New Perspectives in GH Research: Davos, March 2002 (Hormone Research)

New Perspectives in GH Research Davos, Switzerland March 7–10, 2002 Editors: Helmut-Günther Dörr, Erlangen Michael B...

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New Perspectives in GH Research Davos, Switzerland March 7–10, 2002

Editors:

Helmut-Günther Dörr, Erlangen Michael B. Ranke, Tübingen Christian Wüster, Mainz

17 figures and 12 tables, 2002

Basel 폷 Freiburg 폷 Paris 폷 London 폷 New York 폷 New Delhi 폷 Bangkok 폷 Singapore 폷 Tokyo 폷 Sydney

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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 or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center (see ‘General Information’). © Copyright 2002 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISBN 3–8055–7523–8

Vol. 58, Suppl. 3, 2002

Contents

1

GH and Kidney

Foreword Molecular Biology of the GH-IGF System

2

Isolated Growth Hormone Deficiency and the GH-1 Gene: Update 2002 Binder, G. (Tübingen)

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30

Zeier, M. (Heidelberg) 35

Clinical Impact of Molecular Diagnostics in Endocrinology. Polymorphisms, Mutations and DNA Technologies

16

20

GH and the Muscular-Skeletal System 39

Auxological, Ophthalmological, Neurological and MRI Findings in 25 Austrian Patients with Septo-Optic Dysplasia (SOD). Preliminary Data Riedl, S.W.; Müllner-Eidenböck, A.; Prayer, D.; Bernert, G.; Frisch, H. (Vienna)

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Effects of Growth Hormone on Skeletal Muscle Weber, M.M. (Köln)

Psychosocial Adaptation to Short Stature – An Indication for Growth Hormone Therapy? Muscle

Fatal Outcome of Sleep Apnoea in PWS during the Initial Phase of Growth Hormone Treatment. A Case Report

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US Experience in Evaluation and Diagnosis of GH Therapy of Intrauterine Growth Retardation/ Small-for-Gestational-Age Children Saenger, P. (Bronx, N.Y.)

Osteoporosis and the Growth Hormone-Insulin-Like Growth Factor Axis Geusens, P.P.M.M. (Maastricht/Diepenbeek); Boonen, S. (Leuven)

Eiholzer, U.; Nordmann, Y.; l'Allemand, D. (Zürich) 27

Systemic Application of Growth Hormone for Enhancement of Secondary and Intramembranous Fracture Healing Bail, H.J.; Kolbeck, S.; Krummrey, G.; Schmidmaier, G.; Haas, N.P.; Raschke, M.J. (Berlin)

Wygold, T. (Lübeck) 24

Effects of Growth Hormone in Patients with Chronic Renal Failure: Experience in Children and Adults Wühl, E.; Schaefer, F. (Heidelberg)

Höppner, W. (Hamburg)

GH in Children

Risk of Mortality in Patients with End-Stage Renal Disease: The Role of Malnutrition and Possible Therapeutic Implications

Hot Topics 56

Growth Hormone Secretagogues and Ghrelin: An Update on Physiology and Clinical Relevance Petersenn, S. (Essen)

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Author Index and Subject Index

Foreword

It has been a tradition for many years for Novo Nordisk to provide a forum at which internationally renowned experts in the field of growth hormone (GH) research and endocrinology meet with experienced paediatricians and adult endocrinologists with the aim of sharing their knowledge. The focus of this year’s symposium was on the latest findings relating to basic research in molecular biology, and on other issues which continue to be challenging although they have been regularly debated in the past. The first part of the symposium dealt with the molecular biology of the GH-IGF system and incorporated a comprehensive update on the genetics of isolated GH deficiency and, in addition, an overview of the clinical impact of molecular diagnostics in the field of endocrinology. Since the quantification of molecular polymorphisms as a prequisite to hormonal treatment has become a favoured practice among clinicians, the need was felt to distinguish clearly between ongoing research and valid, current practice. In the paediatrics session, it became evident that our deepening of clinical knowledge is inextricably linked with the data collection of rare diseases such as septooptic dysplasia. It is interesting to note that although GH treatment was approved by the regulatory authorities several years ago, no consensus has been achieved yet on whether or not short stature is associated with psychologi-

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cal deficits. Advancements in this field will be influenced by the anticipated approval of IUGR (as in France and the USA) as an indication in Europe from 2003 onwards. The experience of paediatric nephrologists who have monitored GH treatment in children with chronic renal failure is valuable to their counterparts who treat adults, as the paediatric experience allows for a better understanding of the pharmacotherapeutics of GH and its efficacy. The current understanding of the anabolic effects of GH on muscle and bone will widen the scope of GH in the treatment of adult patients, and will, hopefully, also serve as a deterrent against the misuse of this hormone among athletes and body builders. The focal point of the final session of the symposium was the recently discovered Ghrelin, which links the lipid, gastrointestinal, and the GH-IGF-1 systems. Ghrelin is also thought to be clinically relevant in the treament of appetite disorders. The guest editors of this supplement to Hormone Research would like to thank the authors, speakers, and all other participants for the fruitful discussions during the meeting. We are also grateful to Novo Nordisk for their continued support in organising this event and in publishing its proceedings. Helmuth G. Dörr Michael B. Ranke Christian Wüster

Molecular Biology of the GH-IGF System Horm Res 2002;58(suppl 3):2–6 DOI: 10.1159/000066476

Isolated Growth Hormone Deficiency and the GH-1 Gene: Update 2002 Gerhard Binder University Children’s Hospital, Tübingen, Germany

Key Words Growth hormone W IGHD W GH-1 gene W GH promotor

Abstract This short review will focus on the mechanisms which are thought to be directly involved in GH expression and particularly on the monogenetic disorders which were shown to cause isolated growth hormone deficiency (IGHD) due to insufficient expression of GH. The overwhelming majority of genetic defects detected in isolated growth hormone deficiency (IGHD) are mutations of the coding region of the GH-1 gene which belongs to a five genes containing gene cluster located on 17q22–24. Depending on the type of the GH-1 gene mutation, the mode of inheritance is recessive or dominant. The promotor region of the GH-1 gene which encompasses the 300 bp of the 5) flanking region is highly polymorphic, but the functionally important cis-acting elements are conserved. This sequence is sufficient to control GH expression in cultured cells, but not in transgenic mice: the human GH locus control region, an enhancer region of the GH-1 gene located approximately 15–32 kB upstream of the GH-1 coding region was shown to direct pituitary-specific, high-level GH expression in vivo. Pro-

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motion of the GH expression needs the coordinate binding of pituitary-specific (i.e., POU1F1) and ubiquitous trans-acting factors to the cis-acting elements. The mutational analysis of trans-acting factors and cis-acting elements of the GH-1 gene has so far not established any defect outside the coding region as the genetic basis of IGHD except for POU1F1 mutations which cause multiple pituitary hormone deficiency including GHD. Several mutations of the GHRH-receptor gene were shown to result in severe IGHD. In the future, the discovery of new defects of the GH expression machinery will add to our understanding of how GH is sufficiently supplied to the organism and will hopefully simplify and improve the diagnostic approach in a subset of children with IGHD. Copyright © 2002 S. Karger AG, Basel

Introduction

This review will briefly describe the known mechanisms involved in GH expression and mainly focus on the monogenetic disorders which were shown to cause IGHD due to an insufficient GH expression. Approximately 1 of 200 short children has an isolated disturbance of GH secretion [1] and needs recombinant GH therapy for nor-

Dr. Gerhard Binder University Children’s Hospital, Hoppe-Seyler-Strasse 1 D–72076 Tübingen (Germany) Tel. +49 7071 2983781, Fax +49 7071 294157 E-Mail [email protected]

Fig. 1. Known GH-1 gene mutations and

mode of inheritance of IGHD. Two microdeletion mutations at codon numbers –10 and +54 of GH-1 gene which cause reading frame shift and the recessive inheritance of IGHD (IA) are not shown.

mal growth. Familiarity of this disorder is much more frequent [2] than the diagnosis of a monogenetic defect. Therefore, our understanding of the genetic basis of IGHD is still limited. Four Mendelian disorders have been described in familial IGHD: two forms transmitted due to an autosomal recessive trait (IGHD IA, with total absence of GH, and IGHD IB, with low GH levels), one due to an autosomal dominant trait (IGHD II) and one due to an X-chromosomal trait (IGHD III) [3]. In general, normal expression of a protein needs a perfect coding sequence, functioning promotor loci (the cis-acting elements) and sufficient amounts of transcription factors (the trans-acting factors).

The gene encoding GH is located in the GH gene cluster which consists of five genes: the GH-1 gene, and the genes encoding CSHP, CSH-1, GH-2 and CSH-2 (in the order from 5) to 3)). This region encompasses 66.5 kB on chromosome 17q22–24 and was one of the first human genome loci completely sequenced, in 1987 [4]. The coding sequences of the five genes as well as the intervening sequences are 92–98% homologous suggesting that this multigene family arose through several duplication events [5]. GH-2 encodes the GH variant, a protein which differs from the GH in 13 amino acids and is expressed in the

placenta during the second half of pregnancy; its bioactivity is comparable to GH, its physiological role unknown [6]. The GH-1 gene consists of 5 exons with short intervening sequences, the complete gene sequence encompasses 1.3 kB [5]. Alternative splicing of the primary RNA results in at least four different mRNA transcripts of which the major one contains the whole coding sequence, while around 10% are processed to an mRNA lacking the first 45 bp of exon 3 and less than 5% to transcripts lacking the whole exon 3, or both exons 3 and 4 [7, 8]. The major GH prohormone of 217 amino acids contains the N-terminal signal peptide which is necessary for the transport into the RES where the prohormone is processed to GH, a 22-kD protein of 191 amino acids. No additional posttranslational modification occurs. 20-kD GH, the main alternative splicing product, has a very similar bioactivity to that of 22-kD GH [9]. The mechanisms involved in the transport of GH through the Golgi complex and the selective storage of GH in densely packed secretory vesicles are still unknown. The overwhelming majority of genetic defects detected in IGHD are mutations of the coding region of the GH-1 gene. The first GH-1 gene mutations described were homozygous deletions of the GH-1 gene locus with heterogeneous sizes of 6.7 kB (frequent), 7.6, 7.0 and 45 kB (IGHD IA) [10, 11]. The clinical consequence is the immunological intolerance to GH and therefore, fre-

IGHD: Update 2002

Horm Res 2002;58(suppl 3):2–6

The Human GH-1 Gene

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Fig. 2. 5) promotor region of the GH-1 gene

and cis-acting elements.

quently the failure of growth-promoting therapy with GH [12]. Interestingly, the parents of these children who are heterozygous for this defect are not GH deficient indicating that loss of one GH-1 allele is well tolerated by the GH-producing machinery. In rare instances, microdeletions or point mutations of the GH-1 gene were reported to cause IGHD IA [13]. The mutant products of these cases were predicted to be absent (stop codon inside the signal peptide) (fig. 1) or severely altered due to truncation and reading frame shift which might induce the early intracellular clearance due to the so-called unfolded protein response [14]. However, recessively inherited mutations of the GH-1 gene do not always result in a complete lack of GH, and then they are accounted to IGHD IB: The only example so far is a homozygous mutation of the GH1 gene donor splice site of intron 4 which also results in an altered GH due to deletion of amino acids 103–126 and a complete different amino acid sequence thereafter [3, 15] (fig. 1). This mutant GH is released from the somatotrophs, but not in sufficient amounts; recombinant GH therapy is well tolerated. Diverse GH-1 gene point mutations of the donor splice site of intron 3 are the main cause of autosomal dominant inherited IGHD II [16] (fig. 1). These mutations cause skipping of exon 3 during splicing with the effect of the loss of amino acids 32–71 of GH. Interestingly, the wildtype GH from the normal allele do not compensate for the del32–71GH suggesting a dominant negative effect of the del32–71GH. The exact mechanism causing insufficient GH secretion is still cryptic. In vivo and in vitro data indi-

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cate that del32–71GH might not be cell-toxic, but interfere with the intracellular transport and storage of the wild-type GH in neuroendocrine cells [17–19]. Recently, three missense mutations of the GH-1 gene (R183H, P89L, V110F) have been described which also cause a dominant inheritance of IGHD whose clinical phenotype, however, is milder than in the children affected by the intron 3 splicing mutations [16, 20, 21] (fig. 1). Interestingly, there exist also two unique missense mutations of the GH-1 gene (R77C, D112G) which were reported to cause a high-normal secretion of a bioinactive GH molecule conferring Kowarski syndrome [22, 23].

Cis-Acting Transcription Elements

The transcription machinery needs cis-acting elements – short stretches of DNA sequences mainly 5) of the gene – to which the transcription factors, also called transacting factors, bind. This binding is a prerequisite for normal regulated transcription by RNA polymerase. The promotor sequence of the GH-1 gene which is sufficient to control GH expression in cultured cells, consists of around 300 bp of the 5) flanking region of the GH-1 gene which contains two cis-acting elements for the pituitary-specific transcription factor Pit-1 (official nomenclature: POU1F1) at –65 to –92 and at –105 to –130 [24] and two nonclassical cAMP-response element motifs at –95 to –99 and at –183 to –187 [25] (fig. 2). In addition, cis-acting elements for the binding of ubiquitous transcription fac-

Binder

tors like Zn-15, SP1, USF, NF-1 have been described, whereby the functional importance of these loci for human GH expression has yet to be tested [24, 26] (fig. 2). While deleterious mutations of the main cis-acting elements of the GH-1 gene promotor have not been detected so far, sequencing analysis of the human GH promotor region revealed a surprisingly high variability [27, 28], which might – at least in part – have been generated by intergenic gene conversion between the five genes of the GH gene locus [29]. In contrast to cultured cells, the proximal promotor of the human GH-1 gene is not sufficient to establish an appropriate expression of GH in pituitary of transgenic mice: When the GH-1 gene was integrated in the mouse genome, it was expressed only sporadically and at low levels in the transgenic target organ [30]. Two loci, approximately 15 and 30 kB upstream of the GH-1 gene, were identified by DNase-I hypersensitivity experiments as directing site of integration-independent, pituitary-specific and high-level GH expression and were therefore named human GH locus control region [30]. Such a region is thought to contain cis-acting elements which bind trans-acting factors inducing an initial alteration of the chromatin structure with subsequent opening of the proximal promotor for transcriptional activation. Recently, three A/T-rich regions which could serve as a cis-acting element for Pit-1 were detected in the human GH locus control region [31].

partly the thyrotrophs due to binding to several other target genes [34]. GHRH is the main extracellular stimulatory regulator of GH expression and release. Its binding to the Gs protein-coupled GHRH receptor causes an elevation of intracellular cAMP levels and activation of protein kinase A [35]. GHRH-receptor mutations have been described to result in severe hypoplasia of the pituitary gland and severe IGHD (type IB) [36], while constitutional activating mutations of the GHRH-receptor-associated G protein (GS) cause growth hormone excess [37]. A clear answer to the question as to which trans-acting factor(s) are finally responsible for the GHRH-induced upregulation of human GH expression is still missing but Pit-1 is very likely to be involved in this pathway [38]. Basal expression of human GH is assumed to need additional trans-acting factors like Sp1, CTF/NF-1, USF, C/EBP, which act in concert with Pit-1. In the future, the discovery of new defects of the GH expression machinery will add to our understanding of how GH is sufficiently supplied for the organism and will hopefully simplify and optimize the diagnostic approach in a subset of children with IGHD.

Acknowledgements I would like to thank P.E. Mullis (Bern, Switzerland) for critical reading of the manuscript and D. Martin (Tübingen) for language editing.

Trans-Acting Factors

The pituitary-specific transcription factor Pit-1, also called GHF1 (GH factor 1), now officially named POU1F1 (POU domain, class 1, transcription factor 1) belongs to the family of POU-domain transcription factors regulating mammalian development. Binding of Pit1 as a dimer to the two cis-acting elements of the GH-1 gene promotor is essential for GH expression. The two protein domains of Pit-1 – POU-specific and POU-homeo – are necessary for high-affinity DNA binding to the GH promotor [32]. After binding, GH gene expression is activated through the N-terminal transactivation domain of Pit-1; however, Pit-1 alone is probably not sufficient for regulated GH expression [33]. Mutations of the functional domains of Pit-1 which are inherited through a dominant or recessive trait cause a combined deficiency of GH, TSH and PRL, because the POU transcription factor is additionally involved in the embryological proliferation and differentiation of the somatotrophs, lactotrophs and

IGHD: Update 2002


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References 1 Vimpani GV, Vimpani AF, Lidgard GP, Cameron EH, Farquhar JW: Prevalence of severe growth hormone deficiency. Br Med J 1977; 2:427–430. 2 Tanner JM: Human growth hormone. Nature 1972;237:433–439. 3 Phillips JA III, Cogan JD: Genetic basis of endocrine disease 6:Molecular basis of familial human growth hormone deficiency. J Clin Endocrinol Metab 1994;78:11–16. 4 Hirt H, Kimelman J, Birnbaum MJ, Chen EY, Seeburg PH, Eberhardt NL, Barta A: The human growth hormone gene locus: Structure, evolution and allelic variations. DNA 1987;6: 59–70. 5 Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH: The human growth hormone locus: Nucleotide sequence, biology and evolution. Genomics 1989;4:479– 497. 6 MacLeod JN, Worsley I, Ray J, Friesen HG, Liebhaber SA, Cooke NE: Human growth hormone variant is a biologically active somatogen and lactogen. Endocrinology 1991;128:1298– 1302. 7 Baumann G: Growth hormone heterogeneity: Genes, isohormones, variants and binding proteins. Endocr Rev 1991;12:424–449. 8 Binder G, Ranke MB: Screening for growth hormone (GH) gene splice site mutations in sporadic cases with severe isolated GH deficiency using ectopic transcript analysis. J Clin Endocrinol Metab 1995;81:1247–1252. 9 Tsunekawa B, Wada M, Ikeda M, Banba S, Kamachi H, Tanaka E, Honjo M: The binding between the stem regions of human growth hormone (GH) receptor compensates for the weaker site 1 binding of 20 kDa human GH (hGH) than that of 22 kDa hGH. J Biol Chem 2000; 275:15652–15656. 10 Phillips JA III, Hjelle B, Seeburg PH, Zachmann M: Molecular basis for familial isolated growth hormone deficiency. Proc Natl Acad Sci USA 1981;78:6372–6375. 11 Akinci A, Kanaka C, Eble A, Akar N, Vidinlisan S, Mullis PE: Isolated growth hormone (GH) deficiency type IA associated with a 45kilobase gene deletion within the human GH gene cluster. J Clin Endocrinol Metab 1992;75: 437–441. 12 Illig R, Prader A, Ferrandez A, Zachmann M: Hereditary prenatal growth hormone deficiency with increased tendency to growth hormone antibody formation (‘A type’ isolated growth hormone deficiency). Acta Paediatr Scand 1971;60(suppl):607. 13 Procter AM, Phillips JA III, Cooper DN: The molecular genetics of growth hormone deficiency. Hum Genet 1998;103:255–272. 14 Mori K: Tripartite management of unfolded proteins in the endoplasmatic reticulum. Cell 2000;101:451–454.

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15 Wagner JK, Eble A, Hindmarsh PC, Mullis PE: Prevalence of human GH-1 gene alterations in patients with isolated growth hormone deficiency. Pediatr Res 1998;43:105–110. 16 Binder G, Keller E, Mix M, Massa GG, Stokvis-Brantsma WH, Wit JM, Ranke MB: Isolated growth hormone deficiency with dominant inheritance (IGHD II): New mutations, new insights. J Clin Endocrinol Metab 2001; 86:3877–3881. 17 Binder G, Brown M, Parks JS: Mechanisms responsible for dominant expression of human growth hormone gene mutations. J Clin Endocrinol Metab 1996;81:4047–4050. 18 Hayashi Y, Yamamoto M, Ohmori S, Kamijo T, Ogawa M, Seo H: Inhibition of growth hormone (GH) secretion by a mutant GH-1 gene product in neuroendocrine cells containing secretory granules: An implication for isolated GH deficiency inherited in an autosomal dominant manner. J Clin Endocrinol Metab 1999; 84:2134–2139. 19 Lee MS, Wajnrajch MP, Kim SS, Plotnick LP, Wang J, Gertner JM, Leibel RL, Dannies PS: Autosomal dominant growth hormone (GH) deficiency type II: The del32–71-GH deletion mutant suppresses secretion of wild-type GH. Endocrinology 2000;141:883–890. 20 Gertner JM, Wajnrajch MP, Leibel RL: Genetic defects in the control of growth hormone secretion. Horm Res 1998;49(suppl 1):9–14. 21 Duquesnoy P, Simon D, Netchine I, Dastot F, Sobrier ML, Goosens M, Czernichow P, Amselem S: Familial isolated growth hormone deficiency with slight height reduction due to a heterozygote mutation in GH gene (abstract). Proc 80th Annual Meeting of the Endocrine Society, 1998, P02–202. 22 Takahashi Y, Kaji Y, Okimura H, Goji H, Abe H, Chihara K: Short stature caused by a mutant growth hormone. N Engl J Med 1996;312:214– 217. 23 Takahashi Y, Shirono H, Arisaka O, Takahashi K, Yagi T, Koga J, Kaji H, Okimura Y, Abe H, Tanaka T, Chihara K: Biologically inactive growth hormone caused by an amino acid substitution. J Clin Invest 1997;100:1159–1165. 24 Theill LE, Karin M: Transcriptional control of GH expression and anterior pituitary development. Endocr Rev 1993;14:670–689. 25 Shepard AR, Zhang W, Eberhardt NL: Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells. J Biol Chem 1994;269:1804–1814. 26 Lipkin SM, Näär AM, Kalla KA, Sack RA, Rosenfeld MG: Identification of a novel zinc finger protein binding a conserved element critical for Pit-1-dependent growth hormone gene expression. Genes Dev 1993;7:1674– 1687.


27 Wagner JK, Eble A, Cogan JD, Prince MA, Phillips JA III, Mullis PE: Allelic variations in the human growth hormone-1 gene promotor of growth hormone-deficient patients and normal controls. Eur J Endocrinol 1997;137:474– 481. 28 Binder G, Kampouridis L, Benz M, Ranke MB: Growth hormone gene promotor polymorphisms in idiopathic short stature (abstract). Horm Res 1997;48:93. 29 Giordano M, Marchetti C, Chiorboli E, Bona G, Richiardi PM: Evidence for gene conversion in the generation of extensive polymorphism in the promotor of the growth hormone gene. Hum Genet 1997;100:249–255. 30 Jones BK, Monks BR, Liebhaber SA, Cooke NE: The human growth hormone gene is regulated by a multicomponent locus control region. Mol Cell Biol 1995;15:7010–7021. 31 Jin Y, Surabhi RM, Fresnoza A, Lytras A, Cattini PA: A role for A/T-rich sequences and Pit1/GHF-1 in a distal enhancer located in the human growth hormone locus control region with preferential pituitary activity in culture and transgenic mice. Mol Endocrinol 1999;13: 1249–1266. 32 Parks JS, Brown MR, Hurley DL, Phelps CJ, Wajnrajch MP: Heritable disorders of pituitary development. J Clin Endocrinol Metab 1999; 84:4362–4370. 33 Lira SA, Kalla KA, Glass CK, Drolet DW, Rosenfeld MG: Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice. Mol Endocrinol 1993;7:694–701. 34 Pfäffle RW, Kim C, Otten B, Wit JM, Eiholzer U, Heimann G, Parks J: Pit-1:Clinical aspects. Horm Res 1996;45(suppl):25–28. 35 Mayo KE, Godfrey PA, Suhr ST, Kulik DJ, Rahal JO: Growth hormone-releasing hormone: Synthesis and signaling. Recent Prog Horm Res 1995;50:35–73. 36 Wajnrajch MP, Gertner JM, Harbison MD, Chua SC Jr, Leibel RL: Nonsense mutation in the growth hormone-releasing hormone receptor causes growth failure analogous to the little mouse. Nat Genet 1996;12:88–90. 37 Landis CA, Masters SB, Spada A: GTPaseinhibiting mutations activate the · chain of Gs and stimulate adenyl cyclase in human pituitary tumors. Nature 1989;340:692–696. 38 Cohen LE, Hashimoto Y, Zanger K, Wondisford F, Radovick S: CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression. J Clin Invest 1999;104:1123–1130.

Binder

Molecular Biology of the GH-IGF System Horm Res 2002;58(suppl 3):7–15 DOI: 10.1159/000066477

Clinical Impact of Molecular Diagnostics in Endocrinology Polymorphisms, Mutations and DNA Technologies

Wolfgang Höppner Institute of Hormone and Fertility Research, University of Hamburg, Germany

Key Words Molecular diagnostics W Adrenogenital syndrome W Endocrine tumors W Multiple endocrine neoplasia W Mutations W Molecular genetics

Abstract Genetic defects in genes encoding hormones, hormone receptors or polypeptides of the signaling pathways usually cause complex disease manifestations characterized by the involvement of several tissues and variable expression. Genetic aberrations, like chromosome aneuploidy, gene translocations or mutations in key regulatory proteins (even if not directly affecting genes of the endocrine system) often lead to clinical symptoms, including central endocrine functions like sexual differentiation or metabolic disturbances, like diabetes mellitus. But also minor genetic alterations like point mutations can affect the function of gene products to cause endocrine diseases. If the underlying molecular defects of endocrinopathies are known, direct molecular diagnosis can be performed. This is particularly useful if it helps to solve difficult differential diagnosis problems or if there exist effective preventive therapeutic options. The present paper presents examples for endocrine diseases in

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which molecular testing significantly increases the specificity and sensitivity of diagnostics and demonstrates the benefits for the patients and the healthcare system. In multiple endocrine neoplasia type 2, an unambiguous identification of gene carriers in affected families can be achieved by genetic testing. As a preventive measure to avoid medullary thyroid carcinoma, prophylactic thyroidectomy is recommended for individuals carrying the disease causing mutation. In adrenogenital syndrome, sequence analysis of the steroid 21-hydroxylase gene has become an important tool to confirm or exclude suspected late-onset forms of the disease, where hormone measurements are not informative. The major benefit, however, lies in identifying heterozygous carriers and providing a reliable prenatal test for couples carrying a defect in the 21-hydroxylase gene. Today, prenatal treatment with dexamethasone, which prevents the virilization in female fetuses, should always be based on results from molecular diagnosis performed from chorionic villus samples. Copyright © 2002 S. Karger AG, Basel

Prof. Wolfgang Höppner Institute for Hormone and Fertility Research University of Hamburg, Grandweg 64 D–22529 Hamburg (Germany) Tel. +49 40 56190813, Fax +49 40 56190864, E-Mail [email protected]

Genes and Defects

Genomic DNA constitutes the total genetic information of an organism. Together with proteins, doublestranded DNA forms multiple linear chromosomes of different sizes. Most genomic DNA is located within the nucleus (human nuclear DNA: 3–5 W 1010 basepairs). Additionally, genomic DNA is also present in the mitochondria as a circular molecule (16,000 basepairs, encoding 37 genes) and in multiple copies. Human genomic DNA contains approximately 30,000–40,000 genes, discrete regions that encode a protein or RNA. A gene comprises the coding DNA sequence as well as the associated regulatory elements that control gene expression. Nuclear eukaryotic genes also contain introns, non-coding regions of hitherto unknown function. Coding DNA represents only 3% of the genomic DNA, the bulk of the DNA is non-coding, much of which is made up of repetitive sequences. The somatic cells are usually diploid, having two sets of homologous chromosomes and hence two copies of each genetic locus, while the germ cells are haploid and have only one copy of each chromosome. Nature has developed efficient tools to control the correct flow of genetic information. Proof-reading and DNArepair systems can detect and correct genetic errors which may occur during replication or can be caused by DNA damage through environmental factors (chemical or physical influence). Despite these repair mechanisms, genetic variations still occur due to events escaping the control mechanisms. These variations in combination with the natural mechanisms of selection are one of the driving forces for evolution. Genetic differences between individuals within a species have most likely also arisen from errors that escaped from the control system. Although most of these variations are probably without any effect, some may predispose for diseases. In rare cases, mutations cause severe genetic diseases.

