The Somatotrophic Axis in Brain Function

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

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THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION Edited by FRED J. NYBERG Uppsala University, Sweden

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Elsevier Academic Press 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright © 2006, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (⫹44) 1865 843830, fax: (⫹44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Customer Support” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data Application submitted British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 13: 978-0-12-088484-1 ISBN 10: 0-12-088484-4 For all information on all Elsevier Academic Press publications visit our Web site at www.books.elsevier.com Printed in the United States of America 05 06 07 08 09 10 9 8 7

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Contents

Preface vii Contributors ix Pioneers in the growth hormone field xi

6. Growth Hormone, Insulin, and IGF-I: Do They Interact at the Blood–Brain Barrier? 75 Weihong Pan, Yongmei Yu, Fred Nyberg, and Abba J. Kastin

Introduction 1 Fred J. Nyberg

7. Growth Hormone and Insulin-like Growth Factors in the CNS: Localisation in Mammalian Species 81 Vincenzo C. Russo and George A. Werther

I. Biosynthesis of GH and IGF-I and Regulation of Their Secretion 5

8. Purification of Growth Hormone Receptors from Human Brain Tissues 91 Zhennan Lai and Fred Nyberg

1. Regulation and Mechanism of Growth Hormone and Insulin-like Growth Factor-I Biosynthesis and Secretion 7 C. B. Chan, Margaret C. L. Tse, and Christopher H. K. Cheng

9. Growth Hormone Receptor Message in the Rat and Human CNS: Structure and Function 99 Madeleine Le Greves

2. Ghrelin, an Endogenous Ligand for the Growth Hormone Secretagogue Receptor 25 Masayasu Kojima and Kenji Kangawa

IV. CNS Action of Growth Hormone: Basic Studies Using Animal Models 109

II. GH and IGF-I and their Function at Receptor Level 37

10. Interaction of Growth Hormone and Prolactin in Brain Circuits 111 David R. Grattan and Tanja A. E. Möderscheim

3. Mechanisms of Signal Transduction Utilized by Growth Hormone 39 Farhad Shafiei, Adrian C. Herington, and Peter E. Lobie

11. Growth Hormone and Insulin-like Growth Factor-I and Cellular Regeneration in the Adult Brain 125 N. David Åberg, Maria A. I. Åberg, and Peter S. Eriksson

4. Insulin-like Growth Factor-I and its Binding Proteins: Regulation of Secretion and Mechanism of Action at Receptor Level 51 Mark G. Slomiany and Steven A. Rosenzweig

12. Growth Hormone and Insulin-like Growth Factor-I and Their Effects on Astroglial Gap Junctions 147 N. David Åberg

III. Growth Hormone and Insulin-like Growth Factors and Their Receptors in the Central Nervous System 67

13. Rodent Models for the Study of the Role of GH–IGF-I or the Insulin Axis in Aging and Longevity: Special Reference to a Transgenic Dwarf Rat Strain and Calorie Restriction 173 Isao Shimokawa

5. Growth Hormone and Insulin-like Growth Factor-I in Human Cerebrospinal Fluid 69 Fred Nyberg

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14. Growth Hormone and Insulin-like Growth Factor-I (IGF-I) and Their Interactions with Brain Circuits Involved in Cognitive Function 185 Melinda Ramsey and William E. Sonntag 15. IGF-I and Neuroprotection 209 E. Carro, J. L. Trejo, S. Fernandez, A. M. Fernandez, and I. Torres-Aleman

V. Immediate and Long-Term Benefits on the CNS and GH Replacement Therapy 217 16. Quality of Life in Adult GH Deficiency 219 Robert D. Murray and Stephen M. Shalet 17. The Somatotropic Axis in Psychologic Functioning: Effects on Sleep and Psychopathology 231 Harald Jörn Schneider and Günter Karl Stalla 18. Growth Hormone Replacement Therapy in Adults: Responsiveness Related to Life Quality 237 Åse Krogh Rasmussen and Ulla Feldt-Rasmussen 19. Psychological Importance to the Child of Growth Hormone Replacement 249 John Eric Chaplin, Jovanna Dahlgren, Berit Kriström, and Kerstin Albertsson Wikland 20. Acromegaly and Brain Function: Effects on the Human Brain during Conditions with Increased Growth Hormone Concentrations 267 Sigbritt Werner

