calorierestricted mouse survival 27 Ghrelin, stomach ghrelinsecreting cells as food-entrainable circadian clocks 216 GHRH, see Growth hormonereleasing hormone Giucagon ATP-sensitive potassium channel role in response during glucose deprivation 119, 120 testing for growth hormone deficiency 195 ventromedial hypothalamus insulin and pancreatic glucagon secretion regulation 10 GLP-1, see Glucagon-like peptide-1 Glucagon-like peptide-1 (GLP-1), receptor agonist activation of thyroid C-cells 46, 47 alleles in glucose and insulin response 156, 157 Glucocorticoid receptor (GR) CLOCK/BMAL regulation 99
Subject Index
Gas5 repression 111 gonadotropin-releasing hormone receptor crosstalk 103, 104 ultradian hormone stimulation and gene transcription pulses 102 Glucose breath test, estimation of insulin resistance in obese prepubertal children 165 Glucose-regulated protein 78 (GRP78), murcomycosis pathogenesis role in diabetic mice 224, 225 GnIH, see Gonadotropininhibitory hormone GnRH, see Gonadotropinreleasing hormone GOAT, see Ghrelin O-acyltransferase Gonadotropin-inhibitory hormone (GnIH), RFRP-1/3 homologs and GPR147 distribution in hypothalamus 214, 215 Gonadotropin-releasing hormone (GnRH) GnRH1, mutation in idiopathic hypogonadotropic hypogonadism 1, 2 melanin-concentrating hormone inhibition of neurons and kisspeptin activation 7, 8 neurokinin B and dynorphin A in arcuate nucleus kisspeptin neurons regulation of pulsatile secretion 6 neurokinin B mutation and release 94, 95 neuron origin studies with mutant mice 28, 29 receptor adrenal tumor expression 103, 104 crosstalk with glucocorticoid receptor 103, 104 GPCR, see G protein-coupled receptor GPR147, distribution in hypothalamus 214, 215 G protein-coupled receptor (GPCR), functional rescue by intermolecular cooperation 97
Subject Index
GR, see Glucocorticoid receptor GRB10, reciprocal imprinting 185 Growth first-year rapid growth relationship to cardiovascular and metabolic risk profile in early adulthood 163, 164 height growth velocity and diabetes type 1 risks 121, 122 heritability of childhood weight gain and adult metabolic disease risk 163 prolonged juvenile state relationship to cardiovascular and metabolic risk profile 163, 164 Growth hormone (GH) cell in vivo imaging of pituitary microcirculation and somatotrope function 29, 30 ghrelin O-acyltransferase in growth hormone mediation of calorierestricted mouse survival 27 insulin-like growth factor-1 promoter chromatin organization in hormoneinduced activation 62, 63 therapy, see also Growth hormone deficiency Prader-Willi syndrome cardiovascular and metabolic risk profile 199 efficacy and safety 40, 41 small for gestational age outcomes 51 Turner syndrome metabolic consequences 203, 204 oxandrolone efficacy and safety 201, 202 Growth hormone deficiency cardiac function and growth hormone therapy effects 198, 199 dominant-negative growth hormone isoform induction by growth
hormone-releasing hormone 25, 26 glucagon test 195 growth hormone therapy adult height outcomes after high-dose therapy in prepubertal period 204, 205 inhaled versus subcutaneous administration 58 patient, physician, and consumer drivers 210 insulin tolerance test 195, 196 radiation induction in cancer survivors 202, 203 Growth hormone-releasing hormone (GHRH) growth hormone deficiency and dominant-negative growth hormone isoform induction 25, 26 receptor hyperactivation and somatotrope hyperplasia suppression 22, 23 GRP78, see Glucose-regulated protein 78
H Height, see also Growth hormone deficiency mate selection in Hadza foragers 209 specialty drug drivers in short stature 210 Hematopoietic stem cell (HSC) gene therapy with lentiviral vector for X-linked adrenoleukodystrophy 219, 220 osteoclast differentiation 68, 69 systemic signals in niche regulation 60, 61 HES1, pituitary development regulation 17 HESX1, transcription factor interactions in pituitary development 18 Hibernation, bone loss prevention in bears 70, 71 Hi-C, genome dynamic mapping 184 High-altitude adaptation, genetics 186
257