Genes and Diseases

Genetic diseases are caused by different types of DNA defects. One extreme is the complete (numeric aneuploidy) or partial (segmental aneuploidy) deletion or duplication of a chromosome. The other extreme is the exchange of a base in a single nucleotide (intragenetic point mutations). Chromosomal aberrations are usually detected by cytogenetic methods. The classical methods are complemented by modern hybridization techniques (FISH: fluo-

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rescence in situ hybridization) and also methods based on polymerase chain reaction (PCR). Most chromosomal aberrations occur as de novo mutations during meiosis of germ cells and are rarely inherited. Inherited genetic diseases are most commonly due to intragenetic mutations, which are detected by modern methods of molecular biology. Deletion and Insertions If small parts of DNA are deleted or inserted (e.g. duplicated parts), often a shift in the reading frame occurs, which disrupts the genetic three-letter code resulting in a truncated or otherwise severely damaged protein. Point Mutations Mutations of a single nucleotide base in the coding region of genes may be grouped into four categories due to their consequences on the genetic information: (1) Mutations which do not cause an amino acid exchange are called silent mutations. Due to the degenerated genetic code most amino acids can be encoded by more than one (three-letter) codon. These mutations usually represent polymorphisms without phenotypic consequences. (2) Mutations resulting in a codon for another amino acid are called missense mutation. Usually an altered activity of the corresponding protein and subsequently a phenotypic change results. (3) Mutations converting an amino acid codon into a stop codon (terminating protein synthesis) result in an truncated, usually inactive protein. (4) Mutations in splice donor or acceptor sites at exonintron junctions lead to aberrant splicing and result usually in inactive proteins. Instability of Repetitive Sequences Repetitive sequences are found in many parts of the genome, most often in regions where no genes are encountered. Some genes, however, contain trinucleotide repeats (e.g. polyglutamine stretch in the androgen receptor) within their coding or their regulatory regions. These repeats may expand during inheritance, as DNA polymerase may have problems to replicate these motifs correctly (slippage) and lead to pathological conditions. Mutations in the Mitochondrial Genome Mutations in the mitochondrial genome may be responsible or at least contribute to a number of chronic degenerative diseases, which affect e.g. brain, heart, muscle, liver, kidney and also endocrine glands. A eukaryotic cell may harbor a few up to several hundreds of mitochondria. Each mitochondrion may contain up to several

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thousands of copies of the circular mitochondrial genome. Mitochondria have a less efficient DNA-repair system. Therefore, mutations may accumulate in the mitochondrial genome during a lifetime and contribute to aging. Single Nucleotide Polymorphisms The comparison of sequencing data of genomic DNA from many probands revealed approximately 1 variant nucleotide base in 1,000 bases [1, 2]. As these polymorphisms usually represent single basepair exchanges they are designated single nucleotide polymorphisms (SNP or snip). These SNPs occur in extragenetic regions as well as also intragenetically and hence may modulate the activity of genes and gene products. Thus some SNPs may contribute to polygenetic diseases or may be associated with risks to develop pathological conditions (e.g. thromboembolism). Individual responses to toxic substances or to therapeutic medications may be determined by SNPs in genes that activate or metabolize these substances or are otherwise involved in their mechanism of action.

Molecular Diagnostic

Before the advent of molecular genetics, the diagnosis of genetic diseases was based on clinical facts and family anamnesis. In addition to clinical criteria, the definitive confirmation was usually based on biochemical parameters taking into account uncertainties due to large individual variations. Some genetic diseases can be reliably predicted on the basis of biochemical measurements, as it is general practice in neonatal screening for inborn errors of metabolism. Molecular diagnostic does not look for biochemical consequences of a genetic defect but rather tries to find directly the defective gene and to characterize the individual mutation of a patient or an affected family. Molecular diagnostic starts with genomic DNA prepared from blood or fetal cells (chorionic villi samples), followed by a PCR to amplify the specific parts of the gene suspected to be responsible. Several methods can be applied for post-PCR analysis to actually identify the mutation. As the identification of a pathological mutation definitely confirms a genetic disease, this result is not only relevant for the individual tested but also for the relatives who also may be carriers of the genetic defect. Genetic testing should therefore always be accompanied by genetic counseling.

Clinical Impact of Molecular Diagnostics in Endocrinology

Preparation of DNA The methods to prepare DNA from biological samples has become a well-standardized procedure due to commercially available systems like the Qiagen Blood Kit (Qiagen GmbH, Hilden, Germany). Cells are lysed with detergent and proteins are digested with proteinase K. DNA is then bound and washed on a silica matrix in microcolumns. By changing the salt concentration the nucleic acid is eluted from the matrix and can directly be used for amplification in the PCR. Polymerase Chain Reaction The first and still most often applied method to amplify the DNA region of interest is the PCR. With specific oligonucleotides flanking the gene region to be amplified, and a DNA polymerase from thermophilic bacteria, a temperature program is repeated up to 40 times, each time doubling the sequence of interest. Analysis of PCR Products – Post-PCR Methods After the part of a gene has been amplified by PCR, additional analytical steps are necessary to discover a mutation. In some cases only one or very few mutations are expected. Hereditary hemochromatosis is in almost 90% of the cases due to a mutation in codon 282. In this case, sequence-specific enzymes (restriction endonucleases) or mutation-specific amplification can be used as well as hybridization with allele-specific oligonucleotide probes (ASO – usually performed as reverse dot-blot hybridization). If mutations can occur anywhere in the candidate gene, DNA sequencing of the complete coding region including the exon-intron junctions as well as the regulatory region is necessary. This makes molecular diagnosis expensive and time-consuming. Efficient and reliable methods are needed. Biotechnology and diagnostic companies currently developing innovative technologies. The aims of these technologies are: (a) miniaturization – to reduce cost of reagents; (b) automation – to reduce hands-on time; (c) parallelization – to increase throughput, and (e) detection technologies – to assure sensitivity and specificity. A number of methods which fulfill some of these criteria have emerged (e.g. DNA chip technology, Affymetrix; mass spectrometry, Sequenom). However, an evolutionary process among the different technologies is still going on.


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Molecular Diagnostic in Endocrinology

Genetic defects in genes encoding hormones, hormone receptors or polypeptides of the signaling pathways usually cause complex disease manifestations characterized by the involvement of several tissues and variable expression. Genetic aberrations, like chromosome aneuploidy, gene translocations or mutations in key regulatory proteins (even if not directly affecting genes of the endocrine system) often lead to clinical symptoms, including central endocrine functions like sexual differentiation or metabolic disturbances, like diabetes mellitus. If the underlying molecular defects of endocrinopathies are known, direct molecular diagnosis can be performed. This is particularly useful if it helps to solve difficult differential diagnosis problems or if there exist effective preventive therapeutic options. The present paper presents examples for endocrine diseases in which molecular testing significantly increases the specificity and sensitivity of diagnostics and demonstrates the benefits for the patients and the healthcare system. In these cases, DNA testing has found its place in clinical routine. The analysis of SNPs in the context of polygenetic diseases is a novel field of application of molecular diagnostics. However, the proof of an association between polymorphisms and a disease is in many cases still lacking. Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency Congenital adrenal hyperplasia (CAH; also called adrenogenital syndrome: AGS) is due to inherited defects in one of the enzymes of cortisol synthesis. With the lack of cortisol the feedback regulation for CRH and ACTH at the hypothalamus and the pituitary is impaired. The result is a hyperplastic adrenal gland producing excess precursors of steroid hormones. The most common cause of CAH is 21-hydroxylase deficiency (90–95% [for a review, see 3]). Precursors that accumulate when 21hydroxylase activity is diminished or abolished are secreted and metabolized to active androgens. Depending on the severity of the genetic defect, a prenatal virilization of girls, a rapid somatic growth with early epiphyseal fusion in both sexes and in severe forms a lack of aldosterone a predisposition to episodically salt wasting in the first month of life can result. Mild mutations or possibly severe mutations in a heterozygous state can result in late-onset AGS with premature pubarche, hirsutism, oligomenorrhea, infertility and polycystic ovary.

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The inheritance of CAH follows an autosomal recessive genetic trait and occurs with a frequency of 1 in 10,000–12,500 life births. The gene encoding 21-hydroxylase (CYP21B) and a pseudogene (CYP21B) are located on the short arm of chromosome 6 in close vicinity to the HLA major histocompatibility complex (6p21.3) approximately 30 kb apart from each other. Adjacent the gene for the fourth component of serum complement (C4B) is found and alternating with a corresponding pseudogene (C4A) (fig. 1a). The high frequency (1:50 among Europeans) of mutations in the CYP21 gene is due the presence of the pseudogenes CYP21A and C4A. Due to the high sequence homology between the active genes and pseudogenes, pairing of sister chromosomes can lead to unequal crossing over during meiosis and parts of the pseudogenes can be converted to the active gene and vice versa. Also, partial or complete deletions or duplications can occur. In fact more than 99% of 21-hydroxylase deficiency can be explained by these gene conversion events. The 21-hydroxylase deficiency can be classified into salt wasting, simple virilizing and a non-classical phenotype. There is a rough genotype phenotype correlation (fig. 1b) for homozygous gene carriers. Most of the CAH patients are compound heterozygous making a prediction of the phenotype very difficult or even impossible. The molecular diagnostic of 21-hydroxylase deficiency is complicated due to the presence of the highly homologous pseudogene. Oligonucleotide primers for PCR have to be selective for the active gene (CYP21B). To reliably find all possible mutations including novel mutations not derived from the pseudogene, sequencing of the complete gene is the best method. In addition, complete or partial deletions of the active gene have to be detected (up to 40% of patients). This is achieved by a (semi)quantitative PCR using primers amplifying active gene and pseudogene in the same reaction. Molecular diagnostic is useful in the diagnosis of nonclassical 21-hydroxylase deficiency when clinical and biochemical data are ambiguous. When carriers of mutations in the 21-hydroxylase gene are identified, testing of partners as well as first- and second-degree relatives is strongly recommended. As the prenatal virilization can be prevented by treatment with dexamethasone, a prenatal diagnosis is strongly recommended for those cases where an affected child was born before or where both parents are carriers of mutations (fig. 2).

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Fig. 1. a Structure of the gene locus of the 21-hydroxylase gene (CYP21). b Mutation in the active 21-hydroxylase gene with the corresponding phenotype. SW = Salt wasting; SV = simply virilizing; NC = non-classical.



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Fig. 2. Diagnostic scheme for the prenatal therapy.

Multiple Endocrine Neoplasia Type 2 Twenty-five percent of medullary thyroid carcinomas (MTC, a neoplasm of parafollicular C-cell origin) occur on a hereditary basis. This autosomal dominant tumor syndrome may display medullary thyroid carcinoma only (FMTC: familial medullary thyroid carcinoma) or may be associated with pheochromocytoma (40–50%) or hyperparathyroidism (15–20%) designated multiple endocrine neoplasia (MEN) type 2A. In MEN2B, an extremely aggressive variant of MTC appears in conjunction with a marfanoid habitus, ganglioneuromatosis, and bumpy lips [4, 5]. In 1993, the genetic background was elucidated. Germline mutations in the RET proto-oncogene are found in almost all cases of MEN2 and form the basis for molecular diagnostic (fig. 3). In MEN2A patients activating mutations in the RET proto-oncogene are usually found in one of the cysteine codons 609, 611, 618, 620 in exon 10 or in 630 and 634 in exon 11 (mutations in 630 are rare). If the cysteine 634 (exon 11) is mutated into any other amino acid, most likely all organ manifestations of MEN2A occur. The penetrance of this mutation is 190%. The mutations in the cysteines in exon 10 more often lead to the FMTC phenotype (70%) but in a significant number of gene carriers also pheochromocytoma occur. The mutations in exon 10 show a tendency to cause a later onset and a milder course

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of the disease than the mutation in codon 634. This correlates also with in vitro results, in which mutations in codon 634 display the highest transforming activity. It has been shown in vitro that the classical cysteine mutations lead to a ligand-independent dimerization of receptor molecules, autophosphorylation and activation of intracellular signaling pathways. A similar activation mechanism exists for an unusual mutation in exon 11 which was found in a patient with a complete MEN2A phenotype. An in-frame duplication creates an additional cysteine residue in the cysteine-rich domain. This event also results in a ligand-independent receptor dimerization. Another rare mutation is a 12-bp out-of-frame duplication in exon 11 also associated with an additional cysteine residue. For this mutation a phenotype distinct from the classical MEN2A has been described. Among gene carriers in this family a high incidence of medullary thyroid carcinoma and parathyroid hyperplasia but no evidence for pheochromocytoma has been found. Although the mechanism of activation through a ligand-independent receptor dimerization has been demonstrated in vitro, the phenotype associated with this mutation is distinct from other mutations in the cysteine-rich domain. Screening for RET mutations in apparently sporadic medullary thyroid carcinoma patients revealed 11.7% of the cases as hereditary. 3.7% had one of the classical extracellular cysteine mutations, whereas in 8% an intracellular

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mutation was found in exon 13 or 14. An FMTC phenotype with a mild course of the disease and low penetrance can be postulated for the mutations in codons 768 and 804. In one family a double mutation in codons 804 and 844 was found. However, the phenotype is not significantly different from families with the 804 mutation. A new hot spot for mutations has been described for the amino acids 790 and 791 (Leu790Phe, Tyr791Phe) encoded in exon 13 of the RET proto-oncogene [6]. The mutations in codons 790 and 791 lead to medullary thyroid carcinoma in most of the cases, but pheochromocytomas have also been found and were at least in two gene carriers diagnosed as the first manifestation. Parathyroid hyperplasia does not seem to be associated. The most aggressive form of multiple endocrine neoplasia is the subtype MEN2B, in which addition to the medullary thyroid carcinoma (100%) and pheochromocytoma (50%) mucutaneous neuromas (100%) and intestinal ganglioneuromatosis (40%) develop. The onset of the disease in this subtype is usually in the first or second decade of life (approximately 20 years earlier than in MEN2A). The tumors have a high tendency to form metastasis. However, if the diagnosis is made early enough and the tumor is resected before the formation of metastasis, the prognosis is not different from MEN2A associated or sporadic medullary thyroid carcinoma. The MEN2B-associated tumors are caused by mutations in the activation loop of the TK2 domain of the RET protooncogene. In 95% of the cases a mutation in codon 918 (Thr918Met) and in 5% a mutation in codon 883 or 922 occurs. The mutation in codon 918 occurs in more than 50% of the cases of MEN2B as de novo mutation. It has been demonstrated in vitro that the transforming activity of MEN2B mutations is not due to a constitutive receptor dimerization but rather to the phosphorylation of different tyrosine residues resulting in the transduction of signals to other intracellular substrates. Mutations in the RET proto-oncogene are also responsible for 50% of the cases of Hirschsprung disease. In contrast to the activating mutations in MEN2, Hirschsprung is due to inactivating mutations in the intra- or extracellular part of the RET receptor. Molecular biology now enables us to easily distinguish between sporadic and hereditary MTC and affords an early identification of gene carriers of RET mutations who are bound to develop MTC later in life. For these patients early prophylactic thyroidectomy is recommended to ensure definitive cure. At the International MEN Workshop (1999) it was agreed that prophylactic surgery, with the objective of removing the thyroid gland before malignant


Fig. 3. Molecular diagnosis in the management of MTC patients (a) and MEN2 families (b).

progression from C-cell hyperplasia to medullary carcinoma has occurred, should be performed at an age of 5–6 years. This strategy would also eliminate the need for additional lymph node dissection, which results in increased morbidity [7]. In all cases where prophylactic thyroidectomy was performed, C-cell hyperplasia or microcarcinoma have been detected in the resected gland. Molecular Diagnostics of SNPs SNPs are single nucleotide polymorphisms with a frequency higher than 1% in the population. Approximately


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Table 1. Genes with clinical relevant SNPs (selection)

Thromboembolism Factor V Leiden Prothrombin (factor II) Methylenetetrahydrofolate reductase Glycoprotein IIIa Glycoprotein IIb Osteoporosis Vitamin D receptor Collagen type I·1 Interleukin-6 Cancer Breast Cytochrome P450 aromatase (CYP19) Glutathione-S-transferase P1 Tumor suppressor gene p53 Glutathione-S-transferase M1 (gene deletion) Progesterone receptor (PROGINS allele, protective) Cervix Interleukin-10 Habitual abortion, subfertility Factor V Leiden Prothrombin (factor II) MTHFR (C677T) Glycoprotein IIIa Plasminogen activator inhibitor-1 Endothelial NO synthase-3 Glutathione-S-transferase P1 Interleukin-l receptor antagonist Metabolism and action of steroid hormones Catechol-O-methyltransferase Cytochrome P450 aromatase (CYP19) Estrogen receptor Androgen receptor 17ß-Steroid dehydrogenase (EDH 17B2) Cytochrome P450 aryl hydrocarbon Hydroxylase (CYP1A1) Steroid 5·-reductase type II (SRD6A2)

3 billion variable positions are present in the human genome and determine the phenotypic multiplicity of the human race as well as the individual predisposition to certain diseases. A number of candidate genes for clinical relevant polymorphisms have been published in the recent years (table 1). Some SNPs have a high phenotypic penetrance (e.g. HFE gene variant C282Y predisposing for hereditary hemochromatosis) whereas in many cases a statistically significant but weak association, or even contradictory results have been published (e.g. BsmI polymorphism in the vitamin-D-receptor gene). Many common diseases have a polygenetic background, as can be recognized from careful analysis of large

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families or from studies with monozygotic twins or siblings. However, a mendelian inheritance with a dominant or recessive genetic trait cannot be demonstrated. In the classical monogenetic diseases the genetic defect usually has a high penetrance, which means that gene carriers have a high statistical probability to develop the corresponding disease. Genetic variants contributing to polygenetic disease usually have a low penetrance. Several predisposing conditions as well as acquired factors have to come together to cause a disease, and that gives rise to the hope that the knowledge of genetic, predisposing risk factors and the avoidance of acquired risks can prevent or delay the disease. Endocrinology and SNPs Endocrine systems consist of many components. The likelihood that relevant SNPs exist in more than one component of an endocrine system is considerably high. Several pathological conditions related to corresponding SNPs are described in the literature. Osteoporosis Approximately 70% of peak bone mass is determined genetically. However, it is obvious that several genetic factors may contribute. The first genetic factor described in the literature was a polymorphism in the vitamin-Dreceptor gene (BsmI). Many publications followed and reported data which were in part opposite to the initial findings. However, it turned out that the regulation of bone metabolism and bone mineral density differs from bone to bone, differs with age and furthermore the results depend very much on the method used to determine bone density. The collagen I-·1 gene contains a SNP also contributing to bone density and to fracture risk as well as the estrogen-receptor gene and the interleukin-6 gene (IL-6). PCO Syndrome and Hyperandrogenemia The polycystic ovary syndrome can occur on the background of genetic variations in several regulatory pathways. It is often associated with hyperandrogenemia, hyperinsulinemia, insulin resistance. Symptoms like androgenic alopecia, hirsutism and visceral adipositas are common in women with PCO. Obviously, SNPs in the enzymes of biosynthesis of androgenic steroid hormones or precursors are contributing to the disposition for PCO, e.g. cholesterol side chain cleavage enzyme, 17·-hydroxylase, and the androgen receptor (CAG repeat). But also variants of the insulin and insulin receptor gene, tumor necrosis factor ·, and the follicle-stimulating hormone ßsubunit may predispose to PCO syndrome.

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Habitual Abortion and Subfertility Loss of an embryo early in pregnancy affects up to 5% of couples. A predisposition can be related to genes encoding components of the clotting system. Common polymorphisms exist in the factor V gene, the gene for pro-

thrombin (factor 2) and the gene for the plasminogen activator inhibitor-1 (PAI-1). These polymorphisms are also associated with the risk for venous thromboembolism. A variant of the glutathione-S-transferase P1 gene is more frequent in women who have had abortions.

References 1 Buetow KH, Edmonson M, MacDonald R, et al: High-throughput development and characterization of a genomewide collection of genebased single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ ionization time of flight mass spectrometry. Proc Natl Acad Sci USA 2001;98:581–584. 2 Cargill M, Altshuler D, Ireland J, et al: Characterization of single nucleotide polymorphisms in coding regions of human genes. Nat Genet 1999;22:231–238.


3 White PC, Speiser PW: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245–291. 4 Ponder BAJ: Multiple endocrine neoplasia type 2; in Scriver CR, Beaudet AL, Sly WS, Valle D (eds): The Metabolic and Molecular Bases of Inherited Disease, ed 8. New York, McGraw-Hill, 2001, pp 931–942. 5 Ritter MM, Hoeppner W: Multiple endocrine Neoplasie Typ 2; in Ganten D, Ruckpaul K (eds): Molekularmedizinische Grundlagen von hereditären Tumorerkrankungen. Berlin, Springer, 2001.

6 Berndt I, Reuter M, Saller B, Frank-Raue K, Groth P, Grussendorf M, Raue F, Ritter MM, Höppner W: A new hot spot for mutations in the RET protooncogene causing familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 1998;83:770–774. 7 Brandi ML, Gagel FR, Angeli A, et al: Consensus: Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab 2001;86:5658–5671.


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GH in Children Horm Res 2002;58(suppl 3):24–26 DOI: 10.1159/000066478

Fatal Outcome of Sleep Apnoea in PWS during the Initial Phase of Growth Hormone Treatment A Case Report

Urs Eiholzer Yves Nordmann Dagmar l’Allemand Foundation Growth Puberty Adolescence, Zürich, Switzerland

Key Words Prader-Willi syndrome W Respirational abnormalities W Hypoventilation W Sudden death

Abstract The case of a boy with Prader-Willi syndrome (PWS) who suffered from respiratory problems since birth and suddenly died at the age of 6.5 years, 4 months after initiation of GH therapy, is presented. This case indicates the possibility of fatal courses in infants and children with PWS as a consequence of respiratory problems and raises the question as to a causal connection between the initiation of GH therapy and the sudden death of this child. Copyright © 2002 S. Karger AG, Basel

Introduction

Prader-Willi syndrome (PWS), first described in 1956 [1], has an estimated prevalence of 1:5,000 to 1:16,000 [2]. The syndrome is characterized by marked muscular hypotonia and feeding difficulties in infancy. Obesity, short stature, hypogonadism, mental retardation and behavioural difficulties become apparent in childhood. Respiratory abnormalities are well known. An increased

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© 2002 S. Karger AG, Basel 0301–0163/02/0589–0024$18.50/0



incidence of sleep-related breathing disorders has been reported in obese adults with PWS [3–8] and only recently, a primary disturbance of central respiratory control was demonstrated in young, not yet obese children with PWS [9]. The actual link between the chromosomal disorder – mostly deletion or maternal disomy of chromosome 15 – and the clinical symptoms is not yet fully understood. Hypothalamic dysfunction, as already originally presumed by Prader et al., appears to underlie many of the features of PWS, including hypogonadism [10, 11], abnormal appetite control, high pain threshold and sleep disorders, but no overt structural abnormalities of the hypothalamus have been found yet. In recent years, it was shown that growth hormone (GH) deficiency due to hypothalamic dysregulation contributes not only to the abnormal growth pattern and osteopenia, but also to the excess of body fat and to the deficit of lean body mass, with consequently reduced energy expenditure. GH therapy was evaluated in several studies and administration of GH, in combination with restriction of food intake, was shown to have a remarkable impact on growth and body composition, resulting in a dramatic change of the phenotype of affected individuals. Therefore a growing number of children with PWS are treated with exogenous GH. To date, only few potential side effects of GH therapy are known: aggravation of scoliosis or kyphosis [12] under GH-

Urs Eiholzer, MD Foundation Growth Puberty Adolescence Möhrlistrasse 69, CH–8006 Zürich (Switzerland) Tel. +41 1 3643700, Fax +41 1 3643701 E-Mail [email protected]

induced catch-up growth, accelerated manifestation of type 2 diabetes mellitus in predisposed individuals [13], manifestation of hypothyroidism due to the hypothalamic dysfunction and, most importantly in this particular case, fluid retention in the initial phase of GH treatment [14, 15]. In this paper, we present the case of a prematurely born boy with PWS who suffered from respiratory problems since birth and died suddenly at the age of 6.5 years 4 months after initiation of GH therapy. The aim of this case report is to sensitize paediatricians and endocrinologists to the fact that a subgroup of children with PWS suffering from hypoventilation and other respiratory problems may be at risk for a sudden deterioration of respiratory function at the beginning of GH therapy.

as seen during the last hospitalization. Following these reports, the physicians in charge were immediately asked to monitor the boy’s respiratory function, to perform echocardiography and to initiate CPAP treatment during night. With the parents’ consent, GH therapy was continued, because not only beneficial effects of GH on respiratory function described before [16] were seen, but also growth and body composition had already improved. At the age of 6.5 years, an echocardiography showed slightly elevated pulmonary pressure and hypertrophy of the right ventricle. The cardiologist also suggested a nocturnal CPAP or oxygen therapy, provided, however, that other possibilities for an improvement of the respiratory function achieved through interventions conducted by an ENT specialist (e.g. tonsillectomy) had been taken into consideration beforehand. Ten days later, the ENT examination revealed in fact enlarged amygdala and tonsillectomy was proposed as the next step. CPAP therapy seemed indicated to the ENT specialist only after failure of tonsillectomy. Fourteen days later, 3 weeks before the day of the planned tonsillectomy, the boy was unexpectedly found dead in his bed by his parents at 6 o’clock in the morning. No post-mortem examination was conducted.

Case Report The boy was born prematurely at 326/7 weeks of pregnancy by caesarean section because of a pathologic CTG and intrauterine growth retardation. Birth weight was 940 g (p ! 3), length 38 cm (p ! 3) and the Apgar score 5/7/7. Already shortly after birth, the infant presented with hypoventilation, which was attributed by the physicians in charge to severe hypotonia and muscular hypotrophy of the child. This condition persisted and CPAP treatment and additional oxygen administration proved necessary for the following 6 months. During this time, repeated atelectases of the left lung were striking. Because of the severe hypotonia, PWS was suspected already in the first days and diagnosed genetically (deletion of chromosome 15). During the following 5 months, due to feeding difficulties, the infant had to be fed via a nasogastric tube. A persistent ductus arteriosus had to be operated because of an acute cardiac decompensation at the age of 23 days. At the age of 6 months the boy was discharged from hospital without CPAP, but ongoing oxygen therapy. His weight was 6,050 g (P 3–10) and his length 64 cm (P 3–10). A cardiologic follow-up at the age of 11 months revealed normal function of the left ventricle and no signs of pulmonary hypertension. Treatment with digoxin and diuretics were stopped. The next 4 years were characterized by rapid weight gain. At the age of 5.5 years, weight had already increased to 24.8 kg (P 90), and the boy had to be hospitalized because of a pneumonia with respiratory insufficiency and subsequent antibiotic treatment. At the age of 5.7 years, the boy was seen for the first time in our institute. Weight had further increased to 27 kg (P 1 97) and height was 109.5 cm (P 15). He showed typical signs of PWS in a distinct manner. Severe obesity and hypoactivity were present. His developmental delay amounted to about 1 year. In accordance with our PWS study design, GH therapy was started at the age of 6.0 years. Only a short time later, at the age of 6.3 years, the boy suffered a fracture of his left tibia and was hospitalized. At the mother’s request, who had noticed episodes of nocturnal apnoeas, transcutaneous O2 saturation was monitored and significant drops to below 87% were seen, but no further action was taken. A few days later, at the age of 6.33 years, the boy was seen again in our institute. The mother reported that her son snored heavily at night and that, also at home, he suffered from apnoeas during sleep,

Growth Hormone Treatment and Apnoea in PWS

Discussion

We present the case of a boy with PWS who suffered from respiratory problems since his premature birth and suddenly died at the age of 6.5 years. At the age of 6.0 years, GH therapy was initiated. At the age of 6.3 years, sleeping apnoea with significant drops in O2 saturation were documented. Before appropriate action was taken, the boy died suddenly in his sleep. The presented case indicates the possibility of fatal courses in infants and children with PWS as a consequence of respiratory problems and raises the question as to a causal connection between the initiation of GH therapy and the sudden death of this child. The pathogenesis of the well-known respiratory problems in PWS seems to be multifactorial, including peripheral and central mechanisms, like muscular hypotonia, tonsillar hyperplasia and hypothalamic and chemoreceptor dysfunction [17]. The decreased lean body mass, already present in infants with PWS [18], might be another important factor involved. The resulting decrease in respiratory muscle mass [19], together with a defect in the function and architecture of the throat due to hypotonia, may be the main reason for disturbed respiration leading to an increase of the respiratory drive set-point in the brain stem and to central hypoventilation. Indeed, as shown in two recent studies, GH therapy in children with PWS improves not only lean body mass [20], but also respiratory function [21], leading to an increase in CO2 sensitivity [16, 22]. In that respect, a causal connection between initiation of GH therapy and deterioration of

Horm Res 2002;58(suppl 3):24–26

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respiratory function seems unlikely. However, it is possible that the known GH side effect of fluid retention [15] could outweigh the positive effects during the first months, leading to a fatal course in some children with PWS, who are already suffering from chronic hypoventilation, as in the present case of this prematurely born infant with primary respiratory insufficiency. We recently published another case report of an infant with PWS who died during the first months of GH therapy [23] and we know of a similar case of a boy in Spain (personal communication of the Spanish PWS Association). It therefore cannot be excluded that some children with PWS are at risk for a sudden death in the first months after initiation of GH therapy, most probably because of a right heart failure.

turity (this case) or obesity [14, 23]. They are at risk for the development of pulmonary hypertension. In the case of respiratory infections in these children, monitoring respiratory function and rigorous treatment of the infection is mandatory. If adenoid hyperplasia is present, tonsillectomy can improve respiratory function. In severe cases, CPAP therapy is needed. In our opinion, examination by polysomnography and echocardiography is mandatory in those children, before GH therapy is initiated. In addition, during the first months after initiation of GH therapy, increased awareness for its possible side effects is necessary in order to avoid fatalities as described.

