IGF-i And IGF Binding Proteins: Basic Research And Clinical Management (Endocrine Development)

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Author: Stefano Cianfarani (Editor) | David R. Clemmons (Editor) | M. O. Savage (Editor)

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IGF-I and IGF Binding Proteins Basic Research and Clinical Management

Endocrine Development Vol. 9

Series Editor

Martin O. Savage

London

IGF-I and IGF Binding Proteins Basic Research and Clinical Management

Volume Editors

Stefano Cianfarani Rome David R. Clemmons Chapel Hill, N.C. Martin O. Savage London

42 figures, 1 in color, and 7 tables, 2005

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

Prof. Stefano Cianfarani

Prof. David R. Clemmons

Rina Balducci Center of Paediatric Endocrinology Department of Public Health and Cell Biology, Tor Vergata University Rome, Italy

Division of Endocrinology Department of Medicine School of Medicine University of North Carolina Chapel Hill, N.C., USA

Prof. Martin O. Savage Paediatric Endocrinology Section Department of Endocrinology St Bartholomew’s Hospital West Smithfield, London, UK Library of Congress Cataloging-in-Publication Data IGF-I and IGF binding proteins: basic research and clinical management / volume editors, Stefano Cianfarani, David R. Clemmons, Martin O. Savage. p. ; cm. – (Endocrine development, ISSN 1421-7082; v. 9) Includes bibliographical references and index. ISBN 3-8055-7926-8 (hard cover : alk. paper) 1. Somatomedin. 2. Insulin-like growth factor-binding proteins. [DNLM: 1. Insulin-Like Growth Factor Binding Proteins–physiology. 2. Insulin-Like Growth Factor Binding Proteins–genetics. 3. Insulin-Like Growth Factor Binding Proteins–therapeutic use. QU 55 I24 2005] I. Cianfarani, Stefano. II. Clemmons, D. R. III. Savage, M. O. (Martin O.) IV. Series. QP552.S65I324 2005 612⬘.015756–dc22 2005006227 Cover illustration: First three domains of the human IGF-I receptor 3-D structure. From Nature 1998;394: 395–399, with permission. Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2005 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 3–8055–7926–8

Contents

VII Foreword Savage, M.O. (London) IX Preface Cianfarani, S. (Rome); Clemmons, D.R. (Chapel Hill, N.C.) Savage, M.O. (London) Basic Research 1 The IGF System: New Developments Relevant to Pediatric Practice Rosenfeld, R.G. (Palo Alto, Calif.) 11 Clinical Relevance of Systemic and Local IGF-I Yakar, S.; Pennisi, P.; Wu, Y.; Zhao, H.; LeRoith, D. (Bethesda, Md.) 17 Cartilage Disorders: Potential Therapeutic Use of Mesenchymal Stem Cells Spagnoli, A.; Longobardi, L.; O’Rear, L. (Nashville, Tenn.) 31 Treatment in Animal Models Guan, J.; Bennet, L.; Gluckman, P.D.; Gunn, A.J. (Auckland) 44 Insulin-Like Growth Factor-I and Risk of Type 2 Diabetes and Coronary Heart Disease: Molecular Epidemiology Sandhu, M.S. (Cambridge)

V

Clinical Management 55 Quantitative Measurement of IGF-I and Its Use in Diagnosing and Monitoring Treatment of Disorders of Growth Hormone Secretion Clemmons, D.R. (Chapel Hill, N.C.) 66 IGF-I and IGFBP-3 Assessment in the Management of Childhood Onset Growth Hormone Deficiency Cianfarani, S.; Liguori, A.; Germani, D. (Rome) 76 IGFs and IGFBPs in Adult Growth Hormone Deficiency Aimaretti, G.; Baldelli, R.; Corneli, G.; Croce, C.; Rovere, S.; Baffoni, C.; Bellone, S.; Gasco, V.; Granata, R.; Grottoli, S.; Ghigo, E. (Turin) 89 Monitoring of Insulin-Like Growth Factors during Growth Hormone Treatment: Adulthood Growth Hormone Deficiency Monson, J.P. (London) 100 IGFs and IGFBPs in GH Insensitivity Savage, M.O.; Blair, J.C.; Jorge, A.J. (London); Street, M.E. (Parma); Ranke, M.B. (Tübingen); Camacho-Hübner, C. (London) 107 Childhood and Adolescent Diabetes Dunger, D.B.; Regan, F.M.; Acerini, C.L. (Cambridge) 121 Insulin Resistance and Type 2 Diabetes mellitus: Is There a Therapeutic Role for IGF-1? Moses, A.C. (Princeton, N.J.) 135 Insulin-Like Growth Factors in the Treatment of Neurological Disease Leinninger, G.M.; Feldman, E.L. (Ann Arbor, Mich.) 160 Insulin-Like Growth Factor System in Amyotrophic Lateral Sclerosis Wilczak, N.; de Keyser, J. (Groningen)

170 Author Index 171 Subject Index

Contents

VI

Foreword

I am delighted to welcome this volume, covering new and topical areas of the IGF field, to the growing family of Endocrine Development publications. As a co-editor, I have carefully supervised the production of this edition. The IGF system pervades the fields of development, linear and cellular growth and metabolism. Consequently, a volume discussing key new developments fits well into the overall aims of the Endocrine Development series. Not only is the functional integrity of the IGF system crucial for normal linear growth and metabolism, it is also of fundamental importance for normal fetal development. This volume covers broad areas of normal IGF and IGF binding protein physiology and genetics. Molecular and developmental defects are discussed, as are the contributions to clinical diagnosis and management of measurement of IGFs and IGFBPs. The therapeutic potential of recombinant human IGFs and IGFBPs in a range of disorders is also covered. This book provides a welcome addition to other Endocrine Development volumes and will hopefully be of interest to scientists and clinicians working in the broad fields of paediatric and adult endocrinology and metabolism. Martin O. Savage, London

VII

Preface

This book consists of a compilation of chapters based on presentations at a symposium entitled ‘IGFs and IGFBPs: Assessment and Therapeutic Benefit’. The symposium was sponsored by Serono Symposia and held in New Orleans in June 2004. The aim of the meeting was to report new advances in the broad field of IGF physiology and pathophysiology, related to clinical medicine. The meeting was considered to have achieved its objectives and we feel that the present volume presents valuable ‘state-of-the-art’ accounts of the range of topics discussed. We were fortunate to have a very high quality faculty consisting of basic scientists and endocrinologists. The areas covered are broad, but focus particularly on clinically relevant topics related to growth, paediatric and adult growth hormone deficiency, metabolic disorders, diabetes mellitus and neurology. We hope that this volume will prove to be helpful for scientists working in IGF and IGF-related fields and to clinicians working in both paediatric and adult endocrinology. The potential therapeutic benefit of IGF and IGFBP therapy in a broad range of disorders is also discussed in this book. Stefano Cianfarani, Rome David R. Clemmons, Chapel Hill, N.C. Martin O. Savage, London

IX

Basic Research Cianfarani S, Clemmons DR, Savage MO (eds): IGF-I and IGF Binding Proteins. Basic Research and Clinical Management. Endocr Dev. Basel, Karger, 2005, vol 9, pp 1–10

The IGF System: New Developments Relevant to Pediatric Practice Ron G. Rosenfeld Lucile Packard Foundation for Children’s Health, Palo Alto, Calif., USA

Abstract In the 50 years since the initial report of the ‘sulfation factor’ or ‘somatomedin’ hypothesis, the IGF system has established itself as the primary mediator of both intrauterine and postnatal growth in mammals. IGF deficiency (IGFD) has emerged as an important clinical diagnosis: secondary IGFD results from insufficient production of GH and is characterized by postnatal growth failure; primary IGFD can result from abnormalities of the GH receptor or GH signaling cascade, or from mutations or deletions of the IGF-I gene. Monitoring IGF production during short-term IGF generation tests or during chronic GH therapy can provide a means for evaluating the efficacy and safety of GH treatment. IGF-I, either alone or in combination with IGF binding proteins, is the treatment of choice for primary IGFD and may have a role in treatment of idiopathic short stature when accompanied by decreased serum concentrations of IGF-I. Copyright © 2005 S. Karger AG, Basel

The IGFs were first identified almost 50 years ago, when Salmon and Daughaday [1] reported that, while GH, itself, could not directly stimulate sulfation in chondrocytes, serum from GH-deficient rats that had been treated with GH could stimulate sulfation successfully. While these seminal studies demonstrated the importance of the IGFs in mediating GH action, the last decade has seen a growing realization of the central role of the IGFs in the fundamental physiology of human growth [2]. A number of these observations, enumerated below, have proven to have direct relevance to the diagnostic management and therapy of children with growth disorders: (1) a growing appreciation of the critical role of the IGF system in both intrauterine and postnatal growth; (2) the concept of ‘IGF deficiency’ (IGFD) as both a physiological

condition and a clinical state; (3) the replacement of ‘GH deficiency’ (GHD) as a clinical diagnosis, with ‘GH responsiveness’ as a pragmatic consideration in choice of therapy; (4) a growing appreciation of the potential utility of IGF generation tests in the management of children with growth disorders; (5) the use of IGF monitoring of GH therapy, and (6) the emergence of IGF-I as a potential therapeutic agent. Collectively, these observations have contributed to a progressive shift in emphasis from an approach characterized by the use of IGF assays as ‘screening tests’ for children with short stature (the era from 1960 to 1990) to an appreciation of the central role of the IGF system in mammalian growth and in optimization of therapy directed at children with growth failure.

Central Role of the IGF System in Both Intrauterine and Postnatal Growth

The critical role of the IGF axis in both prenatal and postnatal growth has been demonstrated convincingly by the combination of targeted gene knockout studies in mice and the careful elucidation of prismatic cases of growth failure [2, 3]. While both prenatal and postnatal growth are profoundly IGF-dependent, only postnatal growth is strongly regulated by GH. Thus, patients with congenital defects of GH secretion or with abnormalities of GH receptors typically have birth lengths within 95% of the norm, presumably reflecting relatively normal IGF production and action in utero. Postnatally, however, their growth characteristics are strikingly similar to those observed in patients with primary IGFD (see below). These findings provide strong support for the mediation of postnatal growth by GH-dependent production of IGFs. On the other hand, prenatal growth appears to be largely GH-independent, but still strongly IGF-dependent. Targeted disruption of the gene for either IGF-I or IGF-II in mice results in a 40% reduction of fetal growth. Knockout of the gene for the IGF-I receptor, the transmembrane tyrosine kinase that mediates the growth-promoting actions of both IGF-I and IGF-II, results in ⬃55% reduction in fetal size. These findings are supported in humans, where deletions of chromosome 15q resulting in haploinsufficiency for the IGF-IR gene are associated with prenatal and postnatal growth retardation. Similarly, compound heterozygosity for point mutations of the IGF-IR gene has been observed in a patient with intrauterine and postnatal growth failure [4]. In sum, these observations support the concept that the IGF system is the major mediator of embryonic, fetal and postnatal growth, and that its actions in the fetus are largely GH-independent. After birth, through mechanisms still unknown, the regulation of IGF-I production becomes primarily GH-dependent, with

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additional contributions from nutrition and sex steroids. In light of these observations, any assessment of growth in mammals must address the role of the IGF system.

Concept of IGF Deficiency

The growing appreciation of the central role of the IGF system in mammalian growth was accompanied by an increasing recognition of the limitations implicit in the conventional diagnosis of GHD [5–7]. Increasingly sophisticated genetic studies of isolated GHD and combined pituitary hormone deficiency, combined with neuroimaging studies of the hypothalamus and pituitary, have indicated that only a minority of children labeled as GHD have identifiable molecular or organic etiologies for their growth failure. This observation was of particular relevance to children carrying a diagnosis of ‘idiopathic, isolated GHD’, which has emerged as an increasingly questionable diagnosis. In early postmarketing studies, 59% of children labeled as GHD were listed as ‘idiopathic’, and only 22% were identified as having organic etiologies. While it is possible that some of these children had unrecognized molecular disorders of the GH gene or various pituitary transcription factors, the current view is that the majority of such cases represent overdiagnosis of GHD. Indeed, the reported prevalence of GHD (1:4,000 to 1:10,000 children) is almost certainly an overestimate. This conclusion is supported by the report of Tauber et al. [8], who showed that when provocative GH testing was repeated in 131 adults who carried a diagnosis of idiopathic childhood-onset GHD on the basis of GH stimulation tests, two-thirds had peak GH levels ⬎10 ng/ml, while only 17% had peak serum concentrations ⬍5 ng/ml. Consistent with these observations were studies from Maghnie et al. [9], who reinvestigated 35 young adults carrying a diagnosis of childhood-onset GHD, and found that 100% of patients with isolated GHD and normal or low pituitary volume on imaging studies achieved normal peak GH concentrations on repeat GH provocative tests. This overdiagnosis of GHD in children with growth failure reflects two major factors: a desire on the part of endocrinologists to be able to justify GH therapy in children with significant growth failure, combined with the recognized inadequacies of conventional GH stimulation tests. Unfortunately, no single physiologic or provocative test or, indeed, combination of tests, has proven to have sufficient specificity for the diagnosis of GHD, despite the fact that a minimum of 34 different provocative methods resulting in 189 different combination protocols have been reported in the literature [10]. The inadequacies of GH stimulation tests have been described extensively in the literature

The IGF System: New Developments Relevant to Pediatric Practice

3

and include the recognition that such tests: (1) are nonphysiologic; (2) rely on totally arbitrary definitions of what constitutes a ‘normal’ and ‘subnormal’ response; (3) do not account for either the age-dependency of GH secretion nor the effect of pubertal levels of sex steroids on GH production; (4) employ GH assays of limited accuracy and reproducibility, and (5) are expensive, cumbersome, and may have associated risks. Given these diagnostic limitations and the growing recognition of the central role of the IGF system in growth, it has been proposed that the concept of IGFD may provide a more meaningful and useful diagnosis [5, 6]. This has been supported by the development of highly sensitive assays for IGF-I, IGF-II and the various IGF binding proteins (IGFBPs), and the demonstration that serum concentrations of IGF-I and IGFBP-3, in particular, are highly predictive of the GH status of patients with childhood-onset GHD. IGF assays, on the other hand, do have some important limitations: (1) there is a marked age and puberty dependence of serum IGF-I, which must be accounted for in evaluating serum concentrations in children and adolescents; (2) IGF-I concentrations may be affected by nutritional status, and (3) serum IGF-I concentrations may decline slowly (over months or years) in some patients with evolving GHD (as observed following cranial irradiation). Despite these caveats, the concept of IGFD as a clinical diagnosis has proven to have considerable value. It has been proposed that IGFD can be divided into primary and secondary causes [11]. Secondary IGFD results from GHD, either on a hypothalamic or pituitary basis, or can result from production of biologically inactive GH as a consequence of mutations of the GH gene. Therapy for secondary IGFD, thus, should be commenced with GH, unless there is evidence of GH-inactivating antibodies. Primary IGFD represents a constellation of disorders characterized by decreased IGF production in the presence of normal or, even, elevated GH secretion. To date, three distinct molecular abnormalities have been identified as causes of primary IGFD: (1) mutations or gene deletions of the GH receptor gene [12]; (2) mutations affecting the post-GHR signaling cascade, as observed in a patient homozygous for a point mutation of the gene for signal transducer and activator of transcription (STAT)-5b [13], and mutations or deletions of the gene for IGF-I [14]. In such cases, GH would be expected to be relatively (or absolutely) ineffective, and replacement with IGF-I would appear to be the therapy of choice. As useful as this classification may be in describing the underlying pathophysiology of the GH-IGF axis, it leaves unresolved the question of optimal therapy for children labeled as idiopathic short stature who have low serum concentrations of IGF-I. While GH treatment has proven to be effective in at least some such patients, the argument has been made that abnormal serum

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concentrations of IGF-I reflect some degree of GH resistance, and that therapy with IGF-I is the more appropriate and ‘physiologic’ treatment. Unfortunately, no data exist to date on the efficacy of IGF-I treatment in such individuals, let alone a direct clinical comparison of GH vs. IGF therapy.

Growth Hormone Deficiency vs. Growth Hormone Responsiveness

If, as stated above, the diagnosis of GHD is clinically problematic, especially for ‘idiopathic, isolated GHD,’ it becomes worthwhile to explore the rationale for establishing this diagnosis. In the case of organic GHD, whether resulting from a congenital malformation of the hypothalamus/pituitary or from an intracranial tumor compromising pituitary function, the diagnosis is best established by neuroimaging studies. In the case of genetic etiologies, whether of isolated GHD or combined pituitary hormone deficiencies, molecular studies are required to establish a definitive diagnosis. Accordingly, it has been proposed by some that conventional GH stimulation tests are not warranted, and can be replaced by a combination of IGF-related screening tests, neuroimaging studies, and, when appropriate, molecular studies. While there is some justification for this approach, it may be unnecessarily draconian. GH provocative tests can play a useful role is establishing the clinical basis of growth failure and, when used properly (and selectively) can contribute to an understanding of underlying pathophysiology. On the other hand, they have proven to be of only modest value in predicting the clinical response of patients to GH therapy and, thus, are of limited utility in determining what patients are the most appropriate recipients of GH therapy. This point is underscored by the recent approval of GH treatment of idiopathic short stature by the Food and Drug Administration in the USA. Ultimately, the more relevant question may be not whether a patient is ‘GHD,’ but whether he is GH-responsive. Unfortunately, limited data are available concerning biomarkers of GH responsiveness, other than the observation that patients with unequivocal GHD respond well to GH replacement, at least in the first year of treatment. Studies from over 20 years ago, exploring IGF generation tests (i.e., short-term IGF responsiveness to the administration of GH) were hampered by: (1) a limited number of subjects; (2) IGF assays of limited specificity and sensitivity; (3) the lack of age-related normative data for IGF assays, and (4) the lack of normative data on IGF generation tests [15]. Nevertheless, such tests, if performed properly and with adequate normative data, might provide a dynamic test of GH sensitivity.


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IGF Generation Tests

The potential value of standardized IGF generation tests was established with the identification of a molecular basis for GH insensitivity (GHI) and the development of alternative therapies to GH, such as the administration of IGF-I. Blum et al. [16] developed and published a set of diagnostic criteria for GHI, which included the failure to raise serum IGF-I and IGFBP-3 concentrations above empirically derived set points. The utility of IGF generation criteria has been corroborated in patients with classical GHI, but never has been adequately tested in other forms of short stature, which may encompass partial GHI. Furthermore, the criteria employed by Blum et al. [16] for the diagnosis of GHI (i.e., a failure to raise serum concentrations of IGF-I and IGFBP-3 by at least 15 and 400 ng/ml, respectively, with GH administration) may prove to be overly restrictive in evaluating patients with less severe forms of GHI. In one study, for example, of genotype:phenotype correlations in GHI, in 63 patients with GHI and ‘negative’ GH binding protein, the mean post-GH rise in serum IGF-I was ⬍20 ng/ml, but with a range of ⬍20–58 ng/ml, while the mean rise in serum IGFBP-3 concentrations was 371 ng/ml, with a range of 95–1762 ng/ml [17]. In patients with a clinical diagnosis of GHI and ‘positive’ GHBP, the rise in serum IGF-I concentrations ranged from ⬍20 to 82 ng/ml, while the increase in serum IGFBP-3 levels ranged from 180 to 1,679 ng/ml. These results corroborated the restrictive nature of the Blum criteria, particularly in GHBP-positive cases of GHI. In an effort to expand the potential utility of IGF generation tests, as well as develop the necessary normative data, investigations were conducted in Ecuador, where the largest cluster of classical GHI patients resides, all of whom are homozygous for an E180 splice mutation affecting the extracellular domain of the GH receptor, thereby providing a ready means of genotype confirmation [18–20]. The study encompassed 198 subjects, including 72 normal statured children and adults, 23 patients with classical GHI, 65 heterozygous relatives, 22 patients with GHD, and 16 children with idiopathic short stature. Subjects were randomly assigned to either ‘low-dose’ GH (0.025 mg/kg/day) or ‘highdose’ GH (0.05 mg/kg/day) for 7 days, with fasting blood samples obtained on the mornings of days 1, 5 and 8. After a 2-week washout period, subjects were then placed on the alternate GH dose, and the protocol repeated. The results of IGF-I and IGFBP-3 generation in the normal subjects provided a template for comparing observations in patients with various growth disorders. As anticipated, patients with the E180 GHR splice mutation, having no functional GHR, had remarkably low basal and stimulated serum IGF-I and IGFBP-3 concentrations. Nevertheless, 5 of the 22 GHI subjects failed to meet the Blum criteria for IGF-I (a rise in serum IGF-I ⬍15 ng/ml after 4 days of GH), demonstrating an increase of serum IGF-I of 16–63 ng/ml (table 1). On the other hand, only 1 of

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Table 1. Sensitivity and specificity of low-dose (a) and high-dose (b) generation tests

a

b

Day 5 Sensitivity Day 5 Specificity Day 8 Sensitivity Day 8 Specificity Day 5 Sensitivity Day 5 Specificity Day 8 Sensitivity Day 8 Specificity

IGF-I ⌬ ⬍15 ng/ml

IGFBP-3 ⌬ ⬍400 ng/ml

Either

Both

86% 97% 82% 97% 86% 92% 91% 97%

95% 69% 100% 85% 100% 87% 100% 92%

95% 69% 100% 82% 100% 82% 100% 90%

86% 97% 82% 100% 86% 97% 91% 100%

Subjects (n ⫽ 61) included in the analysis were any with short stature [height ⬍–2 SD (GHD, n ⫽ 23; GHI, n ⫽ 22; ISS, n ⫽ 16)]. The criteria of Blum et al. [16] were employed to define a positive test for GHI (⌬ IGF-I ⬍15 ng/ml and/or ⌬ IGFBP-3 ⬍400 ng/ml).

the 22 subjects raised serum IGFBP-3 by more than 400 ng/ml by day 5. The highest degree of sensitivity and specificity for GHI was observed with the 7-day high-dose GH IGFBP-3 generation test, which yielded 100% sensitivity and 91% specificity. Maximal sensitivity in the diagnosis of GHI is achieved by defining GHI as the failure to raise either IGF-I or IGFBP-3 on day 8 of either low-dose or high-dose GH (100% sensitivity and 82% specificity) (table 1). Maximal specificity in the diagnosis of GHI is attained by defining ‘non-GHI’ as the failure to raise both IGF-I and IGFBP-3 on day 8 of high-dose GH (100% specificity and 82% sensitivity). It is important to note, however, that these results are restricted to one specific mutation of the GHR gene, and cannot necessarily be extrapolated to other cases of GHI. The patient with the STAT-5b mutation, who was unquestionably GH insensitive, had a rise in serum IGF-I of 17 ng/ml and a rise in serum IGFBP-3 of 174 ng/ml. It is clear that, as efforts are made to define partial GHI, diagnostic criteria will need to be refined. The use of dynamic tests, such as IGF generation tests, should prove of particular value in determining which patients currently identified as ‘idiopathic short stature,’ actually have various degrees of GH resistance. Furthermore, the results of such tests should help determine which patients are more likely to benefit from GH vs. IGF-I treatment.

Use of IGF Assays for Monitoring GH Therapy

Although assays for IGF-I and IGFBP-3 have been employed as part of the diagnostic strategy for pediatric GHD and GHI for decades, they had not been


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used routinely for monitoring GH therapy, in part because the growth response to GH administration was both a clinically relevant and readily determined measure of the efficacy of GH treatment in children. Indeed, it was the use of GH in adults with GHD which provided the first clear rationale for dose titration based upon serum concentrations of IGF-I. A further rationale for IGF monitoring emerged from a series of epidemiologic studies which suggested that the risk of a number of adult cancers, particularly prostate, breast and colon, in normal adults was directly related to serum concentrations of IGF-I and, in some situations, inversely correlated with serum levels of IGFBP-3 [21]. In GH dose-response studies in pediatric GHD, it was noted that many prepubertal children receiving the conventional GH dose of 0.05 mg/kg/day and, particularly, in children receiving 0.1 mg/kg/day, serum IGF-I concentrations frequently exceeded ⫹2 SD for age and sex [22]. Furthermore, such studies have indicated considerable inter-subject variability in GH sensitivity. Some of such variability may be gender-related, as endogenous estrogen levels impact IGF generation. It is likely, however, that multiple other factors, including GH receptor levels, efficiency of GH signaling, IGF transcription and translation rates, and IGF half-life all impact serum IGF concentrations following GH administration. The long-term ramifications of high serum IGF-I levels in GH recipients remain uncertain, but, given the association of high serum IGF-I concentrations and cancer, as well as the IGF-correlated side effects of longterm acromegaly, it has been proposed that monitoring pediatric GH therapy by measuring serum levels of IGF-I and, possibly, IGFBP-3 represents a prudent approach. Having said that, there exist essentially no data to provide a solid evidence-based rationale for targeting any specific serum IGF-I concentration as a goal. In the absence of additional data, the best recommendation would appear to be that the clinical response of the patient (i.e., the growth rate in children) should be balanced against serum levels of IGF-I. If the patient demonstrates a satisfactory growth response while maintaining a serum IGF-I concentration within the normal range, GH dosage can be held constant. In patients growing suboptimally, serum IGF-I levels may be used to increase GH dosage, titrating the growth response against serum levels of IGF-I and IGFBP-3.

IGF-I as a Potential Therapeutic Agent

The use of IGF-I for stimulation of skeletal growth in children will be addressed in depth elsewhere in this volume. It is probably worthwhile, however, in the context of this chapter, to make the following brief points: (1) For patients with severe primary IGFD, resulting from abnormalities of the GH receptor, GH signaling cascade, or IGF-I gene, IGF-I would appear to

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be the therapy of choice. In such cases, IGF generation tests have demonstrated the ineffectiveness of GH in increasing IGF production. (2) For patients with milder forms of IGFD (as, for example, in the ⬃25% of idiopathic short stature patients with low serum concentrations of IGF-I), a legitimate rationale exists currently for either GH or IGF-I therapy. Carefully performed controlled studies will be necessary to determine the relative merits and safety of each therapeutic modality. (3) On theoretical grounds, a rationale can be developed for the efficacy of either IGF-I alone, or IGF-I in combination with IGFBP-3. Once again, clinical trials will be required before judgment can be pronounced on the relative merits of each therapeutic approach.

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Salmon WD Jr, Daughaday WH: A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. 1956. J Lab Clin Med 1990;116:408–419. Rosenfeld RG: Insulin-like growth factors and the basis of growth. N Engl J Med 2003;349: 2184–2186. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A: Role of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 2001;229:141–162. Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, Keller E, Kiess W, Klammt J, Kratzsch J, Osgood D, Pfaffle R, Raile K, Seidel B, Smith RJ, Chernausek SD: IGF-I receptor mutations resulting in intrauterine and postnatal growth retardation. N Engl J Med 2003;349: 2211–2222. Rosenfeld RG, Cohen PC: Disorders of growth hormone/insulin-like growth factor secretion and action; in Sperling MA (ed): Pediatric Endocrinology. Philadelphia, Saunders, 2002, pp 211–288. Reiter EO, Rosenfeld RG: Normal and aberrant growth; in Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology. Philadelphia, Saunders, 1998, pp 1427–1507. Rosenfeld RG: Is growth hormone deficiency a viable diagnosis? J Clin Endocrinol Metab 1997;82:349–351. Tauber M, Moulin P, Pienkowski C, Jouret B, Rochiccioli P: Growth hormone (GH) retesting and auxological data in 131 GH-deficient patients after completion of treatment. J Clin Endocrinol Metab 1997;82:353–356. Maghnie M, Strigazzi C, Tinelli C, Autelli M, Cisternino M, Loche S, Severi F: Growth hormone (GH) deficiency (GHD) of childhood onset: Reassessment of GH status and evaluation of the predictive criteria for permanent GHD in young adults. J Clin Endocrinol Metab 1999;84: 1324–1328. Sizonenko PC, Clayton PE, Cohen P, Hintz RL, Tanaka T, Laron Z: Diagnosis and management of growth hormone deficiency in childhood and adolescence. I. Diagnosis of growth hormone deficiency. J Clin Endocrinol Metab 2001;86:3323–3327. Rosenfeld RG, Hwa V: Toward a molecular basis for idiopathic short stature. J Clin Endocrinol Metab 2004;89:1066–1067. Rosenfeld RG: Broadening the growth hormone insensitivity syndrome. N Engl J Med 1995;333: 1145–1146. Kofoed EM, Hwa V, Little B, Woods KA, Buckway CK, Tsubaki J, Pratt KL, Bezrodnik L, Jasper H, Tepper A, Heinrich JJ, Rosenfeld RG: Growth hormone insensitivity associated with a STAT5b mutation. N Engl J Med 2003;349:1139–1147.


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Woods KA, Camacho-Hubner C, Savage MO, Clark AJL: Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med 1996;335:1363–1365. Rosenfeld RG, Kemp SF, Hintz RL: Constancy of somatomedin response to growth hormone treatment of hypopituitary dwarfism and lack of correlation with growth rate. J Clin Endocrinol Metab 1981;53:611–617. Blum WF, Cotterill AM, Postel-Vinay MC, Ranke MB, Savage MO, Wilton P: Improvement of diagnostic criteria in growth hormone insensitivity syndrome: Solutions and pitfalls. Acta Paediatr 1994;399(suppl):117–124. Woods KA, Dastot F, Preece MA, Clark AJL, Postel-Vinay M-C, Chatelain P, Ranke MB, Rosenfeld RG, Amselem S, Savage MO: Phenotype:genotype relationships in growth hormone insensitivity syndrome. J Clin Endocrinol Metab 1997;82:3529–3535. Buckway CK, Guevara-Aguirre J, Pratt KL, Burren CP, Rosenfeld RG: The IGF-I generation test revisited: A marker of GH sensitivity. J Clin Endocrinol Metab 2001;86:5176–5183. Buckway CK, Selva KA, Pratt KL, Tjoeng E, Guevara-Aguirre J, Rosenfeld RG: Insulin-like growth factor binding protein-3 generation as a measure of GH sensitivity. J Clin Endocrinol Metab 2002;87:4754–4765. Selva KA, Buckway CK, Sexton G, Pratt KL, Tjoeng E, Guevara-Aguirre J, Rosenfeld RG: Reproducibility in patterns of IGF generation tests with special reference to idiopathic short stature. Horm Res 2003;60:237–246. Ibrahim YH, Yee D: Insulin-like growth factor-I and cancer risk. Growth Horm IGF Res 2004;14:261–269. Cohen P, Bright GM, Rogol A, Kappelgaard AM, Rosenfeld RG: Effects of dose and gender on the growth and growth factor response to GH in GH-deficient children: Implications for efficacy and safety. J Clin Endocrinol Metab 2002;87:90–98.

Ron G. Rosenfeld, MD Senior Vice-President for Medical Affairs Lucile Packard Foundation for Children’s Health 770 Welch Road, Suite 350, Palo Alto, CA 94304 (USA) Tel. ⫹1 650 494 6930, Fax ⫹1 650 498 2619, E-Mail [email protected]

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Clinical Relevance of Systemic and Local IGF-I Shoshana Yakar, Patricia Pennisi, Yiping Wu, Hong Zhao, Derek LeRoith Diabetes Branch, NIDDK, NIH MSC, Bethesda, Md., USA

Abstract The insulin-like growth factor family of ligands, receptors and binding proteins are critical for many normal physiological functions. These include normal development during fetal and post-natal development and maintenance of organ function in adult life. Circulating IGF-I is produced primarily by the liver under GH control, whereas the production of tissue IGF-I has other controls. Recent studies have demonstrated that both circulating and tissue IGF-I are important for maintaining the normal structure-function of complex organs such as bone. Circulating IGF-I is important for maintaining ambient GH levels; in its absence GH elevation is seen leading to insulin resistance. In addition, low levels of circulating IGF-I retard the progression and metastatic potential of a number of cancers. Copyright © 2005 S. Karger AG, Basel

Introduction

Since the event of molecular endocrinology it was established that insulinlike growth factor I (IGF-I) has both an endocrine and a paracrine mode of delivery. Since almost all tissues express IGF-I and its receptor it was of interest to determine the need for both systems. This review will outline the history behind this development and the mouse models created to attempt to answer the question, posed above. We will then extrapolate the findings to the human situation and demonstrate the clinical relevance of these findings to normal physiology and disease states.

Somatomedin Hypothesis Revisited

In the 1950s, Daughaday et al. [1] demonstrated that the effect of pituitary growth hormone (GH) on bone growth (particularly on cartilage growth) was mediated by a liver-derived protein. Naming it somatomedin, he went on to develop the ‘somatomedin hypothesis’ in the 1970s [2] which was revisited over a decade later when molecular biology allowed many investigators to determine that the liver was not the only tissue that expressed somatomedin C (or IGF-I, the new name for somatomedin C). Studies demonstrated that liver IGF-I was regulated largely by the ambient level of GH in the circulation while this was not necessarily the case with non-hepatic tissue production of IGF-I. Furthermore, it was generally accepted that tissue IGF-I acted locally and two systems were considered to be present; the circulating or endocrine delivery of IGF-I primarily liver-derived and the tissue or paracrine form of IGF-I. The immediate obvious question was, why two systems? If every tissue produces IGF-I, why is there the liver-derived endocrine form? This was especially relevant since post-natal liver does not express IGF-I receptors and thus liver-derived IGF-I must affect other tissues. To try to determine the reason for the existence of two systems, we created a mouse model that had the igf1 gene deleted from the liver in a tissue-specific manner, assuming that this would totally remove the endocrine form of IGF-I and potentially answer the question.

Liver-Derived IGF-I Gene-Deleted Mouse Model

Tissue-specific gene deletion models can be created using the Cre lox/P system. We created a mouse with exon 4 of the igf1 gene flanked by two lox/P sequences in tandem using the technique of homologous recombination, described in detail elsewhere. This mouse was mated with a separate transgenic mouse model expressing the Cre-recombinase enzyme in liver; this mouse was created by introducing the Cre under the influence of the albumin promoter/enhancer. As previously described, the mating resulted in a mouse line (liver-specific igf1-deficient, LID) in which IGF-I mRNA in liver was totally abrogated, while IGF-I mRNA in other tissues was perfectly normal. Total circulating IGF-I levels were lowered by 75% and GH levels were correspondingly elevated ⬃4-fold. There was no compensation, whereby tissue expression of IGF-I was increased, nor did IGF-II expression change. The initial conclusion from these studies was that post-natal mouse growth and development did not require liver IGF-I, which is the main source for endocrine IGF-I [3]. However, circulating levels were not completely depleted and the

Yakar/Pennisi/Wu/Zhao/LeRoith

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possibility was still present that ‘free’ IGF-I was ‘normal’, especially in light of the 75% reduction in circulating IGF-binding proteins (IGFBPs). To further evaluate the role of the circulating form of IGF-I, we crossed the LID mice with the acid-labile subunit (ALS) gene-deleted mouse (ALSKO). ALS, which is found primarily in circulation, stabilizes the IGF-I/IGF binding protein 3 complex and prevents a rapid degradation of the IGF-I peptide. The resultant mouse LID/ALSKO showed a further reduction in circulating IGF-I levels (85–90%) with a further reciprocal increase in GH levels. In contrast to the LID mice, the LID/ALSKO mice showed early post-natal growth retardation as measured by body length and femoral length. These results led us to revise our earlier conclusions and strongly suggested that post-natal growth in the mouse was determined to some extent by endocrine IGF-I, though tissue IGF-I may still play a role since the total IGF-I knockout mice are still much more retarded in growth [4]. To further examine the relative roles of endocrine versus paracrine IGF-I, we examined a complex tissue system. Bone length was reduced by 15–20% in the LID/ALSKO mice, while there was a 30% reduction in periosteal circumference and cortical bone density. This led us to conclude that bone length was the result of both endocrine and local IGF-I effects (IGF-I gene expression was maintained in the region of the cartilage), whereas bone density was more dependent on circulating IGF-I, giving us an explanation for two IGF-I systems [4]. Studying other complex tissues should help to prove this hypothesis. Inactivation of GH action in the LID mouse was achieved by crossing the LID mouse to a transgenic mouse expressing a GH antagonist (GHa). The double transgenic mouse LID/GHa exhibited a further reduction in circulating IGF-I levels, suggesting that GHa suppressed extrahepatic IGF-I secretion into the circulation. Body weight of the GHa transgenic mice did not differ significantly from the double gene disrupted mice LID/GHa. However, both groups were significantly smaller than control and LID mice (starting at ⬃2 weeks of age) [5]. Additionally, inactivation of GH action resulted in a marked increase in white adipose tissue, most likely due to inhibition of lipolysis.

Clinical Relevance

Cancer The IGF-I system and its relationship to cancer cell growth has been of great interest to investigators. The ligands (IGF-I and IGF-II) are expressed by many if not most cancers. IGF-II in particular is of great interest since its gene expression is controlled by imprinting and methylation of the promoter and this control maybe released in certain tumors leading to enhanced expression and

Clinical Relevance of Systemic and Local IGF-I

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release of IGF-II resulting in autocrine stimulation of cancer cell growth. The receptors that are involved in mediating the proliferative and anti-apoptotic effects primarily are the IGF-IRs that are generally overexpressed in most tumors. A subtype of the insulin receptor (IR-A) is also found in tumors and binds IGF-II with very high affinity and may play a role in some tumors. Most cells and indeed most tumors express various IGFBPs and their role in modulating cancer cell growth or apoptosis is also being studied. The importance of the role of the IGF system in cancer has led many investigators to search for molecules that will inhibit the IGF-IR, either at the ligand binding domain or the tyrosine kinase region to enhance apoptosis and aid in the therapy of these cancers. Interest in this system has also peaked recently with the findings that individuals with levels of circulating IGF-I in the upper quintile of the normal range have an increased relative risk of developing colon, breast, prostate, lung and bladder cancer. We decided to use the LID mice to study the effect of the markedly reduced IGF-I levels on cancer development and growth. Three cancer models were used: (1) Implanting cells of the mouse colon carcinoma 38 onto the cecum of mice leads to the development of cancer in a high proportion of mice. The number of mice developing these cancer growths was reduced in the LID group and the sizes of the tumors were significantly smaller. The number of mice showing liver metastases was also reduced in the LID group compared to controls and the number of liver nodules was reduced [6]. (2) SV40 large-T antigen (SV40) transgenic female mice develop mammary tumors. When mated with the LID mice, the double transgenic LID/SV40 mice developed fewer tumors, that grew more slowly [7]. (3) The carcinogen DMBA causes mammary tumors in rodents. Fewer tumors developed in LID mice compared to controls [7]. We concluded from the above studies that circulating levels of IGF-I affected tumor development, growth and metastases in the mouse. The effects seem to be specific to the level of circulating IGF-I, since tissue IGF-I gene expression was unaffected in the LID mice (table 1). Insulin Resistance In our initial evaluation of the LID mice we determined that they displayed elevated circulating insulin levels in the face of normoglycemia suggesting that they had developed insulin resistance. This was confirmed using the hyperinsulinemic-euglycemic clamps which showed a failure of insulin to inhibit hepatic glucose production and markedly reduced whole body glucose infusion as a measure of insulin-induced glucose disposal into muscle.


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Table 1. Development of cecal cancer in mice

Control LID

Mice developing tumors n (%)

Mice developing liver metastases n (%)

25 (57%) 16 (31%)

11 (44%) 5 (31%)

The finding that LID mice developed insulin resistance led to the obvious question – Was it due to the reduction in circulating IGF-I levels or secondary to the increased GH levels? This question is pertinent in light of the clinical findings that treatment of patients with severe insulin resistance with recombinant human IGF-I was successful in overcoming the resistance. On the other hand, acromegalic patients commonly demonstrate insulin resistance or worsening of their diabetes. It occurs, interestingly, in the presence of elevated IGF-I levels in the circulation and improves with treatment and reduction in the GH and IGF-I levels. In an attempt to determine whether it was the GH excess or the lack of IGF-I that caused the insulin resistance in the LID mice, we performed three different experiments: (1) rhIGF-I was administered to the LID mice to restore circulating levels of IGF-I and reduce GH levels; insulin sensitivity as measured by an insulin tolerance test was restored. This manipulation did not answer the question since the levels of both IGF-I and GH were restored to normal [8]. (2) Administration of a GH-releasing hormone antagonist reduced the circulating GH levels and despite the low IGF-I levels that remained, insulin sensitivity was restored [8]. (3) To confirm that GH was the primary culprit in causing insulin resistance, we mated the LID mice with the GHa transgenic mouse model to inactivate GH action. In the double-crossed animals (LID/GHa), IGF-I levels fell further. However, insulin resistance was abrogated suggesting that GH plays a major role in causing the insulin resistance [5]. However, we cannot exclude a role for IGF-I in affecting insulin sensitivity, since in the Laron-type human dwarfs that show GH resistance, and may develop insulin resistance, rhIGF-I does relieve the resistance [9]. Furthermore, in our LID/ALSKO mouse model, we noted that insulin sensitivity was restored in muscle and adipose tissue (not in liver). This was apparently due to the increase in free IGF-I in the circulation associated with a marked reduction in IGFBP-3 (and absent ALS). Since this was seen in the presence of very elevated GH levels it suggested a direct IGF-I effect in muscle. In addition, since liver does not express IGF-IRs this confirms an IGF-I-related effect in these mice.

Clinical Relevance of Systemic and Local IGF-I

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Summary and Future Directions

While many of the conclusions from mouse models and extrapolations to the human condition are made, one must do so cautiously for the obvious reasons. Future studies in the mouse models are needed to extend our understanding of normal physiology and disease states and eventually similar studies should be performed in humans. Gene-deletion experiments of nature in humans often bring new questions, that can be studied in the mouse models.

References 1 2 3 4 5 6 7 8 9

Daughaday WH, Hall K, Raben MS, Salmon WD Jr, van den Brande JL, van Wyk JJ: Somatomedin: Proposed designation for sulphation factor. Nature 1972;235:107. Le Roith D, Bondy C, Yakar S, Liu JL, Butler A: The somatomedin hypothesis: 2001. Endocr Rev 2001;22:53–74. Yakar S, Liu JL, Stannard B, et al: Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 1999;96:7324–7329. Yakar S, Rosen CJ, Beamer WG, et al: Circulating levels of IGF-1 directly regulate bone growth and density. J Clin Invest 2002;110:771–781. Yakar S, Setser J, Zhao H, et al: Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1-deficient mice. J Clin Invest 2004;113:96–105. Wu Y, Yakar S, Zhao L, Hennighausen L, LeRoith D: Circulating insulin-like growth factor-I levels regulate colon cancer growth and metastasis. Cancer Res 2002;62:1030–1035. Wu Y, Cui K, Miyoshi K, et al: Reduced circulating insulin-like growth factor I levels delay the onset of chemically and genetically induced mammary tumors. Cancer Res 2003;63:4384–4388. Yakar S, Liu JL, Fernandez AM, et al: Liver-specific IGF-1 gene deletion leads to muscle insulin insensitivity. Diabetes 2001;50:1110–1118. Laron Z, Avitzur Y, Klinger B: Carbohydrate metabolism in primary growth hormone resistance (Laron syndrome) before and during insulin-like growth factor-I treatment. Metabolism 1995;44:113–118.