21. Human Aging and the Growth Hormone/Insulin-like Growth factor-I axis: The Impact of Growth Factors on Dementia 271 E. Arvat, R. Giordano, Micaela Pellegrino, Fabio Lanfranco, Matteo Baldi, Andreea Picu, Lorenza Bonelli, and E. Ghigo 22. Cognitive Status of Adult GHD Patients and GH-Induced Neuropsychological Changes 287 Jan Berend Deijen and Lucia. I. Arwert 23. Growth Hormone and Insulin-like Growth Factor-I in Alzheimer’s Disease 301 José Manuel Gómez

VI. GH Replacement and Related Therapies in CNS Disorders: A Future Perspective 311 24. Growth Hormone Antagonists – A Pharmacological Tool in Present and Future Therapies 313 John J. Kopchick, Lingua Qiu, Elahu Gosney, Chad Keller, Amanda Palmer, and Sudha Sankaran 25. Growth Hormone Replacement Therapy and Life Quality: Future Perspectives 327 Peter Sönksen Index 335

Preface

During the past decade studies have shown that growth hormone (GH) may exert profound effects on the central nervous system (CNS). The hormone thus appears to display an important role in many functions related to the CNS. GH deficiency is known to result in a reduction of several dimensions related to quality of life. However, exogenous compensation of the hormone may counteract these disabilities. For instance, GH replacement therapy is found to improve the psychological capabilities in adult GH-deficient (GHD) patients. Beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation, and working capacity, have been reported. GH treatment of GHD children has also been observed to produce significant improvement in many behavioral problems seen in these individuals. Studies also indicated that GH replacement therapy affects the cerebrospinal fluid (CSF) levels of various hormones and neurotransmitters. Further support for CNS being a target for GH emerges from observations indicating that the hormone may cross the blood–brain barrier (BBB) and from studies confirming the presence of GH receptors (GHR) in the brain. Many effects of GH are mediated through the release of insulinlike growth factor-I (IGF-I). Therefore, studies on effects induced by IGF-I on brain function have been applied in order to obtain increased understanding of the mechanism by which GH exerts effects on brain areas related to various behaviors seen to be affected during GH replacement. The aim of this book is to summarize and highlight some recent knowledge in this area. This includes clinical observations of patients with a reduced ability to produce the hormone along with successful outcomes of GH replacement therapy in GHD patients and neuroprotective effects involving the somatotrophic axis, as well as animal models to explore the mechanisms at the neurochemical level by which the hormone induces its effects.

The first two chapters deal with some fundamental concepts regarding the biosynthesis and chemical nature of GH and IGF-I, focusing on mechanisms behind the regulation of secretion of these hormones to give the reader a brief background of events taking place in the body before the hormones interact with their receptors. The following two chapters describe basic mechanisms of how GH and IGF-I interact with receptors on their target cells. This includes text on receptor structure and mechanisms involved in signal transduction to finally give the GH or IGF-I response. Subsequent to these introductory chapters, the book content addresses questions related to the CNS. Chapters 5 and 6 discuss mechanisms by which GH and IGF-I may reach their targets in the CNS. Also, methods for quantification and analysis of the two hormones in the CSF and their application in clinical studies are highlighted. Models used to study the transport of GH over the BBB are described. It seems that although there is strong evidence that GH may cross the BBB, as judged from human studies, it is difficult to demonstrate an active transport mechanism for the hormone using animal models. In human subjects, administration of GH causes a dose-dependent increase of the hormone in the CSF, whereas in models using rats or cell lines, penetration of the hormone across the BBB seems limited. As for IGF-I, it is demonstrated that this growth factor in conformity with insulin and prolactin may be transported over the BBB through a receptor-mediated mechanism. An important part of the book content is given in Chapters 7–9, dealing with GH and IGF-I receptors in the brain regarding their regional distribution along with their chemical nature. Data on purification of GH receptors from human brain tissues, as well as description of the characteristic of GHR gene transcripts, are included in these chapters.