Acknowledgements Conclusion

A subgroup of children with PWS suffer from respiratory disturbances with chronic hypoventilation. This subgroup may be defined by additional risk factors, as prema-

We gratefully thank Mrs. Karin Stutz for her help in translating. Our special thanks go to the parents of the deceased child who helped us to reconstruct the case by answering our questions despite being deeply in mourning.

References 1 Prader A, Labhart A, Willi H: Ein Syndrom von Adipositas, Kleinwuchs, Kryptorchismus und Oligophrenie nach myotonieartigem Zustand im Neugeborenenalter. Schweiz Med Wochenschr 1956;86:1260–1261. 2 Burd L, Vesely B, Martsolf J, Korbeshian J: Prevalence study of Prader-Willi syndrome in North Dakota. Am J Med Genet 1990;37:97– 99. 3 Cassidy SB, McKillop JA, Morgan WJ: Sleep disorders in Prader-Willi syndrome. Dysmorphol Clin Genet 1990;4:13–17. 4 Hertz G, Cataletto M, Feinsilver SH, Angulo M: Sleep and breathing patterns in patients with Prader-Willi syndrome: Effects of age and gender. Sleep 1993;16:366–371. 5 Kaplan J, Fredrickson PA, Richardson JW: Sleep and breathing in patients with the Prader-Willi syndrome. Mayo Clin Proc 1991;66: 1124–1126. 6 Richards A, Quaghebeur G, Clift S, Holland A, Dahlitz M, Parkes D: The upper airway and sleep apnoea in the Prader-Willi syndrome. Clin Otolaryngol 1994;19:193–197. 7 Sforza E, Krieger J, Geisert J, Kurtz D: Sleep and breathing abnormalities in a case of Prader-Willi syndrome. The effects of acute continuous positive airway pressure treatment. Acta Paediatr Scand 1991;80:80–85. 8 Hall BD, Smith D: Prader-Willi syndrome. J Pediatr 1972;81:286–293.

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9 Schlüter B, Buschatz D, Trowitsch E, Aksu F, Andler W: Respiratory control in children with Prader-Willi syndrome. Eur J Pediatr 1997; 156:65–68. 10 Jeffcoate WJ, Laurance BM, Edwards CRW, Besser GM: Endocrine function in the PraderWilli syndrome. Clin Endocrinol (Oxf) 1980; 12:81–89. 11 Cassidy S, Rubin K, Mukaida C: Genital abnormalities and hypogonadism in 105 patients with Prader-Willi syndrome. Am J Med Genet 1987;28:922–923. 12 Cassidy SB: Prader-Willi syndrome. J Med Genet 1997;34:917–923. 13 Cutfield WS, Wilton P, Bennmarker H, Albertsson-Wikland K, Chatelain P, Ranke MB, Price DA: Incidence of diabetes mellitus and impaired glucose tolerance in children and adolescents receiving growth-hormone treatment. Lancet 2000;355:610–613. 14 Holmes SJ, Shalet SM: Which adults develop side effects of growth hormone replacement? Clin Endocrinol (Oxf) 1995;43:143–149. 15 Wilton P: Safety in growth hormone replacement therapy: A matter of varied responsiveness? Horm Res 2001;55(suppl 2):61–64. 16 Lindgren AC, Hellstrom LG, Ritzen EM, Milerad J: Growth hormone treatment increases CO2 response, ventilation and central inspiratory drive in children with Prader-Willi syndrome. Eur J Pediatr 1999;158:936–940.

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17 Livingston FR, Arens R, Bailey SL, Keens TG, Ward SL: Hypercapnic arousal responses in Prader-Willi syndrome. Chest 1995;108:1627– 1631. 18 Eiholzer U, Blum WF, Molinari L: Body fat determined by skinfold measurements is elevated despite underweight in infants with Prader-Labhart-Willi syndrome. J Pediatr 1999;134:222–225. 19 Hakonarson H, Moskovitz J, Daigle KL, Cassidy SB, Cloutier MM: Pulmonary function abnormalities in Prader-Willi syndrome. J Pediatr 1995;126:565–570. 20 Eiholzer U, l’Allemand D, van der Sluis I, Steinert H, Ellis K: Body composition abnormalities in children with Prader-Willi syndrome and long-term effects of growth hormone therapy. Horm Res 2000;53:200–206. 21 Carrel A, Myers S, Whitman B, Allen D: Growth hormone improves body composition, fat utilization, physical strength and agility in Prader-Willi syndrome: A controlled study. J Pediatr 1999;134:215–221. 22 Lindgren AC, Ritzen EM: Five years of growth hormone treatment in children with PraderWilli syndrome. Swedish National Growth Hormone Advisory Group. Acta Paediatr Suppl 1999;88:109–111. 23 Nordmann Y, Eiholzer U, l’Allemand D, Mirjanic S, Markwalder C: Sudden death of an infant with PWS – Not a unique case? Biol Neonate 2002;82:139–141.

Eiholzer/Nordmann/l’Allemand


US Experience in Evaluation and Diagnosis of GH Therapy of Intrauterine Growth Retardation/ Small-for-Gestational-Age Children Paul Saenger Department of Pediatrics, Division of Pediatric Endocrinology, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, N.Y., USA

Key Words Intrauterine growth retardation W Small for gestational age W Growth hormone W Insulin resistance

Abstract The potential role of exogenous GH in treating short children born small for gestational age (SGA) has been discussed since the early 1960s. Pivotal studies in Europe during the last 10 years have shown that GH treatment of short children born SGA during childhood and early puberty (1) normalizes stature, (2) increases final height above predicted height and (3) allows children to reach their target height. A study now under way in the USA will provide additional much needed data about efficacy and safety of GH treatment in intrauterine growth retardation/SGA. Copyright © 2002 S. Karger AG, Basel

In 2001, continuous growth hormone (GH) treatment of small-for-gestational-age infants (SGA) was approved in the USA. This move by regulatory agencies has focused the attention in the USA on evaluation, diagnosis and treatment of SGA infants with GH.

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Accessible online at: www.karger.com/hre

Definition

SGA represents a statistical grouping of infants. It has been defined as birth weight and/or length of at least 2 SDs below the mean (^2 SD) for gestational age. For the USA, SGA has thus been defined as: (1) birth weight !2,500 g at a gestational age of 137 weeks; (2) birth weight or length less than the 3rd percentile for gestational age, and (3) ponderal weight index (weight in grams ! 100 divided by length in cm) !2 SD. In essence, SGA is defined as birth weight and/or length of at least !2 SD [1, 2]. In the USA, the most accepted curve auxologic reference standard to evaluate SGA is the Usher/McLean growth curve providing data for birth weight and length [3]. These standards were obtained in 1969 in Montreal from measurements in seven dimensions – heel, length, head, chest, abdominal and head circumferences, foot length and double skin thickness. Normal smooth curves were drawn of the mean B 2 SD. Gestational age was calculated to the nearest week for the last normal menstrual period. The prevalence of SGA in the USA is considerable. For 1999, the most recent year for which vital statistics data are now available, there were 3,959,470 live births. By definition, then, 3% of these newborns were SGA, weighing !2,500 g [4]. This would constitute a total number of 118,784 children born SGA per year. Up to 90% of children born SGA experience spontaneous catch-up growth [1]. Indeed, 83% show catch-up by 2 years. Of the remain-

Paul Saenger, MD Division of Pediatric Endocrinology The Children’s Hospital at Montefiore 111 East 210th Street, Bronx, NY 10467 (USA) Tel. +1 718 920 4664, Fax +1 718 405 5609, E-Mail [email protected]

ing SGA infants, about half remain short even in adulthood. If not present by age 2 or 3 years of age, catch-up growth is unlikely, the exception being the very premature infant. Therefore, there will be 12,000–15,000 new patients with SGA per year who will not show catch-up by age 3. Since there are approximately 150,000 children with SGA between 3 years and puberty, the total collective of short SGA children is approximately 160,000– 170,000 children in the USA.

Etiology

Factors influencing intrauterine growth retardation (IUGR) are hormones, growth factors and intrauterine factors provided by the placenta. Among hormonal factors, insulin plays clearly a significant role in the regulation of fetal growth, while GH and thyroid hormone are less unimportant. Leptin has also been added to the list of hormones that influence birth weight. The list of growth factors that play a role in fetal growth include: IGF-1, IGF-2, IGFBP-3, epidermal growth factor, transforming growth factor, platelet-derived growth factor, fibroblast growth factor and nerve growth factor. Placental function and normal production of placental GH are additional other important factors in providing an adequate supply of intrauterine nutrients to the fetus [5]. Considerable evidence has accrued that children born with low birth weight have adverse non-growth correlates in later life. For example, lower cognitive scores when assessed at age 5–6 years of age, school difficulties were uncovered in 20-year-olds born SGA and generally lower school performance was seen when SGA children were evaluated at 12 and 18 years of age. Thus, being born SGA is associated with an increased risk of subnormal intellectual and psychological performance. This was particularly apparent in those who did not experience catch-up growth [6]. In a recent study by Strauss [7], the author arrived at the conclusion that adults who were born SGA had significant differences in academic achievement and professional attainment compared with adults who were of normal birth weight.

Syndrome X

SGA can also lead to significant morbidities in adult life which could have their origin in faulty intrauterine programming secondary to fetal undernutrition. These

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conditions are best characterized as syndrome X – hypertension, hyperlipidemia and type 2 diabetes mellitus. There is also a high prevalence of coronary heart disease. Deprivation of nutrients and/or oxygen during brief, but critical periods of in utero development may permanently change or program the fetus with untoward consequences in adult life [8, 9]. Historical data of studies conducted in SGA infants with GH therapy from the 1960s and 1970s show limited improvement due to short periods of intervention with GH and relatively low doses of GH. There were serious restrictions in the supply of pituitary-derived GH available then [10].

Treatment

Recent studies using higher doses of rhGH have shown that the growth response is better than with conventional doses and that most of the unresponsiveness to GH can be overcome with increased dosing. These studies include data of de Zegher et al. [11, 12], Sas et al. [13, 14] and several other groups. The de Zegher and Sas studies essentially evaluated the effects of two doses of continuous GH treatment over 5–6 years. While responsiveness was documented in both groups, the observed height increments were greater in the group receiving 67 Ìg/kg/day (or approximately 0.45 mg/kg/week) or 6 IU/m2/day and represented near normalization of target midparental height centiles. Notably the auxologic response after 5 years in the Sas study was not related to spontaneous or provoked GH levels or baseline IGF-1 levels. Similarly, the group of Azcona et al. [15] using a lower dose of GH (0.3 mg/kg/week) failed to show a difference between the GH-sufficient and -insufficient group, although each had a response less than those in the studies employing higher doses. Thus, the degree of GH sufficiency did not appear to predict the response either to traditional or higher doses of GH [11–16]. Assessment of treatment data suggests that children with both SGA/IUGR and Russell-Silver syndrome (RSS) are less responsive to GH/IGF-1 than are GHD children. Since IGF-1 unresponsiveness is a function of both IGF-1 production and availability as well as IGF-1 action, the role of IGF-1 and IGFBP-3 in the growth-promoting action of GH in these children merits further investigation. Since nutrition is a key determinant of IGF-1 production, and children with SGA/IUGR who have not experienced catch-up growth tend to have reduced muscle and

Saenger

fat mass and are described as having poor appetites, the role of nutritional status on treatment responses need to be assessed. Similarly, factors potentially determining IGF-1 production in response to GH, such as birth weight SDS, birth length SDS, sex and age may play a role in determining responsiveness. Finally, SGA/IUGR is a consequence of heterogeneous etiologies. These range from genetic disorders including uniparental disomy of chromosome 7 in some patients with RSS and defects in the IGF receptor to exogenous factors like maternal smoking or ethanol ingestion. Thus, attempts to determine the causes of the SGA need to be made and assessed [17, 18].

Outlook

Long-term treatment with GH at a dose of 0.48 mg/ kg/week was approved by regulatory agencies in the USA in the summer of 2001. This therapeutic indication is not

related to spontaneous secretion or dependent on low stimulated GH or IGF-1 levels. To this end, a clinical protocol to study the effect of GH in infants born SGA at two different doses has now been begun in the USA. Prepubertal children with a height SDS ^ 2.0 SD for chronological age will be treated. They have to be at least 3 years old and birth weight and/or length has to be ^2 SD for gestational age. The purpose of treatment intervention in those infants who have not shown spontaneous catch-up growth is to induce catch-up growth, normalize growth during childhood, and hopefully normalize adult height. It is well known that GH therapy may cause insulin resistance during treatments. Since insulin resistance is already a characteristic of treatment-naive SGA patients [20], this is of great concern. Safety data so far show no aggravation of insulin resistance and normalization of insulin levels to pretreatment levels within the 6 months after discontinuation of GH therapy [21].

References 1 Karlberg J, Albertsson-Wikland K: Growth in full-term small-for-gestational infants: From birth to final height. Pediatr Res 1995;38:733– 739. 2 Bakketeig LS: Current growth standards, definitions, diagnosis and classification of foetal growth retardation. Eur J Clin Nutr 1998;52: S1–S4. 3 Usher R, McLean F: Intrauterine growth of live-born Caucasian infants at sea level: Standards obtained from measurements in seven dimensions of infants born between 25 and 44 weeks of gestation. J Pediatr 1969;74:901– 910. 4 Ventura SJ, Martin JA, Curtin SC, et al: Births: Final data for 1999. Natl Vital Stat Rep 2002; 49:1–22. 5 Gluckman PD: The endocrine regulation of fetal growth in late gestation. The role of insulin-like growth factors. J Clin Endocrinol Metab 1995;80:1047–1050. 6 Larroque B, Bertrais S, Czernichow P, Leger J: School difficulties in 20-year-olds who were born small for gestational age in a regional cohort study. Pediatrics 2001;108:111–115. 7 Strauss RS: Adult functional outcome of those born small for gestational age: Twenty-six year follow-up of the 1970 British Birth Cohort. JAMA 2000;283:625–632. 8 Barker DJ, Hales CN, Fall CH, et al: Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidemia (syndrome X): Relation to reduced fetal growth. Diabetologia 1993;36:62–67.

GH in IUGR

9 Barker DJP: The fetal origins of coronary heart disease. Acta Paediatr Suppl 1997;422:78–82. 10 Lanes R, Plotnick LP, Lee PA: Sustained effect of human growth hormone therapy on children with intrauterine growth retardation. Pediatr 1979;63:731–735. 11 De Zegher F, Maes M, Gargosky SE, Heinrichs C, et al: High-dose growth hormone treatment of short children born small for gestational age. J Clin Endocrinol Metab 1996;81:1887–1892. 12 De Zegher F, Albertsson-Wikland K, Wollman HA, Chatelain P, et al: Growth hormone treatment of short children born small for gestational age: Growth responses with continuous and discontinuous regimens over 6 years. J Clin Endocrinol Metab 2000;85:2816–2821. 13 Sas T, de Waal W, Mulder P, Houdijk M, et al: Growth hormone treatment in children with short stature born small for gestational age: 5year results of a randomized, double-blind, dose-response trial. J Clin Endocrinol Metab 1999;84:3064–3070. 14 Sas T, Mulder P, Hokken-Koelega A: Body composition, blood pressure and lipid metabolism before and during long-term growth hormone (GH) treatment in children with short stature born small for gestational age either with or without GH deficiency. J Clin Endocrinol Metab 2000;85:3786–3792. 15 Azcona C, Albanese A, Bareille P, Stanhope R: Growth hormone treatment in growth hormone-sufficient and -insufficient children with intrauterine growth retardation/Russell-Silver syndrome. Horm Res 1998;50:22–27.

16 Fjellestad-Paulsen A, Czernichow P, Brauner R, Bost M, et al: Three-year data from a comparative study with recombinant human growth hormone in the treatment of short stature in young children with intrauterine growth retardation. Acta Paediatr 1998;87:511–517. 17 Price SM, Stanhope R, Garrett C, Preece MA, et al: The spectrum of Silver-Russell syndrome: A clinical and molecular genetic study and new diagnostic criteria. J Med Genet 1999;36:837– 842. 18 Abuzzahab MJ, Goddard A, Grigorescu F, Lautier C, et al: Human IGF-1 receptor modulations associated with intrauterine and postnatal growth retardation. Proc Endocrine Annual Meeting, Toronto 2000, abstr 1947. 19 Saenger P, Attie KM, DiMartino-Nardi J, Hintz R, et al: Metabolic consequences of 5year growth hormone (GH) therapy in children treated with GH for idiopathic short stature. J Clin Endocrinol Metab 1998;83:3115–120. 20 Leger J, Levy-Marchal C, Bloch J, Pinet A, et al: Reduced final height and indications for insulin resistance in 20-year-olds born small for gestational age: Regional cohort study. Br Med J 1997;315:341–347. 21 Van Pareren Y, Sas T, Hokken-Koelega A: Carbohydrate metabolism after long-term growth hormone treatment in short children born small for gestational age. Pediatr Res 2001; 49(suppl 6):74A.

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GH and Kidney Horm Res 2002;58(suppl 3):35–38 DOI: 10.1159/000066480

Effects of Growth Hormone in Patients with Chronic Renal Failure: Experience in Children and Adults Elke Wühl Franz Schaefer Division of Paediatric Nephrology, University Children’s Hospital, Heidelberg, Germany

Key Words Growth hormone treatment W Chronic renal failure W Children W Adults

Abstract Recombinant human growth hormone (GH) has proven effective in promoting growth in short children with chronic renal failure before and after renal transplantation. The action of GH and its mediator insulin-like growth factor 1 on body composition, protein, glucose and bone metabolism offers additional therapeutic options. One might be the improvement of the catabolic state in adults with end-stage renal failure. In few pilot studies and two placebo-controlled studies of 6 months duration, GH treatment in adults on dialysis showed clear anabolic effects resulting in a significant increase in lean body mass.

tion. The drug has been approved in the USA and in Europe for treatment of short stature in prepubertal and pubertal children with chronic renal failure. However, GH and its mediator insulin-like growth factor 1 (IGF-1) not only promote growth, but also have key metabolic actions such as stimulation of protein synthesis, bone and glucose metabolism, and affect body composition by reducing body fat and increasing lean body mass. In 1996, GH was approved by the FDA for treatment of cachexia and muscle wasting in patients with the acquired immune deficiency syndrome (AIDS). Treatment with GH may also be of clinical importance in other patients with chronic degenerative diseases, such as adult patients with chronic renal failure.

Rationale for GH Treatment in Patients with Chronic Renal Failure

Copyright © 2002 S. Karger AG, Basel

Introduction

Recombinant human growth hormone (GH) is very effective in promoting growth in short children with chronic renal failure before and after renal transplanta-

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In patients with uraemia, basal levels and spontaneous secretion of endogenous GH are either normal or even increased. Despite this fact, GH action is decreased in uraemia due to insensitivity to GH. This is caused by a decreased expression of the gene encoding for the GH receptor in liver and growth cartilage, decreased production of IGF-1 and increased serum concentrations of var-

Dr. Elke Wühl Sektion für Pädiatrische Nephrologie Universitäts-Kinderklinik, Im Neuenheimer Feld 150 D–69120 Heidelberg (Germany) Tel. +49 6221 562311, Fax +49 6221 564203, E-Mail [email protected]

ious IGF-binding proteins leading to decreased bioactivity of IGF-1. Furthermore, there is additional target organ hyporesponsiveness due to post-receptor defects downstream of the GH and IGF-1 receptors. This insensitivity of the uraemic organism can be overcome by supraphysiological doses of GH [1].

Effect of GH Treatment in Children with Chronic Renal Failure

Improvement of statural height is the main goal in GH treatment in short children with chronic renal failure. Several short-term placebo-controlled [2, 3] and longterm open-labelled studies [4, 5] have been performed. GH was administered in an average dose of 0.33 mg/kg/ week in daily evening subcutaneous injections. With this supraphysiological dosing a significant increase in serum IGF-1 levels can be induced and catch-up growth can be achieved. During the first 3 treatment years an increment of 1.5 standard deviation scores (SDS) in children on conservative treatment, 0.8 SDS in children on dialysis and 1–1.5 SDS in transplanted children could be observed. Thereafter, growth was parallel to the centiles. Final height data indicated that growth was improved by +1.5 SDS in GH-treated children, whereas in an untreated control group height SDS decreased by –0.8 SDS within the observation period of 8 years [6]. More than 65% of patients obtained an adult height within the normal range. The period of catch-up growth was restricted to the prepubertal growth phase and no influence on the onset and duration of puberty was found. Growth was positively correlated with duration of GH treatment and the degree of growth retardation and inverse with the treatment modality (dialysis). A remodelling of body composition was observed with a decrease of fat mass and an increase of fat-free mass and total body water [7, 8]. There was no acceleration of bone maturation or signs of deteriorating renal osteodystrophy. Evaluation of bone metabolism showed an increase of serum alkaline phosphatase, osteocalcin, serum procollagen type I c-terminal propeptide (PICP) and the carboxy-terminal telopeptide of type I collagen (ICTP) after 24 months of GH treatment compared to baseline levels and to healthy controls. Serum calcium, phosphate, 1,25-vitamin D3 and parathyroid hormone levels did not change significantly [7]. Bone mineral density and content increased significantly under GH treatment [8]. No evidence for a decrease in renal function under GH treatment could be found [9].

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A selective increase in fasting and glucose-stimulated insulin secretion without a change in glycosylated haemoglobin and glucose tolerance was observed in patients with chronic renal failure and after renal transplantation, but also in short normal children in response to GH treatment. This phenomenon was exaggerated in patients on dialysis and after renal transplantation and persisted for up to 5 years of GH treatment. The absence of increased glucose tolerance during long-term GH treatment may be reassuring with respect to the diabetogenic potential of GH, nevertheless persisting hyperinsulinaemia, combined with the dyslipidaemia associated with chronic renal failure may raise concern that GH contributes to the long-term risk for premature atherosclerosis in patients with childhood-onset chronic renal failure [10]. Cholesterol and triglyceride serum levels did not change significantly under GH treatment. No major side effects were noted, with the exception of the occurrence of pseudotumour cerebri (incidence 0. 9%) if the dose was not slowly adjusted.

Effect of GH Treatment in Adults with Chronic Renal Failure

In contrast to paediatric patients, in adults with chronic renal failure the target of GH treatment was improvement of the catabolic state. Some short-term studies (3 days to 6 months) evaluated the effects of GH on glucose and bone metabolism and on body composition in an experimental setting and in small groups of up to 30 adult patients under GH treatment. GH was administered in a similar dosage (0.2–0.33 mg/kg/day) as in children. Body composition showed a significant increase of lean body mass in haemodialysis patients treated with GH, 0.7 mg/kg 3 times weekly over 6 months, compared to untreated controls (fig. 1a). A reduction in the protein catabolic rate as well as an increase of lower leg muscle area and muscle strength was observed (fig. 1b, c) [11]. Hansen et al. [12] found a decrease in body fat mass and an increase in fat-free mass of 25% respectively, while body weight was stable. GH treatment over 6 months induced a significant increase in serum albumin level compared to untreated controls (fig. 2) [11]. Since hypoalbuminaemia is associated with increased risk of mortality in end-stage renal failure [13], GH-induced elevation of serum albumin levels might reduce disease-related mortality risk in this population.

Wühl/Schaefer

Fig. 2. Change in serum albumin concentration during 6 months of

treatment with GH (P) or placebo ($) in 17 elderly hemodialysis patients [11].

Fig. 1. Change in fat-free mass (a), area of lower leg muscle (b) and mean hand-grip strength (c) during 6 months of treatment with GH

(P) or placebo ($) in 17 elderly hemodialysis patients [11].

GH in Chronic Renal Failure

Echocardiographic evaluation showed an increase in left ventricular mass without a change of cardiac function in the study of Jensen et al. [14], but this could not be confirmed by the findings of Johannsson et al. [11]. Although the clinical significance of these findings is not clear yet, careful attention should be paid to left ventricular hypertrophy and cardiac function in adults under GH treatment. Concerning the effect of GH on bone metabolism, no clear statement is possible to date whether GH-induced increased bone turnover results in a beneficial effect on uraemic bone disease. Whereas markers of bone (serum procollagen type I c-terminal peptide) and non-bone collagen synthesis (serum procollagen type III N-terminal propeptide) increased, there was no change in parathyroid hormone, serum calcium, phosphate, alkaline phosphatase and bone-specific phosphatase levels [15]. Short-term treatment with GH over 7 days increased baseline glucose levels in 10 patients under peritoneal dialysis [16]. In contrast, the studies by Johannsson et al. [11] and Jensen et al. [14] did not find alterations of serum glucose and insulin levels during the 6-month study period. As in children, no change in cholesterol and triglyceride levels could be found. In the reported studies only minor side effects as arthralgia, myalgia, and arterial hypertension were reported.

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Conclusion

GH is safe and effective for treatment of growth failure in children with chronic renal failure. In adults an improvement of the catabolic state with increase of fat-free

mass and serum albumin levels during a 6-month treatment period could be achieved. Nevertheless, long-term data on the effect of GH treatment in uraemic adults on bone, glucose and lipid metabolism or the influence on mortality are not available to date.

References 1 Mehls O, Ritz E, Hunziker EB, Eggli P, Heinrich U, Zapf J: Improvement of growth and food utilization by human recombinant growth hormone in uremia. Kidney Int 1988;33:45– 52. 2 Fine RN, Kohaut EC, Brown D, Perlman AJ for the Genentech Cooperative Study Group: Growth after recombinant human growth hormone treatment in children with chronic renal failure: Report of a multicenter, randomized double-blind placebo-controlled study. J Pediatr 1994;124:374–382. 3 Hokken-Koelega ACS, Stijnen T, De Muinck Keizer-Schrama SMPF, et al: Placebo-controlled, double-blind, cross-over trials of growth hormone treatment in prepubertal children with chronic renal failure. Lancet 1991; 338:585–590. 4 Fine RN, Kohaut E, Brown D, Kuntze J, Attie KM: Long-term treatment of growth-retarded children with chronic renal insufficiency with recombinant human growth hormone. Kidney Int 1996;49:781–785. 5 Haffner D, Wühl E, Schaefer F, Nissel R, Tönshoff B, Mehls O, the German Study Group for Growth Hormone Treatment in Chronic Renal Failure: Factors predictive of the short- and long-term efficacy of growth hormone treatment in prepubertal children with chronic renal failure. J Am Soc Nephrol 1998; 9:1899–1907.

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6 Haffner D, Schaefer F, Nissel R, Wühl E, Tönshoff B, Mehls O, the German Study Group for Growth Hormone Treatment in Chronic Renal Failure: Effect of recombinant human growth hormone treatment on adult height or children with chronic renal failure. N Engl J Med 2000;343:923–930. 7 Johnson VL, Wang J, Kaskel FJ, Pierson RN: Changes in body composition of children with chronic renal failure on growth hormone. Pediatr Nephrol 2000;14:695–700. 8 Van der Sluis IM, Boot AM, Nauta J, Hop WJC, de Jong MCJW, Lilien MR, Groothoff JW, van Wijk AE, Pols HAP, Hokken-Koelega ACS, De Muinck Keizer-Schrama SMPF: Bone density and body composition in chronic renal failure: Effects of growth hormone treatment. Pediatr Nephrol 2000:15:221–228. 9 Wühl E, Haffner D, Gretz N, Offner G, van’t Hoff WG, Broyer M, Mehls O: Treatment with recombinant human growth hormone in short children with nephropathic cystinosis: No evidence for increased deterioration rate of renal function. The European Study Group on Growth Hormone Treatment in Short Children with Nephropathic Cystinosis. Pediatr Res 1998;43:484–488. 10 Haffner D, Nissel R, Wühl E, Schaefer F, Bettendorf M, Tönshoff B, Mehls O, and members of the German Study Group for Growth Hormone Treatment in Chronic Renal Failure: Metabolic effects of long-term growth hormone treatment in prepubertal children with chronic renal failure and after kidney transplantation. Pediatr Res 43:209–215.