Derek LeRoith, MD, PhD Diabetes Branch, NIDDK Room 8D12, Bldg 10, NIH MSC 1758 Bethesda, MD 20892–1758 (USA) Tel. ⫹1 301 4968090, Fax ⫹1 301 4804386, E-Mail [email protected]


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Cartilage Disorders: Potential Therapeutic Use of Mesenchymal Stem Cells Anna Spagnolia,b, Lara Longobardia, Lynda O’Reara Departments of aPediatrics and bCancer Biology, Vanderbilt University Medical Center, T-0107 Medical Center North, Nashville, Tenn., USA

Abstract Chondrogenesis is a well-orchestrated process driven by chondroprogenitors that undergo to condensation, proliferation and chondrocyte differentiation. Because cartilage lacks regenerative ability, treatments for cartilage diseases are primarily palliative. Adult bone marrow contains a reservoir of mesenchymal stem cells (MSC) with in vitro and in vivo potential of becoming cartilage. To optimize the potential therapeutic use of MSC in cartilage disorders, we need to understand the mechanisms by which growth factors determine their chondrogenic potential. Insulin-like growth factors (IGFs) play a central role in chondrogenesis as indicated by the severe growth failure observed in animals carrying null mutations of Igfs and Igf1R genes. We have found that IGF-I has potent chondrogenic effects in MSC. Effects are similar to transforming growth factor- (TGF-). Insulin-like growth factor binding protein-3 (IGFBP-3), well characterized as IGF carrier, has intrinsic bioactivities that are independent of IGF binding. IGFBP-3 levels are increased in degenerative cartilage diseases such as osteoarthritis. We have demonstrated that IGFBP-3 has IGF-independent growth inhibitory effects in chondroprogenitors. We now show that IGFBP-3 induces MSC apoptosis and antagonizes TGF- chondroinductive effects in chondroprogenitors. Understanding IGF-I chondroinductive and IGFBP-3 chondroinhibitory effects would provide critical information to optimize the therapeutic use of MSC in cartilage disorders. Copyright © 2005 S. Karger AG, Basel

Chondrogenesis: A Temporally and Spatially Orchestrated Process

In the developing skeleton, chondrogenesis is initiated by the migration of mesenchymal chondroprogenitors to the bone segment sites, where they undergo

condensation, proliferation and then chondrocyte differentiation and hypertrophy. Cell apoptosis at the edges of the mesenchyme condensates determines the skeletal patterning. Factors that promote chondrogenesis include insulin-like growth factors (IGFs), transforming growth factor- (TGF-) and bone morphogenic proteins (BMPs). IGFs and BMPs are critical in chondrocyte differentiation but do not seem to be involved in the condensation process. In vertebrates, cartilage is essential for prenatal and postnatal skeletal growth that results mainly from endochondral ossification, a complex process whereby a cartilage template is replaced with bone. Articular cartilage is essential for the functional integrity of the skeleton providing a cushion between the bones of the joints. Unlike most other tissues, cartilage lacks regenerative ability. Therefore, treatments for traumatic, congenital, degenerative cartilage diseases are primarily palliative. This has led to efforts to develop alternative means to restore damaged cartilage. The appearance of the cartilaginous tissue during fracture callus formation and the capacity of articular cartilage to repair when the damage penetrates the subchondral bone are evidence that there are chondroprogenitors in adult tissues and that these cells can differentiate into chondrocytes under appropriate conditions. The importance of bone marrow (BM) to provide chondroprogenitors to heal articular cartilage defects is seen by the lack of repair when the subchondral bone is not penetrated and BM chondroprogenitors are inaccessible to the site. Unfortunately, the repair of full-thickness articular cartilage defects by BM cells has been less than ideal, with the repair tissue being more of a fibrocartilage than a hyaline cartilage and consequently more fragile. Therefore, strategies need to be developed to deliver appropriate chondroinductive growth factors to optimize the BM-derived chondroprogenitor healing capacity.

Adult BM-Derived Mesenchymal Stem Cells: Potential Source for Cartilage

The evidence that BM contains mesenchymal stem cells (MSC) that can differentiate into chondrocytes emerged in the 1970s. Friedenstein et al. [1] found that BM contained plastic-adherent cells that differentiated into bone and cartilage in vitro and in vivo. These initial observations indicated the therapeutic potential for MSC, resulting in a phenomenal expansion of MSC studies over the last decade. Culture techniques for the isolation and expansion of MSC from numerous species, including humans and rodents, have been developed [2]. The number of MSC in BM is low, but they are easy to isolate and expand; millions of cells can be generated from few milliliters of BM. Furthermore, MSC can be easily transfected with exogenous genes. For these reasons, the use of MSC for gene therapy appears to have several advantages over hematopoietic

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stem cells. Moreover, the use of MSC for cellular therapy offers advantages over embryonic stem cells: MSC can be autologous, are easy to harvest, are more abundant, have no evidence of tumorgenicity, and raise no ethical controversies. Identification of MSC has been mostly based on the absence of hematopoietic markers. Recently, a population of MSC that retains the chondrogenic potential has been characterized by Baddoo et al. [3], who immunodepleted murine BM from the hematopoietic CD34-, CD45-, CD11b-positive cells. The concept of a postnatal stem cell as a discrete cell type with specific molecular markers has been recently challenged. Blau et al. [4] have hypothesized that an adult stem cell, rather than being a discrete cellular entity, is a functional response of many types of diverse cells to microenvironmental regenerative cues. Under specific culture conditions MSC can differentiate into chondrocytes, adipocytes, myocytes, osteocytes, cardiomyocytes and neural cells [2]. MSC cultured in high density in a defined serum-free medium, in the presence of TGF-, express a chondrogenic potential [5–7]. Cultured in these chondrogenic conditions, MSC form pellets of aggregated cells that subsequently acquire markers of chondrogenic differentiation and hypertrophy within 14 days of culture [5]. The chondrogenic potential of the pellet culture is indicated by the fact that more than 90% of the cells recovered after 14 days of culture with TGF- have a chondrocyte phenotype [7]. Neocartilage generated from MSC, cultured in high-density aggregates with TGF- resembles native cartilage immunohistochemically and by gene expression pattern [5, 6]. It has been implied that the in vitro chondrogenic differentiation of adult BM-derived MSC recapitulates embryonic chondrogenesis [6]. The role of IGF-I in the chondrogenic potential of MSC is poorly understood. MSC express IGF-I and several IGFBPs including IGFBP-3 but most studies have reported that the addition of IGF-I to the chondrogenic or expansion medium has no effects on MSC [3, 8]. Although this suggests that IGF-I seem to be dispensable, it is important to emphasize that the insulin content in the ITS (insulin-transferrin-selenious acid) mixture added to the chondrogenic medium is 100 times above physiological levels. Since insulin, as compared to IGF, has an affinity of almost two orders of magnitude lower for the IGF receptor, it is possible that the lack of IGF biological activity was due to the non-specific stimulation of IGF receptor by insulin. In our studies, we have evaluated the IGF effects in the absence of insulin. We have found that IGF-I increases MSC pellet size and induces proteoglycan (PG) synthesis with a potency that equals TGF- effect (see section below). In vivo animal studies have shown that MSC infused systemically or implanted locally home into several tissues (especially damaged tissues) including bone and cartilage [9]. MSC implanted in rodents have been shown to

IGF System in MSC Chondrogenic Potential

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form and reconstitute damaged cartilage and infused in a caprine animal model for osteoarthritis delayed the disease progression [9]. Initial clinical studies, including a study in children with severe osteogenesis imperfecta, have demonstrated that MSC or whole BM infusions can be used to repair damaged tissues, including bone [9, 10]. In the 3 children over 5 with osteogenesis imperfecta in which donor engraftment was achieved, growth velocity and bone density increased significantly [10]. These initial animal and clinical studies although promising showed that the number of engrafted cells was low. In addition, it was not determined whether the clinical improvement was due to the donor cells functionally reconstituting the damaged tissue. The ability of adult stem cells to migrate, engraft and differentiate into at least one cell type of a tissue other than the tissue of origin has been termed stem cell plasticity [11]. Several mechanisms have been proposed to explain adult stem cell plasticity that include: (1) transdifferentiation, the conversion of a dormant cell to different cell types; (2) dedifferentiation, the differentiation of a cell into a more primitive multipotent cell and redifferentiation into a new lineage; (3) pluripotency, the ability to differentiate into cells of multiple germ layers, and (4) fusion, the transfer of the genetic information to another cell. Dedifferentiation has not been found in mammalians [11]. Pluripotency has been demonstrated in a subpopulation of MSC after multiple passages [11]. Cell-cell fusion has been found in several in vivo and in vitro human and rodent systems [11]. While fusion has been implicated in the contributions of transplanted BM hematopoietic stem cells to adult tissues, it does not appear to be involved in the expression of muscle markers in transplanted MSC into injured muscles [2]. To optimize the therapeutic usage of MSC, we need to understand the mechanisms by which growth factors convert them from the process of self-replication to that of chondrogenic potential and differentiation. One rational strategy to identify such factors is to recapitulate the same growth factor environment that is found during cartilage development. This strategy has led us to hypothesize that IGF may play a central role in cartilage formation derived from MSC. Use of MSC in cartilage diseases offers several advantages, MSC can directly repopulate and repair the damaged cartilage, can be used as carriers for gene therapy in genetic diseases, and can be engineered to deliver chondroinductive growth factors. Potential clinical applications of MSC in cartilage disorders include genetic and degenerative diseases, traumatic lesions and fractures.

IGF in Cartilage

IGFs were first discovered by their ‘sulfation’ action on cartilage but the essential role of the IGF system in skeletal growth was definitively demonstrated


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by the severe growth failure observed in animals carrying null mutations of the genes encoding the Igfs and the Igf1R [12]. In rodents and humans, Igf1 gene deletion determines severe prenatal and postnatal growth retardation [12]. Since IGF-II expression (in rodents) is silenced in almost all tissues after birth, IGF2 null mice are born small but have a normal postnatal growth [12]. Igf1–/–Igf2–/– double mutants are born smaller than the single mutants [12]. Mice nullizygotes for the Igf1R die after birth and demonstrate growth retardation [12]. Studies directed at clarifying the precise mechanisms of IGF action in the chondrogenesis process have led to contradictory results and theories [13]. The classical somatomedin hypothesis postulates that growth hormone (GH) stimulates IGF-I synthesis, mainly in the liver. IGF-I is then transported to the growth plate, where it acts as an endocrine factor. The modified somatomedin hypothesis postulates that IGF-I is produced in the growth plate under the stimulation of GH and acts as a paracrine/autocrine factor. In addition to these two hypotheses, is the dual-effector theory that advocates that GH has a cell target that is independent from IGF promoting the differentiation of resting chondrocytes to chondrocytes. The dual-effector theory has been questioned by the report that in double Gh-receptor/Igf1 nullizygotes, GH and IGF-I appear to have independent and overlapping effects on the same cell target, namely chondrocytes [14]. More recently, Yakar et al. [15] have reported that mice that have double disruption of the liver Igf1 (LID) and Als (ALSKO) genes have 85–90% reduction in IGF-I circulating levels and show postnatal growth failure. This finding supports the classical somatomedin hypothesis and indicates that there is a threshold concentration for circulating IGF-I that is essential to sustain a normal growth. Evaluation of the growth plate morphometry in Igf1, Ghr and double mutant nullizygotes have clearly indicated that IGF-I and GH are essential for the differentiation of proliferative into hypertrophic chondrocytes [14, 16]. However, it is unclear the reason that determines the expansion of the germinal zone in all the nullizygotes [14, 16]. In addition to their roles in the skeletal growth process, IGFs are also key players in the adult articular cartilage homeostasis, owing their potent anabolic effects on matrix production [17]. Furthermore, IGF-I blocks inflammatory effects of cytokines that induce cartilage breakdown.

IGFBP-3 in Cartilage

IGFBP-3 is the predominant circulating IGF carrier protein in postnatal life [13, 18]. By binding IGFs, IGFBP-3 modulates their biological functions: it prolongs half-life, protects against the acute insulin-like effects, acts as transport protein across the capillary barrier, and modulates presentation to their


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receptors [13, 18]. Transgenic mice overexpressing IGFBP-3 exhibit prenatal and postnatal skeletal growth retardation, low bone density and insulin resistance and these effects cannot be explained by a decrease of free (bioavailable) IGF-I, increase of GH secretion or adiposity [19]. Well characterized as an IGF carrier, IGFBP-3 has been extensively reported to exert pleiotropic effects on diverse cell types and to have a broad range of functions that are independent of its binding to IGF [13, 18]. Through this IGF-independent action, IGFBP-3 has been shown to control cell proliferation, glucose uptake and to induce or enhance apoptosis [13, 18]. Furthermore, IGFBP-3 mediates the antiproliferative and pro-apoptotic effects of multiple agents, including TGF-, p53, TNF-, retinoid acid, antiestrogens and vitamin D analogs [18]. IGFBP-3 binds to several circulating factors and cellular proteins located in different cell compartments. It binds to extracellular matrix proteins and to a cell-membrane receptor and transported to the nucleus by the importin -subunit, binds to the retinoid X receptor- [18]. In addition, IGFBP-3 binds to fibrinogen, fibrin, plasminogen, transferrin, fibronectin and humanin [18]. We have used the RCJ3.1C5.18 chondrogenic cell line to study the effects of IGFBP-3 in chondrogenesis. The RCJ3.1C5.18 cell system is a wellestablished model to study cartilage formation in vitro; cells, derived from rat calvaria, are not transformed and they sequentially acquire over 14 days of culture markers of chondrocyte differentiation and hypertrophy [20]. Furthermore, RCJ3.1C5.18 cells do not express IGFs and IGFBP-3, and peptide effects can be studied without the interference of endogenous proteins [20]. We have found that in RCJ3.1C5.18 chondroprogenitors, IGFBP-3 has IGF-independent antiproliferative and apoptotic effects [20–22]. The IGFBP-3 growth inhibitory action is related to the differentiation stage of the cells, in fact it is IGF-independent and IGF-dependent in chondroprogenitors while in terminally differentiated chondrocytes the effect is exclusively IGF-dependent [20]. Our laboratory has been the first to identify a functional signaling pathway for IGFBP-3 [21]. We have demonstrated that in RCJ3.1C5.18 chondroprogenitors, IGFBP-3 induces STAT-1 phosphorylation as well as transcription and through STAT-1 activation IGFBP-3 induces cell apoptosis [21]. In addition, we have determined that IGFBP-3, by decreasing chondroprogenitor number, regulates chondrocyte differentiation [21]. Articular chondrocytes from patients with osteoarthritis produce more IGFBPs than cells isolated from normal cartilage [17, 23]. In particular, an IGFBP-3 increase correlates with the severity of the disease [23]. In synovial fluid from patients with rheumatoid arthritis and osteoarthritis, a derangement of the IGF system has been reported: IGFBP-3 was found to be increased up to 24-fold; IGFBP-3 proteolysis was decreased; IGF-I levels were increased (up to 3.5-fold) but primarily associated to the 150-kDa ternary complex [24]. These


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findings indicate that in arthritic diseases, increase of IGFBPs leads to a decrease in IGF-I availability and support the hypothesis that in these conditions disruption of the IGF system renders the cartilage insensitive to the anabolic actions of IGF. Children with chronic renal failure have severe growth retardation and complex disturbances of the IGF system. These include an increase of a 29-kDa IGFBP-3 fragment in the circulation as well as in the extracellular spaces. Purified from peritoneal dialysate, this IGFBP-3 fragment has been shown to have a marked lower affinity for IGF than intact IGFBP-3 and to inhibit IGF-II mitogenic effect in a chondrosarcoma cell line [25]. IGF-I Chondroinductive and IGFBP-3 Chondroinhibitory Effects in the Cartilage Formation Process Derived from Mesenchymal Stem Cells and Chondroprogenitors

Materials and Methods Mouse BM MSC (mMSC) Isolation, Expansion and Chondrogenic Micromass Pellet Formation As schematically presented in figure 1, BM was obtained from 1-month-old mice by flushing the shaft of dissected femoral and tibial diaphyses. Nucleated BM cells were plated in fibronectin-coated plates in 60% DMEM-LG, 40% MCDB201, ITS-Premix 1, LA-BSA 1, ascorbate-2-phosphate (10–4 M), hPDGF-BB (10 ng/ml), EGF (10 ng/ml), mLIF (10 ng/ml), dexamethasone (10–9 M) and 2% FBS (lot selected for maximal growth by CFU assay; Atlanta Biological, Atlanta, Ga., USA) [7]. All products were from Sigma Chemical Co. (St. Louis, Mo., USA), except for DMEM-LG (Invitrogen Life Technologies, Carlsbad, Calif., USA), ascorbate-2-phosphate (Wako Pure Biochemicals, Ltd, Osaka, Japan), hPDGF-BB (R&D Systems, Minneapolis, Minn., USA), ITS-Premix (BD Biosciences, Bedford, Mass., USA) and mLIF (Chemicon International, Temecula, Calif., USA). After 3 days of culture, cells were washed with PBS and adherent cells were expanded for 7–10 days (80% confluence). Cells were used within 1–2 passages. To enrich the population of MSC, a negative selection was performed eliminating the CD34, CD45, and CD11b hematopoietic cells using a MACS magnetic immunodepletion system (Miltenyi Biotec, Auburn, Calif., USA) [3]. Antibodies were obtained from BD Pharmingen. After immunodepletion 95% of the cells have a spindle-shape phenotype. Immediately after selection, mMSC were induced to express the chondrogenic potential using a micromass pellet technique [5]. MSC (2 105) were spun down in 15-ml polypropylene conical tubes and placed in a defined serum-free medium consisting of


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Mouse femur and tibia

Human iliac crest

Isolation of mononuclear cells

Ficoll separation only for human BM

Expansion of adherent cells

FN coated plates 2% FBS hPDGF-BB, mEGF LIF (only for mMSC)

Immunodepletion of CD45/CD34/CD11bcells

7–10 days after harvesting

Chondrogenic micromass pellet

1.5 2 105 15ml tube with SF defined medium 24 hour-old pellet

Fig. 1. Diagram showing hMSC and mMSC isolation, expansion, selection and chondrogenic micromass pellet culture. BM Bone marrow; FN fibronectin; FBS fetal bovine serum; SF serum-free medium.

DMEM-HG, insulin-transferrin-selenious acid (ITS-Premix) 1 mixture (containing 6.2 g/ml of insulin), dexamethasone (10–7 M), L-glutamine (2 mM) (Sigma), pyruvate (100 g/ml) (Sigma), ascorbate-2-phosphate (50 g/ml), porcine TGF-1 (10 ng/ml) (R&D Systems). Chondrogenic medium was changed every 3 days. To determine the effect of IGF-I, without interference with the insulin contained in the ITS, we removed insulin from the mixture (TS). To avoid interference with endogenous IGFBPs, we employed des-IGF-I, an IGF analog that binds the IGF receptor with the same affinity of IGF-I but has ⬃100 times less affinity for IGFBPs. Cells were treated with des-IGF-I (100 ng/ml) (Diagnostic System Laboratories, Inc. Webster, Tex., USA) every 3 days. Human BM MSC (hMSC) Isolation, Expansion and Chondrogenic Micromass Pellet Formation hMSC have been isolated from 5 ml of iliac crest BM aspirate obtained from normal adult donors undergoing iliac crest bone graft harvest, after subjects had signed informed consent (IRB approved by Vanderbilt University). Subjects


24

were 18–50 years old, without signs of infection or cancer. To expand and select hMSC we used protocols similar to mMSC; the only differences were that nucleated BM cells were isolated from BM using Percoll (Amersham, Piscataway, N.J., USA) gradient fractionation and mLIF was not added to the expansion medium used to culture hMSC. RCJ3.1C5.18 Mesenchymal Chondrogenic Cells RCJ3.1C5.18 cells were grown as previously reported [20]. Briefly, cells were plated at a density of 6 104 cells/well in 6-well dishes and grown in MEM supplemented (Gibco) with 15% heat-inactivated fetal bovine serum, 10–7 M dexamethasone, and 2 mM sodium pyruvate. After reaching confluence (4 days of culture), fresh differentiating medium supplemented with 50 g/ml of ascorbic acid and 10 mM -glycerophosphate (Gibco) was added and changed every 3 days. We have previously shown that cells grown in this manner undergo a reproducible, time-dependent progression from chondroprogenitors to early differentiated chondrocytes [20]. Measurement of Cell Apoptosis Immediately after MSC were pelleted for micromass culture, recombinant ND-IGFBP-3 (30 nM) (Celtrix Pharmaceuticals, Inc., Santa Clara, Calif., USA) was added to the chondrogenic medium and cells were incubated for 48 h. Cell apoptosis was determined using a quantitative cell death detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany) as previously reported [21]. Briefly, cell lysates were obtained following the manufacturer’s instruction and subjected to a quantitative sandwich enzyme immunoassay, using antibodies directed against DNA and histones. This allows the specific determination of mono- and oligonucleosomes, which are released into the cytoplasm of apoptotic cells. PG Synthesis Assay PG synthesis was quantified by Alcian Blue staining as previously described [22]. Two days after seeding, RCJ3.1C5.18 cells were treated with or without IGFBP-3 (30 nM) for 5 h in serum-free medium, followed by treatment with or without TGF- (5 ng/ml) that was also added at 4-day culture. At 7-day culture, cells were Alcian Blue assayed. mMSC and hMSC pellets were incubated in defined medium with or without growth factors and stained with Alcian Blue at 2, 7, and 14 days of culture. Defined medium contained TS mixture (without insulin) or ITS mixture (with insulin 6.2 g/ml) as indicated. Medium was changed every 3 days and growth factors were added in accordance. For Alcian Blue assay, cells were rinsed with 95% ethanol (RCJ3.1C5.18 cells) or PBS (MSC), stained with Alcian Blue (1% in 3% acetic acid) for 30–60 min, rinsed in 3% acetic acid and solubilized in 1% SDS. Alcian Blue absorbance is measured at 605 nm.


25

Mouse pellets 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 TGF-

a

Alcian Blue AU605 *p 0.05 vs control without TGF- n 3

2D pellet

7D pellet Human pellets

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 TGF-

b

14D pellet *

*

Alcian Blue AU605 *p 0.05 vs. control without TGF- n 3

2D pellet

*

*

7D pellet

14D pellet

Fig. 2. PG synthesis in mMSC and hMSC chondrogenic pellet: TGF- effect and timecourse. Chondrogenic mMSC (a) and hMSC (b) pellets were incubated in defined chondrogenic medium with or without TGF- (10 ng/ml) over 14 days of culture. Medium was changed every 3 days. To determine PG synthesis, pellets were subjected to Alcian Blue assay after 2, 7, and 14 days of culture (more details in the Materials and Methods section). Alcian Blue absorbance is measured at 605 nm.

Statistics Data are presented as mean SD. Statistical differences between groups were assessed by one-way ANOVA followed by Student-Newman-Keuls test for all pairwise multiple comparisons, or, when necessary, by one-way ANOVA on ranks (Kruskal-Wallis) followed by Student-Newman-Keuls test. Statistical significance was set at p 0.05; analysis was performed using Sigmastat Package (Jandel Scientific, San Rafael, Calif., USA). Results Selected mMSC undergo chondrogenic pellet formation after 1 day of culture (fig. 1). As shown in figure 2, cells treated with TGF- produced an increasing amount of PG over 14 days of culture. At 14 days, a pellet was


26

Cont

TGF-

IGF-I

TGF- IGF-I

Cont

TS

TGF- ITS

1 cm

Fig. 3. IGF-I increases chondrogenic pellet size; the effect is similar to TGF-. mMSC chondrogenic pellets were incubated in defined medium: (1) without peptides (control), or with (2) TGF- (10 ng/ml), or with (3) IGF-I (100 ng/ml), or with (4) IGF-I (100 ng/ml) and TGF- (10 ng/ml). Defined medium contained TS mixture (without insulin) or ITS mixture (with insulin 6.2 g/ml) as indicated. Medium was changed every 3 days and growth factors were added in accordance. Picture depicts MSC pellets at 7-day culture. Relative size of the pellets is indicated by a 1-cm ruler on the bottom.

detectable in only 20% of the cultures incubated without TGF-. hMSC treated with TGF- showed a similar amount of PG synthesis compared with mMSC, with a similar time-course over 14 days (fig. 2). We noted that hMSC were larger than mMSC. We then evaluated the effect of IGF-I on chondrogenic pellet size. We found that mMSC treated with IGF-I or TGF- formed pellets that at 7 days were at least twice bigger than control (fig. 3). IGF-I (added to TS mixture without insulin) was more effective than insulin (ITS mixture with 6.2 g/ml of insulin) in enhancing pellet size (fig. 3). These findings indicate that IGF-I increases pellet size similarly to TGF- and it is more potent than supraphysiological doses of insulin. To determine whether the effect of IGF-I on pellet size was associated with an increase of chondrocyte differentiation, we evaluated PG synthesis by Alcian Blue staining. As shown in figure 4a, in the presence of IGF-I, MSC, grown in pellets for 7 days, synthesized more PG than control, and the effect of IGF-I was similar to TGF-. The two growth factors had combined effects. Similar results were seen in hMSC (fig. 4b). We also found that IGF-I had effects similar to insulin (ITS mixture with 6.2 g/ml of insulin) indicating that the chondroinductive action of insulin may be an IGF effect (fig. 4a). We have previously shown that IGFBP-3 induces cell apoptosis in RCJ3.1C5.18 chondroprogenitors. We now report that IGFBP-3 has a proapoptotic action in BM-derived MSC; we found that IGFBP-3 treatment increased apoptosis to 195 15% of control untreated cells (p 0.05; n 6). Since BMderived MSC produce IGF while RCJ3.1C5.18 cells do not, it is unclear if this effect is related to an IGF-dependent or -independent action of IGFBP-3.


27

% TS control

500 400 300

Mouse pellets *• *p 0.05 vs. TS control •p 0.05 vs. TGF- * and IGF-I * n 3

* *

200 100 0 Control

TGF-

a

IGF-I TS

IGF-I Control TGF-

Human pellets % Control

400 300 200

*•

*p 0.05

vs. control •p 0.05 vs. TGF- * and IGF-I n3

TGF- ITS

*

100 0 Control

TGF-

b

IGF-I

IGF-I TGF-

Fig. 4. IGF-I induces chondrocyte differentiation in mMSC and hMSC pellets as indicated by PG synthesis increase. mMSC (a) and hMSC (b) pellets were incubated in defined medium: (1) without peptides (control), or with (2) TGF- (10 ng/ml), or with (3) IGF-I (100 ng/ml), or with (4) IGF-I (100 ng/ml) and TGF- (10 ng/ml). Defined medium contained TS mixture (without insulin) or ITS mixture (with insulin 6.2 g/ml) as indicated. Medium was changed every 3 days and growth factors were added in accordance. To determine PG synthesis, pellets were subjected to Alcian Blue assay at 7 days of culture (more details in the Materials and Methods section). Alcian Blue absorbance is measured at 605 nm and results are expressed as percentage of absorbance measured in the control, which was given an arbitrary value of 100%.

We next examined if IGFBP-3 had an IGF-independent chondroinhibitory action on TGF-. We found that IGFBP-3, in RCJ3.1C5.18 cells, inhibited PG synthesis and blocked the effect of TGF- (fig. 5).

Conclusion

Understanding the role of the IGF system in the chondrogenic potential of MSC and chondroprogenitors may provide critical information to optimize the therapeutic use of MSC to treat cartilage disorders. Identification of the molecular mechanisms underlying these growth factor effects will help us to develop gene therapy aimed at transferring IGF-I therapeutic effects into MSC or at inhibiting IGFBP-3 chondroinhibitory action.


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140 120

*p 0.05

vs. control •p 0.05 vs. TGF- n3

*

•

% Control

100 80 * 60 40 20 0 Control

IGFBP-3

TGF-

IGFBP-3 TGF-

Fig. 5. IGFBP-3 has a chondroinhibitory action on TGF--induced PG synthesis. Two days after seeding, RCJ3.1C5.18 cells were treated with or without IGFBP-3 (30 nM) for 5 h in serum-free medium, followed by treatment with or without TGF- (5 ng/ml) that was also added at 4-day culture. Cells were subjected to Alcian Blue assay at 7 days of culture (more details in the Materials and Methods section). Alcian Blue absorbance is measured at 605 nm and results are expressed as percentage of absorbance measured in the control, which was given an arbitrary value of 100%.

Acknowledgments This work was supported in part by Lawson Wilkins Pediatric Endocrine Society Clinical Scholar Award and by Vanderbilt University Physician Scientist Development Program Award (to A.S.)

References 1 2 3 4 5 6

7 8

Friedenstein AJ, Chailakhyan RK, Gerasimov UV: Bone marrow osteogenic stem cells: In vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–272. Prockop DJ, Gregory CA, Spees JL: One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci USA 2003;100(suppl 1):11917–11923. Baddoo M, Hill K, Wilkinson R et al: Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem 2003;89:1235–1249. Blau HM, Brazelton TR, Weimann JM: The evolving concept of a stem cell: Entity or function? Cell 2001;105:829–841. Johnstone B, Hering TM, Caplan AI et al: In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265–272. Sekiya I, Vuoristo JT, Larson BL, Prockop DJ: In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA 2002;99:4397–4402. Qi H, Aguiar DJ, Williams SM et al: Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci USA 2003;100:3305–3310. Milne M, Quail JM, Rosen CJ, Baran DT: Insulin-like growth factor binding proteins in femoral and vertebral bone marrow stromal cells: Expression and regulation by thyroid hormone and dexamethasone. J Cell Biochem 2001;81:229–240.


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Barry FP, Murphy JM: Mesenchymal stem cells: Clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568–584. Horwitz EM, Prockop DJ, Gordon PL et al: Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001;97:1227–1231. Wagers AJ, Weissman IL: Plasticity of adult stem cells. Cell 2004;116:639–648. Baker J, Liu JP, Robertson EJ, Efstratiadis A: Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993;75:73–82. Spagnoli A, Rosenfeld RG: The mechanisms by which growth hormone brings about growth. The relative contributions of growth hormone and insulin-like growth factors. Endocrinol Metab Clin North Am 1996;25:615–631. Lupu F, Terwilliger JD, Lee K et al: Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 2001;229:141–162. Yakar S, Rosen CJ, Beamer WG et al: Circulating levels of IGF-1 directly regulate bone growth and density. J Clin Invest 2002;110:771–781. Wang J, Zhou J, Bondy CA: Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy. FASEB J 1999;13:1985–1990. Martel-Pelletier J, Di Battista JA, Lajeunesse D, Pelletier JP: IGF/IGFBP axis in cartilage and bone in osteoarthritis pathogenesis. Inflamm Res 1998;47:90–100. Firth SM, Baxter RC: Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 2002;23:824–854. Modric T, Silha JV, Shi Z et al: Phenotypic manifestations of insulin-like growth factor-binding protein-3 overexpression in transgenic mice. Endocrinology 2001;142:1958–1967. Spagnoli A, Hwa V, Horton WA et al: Antiproliferative effects of insulin-like growth factor-binding protein-3 in mesenchymal chondrogenic cell line RCJ3.1C5.18 relationship to differentiation stage. J Biol Chem 2001;276:5533–5540. Spagnoli A, Torello M, Nagalla SR et al: Identification of STAT-1 as a molecular target of IGFBP-3 in the process of chondrogenesis. J Biol Chem 2002;277:18860–18867. Longobardi L, Torello M, Buckway C et al: A novel insulin-like growth factor (IGF)-independent role for IGF binding protein-3 in mesenchymal chondroprogenitor cell apoptosis. Endocrinology 2003;144:1695–1702. Morales TI: The insulin-like growth factor binding proteins in uncultured human cartilage: Increases in insulin-like growth factor binding protein 3 during osteoarthritis. Arthritis Rheum 2002;46:2358–2367. Whellams EJ, Maile LA, Fernihough JK et al: Alterations in insulin-like growth factor binding protein-3 proteolysis and complex formation in the arthritic joint. J Endocrinol 2000;165:545–556. Durham SK, Mohan S, Liu F et al: Bioactivity of a 29-kilodalton insulin-like growth factor binding protein-3 fragment present in excess in chronic renal failure serum. Pediatr Res 1997;42:335–341.

Anna Spagnoli, MD Department of Pediatrics, Vanderbilt University Medical Center T-0107 Medical Center North Nashville, TN 37232-2579 (USA) Tel. 1 615 9363198, Fax 1 615 9363198, E-Mail [email protected]


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Treatment in Animal Models J. Guan, L. Bennet, P.D. Gluckman, A.J. Gunn The Liggins Institute, Department of Physiology and Departments of Paediatrics and Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, The University of Auckland, Auckland, New Zealand

Abstract It is now well established that neurons and other cell types may die many hours or even days after hypoxic-ischemic injury due to activation of programmed cell death (apoptotic) pathways. The potent anti-apoptotic factor IGF-1 and its binding proteins and receptors are intensely induced within damaged brain regions following brain injury suggesting a possible a role for IGF-1 in endogenous brain recovery. Exogenous administration of IGF-1 within a few hours after brain injury has now been shown to be protective in both grey and white matter, and leads to improved long-term neurological function. The limited window of opportunity for treatment with IGF-1 can be extended by spontaneous mild post-hypoxic hypothermia, probably due to delayed evolution of apoptotic processes. The efficacy of IGF-1 is specific to particular cellular phenotypes and brain regions, and its neuroprotective effects are mediated by IGF-1 receptors and binding proteins. Intriguingly its naturally cleaved N-terminal tripeptide (glycine-proline-glutamate, GPE) has been demonstrated to be neuroprotective after both central and peripheral administration. Peripheral administration of GPE also prevents the loss of dopamine neurons and improves long-term functional recovery following 6-OHDA lesion. However, GPE is unlikely to contribute significantly to the direct effects of IGF-1. Copyright © 2005 S. Karger AG, Basel

IGF-1 Induction and Delayed Neuronal Injury after Ischemic Brain Injury

Hypoxic-ischemic (HI) encephalopathy is generally a consequence of a systemic asphyxial insult leading to hypoxemia, acidosis and cardiovascular compromise causing arterial hypotension and thus impaired cerebrovascular perfusion. The cerebral insult is thus typically global and reversible rather than focal as seen with stroke. The major causes of HI encephalopathy include cardiac arrest, perinatal asphyxia, and near-drowning.

It is now well established that ischemic brain injury is not a single ‘event’ that necessarily occurs during the insult, but rather it is an evolving process precipitated by the insult. Although neurons may die during the actual ischemic or asphyxial event (primary cell death), many neurons only die many hours, or even days later. Recently, intensive research in the factors involved in cell death have made it clear that an acute event activates both protective and damaging endogenous responses, many mediated by glia. The timing of terminal cell death has been defined in many models, and is strongly related to the severity of the primary injury. For example, DNA fragmentation in the hippocampus can be detected as early as 10 h after a 60 min HI injury, whereas DNA fragmentation in the hippocampus was only detectable 3–5 days after a shorter, 15-minute HI injury in infant rats [1]. In parallel to the temporal loss of neurons, a progressive induction of IGF-1 was detected within activated astroglia of damaged brain regions 3–5 days after HI injury in infant rats, which was closely correlated with the severity of brain injury [2]. The induction of IGF-1 in the adult appears to be slower. For example, enhanced immunoreactive IGF-1 protein release was found in microdialysate within the hippocampal region only from 7 to 14 days following an electrolytic lesion. The induction of IGF-1 is specific since, unlike IGF-1, HI injury associated IGF-2 expression in infant rats occurs much later after the insult. Further, IGF-2 was found only in microglia or tissue macrophages [3] both of which play similar roles in removing neuronal debris following infarction. Interestingly, IGFBP-2 and -3 were temporally co-expressed with IGF-1 following HI injury, however they were induced in the penumbral region instead of in the core area of infarction. The similar time courses of delayed neuronal death and endogenous IGF-1 induction suggest the possibility of a window of opportunity for neuronal rescue by IGF-1, whereby early intervention with exogenous IGF-1 prior to endogenous induction may be able to arrest the development of post-ischemic brain injury. In this chapter, we review the evidence for neuroprotection with IGF-1 and its N-terminal tripeptide, glycine-prolineglutamate (GPE).

Neuroprotection and IGF-1

Dose Dependence Using a modified ‘Levine’ preparation (unilateral carotid ligation and 10 min of 6% hypoxia in adult rats), IGF-1 was first tested in adult rats. A single dose of 5 or 50 ␮g, but not 0.5 ␮g rhIGF-1 given intracerebroventricularly (ICV) 2 h following HI injury reduced cortical infarction. Fifty but not 5 ␮g of rhIGF-1 also significantly reduced selective neuronal loss [4]. Consistent with

Guan/Bennet/Gluckman/Gunn

32

this finding, Bergstedt and Wieloch [5] also showed that 2 ␮g of IGF-1 fails to reduce neuronal loss after ischemic injury in rats. A bell-shaped dose response of IGF-1 effects on neuronal survival has also been reported in fetal sheep with a 1-hour ICV infusion started 2 h after a 30 min period of cerebral ischemia and reperfusion. However, the neuroprotective dose range was much lower (1–3 ␮g) [6] than in adult rats (20–50 ␮g), despite the much larger size of a fetal lamb. The much shorter CSF turnover in rats (⬍1 h in the rat vs. 2 h in sheep), and the longer infusion period in the fetal sheep studies (1 h in sheep vs. 10–20 min in rats) may have resulted in better maintained efficacious IGF-1 levels in the CSF. Perhaps more importantly, the homology of the amino acid sequence of rh-IGF-1, which was used in the studies in both species, is much greater for sheep IGF-1 than for rat IGF-1. Selectivity or Brain Regions and Phenotypes Neuroprotection with exogenous IGF-1 after HI injury in the rat was selective, greatest in the lateral parietal cortex, the dentate gyrus of the hippocampus and intermediate in the striatum, the thalamus, and some subregions of the hippocampus, with no effect in the CA1–2 subregion of the hippocampus. This relationship corresponded closely with the distribution of tritiated IGF-1 in the brain after ICV administration, with limited uptake of IGF-1 in the CA1–2 field of the hippocampus [4]. The lower levels of IGF-1 receptor density may explain the lack of treatment effects of IGF-1 in this region. However, in contrast to these results, in the fetal sheep model described above, an infusion of IGF-1 2 h after ischemia was associated with global protection, including in the hippocampus. This suggests that model species and/or maturational factors modify the effects of IGF-1. For example, in the adult rat the CA1–2 subregions of the hippocampus are much more sensitive to HI injury, whereas the most damaged subregion of hippocampus after 30 min ischemia is the CA3 subregion in fetal sheep [4]. Within particular brain regions, the effects of IGF-1 are not homogenous. This has been evaluated in the striatum, which contains many different neuronal phenotypes. Selective loss of striatal phenotypic neurons after ischemic brain injury alters the neurochemical anatomy and impairs the function of the basal ganglia. The neuropeptides, calcium binding proteins and enzymes for neurotransmitters that are used to mark phenotypic striatal neurons can be used to semiquantitatively assess the neurobioactivity of these phenotypes. In the near-term fetal sheep, a single dose of IGF-1 selectively prevented the loss of ChAT, CAD, nNOS, NPY and calbindin-containing neurons in the striatum, but had no effect on the post-ischemic loss of parvalbumin-containing neurons [4].

Treatment in Animal Models

33

Functionally, IGF-1 appears to be involved in the regulation of both peripheral and central cholinergic activity. Peripheral cholinergic blockade results in an increase in circulating IGF-1 and IGFBP-3. In vitro studies have suggested that IGF-1 affects acetycholine (ACh) release differently across brain regions. IGF-1 stimulates ACh release in the cerebral cortex but inhibited ACh release in the hippocampus and had no effect in the striatum. Reduced ACh release from presynaptic neurons can result in post-synaptic neuronal dysfunction and increased secondary neuronal death. Given that IGF-1 reduces the loss of cholinergic neurons after HI injury, IGF-1 may have a specific therapeutic role in neurological disorders due to deficiency of ACh. In the fetal sheep, ischemia resulted in an approximately 50% loss of NPY neurons. Interestingly, there was a threefold increase in the number of NPY neurons after IGF-1 treatment, possibly due to induction of NPY by IGF-1 in non-NPY neurons. In sham control fetal sheep, the population of nNOS-positive neurons was threefold higher than NPY-positive neurons, which are co-localized in adulthood. It is not known whether this relatively high nNOS expression in fetal brains reflects a specific role in brain development. However, NOS appears to play an important role in recovery from ischemia. For example, selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin was protective up to 24 h after HI. Interestingly, neuroprotection with IGF-1 after spinal cord injury was also associated with reduced up-regulation of nNOS immunoreactivity. Parvalbumin immunoreactivity is co-localized with GAD-positive neurons in adulthood. Surprisingly, while IGF-1 failed to protect parvalbumin neurons, the loss of GAD-positive neurons was completely restored by IGF-1 treatment [4]. This suggests that IGF-1 not only protects GAD-containing neurons, but may also promote GAD expression in neurons which normally do not express GAD. Conversely, the loss of parvalbumin immunostaining might be due to a combination of neuronal loss and reduced parvalbumin production in parvalbumin neurons after ischemia, which appears differentially modulated by IGF-1 treatment compared to GAD. Consistent with these speculations, IGF-1 has been found to promote GAD immunoreactivity of striatal neurons in culture; this effect is presumably via IGF-1 receptors, as insulin shows a competitive effect. The extracellular accumulation of glutamate during brain injury is believed to play a key role in the pathogenesis of ischemic neuronal death. With its ability to convert glutamate to GABA, increased GAD expression may help reduce accumulation of excitotoxins and thus protect neurons from glutamateinduced excitotoxicity. Thus, speculatively, alleviation of the excitotoxic effects of glutamate and increased GABA activity after ischemia may be a possible mechanism of neuronal rescue by IGF-1.


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Window of Opportunity for Treatment In animal experimental studies, most neuroprotective compounds are much more effective when given before, rather than after brain injury. In contrast, IGF-1 does not improve neuronal outcome when given before ischemia in rats [4]. Nevertheless, pre-treatment per se with IGF-1 does not seem to be harmful, as IGF-1 still improved outcome when treatment was initiated 30 min before brain injury but was then continued for 7 days [7]. One possible explanation for the lack of a pre-treatment effect of IGF-1 might be less effective tissue penetration into the normal brain, as the tissue penetration of IGF-1 is largely injury-dependent. There are conflicting data on whether there is additional benefit from prolonged infusions over many days compared with short infusions. In adult rats, continuous central (ICV) administration of 50 ␮g IGF-1 per day over a 7-day period has been reported to reduce neuronal death in the CA1–2 subregion of the hippocampus in the rat, whereas this region was not protected after a single dose of 50 ␮g IGF-1 [7]. In contrast, in near-term fetal sheep a further 24-hour infusion of IGF-1, following an initial 1-hour infusion, did not improve neuroprotection further compared with the 1-hour infusion alone [unpubl. data]. It is unclear whether these contrasting results reflect maturational differences in the rate of evolution of neuronal loss or other experimental differences. Further studies are needed to resolve whether multiple doses and/or longer treatment periods may improve long-term neuroprotection following acute ischemic brain injury. A key practical issue is that recruitment of patients in a timely manner is a formidable problem. Despite the proven efficacy of anticoagulation after acute ischemic stroke for example, only 5–15% of the patients are able to be enrolled within the critical time of 2–4 h. Such early administration also appears to be important for IGF-1. Whereas 50 ␮g of IGF-1 was protective when given 2 h after HI, it was not protective when administered 6 h after HI injury [4]. This rapid loss of efficacy is most easily understood in relation to the different phases of post-ischemic neuronal loss. IGF-1 is known to suppress apoptosis through mechanisms involving activation of multiple protein kinase pathways which are most likely to be important early in the latent phase of evolving programmed cell death, prior to initiation of the intranuclear execution phase of apoptosis. Thus it is likely that an additional reason for the failure of pre-insult treatment of IGF-1 is that the relevant mechanisms are not active until a critical time window after reoxygenation. To be clinically useful, a broad window of therapeutic opportunity is highly desirable. One suggested approach that might allow such an extended window of opportunity is mild cerebral hypothermia. Brain injury is commonly associated with a decline in brain temperature during and for a short period after an insult, unless it is actively prevented by peri-insult warming. Several


35

studies in adult and newborn animals have reported that short periods of active post-insult cerebral cooling delay neuronal death without reducing long-term neuronal loss. Similarly, in a recent study, 2 h recovery in a relatively cool (23⬚C) environment was not sufficient for neuroprotection compared to recovery in a warm (34⬚C) environment. Nevertheless, this interval of mild relative brain cooling significantly extended the window for IGF-1 treatment to 6 h after HI. These data confirm that the brief period of injury-induced cerebral cooling mainly acts to delay the development of neuronal loss rather than to prevent it, and that it can prolong the window of opportunity for definitive treatment. Progressive Neuronal Injury and Functional Recovery The experience from large clinical trials of drugs that are neuroprotective in animal experiments has been highly disappointing to date. Loss of early protection in the long-term is one potential factor that may underlie this failure. In many experimental models in addition to the ‘secondary’ phase of neuronal loss in the first week or so after injury, delayed cell death can continue in a progressive ‘tertiary’ phase over many months. It is unknown how these data relate to the long-term evolution of damage in man. When adult rats subjected to unilateral HI were allowed to recover for 20 days, a large area of cavitation developed in the lateral cortex. However, within the penumbra surrounding the cavitated area many cells displayed acute ischemic cell change, with condensation of the nucleus and cytoplasmic eosinophilia [4]. This morphology was the same as that reported in neurons within the core area of damaged cortex 5 days post-injury, suggesting ongoing neuronal loss even 20 days after the injury. The causes of this chronic neuronal injury are not yet well understood. However, the post-ischemic microglial reaction can be associated with induction of by-products such as nitric oxide and cytokines [8]. These factors can benefit neuronal recovery, but can also initiate ‘next wave’ of programmed neuronal injury, and thus contribute to the tertiary neuronal degeneration [8]. Clearly this finding, that injury may not fully resolve for months after injury, makes it highly uncertain whether even dramatic short-term neuroprotection reflects true efficacy or is merely due to delayed evolution of neuronal death. Therefore, evaluation of long-term neuronal outcome and associated functional recovery are critical for determining treatment effects of compounds. The effect of IGF-1 on neurological function has been evaluated using an adhesive removal test in which an adhesive label was applied on the distal portion of the left and right forelimbs, and the time taken for the rat to contact each patch was measured. Because the injury induced by the HI model is unilateral, the effect of treatment can be assessed using the ratio of time taken for


36

each side. This ratio reflects changes in somatosensory-motor function and coordination. A single dose of 50 ␮g of IGF-1 given ICV 2 h after HI injury reduced the prolongation in patch contact time (L/R ratio) compared to the vehicle group, particularly 3 days after injury, when the deficit was greatest. Despite this significant improvement in functional recovery there was no effect of IGF-1 treatment on the extent of cortical infarction at day 20 [4]. In contrast, the percentage of progressive neuronal loss in the penumbra of the infarct was significantly reduced in the IGF-1-treated group, suggesting that this late neuronal loss is a greater contributor to the long-term behavioral deficit than the fixed area of the cortical infarct [4]. As a potent anti-apoptotic agent it is likely that IGF-1 simply reduced ongoing neuronal injury by blocking programmed cell death [9]. An alternative hypothesis is that IGF-1 may promote neurogenesis and neuroplasticity within the somatosensory system [10] that would help compensate for loss of neuronal function. One of the many suggested mechanisms for the gradual recovery of behavioral deficits after stroke is collateralization of neural function. It has been reported that IGF-1 can enhance adult sensory neuronal regeneration in vitro and nerve regeneration in vivo after injury. IGF-1 has also been shown to induce climbing nerve fibre re-innervation of the cerebellum after traumatic injury suggesting that it promotes neuronal plasticity [11]. Unlike the long-term functional recovery, the short-term functional recovery of IGF-1 was found to be associated with reduced cortical infarction 3 days after focal ischemia [12]. This finding also suggests that IGF-1 treatment may only delay the evolution of the core injury rather than preventing it. Despite limited penetration into the brain, peripheral administration of IGF-1 also seems to improve neural outcome after focal ischemia [13] and improve motor and cognitive function after traumatic brain injury. However it is not clear whether the transient hypoglycemia due to IGF-1 treatment may have contributed to the observed improvement [14], as mild hypoglycemia can be neuroprotective (discussed in more detail below).