Hippocampus, volume in diabetes type 1 126, 127 HMGB1, role in early loss of transplanted islets in mice 136, 137 Hodgkin’s lymphoma, thyroid secondary neoplasms after treatment 39, 40 Homo floresiensis, cladistic analysis 233 HRAS, activating mutations in congenital disorders and testicular tumors 222 HSC, see Hematopoietic stem cell Hydrocortisone, congenital adrenal hyperplasia management formulation pharmacology 109, 110 replacement strategies 110 21-Hydroxylase deficiency, see Congenital adrenal hyperplasia Hypercholesterolemia, see Familial hypercholesterolemia Hypertension, aldosterone role in resistant hypertension 115, 116 Hyperthyrotropinemia, see Thyroid-stimulating hormone Hypothyroidism comparative genomic hybridization in congenital hypothyroidism and thyroid dysgenesis 42, 43 DUOX2 mutation in transient congenital hypothyroidism 42 intra-amniotic thyroxine treatment in non-immune fetal goitrous hypothyroidism 35, 36 neonatal screening for central hypothyroidism 36, 37 thyroid hormone regimens for transient hypothyroxinemia in neonates 189, 190
I Idiopathic hypogonadotropic hypogonadism (IHH)
258
GnRH1 mutation 1, 2 hypothalamus, pituitary, and testes defects 94 IGF-1, see Insulin-like growth factor-1 IGF-2, see Insulin-like growth factor-2 IGFBP-2, see Insulin-like growth factor-binding protein-2 IGFBP-3, see Insulin-like growth factor-binding protein-3 IGFBP-5, see Insulin-like growth factor-binding protein-5 IHH, see Idiopathic hypogonadotropic hypogonadism IB␣, mutation and IGF-1 insensitivity of fibroblasts 56, 57 IL-17, see Interleukin-17 IL-22, see Interleukin-22 Imprinting DLK-MEG3 gene region and diabetes type 1 susceptibility 180 DNA methylation patterns for in vitro fertilization 221 reciprocal imprinting of GRB10 185 Insulin closed-loop delivery system 131, 132 diabetes type 1 continuous subcutaneous insulin infusion versus multiple injections 191, 192 gene methylation 127, 128 glucose breath test estimation of insulin resistance in obese prepubertal children 165 receptor interaction with polyoma virus middle T antigen for oncogene activation 59, 60 Insulin-like growth factor-1 (IGF-1) decline and cardiometabolic vulnerability in young Africans 53, 54 elderly bioactivity and metabolic syndrome 54, 55 growth plate chondrocyte effects 72, 73
IB␣ mutation and fibroblast insensitivity 56, 57 mutation effects on development 55, 56 preterm infant combination therapy with insulin-like growth factor-binding protein-3 59 promoter chromatin organization in hormoneinduced activation 62, 63 receptor haploinsufficiency nonsense-mediated messenger ribonucleic acid decay 52, 53 small for gestational age 52 receptor immunoliposomes for cancer treatment 61, 62 interaction with polyoma virus middle T antigen for oncogene activation 59, 60 stem cell niche regulation 60, 61 thyroid gland volume, insulin-like growth factor1, and anthropometric variables in euthyroid prepubertal children 197, 198 Insulin-like growth factor-2 (IGF2), DNA microarray analysis of adrenocortical tumors 108 Insulin-like growth factorbinding protein-2 (IGFBP-2), serum levels in small for gestational age 50, 51 Insulin-like growth factorbinding protein-3 (IGFBP-3) preterm infant combination therapy with insulin-like growth factor-1 59 Insulin-like growth factorbinding protein-5 (IGFBP-5), pregnancy-associated plasma protein-A2 mediation of proteolysis 57, 58 Insulin receptor substrate-1 (IRS1), local alleles and metabolic disorders 176, 177
Subject Index
Insulin tolerance test (ITT), growth hormone deficiency 195, 196 Intercourse, reproduction relationship trends 87 Interleukin-17 (IL-17), autoantibodies in autoimmune polyendocrine syndrome type I 222, 223 Interleukin-22 (IL-22), autoantibodies in autoimmune polyendocrine syndrome type I 222, 223 Iodine, supplementation effects on cognition 43, 44 IRS1, see Insulin receptor substrate-1 ITT, see Insulin tolerance test
J JNK, see Jun N-terminal kinase Jun N-terminal kinase (JNK), brain signaling and glucose metabolism regulation 24, 25
K Kcne2, mutant mouse studies of thyroid hormone synthesis 33, 34 Ki-67, DNA microarray analysis of adrenocortical tumors 108 Kisspeptin estradiol-kisspeptin positive feedback mechanism in puberty onset 4, 5 melanin-concentrating hormone inhibition of neurons and kisspeptin activation 7, 8 neurokinin B and dynorphin A in arcuate nucleus kisspeptin neurons and regulation of gonadotropin-releasing hormone pulsatile secretion 6