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11 Johannsson G, Bengtsson BA, Ahlmen J: Double-blind, placebo-controlled study of growth hormone treatment in elderly patients undergoing chronic hemodialysis: Anabolic effect and functional improvement. Am J Kidney Dis 1999;33:709–717. 12 Hansen TB, Gram J, Jensen PB, Kristiansen JH, Ekelund B, Christiansen JS, Pedersen FB: Influence of growth hormone on whole body and regional soft tissue composition in adult patients on hemodialysis. A double-blind, randomized placebo-controlled study. Clin Nephrol 2000;53:99–107. 13 Owen WF Jr, Lew NL, Liu Y, Lowrie EG, Lazarus JM: The urea reduction ratio and serum albumin concentrations as predictors of mortality in patients undergoing hemodialysis. N Engl J Med 1993;329:1001–1006. 14 Jensen B, Ekelund B, Nielsen FT, Baumbach L, Pedersen FB, Oxhoj H: Changes in cardiac muscle mass and function in hemodialysis patients during growth hormone treatment. Clin Nephrol 2000;53:25–32. 15 Gram J, Hansen TB, Jensen PB, Christensen JH, Ladefoged S, Pedersen FB: The effect of recombinant human growth hormone treatment on bone and mineral metabolism in hemodialysis patients. Nephrol Dial Transplant 1998;13:1529–1534. 16 Ikizler TA, Wingard RL, Breyer JA, Parker RA, Hakim RM: Short-term effects of recombinant human growth hormone in CAPD patients. Kidney Int 1994;46:1178–1183.

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GH and the Muscular-Skeletal System Horm Res 2002;58(suppl 3):39–42 DOI: 10.1159/000066481

Systemic Application of Growth Hormone for Enhancement of Secondary and Intramembranous Fracture Healing Hermann J. Bail Stefan Kolbeck Gert Krummrey Gerhard Schmidmaier Norbert P. Haas Michael J. Raschke Clinic for Trauma and Reconstructive Surgery, Charité – Campus Virchow Clinic, Medizinische Fakultät der Humboldt-Universität zu Berlin, Germany

Key Words Growth hormone W Distraction osteogenesis W Defect fracture W Fracture healing W Micropigs

Abstract Hormones are known to influence bone metabolism and cellular mechanisms of fracture healing. Recent technologies in molecular biology offer recombinant production of hormones, which makes them applicable for pharmacological use. To investigate the effect of systemic growth hormone (GH) application experiments were performed in micropig animal models. Systemic daily subcutaneous injection of species-specific recombinant GH was investigated in Yucatan micropigs to evaluate the effect on secondary fracture healing in a standardized gap model (1 cm) and on intramembranous bone formation in distraction osteogenesis (DO). Quantitative computed tomography (qCT), biomechanical testing, measurement of systemic insulin-like growth factor 1 (IGF-1) levels as well as histomorphometric analyses were performed to investigate differences in regenerate

Supported by Novo Nordisk A/S, Denmark.

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formation. Systemic GH administration significantly increased the torsional stability of the regenerate in comparison to the contralateral side in both experiments. qCT showed accelerated fracture bridging in the GHtreated animals in bone defect healing, while in DO histomorphometry elicited larger callus areas in the case of GH application. Systemic IGF-1 levels were significantly increased in both GH-treated groups. These experiments show that the systemic administration of recombinant GH accelerates fracture healing in standardized animal models. Clinical studies have now been initiated in order to prove the safety and the effectiveness of this therapeutical option. Copyright © 2002 S. Karger AG, Basel

Introduction

Despite all improvements in fracture treatment, complex situations including comminuted fractures and those with bone defects still remain challenging in orthopedic and trauma surgery. Reduced fracture healing such as delayed and non-unions can be seen in about 5–10% of all fractures [1]. Methods accelerating bone formation and fracture healing are therefore the subject of current investigations.

Hermann Josef Bail, MD Trauma and Reconstructive Surgery Charité – Campus Virchow Humboldt, University of Berlin Augustenburger Platz 1, D–13353 Berlin (Germany) Tel. +49 30 450 552373, Fax +49 30 450 552901, E-Mail [email protected]

Fracture healing with the consecutive stages of bone formation, resorption and remodeling is influenced by a complex interaction of numerous hormones, cytokines, the extracellular matrix and several growth factors [2–4]. Recent advances in molecular technology and the sequencing of some of these substances have made their recombinant production and application possible, while a number of studies investigated the genetic expression of these factors and their receptors to understand the underlying mechanisms [5–7]. Growth hormone (GH) is known to have a direct and indirect stimulatory effect on fracture healing. Investigations of GH-related effects on fracture healing have yielded controversial results: in some studies, enhanced fracture healing was achieved in rodents [8–10], while other investigators could not find any GH-related effect on callus formation [11–12]. These differences may partly be explained by the fact, that non-species-specific GH was used in all of these experiments and antibody formation may have occurred, thus producing an inhibitory effect [13, 14]. So far, only few investigators have used the now available species-specific GHs in animal studies dealing with fracture healing [15, 16]. IGF-1 is the main mediator of the GH-related effects on target tissues [17–19] and has been found to stimulate the replication of osteoblasts and the synthesis of bone matrix [20]. To investigate the effect of systemic application of GH on fracture healing, two experiments were performed: Using Yucatan micropigs the effect of GH on secondary fracture healing was investigated in a tibia gap model [16], imitating bone loss after e.g. a comminuted fracture. Furthermore, the effect of GH on intramembranous bone formation in distraction osteogenesis (DO) was investigated. The indications for this surgical procedure are similar to those for traditional bone grafting and transplantation, including limb lengthening and osseous defects caused by trauma, tumor resection or infection [15]. However, the major disadvantage of the method described by Ilizarov [21, 22] is the long treatment period required for distraction and proper ossification of the bone regenerate, sometimes spanning more than 1 year. The studies included (a) the measurement of IGF-1 in the serum, (b) biomechanical testing and (c) quantification of the callus volume and density using quantitative computed tomography (qCT) in the bone defect groups and using histomorphometric analysis in the DO groups, respectively.

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Bone Defect Model

Materials and Methods Twenty-four mature female Yucatan micropigs, matched for age and weight, were divided into two groups (treatment group, n = 12 and placebo group, n = 12). A standardized osteotomy of 1 cm was cut in the right tibia of the hind limb using an oscillating saw. This defect was stabilized with a low contact dynamic compression plate (LCDCP). Micropigs in the treatment group received a single daily subcutaneous injection of 100 Ìg of recombinant porcine GH (r-pGH) per kg body weight (BW), while micropigs in the control group received sodium chloride as a placebo. IGF-1 serum levels were determined with a non-competitive time-resolved immunofluorometric assay (TR-IFMA) as previously described [23]. Animals were sacrificed 42 days after surgery. Both tibiae were harvested and qCT was performed, measuring the total amount of callus in the defect, bone mineral density (BMD) and bone mineral content (BMC). Torsional failure load and torsional stiffness were determined through biomechanical testing. A more detailed description of methods can be found elsewhere [16]. Results Two animals had to be excluded from the study, due to local infection or surgical failure. The mean level of serum IGF-1 in relation to the pre-treatment level increased in GH-treated animals to 382 B 86%, whereas the IGF-1 levels of the control group decreased to 69 B 14% (p ! 0.05). qCT measurements revealed a significant difference in the BMC of the defect zone between the GH group and the placebo group (GH: 2,833 B 679 mg; placebo: 2,215 B 636 mg; p ! 0.05). The BMD in the defect zone was comparable in both groups (GH: 668 B 60 mg/mm2; placebo: 629 B 52 mg/mm2). In relation to the intact contralateral tibia, the defect tibia in the treatment group measured 75.21 B 30.5% of the torsional failure load, while the control group measured 40.57 B 22.1% (p ! 0.05). The values for the torsional stiffness were 134.8 B 67.7% for the GH group and 64.9 B 30.37% for the control animals (p ! 0.05).

Distraction Osteogenesis Model

Materials and Methods Thirty mature female Yucatan micropigs were divided into two groups (treatment group, n = 15 and control group, n = 15) using GH base levels, which were deter-

Bail/Kolbeck/Krummrey/Schmidmaier/ Haas/Raschke

mined prior to the start of the experiment. An external half ring fixator, consisting of two half rings connected by three threaded rods, was mounted under fluoroscopic control to the left tibia using four 4.5-mm half-pins. An osteotomy was carried out in the middle of the tibia using an oscillating saw. The periosteum was carefully opened before osteotomy, protected throughout the procedure and then closed. Following a 4-day latency period, the fixator was distracted at a rate of 2 mm/day (1 mm every 12 h) for 10 days. For another 10 days, the regenerates were allowed to consolidate. Micropigs in the treatment group received a single daily subcutaneous injection of 100 Ìg r-pGH/kg BW, while micropigs in the control group received sodium chloride as a placebo. The serum levels of IGF-1 were monitored analogous to the bone gap groups [23]. The animals were sacrificed on day 25 and both tibiae were harvested. Serial slices from the regenerate were produced using a hard-cutting microtome. For assessment of the calcified tissue, the sections were stained due to the modified von Kossa method. Using an image analysis workstation, the area of the total callus and the area fraction of calcified tissue (callus density) were evaluated. A more detailed description of methods have been published elsewhere [16, 24]. Results A total of 6 animals were excluded from the study, due to local infection or surgical failure. The mean level of serum IGF-1 increased to 442 B 108% of the pre-treatment levels in the r-pGH-treated animals, while mean serum IGF-1 levels remained nearly constant in the control group (93 B 25%, p ! 0.05). Compared to the intact contralateral tibia, the distracted tibiae reached 64 B 22.42% (GH) and 26.15 B 12.88% (placebo) of the final torsional failure load (p ! 0.05). The values for the torsional stiffness were 75.8 B 23.3% (GH) and 24.4 B 15.4% (placebo, p ! 0.05). The histomorphometric measurements showed a significantly larger callus area in the GH group (515.42 B 47.10 mm²), compared to the control group (440.31 B 56.31 mm²). This was significant when data were normalized with the bone end diameters and the width of the distraction gap (p ! 0.05). The histomorphometry exhibited no difference in the callus density of both groups.

Growth Hormone Enhances Fracture Healing

Discussion

The effect of systemic GH application on bone formation in micropigs and an intact GH–IGF-1 axis was demonstrated. The used dosage (100 Ìg r-pGH/kg BW once per day) resulted in a marked and steady increase in IGF1 serum levels during the examination period. This is in contrast to other studies, in which serum IGF-1 levels remained unchanged in response to GH administration [11]. This may partly be explained by antibody formation against the used allogenous GH [13, 14]. In our opinion, the present model of DO in micropigs provides a consistent model of bone formation due to the use of a standardized osteotomy and the similarly sized bone gaps. In DO the regenerate is predominantly formed by osteoblastic bone formation, without the occurrence of cartilage tissue [25, 26]. Although this is different compared to secondary fracture healing, basic events like matrix deposition and tissue calcification, leading to the formation of woven bone, remain the same and therefore results may be compared to other studies. Consecutively, the huge effect of GH on the biomechanical stability found in DO (1100% increase) could be confirmed using a model of secondary fracture healing. In the tibia defect model, the biomechanical testing exhibited a comparable advantage in the GHtreated group regarding the biomechanical stability. The qCT analysis performed in the gap model confirmed the different biomechanical results in the treated and the untreated group, showing a significantly higher BMC in the GH group, while the BMD did not differ. This indicates that a greater amount of newly formed callus was present in GH-treated animals, while the callus properties, as represented by the BMD, appeared to be similar compared to the placebo group. This is in line with the histological findings from the DO model, which showed a significantly larger callus area in the GH group while the bone density did not differ [24]. Mosekilde et al. [27] also found a larger amount of callus after GH application in a fracture model in rats, but the newly formed callus presented a looser structure compared to placebo animals, resulting in a lower bone density. In this animal model of secondary fracture healing established by Bak et al. [10], a very high dosage of recombinant human GH was applied, exceeding the presently used dosage by more than 20 times. In these papers a stimulation of the bone marrow cells could be observed and was discussed as a possible reason for the loose callus structure. Preliminary studies in the present animal models investigating the r-pGH dose dependency were not conducted. The dosage of r-pGH used (100 Ìg/kg/day) was chosen to ensure that

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endogenous GH production was blocked through negative feedback mechanisms. A therapeutically effective dosage was aimed at, which is necessarily higher than the dosage used in GH deficiency [28]. However, the dosage was still lower than in other therapeutic clinical studies, e.g. in burns or polytrauma patients, in which 150– 200 Ìg/kg/day or even higher doses were used [29–31]. Systemic application of GH has shown to be a promising therapeutic option. Serious side effects were not found in these studies. Heterotopic ossification as a possible

indicator of excessive stimulation of regenerate formation were not detected. Systemic application may be beneficial in situations in which direct access to the fracture site is not possible, desirable or necessary (e.g. in conservative fracture treatment). In summary, our findings strongly suggest that systemic GH may be used clinically in the future to accelerate the process of fracture healing. However, clinical studies, which have been initiated already, will have to validate the effectiveness and safety of these methods.

References 1 Einhorn T, Lane J: Significant advances have been made in the way surgeons treat fractures. Clin Orthop 1998;355(suppl):S2–S3. 2 Welch RD, Jones AL, Bucholz RW, Reinert CM, Tjia JS, Pierce WA, Wozney JM, Li XJ: Effect of recombinant human bone morphogenetic protein-2 on fracture healing in a goat tibial fracture model. J Bone Miner Res 1998;13: 1483–1490. 3 Lind M: Growth factors: Possible new clinical tools. A review. Acta Orthop Scand 1996;67: 407–417. 4 Mohan S, Baylink DJ: Bone growth factors. Clin Orthop 1991;17:30–48. 5 Bostrom MP, Lane JM, Berberian WS, Missri AA, Tomin E, Weiland A, Doty SB, Glaser D, Rosen VM: Immunolocalization and expression of bone morphogenetic proteins 2 and 4 in fracture healing. J Orthop Res 1995;13:357– 367. 6 Einhorn TA, Majeska RJ, Rush EB, Levine PM, Horowitz MC: The expression of cytokine activity by fracture callus. J Bone Miner Res 1995;10:1272–1281. 7 Fujii H, Kitazawa R, Maeda S, Mizuno K, Kitazawa S: Expression of platelet-derived growth factor proteins and their receptor · and ß mRNAs during fracture healing in the normal mouse. Histochem Cell Biol 1999;112: 131–138. 8 Ashton IK, Dekel S: Fracture repair in the Snell dwarf mouse. Br J Exp Pathol 1983;64:479– 486. 9 Bab I, Gazit D, Muhlrad A, Shteyer A: Regenerating bone marrow produces a potent growthpromoting activity to osteogenic cells [published erratum appears in Endocrinology 1991; 128:2638]. Endocrinology 1988;123:345–352. 10 Bak B, Jorgensen PH, Andreassen TT: Increased mechanical strength of healing rat tibial fractures treated with biosynthetic human growth hormone. Bone 1990;11:233–239. 11 Carpenter JE, Hipp JA, Gerhart TN, Rudman CG, Hayes WC, Trippel SB: Failure of growth hormone to alter the biomechanics of fracture healing in a rabbit model. J Bone Joint Surg Am 1992;74:359–367.

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12 Northmore-Ball MD, Wood MR, Meggitt BF: A biomechanical study of the effects of growth hormone in experimental fracture healing. J Bone Joint Surg Br 1980;62:391–396. 13 Van Herpen H, Rijnberk A, Mol JA: Production of antibodies to biosynthetic human growth hormone in the dog. Vet Rec 1994;134: 171–171. 14 Zwickl CM, Cocke KS, Tamura RN, Holzhausen LM, Brophy GT, Bick PH, Wierda D: Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys. Fundam Appl Toxicol 1991;16:275–287. 15 Raschke MJ, Bail H, Windhagen HJ, Kolbeck SF, Weiler A, Raun K, Kappelgard A, Skiaerbaek C, Haas NP: Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis. Bone 1999;24: 81–88. 16 Raschke M, Kolbeck S, Bail H, Schmidmaier G, Flyvbjerg A, Lindner T, Dahne M, Roenne I, Haas N: Homologous growth hormone accelerates fracture healing. Trans Orthop Res Soc 1999;45. 17 Green H, Morikawa M, Nixon T: A dual effector theory of growth-hormone action. Differentiation 1985;29:195–198. 18 Isaksson OG, Jansson JO, Gause IA: Growth hormone stimulates longitudinal bone growth directly. Science 1982;216:1237–1239. 19 Trippel S, Coutts R, Einhorn T, Mundy R, Rosenfeld R: Growth factors as therapeutic agents. J Bone Joint Surg Am 1996;78:1272– 1286. 20 Hill PA, Reynolds JJ, Meikle MC: Osteoblasts mediate insulin-like growth factor-1 and -2 stimulation of osteoclast formation and function. Endocrinology 1995;136:124–131. 21 Ilizarov GA: The tension-stress effect on the genesis and growth of tissues. II. The influence of the rate and frequency of distraction. Clin Orthop 1989;239:263–285.

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22 Ilizarov GA: The tension-stress effect on the genesis and growth of tissues. I. The influence of stability of fixation and soft-tissue preservation. Clin Orthop 1989;238:249–281. 23 Frystyk J, Dinesen B, Orskov H: Non-competitive time-resolved immunofluorometric assays for determination of human insulin-like growth factor 1 and 2. Growth Regul 1995;5:169–176. 24 Bail H, Raschke M, Kolbeck S, Windhagen H, Weiler A, Raun K, Mosekilde L, Haas NP: Recombinant species-specific growth hormone increases hard callus formation in distraction osteogenesis. Bone 2002;30:117–124. 25 Aronson J, Harp JHJ: Factors influencing the choice of external fixation for distraction osteogenesis. Instr Course Lect 1990;39:175–183. 26 Ilizarov GA, Barabash AP, Imerlishvili IA, Larionov AA, Kochetkov IS: Morphological characteristics of the formation and reconstruction of bone tissue in the replacement of extensive bone defects (in Russian). Ortop Travmatol Protez 1984;Jan:16–20. 27 Mosekilde L, Bak B: The effects of growth hormone on fracture healing in rats: A histological description. Bone 1993;14:19–27. 28 Moller J, Jorgensen JO, Lauersen T, Frystyk J, Naeraa RW, Orskov H, Christiansen JS: Growth hormone dose regimens in adult GH deficiency: Effects on biochemical growth markers and metabolic parameters. Clin Endocrinol (Oxf) 1993;39:403–408. 29 Lal SO, Wolf SE, Herndon DN. Growth hormone, burns and tissue healing. Growth Horm IGF Res 2000;10(suppl B):S39–S43. 30 Pelzer M, Hartmann B, Blome-Eberwein S, Raff T, Germann G: Effect of recombinant growth hormone on wound healing in severely burned adults. A placebo controlled, randomized double-blind phase II study (in German). Chirurg 2000;71:1352–1358. 31 Rockich KT, Hatton JC, Kryscio RJ, Young BA, Blouin RA: Effect of recombinant human growth hormone and insulin-like growth factor-1 administration on IGF-1 and IGF-binding protein-3 levels in brain injury. Pharmacotherapy 1999;19:1432–1436.

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GH and the Muscular-Skeletal System Horm Res 2002;58(suppl 3):43–48 DOI: 10.1159/000066482

Effects of Growth Hormone on Skeletal Muscle Matthias M. Weber Klinik II und Poliklinik für Innere Medizin der Universität zu Köln und Lehrstuhl II für Innere Medizin des Krankenhauses Köln-Merheim, Deutschland

Key Words Growth hormone W Muscle W Strength

Abstract Human growth hormone (GH) is widely abused as a performance-enhancing anabolic drug by athletes and bodybuilders. However, the effects of GH on skeletal muscle mass, strength and fibre composition remain unclear. We therefore summarize in the following the current knowledge on the physiological role of GH in the regulation of skeletal muscle growth and function and evaluate its potential therapeutic potency as a muscle anabolic hormone. In states of GH deficiency, reduced muscle mass and strength are characteristic findings which can be reversed successfully by the supplementation of GH. In contrast, the currently available data suggest that GH administration alone or in combination with strength exercise has little, if any, effect on muscle volume, strength and fibre composition in non-GH-deficient healthy young individuals. This assumption is supported by the lack of evidence for a significant performanceenhancing effect of GH in athletes. However, further studies will be necessary to define patient populations which might benefit from GH treatment like frail elderly individuals in whom a GH-induced change into a more youthful muscle fibre composition has been reported.

Although the alleged widespread abuse of growth hormone (GH) as a performance-enhancing substance by athletes and bodybuilders suggests that GH is a powerful anabolic drug, the effects of GH on skeletal muscle mass, strength and fibre composition remain unclear. In the following, we summarize the current knowledge on the physiological role of GH in the regulation of skeletal muscle growth and function in different states of GH deficiency (GHD) and GH excess and evaluate its potential therapeutic potency as a muscle anabolic hormone.

Effect of GH on Skeletal Muscle Mass and Function in GHD

The syndrome of GHD is associated with reduced muscle mass, strength and exercise performance [1, 2]. Furthermore, the discontinuation of GH supplementation in patients with GHD leads to a reduction in isometric muscle strength and muscle size by 5% after 1 year [3]. In addition, adolescents with childhood onset of GHD lose lean body mass and do not gain muscle strength upon discontinuation of GH, in contrast to healthy subjects and GH-substituted GHD patients of the same age [4]. Short-term GH replacement has been shown to improve body composition and exercise capacity [1, 5]. In placebo-controlled, short-term studies a significant in-

Copyright © 2002 S. Karger AG, Basel

ABC

© 2002 S. Karger AG, Basel 0301–0163/02/0589–0043$18.50/0



Priv.-Doz. Dr. Matthias M. Weber Lehrstuhl II für Innere Medizin der Universität zu Köln Medizinische Klinik II, Krankenhaus Köln-Merheim Ostmerheimer Strasse 200, D–51109 Köln (Germany) Tel. +49 221 8907 3820, Fax +49 221 8907 3659, E-Mail [email protected]

Table 1. Contractile properties of different

fibre types

Fibre type

Type 1

Type 2a

Type 2b

Colour Contraction Strength Fatigability Energy metabolism

Red Slow twitch Weak Slow Aerobic

Rose Medium twitch Medium Medium Aerobic + anaerobic

White Fast twitch Strong Fast Anaerobic

crease in muscle mass but no increase in strength was observed during up to 6 months of GH substitution [6, 7]. This anabolic effect on muscle mass was sustained during open-labelled long-term GH replacement therapy with a maximum increase of leg muscle mass by 28.7% after 5 years of treatment and a parallel increase in maximal workload by 23% [8]. Furthermore, a significant increase in muscle strength could be shown after prolonged GH substitution in open-labelled studies [7, 9, 10]. Johannsson et al. [10] show that GH-deficient adults have lower isometric knee extensor, knee flexor and hand-grip strength than age-matched healthy controls, and that 2 years of GH treatment increase and normalize mean isometric knee extensor and flexor strength. In this study, the increase in muscle strength was more marked in younger patients and in patients with lower initial strength. Surprisingly, muscle endurance decreased during GH treatment while hand-grip strength remained unchanged. The fact that in most studies with GH-deficient patients intrinsic strength (strength expressed per muscle unit) remained normal, suggests that the decrease in muscle strength in these patients is mainly a function of decreased muscle mass [11, 12]. This is supported by the unchanged bioenergetic status of the muscle in GHD patients as measured by phosphorus nuclear magnetic resonance spectroscopy both at baseline and after GH therapy [12]. In contrast, Cuneo et al. [2, 13] reported a decreased intrinsic isometric strength in GHD patients which did not improve after 6 months of GH therapy.

Effect of GH on Skeletal Muscle Fibre Composition in GHD

The myosin molecule consists of two heavy and four light chains, expressed in a cell-specific manner. Depending on the composition of the myosin heavy chains (MHC), different muscle fibre types can be classified which are important in determining the contractile properties of the skeletal muscle (table 1). Apart from heredity,

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a variety of factors influence the MHC composition of skeletal muscle including age, physical activity, neural input and hormones [14]. Since the proportion of the different fibre types is a major determinant of the contractile properties of the skeletal muscles, the impaired muscle function in GHD patients could at least in part be caused by an altered muscle fibre composition. In skeletal muscle of GH-deficient rats a decreased proportion of MHC I and type 1 fibres and an increased proportion of MHC II and type 2 fibres as compared to normal age-matched control rats has been reported [15, 16]. In some [15–17] but not in all studies [18, 19] this fibre type composition could be normalized by treatment of the GH-deficient rats with GH. Patients with GHD show contractile properties of their quadriceps muscle (shortened half-life relaxation time, rightward shift of force-frequency relation) which are compatible with a greater proportion of fast fatigable type 2 fibres [20]. Therefore, it has been suggested that the greater fatigability of GHD patients might be due to a relative decrease in slow-twitch, fatigue-resistant type 1 muscle fibres within the muscle of patients with GHD. While previous studies in GHD humans failed to demonstrate an effect of GH on muscle fibre composition [3, 21– 23] some recent studies in humans partially support this hypothesis [24, 62]. Possible reasons for these discrepancies are methodological differences, small sample sizes, and different patient populations with childhood- versus adult-onset GHD as well as isolated GHD versus multiple pituitary hormone deficiencies. In older studies, fibre type composition was determined by traditional histochemistry and counting of the different fibres. Recently, MHC isoform composition determined by SDS-PAGE is used as a molecular marker of muscle fibre types, thus taking into account variations in fibre size and reducing the sampling error [23, 62]. Using this method, Bottinelli et al. [23] reported an unaltered MHC isoform pattern in 5 patients with childhood-onset GHD as compared to a normal control group. In these patients, absolute values of quadriceps muscle strength and fibre cross-sectional area

Weber

were significantly lower than in age- and sex-matched controls. However, when normalized for quadriceps muscle area and subjects height, no difference was found between GHD and controls for muscle strength, fibre size, twitch characteristics, fatigue index and fibre type distribution (although GHD patients showed a slightly but non-significant higher percentage of fast type 2b fibres). In contrast to this study, Daugaard et al. [62] reported a significant higher amount of MHC IIX corresponding to fast-twitch type 2b fibres in 20 patients with adult-onset GHD as compared to a normal control group. No correlation was found between the duration or severity of the GHD and the amount of type 2 fibres, and GH treatment for 6 months did not influence the fibre composition in these patients. However, there was a strong negative correlation between the amount of MHC IIX and a significant positive correlation between the amount of MHC I and the physical fitness, measured as VO2max. Therefore, the authors speculate that the high amount of type 2b fibres in GHD patients is secondary to the low physical activity level in these patients. Woodhouse et al. [24] report that treatment with GH for 3 months in a doubleblind, placebo-controlled study with 28 GHD adults had no effect on muscle strength and fibre composition. However, GH treatment significantly stimulated muscular IGF-I mRNA expression and increased skeletal muscle fibre size, primarily involving type 1 fibres. In addition, GH significantly increased the ventilation threshold (VeT) by 18% and decreased the oxygen cost of walking relative to VeT by 14–21%, thus improving the submaximal aerobic capacity and the perception of increased fatigue of the GHD patients.

Effect of GH on Age-Related Changes in Skeletal Muscles

Advancing age is associated with reduced skeletal muscle mass, muscle strength and changes in body composition (increased body fat and decreased lean body mass) which resemble those of GHD in young adults. Since in GHD patients these changes can be reversed by the substitution with recombinant human GH, a potential causal relation between the age-associated decline in GH serum levels and the age-related decline in muscle mass, strength and physical capacity has been proposed. In healthy elderly people, the administration of GH decreases body fat and increases lean body mass, but with the exception of one study [25], no significant effects on muscle mass and function have been found, neither with GH treatment

Effects of Growth Hormone on Skeletal Muscle

Fig. 1. Percentage change in type 2 muscle fibres at 6 months compared to baseline in healthy elderly: No GH = all groups without GH; GH all = all groups receiving GH; Exercise = exercise alone; GH = GH alone; GH/EX = GH in combination with exercise [adapted from 31]. * p = 0.027.

alone, nor in combination of GH with strength training [26–31]. In animal models, GH is an anabolic hormone which is able to increase muscle mass and to restore muscle protein synthesis in old rats [18, 32–37]. While in earlier studies in humans, supraphysiological doses of GH stimulated muscle protein synthesis [38, 39], recent studies using tracer methods indicate that GH at physiological doses does not have a significant effect on myofibrillar protein synthesis or breakdown [25, 29, 40–43]. Furthermore, the GH-induced increase in lean body mass in healthy nonGH-deficient individuals does not seem to be due to a significant increase in muscle mass, as has been speculated [25, 44], but rather due to the water-retaining effect of GH as has been shown by measurement of total body water by deuterium oxide dilution [29] and by the rapid decline in fat-free mass after cessation of GH therapy [45]. This is in accordance with the fact that despite a significant GH-induced increase in fat-free mass, no GHinduced increase in muscle volume could be detected by NMR scans in healthy elderly males [27, 29]. As a consequence, in most studies no effect of GH alone or in combination with strength exercise on muscle function of healthy older individuals could be detected, while

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strength training alone resulted in substantial increases in muscle force [26–31]. Additionally, in elderly individuals, GH-associated side effects (e.g. oedema, arthralgias, myalgias and carpal tunnel syndrome) occurred more frequently as compared to GHD patients [46]. In analogy to these studies, no added benefit on muscular force or protein biosynthesis was reported when weight training was combined with GH administration in healthy young men, power athletes and in experienced weightlifters [41, 42, 47]. In contrast to these studies, Welle et al. [25] reported a significant increase in isokinetic thigh muscle strength in 5 healthy older men after GH treatment for 3 months. However, no consistent effect was observed for individual muscle groups and angular velocity categories, and only when the percent changes from individual categories of knee extension and flexion were combined, the increase in the GH group (+14 B 5%) as compared to the placebo group (+2 B 4%) reached statistical significance (p ! 0.05). The reason for this discrepancy remains unclear but can be explained in part by the small number of individuals investigated and by methodological differences. When GH is combined with endurance training in healthy elderly women, a larger increase in the oxidative enzyme activity of the skeletal muscle was observed as compared to endurance training alone, while peak oxygen uptake (VO2max) did not further increase [45]. During ageing, there seems to be a specific loss of fasttwitch type 2 muscle fibres what is in contrast to the reduction in type 1 muscle fibres reported in patients with GHD [48, 49]. The type 2 fibre atrophy in sarcopenic frail older people has been associated with the age-related reduction in muscle force production, which is required to rapidly correct positional changes and thus is associated with an increased risk of falling and disability. After 12 weeks of modest intensity strength training, elderly individuals show a significant 50–60% increase in muscle strength which is paralleled by skeletal muscle fibre hypertrophy [29, 30, 50, 51]. In contrast to resistance exercise, the administration of GH does not significantly increase muscle fibre size in untrained or exercising elderly men, as determined by quadriceps biopsy [27, 31, 52]. However, in two studies, GH treatment predominantly induced the abundance of fast-twitch type 2 fibres, what might be regarded as a change into a more youthful MHC composition [27, 31] (fig. 1). The physiological relevance of this shift towards more type 2b fibres remains unclear so far, especially since this effect of GH seems to be overcome in the combination with exercise, which induces an decrease in type 1 and an increase in type 2a fibres [27].