White Matter Protection with IGF-1

The great majority of studies of ischemic injury, such as those discussed above, have focused on the mechanisms and potential treatment of neuronal injury. Although injury of immature white matter is well known to be the dominant cause of neural handicap in very premature infants [15], white matter damage in later life has been relatively neglected [16], in large part


37

because white matter is believed to be much less vulnerable to injury than grey matter. Recent studies have demonstrated that differentiated oligodendrocytes and myelinated axons are typically at least as vulnerable to ischemic injury as neurons, and in some conditions may be more vulnerable. For example, after focal ischemia in the adult rat, oligodendrocyte loss is reported to develop earlier than neuronal injury [17]. IGF-1 has potent survival and proliferative effects on myelinating glia in vitro, suggesting that it might be able to prevent post-ischemic demyelination. This has been tested in the fetal sheep model of reversible cerebral ischemia. Ischemia induced by 30 min of bilateral carotid occlusion resulted in a ‘watershed’ distribution of both white and grey matter injury, most severe in the parasagittal fronto-parietal gyrii. Post-ischemic cerebral white matter injury was characterized by profound loss of both myelin and myelinating oligodendrocytes marked using expression of proteolipid protein (PLP) mRNA within the intragyral white matter tracts [4]. There was an intense induction of reactive astrocytes and macrophages in both the intragyral white matter and the coronal radiata, whereas cell loss was almost entirely intragyral. This primarily peripheral pattern of injury is similar to findings in term babies with perinatal asphyxia [17]. Strikingly, cells with apoptotic morphology were widespread within the white matter tracts after ischemia and some of them expressed activated caspase-3. Co-localization between PLP and caspase-3 confirmed that these apoptotic cells were oligodendrocytes. Consistent with its neuroprotective dose range, a 1-hour infusion of IGF-1 (3 ␮g, ICV) reduced the post-ischemic loss of both myelin and bioactive oligodendrocytes (PLP-positive) in the parasagittal white matter tracts compared to the vehicle group. Potentially, a loss of PLP could be the result of either loss of oligodendrocytes or reduced myelin production by surviving but inactive oligodendrocytes. However, the number of cells expressing PLP in the vehicle and IGF-1-treated groups were closely correlated with the density of MBP, particularly in the intragyral white matter tracts. Furthermore, the degree of white matter protection by IGF-1 treatment was closely associated with suppression of activated caspase-3, a major component of the apoptotic pathways, in this region. These findings suggest that the acute post-ischemic demyelination is primary, i.e. due to loss of oligodendrocytes [4]. Furthermore, they demonstrate that exogenous IGF-1 can interrupt critical post-ischemic processes, occurring early in the ‘latent’ phase of recovery, that lead to white matter injury. The reduced demyelination after IGF-1 treatment is associated with a significant increase in numbers of reactive astrocytes, and also, surprisingly, reactive microglia [4], raising the possibility that reactive glia may indirectly mediate some of the protective effects of IGF-1.


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Modes of Action of IGF-1

Mediated by IGF-1 Receptor Insulin binds to the IGF-1 receptor with much weaker affinity than IGF-1. This has been used as a tool to examine the role of the IGF-1 receptor in neuroprotection. Guan et al. have demonstrated that a single dose of insulin, equimolar to the effective dose of IGF-1, failed to prevent neuronal loss and cortical infarction. A much higher dose of insulin than IGF-1 was required for neuroprotection in the cortex, where the density of insulin receptors is low. Mild hypoglycemia in the peri-ischemic period may be protective under some conditions. When insulin-induced hypoglycemia was prevented, high-dose insulin was still partially protective after global cerebral ischemia in the rat [7]. Given that IGF-1 is more protective than insulin in equimolar doses, this suggests that the effects of insulin are at least partially mediated through the IGF-1 receptor, although a contribution from an insulin receptor-mediated effect could not be ruled out [7]. More direct evidence has been provided by studies of co-administration of IGF-1 with an IGF-1 receptor antagonist. The treatment with the antagonist significantly blocked IGF-1 neuroprotection and functional recovery in 6-OHDA-induced parkinsonian rats [18]. Mediated by IGF Binding Proteins IGF-1 and IGF-2 share binding to the six IGF binding proteins (BPs). The precise roles of the IGFBPs in the CNS are not fully understood. They act partly as a buffer, partly in some situations to help translocate IGFs to the site of action, and to target IGFs to specific cell types. The previously noted intense, contemporaneous expression of IGFBP-2 and IGFBP-3 with IGF-1 in injured brain regions indicates a potential role for the IGFBPs either in endogenous neuronal recovery or in wound repair. IGFBP-2 is the predominant IGFBP in the CSF and has a higher affinity for IGF-2 than IGF-1. In contrast to IGF-2, a truncated form of IGF-1, desN-(l-3)IGF-1 has markedly reduced binding affinity to the IGFBPs [19], which lead to a more potent bioactivity in vitro. In equimolar doses to the effective dose of IGF-1, both des-IGF-1 and IGF-2 have been reported not to be neuroprotective in the adult rat after HI injury [4]. Given that des-IGF-1 is more potent than IGF-1 in vitro, it is reasonable to speculate that the effective dose for des-IGF-1 might be considerably lower than that of IGF-1. Alternatively, however, it might be that binding to IGFBP is essential to the neuroprotection of IGF-1, and the reduced affinity to IGFBPs would impair the bioavailability of des-IGP-1. In order to test this hypothesis, the effects of low (2 ␮g) and high (150 ␮g) dose of des-IGF-1 were


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compared. This study demonstrated that there was no effect of low dose desIGF-1, and only a trend to neuroprotection with high-dose treatment [4]. This finding, that low-dose des-IGF-1 is ineffective despite higher local bioavailability, provides strong in vivo evidence that binding of IGF-1 to IGFBPs is essential for the neuroprotective actions of IGF-1. Since IGF-2 has much higher affinity for IGFBPs than IGF-1, particularly IGFBP-2, IGF-2 has been used as a tool to displace exogenous IGF-1 from endogenous IGFBPs. Neuroprotection with IGF-1 was abolished after treatment with the combination of IGF-1 and IGF-2. Further, tissue uptake of tritiated IGF-1 after central administrated was also blocked by combination treatment with IGF-2 [4]. The most obvious explanation for this observation is that the stronger affinity of IGF-2 to the IGFBPs reduces IGFBP/IGF-1 complex formation and therefore, decreases translocation of IGF-1 to the site of action [4]. In conclusion, these results provide evidence that the neuroprotective effects of IGF-1 are dependent on complex interactions with IGFBPs, and particularly IGFBP-2. Possible Contribution of the N-Terminal Tripeptide of IGF-1 The N-terminal tripeptide of IGF-1, GPE, has been suggested to be naturally cleaved from IGF-1 by an acid protease in both serum and brain tissue [20]. Its neurobioactivity was first demonstrated in vitro by showing enhanced dopamine and acetylcholine release [21]. Central administration of GPE (3 ␮g), in a dose equimolar to the effective dose of IGF-1, has been found to be neuroprotective following HI injury in adult rats. Like its parent peptide, GPE has selective effects in certain neuronal phenotypes and brain regions [22]. The most clearly described difference between IGF-1 and GPE in neuroprotection is that GPE protects the CA1–2 subregions of the hippocampus where IGF-1 is not effective, most likely due to the relatively low density of IGF-1 receptors [22]. Using the same treatment regime, GPE also prevents 6-OHDA-induced loss of tyrosine-hydroxylase reactivity in the substantia nigra and the striatum [23]. GPE has a small molecular weight and crosses the blood-brain barrier [24]. Even though it is rapidly broken down in plasma [24], GPE attenuates 6-OHDA-induced long-term parkinsonian deficit after peripheral administration in adult rats [25], although it did not reduce neuronal loss 12 weeks after the lesion. Peripheral administration also improves neuronal outcome after HI injury in infant rats [26], suggesting effective central penetration. The mode of action of GPE is not yet clear. Unlike des-IGF-1, GPE does not interact with IGF-1 receptors and has been found to have some NMDA-like effects [27] however with no evidence of binding to NMDA receptors. Glial


40

orientated cellular localization has been reported by a microautoradiogrphic study using tritiated GPE [26], whereas the majority of IGF-1 signal is located on neurons, particularly neurons with apoptotic morphology [4]. Similar to its parent peptide, tissue penetration of labeled GPE is also markedly increased after injury [4, 26]. Possible Indirect Effects of IGF-1 Other potential indirect mechanisms of action for IGF-1 have been excluded. In particular, treatment with 20 ␮g IGF-1 does not alter cortical temperature for 18 h after HI injury compared to vehicle-treated controls [4], excluding the possibility of a hypothermic mechanism in IGF-1 neuroprotection. Similarly, although mild hypoglycemia can be protective, and IGF-1 can lower blood glucose through cross-reactivity with the insulin receptor, in practice, central administration of neuroprotective doses of IGF-1 does not significantly alter systemic glucose concentrations, excluding the possibility of a hypothermic mechanism in IGF-1 neuroprotection.

Conclusions

Reversible HI cerebral injury is now well established to be an evolving process, characterized by an early ‘latent’ phase followed by secondary deterioration and the ultimately the development of delayed neuronal and oligodendroglial death. This delayed cell loss involves both apoptotic and necrotic processes, and thus anti-apoptotic agents have the potential to interrupt the development of cell death. We and others have now shown that the IGF system and other endogenous growth factors are induced many days after injury, but that in the first few hours after HI, endogenous IGF-1 expression is actually reduced, suggesting that inadequate levels of IGF-1 in that critical early period favours the development of apoptosis. Consistent with this hypothesis, exogenous administration of IGF-1 started early in the latent phase in a range of models and species greatly reduced delayed neuronal death as well as oligodendrocyte loss and demyelination, and improved functional recovery. These effects are mediated by the IGF-1 receptor and the IGFBPs. The key finding that the neuroprotective actions of IGF-1 are mediated by blocking early apoptotic events explains why its effects are highly dependent on the timing of administration, with a relatively narrow window of opportunity after severe HI injury. Such early recruitment of patients poses considerable practical difficulties. The demonstration that early mild post-insult cooling delays the progression of apoptotic or programmed cell death provides a practical means of widening the window of opportunity for treatment. GPE, with its


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small molecular weight and greater ability to cross the blood-brain barrier, may have significant advantages for clinical application. While the experimental studies to date are very promising, a very cautious approach to clinical application is needed. We can be optimistic, however, that this knowledge will ultimately lead to effective therapies which will probably combine multiple strategies for optimal effectiveness, such as mild early hypothermia followed by an anti-apoptotic agent such as IGF-1 or GPE.

Acknowledgements The authors’ work presented in this chapter was supported by grants from the Health Research Council of New Zealand, USPHS grant R01 HD-32752, the Neurological Foundation of New Zealand, the Auckland Medical Research Foundation, the New Zealand Lottery Grants Board and NeuronZ Limited (Auckland, New Zealand).

References 1

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Beilharz EJ, Williams CE, Dragunow M, Sirimanne ES, Gluckman PD: Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: Evidence for apoptosis during selective neuronal loss. Mol Brain Res 1995;29:1–14. Beilharz EJ, Russo VC, Butler G, Baker NL, Connor B, et al: Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic-ischemic injury. Mol Brain Res 1998;59:119–134. Lee WH, Bondy C: Insulin-like growth factors and cerebral ischemia. Ann NY Acad Sci 1993;679:418–422. Guan J, Bennet L, Gluckman PD, Gunn AJ: Insulin-like growth factor-1 and post-ischemic brain injury. Prog Neurobiol 2003;70:443–462. Bergstedt K, Wieloch T: Changes in insulin-like growth factor 1 receptor density after transient cerebral ischemia in the rat. Lack of protection against ischemic brain damage following injection of insulin-like growth factor 1. J Cereb Blood Flow Metab 1993;13:895–898. Johnston BM, Mallard EC, Williams CE, Gluckman PD: Insulin-like growth factor-1 is a potent neuronal rescue agent after hypoxic-ischemic injury in fetal lambs. J Clin Invest 1996;97:300–308. Zhu CZ, Auer RN: Centrally administered insulin and IGF-1 in transient forebrain ischaemia in fasted rats. Neurol Res 1994;16:116–120. Sharp FR, Lu A, Tang Y, Millhom DE: Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab 2000;20:1011–1032. Russell JW, Windebank AJ, Schenone A, Feldman EL: Insulin-like growth factor-1 prevents apoptosis in neurons after nerve growth factor withdrawal. J Neurobiol 1998;36:455–467. Anderson MF, Aberg MAI, Nilsson M, Eriksson PS: Insulin-like growth factor-1 and neurogenesis in the adult mammalian brain. Dev Brain Res 2002;134:115–122. Sherrard RM: Insulin-like growth factor 1 induces climbing fibre re-innervation of the rat cerebellum. Neuroreport 1997;8:3325–3328. Liu XF, Fawcett JR, Thome RG, Frey WH: Non-invasive intranasal insulin-like growth factor-1 reduces infarct volume and improves neurologic function in rats following middle cerebral artery occlusion. Neurosci Lett 2001;308:91–94. Schabitz WR, Hoffmann TT, Heiland S, Kollmar R, Bardutzky J, et al: Delayed neuroprotective effect of insulin-like growth factor-1 after experimental transient focal cerebral ischemia monitored with MRI. Stroke 2001;32:1226–1233.


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Saatman KE, Contreras PC, Smith DH, Raghupthi R, Me Dermott KL, Femandes SC, Sanderson KL, Voddi M, Mclntoch TK: Insulin-like growth factor-1 improves both neurological motor and cognitive outcome following experimental brain injury. Exp Neurol 1997;147:418–427. Inder T, Huppi PS, Zientara GP, Maier SE, Jolesz FA, et al: Early detection of periventricular leukomalacia by diffusion-weighted magnetic resonance imaging techniques. J Pediatr 1999;134: 631–634. Petty AM, Wettstein JG: White matter ischaemia. Brain Res Brain Res Rev 2000;31:58–64. Pantoni L, Garcia JH, Gutierrez JA: Cerebral white matter is highly vulnerable to ischemia. Stroke 1996;27:1641–1646. Quesada A, Micevych PE: Estrogen interacts with the IGF-1 system to protect nigrostriatal dopamine and maintain motoric behavior after 6-hydroxdopamine lesions. J Neurosci Res 2004;75:107–116. Sara VR, Hall K: Insulin-like growth factors and their binding proteins. Physiol Rev 1990;70:591–614. Yamamoto H, Murphy LJ: Enzymatic conversion of IGF-1 to des(l-3)IGF-I in rat serum and tissues: A further potential site of growth hormone regulation of IGF-1 action. J Endocrinol 1995;146: 141–148. Sara VR, Carlsson-Skwirut C, Drakenberg K, Giacobini MB, Hakansson L, et al: The biological role of truncated insulin-like growth factor-1 and the tripeptide GPE in the central nervous system. Ann NY Acad Sci 1993;692:183–191. Guan J, Waldvogel HJ, Faull RLM, Gluckman PD, Williams CE: The effects of the N-terminal tripeptide of insulin-like growth factor-1, glycine-proline-glutamate in different regions following hypoxic-ischemic brain injury in adult rats. Neuroscience 1999;89:649–659. Guan J, Krishnamurthi RV, Waldvogel HJ, Faull RLM, dark RG, et al: N-terminal tripeptide of IGF1 (GPE) prevents the loss of TH-positive neurons after 6-OHDA induced nigral lesion in rats. Brain Res 2000;859:286–292. Batchelor DC, Lin H, Wen JY, Keven C, van Ziji PL, et al: Pharmacokinetics of glycineproline-glutamate, the N-terminal tripeptide of insulin-like growth factor-1, in rats. Anal Biochem 2003;323:156–163. Krishnamurthi R, Stott S, Maingay M, Faull RLM, McCarthy D, et al: N-terminal tripeptide of IGF-1 improves functional deficits after 6-OHDA lesion in rats. Neuroreport 2004;15:1601–1604. Sizonenko SV, Sirimanne ES, Gluckman PD, Williams CE: Neuroprotective effects of the N-terminal tripeptide of IGF-1, glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury. Brain Res 2001;922:42–50. Bourguignon JP, Gerard A: Role of insulin-like growth factor binding proteins in limitation of IGF-1 degradation into the N-methyl-D-aspartate receptor antagonist GPE: Evidence from gonadotrophin-releasing hormone secretion in vitro at two developmental stages. Brain Res 1999;847:247–252.

Dr. Jian Guan The Liggins Institute, Faculty of Medicine and Health Sciences The University of Auckland, Private Bag 92019 Auckland (New Zealand) Tel. ⫹64 9 3737599 (ext 86134), Fax ⫹64 9 3737497, E-Mail [email protected]


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Insulin-Like Growth Factor-I and Risk of Type 2 Diabetes and Coronary Heart Disease: Molecular Epidemiology Manjinder S. Sandhu Department of Public Health and Primary Care, Institute of Public Health, University of Cambridge, Strangeways Research Laboratory, Cambridge, UK

Abstract Insulin-like growth factors (IGFs) play a fundamental role in somatic growth and cellular differentiation, metabolism and survival. Indeed, the processes linking nutrition, metabolism and growth are thought to involve a complex interrelation among insulin, growth hormone (GH), IGFs and their binding proteins (IGFBPs). However, accumulating data from both experimental and molecular epidemiological studies indicate that these growth factors may also be important in the pathophysiological processes underlying chronic disease, including type 2 diabetes mellitus, coronary heart disease and cancer. Experimental and observational studies suggest that higher levels of circulating IGF-I may increase risk of several cancers. By contrast, recent prospective epidemiological studies suggest that relatively higher IGF-I levels may reduce the risk of type 2 diabetes and coronary heart disease. However, these relatively small-scale observational studies are susceptible to chance, reverse causality and residual or unmeasured confounding. A ‘Mendelian randomization’ approach based on large-scale gene association and prospective observational studies might help determine the possible causal role of IGF-I and its binding proteins in the aetiology of type 2 diabetes, coronary heart disease and cancer. Copyright © 2005 S. Karger AG, Basel

Introduction

Insulin-like growth factor (IGF)-I and -II are peptide hormones that share sequence homology with insulin [1]. The biological actions of the IGFs are primarily mediated by the IGF-I cell-surface receptor (IGF-IR). Most circulating

IGF-I and IGF-II are bound to the insulin-like growth factor binding proteins (IGFBPs), especially IGFBP-3, which binds 75–90% of circulating IGFs. Thus IGFBPs are important regulators of IGF bioactivity; however, IGFBPs may also have biological actions that are independent of IGF-I and IGF-II regulation [2]. The IGFs play a fundamental role in somatic growth and cellular differentiation, metabolism and survival. Indeed, the processes linking nutrition, metabolism and growth are thought to involve a complex interrelation among insulin, growth hormone (GH), IGFs and their binding proteins (IGFBPs) [3]. However, accumulating data from both experimental and observational studies indicate that these growth factors may also be important in the pathophysiological processes underlying chronic disease, including type 2 diabetes mellitus, coronary heart disease and cancer [4–6]. This review discusses the possible association between IGF-I and risk of type 2 diabetes and coronary heart disease.

Type 2 Diabetes

The structural homology with insulin and IGFBP-regulated hypoglycaemic activity suggest that IGF-I and its binding proteins play an intrinsic role in glucose homeostasis and metabolism [7]. Results of experiments with transgenic animals have shown that inactivation of the IGF-I gene specific to the liver, resulting in marked decreases in circulating concentrations of IGF-I, is associated with hyperinsulinaemia and insulin insensitivity [8, 9]. Consistent with these observations, infusions of recombinant human IGF-I (rhIGF-I) in animals and humans are characterised by acute decreases in blood glucose and insulin concentrations [10]. Studies of patients with extreme insulin resistance as a result of insulin receptor abnormalities and type 2 diabetes have shown a more than threefold improvement in insulin sensitivity and glucose tolerance following infusions of rhIGF-I [11–13]. Compared with placebo, a large trial of patients with type 2 diabetes showed that IGF-I monotherapy was related to a significant reduction in haemoglobin A1c [14]. However, the high doses used in these studies, usually above the normal physiological range, were associated with a number of sideeffects [11]. More recently, trials using a combination of IGF-I and IGFBP-3 have led to a reduction in insulin treatment and improvement in glucose tolerance in patients with type 1 and 2 diabetes [15, 16]. Notably, there were fewer side-effects using this combination therapy. IGF-I could contribute to changes in insulin sensitivity and glucose uptake via direct IGF-I receptor-mediated effects on skeletal muscle [8, 9, 16]. In vitro and in vivo data have also shown that the IGF-I receptor in skeletal muscle can

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represent an alternative receptor for metabolic signalling in insulin receptordeficient muscle cells, suggesting that IGF-I can mimic insulin’s effect on glucose uptake by skeletal muscle through its own receptor [17, 18]. Furthermore, a direct effect of IGF-I is consistent with the observation that, following rhIGFI administration, insulin requirements in type 1 diabetics are reduced without showing any changes in overnight GH pulsatility [19]. Moreover, a recent study has shown that combined therapy with a GH receptor antagonist and rhIGF-I is associated with a greater improvement in insulin sensitivity than that following treatment with the GH receptor antagonist alone, suggesting that IGF-I has insulin-sensitising actions that may be independent of its ability to suppress GH [16]. Alternatively, the glucose-lowering and insulin-sensitising actions of IGF-I may be indirect via suppression of glucose counter-regulatory hormones, such as glucagon and GH [9, 20]. Indeed, GH is well known to induce in vivo insulin resistance and stimulates IGF-I production, whereas circulating IGF-I inhibits pituitary GH release via a classical negative feedback mechanism [21]. Hence IGF-I may be associated with glucose homeostasis through its effects on GH suppression. The high doses of IGF-I given in some intervention studies may also activate the insulin receptor [11, 22]. However, this mechanism would not explain the glucose-lowering and insulin-sensitising effect of IGF-I in people with defects in the insulin receptor [23]. An animal model of liver-specific IGF-I gene deletion (LID) and overexpression of a mutant form of GH that prevents GH activation of its receptor has provided further insights into the relative roles of GH and IGF-I in the regulation of insulin sensitivity [24]. In this double mutant model, blocking the action of GH in the presence of low circulating IGF-I levels results in a substantial increase in insulin sensitivity compared with control or LID animals. These results suggest that GH expression may be a primary factor in regulating insulin sensitivity, whereas IGF-I may play a lesser and more subtle role in the regulation of glucose homeostasis. Investigations in animals also indicate that IGF-I may regulate pancreatic ␤-cell growth, survival and function [25]. A study using intercrossed mice heterozygous for null alleles of the IGF-I receptor and IRS-2 revealed that IGF-I receptors promote ␤-cell development and survival through the IRS-2 signalling pathway [26, 27]. However, similar investigations of animals with lossof-function mutations in IGF-I and insulin receptors indicated that, as long as the insulin receptor system remains intact, IGF-I receptor haploinsufficiency does not affect insulin action and alter glucose homeostasis [28]. More recent investigations have examined the effect of ␤-cell-specific deletion of the IGF-I receptor on ␤-cell function and glucose regulation [29, 30]. These studies found that lack of functional IGF-I receptors in ␤-cells did not affect ␤-cell mass, but

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resulted in impaired insulin secretion and glucose tolerance. These data suggest that IGF-I might be implicated in the development of adult diabetes through its anti-apoptotic action on pancreatic ␤-cells. Observational studies in humans examining the association between IGF-I levels and glucose tolerance are limited. Previous cross-sectional reports of the association between circulating IGF-I concentrations and glucose tolerance have been contradictory [31, 32]. However, because secondary effects may confound them, interpretation of these cross-sectional data is difficult [32], particularly in relation to changes in circulating IGFBPs, GH and insulin, which may lead to changes in circulating IGF-I levels. By contrast, prospective observational studies assess the association between circulating IGF-I levels in apparently healthy populations and the subsequent development of glucose intolerance in these individuals. To date, one small, prospective observational study has shown that low IGF-I levels are associated with an increased risk of IGT/type 2 diabetes [33]. This preliminary investigation also suggested that IGFBP-1 levels might modify the association between IGF-I and glucose tolerance. However, results from small-scale observational data are susceptible to chance and unmeasured or residual confounding [34, 35], and therefore require independent replication in large-scale observational studies. A gene-association study by Vaessen et al. [4] found that participants who did not carry the wild-type (192 bp) allele of a microsatellite polymorphism in the promoter region of the IGF-I gene had lower circulating IGF-I concentrations, shorter stature and an increased risk of type 2 diabetes, compared with participants who were homozygous for the wild-type allele. By contrast, a subsequent investigation of this microsatellite polymorphism in the IGF-I gene found that, compared with participants who were homozygous or heterozygous for the wild-type allele (192 bp), non-carriers of the wild-type allele had a reduced risk of type 2 diabetes [36]. Individuals homozygous for the wild-type allele also had lower IGF-I levels compared with those who were non-carriers or heterozygous for the wild-type allele. Thus both studies reported that the IGF-I allele associated with low IGF-I levels was also associated with an increased risk of type 2 diabetes. These discrepant observations might be due to chance or variations in patterns of linkage disequilibrium between populations [36]. However, differences in other genetic and environmental factors between study populations, particularly in relation to selection and inclusion of participants and their demographic, metabolic and clinical characteristics, may be alternative explanations for these contrasting findings [4, 36]. A more systematic approach to identifying common variation in the IGF-I gene and their functional properties in large-scale gene association studies may help explain these discrepant observations [37].


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Coronary Heart Disease

IGF-I may be involved in the pathogenesis of coronary heart disease [5]. Its main function in the heart is to stimulate cardiac growth and contractile function. IGF-I also has a potent anti-apoptotic action, and experimental data indicate that overexpression of IGF-I in transgenic mice prevents cell death of myocardium after infarction [38]. Similarly, administration of IGF-I has been found to improve cardiac function and structure in post-infarct swine [39]. Likewise, exogenous administration of IGF-I has been shown to improve cardiac function in cardiomyopathy, ischaemia and heart failure [39–41]. More recently, cardiac-specific overexpression of the IGF-I receptor has been associated with physiological cardiac hypertrophy and enhanced systolic function [42]. These effects were found to be PI3K(p110␣)-dependent, suggesting that this pathway is the critical downstream signalling molecule of the IGF-I receptor pathway for the regulation of cardiac function [42]. These results suggest that IGF-I may have a cardioprotective effect by reducing myocardial apoptosis and injury in response to ischemia [43]. In humans, several studies have demonstrated that growth-hormone-deficient adults have increased intima-media thickness [44]. Adults with GH deficiency also display cardiac dysfunction and decreased ventricular mass, which can be reversed by short-term IGF-I therapy [45]. Consistent with this observation, intravenous administration of rhIGF-I is associated with improved cardiac performance in patients with chronic heart failure [46]. Cross-sectional observational studies have also shown that circulating serum IGF-I levels are lower in people with coronary artery disease or atherosclerotic plaques compared with healthy controls [47], although these findings have been inconsistent [48]. Lower free IGF-I concentrations are also associated with increased intimamedia thickness of the carotid artery wall in men with and without prevalent cardiovascular disease [49]. Similarly, compared with control participants, patients with acute myocardial infarction have significantly lower levels of circulating IGF-I [50]. One investigation has also shown that low IGF-I levels in patients with acute myocardial infarction were related to increased 2-year allcause mortality [51]. Studies examining the prospective association between circulating levels of IGF-I and subsequent risk of coronary heart disease in apparently healthy populations are limited. Juul et al. [5] showed that individuals with low IGF-I levels and high IGFBP-3 levels have a significantly increased risk of dying from ischaemic heart disease, providing the first prospective observational evidence for a possible role of IGF-I and IGFBP-3 in the pathogenesis of coronary heart disease. Two more reports have been recently published, which also suggest that circulating IGF-I is inversely associated with risk of mortality from

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Juul et al., 2002 [5] Vasan et al., 2003 [52] Laughlin et al., 2004 [53]

Combined 0.5

0.75

1

1.25

Odds ratio

Fig. 1. Meta-analytical summary of the relative risk of coronary heart disease or heart failure per 1 SD increase in circulating levels of IGF-I.

CHD and heart failure [52, 53]. Although these studies are only broadly comparable, and summary meta-analytical estimates must be interpreted cautiously, we have calculated a pooled summary estimate of relative risk of the published results in figure 1. The prospective observational evidence suggests that relatively higher circulating IGF-I levels might be associated with a lower risk of mortality from CHD or heart failure with a summary effect size (odds ratio) of 0.74 (95% CI 0.64–0.85) with every standard deviation increase in circulating IGF-I levels, suggesting that circulating IGF-I is associated with a decreased risk of coronary heart disease. There was no detectable heterogeneity between studies (Q (2 d.f.) ⫽ 0.07; p ⫽ 0.97). Only one prospective study has assessed the association between circulating IGFBP-3 and risk of CHD, finding a significant positive association (OR ⫽ 1.35 (95% CI 1.05–1.72) for every SD increase in IGFBP-3 following adjustment for confirmed risk factors for CHD) [5]. This observation suggests that elevated IGFBP-3 may reduce IGF-I bioavailability or have an independent effect on CHD risk. The three studies that have examined the prospective association between circulating IGF-I levels and subsequent risk of mortality from CHD or heart failure collectively amount to 392 cases of CHD. Furthermore, only one study (231 cases) has assessed the association between IGFBP-3 levels and subsequent CHD risk. It has now become clearly recognised that larger epidemiological studies are required to reliably assess moderate effects. For example, cumulative data have recently shown a marked attenuation in the magnitude of the association between circulating levels of C-reactive protein and risk of CHD, which has implications for its predictive utility [54]. Importantly, it is


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unclear whether these modest associations among IGF-I and coronary heart disease are simply due to chance, reverse causality or residual or unmeasured confounding. Hence data from larger and more rigorous studies are required to confirm or refute the possible inverse association between IGF-I levels and risk of coronary heart disease. One population-based gene-association study has reported that participants who did not carry the wild-type (192 bp) allele of a microsatellite polymorphism in the promoter region of the IGF-I gene had lower circulating IGF-I concentrations, shorter stature and an increased risk of myocardial infarction [4]. This polymorphism was also found to be strongly associated with myocardial infarction in participants with type 2 diabetes. Specifically, individuals who did not carry the wild-type allele had a more than threefold higher risk of myocardial infarction compared with individuals homozygous for the wildtype allele, suggesting that IGF-I may be an important factor in the development of diabetic-vascular complications. However, subsequent studies have failed to find any consistent functional associations between this gene variant and circulating levels of IGF-I [36, 55].

Role of ‘Mendelian Randomization’

The possible association between IGF-I levels and risk of type 2 diabetes and coronary heart disease may be due to residual confounding (measurement error) or unknown confounders. As a result, classical observational epidemiology cannot resolve these limitations. Indeed, other unmeasured biomarkers correlated with circulating levels of IGF-I may explain the apparent inverse association between IGF-I and risk of type 2 diabetes and coronary heart disease. Furthermore, these associations may be the result of reverse causation – that is, the relation between risk factors and disease could be the result of latent disease rather than the risk factors themselves. However, ‘Mendelian randomization’ – ‘the random assortment of genes from parents to offspring that occurs during gamete formation and conception’ – may provide an epidemiological approach that is much less susceptible to reverse causality and confounding, and thus a method of assessing whether certain risk factors might be causally related to disease [56, 57]. In this scenario, the interrelation among IGF-I gene variants, IGF-I levels and risk of type 2 diabetes and coronary heart disease may reflect the true magnitude of association between IGF-I expression and disease risk. Thus a comparison of groups of individuals defined by genotype, based on a Mendelian randomization design, is equivalent to a randomised comparison. However, linkage disequilibrium may be one limitation of this approach [57]. Furthermore, to assess these associations reliably and with

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appropriate statistical power, large-scale molecular epidemiological studies are required [58].

Conclusions

The increasing awareness of the importance of the IGFs and their binding proteins in regulating growth and metabolism has led to recent investigations examining the possible role of these growth factors in the aetiology of chronic disease. The IGFs are ubiquitous peptide hormones involved in controlling normal physiology throughout life. Specifically, their underexpression may be associated with an increased risk of type 2 diabetes and coronary heart disease, whereas overexpression may be linked to an increased risk of several cancers. Current observational epidemiology is susceptible to reverse causality and residual and unmeasured confounding. A Mendelian randomization approach based on large-scale gene association and prospective observational studies might help determine the possible causal role of IGF-I and its binding proteins in the aetiology of type 2 diabetes, coronary heart disease and cancer. References 1 2 3 4

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Clemmons DR, Busby WH, Arai T, Nam TJ, Clarke JB, Jones JI et al: Role of insulin-like growth factor binding proteins in the control of IGF actions. Prog Growth Factor Res 1995;6:357–366. Ferry RJ Jr, Cerri RW, Cohen P: Insulin-like growth factor binding proteins: New proteins, new functions. Horm Res 1999;51:53–67. Underwood LE, Thissen JP, Lemozy S, Ketelslegers JM, Clemmons DR: Hormonal and nutritional regulation of IGF-I and its binding proteins. Horm Res 1994;42:145–151. Vaessen N, Heutink P, Janssen JA, Witteman JC, Testers L, Hofman A et al: A polymorphism in the gene for IGF-I: Functional properties and risk for type 2 diabetes and myocardial infarction. Diabetes 2001;50:637–642. Juul A, Scheike T, Davidsen M, Gyllenborg J, Jorgensen T: Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: A population-based case-control study. Circulation 2002;106:939–944. Renehan AG, Zwahlen M, Minder C, O’Dwyer ST, Shalet SM, Egger M: Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk: Systematic review and meta-regression analysis. Lancet 2004;363:1346–1353. Jones JI, Clemmons DR: Insulin-like growth factors and their binding proteins: Biological actions. Endocr Rev 1995;16:3–34. Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J et al: Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 2001;50:1110–1118. Sjogren K, Wallenius K, Liu JL, Bohlooly Y, Pacini G, Svensson L et al: Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism. Diabetes 2001;50:1539–1545. Dunger DB, Acerini CL: Does recombinant human insulin-like growth factor-1 have a role in the treatment of diabetes? Diabet Med 1997;14:723–731. Moses AC, Young SC, Morrow LA, O’Brien M, Clemmons DR: Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes. Diabetes 1996;45:91–100.


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Sandhu MS, Heald AH, Gibson JM, Cruickshank JK, Dunger DB, Wareham NJ: Circulating concentrations of insulin-like growth factor-I and development of glucose intolerance: A prospective observational study. Lancet 2002;359:1740–1745. Lawlor DA, Davey SG, Kundu D, Bruckdorfer KR, Ebrahim S: Those confounded vitamins: What can we learn from the differences between observational versus randomised trial evidence? Lancet 2004;363:1724–1727. Concato J, Horwitz RI: Beyond randomised versus observational studies. Lancet 2004;363: 1660–1661. Frayling TM, Hattersley AT, McCarthy A, Holly J, Mitchell SM, Gloyn AL et al: A putative functional polymorphism in the IGF-I gene: Association studies with type 2 diabetes, adult height, glucose tolerance, and fetal growth in UK populations. Diabetes 2002;51:2313–2316. Johnson GC, Esposito L, Barratt BJ, Smith AN, Heward J, Di Genova G et al: Haplotype tagging for the identification of common disease genes. Nat Genet 2001;29:233–237. Li Q, Li B, Wang X, Leri A, Jana KP, Liu Y et al: Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J Clin Invest 1997;100:1991–1999. Kotlyar AA, Vered Z, Goldberg I, Chouraqui P, Nas D, Fridman E et al: Insulin-like growth factor I and II preserve myocardial structure in post-infarct swine. Heart 2001;86:693–700. Duerr RL, Huang S, Miraliakbar HR, Clark R, Chien KR, Ross J Jr: Insulin-like growth factor-1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure. J Clin Invest 1995;95:619–627. Redaelli G, Malhotra A, Li B, Li P, Sonnenblick EH, Hofmann PA et al: Effects of constitutive overexpression of insulin-like growth factor-1 on the mechanical characteristics and molecular properties of ventricular myocytes. Circ Res 1998;82:594–603. McMullen JR, Shioi T, Huang WY, Zhang L, Tarnavski O, Bisping E et al: The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110␣) pathway. J Biol Chem 2004;279:4782–4793. Buerke M, Murohara T, Skurk C, Nuss C, Tomaselli K, Lefer AM: Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci USA 1995;92:8031–8035. Capaldo B, Patti L, Oliviero U, Longobardi S, Pardo F, Vitale F et al: Increased arterial intimamedia thickness in childhood-onset growth hormone deficiency. J Clin Endocrinol Metab 1997;82:1378–1381. Ren J, Samson WK, Sowers JR: Insulin-like growth factor I as a cardiac hormone: Physiological and pathophysiological implications in heart disease. J Mol Cell Cardiol 1999;31:2049–2061. Donath MY, Sutsch G, Yan XW, Piva B, Brunner HP, Glatz Y et al: Acute cardiovascular effects of insulin-like growth factor I in patients with chronic heart failure. J Clin Endocrinol Metab 1998;83:3177–3183. Janssen JA, Stolk RP, Pols HA, Grobbee DE, Lamberts SW: Serum total IGF-I, free IGF-I, and IGFB-1 levels in an elderly population: Relation to cardiovascular risk factors and disease. Arterioscler Thromb Vasc Biol 1998;18:277–282. Janssen JA, Lamberts SW: The role of IGF-I in the development of cardiovascular disease in type 2 diabetes mellitus: Is prevention possible? Eur J Endocrinol 2002;146:467–477. Van den Beld AW, Bots ML, Janssen JA, Pols HA, Lamberts SW, Grobbee DE: Endogenous hormones and carotid atherosclerosis in elderly men. Am J Epidemiol 2003;157:25–31. Conti E, Andreotti F, Sciahbasi A, Riccardi P, Marra G, Menini E et al: Markedly reduced insulinlike growth factor-1 in the acute phase of myocardial infarction. J Am Coll Cardiol 2001;38: 26–32. Friberg L, Werner S, Eggertsen G, Ahnve S: Growth hormone and insulin-like growth factor-1 in acute myocardial infarction. Eur Heart J 2000;21:1547–1554. Vasan RS, Sullivan LM, D’Agostino RB, Roubenoff R, Harris T, Sawyer DB et al: Serum insulinlike growth factor I and risk for heart failure in elderly individuals without a previous myocardial infarction: The Framingham Heart Study. Ann Intern Med 2003;139:642–648. Laughlin GA, Barrett-Connor E, Criqui MH, Kritz-Silverstein D: The prospective association of serum insulin-like growth factor I (IGF-I) and IGF-binding protein-1 levels with all cause and


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cardiovascular disease mortality in older adults: The Rancho Bernardo Study. J Clin Endocrinol Metab 2004;89:114–120. Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A et al: C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med 2004;350:1387–1397. Allen NE, Davey GK, Key TJ, Zhang S, Narod SA: Serum insulin-like growth factor I (IGF-I) concentration in men is not associated with the cytosine-adenosine repeat polymorphism of the IGF-I gene. Cancer Epidemiol Biomarkers Prev 2002;11:319–320. Davey SG, Ebrahim S: ‘Mendelian randomization’: Can genetic epidemiology contribute to understanding environmental determinants of disease? Int J Epidemiol 2003;32:1–22. Clayton D, McKeigue PM: Epidemiological methods for studying genes and environmental factors in complex diseases. Lancet 2001;358:1356–1360. Davey SG, Harbord R, Ebrahim S: Fibrinogen, C-reactive protein and coronary heart disease: Does Mendelian randomization suggest the associations are non-causal? QJM 2004;97:163–166.

Dr. Manjinder S. Sandhu Department of Public Health and Primary Care Institute of Public Health, University of Cambridge, Strangeways Research Laboratory Wort’s Causeway, Cambridge CB1 8RN (UK) Tel. ⫹44 1223 740168, Fax ⫹44 1223 740177, E-Mail [email protected]

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Clinical Management Cianfarani S, Clemmons DR, Savage MO (eds): IGF-I and IGF Binding Proteins. Basic Research and Clinical Management. Endocr Dev. Basel, Karger, 2005, vol 9, pp 55–65

Quantitative Measurement of IGF-I and Its Use in Diagnosing and Monitoring Treatment of Disorders of Growth Hormone Secretion David R. Clemmons Division of Endocrinology, Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, N.C., USA

Abstract Quantitative measurements of serum IGF-I concentrations are extremely useful in the diagnosis of growth hormone deficiency in children and young adults and in the diagnosis of acromegaly. Similarly, IGF-I measurements have been of great use to clinical investigators who are monitoring the effect of either growth hormone or IGF-I in various disorders. Several guidelines have proven very useful for assaying IGF-I and defining high-quality assays. Since IGF-I circulates associated with IGF binding proteins it is essential that a highquality assay remove binding protein interference. Similarly, it is essential that the antibodies that are utilized in the assay have both high specificity and high affinity for IGF-I. Assay reproducibility is effected by such factors as quality of sample collection, utilization of proper internal and external standards and published validation using not only normal samples but samples from a variety of pathophysiologic conditions. Interlaboratory variability often is due to differences in one of more of these characteristics. When assessing the validity of abnormal assay results it is important to be able to compare the value to a normative dataset that has been developed on a large number of subjects. Recently a normative dataset was published on 2,812 subjects and this provides an important reference standard by which other assays should be judged. Careful attention to these characteristics is likely to result in valid and useful IGF-I assay results. Copyright © 2005 S. Karger AG, Basel

Introduction

Growth hormone (GH) is secreted in a pulsatile manner by the normal pituitary gland. Several of the variables that control GH secretion vary greatly

during the lifespan such as secretion of sex steroids, therefore GH secretory rates show wide variability across a 24-hour interval and at different times in the lifespan being highest at puberty and lowest at elderly adults. These variations have made it difficult to define normal GH secretion even when standardized stimulation and suppression tests are utilized. Likewise, monitoring GH values in patients who are receiving treatment for GH deficiency or acromegaly has proven to be of minimal value in predicting as successful response to therapy. Therefore, attempts to use insulin-like growth factor-I (IGF-I), a peptide whose synthesis and secretion is stimulated by GH, as in index of 24-hour GH blood production rates, have shown that measurement of this peptide can provide useful information [1, 2]. The major problem in interpreting IGF-I values has been difficulty in obtaining standardized measurements. Some assays have utilized inadequate numbers of normal subjects to define the normal range of IGF-I values and others have had difficulties in reproducibility. These IGF-I assay problems have led to difficulties in the interpretation of IGF-I values [3]. Therefore, utilization of IGF-I assays depends upon a thorough knowledge of the problems that may be encountered and a high enough degree of standardization that the clinician can be reassured that the information that is obtained is of high quality.