L Lactase persistence (LP), origins in Europe 230, 231 Lactose intolerance, prehistoric hunter-gatherer population in northern Europe 230
Subject Index
Leptin early overnutrition and resistance 143, 144 gonadotropin-releasing hormone neuron function regulation 8, 9 neuropeptide W regulation 9 receptor signaling through Tyr985 in regulation of energy balance 143 serotonin mediation of bone effects 79 Letrozole, vertebral morphology effects in male idiopathic short stature or constitutional delay of puberty 96 LHX2, see LIM homeobox 2 LHX3, see LIM homeobox 4 LIM homeobox 2 (LHX2), pituitary development regulation 15, 16 LIM homeobox 3 (LHX3), mutation, pituitary hormone deficiency, hearing impairment, and vertebral malformations 14, 15 LIN28B, variants in constitutional delay of growth and puberty 93 Liposome, insulin-like growth factor-1 receptor immunoliposomes for cancer treatment 61, 62 LKB1, energy expenditure control and body weight determination 147, 148 Losartan, see Angiotensinconverting enzyme inhibitors LP, see Lactase persistence 17,20-Lyase, cytochrome b5 mutation in deficiency 114
M MAPK, see Mitogen-activated protein kinase Mate selection, body size effects in Hadza foragers 209 MCH, see Melanin-concentrating hormone Median eminence, tanycyte plasticity 4 Melanin-concentrating hormone (MCH), inhibition
of gonadotropin-releasing hormone neurons and kisspeptin activation 7, 8 Melanocortin receptor MC2R accessory proteins 30 Sim1 deficiency, MC4R decrease, and hyperphagic obesity 7 Metabolic syndrome aldosterone role 115, 116 elderly insulin-like growth factor-1 bioactivity 54, 55 ethnic differences in diagnosis 161, 162 Toll-like receptor 5 knockout mouse and gut microbiota 149, 150 Metformin, cancer mortality reduction 159, 160 Methimazole, hepatotoxicity in children 34, 35 Miller syndrome exome sequencing 172, 173 family-based genome sequencing 173 MiR-33, cholesterol homeostasis regulation 234, 235 Mitogen-activated protein kinase (MAPK), pituitary ERK signaling in female fertility 23, 24 Multiple sclerosis, discordant monozygotic twin studies 179 Murcomycosis, GRP78 pathogenesis role in diabetic mice 224, 225
N Natural selection, contemporary humans 229, 232 Neurokinin B arcuate nucleus discovery of neurons 5 kisspeptin neurons in pulsatile gonadotropinreleasing hormone secretion 6 mutation and neonatal gonadotropin-releasing hormone release 94, 95 Neuropeptide W (NPW), anorectic activity and leptin regulation 9
259
Neuropeptide Y (NPY), receptor variants and nutrient-specific food intake in Europeans 231, 232 NF-B, see Nuclear factor-B NKCC1, mediation of chondrocyte volume increase in growth plate 74, 75 NKX2-1, mutations in brainlung-thyroid syndrome 41, 42 NPW, see Neuropeptide W NPY, see Neuropeptide Y Nuclear factor-B (NF-B) curcumin effects in Cushing's disease 30, 31 inhibition, see IB␣ Nutrition, fertility impact in children 85, 86
O Obesity adult mortality impact of childhood obesity 162, 163 ATP-sensitive potassium channel in energy expenditure control and body weight determination 146, 147 Australia active-afterschool-communities program 151 bariatric surgery laparoscopic gastric banding in severely obese adolescents 150, 151 maternal intergenerational transmission of obesity 144, 145 bombesin receptor subtype-3 agonist therapy 142 chemerin and angiogenesis stimulation 211, 212 chromosomal deletions chromosome 16p11.2 and penetrance 145, 146 severe early-onset obesity 145 diabetes type 2 screening 160, 161 dopamine D2 receptor role in addiction and compul-
260
sive eating in obese rats 141, 142 FTO locus studies in African-derived populations 183 Fyn in energy expenditure control and body weight determination 147, 148 glucose breath test estimation of insulin resistance in obese prepubertal children 165 Gs␣ deficiency and lean phenotype 148 leptin early overnutrition and resistance 143, 144 receptor signaling through Tyr985 in regulation of energy balance 143 maternal obesity effects on fetal skeletal muscle development 85 pregnancy outcome 84 molecular targets for therapy 152 Santa Claus, impact on childhood obesity 169 school-based intervention meta-analysis 200, 201 Sim1 deficiency and hyperphagic obesity 7 thyroid function 152 Zfp423 transcriptional control of preadipocyte determination 139, 140 OCs, see Oral contraceptives OI, see Osteogenesis imperfecta OPG, see Osteoprotegerin Oral contraceptives (OCs), serum lipid response 166 Orthodenticle homeobox 2 (OTX2), mutation and pituitary phenotype 13, 14 Osteoblast, FoxO1 effects 69, 70 Osteoclast, differentiation 68, 69 Osteogenesis imperfecta (OI), cyclophilin B human deficiency studies 67 knockout mice 66 Osteoporosis osteoprotegerin neutralizing antibodies 65, 66