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The outline of a procedure combining affinity chromatography and zone electrophoresis for the recovery of a purified preparation of GHR from the human choroid plexus is described in Chapter 8, and Chapter 9 deals with the characteristics of the GHR message in various regions of the rat brain. A comprehensive review on the localization of GH and IGF-I receptors is given in Chapter 7. In the next section of the book there are a series of chapters (10–15) dealing with functional aspects of the role of the somatotrophic axis in the brain. Various types of animal models and cell systems have been used to establish a comparatively deep knowledge in functions that GH and IGF-I have in CNS injury, neurogenesis, and cognitive behaviors. Also, the interaction in various brain circuits of GH with prolactin is highlighted. A major part of this book (Chapters 16–23) is directed to clinical studies. Hence, the role of the somatotrophic axis in acromegaly, life quality, psychiatric diseases, aging, and neurodegenerative diseases, e.g., Alzheimer’s disease, is discussed. Also, the beneficial effects of hormone replacement GHD in children and GH-induced improvement on cognitive functions in elderly GHD patients are highlighted.

Finally, the remaining chapters focus on future perspectives, e.g., on the potential use of GH antagonists as possible pharmacological tools in future therapy and on future perspectives with respect to GH replacement therapy and life quality. Taken together, it has been interesting to see that it has been possible to get contributors to this book with such a good representation of geographical districts around the world, but also a good representation of the research around the world on the somatotrophic axis in relation to CNS. It is believed that with the current advances in research on GH and IGF-I, this volume will represent a timely book that contributes an important topic that will be of interest to a variety of scientists from students and medical practitioners to basic and clinical researchers. I am grateful to all my colleagues who have contributed such excellent chapters to this volume. As editor, I would like to point out that all these chapters were submitted within a time period of 6–7 months and should therefore contribute the very recent knowledge in the respective field covered within the volume. Fred Nyberg Uppsala in June, 2005

Contributors

Maria A. Åberg, Goteborg University, The Arvid Carlsson Institute for Neuroscience, Institute of Clinical Neuroscience, Goteborg, Sweden

Jovanna Dahlgren, Sahlgrenska Academy at Goteborg University, Institute for the Health of Women and Children, Goteborg Pediatric Growth Center, Goteborg, Sweden

N. David Åberg, Goteborg University, Research Center of Endocrinology and Metabolism, Institute of Internal Medicine, Goteborg, Sweden

Jan B. Deijen, Vrije Universiteit, Department of Clinical Neuropsychology, Amsterdam, The Netherlands Peter S. Eriksson, Goteborg University, The Arvid Carlsson Institute for Neuroscience, Institute of Clinical Neuroscience, Goteborg, Sweden

Kerstin Albertsson-Wikland, Sahlgrenska Academy at Goteborg University, Institute for the Health of Women and Children, Goteborg Pediatric Growth Center, Goteborg, Sweden

Ulla Feldt-Rasmussen, National University Hospital, Medical Endocrinology PE 2132, Copenhagen, Denmark

Emanuela Arvat, University of Turin, Department of Internal Medicine, Torino, Italy

S. Fernandez, Instituto Cajal, CSIC, Laboratory of Neuroendocrinology, Madrid, Spain

Lucia I. Arwert, VU University Medical Center, Department of Endocrinology, Amsterdam, The Netherlands

Ana M. Fernandez, Instituto Cajal, CSIC, Laboratory of Neuroendocrinology, Madrid, Spain

Matteo Baldi, University of Turin, Department of Internal Medicine, Torino, Italy

Ezio Ghigo, University of Turin, Department of Internal Medicine, Torino, Italy

Mario Bo, University of Turin, Department of Internal Medicine, Torino, Italy

Roberta Giordano, University of Turin, Department of Internal Medicine, Torino, Italy

Lorenza Bonelli, University of Turin, Department of Internal Medicine, Torino, Italy