Discrepancies among Various Assays and the Etiology of These Discrepancies

Contradictory conclusions from three studies that have attempted to determine the value of measuring IGF-I in predicting long-term outcomes, specifically mortality in acromegaly, serve as an example of how failure to adjust study design to deal with variability in IGF-I measurements can lead to erroneous conclusions. One epidemiologic study concluded IGF-I was of no value in predicting mortality [4] and two other studies concluded IGF-I was of a great deal of value in making this prediction [5, 6]. Obvious reasons for this discrepancy could be such factors as patient selection and the methodologies used to monitor outcome. However it is more likely that the differences are due to the types of IGF-I assays that were utilized and the ways in which they were used; i.e., the two studies which found a positive correlation between elevations in IGF-I and increased mortality utilized multiple IGF-I measurements, e.g. one study utilized four measurements over 10 years. In contrast, the study that found no correlation used a single IGF-I measurement for each subject. Recently, Milani et al. [7] reported that there is up to 32% biologic variability in IGF-I measurements, that is if the values are assayed in the same assay thus eliminating interassay variability, the difference in results can be as great as

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IGF-l (U/ml)

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10 50 loge (GH (mU/l))

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Fig. 1. Relationship between GH and IGF-I. The figure shows GH and IGF-I values from normal subjects and patients with acromegaly. IGF-I increases linearly as a function of log base 10 increase in GH.

32% for two samples obtained from the same subject on different days. This means that a given subject may have an IGF-I value on one morning and that can change by as much as 32% if they are sampled the next morning. A second problem with the study that found no correlation between IGF-I and mortality illustrates the need for an adequate numbers of normal subjects. In that study only three age ranges were utilized for normative subjects and the number of subjects that were ⬎50 years of age was not stated. Likewise the numbers of subjects in the two other age ranges was minimal. Therefore, the normative data were analyzed in a manner that made it difficult to distinguish acromegaly from normal. Finally, the IGF-I assay method utilized in the study clearly did not remove all IGF binding proteins (IGFBPs) from the sample. When that method is compared to another method of removing IGFBPs [8], residual binding protein in the sample is easily detected. This would be expected to interfere with the value and give falsely elevated values which would alter the ability of the assay to detect a difference between acromegaly and normal. Therefore, the quality of IGF-I assays and the adequacy of the normative data can lead to very different results and interpretations.

Relationship of GH Secretion to Plasma IGF-I

When IGF-I values obtained from normal subjects and those with GH hypersecretion are compared to GH values the two measurements show a logarithmic relationship [9], that is there is a linear increase in IGF-I as a function of the logarithmic increase in GH (fig. 1). Multiple different studies have shown that there is a clear positive relationship between these two peptides and changes in IGF-I that

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reflect changes in GH secretion. IGF-I measurements also correlate with clinical measurements that reflect changes in GH biologic activity. Correlating height SDS scores with IGF-I has shown that there is a correlation and studies that have used increased stature in response to GH as an endpoint have established that there is an unambiguous relationship [10, 11]. Similarly, the degree of improvement in signs and symptom scores in acromegaly in response to treatment has been shown to correlate with a change in IGF-I, suggesting that these IGF-I values are providing a clear index of the degree of abnormality of GH secretion [12, 13]. In summary, large clinical studies have shown have that there is a strong correlation between symptoms and signs of GH action at the tissue level and changes in IGF-I.

Variation in IGF-I Values in Normal Subjects

Several factors other than GH function to regulate IGF-I concentrations. In GH deficiency, these factors are the predominate determinates of the total serum IGF-I concentration and the influence of these factors relative to GH is much greater as compared to normal state (because GH secretion is low). Even in normal subjects, GH secretion accounts for less than 50% of the variability in IGF-I. In contrast, in acromegaly the contribution of GH secretion to the total IGF-I value is probably greater than 80% in most subjects, therefore IGF-I measurements provide a very accurate estimation of changes in GH secretion and/or activity in patients with acromegaly. These non-GH-related determinants of IGF-I include an undefined genetic determinant which in identical twin studies has been shown to account for 40% of the variability in IGF-I [14], age-related changes in GH secretion [15], changes in sex steroid secretion, discordant changes in IGFBPs [16] and changes in nutritional status [17]. Age is clearly an important variable in determining IGF-I values and since GH secretion declines between ages 20 and 80 years and there is substantial reduction in serum IGF-I (fig. 2). Likewise, discordant changes in IGFBPs are an important determinant of this problem. As an example in GH deficiency, IGFPB-2 concentrations increase 2- to 3-fold. This results in slowing of the rate of clearance of IGF-I and thus elevates values beyond what they would be expected to be if no increase in IGFBP-2 had occurred. Changes in nutritional status, while important determinants of low IGF-I in the third world, are largely irrelevant in the USA and Europe. However, patients with poorly controlled diabetes may have suppressed values due to poor nutrition since they do not adequately synthesize IGF-I in the liver due to poor insulin action in this tissue [18]. Therefore, these patients have the equivalent of a nutritional deficient since hepatic synthesis of IGF-I is impaired. To deal with the problem of age-related changes in GH

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Fig. 2. IGF-I declines with age. GH secretion declines with age which is associated with a parallel decrease in IGF-I values.

secretion, the only adequate solution is to use reference ranges that have adequate numbers of normal subjects. Recently a study was completed in Europe that analyzed the IGF-I values 2,812 adults. These adults were then subdivided into 5-year age intervals between birth and 75 years of age. As shown in figure 3, the 95% confidence interval (CI) for this number of subjects is quite narrow compared to those previously published. Therefore, utilization of these normative ranges is likely to make the differentiation between states of GH deficiency and/or GH excess and normal more precise.

Technical Problems with IGF-I Assays

The major technical problem that has been encountered is interference by IGFBPs. The initial attempts to deal with this problem included various extraction methods. The most commonly utilized is a method termed ‘acid ethanol extraction’. The principle has been to separate IGF-I that is bound to IGFBPs


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Fig. 3. Normal ranges for IGF-I values. The 95% CIs for normal IGF-I values for several age ranges shown. The number of subjects for each age interval is shown.

Acid/ethanol

SepPak

Fig. 4. Residual IGF-I binding protein in sera after extraction. Serum from patients with acromegaly was extracted using acid ethanol precipitation lanes 1–5 and then analyzed by ligand blotting using radiolabelled IGF-I to detect the residual binding protein that remained after extraction. The same serum specimens shown in lanes 1 and 2 were chromotographed using SepPak C-18 and the results are shown in lanes 6 and 7. The band denoted by the upper arrow is IGFBP-3 and the band denoted by the lower arrow is IGFBP-4.

and therefore present as a high molecular weight form, from unbound IGF-I. Acid is added to disassociate the IGF-I from the binding protein so that the total IGF-I concentration can be measured. The problem with this method as shown in figure 4 is that it often does not remove all of the binding proteins. A second method in which an acidified sample is separated from binding proteins following chromatography over a hydrophobic column shows that the chromatographic

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method was far more effective in removing IGFBPs from these particular serum samples. Since the radioimmunoassay for IGF-I is performed after the binding proteins have been removed, it is clear that the acid ethanol method in this case resulted in substantial retention of binding proteins which would then interfere with the ability of the IGF-I in the serum sample to bind to antibodies appropriately and thus lead to inaccurate measurements. Several assays have overcome this problem by using two additional methodologic changes that do not require an extraction step and can be performed as direct measurements. In the first, a two-site capture assay technology is utilized. That is, instead of using a single antibody and measuring the ability of endogenous IGF-I in the serum to compete with radiolabelled IGF-I for binding to that antibody (the traditional radioimmunoassay method), these methods rely on two antibodies that are prepared to two separate binding sites within IGF-I. One antibody is generally fixed to a solid support, either the walls of an assay tube or a solid matrix such as plastic beads that can be incubated with the serum sample. This is termed the capture antibody because it will bind to the IGF-I in solution. A second antibody is labelled with some type of radiolabelled probe that can be detected; for the most sensitive assays this is generally a chemiluminescence probe. This antibody is also added and the IGF-I that has bound to the capture antibody is detected. By washing the sample extensively and removing the unbound IGF-I, only the residual IGF-I that is bound to the solid support is measured. Of note, this method as described does not completely eliminate binding protein interference because the residual binding protein can compete with the capture antibody and limit the amount of endogenous IGF-I that binds to it. To eliminate this problem, either one of the extraction methods described previously is used prior to assay to remove the binding proteins or the second antibody that is labelled has to be monospecific for IGF-I, and have no affinity for IGF-II. IGF-II which binds the binding proteins with affinity that is equal or greater to IGF-I is then added in excess [19]. This saturates the endogenous binding proteins eliminating their capacity to bind to the IGF-I and therefore eliminates binding protein interference. This latter method has found a high degree of acceptance. It is entirely dependent upon the specificity of the antibody. If such an antibody is available, this is a very effective method for removing binding protein interference and eliminates the time-consuming and sometimes non-producible extraction steps. An additional technical problem has been assay validation. Generally for validation, reference laboratories have simply performed correlation coefficients using several serum specimens. That is they have not redefined the normative range for their new assay based on samples obtained from normal volunteers and patients with GH excess and/or deficiency. Instead they have simply taken samples that span the normal range of values that were quantified with the old assay


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methodology that is being replaced and compared them to values obtained using the new assay methodology. If the correlation is coefficient high (i.e. ⬎0.95), they have assumed that the assays are measuring the same thing. However it is clear from examination from some of the correlation coefficient data that there are subjects who do not have a 0.95 correlation and therefore although the extrapolation may be strong for most values, it is not uniformally effective. Other problems that occur are failure to assay samples in duplicates and failure to repeat samples that fall at the high or low end of the standard curve. This can result in significant errors in the actual measurement. Similarly, standard curve drift is a problem, that is the assay standards that are utilized may be measured as different values over time, therefore the clinician may receive information that is not useful in monitoring patients progressively because the absolute number that is reported changes over time as a result change in the detection of the standard. Other problems that have occurred are due to special problems with the US healthcare system. That is because patients in managed care systems often change providers and each provider may use a different reference laboratory, there is a need to be able to compare values across different reference laboratories so that clinicians are able to make treatment decisions regarding past information from a reference laboratory that is different from the one the patient’s health plan currently utilizes. However, there is no acceptable universal standard that is utilized across reference laboratories, therefore this type comparison is impossible. Furthermore, the reference laboratories do not publish conversion factors therefore there is no mathematical way to correct for differences in values from different laboratories. Finally, some reference laboratories are so large that there may even be two different methods used within a given reference laboratory but the results are reported without informing the clinician of this information so that the clinician may not even know that two different assays were used within the same reference laboratory to obtain two different results. Examples of different results for the same serum pools assayed in different laboratories for IGF-I have been reported to vary by as much as 68%. Clearly, standardization of assays across laboratories would improve quality control and provide normative data that would be more generally available and patients would benefit from less need to perform repetitive assays in one reference laboratory in order to establish a stable baseline of values on which to base treatment decisions.

Clinical Use of IGF-I Assays

With the development of more accurate and precise IGF-I measurements, it has been possible to use these values to diagnose both GH deficiency and acromegaly and accurately monitor treatment in these subjects. In acromegaly,

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IGF-I is an excellent way to both diagnose GH secretion and monitor the response to treatment. Studies have shown that greater than 98% of patients with proven acromegaly have elevated IGF-I values [20, 21]. Similarly, monitoring studies have shown that changes in IGF-I strongly predict changes in improvement in signs and symptoms in acromegaly in patients that are treated surgically, with radiotherapy, or with medications designed to lower GH secretion or block GH action. In contrast in GH deficiency because of these other variables that regulate IGF-I when GH secretion is low, there is significant overlap between normal subjects and those with GHD [22]. In patients 40 years of age or less where there is a reasonable difference between mean normal subjects and patients with GH deficiency, IGF-I is useful diagnostic tool, that is approximately 80% of patients with proven severe GH deficiency will have IGF-I levels below the 95% CI level if adequate numbers of normative subjects are used to define this interval. Between age 40 and 60 years this percentage drops to approximately 60% and ⬎60 years of age IGF-I values do have not proven utility in diagnosing GH deficiency. Looking at a large population of subjects who encompassed the entire adult age range, Hartman et al. [2] found that an IGF-I value ⬍84 ng/ml was strongly predictive of GH deficiency. However in his study a substantial number of subjects who had normal IGF-I values had GHD. Stated another way, he concluded that normal subjects almost never had a value ⬍84 ng/ml, however a substantial number of his severely GHdeficient subjects, particularly aged ⬎40 years, had IGF-I values that were higher than this level. Very few subjects with severe GHD have been shown to have IGF-I values ⬎1 SD below the mean. Therefore it has been recommended in screening for GH deficiency if IGF-I values are ⬎1 SD below the mean it is likely that GHD will be confirmed by stimulation testing. However, if they are less than this value, a stimulation test should be performed to confirm the presence of GH deficiency and most subjects who are treated will be normal. Some other questions that are important to ask in terms of evaluating IGF-I assays are at what range on the standard curve are assay results automatically repeated? How often is this standard curve recalibrated? What is interassay variability? Was the published reference value that contains data that prove GH dependence actually used in a given laboratory? IGF-I values are also extremely useful in monitoring in response to GH replacement therapy. Specifically they have been a great deal of help in predicting GH toxicity [23]. The general recommendation has been to achieve IGF-I target values that are within the normal range and when this is done the manifestations of GH toxicity are extremely low, therefore the assay has been very useful in preventing GH overdosage and avoiding the development of these complications. In summary, the major problems in interpreting IGF-I values have been as follows: (1) Factors other than GH control IGF-I concentrations in normal


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serum. (2) Changes in IGFBPs that occur in GH deficiency or GH excess may lead to change in total IGF-I values that make clinical results more difficult to interpret. (3) Lack of adequate numbers of normative subjects in various age ranges to tightly define 95% CIs on which to base a decision as to whether a patient’s value is normal or abnormal. (4) Lack of acceptable universal standard and inadequate knowledge of how different laboratories establish their absolute standard and relative normative values. Based on these difficulties, the following criteria are recommended for selecting IGF-I assays: (1) Adequate and ageadjusted normative data. (2) A knowledge of the interassay variability and a published interassay variability that is within the acceptable ranges. (3) Utilization of a method that substantially eliminates IGFBP interference. (4) The assay results should show proven GH dependence, that is they should be increased in acromegalic subjects and low in most subjects ⬍40 years of age with GH deficiency. (5) Finally, there should be reference values that are available to allow comparison of results to other commercial assays. It is not practical at present to demand this from every reference laboratory but it should be strongly encouraged to become a standardize criteria in the future.

Acknowledgments The author wishes to thank Ms. Laura Lindsey for her help in preparing the manuscript. This work was supported by a grant from the National Institutes of Health HL56850.

References 1 2 3 4

5

6

7

8

Freda PU: Current concepts in the biochemical assessment of the patient with acromegaly. Growth Horm IGF Res 2003;13:171–184. Furlanetto RW: The somatomedin C binding protein: Evidence for a heterologous subunit structure. J Clin Endocrinol Metab 1980;51:12–19. Mitchell H, Dattani MT, Nanduri V, Hindmarsh PC, Preece MA, Brook CG: Failure of IGF-I and IGFBP-3 to diagnose growth hormone insufficiency. Arch Dis Child 1999;80:443–447. Ayuk J, Clayton RN, Holder G, Sheppard MC, Stewart PM, Bates AS: Growth hormone and pituitary radiotherapy, but not serum insulin-like growth factor-I concentrations, predict excess mortality in patients with acromegaly. J Clin Endocrinol Metab 2004;89:1613–1617. Biermasz NR, Dekker FW, Pereira AM, van Thiel SW, Schutte PJ, van Dulken H, Romijn JA, Roelfsema F: Determinants of survival in treated acromegaly in a single center: Predictive value of serial insulin-like growth factor I measurements. J Clin Endocrinol Metab 2004;89:2789–2796. Swearingen B, Barker FG, 2nd, Katznelson L, Biller BM, Grinspoon S, Klibanski A, Moayeri N, Black PM, Zervas NT: Long-term mortality after transsphenoidal surgery and adjunctive therapy for acromegaly. J Clin Endocrinol Metab 1998;83:3419–3426. Milani D, Carmichael JD, Welkowitz J, Ferris S, Reitz RE, Danoff A, Kleinberg DL: Variability and reliability of single serum IGF-I measurements: Impact on determining predictability of risk ratios in disease development. J Clin Endocrinol Metab 2004;89:2271–2274. Conover CA, Johnstone EW, Turner RT, Evans GL, John Ballard FJ, Doran PM, Khosla S: Subcutaneous administration of insulin-like growth factor (IGF)-II/IGF binding protein-2

Clemmons

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complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis. Growth Horm IGF Res 2002;12:178–183. Barkan AL, Beitins IZ, Kelch RP: Plasma insulin-like growth factor-I/somatomedin-C in acromegaly: Correlation with the degree of growth hormone hypersecretion. J Clin Endocrinol Metab 1988;67:69–73. Argente J, Barrios V, Pozo J, Munoz MT, Hervas F, Stene M, Hernandez M: Normative data for insulin-like growth factors (IGFs), IGF-binding proteins, and growth hormone-binding protein in a healthy Spanish pediatric population: Age- and sex-related changes. J Clin Endocrinol Metab 1993;77:1522–1528. Jaruratanasirikul S, Sriplung H, Leethanaporn K: Serum insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) in healthy Thai children and adolescents: Relation to height, weight, and body mass index. J Med Assoc Thai 1999;82:984–990. Parkinson C, Scarlett JA, Trainer PJ: Pegvisomant in the treatment of acromegaly. Adv Drug Deliv Rev 2003;55:1303–1314. Kopchick JJ, Parkinson C, Stevens EC, Trainer PJ: Growth hormone receptor antagonists: Discovery, development, and use in patients with acromegaly. Endocr Rev 2002;23:623–646. Harrela M, Koistinen H, Kaprio J, Lehtovirta M, Tuomilehto J, Eriksson J, Toivanen L, Koskenvuo M, Leinonen P, Koistinen R, Seppala M: Genetic and environmental components of interindividual variation in circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3. J Clin Invest 1996;98:2612–2615. Lofqvist C, Andersson E, Gelander L, Rosberg S, Blum WF, Albertsson Wikland K: Reference values for IGF-I throughout childhood and adolescence: A model that accounts simultaneously for the effect of gender, age, and puberty. J Clin Endocrinol Metab 2001;86:5870–5876. Meyers KM, Huston LY, Clemmons RM: Regulation of canine platelet function II. Catecholamines. Am J Physiol 1983;245:R100–R109. Khorsandi MJ, Fagin JA, Giannella-Neto D, Forrester JS, Cercek B: Regulation of insulin-like growth factor-I and its receptor in rat aorta after balloon denudation. Evidence for local bioactivity. J Clin Invest 1992;90:1926–1931. Baxter RC, Binoux M, Clemmons DR, Conover C, Drop SL, Holly JM, Mohan S, Oh Y, Rosenfeld RG: Recommendations for nomenclature of the insulin-like growth factor binding protein (IGFBP) superfamily. Growth Horm IGF Res 1998;8:273–274. Blum WF, Breier BH: Radioimmunoassays for IGFs and IGFBPs. Growth Regul 1994;4(suppl 1): 11–19. Clemmons DR: Commercial assays available for insulin-like growth factor I and their use in diagnosing growth hormone deficiency. Horm Res 2001;55(suppl 2):73–79. Thissen JP, Ketelslegers JM, Maiter D: Use of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 in the diagnosis of acromegaly and growth hormone deficiency in adults. Growth Regul 1996;6:222–229. Aimaretti G, Corneli G, Baldelli R, Di Somma C, Gasco V, Durante C, Ausiello L, Rovere S, Grottoli S, Tamburrano G, Ghigo E: Diagnostic reliability of a single IGF-I measurement in 237 adults with total anterior hypopituitarism and severe GH deficiency. Clin Endocrinol (Oxf) 2003;59:56–61. Tillmann V, Patel L, Gill MS, Whatmore AJ, Price DA, Kibirige MS, Wales JK, Clayton PE: Monitoring serum insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and leptin during growth hormone treatment for disordered growth. Clin Endocrinol (Oxf) 2000;53:329–336.

Prof. David R. Clemmons, MD Division of Endocrinology, Department of Medicine University of North Carolina School of Medicine, CB #7170 6111 Thurston-Bowles, Chapel Hill, NC 27599–7170 (USA) Tel. ⫹1 919 9664735, Fax ⫹1 919 9666025, E-Mail [email protected]


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IGF-I and IGFBP-3 Assessment in the Management of Childhood Onset Growth Hormone Deficiency S. Cianfarani, A. Liguori, D. Germani Rina Balducci Center of Pediatric Endocrinology, Department of Public Health and Cell Biology, Tor Vergata University, Rome, Italy

Abstract The diagnosis of growth hormone (GH) deficiency (GHD) in childhood is not straightforward, being still based on a comprehensive clinical, anthropometric, endocrine and neuroradiological assessment. Due to their GH dependency and relative stability in circulation, IGF-I and IGFBP-3 serum concentrations were proposed as reliable indicators of daily GH secretion. However, the sensitivity of assays for both IGF-I and IGFBP-3 is inadequate to exclude the diagnosis of GHD merely on the basis of a normal value of the two parameters, although it seems likely that IGF-I values higher than –1 SD reflect a normal GH secretion. On the other hand, as the specificity of both measurements is over 90%, subnormal concentrations strongly support the diagnosis of GHD. Finally, combining the evaluation of growth velocity with IGF-I measurement, sensitivity and specificity reach a value ⱖ95%, implying that two subnormal values strongly suggest and two normal values strongly oppose the diagnosis of GHD. Copyright © 2005 S. Karger AG, Basel

Pitfalls in the Diagnosis of Growth Hormone Deficiency

The diagnosis of growth hormone (GH) deficiency (GHD) in childhood is not straightforward and though there are well-defined auxological, biochemical and radiological criteria to define a child with severe GHD, in clinical practice many children who have moderate GHD do not fit all the set criteria [1]. At the same time, each criterion has its own pitfalls. Indeed, a child with idiopathic short stature may auxologically resemble a GHD patient, as well as a GHD patient may show wide fluctuations of growth rate [1, 2].

Although GH stimulation tests are invasive, non-physiological and hazardous, the finding of peak GH responses ⬍10 ␮g/l to at least two different provocative tests is traditionally considered the gold standard for the diagnosis of GHD. However, the threshold level used to define a normal GH response was defined arbitrarily and each stimulation test, including the combination of GHRH with somatostatin release inhibitors such as arginine and pyridostigmine, has a high false positive rate [3, 4]. Therefore, with rare exception, the diagnosis of GHD is made on arbitrary grounds, and it is not surprising that a high proportion of children diagnosed as GH-deficient will have a normal GH response when re-tested at the end of growth [5–8]. In conclusion, the diagnosis of GHD must be based on a comprehensive clinical auxological, endocrine and neuroradiological assessment, but ‘even under ideal circumstances, errors of both overdiagnosis and underdiagnosis of GHD still are likely’ [9].

Insulin-Like Growth Factor-I Evaluation

The growth-promoting action of GH is mainly mediated by the GH-dependent peptide insulin-like growth factor-I (IGF-I), whose serum concentrations are stable during the day due to the complexing with a family of IGF-binding proteins (IGFBPs). The measurement of IGF-I levels, due to the close GH dependency and stability in circulation, was proposed as a simple and reliable method for diagnosing or excluding GHD [10]. However, multiple factors may affect the IGF-I assessment. An initial technical problem was represented by the necessity of IGF extraction to avoid the interference of IGFBPs. A variety of technical approaches have been successfully applied to overcome this problem such as the extraction by acid size exclusion chromatography or the blockade of IGFBP-binding sites with an excess of IGF-II. An additional limitation of IGF-I assessment is the age dependency, which determines an overlap between the physiological low concentrations of children aged ⬍6 years and the abnormal levels of GHD patients. Finally, additional factors affecting IGF-I levels independently of GH are represented by degree of sexual maturation, nutritional status, intestinal absorption, hepatic function and thyroid hormones [3, 11, 12]. There is also a significant physiological interindividual heterogeneity secondary to genetic determinants. Results from studies carried out on animal models, twins and population cohorts suggest that there are heritable determinants of the IGF-I concentrations [13]. The overlap of IGF-I values between non-GHD and GHD children represents another important limitation of the IGF-I measurement. Figure 1a and b

IGF-I/IGFBP-3 Assessment in Childhood Onset GHD Management

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Years

⫺2 SD ⫺1.5 SD ⫺1 SD

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900

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a Fig. 1a. IGF-I levels in males with GHD plotted on standard reference curves.

shows that GHD patients may have only slightly subnormal IGF-I levels, though never higher than –1 SD. Consistent with this, it has recently been proposed to raise the cut-off value to –1.65 SD to obtain the best diagnostic efficiency of IGF-I assessment [14]. In table 1 we have summarized sensitivity and specificity of IGF-I measurement as reported by different studies.

Cianfarani/Liguori/Germani

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b Fig. 1b. IGF-I levels in females with GHD plotted on standard reference curves.

IGF-Binding Protein-3 Evaluation

Among the six known IGFBPs, IGFBP-3 is the major serum carrier of IGFs and circulates as part of a ternary complex consisting of the binding protein, an IGF peptide, and an acid-labile subunit [25].


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Table 1. Reported sensitivity and specificity of IGF-I evaluation in diagnosing GHD Group (first author)

Sensitivity, %

Specificity, %

Cianfarani [15] Nunez [16] Juul [17] Tillman [18] Rikken [19] Mitchell [12] Weinzimer [20] Granada [21] Cianfarani [4] Bussieres [22] Lissett [23] Das [24] Boquete [14]

75 69 53 34 65 62 73 70 73 72 86 86 68

90 76 98 72 78 47 NA 95 95 95 NA 100 97

NA ⫽ Not assessed.

IGFBP-3 serum levels are constant throughout the day and are closely GH-dependent [25]. These characteristics led to propose the IGFBP-3 assessment as a reliable and simple screening test in the work-up of children with short stature. Preliminary results were encouraging, Blum et al. [26] showing a sensitivity of 97% and a specificity of 95% in the diagnosis of GHD. Moreover, IGFBP-3 measurement seems to offer several important advantages over IGF-I determination: (1) no extraction step is required before measurement, improving the precision and facilitating the procedure; (2) IGFBP-3 normally circulates in the serum at high concentrations, so that assay sensitivity is not problematic; (3) IGFBP-3 serum concentrations, like those of IGF-I, are agedependent, but the normal range varies only modestly with age and pubertal status; (4) the impact of nutritional status is not as great as it is with IGF-I, and, finally, (5) the molar concentrations of IGFBP-3 approximate the sum of the molar concentrations of IGF-I ⫹ IGF-II, making it possible to estimate the total IGF content. However, the preliminary promising results were not confirmed in subsequent studies (table 2). A reason of the low sensitivity of IGFBP-3 assessment is that its concentrations reflect the total IGF concentrations, IGF-I ⫹ IGF-II, IGF-II being less GH-dependent than IGF-I alone [25]. In addition, the measurement of IGFBP-3 serum concentrations is complicated by the existence of a family of proteases that are capable of degrading IGFBP-3. These proteases,


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Table 2. Reported sensitivity and specificity of IGFBP-3 evaluation in diagnosing GHD Group (first author)

Sensitivity, %

Specificity, %

Blum [26] Hasegawa [28] Cianfarani [15] Nunez [16] Juul [17] Tillman [18] Rikken [19] Mitchell [12] Weinzimer [20] Granada [21] Cianfarani [4] Das [24] Boquete [14]

97 92 50 50 60 22 53 61 50 33 30 79 90

95 69 92 69 98 92 81 68 NA 100 98 86 60

NA ⫽ Not assessed.

kDa

Prepubertal

Pubertal

Adult

42 39

kDa 42 39 29

29 21

21

18

18

Fig. 2. A representative autoradiogram of Western immunoblot of IGFBP-3 in serum from prepubertal, pubertal and adult patients with GHD. The intact form of IGFBP-3 is seen at approximately 42–39 kDa, the major IGFBP-3 fragment is seen at about 29 kDa, additional smaller IGFBP-3 fragments are seen at approximately 21 and 18 kDa.

which include plasmin, matrix metalloproteinases, and prostate-specific antigen, degrade IGFBP-3, thereby reducing its affinity for IGFs and eventually leading to increased IGF bioavailability. Proteolysis can profoundly affect the IGFBP-3 measurement [27]. Indeed, the anti-IGFBP-3 antibodies recognize all the IGFBP-3 circulating forms including IGFBP-3 fragments, whose relative amount increases in GHD due to the presence of high proteolytic activity, as shown in figure 2 [29, 30].


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Is IGF-I and IGFBP-3 Assessment a Helpful Diagnostic Tool or a Confounding Factor?

To test the sensitivity and specificity of IGF-I and IGFBP-3 measurement in the diagnosis of GHD, we carried out a retrospective study on 33 GHD children selected according to restrictive diagnostic criteria such as (1) stature ⬍–2 Z-score, (2) delayed bone age (at least 1 year), (3) GH peak response to two different provocative tests ⬍10 ␮g/l, (4) brain MRI positive for hypothalamuspituitary abnormalities such as pituitary hypoplasia, stalk agenesis and ectopic posterior lobe, (5) catch-up growth during the first year of GH replacement therapy (growth rate ⱖ75th centile). The cut-off value of –1.9 SD was chosen for both IGF-I and IGFBP-3 measurements. We also evaluated IGF-I and IGFBP-3 concentrations in a group of 56 children with idiopathic short stature who were referred to our Centre for short stature and/or reduced growth velocity but normal GH peak responses to stimulation tests [4]. In our experience, sensitivity of IGF-I measurement ranged from 73 to 75%, whereas specificity was from 90 to 95% [4, 15]. Sensitivity of IGFBP-3 measurement was even less, ranging from 30 to 50%, whereas specificity was from 92 to 98% [4, 15].

IGF-I and IGFBP-3 Assessment in Clinical Practice

The low sensitivity of the tests suggests that measurements of IGF-I and IGFBP-3 do not discriminate between short children with or without GHD, as a normal value does not rule out GHD. It seems likely that an IGF-I value ⬎–1 SD is incompatible with a condition of GHD. On the other hand, due to the high specificity, low concentrations of IGF-I and/or IGFBP-3 are highly suggestive of GHD and, when associated to normal GH responses to stimulation tests, uncommon conditions such as neurosecretory dysfunction [31] or GH receptor deficiency [32] should be suspected.

Combining Auxological and Biochemical Criteria, a Comprehensive Approach

Among auxological criteria for the diagnosis of GHD, height velocity has always been considered the more reliable parameter: the lower the growth rate the more likely the diagnosis of GHD [9]. In our experience, height velocity assessment has a high sensitivity (82%) but a low specificity (43%). Combining the IGF-I with growth rate measurement we found that sensitivity reached 95%


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IGF-I

⫺1.9 SD

(44%)

a

(26%) b GHD ISS

(23%) c Cut-off (25th centile)

(7%) d HV

Fig. 3. Individual data for IGF-I, expressed as values above or below the cut-off point of –1.9 SD, and HV, expressed as values above or below the cut-off point of 25th centile. a Subjects with normal IGF-I concentrations and low HV. b Children with normal values of both IGF-I and HV. c Patients with low values of both IGF-I and HV. d Subjects with normal HV but low IGF-I concentrations. ⫽ ISS; ⫽ GHD [reproduced from 4, with permission].

and specificity 96% [4]. Our results suggest that normal IGF-I and growth rate (⬎25th centile) rule out the diagnosis of GHD, whereas when both the indices are subnormal, GHD is so likely that only one GH provocative test might be sufficient, together with brain MRI, to make the diagnosis of GHD (fig. 3). In case of discrepancy between the two parameters, the standard diagnostic procedure should be followed. References 1

2 3 4

5 6

Growth Hormone Research Society: Consensus guidelines for the diagnosis and treatment of growth hormone deficiency in childhood and adolescence. J Clin Endocrinol Metab 2000;85: 3990–3993. Voss LD, Wilkin TJ, Bailey BJR, Betts PR: The reliability of height and height velocity in the assessment of growth (the Wessex Growth Study). Arch Dis Child 1991;66:833–837. Shalet SM, Toogood A, Rahim A, Brennan BMD: The diagnosis of growth hormone deficiency in children and adults. Endocr Rev 1998;19:203–223. Cianfarani S, Tondinelli T, Spadoni GL, Scirè G, Boemi S, Boscherini B: Height velocity and IGF-I assessment in the diagnosis of childhood onset GH insufficiency: Do we still need a second GH stimulation test? Clin Endocrinol 2002;57:161–167. Clayton PE, Price DA, Shalet SM: Growth hormone state after completion of treatment with growth hormone. Arch Dis Child 1987;62:222–226. Cacciari E, Tassoni P, Parisi G, Pirazzoli P, Zucchini S, Mandini M, Cicognani A, Balsamo A: Pitfalls in diagnosing impaired growth hormone secretion: Retesting after replacement therapy of 63 patients defined as growth hormone deficient. J Clin Endocrinol Metab 1992;74:1284–1289.


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Nicolson A, Toogood AA, Rahim A, Shalet SM: The prevalence of severe growth hormone deficiency in adults who received growth hormone replacement in childhood. Clin Endocrinol 1996;44:311–316. Tauber M, Moulin P, Pienkowski C, Jouret B, Rochiccioli P: Growth hormone retesting and auxological data in 131 growth hormone-deficient patients after completion of treatment. J Clin Endocrinol Metab 1997;82:352–356. Hintz RL: The role of auxologic and growth factor measurements in the diagnosis of growth hormone deficiency. Pediatrics 1998;102:524–526. Moore DC, Ruvalcaba RHA, Smith EK, Kelly VC: Plasma somatomedin-C as a screening test for growth hormone deficiency in children and adolescents. Hormone Res 1982;16:49–55. Rosenfeld RG, Wilson DM, Lee PDK, Hintz RL: Insulin-like growth factors I and II in evaluation of growth retardation. J Pediatr 1986;109:428–433. Mitchell H, Dattani MT, Nanduri V, Hindmarsh PC, Preece MA, Brook CGD: Failure of IGF-I and IGFBP-3 to diagnose growth hormone insufficiency. Arch Dis Child 1999;80:443–447. Rosen CJ, Pollack MP: Circulating IGF-I: New perspectives for a new century. Trends Endocrinol Metab 1999;10:136–141. Boquete HR, Sobrado PGV, Fideleff HL, Sequera AM, Giaccio AV, Suarez MG, Ruibal GF, Miras M: Evaluation of diagnostic accuracy of insulin-like growth factor (IGF)-I and IGF-binding protein-3 in growth hormone-deficient children and adults using ROC plot analysis. J Clin Endocrinol Metab 2003;88:4702–4708. Cianfarani S, Boemi S, Spagnoli A, Cappa M, Argirò G, Vaccaro F, Manca Bitti ML, Boscherini B: Is IGF binding protein-3 assessment helpful for the diagnosis of GH deficiency? Clin Endocrinol 1995;43:43–47. Nunez AB, Municchi G, Barnes KM, Rose SR: Insulin-like growth factor (IGF)-I and IGFbinding protein-3 concentrations compared to stimulated and night growth hormone in the evaluation of short children – A clinical research centre study. J Clin Endocrinol Metab 1996;81: 1927–1932. Juul A, Skakkebaek NE: Prediction of the outcome of growth hormone provocative testing in short children by measurement of serum levels of insulin-like growth factor I and insulin-like growth factor binding protein-3. J Pediatr 1997;130:197–204. Tillman V, Buckler JMH, Kibirige MS, Price DA, Shalet SM, Wales JKH, Addison MG, Gill MS, Whatmore AJ, Clayton PE: Biochemical tests in the diagnosis of childhood growth hormone deficiency. J Clin Endocrinol Metab 1997;82:531–535. Rikken B, van Doorn J, Ringeling A, van den Brande JL, Massa G, Wit JM: Plasma levels of insulin-like growth factor (IGF)-I, IGF-II and IGF-binding protein-3 in the evaluation of childhood growth hormone deficiency. Horm Res 1998;50:166–176. Weinzimer SA, Homan SA, Ferry RJ, Moshang T: Serum IGF-I and IGFBP-3 concentrations do not accurately predict growth hormone deficiency in children with brain tumours. Clin Endocrinol 1999;51:339–345. Granada ML, Murillo J, Lucas A, Salinas I, Lopis MA, Castells I, Foz M, Sanmartì A: Diagnostic efficiency of serum IGFI, IGF-binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and urinary GH measurements in the diagnosis of adult GH deficiency: Importance of an appropriate reference population. Eur J Endocrinol 2000;142:243–253. Bussieres L, Souberbielle JC, Pinto G, Adan L, Noel M, Brauner R: The use of insulin-like growth factor 1 reference values for the diagnosis of growth hormone deficiency in prepubertal children. Clin Endocrinol 2000;52:735–739. Lissett CA, Jonsson P, Monson JP, Shalet SM: Determinants of IGF-I status in a large cohort of growth hormone-deficient subjects: The role of timing of onset of growth hormone deficiency. Clin Endocrinol 2003;59:773–778. Das U, Whatmore AJ, Khosravi J, Wales JKH, Butler G, Kibirige MS, Diamandi A, Jones J, Patel L, Hall CM: IGF-I and IGF-binding protein-3 measurements on filter paper blood spots in children and adolescents on GH treatment: Use in monitoring and as markers of growth performance. Eur J Endocrinol 2003;149:179–185. Baxter RC: Circulating binding proteins for the insulin-like growth factors. Trends Endocrinol Metab 1993;4:91–96.


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Blum WF, Ranke MB, Kietzmann K, Gauggel E, Zeisel HJ, Bierich JR: A specific radioimmunoassay for the growth hormone (GH)-dependent somatomedin-binding protein: Its use for diagnosis of GH deficiency. J Clin Endocrinol Metab 1990;70:1292–1298. Rosenfeld RG, Gargosky SE: Assays for insulin-like growth factors and their binding proteins: Practicalities and pitfalls. J Pediatr 1996;128:S52–S57. Hasegawa Y, Hasegawa T, Aso T, Kotoh S, Nose O, Ohyama Y, Araki K, Tanaka T, Saisyo S, Yokoya S, Nishi Y, Miyamoto S, Sasaki N, Kurimoto F, Stne M, Tsuchiya Y: Clinical utility of insulin-like growth factor binding protein-3 in the evaluation and treatment of short children with suspected growth hormone deficiency. Eur J Endocrinol 1994;131:27–32. Jones JL, Clemmons DR: Insulin-like growth factors and their binding proteins. Endocr Rev 1995;16:3–34. Spagnoli A, Gargosky SE, Spadoni GL, MacGillivray M, Oh Y, Boscherini B, Rosenfeld RG: Characterisation of a low molecular mass form of insulin-like growth factor binding protein-3 (17.7 kDa) in urine and serum from healthy children and growth hormone-deficient patients: Relationship with GH therapy. J Clin Endocrinol Metab 1995;80:3668–3676. Spiliotis BE, August GP, Hung W, Sonis W, Mendelson W, Bercu BB: Growth hormone neurosecretory dysfunction. A treatable cause of short stature. JAMA 1984;251:2223–2230. Blum WF, Ranke MB, Savage MO, Hall K and the Kabi Pharmacia Study Group on Insulin-like Growth Factor I Treatment in Growth Hormone Insensitivity Syndromes: Insulin-like growth factors and their binding proteins in patients with growth hormone receptor deficiency: Suggestions for new diagnostic criteria. Acta Paediatr 1992;383:125–126.

Prof. S. Cianfarani, MD Rina Balducci Center of Pediatric Endocrinology Department of Public Health and Cell Biology Tor Vergata University Via Montpellier 1, Room E-178, IT–00133 Rome (Italy) Tel. ⫹39 06 5100 2314, Fax ⫹39 06 59 17415, E-Mail [email protected]


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IGFs and IGFBPs in Adult Growth Hormone Deficiency Gianluca Aimaretti, Roberto Baldelli, Ginevra Corneli, Chiara Croce, Silvia Rovere, Claudia Baffoni, Simonetta Bellone, Valentina Gasco, Riccarda Granata, Silvia Grottoli, Ezio Ghigo Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy

Abstract In the current guidelines for the diagnosis of adult GH deficiency (GHD) it is stated that, within the appropriate clinical context, it has to be shown by provocative tests only. But the diagnostic value of measuring IGF-I levels has been recently revisited. It has been confirmed that normal IGF-I levels do not rule out severe GHD in adults. However, it has also been emphasized that very low IGF-I levels in patients highly suspected for GHD (and without malnutrition, liver disease or hypothyroidism) could be considered definite evidence for severe GHD. This assumption particularly applies to patients with childhood-onset, severe GHD or with multiple hypopituitarism acquired in adulthood. The value of measuring IGF-I levels for monitoring the efficacy and the adequacy of rhGH replacement remains definitely accepted. Copyright © 2005 S. Karger AG, Basel

Introduction

Following the consensus guidelines recommendations, within the appropriate clinical context, the primary pituitary pathology, provocative tests are considered the milestones for the diagnosis of adult GH deficiency (GHD) [1, 2]. This statement derives from the evidence that the peak GH response to provocative stimuli such as the insulin tolerance test (ITT), glucagon, GHRH ⫹ arginine or GH secretagogues (GHS) is able to distinguish normal subjects from patients with GHD, if used with appropriate cut-off limits [1, 3–5].

IGF-I can be considered the best marker of the GH secretory status [6–8] as indicated by the concomitant age-related variations of both GH and IGF-I secretion including the typical increase at puberty and the progressive decrease with aging observed in normal subjects [5, 9–13]. GH is the key regulator of IGF-I synthesis and release [5, 6, 8]; as a matter of fact, IGF-I levels are increased in acromegaly and gigantism but reduced in GHD [8, 14]. However, we need to consider that a critical influence on IGF-I synthesis and secretion is also played by the nutritional status and peripheral hormones such as insulin, thyroid hormones, gonadal steroids and glucocorticoids, and, as pointed out by some authors but not by others, also by gender [15–20]. In clinical practice, the existence of clinical conditions (such as energy restriction, fasting and malnutrition, liver disease, diabetes mellitus type 1, hypothyroidism) of peripheral GH insensitivity in which GH hypersecretion is coupled to low IGF-I levels is well known; these are therefore conditions of deficient GH action [17, 21]. IGF-I action is modulated by specific binding proteins among which IGFBP-3 is the most abundant and GH-dependent circulating form [15]; for this reason, IGFBP-3 has been considered another marker of GH secretion although it is less sensitive than IGF-I [8, 22–25]. IGF-I is generally measured as total circulating levels although the assay of free IGF-I levels can be performed [12, 26–31]. Mean IGF-I levels as well as IGFBP-3 and 24-hour GH concentrations in GHD are lower than in normal adults but there is considerable overlap between the two groups in the individual levels so that these parameters have been therefore considered unable to distinguish between normal subjects and GHD adults [1, 5, 32]. Total IGF-I measurement has, however, been recommended as a biochemical parameter for monitoring the efficacy and the adequacy of rhGH replacement in patients with severe GHD [1, 5]. Recent papers have revisited the diagnostic value of measuring IGF-I levels in adult GHD patients. The aim of this paper is to consider more recent studies and personal results indicating that IGF-I measurement has some considerable diagnostic value and that very low levels of circulating total IGF-I levels can sometimes be considered as definite evidence for adult GHD.