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Effects of GH Excess on Skeletal Muscles

Long-term excess of GH leads to acromegaly, a disease which is associated with a myopathy in which the muscles appear to be hypertrophic but are functionally weaker [53, 54]. Especially the well-characterized cardiac myopathy contributes to the significantly elevated mortality in patients with uncontrolled acromegaly. Muscle biopsies in patients with long-standing acromegaly reveal inconsistent changes with normal or hypertrophic type 1 muscle fibres, atrophy mainly of type 2 muscle fibres, as well as various ultrastructural changes [53, 55–57]. Despite the lack of valid evidence for its effectiveness and its potentially serious side effects, GH is believed to be a powerful muscle anabolic hormone among athletes. The abuse of GH in sports is considered to be a significant and increasing problem [58]. The only controlled studies on the effects of GH on muscle function in experienced weightlifters or power athletes have not been able to show a significant positive effect of GH on muscular protein biosynthesis or strength [42, 47, 59, 60]. Thus, valid data on the effectiveness of GH as a performance-enhancing anabolic drug are missing and all information is based on rumours, anonymous surveys and anecdotal reports, with some individuals attesting spectacular results while others found no change [61]. However, it cannot be excluded that elite athletes in whom very small increases in performance result in dramatic improvement in ranking may benefit from additional GH administration. Furthermore, ethical guidelines do not allow to perform controlled studies with prolonged and excessive doses of GH which might be necessary for improvements in athletic performance but which might also be associated with potentially serious side effects [60]. In conclusion, reduced muscle mass and strength are characteristic findings of GH deficiency which can be reversed by the supplementation of GH. In contrast, the currently available data on the effects of GH in non-GHdeficient individuals suggest that GH administration alone or in combination with strength exercise has little, if any, effect on muscle volume, strength and fibre composition. This assumption is supported by the findings in acromegaly where the muscles appear hypertrophic but are weaker and by the lack of evidence for a significant performance-enhancing effect of GH in athletes. Therefore, further studies will be necessary, to define patient populations which might benefit from GH treatment like frail elderly or malnourished individuals.

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References 1 De Boer H, Blok GJ, van der Veen EA: Clinical aspects of growth hormone deficiency in adults. Endocr Rev 1995;16:63–86. 2 Cuneo RC, Salomon F, Wiles CM, Sonksen PH: Skeletal muscle performance in adults with growth hormone deficiency. Horm Res 1990;33(suppl 4):55–60. 3 Rutherford OM, Jones DA, Round JM, Preece MA: Changes in skeletal muscle after discontinuation of growth hormone treatment in young adults with hypopituitarism. Acta Paediatr Scand Suppl 1989;356:61–63. 4 Hulthen L, Bengtsson BA, Sunnerhagen KS, Hallberg L, Grimby G, Johannsson G: GH is needed for the maturation of muscle mass and strength in adolescents. J Clin Endocrinol Metab 2001;86:4765–4770. 5 Carroll PV, Christ ER, Bengtsson BA, Carlsson L, Christiansen JS, Clemmons D, Hintz R, Ho K, Laron Z, Sizonenko P, Sonksen PH, Tanaka T, Thorne M: Growth hormone deficiency in adulthood and the effects of growth hormone replacement: A review. Growth Hormone Research Society Scientific Committee. J Clin Endocrinol Metab 1998;83:382–395. 6 Jorgensen JO, Pedersen SA, Thuesen L, Jorgensen J, Ingemann-Hansen T, Skakkebaek NE, Christiansen JS: Beneficial effects of growth hormone treatment in GH-deficient adults. Lancet 1989;i:1221–1225. 7 Beshyah SA, Freemantle C, Shahi M, Anyaoku V, Merson S, Lynch S, Skinner E, Sharp P, Foale R, Johnston DG: Replacement treatment with biosynthetic human growth hormone in growth hormone-deficient hypopituitary adults. Clin Endocrinol (Oxf) 1995;42:73–84. 8 Ter Maaten JC, de Boer H, Kamp O, Stuurman L, van der Veen EA: Long-term effects of growth hormone (GH) replacement in men with childhood-onset GH deficiency. J Clin Endocrinol Metab 1999;84:2373–2380. 9 Jorgensen JO, Thuesen L, Muller J, Ovesen P, Skakkebaek NE, Christiansen JS: Three years of growth hormone treatment in growth hormone-deficient adults: Near normalization of body composition and physical performance. Eur J Endocrinol 1994;130:224–228. 10 Johannsson G, Grimby G, Sunnerhagen KS, Bengtsson BA: Two years of growth hormone (GH) treatment increase isometric and isokinetic muscle strength in GH-deficient adults. J Clin Endocrinol Metab 1997;82:2877–2884. 11 Sartorio A, Narici M, Conti A, Monzani M, Faglia G: Quadriceps and hand-grip strength in adults with childhood-onset growth hormone deficiency. Eur J Endocrinol 1995;132:37–41. 12 Janssen YJ, Doornbos J, Roelfsema F: Changes in muscle volume, strength, and bioenergetics during recombinant human growth hormone (GH) therapy in adults with GH deficiency. J Clin Endocrinol Metab 1999;84:279–284. 13 Cuneo RC, Salomon F, Wiles CM, Hesp R, Sonksen PH: Growth hormone treatment in growth hormone-deficient adults. I. Effects on muscle mass and strength. J Appl Physiol 1991; 70:688–694.

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14 Staron RS, Johnson P: Myosin polymorphism and differential expression in adult human skeletal muscle. Comp Biochem Physiol B 1993;106:463–475. 15 Daugaard JR, Laustsen JL, Hansen BS, Richter EA: Growth hormone induces muscle fibre type transformation in growth hormone-deficient rats. Acta Physiol Scand 1998;164:119– 126. 16 Ayling CM, Moreland BH, Zanelli JM, Schulster D: Human growth hormone treatment of hypophysectomized rats increases the proportion of type-1 fibres in skeletal muscle. J Endocrinol 1989;123:429–435. 17 Loughna PT, Bates PC: Interactions between growth hormone and nutrition in hypophysectomised rats: Skeletal muscle myosin heavy chain mRNA levels. Biochem Biophys Res Commun 1994;198:97–102. 18 Roy RR, Tri C, Grossman EJ, Talmadge RJ, Grindeland RE, Mukku VR, Edgerton VR: IGF-1, growth hormone, and/or exercise effects on non-weight-bearing soleus of hypophysectomized rats. J Appl Physiol 1996;81:302–311. 19 Grossman EJ, Grindeland RE, Roy RR, Talmadge RJ, Evans J, Edgerton VR: Growth hormone, IGF-1, and exercise effects on nonweight-bearing fast muscles of hypophysectomized rats. J Appl Physiol 1997;83:1522– 1530. 20 Rutherford OM, Beshyah SA, Schott J, Watkins Y, Johnston DG: Contractile properties of the quadriceps muscle in growth hormone-deficient hypopituitary adults. Clin Sci (Lond) 1995;88:67–71. 21 Whitehead HM, Gilliland JS, Allen IV, Hadden DR: Growth hormone treatment in adults with growth hormone deficiency: Effect on muscle fibre size and proportions. Acta Paediatr Scand Suppl 1989;356:65–67. 22 Cuneo RC, Salomon F, Wiles CM, Round JM, Jones D, Hesp R, Sonksen PH: Histology of skeletal muscle in adults with GH deficiency: Comparison with normal muscle and response to GH treatment. Horm Res 1992;37:23–28. 23 Bottinelli R, Narici M, Pellegrino MA, Kayser B, Canepari M, Faglia G, Sartorio A: Contractile properties and fiber type distribution of quadriceps muscles in adults with childhoodonset growth hormone deficiency. J Clin Endocrinol Metab 1997;82:4133–4138. 24 Woodhouse LJ, Asa SL, Thomas SG, Ezzat S: Measures of submaximal aerobic performance evaluate and predict functional response to growth hormone (GH) treatment in GH-deficient adults. J Clin Endocrinol Metab 1999;84: 4570–4577. 25 Welle S, Thornton C, Statt M, McHenry B: Growth hormone increases muscle mass and strength but does not rejuvenate myofibrillar protein synthesis in healthy subjects over 60 years old. J Clin Endocrinol Metab 1996;81: 3239–3243.

26 Papadakis MA, Grady D, Black D, Tierney MJ, Gooding GA, Schambelan M, Grunfeld C: Growth hormone replacement in healthy older men improves body composition but not functional ability. Ann Intern Med 1996;124:708– 716. 27 Lange KH, Andersen JL, Beyer N, Isaksson F, Larsson B, Rasmussen MH, Juul A, Bulow J, Kjaer M: GH administration changes myosin heavy chain isoforms in skeletal muscle but does not augment muscle strength or hypertrophy, either alone or combined with resistance exercise training in healthy elderly men. J Clin Endocrinol Metab 2002;87:513–523. 28 Thompson JL, Butterfield GE, Gylfadottir UK, Yesavage J, Marcus R, Hintz RL, Pearman A, Hoffman AR: Effects of human growth hormone, insulin-like growth factor 1, and diet and exercise on body composition of obese postmenopausal women. J Clin Endocrinol Metab 1998;83:1477–1484. 29 Yarasheski KE, Zachwieja JJ, Campbell JA, Bier DM: Effect of growth hormone and resistance exercise on muscle growth and strength in older men. Am J Physiol 1995;268:E268– E276. 30 Taaffe DR, Pruitt L, Reim J, Hintz RL, Butterfield G, Hoffman AR, Marcus R: Effect of recombinant human growth hormone on the muscle strength response to resistance exercise in elderly men. J Clin Endocrinol Metab 1994; 79:1361–1366. 31 Hennessey JV, Chromiak JA, DellaVentura S, Reinert SE, Puhl J, Kiel DP, Rosen CJ, Vandenburgh H, MacLean DB: Growth hormone administration and exercise effects on muscle fiber type and diameter in moderately frail older people. J Am Geriatr Soc 2001;49:852–858. 32 Linderman JK, Gosselink KL, Booth FW, Mukku VR, Grindeland RE: Resistance exercise and growth hormone as countermeasures for skeletal muscle atrophy in hindlimb-suspended rats. Am J Physiol 1994;267:R365– R371. 33 Carmeli E, Hochberg Z, Livne E, Lichtenstein I, Kestelboim C, Silbermann M, Reznick AZ: Effect of growth hormone on gastrocnemius muscle of aged rats after immobilization: Biochemistry and morphology. J Appl Physiol 1993;75:1529–1535. 34 Goldberg AL, Goodman HM: Relationship between growth hormone and muscular work in determining muscle size. J Physiol 1969;200: 655–666. 35 Ullman M, Oldfors A: Effects of growth hormone on skeletal muscle. I. Studies on normal adult rats. Acta Physiol Scand 1989;135:531– 536. 36 Everitt AV, Terry V, Phillips MJ, Kerry HM, Shorey CD: Morphometric analysis of gastrocnemius muscle fiber size and fiber proportions in the hypophysectomized rat after prolonged administration of growth hormone or thyroxine. Growth Dev Aging 1996;60:85–93.

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37 Sonntag WE, Hylka VW, Meites J: Growth hormone restores protein synthesis in skeletal muscle of old male rats. J Gerontol 1985;40: 689–694. 38 Fryburg DA, Gelfand RA, Barrett EJ: Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans. Am J Physiol 1991;260:E499–E504. 39 Fryburg DA, Barrett EJ: Growth hormone acutely stimulates skeletal muscle but not whole-body protein synthesis in humans. Metabolism 1993;42:1223–1227. 40 Copeland KC, Nair KS: Acute growth hormone effects on amino acid and lipid metabolism. J Clin Endocrinol Metab 1994;78:1040–1047. 41 Yarasheski KE, Campbell JA, Smith K, Rennie MJ, Holloszy JO, Bier DM: Effect of growth hormone and resistance exercise on muscle growth in young men. Am J Physiol 1992;262: E261–E267. 42 Yarasheski KE, Zachweija JJ, Angelopoulos TJ, Bier DM: Short-term growth hormone treatment does not increase muscle protein synthesis in experienced weightlifters. J Appl Physiol 1993;74:3073–3076. 43 Manson JM, Smith RJ, Wilmore DW: Growth hormone stimulates protein synthesis during hypocaloric parenteral nutrition. Role of hormonal-substrate environment. Ann Surg 1988; 208:136–142. 44 Rudman D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L, Rudman IW, Mattson DE: Effects of human growth hormone in men over 60 years old. N Engl J Med 1990;323:1–6.

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45 Lange KH, Isaksson F, Juul A, Rasmussen MH, Bulow J, Kjaer M: Growth hormone enhances effects of endurance training on oxidative muscle metabolism in elderly women. Am J Physiol Endocrinol Metab 2000;279:E989– E996. 46 Yarasheski KE, Zachwieja JJ: Growth hormone therapy for the elderly: The fountain of youth proves toxic. JAMA 1993;270:1694. 47 Deyssig R, Frisch H, Blum WF, Waldhor T: Effect of growth hormone treatment on hormonal parameters, body composition and strength in athletes. Acta Endocrinol (Copenh) 1993;128:313–318. 48 Singh MA, Ding W, Manfredi TJ, Solares GS, O’Neill EF, Clements KM, Ryan ND, Kehayias JJ, Fielding RA, Evans WJ: Insulin-like growth factor 1 in skeletal muscle after weightlifting exercise in frail elders. Am J Physiol 1999;277:E135–E143. 49 Porter MM, Vandervoort AA, Lexell J: Aging of human muscle: Structure, function and adaptability. Scand J Med Sci Sports 1995;5: 129–142. 50 MacDougall JD, Elder GC, Sale DG, Moroz JR, Sutton JR: Effects of strength training and immobilization on human muscle fibres. Eur J Appl Physiol Occup Physiol 1980;43:25–34. 51 Frontera WR, Meredith CN, O’Reilly KP, Knuttgen HG, Evans WJ: Strength conditioning in older men: Skeletal muscle hypertrophy and improved function. J Appl Physiol 1988; 64:1038–1044. 52 Taaffe DR, Jin IH, Vu TH, Hoffman AR, Marcus R: Lack of effect of recombinant human growth hormone (GH) on muscle morphology and GH-insulin-like growth factor expression in resistance-trained elderly men. J Clin Endocrinol Metab 1996;81:421–425.

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53 Brumback RA, Barr CE: Myopathy in acromegaly. A case study. Pathol Res Pract 1983; 177:41–46. 54 Wolf E, Wanke R, Schenck E, Hermanns W, Brem G: Effects of growth hormone overproduction on grip strength of transgenic mice. Eur J Endocrinol 1995;133:735–740. 55 Nagulesparen M, Trickey R, Davies MJ, Jenkins JS: Muscle changes in acromegaly. Br Med J 1976;ii:914–915. 56 Cheah JS, Chua SP, Ho CL: Ultrastructure of the skeletal muscles in acromegaly before and after hypophysectomy. Am J Med Sci 1975; 269:183–187. 57 Stern LZ, Payne CM, Hannapel LK: Acromegaly: Histochemical and electron microscopic changes in deltoid and intercostal muscle. Neurology 1974;24:589–593. 58 Rickert VI, Pawlak-Morello C, Sheppard V, Jay MS: Human growth hormone: A new substance of abuse among adolescents? Clin Pediatr (Phila) 1992;31:723–726. 59 Ehrnborg C, Bengtsson BA, Rosen T: Growth hormone abuse. Baillières Best Pract Res Clin Endocrinol Metab 2000;14:71–77. 60 Jenkins PJ: Growth hormone and exercise: Physiology, use and abuse. Growth Horm IGF Res 2001;11(suppl A):S71–S77. 61 Macintyre JG: Growth hormone and athletes. Sports Med 1987;4:129–142. 62 Daugaard JR, Bramnert M, Manhem P, Endre T, Groop LC, Lofman M, Richter EA: Effect of 6 months of GH treatment on myosin heavy chain composition in GH-deficient patients. Eur J Endocrinol 1999;141:342–349.

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Muscle Horm Res 2002;58(suppl 3):49–55 DOI: 10.1159/000066483

Osteoporosis and the Growth HormoneInsulin-Like Growth Factor Axis Piet P.M.M Geusens a Steven Boonen b a Department of Rheumatology, University Hospital, Maastricht, The Netherlands and Biomedical Research Institute, Limburgs Universitair Centrum, Diepenbeek, Belgium; b Leuven University Center for Metabolic Bone Diseases, Katholieke Universiteit Leuven, Belgium

Key Words Osteoporosis W Insulin-like growth factor W Growth hormone W Hip fracture

Abstract Osteoporosis is the result of an imbalance between bone resorption and bone formation. Currently, mainly drugs that inhibit bone resorption are available for the treatment of osteoporosis. A new approach in the treatment of osteoporosis is the use of anabolic agents that increase bone turnover, both bone formation and resorption. Growth hormone (GH) and insulin-like growth factors (IGFs) are essential in the development and growth of the skeleton and for the maintenance of bone mass and density. We will review the evidence of GH and IGF-I in the pathophysiology and treatment of osteoporosis. Copyright © 2002 S. Karger AG, Basel

Introduction

There are many arguments that growth hormone (GH) and insulin-like growth factors (IGF) play an important role in bone metabolism [1–5]. GH and IGF-I are essen-

ABC

© 2002 S. Karger AG, Basel 0301–0163/02/0589–0049$18.50/0



tial for the development and growth of the skeleton and for the maintenance of bone mass and density [1–5]. In this review, we will summarize studies that have analyzed the correlations between GH and IGF-I and bone density or have assessed the effects of GH and IGF-I in growth hormone deficiency (GHD), in elderly subjects and in osteoporotic patients.

GH and IGF-I Administration in Animal Models

Several studies have been performed in aged rat models [6–9]. In these animal models, GH administration accelerates bone remodelling [6], increases bone mass, especially by periosteal bone apposition, increases bone strength [7, 8], and enhances fracture healing [9]. In GHdeficient rats, bone growth is reduced [10], and growth rate can be normalized by exogenous GH [11]. In line with these findings, transgenic mice that secrete supernormal levels of GH show enhanced bone growth [12]. IGF-I administration increases bone mass, but not density in growing rats, indicating an effect on bone size [13]. In ovariectomized rats, bone density and bone strength are preserved by exogenous IGF-I [8].

Prof. Dr. Piet Geusens Department of Rheumatology, University Hospital P. Debyelaan 25, Postbus 5800 NL–6202 AZ Maastricht (The Netherlands) Tel. +32 89362977, Fax +32 89304186, E-Mail [email protected]

GH and IGF-I and Bone Remodelling

The effect of GH on bone remodelling in humans is complex and not fully understood [1, 2, 14]. GH has a direct effect on osteoblasts in vitro [15]. GH binding to its receptors leads to activation of JAK2, that activates MAP kinase and STATs [14]. GH stimulates mRNA expression of the protooncogen c-fos [16] and stimulates the secretion of collagen-I, alkaline phosphates and osteocalcin [15]. Bone is a major reservoir for IGF-I and could in this way be a coupling factor between bone resorption and formation [1, 17, 18]. The IGFs embedded in newly formed bone by osteoblasts are released when bone is resorbed, thus enabling stimulation of new bone formation. IGFs strongly inhibit osteoblast apoptosis in vitro [19]. On the other hand, IGFs may increase osteoclastogenesis [20]. One explanation could be that IGF-I suppresses the secretion of osteoprotegerin, allowing enhanced bone resorption [17]. Additionally, GH and IGF-I have effects on muscle mass and on calcium transport in the gut [17], and these, too, may contribute to their skeletal impact.

GH Excess

Aging and the GH-IGF-I Axis

Patients with acromegaly have cortical bones of greater size with a higher BMD [21], although hypogonadism and disturbed vitamin D metabolism – frequent findings in acromegaly – can obscure these changes [22]. Trabecular BMD can be increased, normal or decreased [23–25]. Bone markers are increased (osteocalcin, alkaline phosphatase, urinary calcium and hydroxyproline excretion) [26], but concomitant hypogonadism can play a role in these changes [22]. Of pathophysiological interest is the finding that IGF-I is increased in patients with osteoarthritis, a condition associated with increased generalized and subchondral bone density and new bone formation in osteophytes [26]. This observation is of particular interest, as osteoarthritis is also a feature of acromegaly.

GH Deficiency and Bone

Children with GH deficiency have short stature and have a decreased bone mass in the forearm [27] and decreased serum levels of osteocalcin [28]. The lower bone mass in young adults with GHD has been related to decreased bone acquisition, as eroded surfaces, osteoid

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thickness and mineral lag time were increased together with decreased serum osteocalcin during adolescent years [29]. The concomitant role of hypogonadism has not been clarified. Adults with GH deficiency have a lower bone density [26]. After correction for BMI, BMD is still significantly decreased in the spine and forearm [27]. However, no differences are found when assessing apparent density by phalangeal ultrasound transmission [30]. Several studies have reported increased fracture prevalence in GHD [3, 31]. Adult hypopituitary patients with GHD have a more than threefold higher fracture prevalence compared with controls [31]. In a large-scale analysis, including 2,024 adult patients between 18 and 82 years of age with hypopituitarism, an increased fracture risk was confirmed (relative risk 2.7 versus controls) [3]. This increased fracture risk appears to be attributable to GHD alone, as there is no additional effect of other pituitary hormone deficiencies or to their replacement therapy [3]. In patients older than 50 years and in men younger than 30 years, the fracture risk is higher in patients that had no GH treatment.

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Serum levels of GH and IGF-I decrease with advancing age [32], although this was not confirmed in all studies [33]. Maximal stimulation capacity of GH is not affected [34]. IGF-I, IGF-II, IGFBP-3 (the major IGF binding protein) and IGFBP-5 (a binding protein known to stimulate bone formation) decrease with age in femoral cortical and trabecular bone [35, 36]. The decrease of IGF-I with age can in part be due to changes in nutritional status [37]. Protein intake is indeed a major determinant of GH-IGF-I [37]. Sufficient protein intake is essential for bone health. Protein malnutrition during childhood and adolescence leads to growth retardation and to low peak bone mass, the extreme example in modern Western societies being anorexia nervosa. The positive effect of protein intake on bone is partially explained by its stimulatory effect on the secretion of IGFs [37]. Numerous studies have even provided evidence for a direct role of the IGF system in mediating changes in the anabolic response to nutrient intake, suggesting that it may be particularly sensitive to nutritional deprivation. During adulthood and senescence, the needs of proteins are constant, but intake declines with age. Low protein intake in the elderly, which is a frequent phenomenon, could therefore contribute to bone loss. Protein sup-

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plementation in elderly after hip fracture is indeed associated with a more favorable outcome after hip fracture, including shorter hospital stay [37].

Osteoporosis, GH and IGF-I

GH secretion is decreased in osteoporosis [38]. As previously discussed, GHD is associated with an increased risk for fractures [3, 31]. Prospective data from the OFELY study have provided evidence that low levels of serum IGF-I may increase the risk of sustaining a hip fracture [39]. In this study, the risk for fracture was 2.9 times higher in subjects with serum levels of IGF-I below the mean [39]. Postmenopausal women with osteoporosis have lower serum levels of IGF-I, IGF-II and IGFBP-3 [40]. Patients with a recent hip fracture have decreased concentrations of GH and IGF-I as well [41]. In these patients, serum PTH and bone markers of bone resorption are increased, and 25(OH)D3 and markers of bone formation are decreased [41]. Moreover, IGF-I, IGF-II, IGFBP-3 and IGFBP-5 in hip fracture patients are correlated with serum osteocalcin and bone density in the femoral neck, suggesting that a deficit in these anabolic components of the IGF system may contribute to the decline in bone formation and the rate of loss in older individuals. In addition to increased bone resorption resulting from hyperparathyroidism secondary to calcium and vitamin D deficiency, impaired bone formation associated with deficiency of the IGF system might therefore be another mechanism that contributes to fragility fracture of the hip in elderly women [41]. Strong correlations between IGF-I and BMD have been observed in older men and women [33]. Also, in male osteoporosis, IGF-I and IGFBP-3 are associated with BMD in the spine [42]. In keeping with these findings, men with idiopathic osteoporosis have decreased serum levels of IGF-I and low rates of bone turnover [42– 44]. Women with anorexia nervosa have decreased serum levels of IGF-I [45]. The relative importance of low protein intake is not yet clear [37].

GH Treatment in GHD Children and Adults

It is not the aim to review the effect of GH in GHD. In short, in children with GHD, rhGH results in an increase in bone formation [46]. In one particular study, BMC in the radius increased after 1 year and Z-score normalized

Osteoporosis and the Growth HormoneInsulin-Like Growth Factor Axis

Fig. 1. Changes in BMD in the spine after GH therapy in adults. Each point refers to the changes in BMD in several studies with different duration [14, 51–56]. The line represents the mean of these changes.

in nearly 50% of the subjects [47]. In adults with GHD, short-term treatment with GH raises serum IGF-I and muscle mass [48, 49]. Fat mass consistently decreases [48, 49]. Markers of bone resorption increase significantly [50]. Recently, studies of longer duration indicate that changes in bone mass do not occur after 12–36 months, but only after several years of treatment (fig. 1) [51–56]. Further long-term studies are needed to evaluate the effect of long-term therapy on other skeletal sites, such as the hip.

GH Administration in Healthy Adults

The effect of GH in healthy adults has been studied in several small and short-term studies [57–60]. While the effects on bone mass and density have been inconsistent (table 1), most studies have shown a stimulation of bone formation and resorption.

GH Therapy in Osteoporosis

Postmenopausal women with osteoporosis have no skeletal resistance to GH as measured by the increase of IGF-I and markers of bone turnover after application of GH [61]. In a 2-year study in elderly women with low bone mass using cyclical rhGH, bone density increased in the spine by 3%, and lesser changes were found in the hip [62]. In elderly women over 75 years of age with a recent hip fracture, a 6-week treatment with rhGH significantly

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Table 1. Studies reporting on effects

of rhGH on bone metabolism in adult humans

Table 2. Studies reporting on effects

of rhIGF-I on bone metabolism in adult humans

Study

Subjects (male/ female)

Duration

BMD

Markers (formation, resorption)

Rudman [57]

21 M

6–12 months

Not available

Marcus [58] Halloway [59] Rosen [60]

16 M+F 27 F M+F

7 days 12 months 12 months

Spine +1.6% (6 months) Not available No change No change

M+F–

Study

Subjects (male/ female)

Duration

BMD

Markers (formation, resorption)

Ebeling [64] Grinspoon [45] Johanssen [66] Boonen [67]

18 F 14 F 24 M 30 F

6 days 6 days 6 weeks 8 weeks

Not available Not available Not available Hip: no change

F+R+ F+R0 F+R+ F+R+

increased the chance of returning to their pre-fracture living situation, but no effect was found below 75 years of age [63].

IGF-I Administration in Healthy Adults

IGF-I increases markers of bone formation and resorption as soon as after 6 days after administration in early postmenopausal women [64] and after 28 days in elderly postmenopausal women [65]. Most studies showed an increase of both bone formation and resorption [45, 66, 67] (table 2). Of interest, only bone formation appears to be stimulated when low-dose IGF-I is used [65].