José Manuel Gomez, Hospital Princeps d’Espana, Endocrine Unit, Barcelona, Spain

Eva Carro, Instituto Cajal, CSIC, Neuroendocrinology, Madrid, Spain

Elahu Gosney, Ohio University, College of Osteopathic Medicine, Edison Biotechnology Institute, Department of Biomedical Sciences, Athens, OH

Laboratory

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Chi-Bun Chan, The Chinese University of Hong Kong, Department of Biochemistry, Hong Kong, China

David Grattan, University of Otago, Department of Anatomy and Structural Biology, Dunedin, New Zealand

John Eric Chaplin, Sahlgrenska Academy at Goteborg University, Institute for the Health of Women and Children, Goteborg Pediatric Growth Center, Goteborg, Sweden

Adrian C. Herington, Queensland University of Technology, School of Life Sciences Brisbane, Australia Kenji Kangawa, National Cardiovascular Center Research Institute, Department of Biochemistry, Suita Osaka, Japan

Christopher H. K. Cheng, The Chinese University of Hong Kong, Department of Biochemistry, Hong Kong, China

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Abba J. Kastin, Pennington Biomedical Research Center, LSU Systems, Baton Rouge, LA

Ase Krogh Rasmussen, National University Hospital, Medical Endocrinology PE 2132, Copenhagen, Denmark

Chad Keller, Ohio University, College of Osteopathic Medicine, Edison Biotechnology Institute, Department of Biomedical Sciences, Athens, OH

Steven A. Rosenzweig, Medical University of South Carolina, Department of Cell and Molecular Pharmacology, Hollings Cancer Center, Charleston, SC

Masayasu Kojima, Kurume University, Institute of Life Science, Molecular Genetics, Kurume Fukuoka, Japan

Vincenzo Russo, Royal Children’s Hospital, Murdoch Childrens Research Institute, Centre for Hormone Research, Parkville, Victoria, Australia

John J. Kopchick, Ohio University, College of Osteopathic Medicine, Edison Biotechnology Institute, Department of Biomedical Sciences, Athens, OH Berit Kristrom, Sahlgrenska Academy at Goteborg University, Institute for the Health of Women and Children, Goteborg Pediatric Growth Center, Goteborg, Sweden Zhennan Lai, National Institutes of Health, National Institute of Neurological Disorders and Stroke, Developmental and Metabolic Neurology Branch, Bethesda, MD

Sudha Sankaran, Ohio University, College of Osteopathic Medicine, Edison Biotechnology Institute, Department of Biomedical Sciences, Athens, OH Harald Schneider, Max Planck Institute of Psychiatry, Internal Medicine, Endocrinology and Clinical Chemistry, Munich, Germany Farhad Shafiei, The Liggins Institute and National Research Centre for Growth and Development, The University of Auckland, Auckland, New Zealand

Lusia Lanfranco, University of Turin, Department of Internal Medicine, Torino, Italy

Stephen M. Shalet, Christie Hospital, Manchester, UK

Madeleine Le Greves, Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden

Isao Shimokawa, Nagasaki University, School of Biomedical Sciences, Department of Pathology & Gerontology, Nagasaki City, Japan

Peter Lobie, The Liggins Institute and National Research Centre for Growth and Development, The University of Auckland, Auckland, New Zealand Tanja A. E. Möderscheim, University of Aukland, Faculty of Medicine & Health Sciences, Liggins Institute and National Research Centre for Growth and Development, Auckland, New Zealand Robert Murray, Christie Hospital NHS Trust, Medicine & Endocrinology, Manchester, UK Fred Nyberg, Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden Amanda Palmer, Ohio University, College of Osteopathic Medicine, Edison Biotechnology Institute, Department of Biomedical Sciences, Athens, OH Weihong Pan, Pennington Biomedical Research Center, LSU Systems, Baton Rouge, LA Micaela Pellegrino, University of Turin, Department of Internal Medicine, Torino, Italy Andreea Picu, University of Turin, Department of Internal Medicine, Torino, Italy Lingua Qiu, Ohio University, College of Osteopathic Medicine, Edison Biotechnology Institute, Department of Biomedical Sciences, Athens OH Melinda Ramsey, Wake Forest University, School of Medicine, Department of Physiology and Pharmacology, Winston-Salem, NC