Diagnosis of Adult GHD: Current Guidelines

In adult age, classical hypopituitarism and GHD are consequences of primary hypothalamus-pituitary diseases [1, 7, 33–41] or represent the persistence of a congenital, idiopathic or acquired pituitary defect that had been diagnosed and treated in childhood [1, 34–37]. More often, multiple pituitary deficits are present but isolated pituitary deficits are not so uncommon.

Diagnostic Value of IGF-I

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In multiple hypopituitarism, GH is usually the first of the anterior pituitary hormones to be affected [7, 34, 35]. GHD can be considered like a hallmark indicating possible impaired pituitary function, which could evolve and spread to other pituitary axes. Adult patients who have hypothalamus-pituitary masses either before or even more after therapy (neurosurgery and/or radiotherapy and/or medical therapy) are at extremely high risk for severe GHD (⬎80%); thus, these patients are at ‘obvious’ rather than high risk for severe GHD and routine evaluation of their pituitary function is mandatory [33, 35]. The same statement applies to patients who had been diagnosed as having either congenital or acquired GHD in childhood. The large majority (⬎90%) of patients with childhood-onset multiple hypopituitarism show persistent severe GHD at retesting in adulthood; on the other hand, among patients who had been treated with rhGH for isolated GHD in childhood, a lower percentage (varying between 30 and 70%) shows persistence of severe GHD at retesting in adulthood [36–38]. Regarding the methodology to assess the diagnosis of adult GHD, consensus guidelines recommend that it should be demonstrated biochemically by provocative test of GH secretion [1]. The ITT test is considered the golden standard test for the diagnosis of adult GHD [1, 3], but due to insulin-induced hypoglycemia, it would be potentially hazardous in patients with central nervous system disorders and ischemic heart disease, conditions often associated with GHD. For this reason, alternative tests have been recommended and GHRH in combination with arginine or GHS have been shown as the most potent and reliable alternatives to ITT. These tests show the highest intra-individual reproducibility, high specificity and sensitivity being able to distinguish normal subjects from GHD adults [1, 4, 5, 42, 43]; the cut-off limits at the 1st centile for these tests is approximately three times higher than that of ITT (9 vs. 3 ␮g/l) [43]. Differently from the classical provocative tests, GHRH ⫹ arginine or GHS have normative limits of GH response that are age- and gender-independent [5]. These diagnostic guidelines do not attribute definitive diagnostic value to other hormonal parameters. In fact, the study of spontaneous GH secretion and the measurement of its 24-hour mean concentration does not distinguish between normal and GHD adults. The mean 24-hour GH concentration in GHD adults is, as expected, lower than in normal adults but there is a considerable overlap between the two groups even when ultra-sensitive methods for GH assay are used [3–4, 44]. Also, IGF-I and IGFBP-3 levels, two main markers of the GH status, are lower in GHD patients than in normal adults if we consider a mean value, but if we consider an individual value a considerable overlap between the two groups is present [1, 5]. Based on this evidence, the measurement of either IGF-I or IGFBP-3 has been considered an unreliable definitive diagnostic tool because normal levels of these parameters do not rule out the existence of severe adult GHD [8, 29, 31].

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The indubitable reference value of measuring IGF-I levels, for monitoring the efficacy and the adequacy of rhGH replacement was, however, recommended and it is widely accepted in clinical practice [1, 24].

Diagnostic Value of IGF-I Measurement

As previously mentioned, the measurement of total IGF-I levels shows a considerable overlap between GHD patients and control subjects [1, 3, 5]. Thus, normal IGF-I levels do not rule out the existence of severe adult GHD [1, 5, 8, 29, 31, 45]. The same picture has been reported by authors [10, 46, 47] who evaluated free IGF-I levels – once again, the overlap between patients and control subjects so that it was concluded that there is no advantage by measuring free instead of total IGF-I levels. In our experience, in a large population of panhypopituitary adults with severe GHD (n ⫽ 214; demonstrated by the severe impairment of the GH response to GHRH ⫹ arginine test, i.e. peak GH ⬍9 ␮g/l), we have recently found that nearly 58.5% of the patients have total IGF-I levels within the age-related normal limits. Mean total IGF-I levels in patients were significantly lower than those in controls in every decade of life; nevertheless, 34.8, 60.8 and 84.5% had individual IGF-I levels above the age-related 3rd centile limits for 20–39, 40–59 and ⬎60 years of age, respectively. Even considering the age-related 25th centile limit 7, 20.2 and 56.8% of GHD patients had individual IGF-I levels above the limits for 20–39, 40–59 and ⬎60 years of age, respectively. The explanation for such a high percentage of normal levels of total IGF-I in the presence of clear and demonstrated severe GHD in adults is unclear. Aging could be a variable partially explaining the remarkable overlap between total IGF-I levels in normal and severe GHD subjects [7, 48, 49]. As a matter of fact, total IGF-I levels undergo progressive decrease with aging reflecting the age-related decrease in GH secretion [48–50]; in fact, the most remarkable overlap between normal and GHD subjects in term of total IGF-I levels occurs in aging (⬎60 years of age) [29, 48, 49]. Taking into account the role played by insulin and nutritional status in the regulation of IGF-I synthesis and secretion [15, 16], the presence of overweight and/or obesity and hyperinsulinism in hypopituitaric patients with GHD would play a critical and important role. It is well known that patients with simple obesity (without pituitary pathology) have generally normal levels of total IGF-I and even increased levels of free IGF-I despite marked GH insufficiency that is sometimes as marked as in hypopituitaric patients with severe GHD [8, 9, 51].


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Mean total IGF-I levels (␮g/l)

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Fig. 1. Mean total IGF-I levels in patients with total hypopituitarism (HP) and severe GHD or isolated severe GHD as function of BMI (lean HP ⫽ BMI ⬍25 kg/m2, overweight and obese HP ⫽ BMI ⬎25 kg/m2).

In agreement with this hypothesis, we have found that mean total IGF-I levels in total hypopituitaric patients with severe GHD and in that with isolated severe GHD and overweight or truly obesity are higher than in total hypopituitaric patients with severe GHD and in that with isolated severe GHD with normal body weight (fig. 1). Concordantly, mean insulin levels in overweight and truly obese patients were higher than in lean patients. Moreover, the percentage of IGF-I levels below the age-related normal limits in GHD adults with normal body weight (52.3%) was higher than in overweight patients (34.6%) and a slight but positive correlation between total IGF-I levels and body mass index (BMI) in hypopituitaric patients with GHD was observed (r ⫽ 0.2, p ⬍ 0.02) (fig. 2, 3). Thus, it is possible that the common occurrence of overweight, insulin resistance and hyperinsulinism in GHD adults, despite the severe impairment of somatotroph function, is able to hypersensitize to very low circulating levels of GH and, in turn, to allow ‘low normal’ IGF-I synthesis and secretion. Other factors would explain such a high percentage of severe GHD adults with total IGF-I levels within the normal range. For instance, it has been reported that adults with severe GH but normal IGF-I levels have a peculiar feature of 24 hours’ spontaneous GH secretion including increase in GH burst frequency coupled with reduced GH burst amplitude and positive association between approximate entropy and total IGF-I levels [52]. Moreover, the peripheral sensitivity to GH in adult GHD shows a great intra-individual variability that is likely to reflect constitutional variability as


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Percentage of low IGF-I levels

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Fig. 2. Percentage of low age-related IGF-I levels in adult hypopituitaric (HP) patients with severe GHD as function of BMI.

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Fig. 3. Positive correlation between BMI and IGF-I levels in total hypopituitaric patients with severe GHD (r ⫽ 0.22; p ⬍ 0.0001).

well as the influence of other factors including gonadal steroids [53]. The sensitivity to GH as function of body weight in GHD adults is still unclear. However, it has been demonstrated that patients with simple obesity have increased IGF-I response to very low rhGH dose indicating that obesity increases GH sensitivity [51]. Again, also a short duration of the disease and a low number of other anterior pituitary deficits are associated to higher IGF-I levels. On the contrary, the higher the number of pituitary deficits, the lower the IGF-I levels [54].


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However, from the clinical and regulatory point of view, there is general agreement that within the appropriate clinical context (the pituitary pathology), despite normal levels of total IGF-I, adults suspected for GHD must undergo provocative tests to clarify the diagnosis [1, 5, 32]. But, before ruling out the diagnostic value of the measurement of total IGF-I, it has to be considered that, at least in well-nourished subjects without liver disease or hypothyroidism, evidence of low, age-related IGF-I levels strongly predicts severe GHD [31, 45]. In adult GHD it has been demonstrated that multiple pituitary deficits and patients with childhood-onset GHD (CO-GHD) are associated to the lowest levels of total IGF-I [55, 56]. Moreover, it has been shown that total IGF-I is a diagnostic parameter with good intra-individual reproducibility, positively associated to the peak GH response to provocative tests [29]. The possibility that very low IGF-I levels could represent definite and single evidence for severe adult GHD was therefore suggested [45]. By analyzing the clinical characteristics and biochemical testing results of 817 patients with a history of either adult-onset hypothalamic or pituitary disease or childhood-onset GHD, Hartman et al. [45] found that adult GHD could be predicted with 95% accuracy by the presence of either three or four other pituitary hormone deficits or a serum IGF-I concentration ⬍84 ␮g/l (11 nmol/l). Based on these results, the authors proposed that adult patients with three or four pituitary deficits and low IGF-I levels do not require a GH stimulation test to make the diagnosis of adult GHD. These clinical predictors are considered at least as accurate as GH stimulation tests performed in routine clinical practice. The diagnostic utility of a cut-off limit as IGF-I ⬍84 ␮g/l to predict adult GHD is limited to the particular IGF-I assay employed in this study. The same diagnostic concept for IGF-I measurement has been followed by other authors who recommended that the IGF-I cut-off limit should be expressed in standard deviation scores (SDS) evaluated in an appropriate reference population of normal subjects [30]. By following this approach, cut-off limits of –2 SDS [30, 57] or ranging from –1.65 to –1.80 for GHD adults whose disease had adult- or childhood-onset respectively [58] have been proposed. Once again, this diagnostic approach was particularly satisfactory if patients with CO-GHD or multiple pituitary deficits were considered [30, 56–58]. Also, the hypothesis that the combined evaluation of IGF-I and IGFBP-3 and/or its ratio provide further diagnostic power has been proposed by some but ruled out by others [58, 59]. By reconsidering IGF-I measurement as a diagnostic approach in our study population of total hypopituitaric adults with severe GHD (as demonstrated by the severe impairment of the GH response to GHRH ⫹ arginine test), we can


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Fig. 4. Individual IGF-I SDS levels in total hypopituitaric adults with severe GHD as demonstrated by the severe impairment of the GH response to GHRH ⫹ arginine test (⬍9 ␮g/l).

say that approximately 41.5% of the patients have low total IGF-I levels below the age-related normal limits. Specifically, 65.2, 39.2 and 15.5% had IGF-I levels below the age-related 3rd centile limits for 20–40, 40–60 and ⬎60 years of age, respectively; this percentage remarkably increases by considering the age-related 25th centile limit that allows to see 93, 79.7 and 43.1% of GHD adults with IGF-I levels below the limits for 20–40, 40–60 and ⬎60 years of age, respectively. Taking into account that we studied total hypopituitaric patients only, this picture partially disagrees with such a remarkable diagnostic power for the total IGF-I measurement. In fact, by translating IGF-I levels in SDS, only 20% of the patients showed an IGF-I SDS value ⬍–2.0 SDS (fig. 4). We anyway agree that the current diagnostic guidelines would be amended providing the IGF-I measurement more diagnostic value. It seems reasonable to state that very low total IGF-I levels (⬍–2 SD) in subjects highly suspected for GHD (particularly in patients with childhood-onset, isolated GHD or multiple hypopituitarism as well as in patients with long-lasting adult-onset multiple or total hypopituitarism) could be considered as definite evidence of severe GHD; these patients would therefore skip provocative tests. On the other hand, the value of measuring total IGF-I levels for monitoring the efficacy and the adequacy of rhGH replacement [1, 2] remains definitely accepted. Very recently, an optimal target range for IGF-I during the treatment of adult GH disorders has been generated based on the KIMS database [60].


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Other Biochemical Parameters for the Diagnosis of Adult GHD

As anticipated above, the measurement of free IGF-I levels does not offer any significant advantage compared to determination of total IGF-I levels for the diagnosis of adult GHD [46, 47]. In a study where the measurement of free IGF-I levels was also compared with IGFBP-3, as another potential diagnostic parameter, it was concluded that free IGF-I measurement did not offer an advantage even over IGFBP-3 [8]. IGFBP-3 is the most abundant circulating IGFBP, mostly GH-dependent and has been considered by some authors as another reliable diagnostic marker of the GH status [8, 15, 22–25]. However, it is less sensitive to GH than IGF-I and the majority of the authors agree that the measurement of IGFBP-3 does not provide any advantage over total IGF-I for the diagnosis of adult GHD [1, 5, 8, 32]. The urinary measurement of IGFBP-3 as well as of IGF-I offered results that were even more disappointing [61–63]. Also, the hypothesis that the combined evaluation of IGF-I and IGFBP-3 and/or its ratio provide further diagnostic power for adult GHD has been proposed by some but ruled out by many others [30, 58]. The acid-labile subunit (ALS) is a glycoprotein that binds IGF-I together with IGFBP-3 to form a trimeric complex [15, 64]. It is GH-dependent but it as well as IGFBP-3 does not reflect the GH status as accurately as IGF-I. ALS measurement has been proposed as an alternative parameter for the diagnosis of adult GHD, but, at present, the large majority of the authors agree that it does not offer any advantage over total IGF-I, at least for the diagnosis of adult GHD [59].

Final considerations

(1) Normal levels of total IGF-I do not rule out severe adult GHD even in panhypopituitary patients, who must undergo a provocative test to demonstrate or exclude severe GHD. (2) However, within the appropriate clinical context, more diagnostic value should be attributed to the measurement of total IGF-I. Very low total IGF-I levels (⬍–2.0 SDS) in subjects highly suspected for GHD (childhood-onset GHD, long-lasting adult-onset multiple or total hypopituitarism) could be considered as definite evidence of severe GHD, even avoiding provocative tests. (3) Measuring IGF-I levels for monitoring the adequacy of rhGH replacement remains undiscussed and useful. (4) The measurements of free IGF-I as well as that of IGFBP-3 and ALS do not provide any advantage over total IGF-I.


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Leong GM, Johannsson G: Growth hormone deficiency: Strategies and indications to continue growth hormone therapy in transition from adolescence to adult life. Horm Res 2003;60(suppl 1): 78–85. Benvenga S, Campenni A, Ruggeri RM, Trimarchi F: Clinical review 113: Hypopituitarism secondary to head trauma. J Clin Endocrinol Metab 2000;85:1353–1361. Gasperi M, Aimaretti G, Cecconi E, Colao A, Di Somma C, Cannavo S, Baffoni C, Cosottini M, Curto L, Trimarchi F, Lombardi G, Grasso L, Ghigo E, Martino E: Impairment of GH secretion in adults with primary empty sella. J Endocrinol Invest 2002;25:329–333. Gasperi M, Cecconi E, Grasso L, Bartalena L, Centoni R, Aimaretti G, Broglio F, Miccoli P, Marcocci C, Ghigo E, Martino E: GH secretion is impaired in patients with primary hyperparathyroidism. J Clin Endocrinol Metab 2002;87:1961–1964. Fisker S, Jorgensen JO, Christiansen JS: Variability in growth hormone stimulation tests. Growth Horm IGF Res 1998;8(suppl A):31–35. Aimaretti G, Corneli G, Razzore P, Bellone S, Baffoni C, Arvat E, Camanni F, Ghigo E: Comparison between insulin-induced hypoglycemia and growth hormone (GH)-releasing hormone ⫹ arginine as provocative tests for the diagnosis of GH deficiency in adults. J Clin Endocrinol Metab 1998;83:1615–1618. Reutens AT, Hoffman DM, Leung KC, Ho KK: Evaluation and application of a highly sensitive assay for serum growth hormone (GH) in the study of adult GH deficiency. J Clin Endocrinol Metab 1995;80:480–485. Hartman ML, Crowe BJ, Biller BM, Ho KK, Clemmons DR, Chipman JJ; HyposCCS Advisory Board; US HypoCCS Study Group: Which patients do not require a GH stimulation test for the diagnosis of adult GH deficiency? J Clin Endocrinol Metab 2002;87:477–485. Skjaerbaek C, Vahl N, Frystyk J, Hansen TB, Jorgensen JO, Hagen C, Christiansen JS, Orskov H: Serum free insulin-like growth factor-I in growth hormone-deficient adults before and after growth hormone replacement. Eur J Endocrinol 1997;137:132–137. Musolino NR, Da Cunha Neto MB, Marino Junior R, Giannella-Neto D, Bronstein MD: Evaluation of free insulin-like growth factor-I measurement on the diagnosis and follow-up treatment of growth hormone-deficient adult patients. Clin Endocrinol (Oxf) 1999;50:441–449. Corpas E, Harman SM, Blackman MR: Human growth hormone and human aging. Endocr Rev 1993;14:20–39. Ghigo E, Arvat E, Gianotti L, Ramunni J, DiVito L, Maccagno B, Grottoli S, Camanni F: Human aging and the GH-IGF-I axis. J Pediatr Endocrinol Metab 1996;9(suppl 3):271–278. Svensson J, Johannsson G, Bengtsson BA: Insulin-like growth factor-I in growth hormone-deficient adults: Relationship to population-based normal value, body composition and insulin tolerance test. Clin Endocrinol (Oxf) 1997;46:579–586. Maccario M, Tassone F, Gauna C, Oleandri SE, Aimaretti G, Procopio M, Grottoli S, Pflaum CD, Strasburger CJ, Ghigo E: Effects of short-term administration of low-dose rhGH on IGF-I levels in obesity and Cushing’s syndrome: Indirect evaluation of sensitivity to GH. Eur J Endocrinol 2001;144:251–256. Svensson J, Veldhuis JD, Iranmanesh A, Bengtsson BA, Johannsson G: Increased orderliness of growth hormone (GH) secretion in GH-deficient adults with low serum insulin-like growth factor I. J Clin Endocrinol Metab 2002;87:2863–2869. Aimaretti G, Fanciulli G, Bellone S, Maccario M, Arvat E, Delitala G, Camanni F, Ghigo E: Enhancement of the peripheral sensitivity to growth hormone in adults with GH deficiency. Eur J Endocrinol 2001;145:267–272. Toogood AA, Beardwell CG, Shalet SM: The severity of growth hormone deficiency in adults with pituitary disease is related to the degree of hypopituitarism. Clin Endocrinol 1994;41:511–516. Attanasio AF, Lamberts SW, Matranga AM, Birkett MA, Bates PC, Valk NK, Hilsted J, Bengtsson BA, Strasburger CJ: Adult growth hormone (GH)-deficient patients demonstrate heterogeneity between childhood onset and adult onset before and during human GH treatment. Adult Growth Hormone Deficiency Study Group. J Clin Endocrinol Metab 1997;82:82–88. Attanasio AF, Howell S, Bates PC, Frewer P, Chipman J, Blum WF, Shalet SM: Body composition, IGF-I and IGFBP-3 concentrations as outcome measures in severely GH-deficient (GHD) patients


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after childhood GH treatment: A comparison with adult-onset GHD patients. J Clin Endocrinol Metab 2002;87:3368–3372. Hilding A, Hall K, Wivall-Helleryd IL, Saaf M, Melin AL, Thoren M: Serum levels of insulin-like growth factor-I in 152 patients with growth hormone deficiency, aged 19–82 years, in relation to those in healthy subjects. J Clin Endocrinol Metab 1999;84:2013–2019. Boquete HR, Sobrado PG, Fideleff HL, Sequera AM, Giaccio AV, Suarez MG, Ruibal GF, Miras M: Evaluation of diagnostic accuracy of insulin-like growth factor (IGF)-I and IGF-binding protein-3 in growth hormone-deficient children and adults using ROC plot analysis. J Clin Endocrinol Metab 2003;88:4702–4708. Fukuda I, Hizuka N, Itoh E, Yasumoto K, Ishikawa Y, Murakami Y, Sata A, Takano K: Acid-labile subunit in growth hormone excess and deficiency in adults: Evaluation of its diagnostic value in comparison with insulin-like growth factor (IGF)-I and IGF-binding protein-3. Endocr J 2002;49:379–386. Mukherjee A, Monson JP, Jonsson PJ, Trainer PJ, Shalet SM, KIMS International Board: Seeking the optimal target range for insulin-like growth factor I during the treatment of adult growth hormone disorders. J Clin Endocrinol Metab 2003;88:5865–5870. Gargosky SE, Hasegawa T, Tapanainen P, MacGillivray M, Hasegawa Y, Rosenfeld RG: Urinary insulin-like growth factors (IGF) and IGF-binding proteins in normal subjects, growth hormone deficiency, and renal disease. J Clin Endocrinol Metab 1993;76:1631–1637. Spagnoli A, Gargosky SE, Spadoni GL, MacGillivray M, Oh Y, Boscherini B, Rosenfeld RG: Characterization of a low molecular mass form of insulin-like growth factor binding protein-3 (17. 7 kDa) in urine and serum from healthy children and growth hormone (GH)-deficient patients: Relationship with GH therapy. J Clin Endocrinol Metab 1995;80:3668–3676. Gill MS, Toogood AA, O’Neill PA, Thorner MO, Shalet SM, Clayton PE: Urinary growth hormone (GH), insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 measurements in the diagnosis of adult GH deficiency. J Clin Endocrinol Metab 1998;83:2562–2565. Baxter RC: IGF binding protein-3 and the acid-labile subunit: Formation of the ternary complex in vitro and in vivo. Adv Exp Med Biol 1993;343:237–244.

Gianluca Aimaretti, MD Division of Endocrinology and Metabolism Department of Internal Medicine, University of Turin C.so Dogliotti, 14, IT–10126 Turin (Italy) Tel. ⫹39 011 6334317/6334336, Fax ⫹39 011 6647421, E-Mail [email protected]


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Monitoring of Insulin-Like Growth Factors during Growth Hormone Treatment: Adulthood Growth Hormone Deficiency John P. Monson Centre for Clinical Endocrinology, William Harvey Research Institute, St Bartholomew’s and The Royal London Hospital, Queen Mary’s School of Medicine and Dentistry, University of London, London, UK

Abstract Decisions on growth hormone (GH) dosing in paediatric practice have depended on body weight or surface area calculations. Although this policy was used initially in the treatment of adult patients, it has become apparent both in clinical studies and longitudinal observation that this results in relative overtreatment of male patients, with a high incidence of immediate side effects, and undertreatment of females. Use of individualized GH dosing against serum IGF-I has proven useful in optimizing therapy across the whole adult age range, including the elderly and in patients who are in transition from post-puberty to full somatic development. It also eliminates gender differences in susceptibility. In this respect, serum IGF-I is superior to other GH-dependent peptides. However, on present evidence, serum IGF-I should be regarded primarily as a safety marker rather than a specific therapeutic marker. Copyright © 2005 S. Karger AG, Basel

Introduction

The initial approach to growth hormone (GH) dosing in hypopituitary GH-deficient adults was based on body weight or surface area as an extension of established paediatric practice [1–3]. This methodology was undoubtedly successful in terms of proving the concept that GH replacement exerted beneficial effects on body composition, lipoprotein profiles and psychological well-being. However, the therapeutic strategy was flawed because it failed to

Table 1. Candidate biochemical markers of GH action Serum insulin-like growth factor-I Serum IGF binding protein-3 Serum acid-labile subunit Serum bone-specific alkaline phosphatase Serum osteocalcin Serum carboxy-terminal propeptides of type 1 collagen Urine or serum pyridinoline and deoxypyridinoline Serum carboxy-terminal cross-linked telopeptides of type 1 collagen Serum insulin Serum leptin

take account of interindividual variations in GH responsiveness and in particular made no adjustment for gender differences in GH sensitivity. As a consequence, weight-based dosing regimens were associated with a high incidence of early side effects, predominantly related to fluid retention, and resulted in obese patients receiving excessive GH doses, relative overtreatment of male patients and undertreatment of female patients [4]. These considerations have prompted a re-evaluation of the methods of optimising GH replacement in adults [4]. This chapter examines these issues in relation to the use of subjective indices of appropriate GH replacement and the utility of measurement of circulating GH-dependent markers of GH action in the clinical management of the GH-deficient adult.

Candidate Biochemical Markers of Growth Hormone Action

Potential methods for monitoring the effects of exogenous GH administration are listed in table 1. These include measurement of the GH-dependent peptides, insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). These assays provide a measurement of the direct effects of GH on hepatic production of these peptides. In addition, measurement of markers of bone remodelling (bone formation: bone-specific alkaline phosphatase, osteocalcin, carboxy-terminal propeptides of type I collagen; bone resorption: serum and urine pyridinoline and deoxypyridinoline, serum carboxy-terminal cross-linked telopeptide of type 1 collagen) may have some utility in the documentation of response [4, 5]. However, despite the fact that bone remodelling activity is decreased in the GH-deficient state, there is considerable overlap with normal individuals. Thus, whilst measurements of

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markers of bone remodelling may be useful indices of therapeutic response in individual patients, they cannot provide a biochemical target indicative of normalisation of GH status [4]. Both serum insulin and leptin are influenced by the adverse changes in body fat distribution which characterise the GH-deficient state and tend to be elevated. Decreases in central adiposity during GH replacement are paralleled by decreases in serum leptin but the overlap with normal levels and interindividual variation limits the value of these measurements for optimisation of therapy [6]. Serum insulin is influenced additionally by the direct effects of GH on carbohydrate metabolism [7]; the evolving effect of GH on insulin sensitivity and serum insulin concentrations represents a summation of these effects and wide interindividual variation is encountered. The GH-dependent proteins, IGF-I, IGFBP-3 and ALS, demonstrate predictably tight interrelationships in healthy subjects [8] (fig. 1) but are limited in terms of their ability to distinguish the baseline GH-deficient state from normal subjects. This is least problematic for IGF-I but, nonetheless, a substantial proportion of GH-deficient patients demonstrate a serum IGF-I within the lower half of the normal reference range [3, 9], presumably under the influence of nutrition, adiposity and gonadal status amongst other poorly defined factors. This overlap increases with advancing age so that a majority of GH-deficient patients aged over 60 years have a serum IGF-I within the normal reference range (fig. 2). In contrast, younger patients, especially those with GH deficiency of childhood onset, are likely to demonstrate a subnormal serum IGF-I [10]. Burman et al. [11] have demonstrated negative correlations between changes in serum IGF-I and fat mass during weight-based GH replacement in male patients; the absence of a significant relationship in females may be related to

Monitoring of IGFs during GH Treatment: Adulthood GH Deficiency

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relative undertreatment using this dosing strategy (fig. 3). A positive relationship between serum IGF-I and gain in lean body mass over 12 months of therapy has also been demonstrated by Thorén et al. [12].

Relative Value of Symptoms and Biochemical Markers in the Assessment of GH Replacement – Use of Serum IGF-I as a Safety Marker

Studies examining the impact of body weight- or surface area-based GH dosing on clinical signs and serum markers of GH action have demonstrated a high prevalence of abnormally elevated serum IGF-I [4, 13]. Furthermore, pathological increments in serum IGF-I are not necessarily associated with a proportional increment in serum IGFBP-3 and ALS indicating that serum IGF-I is a more sensitive potential marker of excess GH exposure. De Boer et al. [13] performed detailed studies examining these aspects and also went on to show that symptoms of GH excess were associated with an elevated serum IGF-I in less than 50% of patients treated with a varying GH dose schedule based on body surface area. In addition, they demonstrated that symptoms of overtreatment with GH were rarely associated with abnormal elevation of serum IGFBP-3 or ALS (fig. 4). Critically, the majority of patients with abnormal elevation of serum IGF-I did not report adverse symptoms confirming that subjective responses are insensitive in the determination of inappropriately high GH exposure [13].

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Fig. 4. Serum IGF-I, IGFBP-3 and ALS SD scores in GH-deficient patients after 6 months of GH replacement, ranked in ascending order of increasing GH dose. Data are taken from De Boer et al. [13] and reproduced with permission.

Influence of Gender on GH Sensitivity

In health, females demonstrate higher GH secretion in terms of pulse amplitude than males [4]. This is associated with quantitatively similar serum IGF-I concentrations suggesting that females are relatively GH resistant, at least in terms of IGF-I generation. This would predict a greater GH dose requirement in female hypopituitary patients, but because initial studies of GH


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replacement in adults utilised the paediatric practice of weight or surface areabased dosing, without gender adjustment, this issue was ignored. In support of this hypothesis, female GHD patients demonstrate lower serum IGF-I concentrations than males [14]. Several studies have indicated significantly lower serum IGF-I generation for a given GH dose in females compared with males [15–18], an effect which appears to be influenced further by oestrogen administration (fig. 5). This is most apparent in patients who are on oral oestrogen replacement, presumably reflecting the direct influence of hepatic first-pass effects on IGF-I production [16, 18], but is also evident in females on transdermal or depot oestrogen replacement and in those with preserved endogenous oestrogen secretion. Interestingly, exogenous androgen administration appears to have an opposite effect, resulting in some enhancement of IGF-I generation for a given GH dose [17] (fig. 5).

Comparisons of Higher and Lower Doses of GH on Clinical Response

Although the initial proof of concept studies of GH replacement in hypopituitary adults utilised supraphysiological dosing, it has become abundantly clear that lower doses of GH produce broadly similar outcomes. Thus, Carroll et al. [19] demonstrated that halving the then conventional weight-based dosing regimen resulted in similar beneficial changes in body composition and quality-of-life measures. These lower doses of GH in this study were associated

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with a predictable reduction in serum IGF-I and provided parallel validation for titration of GH dose against serum IGF-I (see below).

Use of GH Dose Titration against Serum IGF-I

The need to move towards individualised dosing of GH replacement was prompted essentially by two sets of observations. Firstly, the fact that GH dose could be reduced without loss of efficacy and secondly, the clear gender differences in GH sensitivity favouring males. Individualised dosing has employed two strategies which may be combined. Some groups have advocated the individual adjustment of GH dose on the basis of serum IGF-I and body composition measurements with priority being given to the factor demonstrating the greatest deviation from normal [20]. This strategy was associated with reduction in mean GH dose without loss of efficacy but resulted in elevation of serum IGF-I above the reference range in approximately 20% of patients [20]. Alternatively, serum IGF-I may be used as the sole determinant of GH dose adjustment. Since serum IGF-I is within the age-related reference range in a substantial proportion of middle-aged and older hypopituitary patients, our group has advocated targeting serum IGF-I to between the median and upper end of the age-related reference range provided adverse symptoms do not occur [15]. This approach maintains the efficacy of treatment, results in lower GH doses and importantly, uses serum IGF-I as a safety marker. Although it would be inappropriate to regard serum IGF-I as a therapeutic marker, it remains the single most sensitive marker of GH action and is therefore critical from a safety standpoint, bearing in mind the potential longer term effects of IGF-I elevation in inducing cardiac changes, increasing risk of neoplasia and affecting adversely carbohydrate metabolism [4]. During GH dose titration, at a median starting dose of 0.27 mg/day, serum IGF-I reaches the target range in a median time of 4 weeks in males and 8 weeks in females [15] (fig. 6). Steady-state serum IGF-I concentrations are reached within 2 weeks of a change in GH dose. In the same studies, measurements of serum IGFBP-3 and ALS demonstrated their relative insensitivity to changes in GH dose in comparison with serum IGF-I [15]. For this reason, IGFBP-3 and ALS are not recommended for GH dose titration.

Value of Serum IGF-I in Monitoring GH Replacement in Older Patients

The use of GH dose titration against serum IGF-I has facilitated the safe treatment of GHD in older adults. Analyses of GH dose and serum IGF-I in the


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KIMS database (pharmacoepidemiological study of GH replacement in adults sponsored by Pfizer Inc.) have indicated a predictable and progressive reduction in GH dose requirement in middle-aged and older adults (65 years) whilst achieving a serum IGF-I SD score within the declining age-related reference range [21, 22]. The beneficial changes in lipoprotein profiles and in quality of life were similar in patients aged 45, 45–55, 55–65 and 65 years, indicating that initiation or continuation of GH replacement in older GHD adults is justified [22]. Furthermore, these data emphasise the importance of GH dose titration as a means of ensuring minimum effective doses of GH.

Role of Serum IGF-I in Monitoring GH Replacement in the Adolescent to Adult Transition Phase

Recent studies examining the impact of discontinuation of GH at completion of linear growth in young adults with persisting GHD have highlighted adverse effects on body composition and lipoprotein profiles [23, 24]. However, the interpretation of these studies requires critical examination of serum IGF-I levels during GH replacement in childhood in order to ensure that the observed effects were not attributable to a decrement in supraphysiological serum IGF-I concentrations. Subsequent randomised intervention studies have demonstrated beneficial effects of continuation of GH replacement, achieving normal serum IGF-I concentrations, on accrual of bone mass and lean body mass [25, 26]. In the latter studies, discontinuation of GH was associated with an increase in insulin sensitivity in contrast with the relative insulin resistance

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which characterises continuation of GH replacement [26]. The latter phenomenon may be critical in optimising the continuing anabolic effect of GH.

IGF-I Status in Relation to the Age and Timing of Onset of GH Deficiency – Implications for GH Dosing in Young Adults

Differences in serum IGF-I concentrations have been reported between patients with childhood-onset (CO) GHD and adult-onset (AO) GHD with lower levels being reported in the former [27]; this may have implications for GH dosing in these patients. However, a number of confounding factors including differences in body mass index, body composition and severity of hypopituitarism may have influenced the results of these studies. This issue has been evaluated recently utilising the large resource of data on both CO-GHD and AO-GHD patients available in the KIMS database [10]. This analysis has demonstrated that although age of onset was the most important factor in determining serum IGF-I standard deviation score (SDS), patients with CO-GHD had significantly lower serum IGF-I SDS than AO-GHD patients after allowing for all other confounding factors including age, numbers of additional hormone deficiencies and gender [10]. The mechanism underlying this phenomenon remains unclear but possible factors include differences in body composition between CO-GHD and AO-GHD and the effects of GHD at critical stages of development. From a practical clinical perspective, these findings indicate the importance of careful follow of serum IGF-I in adults with CO-GHD to ensure that GH treatment dosing is adequate. The latter point is particularly relevant to females who have a lower mean baseline serum IGF-I SDS than males [14].

General Conclusions: Value and Limitation of Serum IGF-I Monitoring during GH Replacement Therapy

The extensive data on use of serum IGF-I for GH dose titration has confirmed the value of this approach in terms of achieving the lowest effective GH dose, avoidance of early side effects and reduction in gender differences in susceptibility [4]. In this respect, serum IGF-I is superior to other GH-dependent peptides. However, measurement of serum IGF-I has clear limitations, not least because of the overlap between baseline serum IGF-I measurements in hypopituitary patients and the normal reference range particularly in middle-aged and older patients. Furthermore, the suboptimal correlation between serum IGF-I and some metabolic functions indicates that serum IGF-I should not be viewed as a therapeutic marker [28, 29]. Rather, measurements of serum IGF-I should


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Salomon F, Cuneo RC, Hesp R, Sönksen P: The effects of treatment with recombinant human growth hormone on body composition and metabolism in patients with growth hormone deficiency. N Engl J Med 1989;321:1979–1803. Jørgensen JOL, Pedersen SA, Thuesen L, Jorgensen J, Ingemann-Hansen T, Skakkabæk N, Christiansen JS: Beneficial effects of growth hormone treatment in GH-deficient adults. Lancet 1989;i:1221–1225. Carroll PV, Christ ER, Bengtsson BÅ, Carlsson L, Christiansen JS, Clemmons D, Hintz R, Ho K, Laron Z, Sizonenko P, Sönksen PH, Tanaka T, Thorner MO: Growth hormone deficiency in adulthood and the effects of growth hormone replacement: A review. J Clin Endocrinol Metab 1998;83:382–395. Drake WM, Howell SJ, Monson JP, Shalet SM: Optimising GH therapy in adults and children. Endocr Rev 2001;22:425–450. Johansen JS, Pedersen SA, Jørgensen JOL, Riis BJ, Christiansen C, Christiansen JS, Skakkebæk NE: Effects of growth hormone (GH) on plasma bone Gla protein in GH-deficient adults. J Clin Endocrinol Metab 1990;70:916–919. Gill MS, Toogood AA, Jones J, Clayton PE, Shalet SM: Serum leptin response to the acute and chronic administration of growth hormone (GH) to elderly subjects with GH deficiency. J Clin Endocrinol Metab 1999;84:1288–1295. Weaver JU, Monson JP, Noonan K, John WG, Edwards A, Evans KA, Cunningham JC: The effect of low dose recombinant growth hormone replacement on regional fat distribution, insulin sensitivity and cardiovascular risk factors in hypopituitary adults. J Clin Endocrinol Metab 1995;80: 153–159. Khosravi MJ, Diamandi A, Mistry J, Krishna RG, Khare A: Acid-labile subunit of human insulinlike growth factor-binding complex: Measurement, molecular and clinical evaluation. J Clin Endocrinol Metab 1997;82:3944–3951. Hilding A, Hall K, Wivall-Helleryd IL, Sääf M, Melin AL, Thorén M: Serum levels of insulin-like growth factor-I in 152 patients with growth hormone deficiency, aged 19–82 years, in relation to those in healthy subjects. J Clin Endocrinol Metab 1999;84:2013–2019. Lissett CA, Jönsson PJ, Monson JP, Shalet SM: IGF-I status in age-matched cohorts with adult onset and childhood onset growth hormone deficiency. Clin Endocrinol 2003;59:773–778. Burman P, Johansson AG, Siegbahn A, Vessby B, Karlsson FA: Growth hormone (GH)-deficient men are more responsive to GH replacement therapy than women. J Clin Endocrinol Metab 1997;82:550–555. Thorén M, Hilding M, Baxter RC, Degerblad M, Wivall-Helleryd IL, Hall K: Serum IGF-I, IGFBP-1 and -3 and the acid-labile subunit as serum markers of body composition during GH therapy in adults with GH deficiency. J Clin Endocrinol Metab 1997;82:223–228. De Boer H, Blok GJ, Popp-Snijders C, Stuurman L, Baxter RC, van der Veen E: Monitoring of serum GH replacement in adults based on measurement of serum markers. J Clin Endocrinol Metab 1996;81:1371–1377. Murray RD, Howell SJ, Lissett CA, Shalet SM: Pre-treatment IGF-I level is the major determinant of GH dosage in adult GH deficiency. Clin Endocrinol 2000;52:537–542. Drake WM, Coyte D, Camacho-Hübner C, Jivanji NM, Kaltsas G, Wood DF, Trainer PJ, Grossman AB, Besser GM, Monson JP: Optimising growth hormone replacement therapy by dose titration in hypopituitary adults. J Clin Endocrinol Metab 1998;83:3913–3919. Cook DM, Ludlam WH, Cook MB: Route of estrogen administration helps to determine growth hormone replacement dose in GH-deficient adults. J Clin Endocrinol Metab 1998;84:3956–3960.

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Span JPT, Pieters GFFM, Sweep CGJ, Hermus ARMM, Smals AGH: Gender difference in insulinlike growth factor-I response to growth hormone treatment (GH) in GH-deficient adults: Role of sex hormone replacement. J Clin Endocrinol Metab 2000;85:1121–1125. Janssen YJH, Helmerhorst F, Frölich M, Roelfsema F: A switch from oral (2 mg/day) to transdermal (50 g/day) 17 -estradiol therapy increases serum insulin-like growth factor-I levels in recombinant human growth hormone (GH)-substituted women with GH deficiency. J Clin Endocrinol Metab 2000;85:464–467. Carroll PV, Littlewood R, Weissberger AJ, Bogalho P, McGauley G, Sönksen PH, Russell-Jones DL: The effects of two doses of replacement GH on the biochemical, body composition and psychological profiles of GH-deficient adults. Eur J Endocrinol 1997;137:146–153. Johannsson G, Rosén T, Bengtsson BÅ: Individual dose titration of GH during GH replacement in hypopituitary adults. Clin Endocrinol 1997;47:571–581. Monson JP, Abs R, Bengtsson BÅ, Bennmarker H, Feldt-Rasmussen U, Hernberg-Ståhl E, Thorén M, Westberg B, Wilton P, Wüster C: Growth hormone deficiency and replacement in elderly hypopituitary adults. Clin Endocrinol 2000;53:281–289. Monson JP, Jönnsson P: Aspects of growth hormone (GH) replacement in elderly patients with GH deficiency: Data from KIMS. Horm Res 2003;60(suppl 1):112–120. Johannsson G, Albertsson-Wikland K, Bengtsson BÅ: Discontinuation of growth hormone (GH) treatment: metabolic effects in GH-deficient and GH-sufficient adolescent patients compared with control subjects. Swedish Study Group for Growth Hormone Treatment in Children. J Clin Endocrinol Metab 1999;84:4516–4524. Colle M, Auzerie J: Discontinuation of growth hormone therapy in growth-hormone-deficient patients: Assessment of body fat mass using bioelectrical impedance. Horm Res 1993;39:192–196. Drake WM, Carroll PV, Maher KT, Metcalfe KA, Camacho-Hubner C, Shaw NJ, Dunger DB, Cheetham TD, Savage MO, Monson JP: The effect of cessation of growth hormone (GH) therapy on bone mineral accretion in GH-deficient adolescents at completion of linear growth. J Clin Endocrinol Metab 2003;88:1658–1663. Carroll PV, Drake WM, Maher KT, Metcalfe KA, Shaw NJ, Dunger DB, Cheetham TD, CamachoHübner C, Savage MO, Monson JP: Comparison of continuation or cessation of growth hormone (GH) therapy on body composition and metabolic status in adolescents with severe GH deficiency at completion of linear growth. J Clin Endocrinol Metabolism 2004;89:3890–3895. Attanassio AF, Lamberts SW, Matranga AM, Birkett MA, Bates PC, Valk NK, Hilsted J, Bengtsson BÅ, Strasburger CJ: Adult growth hormone (GH)-deficient patients demonstrate heterogeneity between childhood onset and adult onset before and during GH treatment. Adult Growth Hormone Deficiency Study Group. J Clin Endocrinol Metab1997;82:82–88. Lucidi P, Lauteri S, Santoni S, Lauteri M, Busciantella-Ricci N, Angeletti G, Santeusiano F, de Feo P: Administration of recombinant human growth hormone on alternate days is sufficient to increase whole body protein synthesis and lipolysis in growth hormone-deficient adults. Clin Endocrinol 2000;52:173–179. Lucidi P, Lauteri M, Lauteri S, Celleno R, Santoni S, Volpi E, Angeletti G, Santeusiano F, de Feo P: A dose-response study of GH replacement on whole body protein and lipid kinetics in GH-deficient adults. J Clin Endocrinol Metab 1998;83:353–357.

Prof. John P. Monson Department of Endocrinology St Bartholomew’s Hospital, West Smithfield London EC1A 7BE (UK) Tel. 44 20 7601 8346, Fax 44 20 7601 8505, E-Mail [email protected]


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IGFs and IGFBPs in GH Insensitivity M.O. Savagea, J.C. Blaira, A.J. Jorgea, M.E. Streetb, M.B. Rankec, C. Camacho-Hübnera a

Department of Endocrinology, St Bartholomew’s Hospital, London, UK; Children’s Hospital, Parma, Italy, and cUniversity Children’s Hospital, Tübingen, Germany b

Abstract IGF-I, IGFBP-3 and ALS are GH-dependent peptides and their production is disturbed in states of GH insensitivity. This chapter explores the relative degrees of IGF-I, IGFBP-3 and ALS deficiency across the spectrum of GH insensitivity. In classical GH insensitivity syndrome (GHIS), known as Laron syndrome, due to GH receptor (GHR) deficiency, serum IGF-I, IGFBP-3 and ALS are severely reduced with inability to produce these peptides during an IGF-I generation test. Across the spectrum of severity of GHR defects, some patients have short stature and normal facial appearance, so-called partial or non-classical GH insensitivity. In these cases the IGF-I, IGFBP-3 deficiency is less severe. A positive relationship exists between height SDS and IGFBP-3 SDS (r2 ⫽ 0.45, p ⬍ 0.001) in patients from the European series with GHIS. In a new series of GHIS cases (n ⫽ 36) there was a significant difference in IGFBP-3 and ALS (p ⬍ 0.05) between classical (n ⫽ 25) and non-classical cases (n ⫽ 11). IGF-I, IGFBP-3 and ALS were significantly higher (p ⬍ 0.05) in pubertal compared with pre-pubertal subjects in the same series. In idiopathic short stature (ISS), heterozygous mutations of the GHR may have a dominant negative effect. ISS patients have lower IGF-I levels than the normal population. In 21 cases, mean IGF-I SDS was ⫺1.39 (⫺2.4 to ⫺1.16) and IGFBP-3; ⫺0.45 (⫺1.13 to 0.38). However, IGF-I and IGFBP-3 responses in the IGF-I generation test were generally normal. In acquired GHI due to chronic illness such as Crohn’s disease, juvenile arthritis and cystic fibrosis, IGF-I deficiency is present, although IGFBP-3 is usually normal. In summary, assessment of IGF-I, IGFBP-3 and ALS contributes to diagnosis in GH insensitivity states. In our experience, IGF-I is more sensitive to disturbance of GH action that IGFBP-3, however in severe GHIS cases, IGF-I is usually undetectable and measurement of IGFBP-3 is valuable as a guide to the severity of the biological defect. Copyright © 2005 S. Karger AG, Basel

Introduction

Growth hormone insensitivity (GHI) is associated with disturbance of the GH-IGF-I axis. Consequently, clinical syndromes of GHI are likely to be accompanied by abnormalities of the constituent peptides of the IGF system. This chapter will focus on three aspects of GHI, namely the spectrum of genetic GHI [1], the assessment of possible GHI in children with idiopathic short stature (ISS) [2] and forms of acquired GHI associated with IGF-I deficiency and abnormalities of linear growth. The contributions of measurement of IGF-I, IGFBP-3 and acid-labile subunit (ALS) to the diagnosis of GHI in varying degrees of severity will also be discussed.

The Spectrum of Genetic GH Insensitivity

Growth hormone insensitivity syndrome (GHIS), or Laron syndrome [3, 4], is the most severe form of genetic GHI and is usually caused by an inherited defect of the GH receptor (GHR) [5]. A post-receptor mutation of the signalling molecule STAT-5b resulting in impaired GH signal transduction has recently been described [6], as has the first mutation of the ALS gene [7]. GHIS is associated with a phenotype of severe post-natal growth failure, adult short stature, impaired cranio-facial development and asymptomatic hypoglycaemia [4, 8]. IGF-I, IGFBP-3 and ALS in GH Insensitivity Syndrome Impaired GHR function or GH signal transduction is associated with a severe decrease in synthesis and circulating concentrations of GH-dependent peptides. In the European GHI series, IGF-I was frequently below the detection limit of the assay [9, 10]. Severe IGF-I deficiency has also been a characteristic finding in other cohorts of GHIS patients [4, 11, 12] and is likely to be the principle cause of abnormal cranio-facial development and linear growth failure. IGFBP-3 deficiency was also present in all GHIS patients and together with IGF-I was used as an entry criterion for the European GHI series [13]. However, IGFBP-3 may be less decreased than IGF-I and, when converted to SDS scores [10], was a sensitive index of the degree of severity of GHI, demonstrated by the significant positive relationship between IGFBP-3 and height SDS values across a spectrum of GHI in the European series [10]. IGFBP-3 values also correlated directly with facial height SDS [14]. In a small series of GHIS patients treated with rhIGF-I, IGFBP-3 SDS values correlated negatively

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Fig. 1. Variation in phenotype from classical Laron syndrome (triangles) to idiopathic short stature (circles) and correlation of height SDS and IGFBP-3 SDS in 59 patients with GH insensitivity.

with the growth response to treatment, indicating that the more severe the biological defect the greater the response to rhIGF-I treatment [15]. Hence, as IGF-I treatment becomes more available, IGFBP-3 levels may become a useful predictor of response in GHI patients [16]. IGF-II levels are also low in GHIS patients, reflecting the deficiency of circulating IGFBP-3 [9]. There is little published data on ALS levels in GHIS patients. Predictably, when measured, ALS is decreased [17] and may reflect the severity of GHR dysfunction [Camacho-Hübner, unpubl. data]. Partial (Non-Classical) GH Insensitivity The European population of GHI patients demonstrated the spectrum of genotype and phenotype heterogeneity [10]. An analysis of 59 of these patients from the total of 82, separated those with typical Laron syndrome features (n ⫽ 50) from those with normal facial appearance (n ⫽ 9) [17]. All these patients fulfilled the strict criteria for GHI [13], which demonstrates that partial or non-classical GHI exists as a clinical entity. As stated above, many had undetectable IGF-I levels but IGFBP-3 values correlated positively with height SDS. IGF-I and IGFB-3 levels in classical compared with non-classical patients are shown in figure 1. All the Laron syndrome patients had both height and IGFBP-3 SD less than –4 SD. Well-characterised patients with partial GHI have been reported less frequently than those with classical Laron syndrome. Nevertheless, several case

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IVS6 pseudoexon IVS8 insertion as-1 G to C IVS9 ds⫹1 G to A ds⫹2 T to C

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reports demonstrate a range of IGF-I and IGFBP-3 abnormalities [18–20]. In the above patients, defects of the GHR have also been identified [21].

Idiopathic Short Stature

The possibility of GHI being the cause of abnormal growth in some ISS patients stemmed from the reports of heterozygous GHR mutations [22], decreased GHBP levels [23] and low IGF-I levels with elevated GH secretion [24] in short children from the NCGS, Sub-Study VI database. Investigation of the GH-IGF-I axis in ISS has recently been reviewed and GHR mutations are summarised in figure 2. Since these reports, there have been extensive studies of IGF-I and IGFBP-3 in populations of children with ISS. Most have shown low IGF-I levels [25–27], but far from the degree seen in GHIS. In IGF-I generation tests, quantitatively inferior responses of IGF-I and IGFBP-3 have been demonstrated compared to control subjects, supporting the hypothesis of mild GHI in some patients. However, most ISS patients show IGF-I and IGFBP-3 responses to GH stimulation. We have found measurement of IGFBP-3 to be relatively unhelpful in the diagnosis of mild GHI in these patients [26]. However in individual patients with severe short stature, measurement of IGF-I, IGFBP-3 and ALS in association with assessment of GH secretion may contribute to the understanding of the pathogenesis of growth failure [28].


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Acquired GH Insensitivity due to Chronic Paediatric Illness

GHI has been reported in a number of chronic diseases such as Crohn’s disease, juvenile chronic arthritis, cystic fibrosis and anorexia nervosa. The list of potential non-endocrine diseases associated with abnormal growth is long, but most have been poorly studied for defects of the GH-IGF-I axis. In chronic illness the major mechanisms contributing to abnormal growth are impaired intake of nutrients, interference by cytokines in the endocrine-growth axis and increased energy expenditure. A combination of these factors may contribute to GHI. In Crohn’s disease, IGF-I deficiency is now well documented [28, 29]. Concentrations of IGFBP-3 may be less abnormal [28]. Normalisation of IGF-I and IGFBPs, in association with decrease in interleukin-6 levels and disease activity score has been reported during treatment with enteral nutrition [30]. The increase in IGF-I has been shown to pre-date changes in nutritional parameters, suggesting that the inflammatory process on its own plays a significant part in causing IGF-I deficiency [30]. Juvenile chronic arthritis is another paediatric illness associated with growth failure and IGF-I deficiency. There are few studies in such patients, however low IGF-I and IGFBP-3 levels are well documented [31] and growth failure with significant decrease in height SDS after diagnosis is a major complication of this disorder. Encouraging results with GH therapy to arrest the evolution of severe short stature have been reported.

Conclusions

Serum IGF-I, IGFBP-3 and ALS in the presence of normal GH secretion have been shown to be useful clinical markers of the severity of GHI. In extreme or classical genetic GHIS, basal determination of these peptides is of major importance for diagnosis. As GHI becomes less severe, the value of these measurements diminishes. For example in ISS, where GHI is said to contribute to the aetiology in some cases, we have found IGF-I to be slightly decreased, but generally responding to GH stimulation. IGFBP-3 on the other hand has been essentially normal and does not appear to contribute to diagnosis [2]. The difficulty in diagnosing mild GHI relates to the lack of diagnostic criteria for such patients. The strict criteria used for diagnosis of Laron syndrome [13] are clearly inappropriate. In chronic illness, and particularly in association with excess cytokine production, measurement of IGF-I and IGFBPs will indicate the presence of GHI. However, this is usually a subtle form of mild GHI, which is far removed from the extreme disturbance of the GH-IGF axis in classical genetic GHIS.


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Johnston LB, Woods KA, Rose S, Clark AJ, Savage MO: The broad spectrum of inherited growth hormone insensitivity. Trends Endocrinol Metab 1998;9:228–232. Blair JC, Savage MO: Investigation of the growth hormone–IGF-I axis in idiopathic short stature. Trends Endocrinol Metab 2002;13:325–330. Laron Z, Pertzelan A, Mannheimer S: Genetic pituitary dwarfism with high serum concentrations of growth hormone – A new inborn error of metabolism? Isr J Med Sci 1966;2:152–155. Laron Z: Laron syndrome (primary growth hormone resistance or insensitivity): The personal experience 1958–2003. J Clin Endocrinol Metab 2004;89:1031–1044. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J: Growth hormone insensitivity due to primary growth hormone receptor deficiency. Endocr Rev 1994;15:369–390. Kofoed EM, Hwa V, Little B, Woods KA, Buckway CK, Tsubaki J, Pratt KL, Bezrodnik L, Jasper H, Tepper A, Heinrich JJ, Rosenfeld RG: Growth hormone insensitivity associated with a STATb mutation. N Engl J Med 2003;349:1110–1112. Domene HM, Bemgolea SV, Martinez AS, Ropelato MG, Pennisi P, Scaglia P, Heinrich JJ, Jasper HG: Deficiency of the circulating insulin-like growth factor system associated with inactivation of the acid-labile subunit gene. N Engl J Med 2004;350:570–577. Rosenbloom AL, Guevara-Aguirre J, Rosenfeld RG, Fielder PJ: The little women of Loja – Growth hormone receptor deficiency in an inbred population of Southern Ecuador. N Engl J Med 1990;323:1367–1374. Savage MO, Blum WF, Ranke MB, Postel-Vinay MC, Cotterill AM, Hall K, Chatelain PG, Preece MA, Rosenfeld RG: Clinical features and endocrine status in patients with growth hormone insensitivity (Laron syndrome). J Clin Endocrinol Metab 1993;77:1465–1471. Woods KA, Dastot F, Preece MA, Clark SJL, Postel-Vinay MC, Chatelain PG, Ranke MB, Rosenfeld RG, Amselem S, Savage MO: Phenotype: Genotype relationships in growth hormone insensitivity syndrome. J Clin Endocrinol Metab 1997;82:3529–3535. Guevara-Aguirre J, Rosenbloom AL, Fielder PJ, Diamond FB, Rosenfeld RG: Growth hormone receptor deficiency in Ecuador: Clinical and biochemical phenotype in two populations. J Clin Endocrinol Metab 1993;76:417–423. Backeljauw PF, Underwood LE: The GHIS collaborative group. Prolonged treatment with recombinant insulin-like growth factor-I in children with growth hormone insensitivity syndrome – A clinical research centre study. J Clin Endocrinol Metab 1996;81;3312–3317. Blum WF, Cotterill AM, Postel-Vinay MC, Ranke MB, Savage MO, Wilton P, Improvement of diagnostic criteria in growth, hormone insensitivity syndrome: Solutions and pitfalls. Acta Paediatr Suppl 1994;399:117–124. Leonard J, Samuels M, Cotterill AM, Savage MO: Effects of recombinant insulin-like growth factor-I on cranio-facial morphology in growth hormone insensitivity. Acta Paediatr Suppl 1994;399:140–141. Azcona C, Preece MA, Rose SJ, Fraser N, Rappaport R, Ranke MB, Savage MO: Growth response to rhIGF-I 80l ␮g/kg twice daily in children with growth hormone insensitivity syndrome: Relationship to severity of clinical phenotype. Clin Endocrinol 1999;51:787–792. Savage MO, Camacho-Hübner C, Dunger DB: Therapeutic applications of the insulin-like growth factors. Growth Horm IGF Res 2004;14:301–308. Burren CP, Woods KA, Rose SJ, Tauber M, Price DA, Heinrich U, Gilli G, Razzaghy-Azar M, Al-Ashwal A, Crock PA, Rochiccioli P, Yordam N, Ranke MB, Chatelain PG, Preece MA, Rosenfeld RG, Savage MO: Clinical and endocrine characteristics in atypical and classical growth hormone insensitivity syndrome. Horm Res 2001;55:125–130. Ayling RM, Ross R, Towner P, Von Laue S, Finidori J, Moutoussamy S et al: A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nat Genet 1997;16:13–14. Iida K, Takahashi Y, Kaji H, Nose O, Okimura Y, Abe H et al: Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain. J Clin Endocrinol Metab 1998;83:531–537.


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Bjarnason R, Banerjee K, Rose SJ, Rosberg S, Metherell L, Clark AJL, Albertsson-Wikland K, Savage MO: Spontaneous growth hormone secretory characteristics in children with partial growth hormone insensitivity. Clin Endocrinol 2002;57:357–361. Metherell LA, Akker SA, Munroe PB, Rose SJ, Caufield M, Savage MO, Chew SL, Clark AJL: Pseudoexon activation as a novel mechanism for disease resulting in atypical growth hormone insensitivity. Am J Hum Gen 2001;69:641–644. Goddard AD, Covello R, Louh SM et al: Mutations of the growth hormone receptor in children with idiopathic short stature. N Engl J Med 1995;333:1093–1098. Carlsson LMS: Partial growth hormone insensitivity in childhood. Baillières Clin Endocrinol Metab 1996;10:389–400. Attie KM, Carlsson LM, Rundle AC, Sherman BM: Evidence for partial growth hormone insensitivity among patients with idiopathic short stature. J Pediatr 1995;127:244–250. Selva KA, Buckway CK, Sexton G, Pratt KL, Tjoeng E, Guevara-Aguirre J, Rosenfeld RG: Reproducibility in patterns of IGF generation with special reference to idiopathic short stature. Horm Res 2003;60:237–246. Blair JC, Camacho-Hübner C, Miraki-Moud F, Clayton PE, Burren C, Lim S, Albertsson-Wikland K, Rosberg S, Bjarnason R, Savage MO: Standard and low dose IGF-I generation tests and spontaneous growth hormone secretion in children with idiopathic short stature. Clin Endocrinol 2004;60:159–164. Rogol AD, Blethen, Sy JP, Veldhuis JD (NCGS): Do growth hormone serial sampling, insulin-like growth factor-I or auxological measurements have an advantage over stimulation testing in predicting the linear growth response to growth hormone therapy? Clin Endocrinol 2003;58:229–237. Beattie RM, Camacho-Hübner C, Wacharasindhu S, Cotterill AM, Walker-Smith JA, Savage MO: Responsiveness of IGF-I and IGFBP-3 to therapeutic intervention in children and adolescents with Crohn’s disease. Clin Endocrinol 1998;48:483–489. Street ME, Angelis GL, Camacho-Hübner C, Giovannelli G, Ziveri MA, Bacchini PL, Bernasconi S, Sansebastiona G, Savage MO: Relationships between serum IGF-I, IGFBP-2, interleukin-1␤ and interleukin-6 in inflammatory bowel disease. Horm Res 2004;61:159–164. Banerjee K, Camacho-Hübner C, Babinska K, Dryhurst K, Edwards R, Savage MO, Robinson IR, Croft NM: Anti-inflammatory and growth stimulating effects precede nutritional restitution during enteral feeding in Crohn’s disease. J Pediatr Gastroenterol Nut 2004;38:270–275. Davies UM, Jones J, Reeve J, Camacho-Hübner C, Woo P: Effects of disease activity and recombinant human growth hormone on insulin-like growth factor-I, insulin-like growth factor binding proteins 1 and 3, and osteocalcin. Arthritis Rheum 1997;40:643–650.

Prof. Martin O. Savage Department of Paediatric Endocrinology St Bartholomew’s Hospital, West Smithfield London EC1A 7BE (UK) Tel./Fax ⫹44 20 7601 8468, E-Mail [email protected]


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Childhood and Adolescent Diabetes David B. Dunger, Fiona M. Regan, Carlo L. Acerini Department of Paediatrics, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK

Abstract Circulating levels of insulin-like growth factor-I (IGF-I) and its principal binding protein IGFBP-3 are reduced, whereas those of the inhibitory binding protein, IGFBP-1, tend to be high in children and adolescents with type 1 diabetes mellitus (T1DM). These abnormalities are thought to arise because of relative portal hypoinsulinaemia and partial resistance at the hepatic growth hormone (GH) receptor. During adolescence, reductions in IGF-I and IGF bioactivity lead to feedback for GH hypersecretion and the elevated GH and low IGF-I levels lead to an increase of the normal insulin resistance encountered during puberty. Low IGF-I levels, but in particular elevated GH levels, have been implicated in the pathogenesis of diabetic microangiopathic complications, in particular, renal hypertrophy, glomerular hyperfiltration and the development of microalbuminuria. Early study of IGF-I replacement with recombinant human IGF-I (rhIGF-I) demonstrated, in the short term, reductions in GH hypersecretion with improved insulin sensitivity and, in the longer term, reductions in insulin requirements and improvements in HbA1c levels. However, larger doses of rhIGF-I were associated with retinopathy either due to rapid improvements in glycaemic control or direct effects of high levels of free IGF-I. More recently, pilot studies using the combination of rhIGF-I/rhIGFBP-3 have confirmed the physiological efficacy of IGF-I replacement in T1DM. The combined treatment is better tolerated and may result in reduced tissue exposure to high levels of ‘free’ IGF-I. Longer term clinical studies with this IGF-I/IGFBP-3 combination are needed. Copyright © 2005 S. Karger AG, Basel

Introduction

Abnormalities of the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis have been consistently demonstrated in children and adolescents with type 1 diabetes mellitus (T1DM). Circulating levels of IGF-I and its principal circulatory binding protein IGFBP-3 are reduced, whereas those of the

inhibitory binding protein, IGFBP-1, tend to be increased [1]. These abnormalities are thought to arise because of the relative portal insulinopenia resulting from subcutaneous, rather than direct portal, administration of insulin [2]. Expression of the hepatic GH receptor is partly insulin dependent and low levels of insulin in the portal circulation lead to relative hepatic GH resistance. This is manifested by low circulating levels of GH binding protein (GHBP) and probably reflects reductions in both hepatic GH receptor number and postreceptor signalling [3]. Overnight GH levels may be normal in prepubertal subjects with T1DM, but during adolescence the reduced circulating IGF-I levels and reduced IGF-I bioavailability lead to feedback drive for GH hypersecretion. The amplitude of overnight GH pulses is increased with increased trough levels and a shortening of pulse periodicity from 180 to 90 min [4]. Although there is relative resistance of the hepatic GH receptor, other tissue GH receptors remain sensitive to these high levels of GH. The direct effects of GH hypersecretion, local tissue production of IGF-I, and the low circulating levels of IGF-I have all been implicated in the insulin resistance and microvascular complications of T1DM which may first become evident during puberty.

Insulin Resistance in T1DM

Normal pubertal development is characterised by a decrease in insulin sensitivity and the compensatory hyperinsulinaemia may have a physiological role in promoting anabolism and growth. Increased insulin requirements are attributable to changes in insulin-stimulated glucose metabolism, which is reduced by 30–40% compared to prepubertal values [5, 6], and is due mainly to reductions in peripheral glucose utilisation rather than to changes in hepatic glucose production [7]. These effects are in part due to increases in GH levels [8], but may be modulated by variation in sex steroid levels. The insulin antagonistic actions of GH are thought to be principally mediated in the peripheral tissues [9, 10]. GH may be acting directly through its own receptor, with interaction with insulin signalling at a post-receptor level, or may be acting indirectly through mobilisation of non-esterified free fatty acids (NEFAs) from adipose tissue. NEFAs have suppressive effects on peripheral glucose metabolism resulting in inhibition of peripheral glucose oxidation and glycolysis [11, 12] and have been implicated in regulating hepatic glucose metabolism and mediating the actions of insulin on the liver [13, 14]. In T1DM the normal pubertal increase in insulin resistance is accentuated and seems to correlate with concomitant increases in nocturnal GH secretion [7, 15]. Spontaneous GH secretion is greater than that seen during normal puberty

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(fig. 1) and overnight plasma GH profiles are characterised by increases in both GH pulse amplitude and in baseline concentrations [4]. There is also evidence linking increased overnight GH secretion to the ‘dawn phenomenon’ of increasing early morning insulin requirements in T1DM [4, 16, 17]. Suppression of nocturnal GH release with agents such as somatostatin and pirenzipine reduces the extent of the dawn increase in insulin requirements [18]. The role that low IGF-I levels play in the development of insulin resistance in T1DM mellitus is more controversial. It is known that human IGF-I exhibits a high degree of structural homology (42–50%) with both proinsulin and insulin [19] and has some affinity for binding to the insulin receptor, which is structurally and functionally related to the type 1 IGF receptor [20]. However, IGF-I is known to directly increase rates of glucose disposal in skeletal muscle independent of insulin [21]. Any direct insulin-like effects of IGF-I on either hepatic glucose output and glucose uptake in skeletal muscle might be mediated through activation of hybrid insulin/IGF-I receptors that have high affinity for IGF-I [22]. Whilst many of the metabolic effects of IGF-I closely resemble those of insulin, it is likely that any differential tissue effects between the two are largely explained by the differences in their receptor distribution. IGF-I appears to have a more pronounced effect on glucose uptake by muscle than on hepatic glucose production, perhaps reflecting the paucity of type 1 IGF receptors in the adult liver [23]. Effects on the adipocyte are also less marked than those of insulin, with little or no effect on fat metabolism observed with IGF-I administration. It has been argued that IGF-I could not have a physiological role in glucose homeostasis because it is retained in the circulation in a large

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binding protein complex. However, experimental evidence suggests that the high IGFBP-1 levels characteristically found in patients with T1DM could, by mopping up ‘free’ IGF-I, contribute to the dawn phenomenon [24]. Restoration of circulating IGF-I levels to within the normal physiological range in subjects with T1DM should, in principle, lead to improvements in insulin sensitivity. Administration of physiological replacement doses of recombinant human IGF-I (rhIGF-I) in subjects with T1DM (given as single subcutaneous dose of 40 ␮g/kg) results in significant reductions in overnight GH and improvements in insulin sensitivity when compared to placebo [25]. Similar observations are seen when rhIGF-I is given as a subcutaneous infusion (20 ␮g/kg/h) over 2 days [26], and a strong association between reductions in GH levels and insulin requirements for euglycaemia have been reported [27]. These effects of rhIGF-I have been shown to be dose dependent with reductions in GH levels and insulin requirements observed with doses as low as 20 ␮g/kg [28]. In hyperinsulinaemic euglycaemic clamp studies the effects of rhIGF-I seem to relate more to the suppression of hepatic glucose production than to peripheral glucose uptake and may be attributed to GH suppression, either acting directly on the liver or mediated by reductions in free fatty acids that accompany falls in GH levels [14]. Disentangling the indirect effects of rhIGF-I administration (via reductions in GH hypersecretion) from its direct effects on insulin sensitivity have proved difficult. There are data indicating that following rhIGF-I administration there may be direct effects on insulin sensitivity and glucose homeostasis. Alterations in IGFBP levels have been observed following rhIGF-I administration [29] and circulating IGF-I bioavailability is increased. Studies by Crowne et al. [30] using subcutaneous rhIGF-I replacement where endogenous GH secretion was suppressed with somatostatin yet normal GH pulsatility maintained with exogenously infused recombinant human GH were indicative of a direct effect of IGF-I on insulin sensitivity. Similar data have been reported by Simpson et al. [31] in studies where rhIGF-I replacement was carried out under conditions where GH secretion was completely suppressed with somatostatin. Thus, it is likely that in T1DM mellitus both GH hypersecretion and low IGF-I levels contribute to the insulin resistance and increasing insulin requirements seen during puberty (fig. 2).

Microvascular Complications of T1DM

The onset of microangiopathic complications in children with T1DM has always been considered rare before the onset of puberty. This proposition is being cast in doubt, however, as recent data from several epidemiological studies


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IGF-I bioavailability

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⫺ve FFA

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⫺ve

Fig. 2. Relationship between insulin, the GH/IGF-I axis and insulin sensitivity in T1DM. ⫺ve indicates inhibition. GHBP ⫽ Growth hormone binding protein; IGFBP ⫽ insulin-like growth hormone binding protein.

have shown a significant effect on the development of complications from the prepubertal duration of diabetes and from puberty itself [32–34]. Differences in the rate of development of microalbuminuria (MA), an important early marker of diabetic nephropathy, has been shown to be related to factors such as age of diagnosis, HbA1c, sex and puberty [33]. Puberty in particular was associated with a three-fold increase risk of MA independent of any effect on glycaemic control, thus suggesting that the hormonal changes of puberty may be important [33]. Derangements of the GH/IGF-I axis have been associated with increased urinary albumin excretion in T1DM [35, 36] and with the development of retinopathy [37]. A role for GH in the pathogenesis of microangiopathic complications has been proposed for many years. Early studies indicated that pituitary ablation could retard the progression of proliferative retinopathy in T1DM [38, 39]. Furthermore, more recent studies in diabetic mouse models indicate that high GH levels may contribute to glomerular hypertrophy, hyperfiltration and the development of diabetic nephropathy [40]. GH hypersecretion in T1DM may therefore constitute a risk for the development of these problems. Although there is hepatic resistance to GH, the integrity of GHR expression in peripheral tissues such as the kidney remains intact [40]. In the face of exaggerated GH secretion, increased paracrine production of IGF-I in peripheral tissues occurs [41], although whether IGF-I is directly involved in the development of microangiopathy remains more contentious. Early studies suggested that high IGF-I


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levels in T1DM could contribute to proliferative retinopathy [42], but subsequent epidemiological studies have shown that the development of retinopathy and microangiopathic complications during puberty is invariably associated with low circulating IGF-I levels [43]. Variations in IGFBPs and IGF bioavailability may also contribute to these risks. In longitudinal population studies during puberty, the risks for the development of MA has been associated with low circulating levels of free and total IGF-I [43]. Sexual dimorphism in development of MA in T1DM has also observed, with females more at risk than their male counterparts, and thus may be related to the relatively lower free and total circulating IGF-I levels and higher free androgen index observed in females [43]. These changes seem to be independent of HbA1c and may reflect an underlying predisposition in subjects with MA to hepatic insulin resistance and thus reduced IGF-I generation.

IGF-I Replacement in T1DM

As defects of the GH/IGF-I axis in T1DM seem to arise from portal insulin deficiency, it seems logical that by simply increasing the amount of insulin delivered it should be possible to reverse these abnormalities and restore insulin sensitivity. However, intensification of subcutaneous insulin therapy during puberty whilst leading to improvements in glycaemic control, may be associated with an increased incidence of symptomatic and asymptomatic hypoglycaemia and, particularly in girls, with unacceptable weight gain. This phenomenon was highlighted by studies such as the Diabetes Control and Complications Trial (DCCT) where significant improvements in HbA1c with insulin therapy intensification where achieved at the expense of a greater risk of severe hypoglycaemia and obesity respectively [44]. These side effects are probably related to peripheral hyperinsulinaemia as high insulin doses are required to achieve normalisation of portal insulin concentrations with subcutaneous insulin delivery. Failure to correct abnormalities of the GH/IGF-I axis with intensive insulin therapy may also contribute to the problem, and failure to reduce GH hypersecretion may restrict benefits of improved glycaemic control on risks for retinopathy and MA. The risks of peripheral hyperinsulinism may be reduced by the administration of insulin via the intraperitoneal route, and the improved portal insulin concentrations achieved by this method can restore equilibrium to the GH/IGF-I axis and improve insulin sensitivity [45]. However, complete correction of the GH/IGF-I axis seems only possible by direct portal administration of insulin [46], but this is not currently a therapeutic option. These observations led to the hypothesis that IGF-I replacement as an adjunct to subcutaneous insulin therapy during puberty may lead to reversal of


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insulin resistance and longer term improvements in glycaemic control. In a preliminary, unblinded safety and efficacy study, 6 young adults were treated with a single daily subcutaneous injection of rhIGF-I (40 ␮g/kg/day) for 28 days, in addition to their multiple insulin injection therapy (3–4 injections daily). Sustained reductions in GH secretion and insulin requirements were observed, as well as a significant improvement in glycated haemoglobin (HbA1c) [47]. Subsequently, two randomised double-blind, placebo-controlled trials have been completed. Quattrin et al. [48] treated 40 adolescents with T1DM with rhIGF-I 80 ␮g/k/day for 28 days in addition to conventional twice-daily insulin therapy, and similar significant reductions in insulin requirements and HbA1c were observed. The same group later extended their observations in a larger study of adolescents and adults treated with rhIGF-I at doses of either 80, 120 or 140 ␮g/kg/day (given in two divided doses) for 12 consecutive weeks [49]. Again, significant reductions in HbA1c values and in insulin dose (range 11–19%) were observed with rhIGF-I therapy compared to placebo; with rhIGF-I 80 ␮g/kg/day having the greatest effect on glycaemic control (mean reduction HbA1c ⫽ 1.2%) [49]. A further study in adults with T1DM given rhIGF-I 50 ␮g/kg twice daily for 19 days also noted significant reductions in both GH concentrations and insulin requirements together with improvements in lipid profiles, however, the study was too short to observe any improvements in HbA1c [50]. Although the doses of rhIGF-I used in these studies were small (molar equivalents 0.05–0.1 unit/kg/day of insulin) it has been argued that the same benefits could have been achieved with intensified insulin therapy. This question was addressed by a large randomised double-blind, placebo-controlled trial in the UK and Sweden [51]. The study included 53 subjects with T1DM who were randomised to receive either placebo or rhIGF-I in doses of 20 or 40 ␮g/kg/day. All the subjects were on multiple insulin injection therapy consisting of 3–4 injections a day. Subjects were treated for 6 months during which time significant reductions in HbA1c were seen with the larger dose of rhIGF-I (40 ␮g/kg) compared to placebo. These improvements were achieved without any need to increase insulin dose, and without the increases in body mass index observed with intensified insulin therapy. There was however some loss of efficacy towards the end of the 6-month period which may be a reflection of poor compliance due to the need to administer 5 injections per day, although loss of bioefficacy with prolonged rhIGF-I administration cannot be excluded. This 6-month clinical trial of rhIGF-I therapy was also important in that it demonstrated that low dose rhIGF-I could be safely administered without significant adverse events. Adverse events have been consistently reported in clinical trials of rhIGF-I therapy given in larger doses. In subjects with severe insulin resistance [52] and


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type 2 diabetes [53], adverse events (table 1) were clearly a dose-related phenomenon, largely occurring with doses of 80 ␮g/kg/day or higher. Of particular concern was the observation that optic disc swelling and accelerated progression of retinopathy was seen in some studies. Thrailkill et al. [49] noted an early worsening of retinopathy, but in ⬎80% of the cases this was associated with the two highest doses of rhIGF-I used (i.e. 40 ⫹ 80 or 80 ⫹ 60 ␮g/kg twice daily). It is unclear whether this effect was due a direct effect by rhIGF-I or indirectly to improvements in glycaemic control and the so-called ‘normoglycaemic re-entry phenomenon’ [54]. However, papillitis was also observed in some patients and although this was occasionally encountered in insulin intensification studies, such as the DCCT, the high prevalence of this complication suggested it might have something to do with the high free IGF-I levels within the circulation observed with the higher doses of rhIGF-I [55]. These data have therefore fuelled speculation that rhIGF-I therapy may potentially accelerate the development of microangiopathic complications, but this issue can only be resolved with further long-term studies. In conclusion, those early studies with rhIGF-I suggested that replacement therapy could be beneficial in T1DM during adolescence, but there was only a narrow window of dose where efficacy and safety could be ensured.

rhIGF-1/rhIGFBP-3 Replacement in T1DM

Recently, the therapeutic use of a rhIGF-I/rhIGFBP-3 combination (SomatoKine®) has been explored in subjects with T1DM. The delivery of rhIGF-I along with its principal binding protein in the presence of the normal or excess levels of ALS observed in T1DM should increase the probability that the IGF-I circulates in the ternary complex. Certainly, the rhIGF-I/rhIGFBP-3 complex has a longer half-life (approx. 18 h) [56] and levels of free IGF-I are generally lower than those observed with the use of rhIGF-I alone. The combination of rhIGF-I with rhIGFBP-3 (its natural carrier protein), in equimolar ratio to form a complex may therefore have therapeutic advantages whereby the delivery and efficacy of rhIGF-I can be achieved without the associated risks of toxicity. A pilot study reported by Clemmons et al. [57] provides support for this hypothesis with the combination therapy being better tolerated and yet effective. In this study, 2 weeks’ continuous subcutaneous infusion of SomatoKine® at a large dose of 2 mg/kg/day (equivalent to IGF-I 400 ␮g/kg/day) led to reduced insulin requirements and improved glycaemic control with no side effects in the T1DM subjects studied [57]. A dose-ranging, double-blind, placebo-controlled study of the rhIGF-I/rhIGFBP-3 complex in 15 adolescents


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Table 1. Summary of reported adverse events in clinical trials of rhIGF-I in children and adolescents with T1DM Author Study design (n patients treated)

Patient age range, years

rhIGF-I dose and schedule

Treatment Adverse duration events

Retinopathy

Nephropathy

47

Open label (6)

13–20

40 ␮g/kg/day given s.c. once daily

28 days

Not reported

No change on direct ophthalmoscopy/ fundoscopy

No change in GFR or renal volume; trend for decreased overnight urine; albumin/ creatinine ratio in 5/6 (p ⫽ 0.07)

48

Doubleblind randomised placebocontrolled (43)

8–17

80 ␮g/kg/day given s.c. twice daily

28 days

Syncope ⫻1, Not reported increased hypoglycaemia p ⬍ 0.05

No significant differences in safety measures (including 24 h urine albumin excretion and creatinine clearance)

51


12–21

20–40 ␮g/kg/day given s.c. once daily

24 weeks

Painful injection ⫻1

No significant changes in retinal photographs

No significant changes in overnight urine; albumin/ creatinine ratio or GFR

49


11–66

80–140 ␮g/kg/d given s.c. twice daily

12 weeks

Facial oedema 9–11%; peripheral oedema 14–23%; jaw tenderness 16–26%; arthralgia 18–25%; headache 45–52%; tachycardia 2–7%

Patients on 120–140 ␮g/kg/day early worsening of retinopathy in12% and optic disc swelling in 19%

No significant change in urinary albumin excretion


115

Overnight GH

Insulin requirement 10

30 20

0 % change vs. placebo

% change vs. placebo

10 0 ⫺10 ⫺20 ⫺30 ⫺40 ⫺50

⫺20 ⫺30 ⫺40

⫺60

⫺50

⫺70

⫺60

⫺80 Placebo

a

⫺10

0.1

0.2

0.4

0.8

rhIGF-I/rhIGFBP-3 dose (mg/kg/day)

Placebo 0.1

b

0.2

0.4

0.8

rhIGF-I/rhIGFBP-3 dose (mg/kg/day)

Fig. 3. Changes in (a) overnight GH levels analysed and (b) insulin requirement for euglycaemia following 2-day treatment with rhIGF-I/IGFBP-3 treatment given as a single subcutaneous injection on each day. Data are mean percentage vs. placebo [from 58, with permission].

(age range 13–24 years) with T1DM confirms these observations. Treatment with rhIGF-I/rhIGFBP-3 complex (SomatoKine® dose 0.1–0.8 mg/kg/day) given as a single subcutaneous injection for 2 days led to dose-dependent increases in IGF-I concentrations, as well as reductions in overnight GH secretion and insulin requirements for euglycaemia [58]. Insulin sensitivity as quantified by total body glucose utilisation during a hyperinsulinaemic euglycaemic clamp study was also increased following IGF-I/IGFBP-3 complex [58] (fig. 3). As with the Clemmons study [57], no adverse effects were observed despite the delivery of equivalent doses of rhIGF-I which when given alone have previously been associated with a variety of problems. Combining rhIGF-I with rhIGFBP-3 may avoid these problems and the reduced levels of free IGF-I may improve the balance between safety and efficacy, but is yet to be tested in longer term clinical trials.

Conclusions

Abnormalities of the GH/IGF-I axis in T1DM largely arise because of the peripheral, rather than the portal, administration of insulin. Although intensification of insulin therapy, particularly during puberty, can lead to improvements in glycaemic control, the increased risks of hypoglycaemia and weight gain are unacceptable and may lead to poor patient adherence to therapy, or in the


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extreme, insulin omission. Although studies such as DCCT [44] showed unequivocally that there is a relationship between glycaemic control and risk of complications during puberty, some individuals develop MA with only very low levels of glycaemic exposure. The degree to which this may reflect continuing GH hypersecretion needs to be determined. Further exploration of the use of rhIGF-I or rhIGF-I/IGFBP-3 replacement as an adjunct to standard subcutaneous insulin therapy is required, for this may bring about further improvements in glycaemic control over and above those achieved with intensified insulin therapy with concomitant reductions in insulin dose and perhaps risks for weight gain, hypoglycaemia and diabetic microangiopathic complications. References 1 2

3

4

5 6

7

8

9

10 11 12 13 14

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Kuzuya H, Matsuura N, Sakamoto M, Makino H, Sakamoto Y, Kadowaki T, Suzuki Y, Kobayashi M, Akazawa Y, Nomura M, Yoshimasa Y, Kasuga M, Goji K, Nagataki S, Oyasu H, Imura H: Trial of insulin-like growth factor I therapy for patients with extreme insulin resistance syndromes. Diabetes 1993;42:696–705. Jabri N, Schalch DS, Schwartz SL, Fischer JS, Kipnes MS, Radnik BJ, Turman NJ, Marcsisin VS, Guler HP: Adverse effects of recombinant human insulin-like growth factor I in obese insulinresistant type II diabetic patients. Diabetes 1994;43:369–374. Keen H: Normoglycaemic re-entry and diabetic complications. Diabet Med 1984;1:85–87. Moller N, Orskov H: Does IGF-I therapy in insulin-dependent diabetes mellitus limit complications? Lancet 1997;350:1188–1189. Guler HP, Zapf J, Schmid C, Froesch ER: Insulin-like growth factors I and II in healthy man. Estimations of half-lives and production rates. Acta Endocrinol (Copenh) 1989;121:753–758. Clemmons DR, Moses AC, McKay MJ, Sommer A, Rosen DM, Ruckle J: The combination of insulin-like growth factor I and insulin-like growth factor-binding protein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: Evidence for in vivo biological activity. J Clin Endocrinol Metab 2000;85:1518–1524. Saukkonen T, Amin R, Williams RM, Fox C, Yuen KC, White MA, Umpleby MA, Acerini CL, Dunger DB: Dose-dependent effects of recombinant human IGF-I/IGF binding protein-3 complex on overnight growth hormone secretion and insulin sensitivity in type 1 diabetes. J Clin Endocrinol Metab 2004;89:4634–4641.

Prof. David B. Dunger Department of Paediatrics, Level 8, Box 116 Addenbrooke’s Hospital, Cambridge CB2 2QQ (UK) Tel. ⫹44 1223 762 944, Fax ⫹44 1223 336 996, E-Mail [email protected]


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Insulin Resistance and Type 2 Diabetes mellitus: Is There a Therapeutic Role for IGF-1? Alan C. Moses Novo Nordisk Pharmaceuticals, Inc., Princeton, N.J., USA

Abstract Severe insulin resistance represents a heterogeneous set of conditions with diverse underlying pathophysiology and limited therapeutic options. While recent studies have identified some of the molecular mechanisms resulting in extreme insulin resistance in peripheral tissues and liver, these studies have resulted in relatively few advances in therapy. Insulin like-growth factor 1 (IGF-1) lowers blood glucose while at the same time lowering serum insulin levels in normal volunteers. Its mechanism of action appears to be independent of activation of the insulin receptor although the role of IGF-1 in normal carbohydrate metabolism remains incompletely defined. IGF-1 also improves insulin resistance both in type 2 diabetes and in subjects with more severe insulin resistance. Small-scale clinical trials have demonstrated the potential utility of rhIGF-1 in selected cases of severe insulin resistance and, in these cases, the risk-benefit ratio appears to favor the use of this drug to ameliorate biochemical abnormalities and clinical symptoms. Copyright © 2005 S. Karger AG, Basel

Introduction

Despite intensive efforts over the last two decades, the therapeutic options for treating insulin resistance remain limited. This unmet medical need has enhanced the potential for recombinant human insulin-like growth factor I (rhIGF-1) to serve as a therapeutic agent for the spectrum of insulin resistance states ranging from type 2 diabetes mellitus to rare cases of severe insulin resistance of diverse etiology. This chapter will review the context for this discussion by examining the underlying pathophysiology of insulin resistance in its various forms, by examining the physiologic and pharmacologic properties of rhIGF-1

relative to carbohydrate homeostasis, and by considering some additional issues in the development of this agent as a drug.

The Spectrum of Insulin Resistance

Insulin resistance has become a ‘buzz’ word among both medical experts and among the lay public. The dramatic increase in the incidence and prevalence of type 2 diabetes [1] that is fueled in part by obesity which contributes to insulin resistance, and in part by the potential cardiovascular consequences associated with insulin resistance [2] has created an imperative to find more effective forms of therapy. On the other side of the spectrum are the rare cases of severe insulin resistance that, despite heterogeneous underlying pathophysiologies, share the common trait of being unaffected by most forms of therapy.

Severe Insulin Resistance

Severe insulin resistance may present with a broad range of degrees of glycemia from normoglycemia, to impaired glucose tolerance, to overt diabetes mellitus. There is clear evidence from some cases that, similar to type 2 diabetes, the degree of glycemic dysregulation is progressive and is associated with worsening ␤-cell function [3]. However, there are no large-scale studies to demonstrate that this is a universal finding. When glucose tolerance is normal or mildly impaired, the diagnosis of severe insulin resistance rests on clinical criteria and on fasting insulin levels which in the non-obese individual should be ⬎40–50 ␮U/ml in the fasting state [4]. In the presence of obesity, fasting insulin levels should be even greater. Post-prandial or post-glucose challenge insulin levels may reach extremely high levels and often are ⬎1,000 ␮U/ml while the ␤-cell is still able to compensate for demands on insulin secretion [5]. Ideally, the diagnosis of severe insulin resistance should be made on the basis of a markedly abnormal response to infused exogenous insulin using either an insulin tolerance test, a frequently sampled IVGTT, a euglycemic insulin clamp procedure, or an glucose-insulin-somatostatin infusion [4]. Severe insulin resistance can be present in a diverse group of clinical syndromes ranging from genetic defects in the insulin receptor to forms of lipodystrophy (table 1). Unfortunately, the majority of patients with severe insulin resistance still cannot be classified at the molecular level, particularly if they have the so-called type A syndrome that is associated with hirsutism, hyperandrogenemia, acanthosis nigricans, and varying degrees of dyslipidemia [3]. While originally believed to be due to mutations in the insulin receptor coding

Moses

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Table 1. Marked variation in phenotype of severe insulin resistance syndromes General classification

Specific syndromes

Genetic defects in the insulin receptor

Leprechaunism (Donohue’s syndrome) Rabson-Mendenhall Type A syndrome (minority)

Genetic defects in post-insulin receptor pathways

Type A syndrome (majority)

Lipodystrophies

Autosomal dominant (Kobberling-Dunnigan) Autosomal recessive (Seip syndrome) Acquired Partial lipodystrophy Generalized lipoatrophy (Lawrence syndrome) HIV-HAART therapy

Immunologic insulin resistance

Anti-IR receptor antibodies (type B) Anti-insulin antibodies

domain, only about 10–15% of patients have identified heterozygous mutations that function in a dominant negative fashion in regard to insulin action. The most severe form of insulin resistance occurs in the Donohue syndrome (leprechaunism) in which mutations in either the regulatory domains or the coding domains of the insulin receptor gene result in severe insulin receptor dysfunction including the most severe form with a functional ‘null’ receptor [6]. Attempts to identify specific genetic defects causing dysregulation of post-insulin receptor pathways have been disappointing. Several polymorphisms in a number of genes have been identified but the cause-and-effect relationship to severe insulin resistance has not been proven. There are multiple forms of lipodystrophy that are associated with moderate to severe insulin resistance. Recently, the lamin A/C gene complex has been implicated as causing the phenotype of Kobberling-Dunnigan syndrome, a form of partial lipoatrophy characterized by truncal and limb lipoatrophy and sparing of the face [7]. Acquired forms of lipoatrophy also are associated with clinically significant insulin resistance including the lipodystrophic syndrome that occurs in HIV-positive individuals on highly active retroviral therapy (HAART) [8].