IGF Therapy in Osteoporosis

In men with idiopathic osteoporosis, rhIGF-I increases markers of bone formation and bone resorption [66]. Recently, novel approaches to enhancing IGF-I action on bone have been proposed by administering IGF-I along with its binding protein-3 (IGF-I/IGFBP-3). A pilot study was performed on 30 older women (aged 65–90 years) with recent hip fracture [67]. Within 72 h after the hip fracture, patients received continuous administration of either placebo (n = 10), 0.5 mg/kg/day of rhIGF-I/

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F+R+ R+F+/– R+F+

Tissue (muscle, fat)

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IGFBP-3 (n = 9), or 1 mg/kg/day (n = 11). Treatment was administered by subcutaneous infusion through a portable minipump for a total of 8 weeks following hip fracture surgery, with patient follow-up to 6 months post-surgery. After an initial loss of bone density in the hip following hip fracture surgery, patients treated with 1 mg/kg/day of rhIGF-I/IGFBP-3 regained a substantial portion of their femoral bone mass (fig. 2). At 6 months post-fracture (4 months following the 2-month infusion), they showed a statistically not significant decrease from baseline in bone density in the hip (–2.6%, p = 0.53). Placebo-treated patients, on the other hand, failed to regain lost bone: at 6 months post-fracture, bone density in the placebo group had declined by 6.1% (p = 0.04). Markers of bone formation and resorption increased to higher levels after IGFI/IGFBP-3 than after placebo. Thus, the highest dose of IGF-I/IGFBP-3 resulted in higher bone turnover during treatment than in placebo. This was associated with bone loss during the first 2 months, followed by a catch-up during follow-up (fig. 3). Positive effects were also found on muscle strength and physical recuperation. In patients treated with the highest dose of rhIGF-I/IGFBP-3, grip strength had increased from baseline by 11.4% by the end of the study (p = 0.04) while patients on placebo lost 11.6% from baseline (p = 0.16). This increase in muscle strength in the high-dose group was associated with a positive effect on functional recovery.

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Fig. 2. Changes in BMD of the femoral neck during and after treatment with IGF-I/ IGFBP-3 [67].

reactions, arthralgia, fatigue, headache, and dyspnea. Preliminary evidence suggests that these side effects may not occur when complexing IGF-I to IGFBP-3.

Conclusions

Fig. 3. Differences in markers of bone turnover and BMD in the femoral neck between the patients treated with the highest dose of IGF-I/IGFBP-3 and those treated with placebo [67].

Side Effects

Treatment with GH can be complicated by edema, increase in body weight, carpal tunnel syndrome and glucose intolerance. Administration of free rhIGF-I is also associated with side effects, such as hypoglycemia, edema, intracranial hypertension and papilledema, syncopal


The involvement of GH and IGFs in skeletal metabolism is essential for growth and maintenance of the skeleton. The recently published effects of rhPTH(1–34) [68] indicate that anabolic agents that stimulate bone formation and bone resorption can have a significant impact in the treatment of osteoporosis, including fracture prevention. In addition, GH and IGF-I have also extraskeletal effects, such as the effects on muscle mass and strength, that could be of benefit for patients with osteoporosis. The data presented indicate a potential role of GH and IGF-I in the treatment of bone involvement in GHD and of osteoporosis. However, large-scale prospective controlled long-term studies will be needed to study the effect of GH and IGF-I on bone mass and fracture risk.

Acknowledgements Dr. S. Boonen is senior clinical investigator of the Fund for Scientific Research, Flanders, Belgium (F.W.O.-Vlaanderen). Dr. S. Boonen is holder of the uncommitted Leuven University Chair in Metabolic Bone Diseases, founded and supported by Merck Sharp & Dohme.

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References 1 Bouillon R: Growth hormone and bone. Horm Res 1991;36(suppl 1):49–55. 2 Ohlsson C, Bengtsson BA, Isaksson OG, Andreassen TT, Slootweg MC: Growth hormone and bone. Endocr Rev 1998;19:55–79. 3 Wuster C, Abs R, Bengtsson BA, Bennmarker H, Feldt-Rasmussen U, Hernberg-Stahl E, Monson JP, Westberg B, Wilton P: The influence of growth hormone deficiency, growth hormone replacement therapy, and other aspects of hypopituitarism on fracture rate and bone mineral density. J Bone Miner Res 2001; 16:398–405. 4 Rosen CJ, Donahue LR: Insulin-like growth factors and bone: The osteoporosis connection revisited. Proc Soc Exp Biol Med 1998;219: 1–7. 5 Rosen CJ, Pollak M: Circulating IGF-I: New perspectives for a new century. Trends Endocrinol Metab 1999;10:136–141. 6 Andreassen TT, Melsen F, Oxlund H: The influence of growth hormone on cancellous and cortical bone of the vertebral body in aged rats. J Bone Miner Res 1996;11:1094–1102. 7 Jorgensen PH, Bak B, Andreassen TT: Mechanical properties and biochemical composition of rat cortical femur and tibia after longterm treatment with biosynthetic human growth hormone. Bone 1991;12:353–359. 8 Verhaeghe J, van Bree R, Van Herck E, Thomas H, Skottner A, Dequeker J, Mosekilde L, Einhorn TA, Bouillon R: Effects of recombinant human growth hormone and insulin-like growth factor-I, with or without 17ß-estradiol, on bone and mineral homeostasis of aged ovariectomized rats. J Bone Miner Res 1996; 11:1723–1735. 9 Bak B, Andreassen TT: The effect of growth hormone on fracture healing in old rats. Bone 1991;12:151–4. 10 Dott NM, Fraser J: The influence of experimental pituitary and thyroid derangements upon the developmental growth of bone. Q J Exp Physiol (Suppl) 1923;13:107–108. 11 Charlton HM, Clark RG, Robinson IC, Goff AE, Cox BS, Bugnon C, Bloch BA: Growth hormone-deficient dwarfism in the rat: A new mutation. J Endocrinol 1988;119:51–58. 12 Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM: Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982;300:611–615. 13 Rosen HN, Chen V, Cittadini A, Greenspan SL, Douglas PS, Moses AC, Beamer WG: Treatment with growth hormone and IGF-I in growing rats increases bone mineral content but not bone mineral density. J Bone Miner Res 1995;10:1352–1358. 14 Bouillon R (ed): GH and Bone. London, OCC Ltd, 1998.

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15 Chenu C, Valentin-Opran A, Chavassieux P, Saez S, Meunier PJ, Delmas PD: Insulin-like growth factor I hormonal regulation by growth hormone and by 1,25(OH)2D3 and activity on human osteoblast-like cells in short-term cultures. Bone 1990;11:81–86. 16 Merriman HL, La Tour D, Linkhart TA, Mohan S, Baylink DJ, Strong DD: Insulin-like growth factor-I and insulin-like growth factorII induce c-fos in mouse osteoblastic cells. Calcif Tissue Int 1990;46:258–262. 17 Wuster C, Rosen C: Growth hormone, insulinlike growth factors; in Marcus, Feldman, Kelsey (eds): Osteoporosis. New York, Academic Press, 2001. 18 Xuetzong Q, Gysin R, Mohan S, Baylink D: Bone growth factors; in Marcus, Feldman, Kelsey (eds): Osteoporosis. New York, Academic Press, 2001. 19 Hill PA, Tumber A, Meikle MC: Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 1997;138:3849– 3858. 20 Hill PA, Reynolds JJ, Meikle MC: Osteoblasts mediate insulin-like growth factor-I and -II stimulation of osteoclast formation and function. Endocrinology 1995;136:124–131. 21 Riggs LR, Randall RV, Wahner HW, Jowsey J, Kelly PJ, Singh M: The nature of the metabolic bone disorder in acromegaly. J Clin Endocrinol 1972;34:543–551. 22 Lesse GP, Fraser WD, Farquharson R, Hipkin L, Vora JP: Gonadal status is an important determinant of bone density in acromegaly. Clin Endocrinol (Oxf) 1998;48:59–65. 23 Ezzat S, Melmed S, Endres D, Eyre DR, Singer FR: Biochemical assessment of bone formation and resorption in acromegaly. J Clin Endocrinol Metab 1993;76:1452–57. 24 Diamond T, Nery L, Pose S: Spinal and peripheral bone mineral densities in acromegaly. Ann Intern Med 1989;111:567–573. 25 Seeman E, Wahner HW, Offord KP, Kumar R, Johnson WJ, Riggs BL: Differential effects of endocrine dysfunction on the axial and the appendicular skeleton. J Clin Invest 1982;69: 1302–1309. 26 Wuster C: Growth hormone and bone metabolism. Acta Endocrinol (Copenh) 1993;128 (suppl 2):14–18. 27 Wuster C, Duckeck G, Ugurel A, Lojen M, Minne HW, Ziegler R: Bone mass of spine and forearm in osteoporosis and in German normals: Influences of sex, age and anthropometric parameters. Eur J Clin Invest 1992;22:366– 370. 28 Delmas PD, Chatelain P, Malaval L, Bonne G: Serum bone GLA-protein in growth hormonedeficient children. J Bone Miner Res 1986;1: 333–338. 29 De Boer H, Blok GJ, van Lingen A, Teule GJ, Lips P, van der Veen EA: Consequences of childhood-onset growth hormone deficiency for adult bone mass. J Bone Miner Res 1994;9: 1319–1326.

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30 Kann P, Piepkorn B, Pfutzner A, Peipenburg R, Beyer J: Bone quality in GH-deficient adults. Acta Endocrinol 1993;128(suppl 2):60. 31 Rosen T, Wilhelmsen L, Landin-Wilhelmsen K, Lappas G, Bengtsson BA: Increased fracture frequency in adult patients with hypopituitarism and GH deficiency. Eur J Endocrinol 1997; 137:240–245. 32 Donahue LR, Hunter SJ, Sherblom AP, Rosen C: Age-related changes in serum insulin-like growth factor-binding proteins in women. J Clin Endocrinol Metab 1990;71:575–579. 33 Langlois JA, Rosen CJ, Visser M, Hannan MT, Harris T, Wilson PW, Kiel DP: Association between insulin-like growth factor I and bone mineral density in older women and men: The Framingham Heart Study. J Clin Endocrinol Metab 1998;83:4257–4262. 34 Corpas E, Harman SM, Blackman MR: Human growth hormone and human aging. Endocr Rev 1993;14:20–39. 35 Boonen S, Aerssens J, Dequeker J, Nicholson P, Cheng X, Lowet G, Verbeke G, Bouillon R: Age-associated decline in human femoral neck cortical and trabecular content of insulin-like growth factor I. Potential implications for agerelated (type II) osteoporotic fracture occurrence. Calcif Tissue Int 1997;61:173–178. 36 Nicolas V, Prewett A, Bettica P, Mohan S, Finkelman RD, Baylink DJ, Farley JR: Age-related decreases in insulin-like growth factor-I and transforming growth factor-ß in femoral cortical bone from both men and women: Implications for bone loss with aging. J Clin Endocrinol Metab 1994;78:1011–1016. 37 Bonjour JP, Schurch MA, Chevalley T, Ammann P, Rizzoli R: Protein intake, IGF-1 and osteoporosis. Osteoporos Int 1997;7(suppl 3): S36–S42. 38 Dequeker J, Burssens A, Bouillon R: Dynamics of growth hormone secretion in patients with osteoporosis and in patients with osteoarthrosis. Horm Res 1982;16:353–356. 39 Garnero P, Sornay-Rendu E, Delmas PD: Low serum IGF-I and occurrence of osteoporotic fractures in postmenopausal women (letter). Lancet 2000;355:898–899. 40 Wuster C, Blum WF, Schlemilch S, Ranke MB, Ziegler R: Decreased serum levels of insulinlike growth factors and IGF binding protein 3 in osteoporosis. J Intern Med 1993;234:249– 255. 41 Boonen S, Mohan S, Dequeker J, Aerssens J, Vanderschueren D, Verbeke G, Broos P, Bouillon R, Baylink DJ: Down-regulation of the serum stimulatory components of the insulinlike growth factor (IGF) system (IGF-I, IGF-II, IGF binding protein [BP]-3, and IGFBP-5) in age-related (type II) femoral neck osteoporosis. J Bone Miner Res 1999;14:2150–2158. 42 Ljunghall S, Johansson AG, Burman P, Kampe O, Lindh E, Karlsson FA: Low plasma levels of insulin-like growth factor 1 in male patients with idiopathic osteoporosis. J Intern Med 1992;232:59–64.

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43 Kurland ES, Rosen CJ, Cosman F, McMahon D, Chan F, Shane E, Lindsay R, Dempster D, Bilezikian JP: Insulin-like growth factor-I in men with idiopathic osteoporosis. J Clin Endocrinol Metab 1997;82:2799–2805. 44 Kurland ES, Chan FK, Rosen CJ, Bilezikian JP: Normal growth hormone secretory reserve in men with idiopathic osteoporosis and reduced circulating levels of insulin-like growth factor I. J Clin Endocrinol Metab 1998;83: 2576–2579. 45 Grinspoon S, Baum H, Lee K, Anderson E, Herzog D, Klibanski A: Effects of short-term recombinant human insulin-like growth factor I administration on bone turnover in osteopenic women with anorexia nervosa. J Clin Endocrinol Metab 1996;81:3864–3870. 46 Carey DE, Goldberg B, Ratzan SK, Rubine KR, Rowe DW: Radioimmunoassay for type I procollagen in GHD children before and during treatment with GH. Pediatr Res 1985;19: 8–11. 47 Saggese G, Baroncelli GI, Bertelloni S, Cinquanta L, Di Nero G: Effects of long-term treatment with growth hormone on bone and mineral metabolism in children with growth hormone deficiency. J Pediatr 1993;122:37– 45. 48 Jorgensen JO, Pedersen SA, Thuesen L, Jorgensen J, Moller J, Muller J, Skakkebaek NE, Christiansen JS: Long-term growth hormone treatment in growth hormone-deficient adults. Acta Endocrinol (Copenh) 1991;125:449–453. 49 Cuneo RC, Salomon F, Wiles CM, Hesp R, Sonksen PH: Growth hormone treatment in growth hormone-deficient adults. I. Effects on muscle mass and strength. J Appl Physiol 1991; 70:688–694. 50 Van der Veen EA, Netelenbos JC: Growth hormone (replacement) therapy in adults: Bone and calcium metabolism. Horm Res 1990;33 (suppl 4):65–68. 51 Janssen YJ, Hamdy NA, Frolich M, Roelfsema F: Skeletal effects of two years of treatment with low physiological doses of recombinant human growth hormone (GH) in patients with adult-onset GH deficiency. J Clin Endocrinol Metab 1998;83:2143–2148.


52 Johannsson G, Rosen T, Bosaeus I, Sjostrom L, Bengtsson BA: Two years of growth hormone (GH) treatment increases bone mineral content and density in hypopituitary patients with adult-onset GH deficiency. J Clin Endocrinol Metab 1996;81:2865–2873. 53 Baum HB, Biller BM, Finkelstein JS, Cannistraro KB, Oppenhein DS, Schoenfeld DA, Michel TH, Wittink H, Klibanski A: Effects of physiologic growth hormone therapy on bone density and body composition in patients with adult-onset growth hormone deficiency. A randomized, placebo-controlled trial. Ann Intern Med 1996;125:883–890. 54 Kann P, Piepkorn B, Schehler B, Andreas J, Lotz J, Prellwitz W, Beyer J: Effect of long-term treatment with GH on bone metabolism, bone mineral density and bone elasticity in GH-deficient adults. Clin Endocrinol (Oxf) 1998;48: 561–568. 55 Baum HB, Biller BM, Finkelstein JS, Cannistraro KB, Oppenhein DS, Schoenfeld DA, Michel TH, Wittink H, Klibanski A: Effects of physiologic growth hormone therapy on bone density and body composition in patients with adult-onset growth hormone deficiency. A randomized, placebo-controlled trial. Ann Intern Med 1996;125(11):883–890. 56 Clanget C, Seck T, Hinke V, Wuster C, Ziegler R, Pfeilschifter J: Effects of 6 years of growth hormone (GH) treatment on bone mineral density in GH-deficient adults. Clin Endocrinol (Oxf) 2001;55:93–99. 57 Rudman D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L, Rudman IW, Mattson DE: Effects of human growth hormone in men over 60 years old. N Engl J Med 1990;323:1–6. 58 Marcus R, Butterfield G, Holloway L, Gilliland L, Baylink DJ, Hintz RL, Sherman BM: Effects of short-term administration of recombinant human growth hormone to elderly people. J Clin Endocrinol Metab 1990;70:519–527. 59 Holloway L, Butterfield G, Hintz RL, Gesundheit N, Marcus R: Effects of recombinant human growth hormone on metabolic indices, body composition and bone turnover in healthy elderly women. J Clin Endocrinol Metab 1994;79:470–479. 60 Rosen CJ and al: The RIGHT study: a randomized placebo controlled trial of rhGH in frail elderly. J Bone Miner 1999;14(suppl):208.

61 Kassem M, Brixen K, Blum WF, Mosekilde L, Eriksen EF: Normal osteoclastic and osteoblastic responses to exogenous growth hormone in patients with postmenopausal spinal osteoporosis. J Bone Miner Res 1994;9:1365–1370. 62 Holloway L, Kohlmeier L, Kent K, Marcus R: Skeletal effects of cyclic recombinant human growth hormone and salmon calcitonin in osteopenic postmenopausal women. J Clin Endocrinol Metab 1997;82:1111–1117. 63 Van der Lely AJ, Lamberts SW, Jauch KW, Swierstra BA, Hertlein H, Danielle De Vries D, Birkett MA, Bates PC, Blum WF, Attanasio AF: Use of human GH in elderly patients with accidental hip fracture. Eur J Endocrinol 2000; 143:585–592. 64 Ebeling PR, Jones JD, O’Fallon WM, Janes CH, Riggs BL: Short-term effects of recombinant human insulin-like growth factor I on bone turnover in normal women. J Clin Endocrinol Metab 1993;77:1384–1387. 65 Ghiron LJ, Thompson JL, Holloway L, Hintz RL, Butterfield GE, Hoffman AR, Marcus R: Effects of recombinant insulin-like growth factor I and growth hormone on bone turnover in elderly women. J Bone Miner Res 1995;10: 1844–1852. 66 Johansson AG, Lindh E, Ljunghall S: Insulinlike growth factor I stimulates bone turnover in osteoporosis. Lancet 1992;339:1619. 67 Boonen S, Rosen C, Bouillon R, Sommer A, McKay M, Rosen D, Adams S, Vanderschueren D, Broos P, Lenaerts J, Raus J, Geusens P: Musculoskeletal effects of the recombinant human insulin-like growth factor-I (rhIGF-I)/IGF binding protein-3 (IGFBP-3) complex in osteoporotic patients with proximal femoral fracture. A double-blind placebo-controlled pilot study. J Clin Endocr Metab 2002;87:1593– 1599. 68 Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH: Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 2001;344:1434–1441.

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Auxological, Ophthalmological, Neurological and MRI Findings in 25 Austrian Patients with Septo-Optic Dysplasia (SOD) Preliminary Data

S.W. Riedl a A. Müllner-Eidenböck b D. Prayer c G. Bernert a H. Frisch a Departments of a Pediatrics, b Ophthalmology and c Radiology, University of Vienna, Austria

Key Words Septo-optic dysplasia W Pituitary insufficiency W Optic nerve hypoplasia W Brain midline anomalies

Abstract Septo-optic dysplasia (SOD) comprises ophthalmological, endocrinological and neurological disorders resulting from varying degrees of midline malformation of the forebrain like visual impairment by optic nerve hypoplasia, endocrine deficits due to hypothalamic and/or pituitary anomalies, and psychomental retardation by associated cortical malformation. MRI shows aplasia/hypoplasia of the septum pellucidum and corpus callosum as a radiological hallmark. For etiology, genetic defects (Hesx1/HESX1 gene) as well as vascular disruption during embryonic brain development are discussed. Aim: To perform detailed analysis of morphological findings and clinical symptoms and to improve care of SOD patients by interdisciplinary management. Patients: We investigated 25 patients with a mean age of 5.1 years at diagnosis. Results: Pituitary insufficiency was present in 11/25 patients, multiple deficits in 6 of them. Bilateral optic nerve hypoplasia was found in 70% of patients, unilateral in 20%. Mild or moderate neurological disorders

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were observed in the majority of patients (14/20), EEG was usually normal (12/19). Analysis of MRI films revealed very heterogeneous morphological anomalies, ranging from isolated agenesis of the septum pellucidum to multiple malformations, involving the cortex. Malrotation of the hippocampal structures was a common finding. Conclusion: We conclude that only interdisciplinary management of SOD patients can ameliorate the exact diagnosis and outcome, depending on early visual or developmental support as well as early diagnosis and substitution of potentially life-threatening endocrine deficits. Copyright © 2002 S. Karger AG, Basel

Introduction

Congenital midline malformations of the forebrain comprise different syndromes with distinct clinical symptoms. The triad of agenesis of the septum pellucidum, corpus callosum and hypoplasia of the optic nerve was first described by DeMorsier [1] in 1956 which consecutively led to the term ‘DeMosier syndrome’. In 1970, Hoyt et al. [2] found an additional association with pituitary insufficiency. MRI techniques allowed more detailed morpho-

H. Frisch, MD Pediatric Department University of Vienna, Währinger Gürtel 18–20 A–1090 Vienna (Austria) Tel. +43 1 40400 3218, Fax +43 1 40400 7683, E-Mail [email protected]

logical analysis of involved structures, leading to a respective classification system [3]. In 1998, Dattani et al. [4] found a mutation of the homeobox gene Hesx1/HESX1 in the mouse model with forebrain anomalies that resemble septo-optic dysplasia (SOD) in man. Screening in SOD patients has revealed only 2 siblings with homozyguos mutations so far and 9 cases of heterozyguos mutations with milder phenotypes [5]. On the other hand, embryological brain damage by vascular disruption may also explain some morphological anomalies and associated clinical symptoms [6]. As SOD results in potentially multiple symptoms concerning endocrinological, ophthalmological and neurological or psychomental disorders, clinical diagnosis and follow-up depends on cooperation of various pediatric subspecialties. Our aim was to work up all patients of Eastern Austria, suspected and diagnosed by different specialists, in an interdisciplinary approach.

Table 1. Anamnestic data (n = 25)

Maternal age ! 20 years Drugs in early pregnancy (medications) Pregnancy complications Birth complications (cesarean section) Associated malformations (cardiac)

5 8 (4) 10 8 (8) 12 (4)

Table 2. Hormonal deficits in 11 patients with SOD (total n = 11/25)

GH TSH ACTH FSH/LH AVP

9 5 4 1 4

Patients and Methods Twenty-five patients (14 f) were enrolled in the study according to MRI and/or ophthalmological findings plus endocrinological and/or neurological symptoms. Mean age at diagnosis was 5.1 years, ranging from 0 to 23.7. Careful patient history was collected with regard to possible predictive factors like maternal age and drugs during early pregnancy. Birth complications and associated malformations were recorded. Auxological parameters were investigated using a Harpenden stadiometer. Baseline hormonal investigations were done at the time of diagnosis and at 3- to 12-monthly intervals, follow-up investigations and dynamic tests being performed according to clinical symptoms and age of the patient. Samples were analyzed using commercial kits. For ophthalmological investigation, visual acuity, field changes and motility disturbances were evaluated with agerelated tests. Indirect funduscopy was performed to detect morphological abnormalities of the optic nerve head and retinal vessels. Early visual support was given to all children with delayed visual maturation. MRI was done using 1.5-T superconduction systems. T1- and T2-weighted series were performed in three section planes. For neurological assessment, EEG and VEP beside physical neurological examination were evaluated.

Results

Anamnestic Data Results are given in table 1. There was no striking young maternal age whereas drugs or medications and pregnancy and birth complications were common. Nearly half of the patients had associated malformations, one third had congenital heart disease.

Interdisciplinary Management in SOD

Auxological Data Nine of the 25 patients had a height ^2 SD at the time of diagnosis. Target height was slightly decreased (–0.36 B 0.44 SDS). Correspondingly, length at birth was –0.34 B 1.03 SDS. Endocrinological Findings Nearly half of the patients had variable pituitary insufficiencies (table 2). Multiple deficiencies (n = 6) comprised ACTH and/or AVP insufficiency strikingly often. Gradually developing pituitary insufficiency occurred in 5 patients. Elevated prolactin levels were found in 9 patients. Only 1 patient had gonadotrophin deficiency. Ophthalmological Findings Bilateral optic nerve hypoplasia (ONH) was found in 76.6%, unilateral ONH in 16.6% of the patients, whereas 6.6% had no fundus abnormalities. Of the cases with bilateral ONH, 30% had normal visual development and 53% had severe delayed visual development, whereas 17% were amaurotic. Patients with bilateral ONH usually presented for the first time during the first 6 months of life because of delayed visual maturation and nystagmus, respectively. Children with unilateral ONH were often presented for the first time during pre-school age because of strabismus [7, 8].

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Table 3. Neurological findings

Normal Slight abnormalities Moderate abnormalities Severe abnormalities

Neurostatus EEG (20/25) (19/25)

VEP (14/25)

2 6 8 4

6 2 3 3

12 4 1 2

Table 4. Early symptoms that lead to diagnosis of SOD (total n = 25)

Ophthalmology Endocrinology Neurology Radiology Family history

12 7 3 2 1

Neurological Investigations Data are shown in table 3. Slight or moderate neurological abnormalities were seen in the majority of patients (14/20) whereas EEG was usually normal (12/19). VEP findings correlated to the extent of visual defects. MRI Findings Detailed analysis was partly limited by MRI quality. The septum pellucidum was absent in 12/16 cases analyzed so far, in 12/16 patients other anomalies of midline structures (corpus callosum, fornix and associated structures) were observed. Anomalies of the pituitary/hypothalamic system occurred in 10/16 patients, hemispheric lesions (mainly schizencephalic clefts) or infratentorial anomalies were seen in 10/16, pathological formation of the hippocampus in 7/15 cases. First Suspicion of SOD Data on the early symptoms that lead to diagnosis of SOD are shown in table 4.

In our series, ophthalmological symptoms were most commonly suspected to lead to the diagnosis of SOD, especially in early infancy (12/25, see table 4), followed by endocrinological symptoms, predominantly growth disorders. 11 of 25 patients had pituitary deficits, 6 of them had multiple deficiencies with relatively high incidence of ACTH and/or AVP deficiency (4/11 and 4/11, respectively). One patient with additional hypertrophic cardiomyopathia died shortly after diagnosis of TSH and ACTH deficiency before substitution could be started. Brodsky et al. [12] described 5 cases of sudden death in early childhood probably due to ACTH or AVP deficiency and pointed at careful endocrinological work-up when SOD is suspected. All but 1 patient in whom it was tested showed normal gonadotropic function which may be attributed to later migration of GnRH cells to hypothalamic nuclei when the midline defect has already occurred [13]. Ophthalmological findings included classical ONH in most cases, confirmed by radiological evidence for decreased optic nerve and chiasma width with pathological enhancement of nerve structures. Slight or moderate neurological symptoms were seen in the majority of patients (14/20), which could be correlated to schizencephalic clefts, cortical polymicrogyria or pathological formation of the hippocampus. In terms of etiology, we found a relatively high frequency of drug or medication ingestion in early pregnancy, a known association with SOD etiology due to vasoactive effects [14], and pregnancy and birth complications, probably secondary to endocrine deficits or associated malformations. Hesx1/HESX1 gene mutations were excluded in 8 patients investigated so far. Diagnosis of such a heterogeneous entity like SOD requires definite criteria and classification in order to establish the diagnosis and not to miss symptoms that may be harmful for the individual patient. Our future aim is to put forward a diagnostic score which should be helpful for diagnosis and clinical classification of SOD. However, our number of patients is still too small to elaborate reliable criteria. International cooperation of five Central European countries is under way to overcome this restriction.

Discussion

SOD patients require early interdisciplinary assessment. Various studies have tried to illustrate the heterogeneous phenotype and sharpen diagnostic criteria [9–11].

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References 1 DeMorsier: Etudes sur les dystrophies cranioencéphaliques. III. Agénésie du septum pellucidum avec malformation du tractus optique: La dysplasie septo-optique. Schweiz Arch Neurol Neurochir Psychiatr 1956;77:267–292. 2 Hoyt WF, Kaplan SL, Grumbach MM, Glaser JS: Septo-optic dysplasia and pituitary dwarfism. Lancet 1970;i:893–894. 3 Barkovich AJ, Kuzniecky RI, Dobyns WB, Jackson GD, Becker LE, Evrad P: A classification scheme for malformations of cortical development. Neuropediatrics 1996;27:59–63. 4 Dattani MT, Martinez-Barbera JP, Thomas PQ, Brickman JM, Gupta R, et al: Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse. Nat Genet 1998;19:125–133. 5 Thomas PQ, Dattani MT, Brickman JM, McNay D, Warne G, et al: Heterozygous HESX1 mutations associated with isolated congenital pituitary hypoplasia and septo-optic dysplasia. Hum Mol Genet 2001;10:39–45.