Mark G. Slomiany, Medical University of South Carolina, Department of Cell and Molecular Pharmacology, Hollings Cancer Center, Charleston, SC Peter Sonksen, King’s College London, School of Medicine, St. Thomas’ Campus, Diabetes, Endocrinology & Internal Medicine, Strand, London, UK William E. Sonntag, Wake Forest University, School of Medicine, Department of Physiology and Pharmacology, Winston-Salem, NC Gunter Karl Stalla, Max Planck Institute of Psychiatry, Department of Endocrinology, Munich, Germany Ignacio Torres-Aleman, Instituto Cajal, CSIC, Laboratory of Neuroendocrinology, Madrid, Spain José Luis Trejo, Instituto Cajal, CSIC, Laboratory of Neuroendocrinology, Madrid, Spain Margaret C. L. Tse, The Chinese University of Hong Kong, Department of Biochemistry, Hong Kong, China Sigbritt Werner, Huddinge Hospital, Department of Medicine, Huddinge, Sweden George Werther, Royal Children’s Hospital, Centre for Hormone Research, Endocrinology and Diabetes, Parkville, Victoria, Australia Yongmei Yu, Pennington Biomedical Research Center, LSU Systems, Baton Rouge, LA

Pioneers in the growth hormone field

Professor in Anatomy in 1915 at Berkley, where he remained until his retirement in 1952. During this time he spent a lot of time visiting a large number of laboratories in the United States and also abroad. Dr. Evans remained active in his research until he passed away in 1971. In addition to his pioneering work on growth hormone together with Joseph Long he was recognized for his brilliant work on the estrous cycle in the rat. In 1930 he created the Institute of Experimental Biology and several capable researchers joined his laboratory; Drs W.R. Lyons and Choh Hao Li with whom Dr. Evans carried out considerable work on pituitary hormones, e.g. lactogenic hormones, gonadotrophins as well as adrenocorticotropic hormone. He has contributed more than 600 scientific papers. One excellent example is his last written article a review chapter on growth published in the Pituitary Gland in 1966. Dr. Evans presented a personality characterized by independence of thought, subtle powers of persuasion and an unruffled demeanor, which made him appear as a figure of mystery to his colleague. He was essentially a forceful character with an underlying tenacity of purpose with a splendid intellect, which carried him forward in the pursuit of truth as he saw it. His memory will remain for many years to come.

Herbert McLean Evans is one of the most recognized endocrinologist in the field of growth hormone. In the early 1920-ies he demonstrated the presence of growth promoting material in adeno-pituitary extracts. In 1944 he published for the first time the isolation of growth hormone from bovine pituitaries. In addition to his pioneering work on growth hormone, Dr. Evans has contributed fundamental discoveries within a broad area of physiology but particularly in the field of endocrinology. His remarkable career as scientist is reflected by his development of the dye for cytological staining known as “Evans blue” and by the widely used “Long-Evans” rat strain. Herbert Evans was born in 1882 in Modesto, California. He received his B.S. from the University of California at Berkley in 1904 but moved to Baltimore in 1905 to continue his medical studies. Evans obtained his M. D. at John Hopkins University in 1908 and received a chair as

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Paul Roos was born in 1928 in Alingsås not to far from Gothenburg in western Sweden. He obtained his B.S. at the University in Lund in 1952 and in 1954 he went to Uppsala to perform his PhD studies in the laboratory of Professor Arne Tiselius (a Nobel Prize Laureate for his development of electrophoresis.) In 1958 he received a Licentiate in Biochemistry and a doctoral degree in the same subject in 1967, all at the University in Uppsala. Paul Roos was promoted to Professor in Biochemistry at Uppsala University in 1972 and remained on this chair until he retired in 1994. When Paul Roos arrived in Uppsala he focused his research on pituitary hormones. Together with Professor Jerker Porath, at the same Department, he directed studies on the melanocyte-stimulating hormone. Fit Cambridge University, in collaboration with Dr. Ievan Harris, he determined the primary sequence of this hormone. However, his major contribution to the field of endocrinology came from his outstanding work on the isolation and characterization of growth hormone and gonadotrophins. Paul Roos was the first to outline a procedure for the recovery and isolation of