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While a number of therapies have been tested in the various forms of severe insulin resistance, the results remain disappointing or beyond the reach of the majority of affected individuals. Thiazolidinediones including troglitazone have been demonstrated to improve insulin resistance and hyperglycemia in some subjects but with a substantial risk of hepatic dysfunction [9]. Parenteral leptin administration has improved insulin resistance in some subjects with severe lipoatrophy which is characterized by extremely low endogenous leptin levels [10]. Unfortunately, the majority of these patients are not responsive to ‘standard’ therapies used to treat hyperglycemia, including insulin secretagogues, metformin, or insulin itself.

Type 2 Diabetes mellitus

Type 2 diabetes represents a heterogeneous group of disorders that has largely defied attempts to unravel the underlying genetic pathophysiology. The current epidemic of type 2 diabetes mellitus is being fueled by the rapid increase in adiposity [1] and marked decrease in physical activity that characterize US demography. Both increasing weight and decreasing physical activity contribute to insulin resistance. Family studies have demonstrated that non-diabetic relatives of subjects with type 2 diabetes who are more insulin resistant have a greater tendency to develop type 2 diabetes later in life [11]. It is beyond the scope of this chapter to discuss the pathophysiology of type 2 diabetes except to note that insulin resistance encoded by genetic factors and exacerbated by environmental influences acts in combination with declining ␤-cell function to produce clinical diabetes. It is interesting to note that gene knock-out studies in mice suggest that insulin resistance caused by the tissue-specific knock-out of the insulin receptor in pancreatic ␤-cells causes ␤-cell dysfunction and diabetes [12]. This puts ␤-cell dysfunction back into the middle of the equation of severe insulin resistance. The treatment of type 2 diabetes remains problematic. While many drugs are available and can be utilized singly or in combination, the net effect on diabetes in the population has been disappointing. Over the last decade, despite improved treatment options, there has been little change in the mean hemoglobin A1c of the US population [13]. Even insulin, which if used properly, can bring the A1c down to near normal in the majority of patients, has been unsuccessful as a therapy because of reluctance to use it aggressively and because of lack of training of both patients and physicians. Thus, for both severe insulin resistance and type 2 diabetes mellitus, there is a need for better therapy. The question is: Is IGF-1 such a therapy?

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IGF-1: The Physiologic Rationale for Use in States of Altered CHO Metabolism

The early appreciation for the insulin-like biological effects of IGF-1 in cell culture systems led to a large series of animal experiments to confirm that in vivo IGF-1 could alter glucose homeostasis. With the purification of sufficient quantities of human IGF-1 (initially from outdated human plasma) to conduct human experiments, the era of IGF drug development began. Guler et al. [14] from Zurich demonstrated that human IGF-1 was capable of inducing hypoglycemia in normal males following intravenous injection at a dose of 100 ␮g/kg body weight. The time course and extent of hypoglycemia was similar between insulin and IGF-1, although the latter had a potency only 7–8% that of insulin on a weight basis. Based on these studies and following the successful cloning and expression of the human IGF-1 gene to produce large quantities of peptide, a number of physiologic investigations were carried out using the euglycemic clamp technique to evaluate the effects of IGF-1 on carbohydrate, protein, and lipid metabolism in normal volunteers [15–18]. In summary, these studies demonstrated that IGF-1 effectively stimulates glucose uptake into muscle and increases whole body glucose metabolism, IGF-1 inhibits protein degradation and stimulates protein synthesis, and IGF-1 decreases levels of free fatty acids. IGF-1 also directly inhibits ␤-cell insulin secretion [15]. In these respects, IGF-1 and insulin are similar although with different potencies for the different effects. However, IGF-1 and insulin differ in that the former suppresses pituitary growth hormone and insulin does not and the latter (insulin) suppresses the synthesis and release of IGF BP1 from the liver. They also differ dramatically in the fact that IGFs and not insulin are bound by a family of specific binding (transport?) proteins that alter their bioavailability and that may determine some tissue specificity of action [19]. Thus, based on its pattern of action and its unique cognate receptor, IGF-1 would seem to be an excellent candidate for use in states of altered CHO metabolism. Early studies on the structure and function of both insulin and IGF-1 and their respective cognate cell surface membrane receptors revealed remarkable homology at the level of both the peptides and the receptors [20, 21]. Indeed, each ligand binds to the other ligand’s receptor although with much lower affinity. Since both receptors are transmembrane tyrosine kinases that interact through a similar series of post-receptor signal transduction pathways, it has been complicated to determine whether the similar biological effects of insulin and IGF-1 are due to the innate activity of the cognate receptor or cross-reactivity at the other ligand’s receptor. This uncertainty continues today. The distinct differences between insulin and IGF-1 on IGF BP1 gene transcription (and regulation


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of circulating levels) (IGF BP1) and the different potencies of the two ligands on the ability to suppress insulin secretion, demonstrate that considerable tissuespecific differences in signaling occur or that the distribution of the peptides to various tissues is complicated and controlled. One potential reason to consider IGF-1in the therapy of diabetes mellitus relates to the prolonged half-life of IGF-1 that results from the specific binding of IGF-1 to binding proteins [18]. This is distinctly different that the short half-life of insulin following endogenous secretion or intravenous injection. In addition, the ability of IGF to suppress endogenous insulin secretion while stimulating glucose uptake in peripheral tissues offers the possibility that IGF 1 could ‘substitute’ for insulin in states of insulin resistance from the most severe to that present in type 2 diabetes mellitus. The ability of IGF-1 to bypass insulin resistance requires that IGF-1 works through different pathways than insulin, at least in critical insulin-responsive tissues.

Do the Data Support an Effect of IGF-1 in States of Insulin Resistance?

Soon after the demonstration that hIGF-1 could produce hypoglycemia following intravenous injection in normal volunteers, Schoenle et al. [22] demonstrated that a bolus injection of 100 ␮g IGF-1 per kilogram body weight could also reduce both insulin and blood glucose levels in subjects with severe insulin resistance. One of these subjects had a heterozygous mutation in the insulin receptor consistent with the diagnosis of type A insulin resistance on both genetic and phenotypic criteria. A number of other case reports appeared demonstrating the potential impact of IGF-1 (and IGF-2) in subjects with diverse forms of severe insulin resistance [23, 24]. My laboratory sought to extend these observations and to investigate the mechanisms underlying these effects. We first conducted an open label trial of 1 month duration in a group of subjects with heterogeneous forms of severe insulin resistance [5]. Some subjects had the phenotype of the type A syndrome without defined mutations in the insulin receptor [3], others had various forms of lipodystrophy including 2 sisters with the Kobberling-Dunnigan form, and 2 subjects had the type B syndrome of insulin resistance with anti-insulin receptor antibodies in the setting of autoimmune disease. Subjects received 100 ␮g rhIGF-1 per kilogram body weight twice daily by subcutaneous injection. The initial results were dramatic. Subjects with marked hyperinsulinemia and impaired glucose tolerance or mild diabetes, demonstrated dramatic reductions in hyperinsulinemia, normalization of glucose tolerance (fig. 1), improved insulin sensitivity as measured by the steady-state plasma glucose test (SSPG),

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1,500 1,000 500 0 08:00 11:00 14:00 17:00 20:00 23:00 02:00 05:00

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Fig. 1. IGF-1 reduces blood glucose and serum insulin levels in severe insulin resistance (type A). A subject with the type A syndrome but without a mutation in the insulin receptor was treated for 28 days with rhIGF-1 at 100 ␮g/kg body weight twice daily. Blood glucose and serum insulin levels were measured frequently over 24 h before (open squares) and after (solid square) rhIGF-1 treatment.

and even improvement in first phase insulin secretion. Some of these effects required up to 2–3 weeks to become apparent, but blood glucose concentrations began to decrease within 24–48 h. Not all subjects responded, however. One subject with generalized lipoatrophy and massive hepatomegaly had little evidence of response [unpubl. data]. Each of the 2 men with type B syndrome demonstrated improved glycemic control and substantial reductions in insulin requirements [unpubl. data]. These subjects continued on rhIGF-1 more chronically on an outpatient basis. In 1 subject, insulin resistance resolved over time probably due to spontaneous reduction of his high titer anti-insulin receptor antibody. The overall pattern of response to rhIGF-1 in severe insulin resistance is illustrated in table 2. Of note, hypertriglyceridemia also responded dramatically to IGF-1. Overall, rhIGF-1 was well tolerated in this generally young group of subjects although mild tachycardia occurring 2 h after injection was noted and confirmed by Holter monitor evaluation [5]. There were no serious adverse reactions in this cohort. In a multi-institutional collaborative trial in Japan, Kuzuyu et al. [25] demonstrated that subjects with diverse forms of severe insulin resistance also responded positively to rhIGF-1 administered more chronically by subcutaneous injection. As is often the case with investigational trials in a rare, heterogeneous disease, each study has been conducted with different endpoints, treatment protocols, doses, and entry criteria. In a two-phase study in subjects with the type A syndrome, rhIGF-1 improved glycemic control and reduced


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Table 2. Range of rhIGF-1 effects in severe insulin resistance as demonstrated in subjects with multiple phenotypes (not all subjects respond to rhIGF-1 treatment) Lowers fasting and mean 24 h insulin Lowers mean 24 h blood glucose in hyperglycemic individuals Allows recovery of 1st phase insulin secretion Increases insulin sensitivity Increases insulin clearance Effects are slow on and slow off

serum insulin levels although the latter effect seemed to wane over several weeks [26]. Thus, despite over 10 years of investigation, there is no consensus on the most appropriate phenotype for treatment or upon the optimal treatment protocol. Dose-ranging studies for rhIGF-1 in severe insulin resistance have never been done in a formal way with most studies utilizing 100 ␮g/kg body weight twice daily and reducing the dose if side effects develop. The situation in type 2 diabetes mellitus is much the same as in states of severe insulin resistance. Initial small-scale clinical trials demonstrated that rhIGF-1 could lower blood glucose concentrations, decrease serum insulin levels, and in the few studies in which it was tested directly, improve insulin sensitivity [27–29]. The effects on mean glycemia were impressive. These proof-of-concept trials led to larger scale clinical development programs at Genentech were rhIGF-1 was studied in type 2 diabetes mellitus as monotherapy in subjects inadequately controlled on oral hypoglycemic agents [30]. The initial studies were the first randomized, controlled, dose-ranging studies with rhIGF-1 and demonstrated a clear dose-response relationship between drug and biological effect with maximum tolerated dose at approximately 40 ␮g/kg body weight twice daily. The effect of higher doses was slightly more impressive for efficacy but produced a high rate of side effects. When used in combination with insulin, rhIGF-1 produced a dramatic reduction in A1c and resulted in a reduction in insulin dose despite better control (fig. 2). Cusi and DeFronzo [31] utilized the euglycemic hyperinsulinemic clamp technique in subjects with type 2 diabetes treated with rhIGF-1 and demonstrated that IGF-1 not only increased glucose utilization in muscle but also decreased endogenous glucose production, suggesting a broad range of physiological effects of rhIGF-1. Overall, trials of rhIGF-1 in type 2 diabetes demonstrated clinical efficacy but raised concerns about systemic toxicity. Early trials with rhIGF-1 doses as high as 160 ␮g/kg body weight twice daily encountered substantial side effects of which edema and jaw pain were the most universal [32]. At lower doses,

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Change in HbA1c (%)

1

0 Placebo ⫺1

rhIGF-1 (40/80)

p⫽0.027

rhIGF-1 (40/40)

Baseline A1c⫽ 9.3 ⫾ 1.3 ⫺2 0

2

4

6

p⬍0.01

rhIGF-1 (20/20)

8

10

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Fig. 2. rhIGF-1 improves glycemic control when added to insulin in type 2 diabetes mellitus. rhIGF-1 was utilized in combination with insulin in subjects with type 2 diabetes. Insulin doses were adjusted to improve glycemic control and rhIGF-1 was administered twice daily in the doses indicated as adjunctive therapy. The baseline A1c was 9.3%. Data are previously unpublished and are from a study sponsored by Genentech.

rhIGF-1 appears to be better tolerated but there are clear dose-dependent side effects. Among the less frequent but more disturbing side effects were Bell’s palsy, apparent progression of proliferative diabetic retinopathy [33], arthralgias, and edema [unpubl. data]. Moreover, as epidemiologic data correlating serum levels of IGF-1 with an increased risk for certain human malignancies began to appear (prostate cancer, breast cancer) [34], the enthusiasm for utilizing rhIGF-1 as a therapy in diabetes began to wane particularly as other classes of drugs directed at improving insulin sensitivity (metformin and the thiazolidinediones) were in late stage clinical development. One potential approach to reducing the dose-dependent side effects of rhIGF-1 while still retaining the biologic and pharmacologic actions was to use a more ‘physiological’ preparation, namely, rhIGF-1 complexed to rhIGF BP3 in a 1:1 molar ratio. This preparation has been tested in small-scale, short-term studies in both type 1 and type 2 diabetes [35, 36] but there is very little experience in cases of severe insulin resistance. In general, the complex of rhIGF-1 and IGF BP3 has a favorable pharmacokinetic profile with stable, steady-state levels achieved within 24–48 h after subcutaneous twice daily injections and a low ratio of free to total IGF-1 levels [unpubl. data]. Thus, it appears that rhIGF-1 remains complexed to the IGF binding protein in the circulation and is released slowly to target tissues. The IGF-1/BP3 complex retains biological activity and potently reduces insulin requirements in both type 1 and type 2


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20 % change in insulin dose (U/24 h)

2mg/kg INF

2mg/kg ON

1mg/kg bid

1mg/kg qhs

0 ⫺20 ⫺40 ⫺60 ⫺80 ⫺100

Treatment group

Fig. 3. IGF-1/BP3 reduces daily insulin requirements in type 2 diabetes mellitus. rhIGF-1 complexed in a 1:1 ratio with IGF BP3 was administered to subjects with type 2 diabetes mellitus for 2 weeks utilizing different doses and different methods of administration (subcutaneous bolus injection and continuous subcutaneous infusion). Biological effectiveness was measured by the reduction in exogenous insulin doses compared to the start of the study. Glycemic control also improved in all groups (data not shown).

diabetes mellitus while improving glycemic control [35, 36] (fig. 3). Fluctuations in blood glucose levels appear to be reduced with IGF-1/BP3 treatment compared to insulin. Importantly, this preparation appears to be better tolerated acutely than ‘free’ rhIGF-1 although there are few data on exposure in type 2 diabetes lasting longer than 14 days. Nonetheless, the lower side effect profile despite higher doses of total IGF-1 being administered strongly suggests that acute adverse effects from IGF-1 are related to levels of ‘free’ IGF-1 in the circulation (and tissues). Maintaining biological effects and clinical effectiveness while reducing side effects will remain a major challenge in the continuing development of rhIGF-1 as a therapeutic agent.

Can IGF-1 Make It as a Therapeutic Agent?

The initial excitement over the availability of sufficient quantities of rhIGF-1 following production of recombinant peptide resulted in a series of academically driven, physiological studies and small sample size clinical trials that suffered from lack of coordinated planning and with little insight into the steps necessary to bring a drug from discovery through regulatory approval. The initial trials were particularly disadvantaged because of lack of appropriate dose-finding studies. All trials were based on a dose of rhIGF-1 (or higher) that derived from the initial physiological studies of Guler et al. [14] demonstrating the acute glucose-lowering effect of human IGF-1. Much time

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was lost in development because of the apparent unacceptable toxicity profile associated with rhIGF-1 that to some extent was a function of the high doses of drug chosen for these studies. Subsequent trials have concentrated on defining the most appropriate dose, dosing frequency, formulation (free rhIGF-1 or IGF-1/BP3 combinations), and clinical indication. There is no question that rhIGF-1 is effective in a variety of clinical situations including some cases of severe insulin resistance and some patients with type 2 diabetes. However, this begs the question as to whether rhIGF-1 can be approved by regulatory agencies and, once approved, sold by its manufacturer. Unanswered questions about both acute and chronic side effects need to be addressed and larger scale clinical trials need to be performed. The conduct of larger scale, coordinated clinical trials for severe insulin resistance will be a particular challenge given the rarity of the condition and the lack of an international consortium to study this condition utilizing a common protocol. The linking of IGF-1 and IGF-1 receptor pathways to the risk for certain types of malignancy, the concern that even if IGF-1 does not in itself cause cell transformation, it can stimulate the growth of transformed cells (preexistent cancers), and the proposed relationship between IGF-1-stimulated pathways and angiogenesis, particularly in the retina [37], has complicated the task of seeking approval for this agent in any medical condition. However, ultimately, it is the risk-benefit ratio that determines the clinical usefulness of a drug. In this regard, one should choose potential indications wisely and seek disease states for which alternative therapies do not exist. Severe insulin resistance falls into this category. Drug development for a rare disease is a unique opportunity but it also poses unique challenges in regard to phenotypic characterization, clinical trial coordination, and cooperation among diverse centers each with small numbers of patients. Successful development of a drug for a rare disease also is more likely to assure payment by health agencies (in countries with single-payer systems) and by health insurers in the USA because of the absence of effective, competitive treatments. The situation for type 2 diabetes is more complicated, particularly in defining the phenotype for clinical trials where alternative therapies are not effective and where there can be agreement with regulatory agencies that the safety profile warrants approval for this indication. For the payers, the challenge is even greater, since rhIGF-1 likely will be priced at a premium and must be demonstrated to be more effective than other treatments in order to be supported through reimbursement. The journey of IGF-1 as a potential treatment in states of insulin resistance has been and will remain an interesting one. The initial promise of utilizing this agent has been disrupted by time and poor execution and indecision around clinical development programs. However, there still is some


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promise for the drug if the mistakes of the past can be avoided in future development.

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Mokdad AH, Bowman BA, Ford ES, Vinicor F, Marks JS, Koplan JP: The continuing epidemic of obesity and diabetes in the United States. JAMA 2001;286:1195–2000. Reaven G, Abbasi F, McLaughlin T: Obesity, insulin resistance, and cardiovascular disease. Recent Prog Horm Res 2004;59:207–223. Moller DE, Cohen O, Yamaguchi Y, Assiz R, Grigorescu F, Eberle A, Morrow LA, Moses AC, Flier JS: Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance. Diabetes 1994;43:247–255. Goldfine A, Moses AC: Syndromes of severe insulin resistance; in Kahn CR, Jacobson AM, King GL, Smith RJ, Weir GC, Moses AC (eds): Joslin Textbook of Diabetes, ed 14, 2005, pp 493–504. Morrow LA, O’Brien MB, Moller DE, Flier JS, Moses AC: Recombinant human insulin-like growth-I therapy improves glycemic control and insulin action in the type A syndrome of severe insulin resistance. J Clin Endocrinol Metab 1994;79:205–210. Taylor SI: Lilly Lecture: Molecular mechanisms of insulin resistance. Lessons from patients with mutations in the insulin-receptor gene. Diabetes 1992;41:1473–1490. Garg A, Vinaitheerthan M, Weatherall PT, Bowcock AM: Phenotypic hetereogeneity in patients with familial partial lipodystrophy (Dunnigan variety) related to the site of missense mutations in lamin a/c gene. J Clin Endocrinol Metab 2001;86:59–65. Kino T, Mirani M, Alesci S, Chrousos GP: AIDS-related lipodystrophy/insulin resistance syndrome. Horm Metab Res 2003;35:129–136. Arioglu E, Duncan-Morin J, Sebring N, Rother KI, Gottlieb N, Lieberman J, Herion D, Kleiner DE, Reynolds J, Premkumar A, Sumner AE, Hoofnagle J, Reitman ML, Taylor SI: Efficacy and safety of troglitazone in the treatment of lipodystrophy syndromes. Ann Intern Med 2000;1333: 263–274. Oral EA, Simha V, Ruiz E, Andewalt A, Premkumar A, Snell P, Wagner AJ, DePaoli AM, Reitman ML, Taylor SI, Gorden P, Garg A: Leptin-replacement therapy for lipodystrophy. N Engl J Med 2002;346:570–578. Martin BC, Warram JH, Krolewski AS, Bergman RN, Soeldner JS, Kahn CR: Role of glucose and insulin resistance in development of type 2 diabetes mellitus: Results of a 25-year follow-up study. Lancet 1992;340:925–929. Kulkarni RN, Holzenberger M, Shih DZ, Ozcan U, Stoffel M, Magnusson MA, Kahn CR: ␤-Cell-specific deletion of the IGF-1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter ␤-cell mass. Nat Genet 2002;31:111–115. Koro CE, Bowlin SJ, Bourgeois N, Fedder DO: Glycemic control from 1988 to 2000 among US adults diagnosed with type 2 diabetes: A preliminary report. Diabetes Care 2004;27:17–20. Guler HP, Zapf J, Froesch ER: Short-term metabolic effects of recombinant human insulin-like growth factor I in healthy adults. N Engl J Med 1987;317:137–140. Elahi D, McAloon-Dyke M, Fukagawa N, Sclater AL, Wong GA, Shannon RP, Minaker KL, Miles JM, Rubenstein AH, Vanderpol CJ, et al: Effects of recombinant IGF-1 on glucose and leucine kinetics in men. Am J Physiol 1993;265:E831–E838. Mauras N, Beaufrere B: Recombinant human insulin-like growth factor-I enhances whole body protein anabolism and significantly diminishes the protein catabolic effects of prednisone in humans without a diabetogenic effect. J Clin Endocrinol Metab 1995;80:869–874. Boulware S, Tamborlane W, Rennert N, Gesundheit N, Sherwin R. Comparison of the metabolic effects of recombinant human insulin-like growth factor-1 and insulin. J Clin Invest 1994;93: 1131–1139.

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Giordano M, Castellino P, Carroll CA, DeFronzo RA: Comparison of the effects of human recombinant insulin-like growth factor-I and insulin on plasma amino acid concentrations and leucine kinetics in humans. Diabetologia 1995;38:732–738. Clemmons DR: Role of insulin-like growth factor binding proteins in controlling IGF actions. Mol Cell Endocrinol 1998;140:19–24. Blundell TL, Bedarkar S, Rinderknecht E, Humbel RE: Insulin-like growth factor: A model for tertiary structure accounting for immunoreactivity and receptor binding. Proc Natl Acad Sci USA 1978;75:180–184. Massague J, Czech MP: The subunit structure of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J Biol Chem 1982;257: 5038–5045. Schoenle EJ, Zenobi PD, Torresani T, Werder EA, Zachmann M, Froesch ER: Recombinant human-insulin like growth factor I reduces hyperglycemia in patients with extreme insulin resistance. Diabetologia 1991;34:675–679. Quin J, Fisher B, Paterson K, Inoue A, Beastall G, MacGuish A: Acute responses to recombinant human insulin-like growth factor I in a patient with Mendenhall’s syndrome. N Engl J Med 1990;323:1425–1426. Usala A, Madigan T, Burguera B, Cefalu W, Sinha MK, Powell JG, Usala SJ: High dose intravenous, but not low dose subcutaneous, insulin-like growth factor-I therapy induces sustained insulin sensitivity in severely resistant type I diabetes mellitus. J Clin Endocrinol Metab 1994;79: 435–440. Kuzuya H, Matsuura N, Sakamoto M, Makino H, Sakamoto Y, Kadowaki T, Suzuki Y, Kobayashi M, Akazawa Y, Nomura M, et al: Trial of insulin-like growth factor I therapy for patients with extreme insulin resistance syndromes. Diabetes 1993;42:696–705. Vestergaard H, Rossen M, Urhammer SA, Muller J, Pedersen O: Short- and long-term metabolic effects of recombinant human IGF-I treatment in patients with severe insulin resistance and diabetes mellitus. Eur J Endocrinol 1997;136:475–482. Froesch ER, Bianda T, Hussain MA: Insulin-like growth factor-I in the therapy of non-insulindependent diabetes mellitus and insulin resistance. Diabetes Metab 1996;22:261–267. Moses AC, Young SC, Morrow LA, O’Brien M, Clemmons DR: Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes. Diabetes 1996;45:91–100. Schalch DS, Turman NJ, Marcsisin VS, Heffernan M, Guler HP: Short-term effects of recombinant human insulin-like growth-factor-I on metabolic control of patients with type II diabetes mellitus. J Clin Endocrinol Metab 1993;77:1563–1568. rhIGF-1 in NIDDM Study (RINDS) Group: Evidence from a dose-ranging study that recombinant insulin-like growth factor I effectively and safely improves glycemic control in non-insulindependent diabetes mellitus (abstract). Diabetes 1996;45(suppl 1):27A. Cusi K, DeFronzo R: Recombinant human insulin-like growth factor I treatment for one week improves metabolic control in type 2 diabetes by ameliorating hepatic and muscle insulin resistance. J Clin Endocrinol Metab 2000;85:3077–3084. Jabri N, Schalch DS, Schwartz SL, Fischer JS, Kipnes MS, Radnik BJ, Turman NJ, Marcsisin VS, Guler HP: Adverse-effects of recombinant human insulin-like growth-factor-I in obese insulinresistant type II diabetic patients. Diabetes 1994;43:369–374. Shigetou M, Sagawa T, Ishibashi T, Nakashima N, Umeda F, Nawata H: Exacerbation of diabetic retinopathy following systemic insulin-like growth factor. I. Report of a case. Rinsho Ganka 1997;51:1251–1255. Renehan AG, Zwahlen M, Minder C, O’Dwyer ST, Shalet SM, Egger M: Insulin-like growth factor, IGF binding protein-3, and cancer risk: Systemic review and meta-regression analysis. Lancet 2004;363:1346–1353. Clemmons DR, Moses AC, McKay MJ, Sommer A, Rosen DM, Ruckle J: The combination of insulin-like growth factor I and insulin-like growth factor-binding protein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: Evidence for in vivo biological activity. J Clin Endocrinol Metab 2000;85:1518–1524.


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Moses A, Clemmons D, Sommer A, Rogol A, Jacobson W: Effects of IGF-I/IGF BP3 combination (somatokine) on glycemia and insulin requirements in type 2 diabetes mellitus. Diabetes 2001;50(suppl 2):A127. Burgos R, Hernandez C, Mateo C, Mesa J, Canton A, Simo R: Vitreous levels of IGF-I, IGF binding protein 1, and IGF binding protein 3 in proliferative diabetic retinopathy – A case-control study. Diabetes Care 2000;23:80–83. Alan C. Moses, MD Novo Nordisk Pharmaceuticals, Inc. 100 College Road West, Princeton, NJ 08540 (USA) Tel. ⫹1 609 9197982, Fax ⫹1 609 9873092, E-Mail [email protected]

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Insulin-Like Growth Factors in the Treatment of Neurological Disease Gina M. Leinninger a, Eva L. Feldmana,b a

Neuroscience Program and bDepartment of Neurology, University of Michigan, Ann Arbor, Mich., USA

Abstract Although the functional deficits of neurological diseases vary, they are all pathologically marked by neuronal degeneration. The ability of insulin-like growth factor-I (IGF-I) to support both sensory and motor neuron regeneration has suggested its potential in treatment of neurological diseases. IGF-I is pleiotrophic, stimulating survival, neurite outgrowth and motility in neurons, as well as myelination of neurons by glia. Understanding the intracellular signaling pathways that mediate these pleiotrophic responses to IGF-I is important for tailoring IGF-I treatment to the appropriate neurological deficit. This review surveys the current understanding of IGF-I pleiotrophism, the underlying signaling conferring these effects, and the status of IGF-I in treatment of human neurological disorders. Copyright © 2005 S. Karger AG, Basel

Neurological Diseases

The diverse functions of the central and peripheral nervous systems are maintained by neurons and their supporting glial cells. Neurons are unique from other cells of the body because of their specialized morphology, allowing connectivity and communication between cells. Neurons further differ from other cells of the body in that they do not undergo mitosis, and thus, their initial number cannot be increased. Once the neuronal population is established during development, any neurons lost through injury or aging cannot be replaced. Similarly, neurons damaged or lost due to neurodegeneration, the hallmark of a host of neurological diseases, are also irretrievable. Accumulative neuronal loss interrupts the neural relay system controlling memory, sensory perception, movement, and muscle control, ultimately leading to deficits in these processes. Thus, a wide array of neurological diseases with disparate presentations,

including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, as well as secondary neurological disorders (e.g. myopathy, neuropathy due to diabetes or chemotherapy) all share a unifying pathology; they are caused by accruing, substantive loss of neurons.

Significance of Insulin-Like Growth Factors in Neurological Disease

In order to treat or prevent neurological disease, it is imperative to promote neuronal survival and enhance neurite outgrowth from damaged neurons. Many growth factors satisfy both of these trophic and tropic requirements, including nerve growth factor, neurotrophin-3, vascular endothelial growth factor and the insulin-like growth factors-I and -II (IGF-I and IGF-II) [1, 2]. Interestingly, multiple neurotrophic factors support either sensory or motor neurons of the peripheral nervous system (PNS), but IGF-I is the only neurotrophic factor capable of supporting both sensory and motor neuron regeneration [1]. These actions suggest that IGF-I may be a powerful therapeutic agent for the survival and outgrowth of neurons, and thus, for treatment of neurological disease. The expression of IGF-I and the type I IGF receptor (IGF-IR) throughout the nervous system highlight the key role of these proteins in neuronal maintenance and survival. Of the two IGFs, IGF-I is more broadly distributed throughout the nervous system, and is normally expressed in craniofacial sensory ganglia [3], sciatic nerve, spinal cord, sensory dorsal root ganglia (DRG), and brain [4–8]. The IGF-IR is also widely expressed throughout the neurons and glia of the brain [9] and the PNS [10, 11]. Although the IGF system is required for general fetal and postnatal development [12, 13], it is particularly crucial for proper development of the PNS. For example, mice deficient in IGF-I have abnormal PNS anatomy and physiology [14]. Similarly, functional signaling through the IGF-IR is crucial for development, as demonstrated by the embryonic lethal phenotype of IGF-IR-deficient mice [14]. These findings illustrate the importance of IGF-I and the IGF-IR in development and maintenance of neurons. Research of the past two decades has gone on to demonstrate a role for IGF-I in trophic and tropic support of the injured or diseased nervous system [15, 16]. While it is clear that IGF-I can promote survival, neurite outgrowth, and motility in neurons, and stimulate myelination by glia, the signaling pathways underlying these responses have remained elusive. Understanding the signaling underlying IGF-I pleiotrophism is critical for identifying relevant neuronal targets and in developing effective strategies for treatment of neurological disease. Further, IGF-I signaling must be studied in the context of the

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system, as the effects of IGF-I on a glial cell differ from those evidenced by primary neurons or malignant neuroblastoma cells. Investigation of the divergent signaling properties in these cell types in response to IGF-I will allow for a more finely-tuned understanding of disease pathology and therapeutic potential.

Signaling Pathways that Mediate IGF-I’s Pleiotrophic Effects in the Nervous System

IGF-I activates two signaling pathways in neurons and glia: (1) the extracellular signal-related kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and (2) the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (refer to fig. 1 for a diagram of these pathways). The ERK/MAPK and PI3K/Akt pathways are also activated by other neurotrophins, and both have been implicated in survival, neurite outgrowth and motility [17]. How, then, do these common signaling pathways stimulate such divergent responses? Or, in other words, how can common signaling pathways dictate just one of an array of possible pleiotrophic effects? While the answer to this is not well understood, it is clear that the effect of IGF-I varies across different classes of neurons. Additionally, IGF-I-stimulated signaling differs between primary neurons and transformed neurons [18–20]. Thus, in order to understand IGF-I’s pleiotrophism within neurological disease, it is key to examine IGF-I signaling in the context of various neuronal and glial cell types.

IGF-I Mediates Neurite Outgrowth: Relevance to Nerve Injury

One of the defining morphological characteristics of differentiated neurons is their processes, namely dendrites and axons. These processes are extended during development and must be re-established if damaged due to injury or neurological disease [21, 22]. Functional dendrites and axons are critical for intact synapse formation and proper neuronal signaling. IGF-I initiates neurite outgrowth in many neuronal subtypes of the nervous system, including neurons of the cortex [23], retina, [24], motor neurons [25–27], and sensory and sympathetic neurons [28, 29]. Most of the evidence for IGF-I-promoted neurite extension comes from studies of damaged peripheral nerves. For example, rats treated with IGF-I after sciatic nerve transection exhibit increased motor neuron survival and re-innervation of muscle [30]. Functional sciatic nerve regeneration is also promoted by IGF-I treatment in mice after sciatic nerve crush [31]. Interestingly, sciatic nerve damage upregulates both IGF-I

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Fig. 1. IGF-I signaling pathways in neurons. IGF-I binding to the IGF-IR causes phosphorylation of intracellular receptor tyrosines. When Shc binds to these tyrosines, it activates a cascade of proteins that ultimately activates the ERK/MAPK pathway. IRS binding to a phosphorylated IGF-IR tyrosine leads to activation of the PI3K/Akt pathway. The ERK/MAPK pathway plays a dominant role in IGF-I-mediated neurite outgrowth, while the PI3K/Akt pathway is dominant in motility, myelination, and neuroprotection.

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and IGF-IR [32, 33], suggesting an endogenous attempt to regenerate damaged neurites and to re-establish interrupted neuronal connections. This response is blunted in complication-prone diabetic nerves, which express lower levels of IGF-I mRNA [34] and exhibit delayed upregulation of IGF-I after sciatic nerve crush [22]. A similar situation exists in rats made diabetic by streptozotocin injection, in which serum levels of IGF-I protein and neuronal expression of IGF-I mRNA are reduced compared to control rats [34, 35]. Human patients with diabetic neuropathy also exhibit decreased levels of serum IGF-I and IGF-IR [36]. In sum, this evidence suggests that decreased IGF-I and resultant diminished support of neurites may underlie the degeneration of peripheral sensory neurons that is a pathological hallmark of diabetic neuropathy. Clearly, loss of IGF-I impairs the function of peripheral neurons, demonstrated by transgenic mice lacking IGF-I (IGF /), which have smaller axonal diameters and decreased motor and sensory nerve conduction velocities compared to normal transgenic control mice [14]. Thus, IGF-I is critical for proper formation, extension, and maintenance of neurites, and therefore, for proper nervous system function. While there is ample evidence that IGF-I induces neurite extension, the signaling pathways controlling the process are just beginning to be understood. Studies of SH-SY5Y neuroblastoma cells and other cultured neurons suggest that while IGF-I stimulates both the PI3K/Akt and ERK/MAPK pathways, the latter is more dominant in promoting neurite outgrowth [37]. Inhibition of the ERK/MAPK pathway with the pharmacological pathway inhibitor PD98059 decreases IGF-I-stimulated neurite extension in a dose-dependent manner (fig. 2). The IGF-I signal may be preferentially diverted through the ERK/MAPK pathway due to the cellular expression profile of insulin receptor substrate proteins-1 and -2 (IRS-1, -2). The IRS proteins bind to phosphotyrosine residues of the IGF-IR and act as docking proteins for downstream signaling components. Interestingly, the potential for neurite extension may be determined by which IRS protein the neuron expresses and whether that IRS protein can activate the PI3K/Akt pathway. For example, hippocampal rat neurons expressing IGF-IR and IRS-1 cannot differentiate and express neurites. However, cells expressing IGF-IR, along with a mutant IRS-1 lacking a phosphotyrosine binding domain, can undergo differentiation and extend neurites [38]. Further, inhibitors of the PI3K/Akt pathway do not inhibit neurite outgrowth in these neurons, suggesting that the PI3K/Akt pathway is not imperative for this function of IGF-I [38, 39]. However, studies in SH-SY5Y cells, which only express IRS-2, indicate that PI3K/Akt inhibition suppresses IGF-I-stimulated neurite outgrowth [40]. These results collectively suggest that neurons expressing IRS-1 may signal predominantly through the ERK/MAPK pathway, while neurons expressing IRS-2 may utilize both PI3K/Akt and ERK/MAPK for neurite extension.


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Neurite extension is also regulated by Shc, another protein that binds to the activated IGF-IR and induces downstream activation of the ERK/MAPK pathway [41, 42]. Expression of a Shc mutant decreases IGF-I-induced ERK phosphorylation, resulting in decreased neurite outgrowth in SH-SY5Y cells (fig. 3) [42]. Further, inhibition of the ERK/MAPK pathway inhibits IGF-I-stimulated neurite outgrowth in SH-SY5Y cells and cultured DRG sensory neurons [37, 43]. Understanding the expression profiles of the IRS and Shc proteins in neuronal cell types will enable investigators to not only predict how neurons will respond to IGF-I treatment, but also to manipulate signaling to promote neurite extension versus another response. This level of understanding is crucial for therapies targeted toward regeneration of neuronal processes and ultimate return of neuronal function.

IGF-I Mediates Neuronal Motility: Relevance to Cancers of the Nervous System

Neurons must be motile during development in order to establish the appropriate connections that enable inter-neuronal signaling. However, excessive neuronal motility, such as that seen in transformed cells of the nervous system, can lead to metastasis and cancer dissemination throughout the body. The role of IGF-I in neuronal motility has been studied in cells from neuroblastomas, brain tumors most commonly found in young children [44]. Though neuroblastomas account for only 7–10% of pediatric malignancies [45], the clinical prognosis is poor; only 20% of high-grade neuroblastoma patients survive [46, 47]. Neuroblastoma cells are motile, and can metastasize to bone, leptomeninges and other organs. While neuroblastoma is treatable, current treatment protocols cannot cure advanced-stage metastatic disease [48]. Understanding the signaling underlying neuroblastoma cell motility is therefore key for developing treatment paradigms directed against metastatic cancer dissemination. IGF-I stimulates motility of cultured neuroblastoma cells (fig. 4), which is mediated through IGF-IR and PI3K/Akt activation [49, 50]. Interestingly, increased IGF-IR expression correlates with increased tumorigenicity of neuroblastoma cells, suggesting that upregulation of the IGF-I signaling system is a determinant in neuroblastoma development [51]. IGF-I/IGF-IR stimulates motility in neurons through extension of filopodia and lamellipodia. Filopodia, which are spike-like protrusions composed of parallel actin filaments, form in response to extracellular matrix proteins and soluble factors [52–54]. In contrast, actin-containing lamellipodia are veil-like structures that mediate cellular protrusion [55, 56]. Filopodia and lamellipodia are both


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b Fig. 3. Expression of the p52 Shc mutant inhibits IGF-I-mediated ERK activation and neurite outgrowth. a Vector- or p52 Shc mutant-transfected SH-SY5Y neuroblastoma cells were serum-starved overnight and then treated with 10 nM IGF-I. After 24 h, neurite-bearing cells were scored. Results are the mean SE of at least three separate observations. p 0.01 (by independent Student’s t test) compared with the vector-transfected cells treated with 10 nM IGF-I. b Representative phase-contrast images of cells from the experiment in a. Bar 50 M [reprinted from 42, with permission].

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Fig. 4. The motility of SH-SY5Y cells is increased by IGF-I. SH-SY5Y neuroblastoma cells were plated on coverslips coated with gold particles, treated with or without IGF-I for 6 or 12 h, then were fixed and mounted. a Example of tracks left in the gold particle-coated surface by migrating cells. Arrow indicates the gold internalized by the cell body. Arrowhead points to the area over which the cell moved during the incubation. b Average increase in track area in response to 1 nM IGF-I treatment for 6 and 12 h. * p 0.0001 in comparison to unstimulated controls for both conditions. For all conditions, n 150 cells [reprinted from 50, with permission].

present on structures at neurite tips, or growth cones [57]. Dynamic polymerization or depolymerization of actin filaments at the leading edge of filopodia and lamellipodia regulate growth cone movement and neuronal motility [58]. Advances in filopodia and lamellipodia are morphologically represented by ‘ruffling’ of the plasma membranes. Treatment of SH-SY5Y neuroblastoma cells with IGF-I increases leading edge actin polymerization [59] and lamellipodial advance, marked by increased lamellipodial ruffling [60]. In contrast to neurite extension, which is mediated via the ERK/MAPK


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pathway, IGF-I-stimulated motility is regulated predominantly through the PI3K/Akt pathway. Inhibition of the PI3K/Akt pathway, but not the ERK/ MAPK pathway, blocks IGF-I-stimulated membrane ruffling [51, 61]. At the protein level, IGF-I contributes to ruffling via phosphorylating focal adhesion proteins such as paxillin and focal adhesion kinase (FAK) [51, 61]. Lamellipodial focal adhesions contribute to stability of the structure, and allow for continued lamellipodial advance [62]. IGF-I treatment of SH-SY5Y neuroblastoma cells causes redistribution of FAK and paxillin from the cell tips to punctate regions behind the sites of lamellipodial ruffles (fig. 5) [51]. Thus, the IGF-Iactivated components of focal adhesion complexes stabilize the base of lamellipodia, allowing leading edge advance, and thereby enable neuronal motility. Identifying such key proteins in IGF-I-mediated motility is important for predicting metastasis and identifying possible points of therapeutic intervention.

IGF-I Mediates Myelination: Relevance to Demyelinating Diseases

Neuronal axons are ensheathed with myelin, a multilayered membrane structure that is produced by glia (oligodendrocytes in the CNS and Schwann cells in the PNS) [63]. Ensheathment increases axonal conduction velocity, allowing for rapid signal dispersal between neurons. Loss of myelin sheaths (a pathological hallmark of multiple sclerosis and demyelinating neuropathy) impairs nervous system function [64]. The discovery that IGF-I overexpressing mice have larger brains and express 30% more myelin compared to transgenic controls suggested that the IGF-I system might have therapeutic potential in demyelinating disorders [65]. Both large and small axons are myelinated in IGF-I overexpressing mice [66], contrary to the concept that only large-diameter axons tend to be myelinated [67]. However, IGF-I-mediated myelination is blocked in transgenic mice overexpressing IGFBP-1, which binds free IGF-I, such that these animals have fewer myelinated fibers and thinner myelin sheaths compared to transgenic controls [68]. Further, mice lacking IGF-I have fewer axons and oligodendrocytes, resulting in reduced white matter of the brain and spinal cord [69]. IGF-I-deficient mice also exhibit decreased expression of myelin-specific proteins [70]. Collectively, these results characterize the critical role of IGF-I in endogenous myelination and suggest that IGF-I may be of therapeutic benefit in demyelinating diseases. Animal models demonstrate that IGF-I treatment is beneficial for re-establishing myelination after nerve injury or neurological disease. In the case of spinal cord injury, IGF-I enhances myelination of transected axons [71], which may aid regain of function. Additionally, IGF-I promotes myelination in animal

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Fig. 5. IGF-I regulation of FAK and paxillin. SH-SY5Y neuroblastoma cells were stimulated for 15 min with 10 nM IGF-I. Cells were fixed and stained simultaneously with Texas Red-phalloidin (red) and anti-FAK or anti-paxillin antibodies (green). Arrows indicate the focal adhesion sites [reprinted from 51, with permission].

models of multiple sclerosis, as induced by cuprizone or experimental allergic encephalomyelitis. In cuprizone-induced demyelination, IGF-IR mRNA is increased during early recovery stages [72], though mRNA levels of other growth factors, such as PDGF and FGF, are unaltered [73]. Further, mice overexpressing IGF-I exhibit a nearly full recovery of CNS myelination 5 weeks after cuprizone treatment, while control mice exhibit near complete demyelination


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Fig. 6. IGF-I promotes myelination of DRG axons. a Dissociated DRG neurons cultured for 21 days in serum-free defined media (control) have healthy unmyelinated axons (white arrows). b Higher magnification of a shows the nucleus (N) of an adjacent Schwann cell (SC). There are no SC processes extending and ensheathing the axons despite the close apposition of SC and axon. c Addition of 1 nM IGF-I to control media results in myelination of a few individual axons (black arrow), although many axons remain unmyelinated.

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at the same stage. Myelination is restored due to reduced apoptotic death of mature oligodendrocytes, coupled with restoration of oligodendrocytes that had degenerated [74]. In the experimental allergic encephalomyelitis model of multiple sclerosis, IGF-I decreases inflammation, demyelination and clinical deficits in treated animals [75–77]. In vitro models demonstrate that IGF-I regulates myelination through several mechanisms. First, IGF-I promotes glial cell survival and proliferation [74]. These IGF-I-stimulated effects are mediated by the PI3K/Akt pathway in developing oligodendroctyes [78], a pathway known to function in IGF-Imediated survival signaling (discussed in the section ‘IGF-I Mediates Neuronal Survival: Relevance to Neurodegenerative Disease’). Secondly, IGF-I promotes contact between glia and neurons. For example, IGF-I promotes Schwann cell attachment to axons of co-cultured DRG neurons. This contact results in ensheathment of the axons with myelin (fig. 6) [79, 80]. However, in the absence of IGF-I, Schwann cells survive, but do not myelinate the axons of co-cultured DRG neurons [80]. Thus, IGF-I does not promote myelination solely by maintaining survival of glia, but also by stimulating intracellular cues that induce glial/neuronal contact and myelination. IGF-I also promotes myelination by increasing expression of P0, a major component of myelin [79, 81, 82]. This upregulation contributes to increased myelin production [83]. Thus, IGF-I signaling stimulates neuronal myelination through several mechanisms, but there remains an imperfect understanding of the underlying biochemistry and signaling pathways. IGF-I Mediates Neuronal Survival: Relevance to Neurodegenerative Disease

While other cell types of the body undergo normal cellular turnover, neurons do not. The neuronal number is fixed after development, and any neurons that die after this point cannot be replenished. This fixed number underlies the d Higher magnification of c shows myelinated axons (black arrow) as well as extensive attachment and ensheathment of unmyelinated axons (white arrow) by SC processes. e Addition of 10 nM IGF-I to control media results in extensive axonal myelination (black arrow) compared with a and c. f Higher magnification showing well-myelinated large and small diameter axons with extensive SC (N-SC nucleus) ensheathment of remaining unmyelinated axons (white arrow). The insert shows normal periodicity of compact myelin and formation of major dense lines in the presence of IGF-I. Although IGF-I is seen to myelinate a small diameter axon in this figure, IGF-I does not preferentially myelinate or overmyelinate smaller axons (1 m2) compared with larger axons (1 m2) [reprinted from 80, with permission].


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pathogenesis of neurodegenerative diseases, where accumulative neuronal loss leads to gaps in neuronal circuits, and thus, progressive functional deficits. Neurons affected by neurodegenerative diseases die by a specific form of cell death known as programmed cell death, or, apoptosis [84]. Unlike necrotic death, which is characterized by an immune response, cellular swelling, and lysis, apoptotic cell death is characterized by membrane blebbing, a specific pattern of DNA fragmentation, new transcription and cell shrinkage [85]. Apoptosis is enacted by a number of cellular proteins that are expressed in healthy cells, but can promote deleterious cellular changes and death in response to toxin, stress, or injury signals. Among these apoptotic regulatory proteins are the family of Bcl proteins (such as Bcl-2, Bcl-xL, Bad, Bax, Bim), the cysteine protease family of caspases, and the c-Jun NH2-terminal kinases [86–90]. Thus, the colloquial term for apoptotic death is ‘cellular suicide’, because it represents an active process by which the cell elects to die in response to aversive stimuli. IGF-I protects neurons exposed to various neurodegenerative assaults. For example, IGF-I prevents apoptotic death in neurons treated with -amyloid, the neurotoxic substance that leads to neuronal loss in Alzheimer’s disease [91]. IGF-I also protects neurons after axotomy or spinal transection, thus preventing trauma and injury-induced neurodegeneration [92]. Neuronal death is similarly prevented by IGF-I after hypoxia ischemia, or stroke [93, 94]. Recent evidence suggests a role for IGF-I-mediated neuroprotection in Parkinson’s disease. Rats with lesioned nigrostraital dopamine pathways (which models the neurodegeneration in Parkinson’s disease) exhibit reduced apoptotic death of substantia nigra compacta neurons when treated with IGF-I. This ultimately increases motor function as compared to that of control-treated rats. However, IGF-I protection is abrogated by co-treatment with IGF-IR blockers, resulting in decreased neuronal survival and motor function [95]. Spinal motor neurons, targets of neurodegeneration in amyotrophic lateral sclerosis (ALS), are also rescued from apoptosis by IGF-I in a mouse model of ALS [96]. ALS-induced motor neuron death is caused, in part, by increased glutamate and resultant glutamate toxicity [97, 98]. IGF-I protects motor neurons from glutamateinduced apoptosis in a time- and dose-dependent manner (fig. 7), demonstrating that IGF-I may be therapeutic at the cellular level in ALS [15]. IGF-I is also neuroprotective for neurons subjected to high glucose treatment, a model of the hyperglycemia-induced neurodegeneration that is a pathological hallmark of diabetic neuropathy [20, 99]. Oligodendrocytes and Schwann cells are also protected by IGF-I [74, 100–103], thereby ensuring their survival and support of neurons. Interestingly, IGF-I-mediated neuroprotective signaling is remarkably consistent throughout the spectrum of neurodegenerative diseases. IGF-I protects

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Fig. 7. IGF-I inhibits glutamate-induced DNA fragmentation in motor neurons. a Motor neurons were treated for 24 h with glutamate (100 M) along with various concentrations of IGF-I and then DNA fragmentation was assessed by TUNEL. * TUNEL staining was significantly decreased by IGF-I in the concentration range between 8 and 250 nM compared with motor neurons exposed to 100 M glutamate alone (p 0.05). b Motor neurons were treated with IGF-I (10 nM), applied either concomitantly with glutamate (100 M) or at various times following the application of glutamate. After 24 h, DNA fragmentation was assessed by TUNEL. # Only co-application of IGF-I decreased DNA fragmentation compared with the application of IGF-I alone (p 0.001) [reprinted from 15, with permission].

neurons from apoptosis by binding to the IGF-IR and activating the PI3K pathway, which ultimately activates Akt [15, 20, 99, 104, 105]. The neuroprotective effects of IGF-I are blocked by PI3K inhibitors or expression of a kinaseinactive Akt mutant, proving that the survival signal is mediated predominantly through the PI3K/Akt pathway [105, 106]. IGF-I-induced activation of Akt in turn activates pro-survival effector proteins (such as cyclic AMP response element-binding protein (CREB)) and inhibits pro-apoptotic effector proteins (such as forkhead (FKHR) and glycogen synthase kinase-3 (GSK-3)) (fig. 8) [20]. Additionally, IGF-I enacts neuroprotection by regulating the family of Bcl proteins, which are known to function in apoptosis [107]. IGF-I may bias the


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Fig. 8. IGF-I regulates the Akt effectors GSK-3, CREB and FKHR. a DRG neurons were treated with control media (C) or stimulated with 10 nM IGF-I for 15 min (CI, 15 min) or 30 min (CI, 30 min). DRG neurons were also treated for 15 min with 10 nM IGF-I and 0.1% DMSO (CIDMSO) or LY294002 (CILY), or separately with glucose (G) or glucose 10 nM IGF-I (G I). The phospho-Akt (pAkt) immunoblot verifies that IGF-I activates Akt in IGF-I-stimulated control and glucose media, but that LY294002 inhibits IGF-I-mediated pAkt activation to control levels. IGF-I causes GSK-3 phosphorylation (pGSK-3) in control and high glucose, which is inhibited by LY294002. IGF-I also induces CREB phosphorylation (pCREB) after 15 min, but phosphorylation decreases by 30 min. CREB phosphorylation is inhibited by LY294002, and CREB can be activated in high glucose conditions. The apoptotic FKHR transcription factor is phosphorylated by IGF-I at 15 and 30 min (pFKHR), and this phosphorylation is inhibited by LY294002. IGF-I also phosphorylates FKHR in high glucose media, suggesting that an apoptotic environment does not negate IGF-I protective signaling. These phosphorylation differences are not due to unequal protein loading, as demonstrated by the equivalent GAPDH bands. Immunoblots shown are representative of results from three separate experiments. b Densitometry of pGSK-3, pCREB, and pFKHR from immunoblots described in a. Expression is represented as the average fold change of samples compared to the controls (C), such that C 1. Error bars represent standard error of the mean (SEM). *p 0.05 and **p 0.01 as determined by one-way ANOVA [reprinted from 20, with permission].

neuron toward survival by increasing expression of anti-apoptotic Bcl proteins (e.g. Bcl-2, Bcl-xL) or decreasing the expression of pro-apoptotic Bcl proteins (e.g. Bax, Bim) [108, 109]. Localization of Bcl proteins is also regulated by IGF-I; pro-apoptotic Bcl proteins are sequestered from the mitochondria, preventing them from inducing mitochondrial damage leading to apoptosis [110, 111]. Additionally, IGF-I results in PI3K/Akt-mediated phosphorylation of Bad, inhibiting its apoptotic function [112]. IGF-I also regulates the cysteine protease family of caspases. Caspases are zymogens that are present as inactive

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pro-forms in healthy cells, but are cleaved in response to apoptotic stimuli [113]. Activation of ‘initiator caspases’ (caspases-8 and -9) leads to activation of executioner caspases (such as caspases-3, -6 and -7) that commit cells to apoptotic death [114]. IGF-I prevents initiator and executioner caspase activation in neurons subjected to neurodegenerative stimuli, thus inhibiting apoptotic progression (fig. 9) [15, 20, 99, 108]. Finally, IGF-I mediates neuroprotection through downstream inhibition of the pro-apoptotic c-Jun NH2terminal kinase pathway, also referred to as the stress-activated MAP kinase pathway [115–117]. Thus, IGF-I promotes neuronal survival through several signaling pathways, which collectively converge to inhibit apoptotic death.

From the Bench to the Bedside: IGF-I Clinical Trials

The regenerative and neuroprotective effects of IGF-I demonstrated by in vitro and in vivo disease models suggest therapeutic potential for IGF-I in the treatment of human neurological disease. To date there are only two published clinical trials of IGF-I in neurological disease, both of which pertain to the efficacy of IGF-I in ALS. The two clinical trials produced mixed results. The North American ALS/IGF-I Study Group reported that patients receiving 9 months of daily IGF-I had slower disease progression. Further, IGF-I-treated patients reported having a better quality of life compared to placebo-treated patients [118]. However, these encouraging results were not replicated in a similar clinical trial conducted by the European ALS/IGF-I Study Group [119]. At the time of this writing, ALS patients are being enrolled for a phase III, double-blind, placebo-controlled trial of IGF-I, which is sponsored by the National Institute of Neurological Disorders and Stroke (NINDS) and the ALS Association. This trial differs from the previous two in that IGF-I will be administered subcutaneously twice per day, in contrast to the single dose tested previously. IGF-I has not been clinically tested for therapeutic benefit in other human neurological diseases. Based on in vitro and in vivo models showing IGF-I neuroprotection in hyperglycemia, IGF-I may be a candidate for therapeutic study in diabetic peripheral neuropathy. However, there are no such trials planned at this time and the focus remains on the benefit of IGF-I in motor neuron disorders. Clinical trials of other growth factors in human patients with neuropathies has produced negative to mixed results, which seems to have decreased initial enthusiasm [120–122]. One factor that may have compromised the efficacy of these clinical trials was the method of growth factor delivery: subcutaneous injection. Systemic administration is not ideal, as it requires high doses of growth factors in hopes that a small amount reaches the target neurons. Further, it is currently impossible to determine how much, if any, of the growth


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factor reaches target neurons at all. Because of the specificity for growth factors for particular sets of neurons, targeted growth factor delivery to key degenerative sites is a better therapeutic paradigm. The efficacy of this delivery method is currently being examined in a phase I/II clinical trial of vascular endothelial growth factor in diabetic neuropathy. Naked vascular endothelial growth factor DNA will be injected intramuscularly into affected extremities (foot, calf muscle, or distal thigh) in a dose escalation paradigm over 4 years. Results of this study should be available in 2006 [123] and will be helpful for determining the potential for viral vector delivery of IGF-I in treatment of human neuropathies.

The Future of IGF-I in Neurological Disease Treatment

Neurological diseases have a high degree of morbidity. As such, the search for new therapies, like IGF-I, is essential. While significant inroads have been made in our understanding of IGF-I signaling and physiology in the nervous system, more work is required to complete our understanding of the pleotropic effects of IGF-I. Knowledge of the intracellular signaling pathways and the protein expression profiles of affected neurons are required for tailoring IGF-I treatment to the appropriate disease paradigm. Further, ongoing research toward the development of targeted IGF-I delivery will be key for treating only the neurons affected by neurological disease, thus amplifying therapeutic benefit and reducing side effects. These and other research horizons will expand our understanding of IGF-I as a potential therapy for neurological disorders.

Acknowledgements The authors thank Ms. Judy Boldt for expert secretarial assistance. This work was supported by grants from the National Institutes of Health (NS043023, NS38849 and NS36778), the Juvenile Diabetes Research Foundation Center for the Study of Complications in Diabetes, and the Program for Understanding Neurological Diseases (PFUND).

References 1

2

Leinninger GM, Meyer GE, Feldman EL: IGFs and the nervous system; in LeRoith D, Zumkeller W, Baxter R (eds): Insulin-Like Growth Factors. Eurekah.com/Landes Bioscience & Kluwer Academic/Plenum Publishers, 2003, pp 158–187. Leinninger GM, Vincent AM, Feldman EL: The role of growth factors in diabetic peripheral neuropathy. J Periph Nerv Syst 2004;9:26–53.


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Eva L. Feldman, MD, PhD Department of Neurology, University of Michigan 4414 Kresge III, 200 Zina Pitcher Place Ann Arbor, MI 48109 (USA) Tel. 1 734 7637274, Fax 1 734 7637275, E-Mail [email protected]


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Insulin-Like Growth Factor System in Amyotrophic Lateral Sclerosis Nadine Wilczak, Jacques de Keyser Department of Neurology, Academic Hospital Groningen, Groningen, The Netherlands

Abstract Insulin-like growth factor-I (IGF-I) is a neurotrophic factor with insulin-like metabolic activities, and possesses potential clinical applications, particularly in neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is a chronic progressive devastating disorder of the central nervous system, characterized by the death of upper and lower motor neurons. Both in vivo and in vitro studies have shown that IGF-I promotes motor neuron survival and strongly enhances motor nerve regeneration. Evidence that IGF-I rescues motor neurons has led to clinical trials of human recombinant IGF-I in ALS patients. However, systemic delivery of human recombinant IGF-I in these trials did not lead to beneficial clinical effects in ALS patients and may be due through inactivation of IGF-I by binding to IGF binding proteins (IGFBPs), and or limited delivery of IGF-I to motor neurons. Recently it was shown that both IGF-I receptors and IGFBPs were increased on motor neurons of ALS patients and free levels of IGF-I were decreased by 50%. In this study it was suggested that IGFBPs inactivate IGF-I by forming inactive complexes. The uses of IGF analogues with low affinity for IGFBPs and analogues that are able to displace IGF-I from IGFBPs are better candidates in new clinical trials. Another possibility is to find a way of IGF-I transport without hindrance of circulating and tissue-specific IGFBPs, such as IGF-I delivery based on gene therapy. Copyright © 2005 S. Karger AG, Basel

Introduction

Amyotrophic lateral sclerosis (ALS), a form of motor neuron disease (MND), is a progressive devastating disease of the central nervous system (CNS). The incidence of ALS is 1–2 per 100,000, and predominantly affects middle-aged and elderly people – 90% of the cases are sporadic and the other 10% are familial [1].

The pathologic hallmarks of ALS are the degeneration and loss of both upper and lower motor neurons with reactive astrocytic gliosis. Upper motor neurons are located in the motor cortex and neurodegeneration of these neurons leads to spastic pareses, overactive tendon reflexes, and Babinski signs [1]. Degeneration of lower motor neurons in the brainstem and spinal cord causes muscle atrophy, weakness, and fasciculations. Death usually results from respiratory failure due to weakness of the ventilatory muscles [1]. Until now the precise causes of both familial and sporadic ALS remain unknown. Familial ALS accounts for about 10% of diagnosed cases, of which 25% are associated with missense mutations in the antioxidant enzyme copper/ zinc superoxidase dismutase (SOD1). The remaining 80% of ALS cases are known as sporadic. There are many hypotheses on the cause of sporadic ALS, including autoimmune reactions to calcium channels on motor neurons, glutamate-induced excitotoxic injury, exposure to toxins, latent infections, and loss of neurotrophic support to motor neurons [1].

Potential Usefulness of IGF-I in MND

The use of neurotrophic factors for the treatment of degenerative MNDs are based on two goals. First, to promote motor neuron survival and, second, to enhance motor nerve regeneration from surviving neurons by axon outgrowth and reinnervation of denervated muscle fibers. Insulin-like growth factor-I (IGF-I) possesses both characteristics and is therefore a potential neurotrophic factor for therapeutic purposes in ALS. As a neurotrophic factor, IGF-I promotes the survival of motor neurons in culture, increases their long-term neurite outgrowth, including branching and synapse formation [2], protects developing motor neurons from axotomy-induced cell death, and promotes muscle reinnervation in vivo [3]. In transgenic mice it was shown that local expression of human IGF-I in skeletal muscle is capable of accelerating the regeneration of peripheral nerves and muscles after a nerve injury [4]. Administration of IGF-I in wobbler mouse, an animal model of upper and lower MND, showed also beneficial effects on muscle strength [5]. IGF-I is widely distributed in the developing and mature CNS, and plays an important role in neuronal survival, and the differentiation and maturation of glial cells. Both, IGF-I and IGF-II are single-chain polypeptides with a molecular weight of approximately 7.5 kDa. These growth factors are expressed by neurons and glial cells where they act in a paracrine or autocrine manner. Binding of IGF-I to IGF-I receptors induces receptor autophosphorylation and the activation of the PI3K/serine-threonine protein kinase Akt signaling pathway. IGF-I signaling through Akt promotes survival of neurons and prevents apoptosis [2].

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Motor neurons die in ALS by necrosis, programmed cell death (apoptosis), or a combination of both. The induction of apoptosis is caused by two basic pathways: the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway [6]. Both pathways require the activation of caspases. The regulation of apoptosis is achieved through members of the Bcl-2 family, which modulate the permeability of mitochondrial membranes and the release of cytochrome c an effector molecule for apoptosis. In the intrinsic pathway, mitochondrial dysfunction leads to the release of an apoptotic effector (cytochrome c) into the cytoplasm. Cytochrome c binds to apoptotic protease-activating factor-1 (Apaf-1) to form the apoptosome, which activates the upstream caspase-9, an initiator of apoptosis. The extrinsic pathway requires the binding of TNF-␣ to cell death receptors, which activates upstream caspases (caspase-8 and -10) and downstream caspases (caspase-3, -6 and -7) that leads to the biochemical aspects of apoptosis. IGF-I prevents apoptosis caused by components of the intrinsic deathsignaling pathway. In vitro and in vivo experiments have shown that IGF-I prevents dephosphorylation and nuclear translocation of the forkhead transcription factor FKHRL-1 regulating the induction of Bcl-2 interacting mediator of cell death (Bim) [7]. In rat retinal ganglion cells, IGF-I inhibits the activation of caspase-9 by preventing the release of cytochrome c and the subsequent formation of apoptosomes [8], and protects neuroblastoma cells from peroxynitriteinduced cell death by preventing cytochrome c-induced caspase-3 activation [9]. It has also been suggested that both the lack of growth factors and the release of cytokines influence survival of neurons in the injured brain. Both the brain and spinal cord demonstrate elevated levels of TNF-␣ during motor neuron dysfunction in animal models of MND [10]. TNF-␣ is a proinflammatory cytokine that contributes to neurodegeneration via binding to its p55 receptor and promotes cell death via the extrinsic death-signaling pathway [6]. Cytokines released during brain injury influence IGF-I actions by regulating IGF-I receptor signaling. During neurodegeneration, TNF-␣ inhibits PI3K/AKT signaling in cerebellar neurons, by suppressing IGF-I-induced tyrosine phosphorylation of insulin-related substrate-2 [11]. Glutamate-induced excitoxicity may also contribute to motor neuronal cell death in sporadic ALS [12]. Glutamate levels in serum and cerebrospinal fluid were altered in ALS patients, and glutamate transport was found to be reduced [12]. IGF-I protects embryonic rat motor neurons [13] and cerebellar granule neurons [14] from glutamate-induced cell death through PI3K/Akt signaling. Glutamate is the major excitatory neurotransmitter in the CNS. Although glutamate is crucial for optimal performance, when released in high amounts, it can induce overexcitation of neurons leading to neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is a general agreement

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that it is largely dependent on an accumulation of intracellular calcium concentrations. Recently it was shown that excess glutamate signaling leads to neuronal death through inactivation of PI3K/AKT signaling that is crucially involved in prosurvival actions of IGF-I [15]. In organotypic spinal cord cultures using the glutamate transport inhibitor THA to create chronic glutamateinduced cell death, IGF-I significantly increases choline acetyltransferase (ChAT) activity and provides neuroprotection of motor neurons [15]. The use of organotypic spinal cord cultures is a better model to investigate IGF-I effects on motor neurons, because it provides the advantage of a postnatal neuronal system, preservation of organotrophic morphology with partially preserved connections, and long-term culture viability [15]. IGF-I signaling via the PI3K/AKT signaling pathway is also involved in the response to neuronal injury caused by free radicals. Oxidative stress caused by free radicals plays a role in many degenerative disorders of the CNS. One of the major findings has been that mutations in the gene on chromosome 21 encoding the enzyme copper/zinc SOD1, underlies 20% of the cases of familial ALS and 5% of the cases of sporadic ALS [1]. SOD1 is an enzyme that requires copper, catalyzes the conversion of intracellular toxic superoxide radicals to hydrogen peroxide and oxygen. Hydrogen peroxide is then eliminated by other free radical scavenging enzymes. It has been shown that mutations in SOD1 result in abnormal toxic accumulation of superoxide radicals, promote the formation of nitrotyrosine residues on intracellular proteins, and may form abnormal intracellular aggregates which results in neurotoxicity [1]. Transgenic mice expressing the human mutant SOD1 gene develop a syndrome with many features of ALS, including motor neuron death, progressive muscle weakness, and early death [1]. Retrograde viral transport of IGF-I in the SOD1-transgenic mouse model prolongs life and delays disease progression [16]. IGF-I treatment before disease onset and during disease onset showed a significant improvement in maximal life span compared to controls. IGF-I-treated animals showed 20% increase in muscle mass, motor neuron survival and reduced astrogliosis. In this study IGF-I mediates survival of motor neurons through the PI3K/Akt signaling pathway, which was demonstrated by increased levels of phosphorylated Akt and reduced caspase-9 cleavage.

Disappointing Results in Clinical Studies with IGF-I

Evidence that IGF-I rescues motor neurons in vitro and in animal models has led to clinical trials of human recombinant IGF-I in ALS patients. Two double-blind, placebo-controlled trials with subcutaneous IGF-I in ALS have been performed. In a US study of high (0.1 mg/kg/day) and low


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ALS

CON

Fig. 1. Immunostaining for IGF-I receptors in spinal motor neurons in ALS (right) and control (left) without neurological disease. Motor neurons are studied in ventral horns (C4–C8) and were visualized with peroxidase-diaminobenzidine staining. IGF-I receptors were 50% increased on motor neurons of ALS patients compared to controls (arrowheads). Scale bar ⫽ 50 ␮m.

(0.05 mg/kg/day) doses of human recombinant IGF-I, the average Appel ALS rating scale in 9 months was 9 points lower for the high-dose group compared with the placebo. However, there was no effect on mortality [17]. The results of the European trial (IGF-I 0.1 mg/kg/day vs. placebo) showed no therapeutic benefit [18]. The FDA did not approve the use of IGF-I on the basis of these trials.

IGF-I System in ALS

The IGF-I signaling system is very complex and is regulated by multiple influences. Besides the IGF-I receptor there are at least six distinct insulin-like growth factor binding proteins (IGFBPs). At the level of the extracellular matrix or cell surface of target cells, these IGFBPs can either inhibit or enhance the presentation of IGF-I to their receptors [19, 20]. In addition, some IGFBPs are capable of mediating biological actions that are IGF-I independent [19, 20]. In patients with ALS, serum levels of IGF-I were found to be either normal or slightly decreased [21], and the expression of IGF-I in ALS spinal cords was found to be normal [22]. Previous studies have shown that [125-I]-IGF-I binding sites were increased in number in ALS spinal cord [23, 24]. Recently it was shown that IGF-I receptors (fig. 1) and IGFBP-2, -5 and -6 (fig. 2) were increased on spinal motor neurons of sporadic ALS, and this increase was related to the loss of neurotrophic support by IGF-I, which was reduced by 50% [25]. Different types of lesions affecting lumbar motor neurons in rats produced a similar pattern of increased levels of IGFBP-2, -5 and -6 in these motor neurons [26], and local application of IGF-I failed to prevent motor neuron death [27]. The increases in IGFBP-2, -5 and-6 in ALS spinal cord might be caused by a decreased activity of proteases or an increase in protease inhibitors.

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IGFBP-2

IGFBP-5

IGFBP-6

ALS

CON

Fig. 2. Immunostaining for IGFBP-2, -5 and -6 in spinal motor neurons in ALS patients (ALS) and control patients (CON) without neurological disease. Motor neurons are studied in ventral horns (C4–C8) and were visualized with peroxidase-diaminobenzidine staining. The concentration of IGFBP-2 was 64% higher, of IGFBP-5 was 46% higher, and of IGFBP-6 was 33% higher on motor neurons (arrowheads) in the ventral horns of ALS patients (ALS) compared with control patients (CON). Scale bar ⫽ 50 ␮m.

Circulating and tissue-specific IGFBPs bind IGFs to form biologically inactive complexes. Potentiation of IGF activity may occur under specific circumstances, and involves the slow dissociation of IGFs from IGFBPs. IGFBP fragments generated by the action of cellular endoproteases show a marked loss of IGF binding. Members of several classes of proteases have shown the ability to cleave IGFBPs, including kallikreins, cathepsins and metalloproteinases. Both IGFBP-2 and -5 are proteolytically cleaved by serine proteases; the identity of proteases cleaving IGFBP-6 remains to be determined [19, 20]. The role of serpins (serine protease inhibitors), which form stable complexes with serine proteases, thereby preventing their proteolytic activity, may be of special interest. Serine protease-like complexes have been identified in ALS motor neurons, and an imbalance of serine proteases and serpins has been hypothesized to play a role in the pathogenesis of ALS [28]. Transgenic mice overexpressing protease nexin-1 (PN-1), a secreted serine protease inhibitor, develop disturbances in motor behavior and histopathological changes described in early stages of human MND [29]. Future Therapies for the Treatment of ALS

Systemic delivery of human recombinant IGF-I in clinical trials did not lead to beneficial clinical effects in ALS patients and may be due through


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inactivation of IGF-I by binding to IGFBPs which are highly expressed in ALS spinal cord, and limited delivery of IGF-I to motor neurons. IGF-I analogues with low affinity for IGFBPs, and IGF-I analogues that display very high affinity for IGFBPs which are able to displace endogenous IGF-I from IGFBPs, may be better candidates for stimulating motor neuron IGF-I receptors. A truncated form of IGF-I known as Des(1–3)IGF-I has been found in adult human brain [30]. Des(1–3)IGF-I is the product of differential processing of pro-IGF-I lacking the tripeptide GPE (Gly-Pro-Glu). The biologically potency of Des(1–3)IGF-I is 10 times higher than that of the full length and is explained by reduced binding to IGFBPs [19, 20]. In addition, GPE has also neuroprotective properties in injured brain. Local and systemic administration of GPE inhibits neuronal apoptosis after brain hypoxia-ischemia injury [31], and prevents the loss of tyrosine-hydroxylase neurons in an animal model for Parkinson [32]. Non-small peptides have been developed which act as IGFBP ligand inhibitors and prevent binding of IGFs to IGFBPs [33], resulting in an elevation of free biological available IGF-I at the level of motor neurons. IGF-I analogues, which are able to displace IGF-I from IGFBPs, have been studied in rats with focal cerebral ischemia [34]. In this model, the intracerebroventricular administration of the IGF-I analogue [(Leu24, 59, 60, Ala31)hIGF-I] with no affinity to IGFBPs and no biological activity at the IGF-I receptors, increased the levels of free biological available IGF-I and provided potent neuroprotection. Another approach to stimulate IGF-I receptors is a better delivery of IGF-I to motor neurons by avoiding physiological hindrances such as binding to circulating IGFBPs and degradation of short-living IGF-I during administration. IGF-I delivery based on gene therapy strategy could be more promising. A recent study has shown that retrograde delivery of IGF-I prolongs survival in a mouse ALS model [16]. In this study, adeno-associated viruses (AAV) expressing IGF-I which are retrogradely transported, were injected in transgenic mice expressing the G93A mutant SOD1. The use of AAV vectors for retrograde transport IGF-I will provide a long-term muscular secretion of IGF-I, and spinal motor neurons can be selectively targeted for IGF-I. Another therapeutic target is to find a way of regulating the bioavailability of IGF-I by dissociating it from IGFBPs. It is well known that binding of IGFBPs to the extracellular matrix or cell surface facilitates the release of IGF-I leading to enhanced delivery to specific cell receptors. Heparin, dermatan sulfate and heparin sulfate interact with IGFBPs, reducing their binding affinity for IGF-I, and therefore increasing bioavailability of IGF-I to its receptor. Glycosaminoglycans (GAGs) are constituents of proteoglycans and they

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promote neuritogenises in vitro, and regeneration following peripheral nerve axotomy [35]. Combination therapy of GAGs/IGF-I was studied in the wobbler and Mnd mice and in axotomized animals [35]. In these models combined IGF-I/GAGs treatment arrested the progression of motor neuron degeneration in both the wobbler and the Mnd mice, and gives an effect far superior to that observed when the same animals were treated with the individual components. Administration of IGF-I in combination with GAGs markedly enhanced the pharmacological effects of IGF-I; a dose of 0.2 mg/kg elicited effects far greater than a dose of 1 mg/kg alone. Thus, combined treatment of IGF-I-GAGs strikingly enhanced pharmacological effects of IGF-I in transgene animals, and it has been suggested that GAGs may regulate the interaction between IGF-I and IGFBPs and expression of IGF-I [35].

References 1 2 3

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8

9

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12 13 14

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Dr. Nadine Wilczak Department of Neurology Academic Hospital Groningen, PO Box 30.001 NL–9700 RB Groningen (The Netherlands) Tel. ⫹31 50 3611142, Fax ⫹31 50 361707, E-Mail [email protected]


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Author Index

Acerini, C.L. 107 Aimaretti, G. 76 Baffoni, C. 76 Baldelli, R. 76 Bellone, S. 76 Bennet, L. 31 Blair, J.C. 100 Camacho-Hübner, C. 100 Cianfarani, S. IX, 66 Clemmons, D.R. IX, 55 Corneli, G. 76 Croce, C. 76 de Keyser, J. 160 Dunger, D.B. 107 Feldman, E.L. 135

Gasco, V. 76 Germani, D. 66 Ghigo, E. 76 Gluckman, P.D. 31 Granata, R. 76 Grottoli, S. 76 Guan, J. 31 Gunn, A.J. 31 Jorge, A.J. 100 Leinninger, G.M. 135 LeRoith, D. 11 Liguori, A. 66 Longobardi, L. 17

O’Rear, L. 17 Pennisi, P. 11 Ranke, M.B. 100 Regan, F.M. 107 Rosenfeld, R.G. 1 Rovere, S. 76 Sandhu, M.S. 44 Savage, M.O. VII, IX, 100 Spagnoli, A. 17 Street, M.E. 100 Wilczak, N. 160 Wu, Y. 11 Yakar, S. 11

Monson, J.P. 89 Moses, A.C. 121

Zhao, H. 11

170

Subject Index

Acid-labile subunit (ALS) adult growth hormone deficiency evaluation 84 function 84 growth hormone activity marker 91 growth hormone insensitivity levels 102, 104 Alzheimer’s disease, insulin-like growth factor-I neuroprotection 148 Amyotrophic lateral sclerosis (ALS) epidemiology 160 etiology 161 glutamate excitotoxicity 162, 163 inflammatory cytokines 162 insulin-like growth factor-I binding protein levels 164, 165 neuroprotection 148 serum levels 164 therapy clinical trials 151, 153, 163, 164 prospects 165–167 rationale 161–163 neuronal apoptosis 162 oxidative stress 163 pathology 161 serpins in pathogenesis 165 Anorexia nervosa, insulin-like growth factor-I deficiency 104 Apoptosis amyotrophic lateral sclerosis 162 mesenchymal stem cell assay and IGFBP-3 induction 25, 27

neuronal survival promotion by insulinlike growth factor-I 147–151, 162 regulatory proteins 148, 162 Beta cell, insulin-like growth factor effects on growth, survival, and function 46, 47 Cancer insulin-like growth factor-I knockout mouse studies 14 mediation of neuroblastoma motility 141, 143, 144 insulin-like growth factor system 13, 14 Cartilage IGFBP-3 effects 21–23 insulin-like growth factor-I role 20, 21 mesenchymal stem cell therapy, see Mesenchymal stem cell Caspases, insulin-like growth factor-I regulation 150, 151 Chondrogenesis IGFBP-3 effects 21–23 insulin-like growth factor-I role 20, 21 mesenchymal stem cell therapy, see Mesenchymal stem cell overview 17, 18 Coronary heart disease, insulin-like growth factor-I cardiac benefits 48 deficiency and risks 48–50 Mendelian randomization in risk assessment 50, 51

171

Crohn’s disease, insulin-like growth factor-I deficiency 104 Cystic fibrosis, insulin-like growth factor-I deficiency 104 Diabetes typ 1 growth hormone insulin resistance role 108–110 levels 107, 108 microvascular complication role 110–112 IGFBP-1 levels 108 IGFBP-3 levels 107 replacement therapy 114, 116 insulin-like growth factor-I insulin resistance role 108–110 levels 107 microvascular complication role 110–112 replacement therapy adverse events 113–115 IGFBP-3 combination therapy 114, 116 prospects 116, 117 rationale 112 trials 113 Diabetes type 2 epidemiology 124 insulin-like growth factor-I beta cell growth, survival and function effects 46, 47 deficiency 45, 47 gene polymorphisms 47 insulin sensitivity response 45, 46 knockout mouse studies 46 Mendelian randomization in risk assessment 50, 51 therapy IGFBP-3 combination therapy 129, 130 insulin resistance trials 126–130 prospects 130–132 rationale 125, 126 safety 128, 129 management 124 Diabetic neuropathy, insulin-like growth factor-I neuroprotection 148

Subject Index

ERK/MAPK pathway insulin-like growth factor-I signaling in nervous system 137, 161, 163 neurite outgrowth mediation and nerve injury relevance 139, 141 neuronal motility mediation and neuroblastoma 141, 143, 144 neuroprotective signaling 150 Growth hormone (GH) biochemical markers of action 90–92 diabetes type 1 insulin resistance role 108–110 levels 107, 108 microvascular complication role 110–112 insulin-like growth factor-I role in action 1, 12 secretion 55, 56 secretion relationship to plasma insulin-like growth factor-I 57, 58 therapy, see Growth hormone deficiency Growth hormone deficiency (GHD) auxological and biochemical assessment of children 72, 73 cardiac dysfunction 48 diagnosis adults 77–79 children 66, 67 overview 3–5, 76, 77 epidemiology 3 growth hormone therapy in adult growth hormone deficiency biochemical markers of action 90–92 dose response 94, 95 dosing 89, 90 insulin-like growth factor-I monitoring adolescent-to-adult transition phase 96, 97 advantages and limitations 97, 98 dose titration 95 elderly patients 95, 96 safety marker 92 young adults 97 sex differences in response 93, 94 IGFBP-3 evaluation 69–71

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insulin-like growth factor generation tests adult growth hormone deficiency 79–83 childhood-onset growth hormone deficiency 67, 68 evaluation 6, 7 growth hormone therapy monitoring 7, 8, 92 responsiveness versus deficiency 5 Growth hormone insensitivity (GHI) acid-labile subunit levels 102, 104 acquired insensitivity due to chronic pediatric illness 104 idiopathic short-stature patients 103 IGFBP-3 deficiency 101–104 insulin-like growth factor-I deficiency 101–104 partial growth hormone insensitivity markers 102, 103 receptor mutations 101, 103 Hypertriglyceridemia, insulin-like growth factor-I response 127 Hypoxic-ischemic encephalopathy causes 31 delayed neuronal injury 32, 41 insulin-like growth factor-I induction after injury 32 neuroprotection mechanisms IGFBP mediation 39, 40 indirect effects 41 insulin-like growth factor-I receptor mediation 39 N-terminal peptide neuroprotection studies 40, 41 neuroprotection studies in animal models brain region selectivity 33, 34 dose dependence 32, 33 progressive neuronal injury and functional recovery 36, 37 therapeutic window 35, 36 white matter protection 37, 38 therapeutic prospects 41, 42 Idiopathic short stature (ISS), growth hormone insensitivity 103

Subject Index

IGFBP-1 (insulin-like growth factor binding protein-1) diabetes type 1 levels 108 diabetes type 2 levels 47 IGFBP-2 (insulin-like growth factor binding protein-2) hypoxic-ischemic encephalopathy role 39, 40 levels in growth hormone deficiency 58 IGFBP-3 (insulin-like growth factor binding protein-3) adult growth hormone deficiency evaluation 84 cartilage effects 21–23 childhood-onset growth hormone deficiency evaluation 69–71 diabetes type 1 levels 107 replacement therapy 114, 116 generation tests in growth hormone deficiency evaluation 6, 7, 77 growth hormone therapy monitoring 7, 8 growth hormone activity marker 91, 92 growth hormone insensitivity and deficiency 101–104 insulin-like growth factor-I combination therapy for insulin resistance 129, 130 sensitivity and specificity in growth hormone deficiency evaluation 72 Insulin, growth hormone activity marker 91 Insulin-like growth factor deficiency (IGFD) assays 4 coronary heart disease risks 48–50 diabetes type 2, see Diabetes type 2 insulin-like growth factor-I therapy 8, 9 management 4, 5 primary versus secondary causes 4 Insulin-like growth factor-I (IGF-I) amyotrophic lateral sclerosis trials, see Amyotrophic lateral sclerosis assays adult growth hormone deficiency evaluation 79–83 childhood-onset growth hormone deficiency evaluation 67, 68

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Insulin-like growth factor-I (IGF-I) (continued) assays (continued) clinical use 62–64 discrepancies 56, 57 growth hormone secretion relationship to plasma insulin-like growth factor-I 57, 58 interpretation problems 63, 64 sensitivity and specificity in growth hormone deficiency evaluation 72 technical problems 59–62 variations of levels in normal subjects 58, 59 beta cell growth, survival and function effects 46, 47 binding proteins, see specific IGFBPs cartilage role 20, 21 coronary heart disease role 48–50 diabetes and deficiency, see Diabetes type 1; Diabetes type 2 growth hormone action role 1, 12 activity marker 91, 92 therapy monitoring, see Growth hormone deficiency growth hormone insensitivity and deficiency 101–104 ischemic brain injury studies, see Hypoxic-ischemic encephalopathy knockout mice cancer studies 14 chondrocyte defects 21 insulin resistance studies 14, 15, 46 liver-derived insulin-like growth factor-I gene deletion 12, 13 nervous system effects 139 prospects for study 16 receptor knockout mouse and growth effects 2 nervous system effects, see Nervous system, insulin-like growth factor-I effects Insulin resistance diabetes type 1 pathophysiology 108–110 insulin-like growth factor-I clinical trials 126–130

Subject Index

knockout mouse studies 14, 15, 46 severe 122–124 spectrum 122 Insulin tolerance test (ITT), adult growth hormone deficiency diagnosis 76, 78 Ischemic brain injury, see Hypoxicischemic encephalopathy Juvenile chronic arthritis, insulin-like growth factor-I deficiency 104 Laron syndrome, see Growth hormone insensitivity Leptin, growth hormone activity marker 91 Mesenchymal stem cell (MSC) cartilage disease therapy 20 chondrogenesis studies of insulin-like growth factor system effects apoptosis assay and IGFBP-3 induction 25, 27 chondrocyte differentiation 18, 19 human cell isolation, expansion, and micromass pellet formation 24, 25 IGFBP-3 inhibitory effects 27, 28 insulin-like growth factor-I effects on chondrogenic pellet size 27 mouse cell isolation, expansion, and micromass pellet formation 23, 24 prostaglandin synthesis assay and transforming growth factor-␤ effects 25–27 RCJ3.1C5.18 cell culture 25 statistical analysis 26 gene and cell therapy advantages 18, 19 implantation studies 19, 20 insulin-like growth factor-I in chondrogenic potential 19 markers 19 plasticity 19 Motor neuron disease, see Amyotrophic lateral sclerosis Multiple sclerosis, insulin-like growth factor-I treatment prospects 144, 145, 147 Myelination, insulin-like growth factor-I promotion 144, 145, 147

174

Nervous system, insulin-like growth factor-I effects amyotrophic lateral sclerosis trials, see Amyotrophic lateral sclerosis myelination and demyelinating diseases 144, 145, 147 neurite outgrowth mediation and nerve injury relevance 137, 139, 141 neurological disease treatment prospects 151, 153 neuronal loss in neurological disease 135, 136, 147–151 neuronal motility mediation and neuroblastoma 141, 143, 144 neuronal survival promotion 147–151 signaling pathways 137, 161, 163 trophic support in neurological disease 136, 137 Parkinson’s disease, insulin-like growth factor-I neuroprotection 148

Subject Index

Phosphatidylinositol 3-kinase/Akt pathway insulin-like growth factor-I signaling in nervous system 137 neurite outgrowth mediation and nerve injury relevance 139, 141 neuroprotective signaling 148–150 Prostaglandins, mesenchymal stem cell synthesis assay and transforming growth factor-␤ effects 25–27 RCJ3.1C5.18 cells, culture 25 Serpins, amyotrophic lateral sclerosis pathogenesis 165 Somatomedin hypothesis, historical perspective 12, 21 Transforming growth factor-␤ (TGF-␤), mesenchymal stem cell prostaglandin synthesis effects 25–27

175

IGF-i And IGF Binding Proteins: Basic Research And Clinical Management (Endocrine Development)

Actin-Binding Proteins and Disease

Actin-Monomer-Binding Proteins

Heparin-Binding Proteins

Endocrine Disrupting Chemicals-from Basic Research to Clinical Practice

Heparin-Binding Proteins

Proteins: Membrane Binding and Pore Formation

Xenotransplantation: Basic Research and Clinical Applications

Basic and Clinical Endocrinology

Basic and Clinical Pharmacology

Clinical and Basic Immunodermatology

Combination Vaccines: Development, Clinical Research and Approval

Poxvirus Secreted Chemokine-binding Proteins

Mood Disorders: Clinical Management and Research Issues

Clinical Endocrine Oncology

Asthma and COPD: Basic Mechanisms and Clinical Management

Asthma and COPD, Second Edition: Basic Mechanisms and Clinical Management

Asthma and COPD, Second Edition: Basic Mechanisms and Clinical Management

Endocrinology: Basic and Clinical Principles

Glaucoma - Basic and Clinical Concepts

Photoaging (Basic and Clinical Dermatology)

Endocrinology: Basic and Clinical Principles

Endocrine Neoplasia (Cancer Treatment and Research)

Endocrinology: Basic and Clinical Principles

Management and Leadership Development

Development and Research Activities

Calcium Binding Proteins (Wiley Series in Protein and Peptide Science)

Latex Intolerance: Basic Science, Epidemiology, and Clinical Management

Itch: Basic Mechanisms and Therapy (Basic and Clinical Dermatology)

Essentials of Apoptosis: A Guide for Basic and Clinical Research

Research and Development Management in the Chemical and Pharmaceutical Industry

IGF-i And IGF Binding Proteins: Basic Research And Clinical Management (Endocrine Development)

Actin-Binding Proteins and Disease

Actin-Monomer-Binding Proteins

Heparin-Binding Proteins

Endocrine Disrupting Chemicals-from Basic Research to Clinical Practice

Heparin-Binding Proteins

Proteins: Membrane Binding and Pore Formation

Xenotransplantation: Basic Research and Clinical Applications

Basic and Clinical Endocrinology

Basic and Clinical Pharmacology

Clinical and Basic Immunodermatology

Combination Vaccines: Development, Clinical Research and Approval

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