Interdisciplinary Management in SOD

6 Lubinsky MS: Hypothesis: Septo-optic dysplasia is a vascular disruption sequence. Am J Med Genet 1997;69:235–236. 7 Hellström A: Optic nerve morphology may reveal adverse events during prenatal and perinatal life-digital image analysis. Surv Ophthalmol 1999;44(suppl 1):63–73. 8 Lambert S, Hoyt C, Narahara M: Optic nerve hypoplasia. Surv Ophthalmol 1987;32:1–9. 9 Arslanian SA, Rothfus WE, Foley TP, Becker DJ: Hormonal, metabolic and neuroradiologic abnormalities associated with septo-optic dysplasia. Acta Endocrinol (Copenh) 1984;107: 282–288. 10 Hellström A, Chatelain P, Cutfield WS, Joensson P, Price DA, et al: Septo-optic dysplasia: Experience with 151 cases in KIGS; in Ranke MB, Wilton P (eds): Growth Hormone Therapy in KIGS. 10 Years’ Experience. Heidelberg/ Leipzig, J.A. Barth, 1999, pp 147–157.

11 Hellström A, Aronsson M, Axelson C, Kyllermann M, Kopp S, et al: Children with septooptic dysplasia – How to improve and sharpen the diagnosis. Horm Res 2000;53(suppl 1):19– 25. 12 Brodsky MC, Conte FA, Taylor D, Hoyt CS, Mrak RE: Sudden death in septo-optic dysplasia. Report of 5 cases. Arch Ophthalmol 1997; 115:66–70. 13 Nanduri VR, Stanhope R: Why is the retention of gonadotrophin secretion common in children with panhypopituitarism due to septooptic dysplasia? Eur J Endocrinol 1999;140: 48–50. 14 Dominguez R, Aguirre-Vila-Coro A, Slopis JM, Bohan TP: Brain and ocular abnormalities in infants with in utero exposure to cocaine and other street drugs. Am J Dis Child 1991;145: 688–695.

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Psychosocial Adaptation to Short Stature – An Indication for Growth Hormone Therapy? Thorsten Wygold University Children’s Hospital, Medical University of Lübeck, Germany

Key Words Psychosocial adaptation W Psychological problems W Short stature W Growth W Childhood W Adolescence W Growth hormone therapy

being short in patients and peers is not at hand. As a consequence, psychosocial problems due to short stature have not been exactly classified yet and therefore do not represent an indication for growth hormone therapy. Copyright © 2002 S. Karger AG, Basel

Abstract Although growth hormone does not clearly improve final height in non-growth-hormone-deficient children with short stature, it leads to a temporary acceleration of growth velocity. It is an ongoing discussion whether this effect supports psychosocial adaptation to short stature and therefore could be an indication for growth hormone treatment in children with short stature without growth hormone deficiency. We have reviewed recent literature concerning psychosocial consequences of short stature. Together with own data we can demonstrate that short people regularly adapt well to their height and have a good self-esteem. On the other hand, we focus on the problem that most studies on this subject suffer from methodical problems. A growth-related questionnaire that evaluates subjective and objective perceptions of This article is dedicated to Prof. Dr. J.H. Brämswig, Münster, Germany, on the occasion of his 60th birthday.

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Introduction

When recombinant growth hormone became available in greater amounts, several studies were initialized to extend the indication for growth hormone therapy to normal short children without growth hormone deficiency. The results do not clearly demonstrate that final height of these patients can be improved. On the other hand, it was observed that therapy with growth hormone causes a temporary acceleration of growth velocity in these children [overview in 1]. This led to the discussion whether the effect of accelerated growth velocity could be helpful in stabilizing psychosocial problems due to short stature [2–4]. The debate is still open if normal short children benefit from auxological improvement under growth hormone treatment concerning psychosocial adaptation to their restricted height.

Thorsten Wygold, MD University Children’s Hospital Medical University of Lübeck, Ratzeburger Allee 160 D–23538 Lübeck (Germany) Tel. +49 451 5002959, Fax +49 451 5002590, E-Mail [email protected]

Fig. 1. Model of psychosocial adaptation of

chronically ill children (Steinhausen [12], translated from German by the author).

From the psychological point of view, short stature is a visibly physical conspicuousness leading to stigmatization. Under this condition, children with short stature can be compared to chronically ill children that are often likewise stigmatized. Steinhausen [12] created a model for the psychosocial adaptation of chronically ill children that, in our opinion, is also valid for children with striking height (fig. 1). According to this model, four levels influence the psychosocial adaptation of affected children and medical

therapy, like growth hormone treatment, is just a minor part of it. It is obvious that in studies concerning the psychosocial adaptation of physical stigmata the affected children have to be evaluated as well as their parents and peers. This leads to another problem imposing difficulties upon interpreting psychosocial studies in stigmatized children: the existing difference in self-reported and observed psychosocial functioning. It is a result of former own studies that adolescents with physical stigmata (chronic illness, short stature, respectively) have as good or better scores compared to a healthy reference group [13–15] when answering a questionnaire concerning their self-esteem (Frankfurter Selbstkonzeptskalen, FSKN, [16]). On first view, this result seems to be striking. On the other hand, to those working on the field of coping strategies these results are well known from chronically ill people. Two models of explanation are discussed: (1) The self-esteem of these affected people is idealized and unrealistic. The answers represent wishful thinking. (2) The self-esteem is realistic. To accomplish problems of daily living is sometimes more difficult for these affected persons. Then, successful coping has an individual importance. In this way they might differ from healthy people concerning their selfassessment. With respect to the above mentioned it is not surprising that newer studies in which affected children were

Psychosocial Adaptation to Short Stature

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Do Children with Short Stature Have Any Growth-Related Psychosocial Risks?

Early studies reported severe psychosocial malfunction in children with short stature, e.g. low self-esteem [5, 6], behavioral problems [5, 7], infantilism and social immaturity [5, 8]. Short adolescents were considered to be isolated from peer groups [9, 10]. From the methodical point of view, most of these studies are doubtful. In some studies, self-constructed, non-standardized questionnaires were used, in others examined patients were heterogeneous in age and underlying growth disturbance, respectively. Another problem is that evaluation of possible psychosocial risk factors often depended on reported observations of parents or peers [11].

The Psychosocial Role of Being Short

21

evaluated did not find any psychological disturbance that was related to short stature [17–19].

mone therapy to be a failure relative to their expectations’ [25]. These expectations additionally influence the answers to psychological interviews and questionnaires.

Do Short People Suffer from Their Height? Conclusion

Recent studies could not find any relationship between short stature and the amount of psychosocial risk factors [17–19]. However, there is a major methodical problem remaining that is not solved yet and qualifies these results. Those children that were evaluated are a selected group. In most studies the patient group consists of children who were referred to the hospital/the outpatient department because of their striking height. By definition, 3% of an age group are short. It is not only my personal experience that pediatric endocrinologists just see a minority of these 3% of short children living in the region that is provided for by their pediatric endocrinology unit. Therefore, evaluated children are usually not representative for all short children of this region. We do not know if the majority of short children is affected by their height in any way. It remains unclear if they suffer from their short stature. If so, what are their coping strategies that ‘prevent’ them from being referred to a pediatric endocrinology unit? To my knowledge only a few study groups have paid respect to this problem until now [17, 20–22]. Considering this, we have to respect that those children with short stature that are patients of an endocrinology unit have a desire to change and expect advice and help from their doctors. However, these expectations influence their answers when evaluated by psychological questionnaires and interviews, respectively. These studies have to handle a bias. This bias becomes more obvious in studies dealing with the effect of growth hormone therapy on psychosocial behavior. Studies are not placebo-controlled and double-blinded and there is no untreated control group [3–5, 23]. So we have to doubt if the reported benefits on psychosocial assessment are due to the effects of growth hormone or just to the effect that suffering short children experience medical treatment and feel better because ‘something happens’. Some authors point out the problem of unrealistic expectations in perceived patient height without [24] or during growth hormone treatment [25]. Although children and their parents have received proper medical information concerning growth and growth hormone therapy, their expectations in the success of this therapy are often unrealistic (too optimistic). ‘In spite of accelerated growth, children and parents perceived growth hor-

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Medical and probably economical interests have focused on the broadening of the indication for growth hormone therapy towards psychosocial assessment. As far as results of studies dealing with this complex can be evaluated, no relationship between psychosocial risk factors and short stature has been found. However, the main problem of these studies is a methodical one. In some studies, affected children are not the primary object of evaluation and in those where they are, these patients represent a selected group. Most of the manuals used are limited and therefore not valid for the evaluation of psychological problems due to short stature. Yet we can neither measure nor distinguish between desire to change of affected children and their relatives or peers. Finally, we do not know much about the subjective needs and fears in daily life of short children with respect to their striking height and we certainly do not know if growth hormone therapy is the best of all possible solutions for them. Coping research is difficult to perform. It requires specific subjective and objective assessment tools. Haverkamp et al. [26] pointed out that for evaluation of psychological problems due to short stature, an assessment of both psychological and physical risks associated with short stature should be made. Population-based studies have to be performed to additionally evaluate those short children that are not referred to an endocrinology unit. As long as there is no such standardized, growth-specific approach, the question whether children with normal short stature suffer from their height cannot be answered sufficiently and there is no evidence that growth hormone therapy might prove to be helpful to this problem.

Wygold

References 1 Finkelstein BS, Imperiale TF, Speroff T, Marrero U, Radcliffe DJ, Cuttler L: Effect of growth hormone therapy on height in children with idiopathic short stature: A meta-analysis. Arch Pediatr Adolesc Med 2002;156:230–240. 2 Boulton TJC, Dunn SM, Quigley CA, Taylor JJ, Thompson L: Perceptions of self and short stature: Effects of two years of growth hormone treatment. Acta Paediatr Scand 1991;377 (suppl):20–27. 3 Huisman J, Slijper FME, Sinnema G, Akkerhuis GW, Brugman-Boezeman A, Feenstra J, den Hartog L, Heufel F: Psychosocial effects of two years of human growth hormone treatment in Turner syndrome. Horm Res 1993;39(suppl 2):56–59. 4 Rovet J, Holland J: Psychological aspects of the Canadian randomized controlled trial of human growth hormone and low-dose ethinyl estradiol in children with Turner syndrome. Horm Res 1993;39(suppl 2):60–64. 5 Kusalic M, Fortin C, Gauthier Y: Psychodynamic aspects of dwarfism. Can Psychiatr Assoc J 1972;17:29–34. 6 Rieser PA: Educational, psychologic and social aspects of short stature. J Pediatr Health Care 1992;6:325–332. 7 Gordon M, Crouthamel C, Post EM, Richard RA: Psychosocial aspects of constitutional short stature: Social competence, behaviour problems, self-esteem and family functioning. J Pediatr 1982;101:477–480. 8 Skuse D: The psychological consequences of being small. J Child Psychol Psychiatry 1987;8: 641–650. 9 Money J, Pollit E: Studies in the psychology of dwarfism. II. Personality maturation and response to growth hormone treatment in hypopituitary dwarfs. J Pediatr 1966;68:381–390.

Psychosocial Adaptation to Short Stature

10 Parker JG, Asher SR: Peer relations and later personal adjustments: Are low-accepted children at risk? Psychol Bull 1987;102:357–389. 11 Holmes CS, Thompson RG, Hayford JT: Parent’s and teacher’s differing view of short children’s behaviour. Child Care Health Dev 1982; 8:3. 12 Steinhausen HC: Aspekte der psychosozialen Adaptation bei chronisch kranken Kindern. Z Pers Psychol Psychother 1987;6:225–232. 13 Wygold T, Schmitt GM: Das positive Selbstwerterleben chronisch kranker junger Menschen mit Morbus Crohn: Realistische Selbsteinschaetzung oder Ausdruck von Abwehrverhalten und Normalitaetsdruck? In Braehler E, Schuhmacher J (eds): Psychologie und Soziologie in der Medizin. Giessen, PsychosozialVerlag, 1996. 14 Wygold T, Schmitt GM, Braemswig JH: The self-concept of short and tall children – A comparison between children and parents. Horm Res 1996;46(suppl 2):54. 15 Schmitt GM: Cystische Fibrose. Leben mit einer chronischen Krankheit. Göttingen, Hogrefe, 1991. 16 Deusinger IM: Die Frankfurter Selbstkonzeptskalen (FSKN). Handanweisung. Göttingen, Hogrefe, 1986. 17 Kranzler JH, Rosenbloom AL, Proctor B, Diamond FB, Watson M: Is short stature a handicap? A comparison of the psychosocial functioning of referred and nonreferred children with normal short stature and children with normal stature. J Pediatr 2000;136:96–102. 18 Gilmour J, Skuse D: Short stature – The role of intelligence in psychosocial adjustment. Arch Dis Child 1996;75:25–31.

19 Sandberg DE, Brook AE, Campos SP: Short stature: A psychosocial burden requiring growth hormone therapy? Pediatrics 1994;94: 832–840. 20 Voss LD, Mulligan J: The short normal child in school: Self-esteem, behavior and attainment before puberty (The Wessex Growth Study); in Stabler B, Underwood LE (eds): Growth, Stature and Adaptation. Chapel Hill, University of North Carolina, 1994. 21 Voss LD, Bailey BJR, Mulligan J, Wilkin TJ, Betts PR: Short stature and school performance: The Wessex Growth Study. Acta Paediatr Scand 1991;377(suppl):29–31. 22 Downie AB, Mulligan J, Stratford RJ, Betts PR, Voss LD: Are short normal children at disadvantage? The Wessex Growth Study. BMJ 1997;314:97–100. 23 Stabler B, Siegel PT, Clopper RR, Stoppani CE, Compton PG, Underwood LE: Behavior change after growth hormone treatment of children with short stature. J Pediatr 1998;133: 366–373. 24 Hunt L, Hazen RA, Sandberg DE: Perceived versus measured height. Which is the stronger predictor of psychosocial functioning? Horm Res 200;53:129–38. 25 Rotnem D, Cohen DJ, Hintz R, Genel M: Psychological sequelae of relative ‘treatment failure’ for children receiving human growth hormone replacement. J Am Acad Child Psychiatry 1979;18:505–520. 26 Haverkamp F, Eiholzer U, Ranke MB, Noeker M: Symptomatic versus substitution growth hormone therapy in short children: From auxology towards a comprehensive multidimensional assessment of short stature and related interventions. J Pediatr Endocrinol Metab 2000;13:403–408.

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GH and Kidney Horm Res 2002;58(suppl 3):30–34 DOI: 10.1159/000066486

Risk of Mortality in Patients with End-Stage Renal Disease: The Role of Malnutrition and Possible Therapeutic Implications M. Zeier Department of Medicine, Division of Nephrology, University of Heidelberg, Germany

Key Words Dialysis W End-stage renal disease W Malnutrition W Mortality W Growth hormone

Abstract The mortality rate of dialysis patients is still considerably high. Beside the traditional risk factors, specific uremiarelated risk factors are identified. Among them, hypoalbuminemia and malnutrition have a strong association to mortality in chronic dialysis patients. Various studies document a strong relation between reduced calorie and protein uptake and mortality in uremic patients. Several factors responsible for malnutrition in dialysis patients have been identified. These factors may be dialysis-associated, due to intercurrent illnesses or are associated with uremic complications (e.g. hyperparathyroidism, anemia, acidosis, etc.). Malnutrition is treatable and can be avoided by several means. Beside the increase in the dose of dialysis and adequate protein and calorie intake, intradialytic nutrition is an additional choice. The combination with specific drugs (e.g. growth hormone) may potentiate the success of the modified treatment modalities, particularly in patients who need nutritional support during an intercurrent illness. Further studies are required to measure the impact of for example growth hormone supplementation on mortality rate and quality of life in malnourished patients on chronic dialysis. Copyright © 2002 S. Karger AG, Basel

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Magnitude of the Problem

Despite several technical advances in medical care and dialysis, morbidity and mortality remain high in patients on chronic renal replacement therapy (e.g. hemodialysis, peritoneal dialysis, renal transplantation). The 5-year survival rate of patients aged above 64 years is worse than that of patients affected with malignancies [1]. The ageadjusted death rate of dialysis patients is estimated to be 4–5 times higher than that of the general population [2]. Cardiovascular disease is the leading cause of morbidity and mortality in dialysis patients, accounting for about 50% of deaths and 30% of hospitalizations in most registries [3], and this is shown in table 1. Even after the successful transplantation of a renal allograft, mortality still remains high. After first year post-transplantation the rate of death with functioning graft (49%) is higher than allograft loss due to rejection (41%) [4] as supported by data from the UNOS registry [5].

Table 1. Cardiovascular risk in end-stage renal disease according to

age Decade of age years

Mortality risk in comparison to the general population

25–34 55–64

10! 4!

Martin Zeier, MD Department of Medicine, Division of Nephrology University of Heidelberg, Bergheimerstrasse 56a D–69115 Heidelberg (Germany) Tel. +49 6221 91120, Fax +49 6221 162476, E-Mail [email protected]

Mortality Risk Factors in Patients with End-Stage Renal Disease

Patients undergoing treatment for end-stage renal failure have several risk factors for morbidity and mortality. For the purposes of an overview it is useful to differentiate between traditional and uremia-related risk factors (for details see also table 2). Traditional risk factors merge with uremia-related risk factors such as dyslipidemia and increased lipoprotein(a), prothrombotic factors, hyperhomocystinemia, increased oxidant stress, hypoalbuminemia, inflammation and abnormalities in the calcium and phosphate metabolism including secondary hyperparathyroidism [6].

Protein and Calorie Malnutrition in Dialysis Patients and the Impact on Mortality

What is the extent of protein and calorie malnutrition in dialysis patients? Protein-calorie malnutrition in chronic hemodialysis patients is estimated to occur in at least 12–40% of patients [8–12]. Various surveys among chronic hemodialysis or peritoneal dialysis patients have defined prevalence rates of 25–50% [13]. Increased mortality has been related to protein and calorie malnutrition in patients receiving chronic hemodialysis [7]. Various other studies have shown a strong a association between reduced protein and calorie uptake in patients on chronic dialysis and increased mortality, although a causal relation was not definitely established [8–16]. For instance, a low protein catabolic rate (PCR), a surrogate marker for low protein intake in stable chronic hemodialysis patients, was found to be strongly associated with the probability of treatment failure in the NCDS (National Cooperative Dialysis Study) [17]. In another study it was pointed out that patients with a PCR of !0.65 g/kg/day had a duration of hospitalization that was 3.5 that of patients with a PCR of 11.2 g/kg/day as well as a substantially higher mortality rate [18]. In a longitudinal study from Finland, Oksa et al. [19] found that 17% of the patients on chronic hemodialysis had protein malnutrition by various criteria, and in a 3-year follow-up study, all of the malnourished patients had died, predominantly from sepsis. More recently a strong correlation between mortality and nutritional parameters was demonstrated in chronic hemodialysis patients [8, 15]. A low serum albumin was the most potent predictor of mortality and an independent risk for death. Serum albumin concentrations between 3.5 and 4.0 g/dl increased the relative risk

Malnutrition and Mortality in Dialysis

Table 2. Traditional and uremia-related risk factors for mortality in

patients with end-stage renal disease (modified according to table 1 [6]) Traditional

Uremia-related

Hypertension Dyslipidemia Diabetes mellitus Left ventricular hypertrophy Smoking Physical inactivity

Dyslipidemia and lipoprotein(a) Prothrombotic factors Hyperhomocysteinemia Oxidative stress Hypoalbuminemia and malnutrition Inflammation Disturbances of serum calcium and serum phosphate Hyperparathyroidism

of death twofold, and albumin concentrations between 3.0 and 3.5 increased the mortality risk by a factor of 5, as compared to the risk of death for the reference group with serum albumin concentrations between 4.0 and 4.5 g/dl [15]. US Renal Data Service (USRDS) data suggest that serum albumin is an independent indicator of subsequent mortality [20]. The specific causes of increased morbidity and mortality in patients on hemodialysis with malnutrition include an increased incidence of cardiovascular disease [12], derangements of the immune function and sepsis [14, 15].

Indices of Malnutrition in Chronically Dialyzed Patients

There are several indices of malnutrition available. Anthropometric measurements (e.g. skinfold thickness, mid-arm muscle circumference) are not sensitive indicators for detecting early malnutrition, and the relationship of these parameters to patient mortality is well documented [21]. Some indices which are relatively easily accessible are summarized in table 3. The most convincing link between malnutrition and mortality is provided by albumin concentration, a routinely measured parameter [15]. Because of its relatively long half-life (20 days) and the vast capacity of the liver to synthesize albumin, a decrease in albumin concentration may follow the onset of malnutrition by several months [22]. Transferrin concentrations appears to be a sensitive and early index of malnutrition. However, its fluctuation with recombinant human erythropoietin, iron administration, and malnutrition, has not been well studied [23]. Insulin-like growth factor 1 (IGF-1) has also been shown to be low in cases of

Horm Res 2002;58(suppl 3):30–34

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Table 3. Indices of malnutrition in hemodialysis patients (according to table 1 [16])

Serum albumin ! 4.0 g/dl Cholesterol concentration ! 150 mg/dl (without statins) Transferrin concentration ! 200 mg/dl Body weight ! 80% of ideal weight Marked reduction in anthropometric measurements Low serum creatinine and urea concentrations in patients without residual renal function IGF-1 concentrations ! 300 Ìg/l PCR (protein catabolic rate) ! 0.8 g/kg/day Low predialysis serum potassium (and possibly serum phosphorus) Prealbumin concentration ! 29 mg/dl

Table 4. Factors affecting the nutritional status of dialysis patients

Dialysis associated Inadequate dose of dialysis Short duration of dialysis sessions Inadequate blood flow to the vascular access site Bioincompatible membranes Loss of amino acids and glucose in dialysate Intercurrent illnesses Infection (e.g. dental root, sinus, colonic diverticulitis, cholecystitis) Malabsorption and gastroparesis Gastritis and esophagitis Underlying disease (e.g. malignancy, chronic heart failure, chronic pulmonary disease) Depression Recurrent hospitalizations for various reasons Biochemical factors Acidosis Hyperparathyroidism Anemia Low IGF-1 Insulin resistance, increased gluconeogensis and decreased glycogen stores

malnutrition in dialysis patients, with a level of !300 Ìg/l indicating severe malnutrition [24]. It may be more specific, since it reflects the anabolic activity of growth hormone (GH) and is less subject to diurnal fluctuation. Predialytic serum-urea and serum-creatinine concentrations in chronic hemodialysis patients give additional information about the current nutritional intake. In stable dialysis patients, dietary protein intake correlates with the PCR which is calculated from the net urea appearance interdialytically [25]. PCR values need to be 11.0 g/kg/ day of protein and preferably as high as 1.3–1.4 g/kg/day

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to maintain lean body mass (LBM). Finally, body composition (LBM, fat content and total body water) can be measured serially in a chronic dialysis patient. In summary, several indices of malnutrition can be used to gauge the nutritional state of hemodialysis patients.

Factors Affecting the Nutritional Status in Dialyzed Patients

Several factors related to the uremic state may contribute to the prevalence of protein-energy malnutrition in chronic renal patients. They are listed in table 4. To provide an overview they are subdivided into dialysis-related issues, intercurrent illnesses and biochemical factors. Anorexia is present even before the initiation of hemodialysis with decreased appetite, a negative nitrogen balance and muscle wasting [27, 28], and anorexia becomes even more pronounced as renal failure progresses [29]. In addition, dialysis patients, in particular the elderly, suffer from additional diseases (e.g. chronic heart failure, chronic lung disease, malignancy). Patients with endstage renal disease are prone to occult infections (e.g. dental root, sinus, colonic diverticula) which often are clinically inapparent. Regular screening for underlying infections is mandatory in the anorectic chronic dialysis patient. In the past it has been shown that the dose of dialysis corresponds with the survival of chronic hemodialyzed patients [30]. The dose of dialysis is dependent on several factors (e.g. duration of dialysis, number of dialysis sessions per week, vascular access, dialysis modalities). Lindsay et al. [31] published a prospective study which showed that PCR increases, with an increasing dose of dialysis, up to a mean Kt/V (measurement of adequacy of dialysis, without going into further details) of 1.32. In the past 10 years several studies have supported these results.

Interventions to Treat Malnutrition in Dialyzed Patients

Several means of improving malnutrition in chronic dialysis patients are available to the nephrologist; these are listed in table 5. They comprise dialytic, nutritional and drug interventions. Anorexia is part of the syndrome of uremia and has been observed in patients receiving inadequate doses of hemodialysis. Since studies show a strong relationship between dialysis and mortality [32] and between mortality and malnutrition [18], it is safe to

Zeier

assume that increased doses of dialysis are helpful in reducing anorexia. The second important part is adequate enteral nutrition in dialysis patients, which is often difficult to achieve, particularly when the patients were on low-protein diets in their predialysis course. The specific protein and calorie intake is detailed in table 5 [33]. Concerted action by physician, dietitian and family members is required to achieve adequate enteral nutrition in the chronic dialysis patient. Intradialytic approaches to improve calorie and protein malnutrition have been tried for a long time. Supplementation of the dialysate with glucose or amino acids [34, 35] is well known. Another effective method of supplementation is intradialytic nutrition. Depending on the formulation, approximately 1,000 calories and 0.7 g/kg body weight of protein can be delivered safely during each hemodialysis session, without the risk of fluid overload and hyperkalemia [36, 37]. There are several drug interventions possible. In brief, erythropoietin (EPO) has been shown to have an impact on protein nutrition as documented by urea kinetic dialysis [38]. In another study, weight gain was observed in 16 of 25 patients receiving EPO [39]. With the development of recombinant DNA technology, recombinant human GH (rhGH) has become available. GH induces anabolic responses in maintenance hemodialysis patients with protein-energy malnutrition [40–42]. Garibotto et al. [43] documented increased muscle protein synthesis and a reduction in negative muscle protein balance in malnourished hemodialysis patients treated with rhGH. A doubleblind, placebo-controlled study on rhGH treatment in elderly patients over 6 months documented an increase in serum albumin, improved hand-grip strength and an increase in LBM with a corresponding decrease in body

Table 5. Means of improving malnutrition in chronic dialysis pa-

tients Dialytic intervention Increase dose of dialysis Nutritional intervention Adequate protein intake 1.1–1.2 g/kg body weight/day; hemodialysis 1.2–1.3 g/kg body weight/day; peritoneal dialysis Adequate calorie intake 35 kcal/kg body weight/day Intradialytic nutrition Intradialytic nutrition (parenteral nutrition during hemodialysis session) Glucose supplementation of dialysate Amino acid supplementation of dialysate Drug intervention Erythropoietin Anabolic steroids Antinausea medication Growth hormone

fat [44]. Furthermore, Hansen et al. [45] showed the same effect on body composition in a comparable study treating chronic hemodialysis patients with rhGH for 6 months. However, in a separate analysis they found a significant increase in cardiac muscle mass [46]. Summing up: rhGH has an anabolic effect in malnourished, chronic hemodialysis patients effecting changes in body composition and improvement of muscle synthesis. Nevertheless, there is no proof at this point in time that rhGH actually reduces mortality in chronic dialysis patients. The observed side effect of increased cardiac muscle is of importance.

References 1 Foley RN, Parfrey PS, Sarnack MJ: Clinical epidemiology of cardiovascular disease in chronic renal failure. Am J Kidney Dis 1998; 32(suppl 3):S112–S119. 2 US Renal Data System: Annual Data Report. National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Md, 1998. 3 Locatelli F, Marcelli D, Ferrucio C, D’Amico M, DelVecchio L, Limido A, Malberti F, Spotti D for the Registro Lombardo Dialisi e Trapianto: Cardiovascular disease in chronic renal failure: The challenge continues. Nephrol Dial Transplant 2000;15(suppl 5):69–80.

Malnutrition and Mortality in Dialysis

4 Kavanagh D, Morris ST, Northridge DB, Rodger RS, Jardine AG: Electrocardiogram and outcome following renal transplantation. Nephron 1999;81:109–110. 5 Ojo AO, Hanson JA, Wolfe RA, Leichtman AB, Agodoa LY, Port FK: Long-term survival in renal transplant recipients with graft function. Kidney Int 2000;57:307–313. 6 Parfrey PS: Cardiac disease in dialysis patients: Diagnosis, burden of disease, prognosis, risk factors and management. Nephrol Dial Transplant 2000;15(suppl 5):58–68.

7 Owen WF Jr, Lew NL, Liu Y, Lowrie EG, Lazurus JM: The urea reduction ratio and serum albumin concentrations as predictors of mortality in patients undergoing hemodialysis. N Engl J Med 1993;329:1061–1066. 8 Kaminski MV Jr, Lowrie EG, Rosenblatt SG, Haase T: Malnutrition is lethal, diagnosable and treatable in ESRD patients. Transplant Proc 1991;23:1810–1815. 9 Blagg CR: Importance of nutrition in dialysis patients. Am J Kidney Dis 1991;17:458–461. 10 Allman MA, Allen BJ, Stewart PM, Blagojevic N, Tiller DJ, Gaskin KJ, Truswell AS: Body protein of patients undergoing haemodialysis. Eur J Clin Nutr 1990;44:123–131.

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11 Wolfson M, Strong CJ, Minturn RD, Gray DK, Kopple JD: Nutritional status and lymphocyte function in maintenance hemodialysis patients. Am J Clin Nutr 1984;37:547–555. 12 Degoulet P, Legrain M, Reach I, Aimes F, Devries C, Rojas P, Jacobs C: Mortality risk factors in patients treated with hemodialysis. Nephron 1982;31:103–110. 13 Kopple JD: McCollum Award Lecture, 1996: Protein-enery malnutrition in maintenance hemodialysis patients. Am J Clin Nutr 1997;65: 1544–1557. 14 Mattern MD, Hak LJ, Lamanna RW, Teasley KM, Laffell MS: Malnutrition, altered immune function and the risk of infection in maintenance hemodialysis patients. Am J Kidney Dis 1992;1:206–218. 15 Lowrie EG, Lew NL: Death risk in hemodialysis patients: The predictive value of commonly measured variables and an evaluation of death rate differences between facilities. Am J Kidney Dis 1990;15:458–482. 16 Hakim RM, Levin N: Malnutrition in hemodialysis patients. Am J Kidney Dis 1993;21:125– 137. 17 Parker TF, Laird NM, Lowrie EG: Comparison of the study groups in the National Cooperative Dialysis Study and a description of morbidity, mortality and patient withdrawal. Kidney Int 1983;23:199–203. 18 Acchiardo SR, Moore LW, Latour PA: Malnutrition as the main factor in morbidity and mortality of hemodialysis patients. Kidney Int 1983;24(suppl 16):199–203. 19 Oksa H, Ahonen K, Pasternak A, Marnela KM: Malnutrition in hemodialysis patients. Scand J Urol Nephrol 1991;25:157–161. 20 Levin NW, Held P, Port FK, Agodoa L: Comorbid factors are not the reason for hypoalbuminaemia in incident hemodialysis patients. J Am Soc Nephrol 1992;3:376. 21 Blumenkrantz MJ, Kopple JD, Gutman RA, Chan YK, Barbour GL, Roberts C, Shen FH, Gandhi VC, Tucker CT, Curtis FK, Coburn JW: Methods for assessing nutritional status of patients with renal failure. Am J Clin Nutr 1980;33:1567–1585. 22 Bischel M: Albumin turnover in chronically hemodialyzed patients. Trans Am Soc Intern Organs 1969;15:298–304. 23 Ooi BS, Dacrocy AF, Pollak VE: Serum transferrin levels in chronic renal failure. Nephron 1972;9:200–204.

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24 Jakob V, Le Carpentier JE, Salzano S, Naylor V, Wild G, Brown CB, el-Nahas AM: IGF-1, a marker of undernutrition in hemodialysis patients. Am J Clin Nutr 1990;52:39–44. 25 Borah MF, Schoenfeld PY, Gotch FA: Nitrogen balance during intermittent dialysis therapy of uremia. Kidney Int 1978;14:491–500. 26 Depner TA: Standards for dialysis adequacy. Semin Dial 1991;4:245–252. 27 Modification of Diet in Renal Disease (MDRD) Study Group, prepared by Kopple J, Berg R, Houser H, Steinman T, Teschan P: Nutritional status of patients with different levels of chronic renal insufficiency. Kidney Int 1989;36(suppl 27):S184–S194. 28 El-Lakany S, Eagon PK, Gavaler JS, Schade RR, Whiteside T, Van Thiel DH: Gastrointestinal function, morphology and immune status in uremia. Nutrition 1990;6:461–468. 29 Maroni B, Steinman TI, Mitch NE: A method for estimating nitrogen intake of patients with chronic renal failure. Kidney Int 1985;27:58– 61. 30 Charra B, Hurot JM, Chazot C, VoVan C, Jean G, Terrat JC, Vanel T, Ruffet M, Laurent G: Comparison of survival data. Kidney Int 2000; 58:901–902. 31 Lindsay R, Spanner E, Allison M: Which comes first, Kt/V or PCR – chicken or egg? Kidney Int 1992;42(suppl 38):S32–S37. 32 Hakim RM, Depner TA, Parker TF: Adequacy of hemodialysis. Am J Kidney Dis 1992;20: 107–123. 33 Kopple JD: Therapeutic approaches to malnutrition in chronic dialysis patients: The different modalities of nutritional support. Am J Kidney Dis 1999;33:180–185. 34 Kopple JD, Bernard D, Swartz R, Messana J, Bergstrom J, Lindholm B, Lim V, Brunori G, Leiserowitz M, Bier DM, Stegink LD, Martis L, Algrim-Boyle C, Serkes KD, Vonesh E, Jones MR: Treatment of malnourished CAPD patients with amino acid based dialysate. Kidney Int 1995;47:1148–1157. 35 Chazot C, Shahmir E, Matias B, Laidlaw S, Kopple JD: Dialytic nutrition: Provision of amino acids in dialysate during hemodialysis. Kidney Int 1997;52:1663–1670. 36 Snyder S, Bergen C, Sigler MH, Teehan BP: Intradialytic parenteral nutrition in chronic hemodialysis patients. ASAIO Trans 1991;37: M373–M375.

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37 Madigan KM, Olshan A, Yingling DJ: Effectiveness of intradialytic parenteral nutrition in diabetic patients with end-stage renal disease. J Am Diet Assoc 1990;90:861–863. 38 Canaud B, Bouloux C, Rivory JP, Taib J, Garred LJ, Florence P, Mion C: Erythropoietininduced changes in protein nutrition: Quantitative assessment by urea kinetic modelling analysis. Blood Purif 1990;8:301–308. 39 Barany P, Petterson E, Ahlberg M, Hultman E, Bergstrom J: Nutritional assessment in anemic hemodialysis patients treated with recombinant human erythropoietin. Clin Nephrol 1991;35:270–279. 40 Schulman G, Wingard RL, Hutchinson RL: The effects of recombinant human growth hormone and intradialytic parenteral nutrition in malnourished hemodialysis patients. Am J Kidney Dis 1993;21:527–534. 41 Ziegler TR, Lazarus JM, Young LS, Hakim R, Wilmore DW: Effects of recombinant human growth hormone in adults receiving maintenance hemodialysis. J Am Soc Nephrol 1991;2: 1130–1135. 42 Kopple JD: The rationale for the use of growth hormone or insulin-like growth factor I in adult patients with renal failure. Miner Electrolyte Metab 1992;18:269–275. 43 Garibotto G, Barreca A, Russo R, Sofia A, Arathi P, Cesarone A, Malaspina M, Fiorini F, Minuto F, Tizianello A: Effects of recombinant growth hormone in muscle protein turnover in malnourished hemodialysis patients. J Clin Invest 1997;99:97–105. 44 Johannsson G, Bengtsson BA, Ahlmen J: Double-blind, placebo-controlled study of growth hormone treatment in elderly patients undergoing chronic hemodialysis: anabolic effect and functional improvement. Am J Kidney Dis 1999;33:709–717. 45 Hansen TB, Gram J, Jensen PB, Kristiansen JH, Ekelund B, Christiansen JS, Pederson FB: Influence of growth hormone on whole body and regional soft tissue composition in adult patients on hemodialysis. A double-blind, randomized, placebo-controlled study. Clin Nephrol 2000;53:99–107. 46 Jensen PB, Ekelund B, Nielsen FT, Baumbach L, Pedersen FB, Oxhoj H: Changes in cardiac muscle mass and function in hemodialysis patients during growth hormone treatment. Clin Nephrol 2000;53:25–32.

Zeier

Hot Topics Horm Res 2002;58(suppl 3):56–61 DOI: 10.1159/000066487

Growth Hormone Secretagogues and Ghrelin: An Update on Physiology and Clinical Relevance S. Petersenn Division of Endocrinology, Medical Center, University of Essen, Germany

Key Words Ghrelin W Growth hormone W Growth hormone secretagogues W Energy homeostasis W Growth hormone deficiency

Abstract The pulsatile release of growth hormone (GH) by the anterior pituitary is stimulated by small synthetic molecules termed GH secretagogues (GHS). The receptor for GHS (GHS-R) belongs to the family of G-protein-coupled receptors. An endogenous specific ligand of 28 amino acids has recently been purified from rat stomach, it has been termed ‘ghrelin’. Ghrelin demonstrates potent and reproducible GH-releasing activity, as well as significant prolactin-, ACTH- and cortisol-releasing activity. However, its major physiological relevance may relate to energy homeostasis. Peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. In man, intravenous ghrelin was shown to stimulate food intake. The pathophysiological role and the potential clinical use of ghrelin are reviewed. Copyright © 2002 S. Karger AG, Basel

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Introduction

The secretion of growth hormone (GH) by the anterior pituitary is under complex control. Small synthetic molecules termed GH secretagogues (GHS) act on the pituitary and the hypothalamus to stimulate and amplify pulsatile GH release. These compounds appear to mimic a putative endogenous ligand which activates a receptor distinct from that of GH-releasing hormone (GHRH) and somatostatin and whose function is probably critical in the regulation of normal GH secretion. Analogs studied so far include GHRP-6, hexarelin and MK-0677.

Ghrelin and Its Receptor

The GHS receptor (GHS-R) belongs to the family of G-protein-coupled receptors [1]. It is encoded by a single highly conserved gene in the human, chimpanzee, pig, cow, rat and mouse. The gene is found at the chromosomal location 3q26.2, and spans approximately 4.3 kb [2]. Two types of GHS-R cDNAs 1a and 1b were identified from human and pig and from rat. Their sequences do not show significant homology with other receptors known so far, the closest relatives being the neurotensin receptor

Dr. S. Petersenn Division of Endocrinology Medical Center, University of Essen Hufelandstrasse 55, D–45122 Essen (Germany) Tel. +49 201 7232822, Fax +49 201 7235187, E-Mail [email protected]

Fig. 1. Structure of the G-protein-coupled

receptors GHS-R type 1a and 1b, and of the endogenous ligand ghrelin.

and the TRH receptor with 59 and 56% similarity, respectively. The human GHS-R type 1a consists of 366 amino acids with seven transmembrane regions (fig. 1), the molecular weight was calculated to be about 41 kDa [1]. The type 1b consists of 289 amino acids with only five predicted transmembrane regions, the nucleotide sequence of codon 1–265 is identical to type 1a. Beyond that, the type 1b cDNA diverges in its nucleotide sequence and is fused to a short conserved reading frame of 24 amino acids. The mRNA for the human GHS-R type 1a results from a splicing event that removes 2,152 nucleotides of intronic sequence. In the GHS-R type 1b mRNA, the intron is not removed [2]. Type 1a was demonstrated to confer high-affinity, specific binding of GHS and lead to Ca2+ release through stimulation of the G protein subunit G·11. In contrast, type 1b failed to bind GHS and to respond to GHS. The binding affinity of various structurally different GHS to the GHS-R is correlated with the GH-stimulatory effect. The intracellular signaling is thought to involve activation of phospholipase C (fig. 2). Subsequent generation of inositol triphosphate causes an increase of free intracellular Ca2+ by redistribution from intracellular stores. Activated phospholipase C may also lead to tyrosine phosphorylation of a potassium channel resulting in inhibition of that channel. Depolarization of the membrane and activation of L-type voltage-gated calcium channels elicit GH secretion. Expression of the cloned GHS-R was shown in hypothalamus and pituitary, consistent with its role in regulating GH release. Expression was also demonstrated in various other regions of the central nervous system and in the pancreas, possibly indicating its involvement in yet undefined physiological functions. Studies utilizing a photoac-

GHS and Ghrelin: An Update

Fig. 2. Signal transduction of the GHS-R.

tivatable ligand suggest a second distinct GHS-R subtype in pituitary cells with a molecular weight of 57 kDa and a third subtype in heart with a molecular weight of 84 kDa. The cDNAs of these additional subtypes have not been isolated yet. An endogenous specific ligand of 28 amino acids has recently been purified from rat stomach [3], it has been termed ‘ghrelin’ (fig. 1). The Ser3 residue is modified by n-octanoic acid, a modification necessary for hormonal activity. By using antibodies against the octanoyl-modified serine and the C-terminal portion, two major molecular forms were demonstrated in various tissues – ghrelin

Horm Res 2002;58(suppl 3):56–61

57

Fig. 3. GH levels during GHRP-6 test (1 Ìg/ kg), GHRH test (1 Ìg/kg) or GHRP-6 + GHRH test. Results (mean B SEM) are shown for healthy individuals.

itself and the nonmodified des-n-octanyl form, designated as des-acyl ghrelin [4]. Ghrelin-immunoreactive cells are distributed from the neck to the base of the oxyntic gland of the stomach, less frequently in the upper small intestine, and infrequent in the lower small intestine and the large intestine [5]. Ghrelin-immunoreactive neurons were localized in the hypothalamic arcuate nucleus [3]. By radioimmunoassay, significant amounts of ghrelin were found in rat stomach, duodenum, ileum, cecum, aorta, atrium, and pancreas, but not in hypothalamus, pituitary, thyroid, submaxillary gland, thymus, adrenal gland, ventricle, lung, liver, colon, kidney, or testis [4]. Furthermore, ghrelin was detected in human and rat placenta showing a pregnancy-related time course of expression [6], as well as in a medullary thyroid carcinoma cell line [7]. The physiological role of ghrelin in these tissues remains to be determined.

Physiological Effects of GHS and Ghrelin

GHS (fig. 3) demonstrate potent and reproducible GHreleasing activity, release more GH than GHRH, and truly synergies with GHRH. Furthermore, GHS possess significant prolactin-, ACTH- and cortisol-releasing activ-

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ity, without any effects on LH, FSH, or TSH secretion (fig. 4). Evidence that ghrelin has similar effects on pituitary hormones [8] supports the hypothesis that ghrelin is a true natural ligand of the GHS-R. Ghrelin is involved in energy homeostasis. Peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats, whereas intracerebroventricular administration generated a dose-dependent increase in food intake and subsequently body weight [9]. In man, intravenous ghrelin was shown to stimulate food intake. Visual analogue scores for appetite were greater during ghrelin compared to control [10]. Ghrelin also increased feeding in GH-deficient rats indicating that ghrelin modifies energy homeostasis independently of its GH-releasing activity [11]. Inhibition of neuropeptide Y or agouti-related protein abolished ghrelininduced feeding. Therefore, ghrelin may interact with these two peptides to indicate to the hypothalamus when an increase in metabolic efficiency is necessary. Besides its rapid effects on food intake, ghrelin stimulates gastric acid secretion and gastric motility [12]. Ghrelin may also have direct cardiovascular effects. In healthy volunteers, intravenous ghrelin had beneficial hemodynamic effects via reducing cardiac afterload and increasing cardiac output [13].

Petersenn

Fig. 4. Levels of ACTH, cortisol, LH, FSH, TSH and prolactin (PRL) after administration of GHRP-6 (1 Ìg/kg) in

healthy individuals.

Regulation of Ghrelin

Considerable plasma concentrations of ghrelin are found in healthy human and rat blood. The stomach was identified as a major source. Plasma ghrelin-like immunoreactivity levels in totally gastrectomized patients were reduced to 35% of those in normal controls [14]. Fasting ghrelin levels are negatively correlated with percent body fat or body mass indexes and may reflect chronic feeding states. In patients with anorexia nervosa ghrelin levels were markedly elevated compared with those in normal controls [14], weight gain decreases ghrelin concentrations in these subjects [15]. In contrast, decreased ghrelin levels were determined in obese Caucasians compared with lean Caucasians [16], whereas weight loss increases circulating levels of ghrelin in obesity [17]. Therefore, in obesity an ineffective compensatory modulation of ghrelin levels is suggested rather than a causative role. Plasma ghrelin levels may also reflect acute feeding states. Plasma ghrelin-like immunoreactivity in humans were increased after 12 h fasting and reduced immediately after habitual feeding [14]. In another study, a nearly twofold increase in plasma ghrelin levels was observed

immediately before each meal, followed by a fall to trough levels within 1 h after eating [18]. The pattern was reciprocal to that of insulin. The preprandial rise in ghrelin levels points to a role in meal initiation, fitting with the physiological effects of ghrelin on food intake. Secretion of ghrelin is not affected by stomach expansion. In rats, stomach filling with water did not change ghrelin levels, whereas filling with dextrose significantly reduced serum ghrelin levels [9]. Similarly, the mean plasma ghrelin concentrations in normal human subjects decreased after an administration of an oral glucose load, but not after the administration of the same volume of water [19]. A specific effect of ingested nutrients on suppression of ghrelin levels was suggested. However, plasma ghrelin levels also decreased rapidly after intravenous glucose administration. A system in ghrelin-producing cells may respond to plasma glucose concentration. The results indicate that ghrelin is an appetite-stimulatory peptide from the stomach, signaling to the hypothalamus when an increase in energetic demand or efficiency is encountered [20]. Inter-meal ghrelin levels in humans were found to rise progressively throughout the day, peaking at 01:00 h, then to decrease steadily until shortly before breakfast, exactly in phase with leptin levels. The precise interactions be-


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Fig. 5. Role of ghrelin as a link between hypothalamus and stomach.

tween leptin and ghrelin are currently unclear. There are conflicting data from rodent studies that show both positive [21] and negative [22] regulation of ghrelin by leptin. Due to the strong GH-secreting effects of ghrelin, a feedback of GH on ghrelin levels is conceivable. However, the data are inconsistent. In Pepck-hGH transgenic mice, low levels of ghrelin compared to normal animals were found, possibly due to negative feedback on ghrelin from high GH levels [23]. In contrast, ghrelin levels in GH-deficient subjects were not significantly different from controls. Replacement with recombinant GH in these subjects did not modify circulating ghrelin levels [24]. So far, most published studies rely on measurement of total ghrelin. In rat plasma, levels of the inactive desacyl ghrelin were 54 times higher than active ghrelin [4]. Specific assays recognizing active ghrelin only are desirable to understand regulation of ghrelin in more detail.

Clinical Use of GHS and Ghrelin

GHS and ghrelin may offer practical diagnostic and therapeutic value in humans. The diagnosis of GH deficiency is established by provocative testing. We and others have demonstrated that provocative tests using the GHS GHRP-6 alone or in combination with GHRH are convenient, safe and reliable tools for the diagnosis of GH deficiency in adults [25–28]. For treatment of GH-deficient children, GH, GHRH and IGF-1 have been used so far. GHS or ghrelin have been proposed as an alternative, as most GH-deficient children respond to GHS compounds [29]. GHS or ghrelin may be used as an anabolic

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treatment in elderly patients with somatopause, for patients with anorexia nervosa, or for pathological anorexia that can accompany cancer, tuberculosis and AIDS. Oral administration of a GHS (MK-677) stimulated the GH/ IGF-1 axis in healthy elderly subjects [30] and in selected GH-deficient adults [31]. It is currently unclear whether ghrelin antagonism could reduce food intake and be developed as a treatment for obesity. Administration of ghrelin may also be beneficial in patients with chronic heart failure. Intravenous infusion of ghrelin significantly increased cardiac index and stroke volume index in these patients [32].

Pathological Changes of the Ghrelin Gene in Disease

Due to the role of ghrelin for energy balance, the ghrelin gene is a candidate gene for obesity in humans. Recently, three mutations in the preproghrelin/ghrelin gene were identified which are associated with obesity [33]. Ghrelin and preproghrelin sequences were determined in 96 unrelated female subjects with severe obesity and in 96 non-obese female controls. The Arg51Gln mutation was the only one located in the mature ghrelin product and was found in 6.3% of obese subjects but not among controls. Obese carriers of the mutation had lower self-reported weighs at 20, 30 and 40 years of age compared to those without the mutation although their weight at the examination date was not different. The pathophysiological relevance of these mutations remains to be determined.

Petersenn

To summarize: Characterization of ghrelin and its receptors may allow new insight into the regulation of GH secretion and additional physiological roles of these agents. Of special interest is the role for energy homeostasis, where ghrelin provides a new link between the stomach and the hypothalamus (fig. 5). In addition to a puta-

tive role in disorders with pathological GH secretion, ghrelin could have a role in the pathogenesis of disorders at both ends of the body weight spectrum. Therefore, GHS and ghrelin may have clinical applications beyond the release of GH.

References 1 Howard AD, Feighner SD, Cully DF, et al: A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273:974–977. 2 Petersenn S, Rasch AC, Penshorn M, Beil FU, Schulte HM: Genomic structure and transcriptional regulation of the human growth hormone secretagogue receptor. Endocrinology 2001;142:2649–2659. 3 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 1999;402:656–660. 4 Hosoda H, Kojima M, Matsuo H, Kangawa K: Ghrelin and des-acyl ghrelin: Two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000;279: 909–913. 5 Date Y, Kojima M, Hosoda H, et al: Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000;141:4255–4261. 6 Gualillo O, Caminos J, Blanco M, et al: Ghrelin, a novel placental-derived hormone. Endocrinology 2001;142:788–794. 7 Kanamoto N, Akamizu T, Hosoda H, et al: Substantial production of ghrelin by a human medullary thyroid carcinoma cell line. J Clin Endocrinol Metab 2001;86:4984–4990. 8 Arvat E, Maccario M, Di Vito L, et al: Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: Comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone. J Clin Endocrinol Metab 2001;86:1169– 1174. 9 Tschop M, Smiley DL, Heiman ML: Ghrelin induces adiposity in rodents. Nature 2000;407: 908–913. 10 Wren AM, Seal LJ, Cohen MA, et al: Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 2001;86: 5992. 11 Nakazato M, Murakami N, Date Y, et al: A role for ghrelin in the central regulation of feeding. Nature 2001;409:194–198. 12 Masuda Y, Tanaka T, Inomata N, et al: Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun 2000;276:905–908.


13 Nagaya N, Kojima M, Uematsu M, et al: Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol 2001;280:R1483– R1487. 14 Ariyasu H, Takaya K, Tagami T, et al: Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 2001;86:4753–4758. 15 Otto B, Cuntz U, Fruehauf E, et al: Weight gain decreases elevated plasma ghrelin concentrations of patients with anorexia nervosa. Eur J Endocrinol 2001;145:669–673. 16 Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML: Circulating ghrelin levels are decreased in human obesity. Diabetes 2001;50:707–709. 17 Hansen TK, Dall R, Hosoda H, et al: Weight loss increases circulating levels of ghrelin in human obesity. Clin Endocrinol (Oxf) 2002;56: 203–206. 18 Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS: A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50: 1714–1719. 19 Shiiya T, Nakazato M, Mizuta M, et al: Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002;87:240–244. 20 Inui A: Ghrelin: An orexigenic and somatotrophic signal from the stomach. Nat Rev Neurosci 2001;2:551–560. 21 Toshinai K, Mondal MS, Nakazato M, et al: Upregulation of ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia and leptin administration. Biochem Biophys Res Commun 2001;281:1220–1225. 22 Asakawa A, Inui A, Kaga T, et al: Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 2001;120:337–345. 23 Wright JC, Bartke A: Ghrelin administration decreases insulin secretion in Ames dwarf mice. 83rd Annual Meeting of the Endocrine Society, Denver, Colo 2001, pp P1–98.

24 Janssen JA, van der Toorn FM, Hofland LJ, et al: Systemic ghrelin levels in subjects with growth hormone deficiency are not modified by one year of growth hormone replacement therapy. Eur J Endocrinol 2001;145:711–716. 25 Gasperi M, Aimaretti G, Scarcello G, et al: Low dose hexarelin and growth hormone (GH)releasing hormone as a diagnostic tool for the diagnosis of GH deficiency in adults: Comparison with insulin-induced hypoglycemia test. J Clin Endocrinol Metab 1999;84:2633–2637. 26 Korbonits M, Kaltsas G, Perry LA, et al: Hexarelin as a test of pituitary reserve in patients with pituitary disease. Clin Endocrinol (Oxf) 1999;51:369–375. 27 Petersenn S, Jung R, Beil FU: Diagnosis of growth hormone deficiency in adults by testing with GHRP-6 alone or in combination with GHRH: Comparison with the insulin tolerance test. Eur J Endocrinol 2002;146:667–672. 28 Popovic V, Leal A, Micic D, et al: GH-releasing hormone and GH-releasing peptide-6 for diagnostic testing in GH-deficient adults. Lancet 2000;356:1137–1142. 29 Laron Z: Growth hormone secretagogues. Clinical experience and therapeutic potential. Drugs 1995;50:595–601. 30 Chapman IM, Bach MA, Van Cauter E, et al: Stimulation of the growth hormone (GH)-insulin-like growth factor 1 axis by daily oral administration of a GH secretogogue (MK-677) in healthy elderly subjects. J Clin Endocrinol Metab 1996;81:4249–4257. 31 Chapman IM, Pescovitz OH, Murphy G, et al: Oral administration of growth hormone (GH)releasing peptide-mimetic MK-677 stimulates the GH/insulin-like growth factor-1 axis in selected GH-deficient adults. J Clin Endocrinol Metab 1997;82:3455–3463. 32 Nagaya N, Miyatake K, Uematsu M, et al: Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. J Clin Endocrinol Metab 2001;86: 5854–5859. 33 Ukkola O, Ravussin E, Jacobson P, et al: Mutations in the preproghrelin/ghrelin gene associated with obesity in humans. J Clin Endocrinol Metab 2001;86:3996–3999.

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Author Index

Bail, H.J. 39 Bernert, G. 16 Binder, G. 2 Boonen, S. 49 Eiholzer, U. 24 Frisch, H. 16 Geusens, P.P.M.M. 49 Haas, N.P. 39 Höppner, W. 7 Kolbeck, S. 39 Krummrey, G. 39 l’Allemand, D. 24 Müllner-Eidenböck, A. 16

Nordmann, Y. 24 Petersenn, S. 56 Prayer, D. 16 Raschke, M.J. 39 Riedl, S.W. 16 Saenger, P. 27 Schaefer, F. 35 Schmidmaier, G. 39 Weber, M.M. 43 Wühl, E. 35 Wygold, T. 20 Zeier, M. 30

Subject Index

Adolescence 20 Adrenogenital syndrome 7 Adults 35 Brain midline anomalies 16 Childhood 20 Children 35 Chronic renal failure 35 Defect fracture 39 Dialysis 30 Distraction osteogenesis 39 Endocrine tumors 7 End-stage renal disease 30 Energy homeostasis 56 Fracture healing 39 GH promotor 2 GH-1 gene 2 Ghrelin 56

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

© 2002 S. Karger AG, Basel


Growth 20 – hormone 2, 27, 30, 39, 43, 49, 56 – – deficiency 56 – – secretagogues 56 – – therapy 20 – – treatment 35 Hip fracture 49 Hypoventilation 24 IGHD 2 Insulin-like growth factor 49 Insulin resistance 27 Intrauterine growth retardation 27 Malnutrition 30 Micropigs 39 Molecular diagnostics 7 – genetics 7 Mortality 30

Multiple endocrine neoplasia 7 Muscle 43 Mutations 7 Optic nerve hypoplasia 16 Osteoporosis 49 Pituitary insufficiency 16 Prader-Willi syndrome 24 Psychological problems 20 Psychosocial adaptation 20 Respirational abnormalities 24 Septo-optic dysplasia 16 Short stature 20 Small for gestational age 27 Strength 43 Sudden death 24

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New Developments in Asthma Research

Dyslexia in Children: New Research

New Directions in Action Research

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New perspectives in magnesium research: Nutrition and health

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Commutative Rings: New Research

Europeanization: New Research Agendas

Dietary magnesium: New research

Dietary Magnesium: New Research

New Research Demolishes Evolution

Schizophrenic Psychology, New Research

Tourism Management: New Research

New Research on Semiconductors

Food Allergies: New Research

Commutative rings. New research

Infinity. New research frontiers

Food Allergies: New Research

New Perspectives in GH Research: Davos, March 2002 (Hormone Research)

Neighbourhood Effects Research: New Perspectives

Perspectives in Receptor Research

Organometallic Chemistry Research Perspectives

Human Growth Hormone. Research and Clinical Practice

Obesity and Metabolism (Frontiers of Hormone Research)

New Developments in Catalysis Research

New Topics in Superconductivity Research

New Developments in Asthma Research

Dyslexia in Children: New Research

New Directions in Action Research

New Frontiers in Environmental Research

New Perspectives in Magnesium Research: Nutrition and Health

Standardization Research in Information Technology: New Perspectives (Premier Reference Source)

New perspectives in magnesium research: Nutrition and health

New Perspectives and Approaches in Plant Growth-Promoting Rhizobacteria Research

Computational Biology: New Research

Learning Disabilities: New Research

Commutative Rings: New Research

Europeanization: New Research Agendas

Dietary magnesium: New research

Dietary Magnesium: New Research

New Research Demolishes Evolution

Schizophrenic Psychology, New Research

Tourism Management: New Research

New Research on Semiconductors

Food Allergies: New Research

Commutative rings. New research

Infinity. New research frontiers

Food Allergies: New Research

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