serotonin inhibitors in management 79, 80 Osteoprotegerin (OPG), neutralizing antibodies in osteoporosis 65, 66 OTX2, see Orthodenticle homeobox 2 Ovary chemotherapy protection by c-Abl-TAp63 pathway inhibition 217 folliculogenesis 90 somatic sex reprogramming to testes by Foxl2 ablation 90, 91 Overcommitment, see Work overcommitment Oxandrolone, efficacy and safety in growth hormonetreated Turner syndrome 201, 202 Oxytocin dopamine interactions in maternal behavior 20 social behavior promotion in autism 20, 21 Oxytocin, Sim1 deficiency, MCR4 decrease, and hyperphagic obesity 7
P PAPP-A2, see Pregnancyassociated plasma proteinA2 Parathyroid hormone (PTH), -arrestin-biased agonist and bone formation mechanisms 215 Parathyroid hormone-related protein (PTHrP), gene mutations in brachydactyly type E 71 Period, stomach ghrelinsecreting cells as food-entrainable circadian clocks 216 Peroxisome proliferatoractivated receptor-␣ (PPAR-␣), thermogenic activation of neonatal brown fat via FGF21 212 PGF2␣, see Prostaglandin F2␣ Pheochromocytoma, TMEM127 mutations 113 Pituitary, see also specific hormones
Subject Index
amyloid fibrils in secretory granules and peptide hormone storage 21, 22 ERK signaling in female fertility 23, 24 idiopathic hypogonadotropic hypogonadism defects 94 transcription factors in development HES1 17 interactions of TLE1, TLE3, HESX1, and PROP1 18 LHX2 15, 16 LHX3 14, 15 OTX2 13, 14 overview 16, 17 Pituitary tumor transforming gene (PTTG), E2F1 induction in pituitary tumors 26 PKC, see Protein kinase C Polyoma virus middle T antigen (PyVmT), insulin receptor interaction for oncogene activation 59, 60 Potassium channel, see ATP-sensitive potassium channel; Kcne2 PPAR-␣, see Peroxisome proliferator-activated receptor-␣ PPNAD, see Primary nodular adrenocortical disease Prader-Willi syndrome (PWS), growth hormone therapy cardiovascular and metabolic risk profile 199 efficacy and safety 40, 41 Pramlintide, glucose excursion lowering in type 1 diabetes 133, 134 PRDM16, transcriptional regulation of myoblast to brown fat switch 148, 149 Pregnancy-associated plasma protein-A2 (PAPP-A2), insulin-like growth factorbinding protein-5 proteolysis mediation 57, 58 Preterm infant, combination therapy with insulin-like growth factor-1/insulin-like growth factor-binding protein-3 59 Primary nodular adrenocortical disease (PPNAD),
Subject Index
microRNA signature 104, 105 PROP1, transcription factor interactions in pituitary development 18 Propylthiouracil, hepatotoxicity in children 34, 35 Prostaglandin F2␣ (PGF2␣), adrenal regulation 111, 112 Protein kinase C (PKC) dehydroepiandrosterone sulfate activation of protein kinase C- 107 PTH, see Parathyroid hormone PTHrP, see Parathyroid hormone-related protein PTTG, see Pituitary tumor transforming gene Puberty aromatase inhibitors effects on vertebral morphology in male constitutional delay of puberty 96 birth weight and odds ratio of status 92, 93 body mass index and timing in boys 194 differential diagnosis of late-onset congenital adrenal hyperplasia from premature adrenarche 196, 197 estradiol-kisspeptin positive feedback mechanism in onset 4, 5 4--GABAA receptor in puberty-associated learning deficits 207 LIN28B in constitutional delay of growth and puberty 93 trends in timing and environmental modifiers 92 PWS, see Prader-Willi syndrome PyVmT, see Polyoma virus middle T antigen
R RANK, central control of fever and female body temperature 78 Rickets, autosomal-recessive hypophosphatemic rickets and ectonucleotide
pyrophosphate/phosphodiesterase mutations 77, 78 Rituximab, preservation of beta cell function 132, 133 Rosuvastin, familial hypercholesterolemia management 168