growth hormone from human pituitaries under mild conditions. His highly purified preparation was subsequently used in a number of clinical studies. The procedure of Dr. Roos was used by AB KABI (Stockholm, Sweden) to prepare growth hormone (trade name: Crescormon) from human pituitaries for clinical use. A growing international interest for treatment of pituitary dwarfism prompted the KABI’s research director to get this growth hormone preparation registered as a new pharmaceutical drug and the Roos preparation of growth hormone was registered under the trade name Crescormon in 1971. Unlike most other preparations of growth hormone available for clinical use at that time Crescormon prepared according to the Roos procedure did not give rise to any antibodies against the hormone. Professor Roos also became recognized for his development of excellent methods for the purification of human pituitary FSH, TSH, LH and prolactin. His FSH preparation was also successfully used in clinical therapy in well-recognized studies he did in collaboration with the gynaecologist Professor Carl Gemzell. All the work carried out by Paul Roos was done with high accuracy and skillfulness. All hormone products leaving his laboratory for clinical purpose were proven to be of a high quality and could successfully be used in various clinics in Sweden and abroad.

Introduction FRED J. NYBERG

Growth hormone (GH) has received a distinct profile among all other hormones produced in the pituitary gland. It represents one of the most extensively characterized proteins within the area of biomedical science. In the biochemical perspective GH consists of a single amino acid chain of around 190 amino acids stabilized by two sulfide bridges. It has a molecular weight around 22,000 daltons and exhibit a comparatively high metabolic stability. Its growth-promoting effects have been attractive not only for basic hormonal research but also in clinical therapy. Although most the biological actions attributed to GH relates to its effects on peripheral organs and tissues, the hormone may induce profound effects on functions linked to the central nervous system (CNS). This has become evident from recent studies directed to investigation of GH-induced effects on psychological functions that have seen to be affected and improved during GH replacement therapy. In 1982 I was involved in studies indicating that rat GH given at physiological concentration may affect the catecholamine turnover in the rat hypothalamus (Andersson et al., 1983). It was suggested that rGH inhibits its own secretion partly via reduction of DA synthesis and release in the median eminence leading to increased somatostatin release and partly via reduced noradrenaline synthesis and turnover in the median eminence leading to reduced secretion of a GH releasing factor. Similar observations were made for prolactin (Andersson et al., 1981). At this time studies suggesting that both lactogenic and somatogenic binding sites are present in the brain appeared in the literature (DiCarlo et al., 1985; Posner et al., 1983). These investigations revealed that GH

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

binding sites were present in brain areas such as hypothalamus, pituitary and the choroid plexus. Subsequent research carried out in my laboratory identified specific binding sites for the hormone not only in these brain regions but also in brain regions not directly connected to the hypothalamicpituitary axis. In the late 1980-ies and early 1990-ies clinical researchers observed decreased ratings on psychological well-being in studies of GH deficient patients and that GH replacement therapy reduced this symptom (Bengtsson et al., 1993; McGauley et al., 1990). Of special interest was the observation that GH may improve learning and memory capabilities. However, the mechanisms underlying these effects were not known. We identified and characterized specific binding sites for the hormone in various areas of the human brain (Lai et al., 1991 and 1993) and found that the density of these sites were sex dependent and declined with aging. We suggested that these sites are involved in the mediation of the GH effects on the brain (Nyberg, 1997, 2000). Also, we cloned the GH receptor in various tissues of the rat brain, including choroids plexus, hippocampus, hypothalamus and the spinal cord (see Thörnwall-Le Grevés, 2001 and chapter 9), but also in the human choroid plexus (se Nyberg et al., 2000). These studies indicated that the nucleotide sequence of these receptor gene transcripts were almost identical with the liver variant of the GH receptor. Studies on the distribution of GHR protein and message in the brain revealed the presence of the GH receptors in many tissues related to the functional anatomy of various behaviors known to be associated with the hormone

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(Burton et al., 1992; