Yearbook of Pediatric Endocrinology 2010
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The body of knowledge in most medical specialties is rapidly expanding, making it virtually impossible to follow all advances in clinical and basic sciences that are relevant to a given field. This is particularly true in pediatric endocrinology, at the cross-road of pediatrics, endocrinology, development and genetics. Providing abstracts of articles that report the year’s breakthrough developments in the basic sciences and evidence-based new knowledge in clinical research and clinical practice that are relevant to the field, the Yearbook of Pediatric Endocrinology 2010 keeps busy clinicians and scientists, pediatric endocrinologists, and also pediatricians and endocrinologists informed on new advances. Twelve Associate Editors and their co-authors selected from several thousand papers those that brought the most meaningful new information, summarized them and provided comments to put them into perspective. The papers are classified into those that identify new genes involved in diseases, new hormones, concepts revised or re-centered, important observations for clinical practice, large-scale clinical trials, new mechanisms, new paradigms, important review articles, new fears and new hopes. Because the Yearbook is endorsed by the European Society for Paediatric Endocrinology (ESPE), its publication is linked to the annual meeting of the ESPE. The Yearbook of Pediatric Endocrinology 2010 covers the medical and scientific literature from June 2009 through May 2010.

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Date de parution 13 septembre 2010
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EAN13 9783805596022
Langue English
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Yearbook of Pediatric Endocrinology 2010
Yearbook of
Pediatric Endocrinology 2010
Endorsed by the European Society for Paediatric Endocrinology
Editors
Jean-Claude Carel
Ze’ev Hochberg
Associate Editors
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
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
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 jean-claude.carel@inserm.fr
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 z_hochberg@rambam.health.gov.il
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 g.butler@ich.ucl.ac.uk
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 evangelia.charmandari@googlemail.com
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 chiarelli@unich.it
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 stefano.cianfarani@uniroma2.it
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 mdattani@ich.ucl.ac.uk
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 nicolas.deroux@inserm.fr
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 ken.ong@mrc-epid.cam.ac.uk
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 Orit.Hamiel@sheba.health.gov.il
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 michel.polak@nck.aphp.fr
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 lars.savendahl@ki.se
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 olle.soder@kbh.ki.se
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 martin.wabitsch@uniklinik-ulm.de
Table of Contents
Preface
Ze’ev Hochberg and Jean-Claude Carel
Neuroendocrinology
Carine Villanueva, Lukas Huihjbregts and Nicolas de Roux
Pituitary
Evelien F. Gevers and Mehul T. Dattani
Thyroid
Michel Polak, Gabor Szinnai, Aurore Carré and Mireille Castanet
Growth and Growth Factors
Stefano Cianfarani
Bone, Growth Plate and Mineral Metabolism
Terhi Heino, Dov Tiosano, Aneta Gawlik and Lars Sävendahl
Reproductive Endocrinology
Olle Söder and Lena Sahlin
Adrenals
Erica L.T. van den Akker and Evangelia Charmandari
Type 1 Diabetes: Clinical and Experimental
Francesco Chiarelli and M. Loredana Marcovecchio
Obesity and Weight Regulation
Martin Wabitsch, Daniel Tews, Michaela Keuper, Carsten Posovszky,
Christian Denzer, Anja Moss, Julia von Schnurbein and
Pamela Fischer-Posovszky
Type 2 Diabetes Mellitus, Metabolic Syndrome, Lipids
Orit Pinhas-Hamiel
Population Genetics and Pharmacogenetics
Ken K. Ong and Cathy Elks
Evidence-Based Medicine in Pediatric Endocrinology
Gary E. Butler
Editor’s Choice
Jean-Claude Carel and Ze’ev Hochberg
Science and Medicine
Ze’ev Hochberg and Jean-Claude Carel
Author Index
Subject Index
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.
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
Methods and Results: 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.
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
Methods and Results: 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.
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, β-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-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 Moléculaire-Neurophysiologie 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 male-specific 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 (AR NesCre ), 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 AR NesCre 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 ERα 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 AR NesCre mice would allow to test this hypothesis.
Male AR NesCre 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 AR NsDel . In contrast with AR NesCre mice, male AR NsDel 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.
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 E 2 (PGE 2 ) synthesis. PGE 2 treatment induces an increase of retraction of cultured tanycytes. Ultrastructural analysis of mouse hypothalamic explants of the ME showed that PGE 2 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 PGE 2 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 emin ence, resulting in tanycytes retraction. This retraction could be facilitated by an estradiol-dependent increase of cyclooxygenases COX1 and 2, which metabolize PGE 2 , 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 kiss-peptin expression in the rostral periventricular area of the third ventricule, where kisspeptin neurons 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, kiss-peptin 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 neuroen-docrine 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.
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 κ-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-α 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.
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 α -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 paraven-tricular 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 leptin-melanocortin-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 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 BA 2+ -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 melanin-concentrating 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 intra-ventricular 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 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.
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 α-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 γ-aminobutyric acid A receptor agonist, muscimol.
Conclusion: Insulin inhibits α-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 α-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 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-α. 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.
Pituitary
Evelien F. Gevers a , b and Mehul T. Dattani a
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
tomogata@nch.go.jp
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 trans-activates 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
berit.kristrom@pediatri.umu.se
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.
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
hw@mail.nih.gov
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 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
m.dattani@ich.ucl.ac.uk
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 hypothalamo-pituitary 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 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, III., USA
Traetzman@life.uiuc.edu
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 α-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 cyclin-dependent 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.
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
scamper@umich.edu
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.
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
e.faloia@univpm.it
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.
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
Michael.meaney@mcgill.ca
Endocrinology 2010;151:2276-2286
Background: Variations in maternal behavior among lactating rats associate with differences in estrogen-oxytocin 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 fluoro-gold 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 mesocorti-colimbic 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
sirigu@isc.cnrs.fr
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.
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
roland.riek@phys.chem.ethz.ch
Science 2009;325:328-332
Background: Amyloid fibrils are highly organized cross-β-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 β-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 amyloid-like aggregates on its own but did so in the presence of β-endorphin, which is also processed from pro-opiomelanocortin and secreted together with ACTH in a regulated secretory pathway. These data were 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, β-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-β-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 non-aggregation 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 β-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 GHRH-receptor 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
RDkineman@uic.edu
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 age-matched controls indicating that cell cycle progression was blocked. Further evidence suggested that 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, SST-knockout 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 hyper-plastic 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
MSmsr14@cornell.edu
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 GnRH-mediated 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, LHβ 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.
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-kinase-kinase 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 α-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. LHβ synthesis was reduced in gonadotropes and ovulation occurred in response to exogenous LHβ, implicating LH deficiency as the primary cause of infertility. In males, LHβ 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 gender-specific GnRH pulsatility and LHβ 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
jens.bruening@uni-koeln.de
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 IGF1. Unexpectedly the thyroid axis was also affected, with an increase in TRH, TRH receptors, TSHβ, 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
primus.mullis@insel.ch
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 17.5-kDa 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 iso-form, 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 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 17.5-kDa 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
SMelmed@csmc.edu
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 up-regulation 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.
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
joe.goldstein@southwestern.edu
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 blood-glucose 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.
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
wrays@mail.nih.gov
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 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
patrice.mollard@igf.crns.fr
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 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
a.j.clark@qmul.ac.uk
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
skansra@mcw.edu
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 PPARγ 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 NFκB, and selectively targets 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 NFκB. The authors recently demonstrated that curcumin inhibited growth and induced apoptosis in prolactin-and 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 NFκB 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 NFκB 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 TNFα-induced activation of IKK that leads to the inhibition of TNF-dependent phosphorylation and degradation of I κ Bα and subsequent nuclear translocation of the p65 subunit of NF κ B to regulate expression of genes implicated in cell cycle progression (e.g. cyclin D family), apoptosis (bcl-2) , and cell migration and invasion (e.g. MMP2 and MMP9). Constitutive activation of NF κ B has been reported in cell lines as well as tumor samples, and may be linked to tumor progression as well as drug resistance. Bcl-xl plays a central role in pituitary cell survival and apoptosis, and these studies suggest that Bcl-xl plays a major role in regulating cell survival in pituitary corticotrophs. Curcumin may therefore modify Bcl-xL levels and hence act as a tumor suppressor, offering new hope to the treatment of Cushing disease.
References
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Thyroid
Michel Polak a , Gabor Szinnai b , Aurore Carré a and Mireille Castanet b
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
gwa2001@med.cornell.edu
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 (T 3 ) and tetraiodothyronine (thyroxine, or T 4 ) 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 T 3 and T 4 . 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 (T 4 ) 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 (T 3 ) and T 4 administration to pups, by supplementing dams with T 4 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 thyroid-stimulating 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 KCNQ1-linked 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
scott.rivkees@.yale.edu
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-than-expected 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, 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 methimazole-related 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
michel.polak@nck.aphp.fr
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 amniotic-fluid 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.
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
Johnny.deladoey@umontreal.ca
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
tdnebesi@iupui.edu
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 T 4 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 T 4 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.
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
luca.persani@unimi.it
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-T 4 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
tim.cheetham@nuth.nhs.uk
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.

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