S Santa Claus, childhood obesity impact 169 SCN, see Suprachiasmatic nucleus Serotonin mediation of leptin bone effects 79 osteoporosis management with inhibitors 79, 80 Sertoli cell derivation from embryonic stem cells 89 differentiation gene discovery 89, 90 Sex, see Intercourse Sex determination, gene discovery 89, 90 SGA, see Small for gestational age Short stature, see Growth hormone deficiency; Height Sim1, deficiency and hyperphagic obesity 7 Small for gestational age (SGA) growth hormone therapy outcomes 51 insulin-like growth factor-1 receptor haploinsufficiency 52 insulin-like growth factorbinding protein-2 serum levels 50, 51 SREBP, see Sterol regulatory element-binding protein Statins, alternative summary statistics for communicating risk reduction 226, 227 Sterol regulatory elementbinding protein (SREBP), MiR-33 and cholesterol homeostasis regulation 234, 235 Stress child abuse stress and aging induction 105, 106 fetal corticosteroid metabolite concentrations and maternal effects 211
261
homeostasis 114, 115 Suprachiasmatic nucleus (SCN), vasopressin secretion regulation 10, 11 Surfactant, NKX2-1 mutations in brain-lung-thyroid syndrome 41, 42
T Tanycyte, estrogen plasticity role in median eminence 4 TAp63, pathway inhibition in oocyte chemotherapy protection 217 TASK1, adrenal adenoma expression and aldosterone production role 112, 113 Telomerase, variants in Ashkenazi centenarians 228 Telomere, shortening in child abuse 105, 106 Testes idiopathic hypogonadotropic hypogonadism defects 94 somatic sex reprogramming of ovaries to testes by Foxl2 ablation 90, 91 Testosterone, estrogen aromatization and male sexual behavior 2, 3 TGF-, see Transforming growth factor- TH, see Thyroid hormone THA, see Thyroid hemiagenesis Thyroid cancer medullary thyroid cancer management guidelines 39 papillary thyroid cancer treatment outcomes 40, 42 secondary neoplasms after Hodgkin’s lymphoma treatment 39, 40 Thyroid hemiagenesis (THA), thyroid pathology risks 192, 193 Thyroid hormone (TH) analog and Mct8 transport studies 45, 46 intra-amniotic thyroxine treatment in non-immune fetal goitrous hypothyroidism 35, 36
262
iodine supplementation effects on cognition 43, 44 Kcne2 mutant mouse studies 33, 34 neonatal screening for central hypothyroidism 36 resistance and metabolic effects 44, 45 Thyroid hormone growth plate chondrocyte effects 72, 73 obesity and thyroid function 152 therapy regimens for transient hypothyroxinemia in neonates 189, 190 Thyroid-stimulating hormone (TSH) neonatal hyperthyrotropinemia 38 neonatal screening for central hypothyroidism 37 Thyrotropin-releasing hormone (TRH), tuberoinfundibular dopamine neuron effects 213, 214 TIDA neuron, see Tuberoinfundibular dopamine neuron TLE1, transcription factor interactions in pituitary development 18 TLE3, transcription factor interactions in pituitary development 18 TLR5, see Toll-like receptor 5 TMEM127, pheochromocytoma mutations 113 Tolerance, perinatal window for Aire control of autoimmunity 223, 224 Toll-like receptor 5 (TLR5), knockout mouse and gut microbiota in metabolic syndrome 149, 150 Transforming growth factor- (TGF-), bone remodeling role 67, 68 Transsexuals, endocrine treatment guidelines 85 TRH, see Thyrotropin-releasing hormone TSH, see Thyroid-stimulating hormone
Tuberoinfundibular dopamine (TIDA) neuron, thyrotropinreleasing hormone effects 213, 214 Turner syndrome, growth hormone therapy metabolic consequences 203, 204 oxandrolone efficacy and safety 201, 202 Turner syndrome, estrogen replacement therapy 190, 191
V Vasopressin, clock regulation 10, 11 Ventromedial hypothalamus (VMH), insulin and pancreatic glucagon secretion regulation 10 Vitamin D maternal status and neonatal bone effects 76 status effects on plasma glucose and lipid levels 167 VMH, see Ventromedial hypothalamus
W Wnt growth plate chondrocyte effects 72, 73 primary nodular adrenocortical disease role 104, 105 Work overcommitment, stress hormone studies 116, 117
X X-linked adrenoleukodystrophy (ALD), hematopoietic stem cell gene therapy with lentiviral vector 219, 220
Z Zfp423, transcriptional control of preadipocyte determination 139, 140
Subject Index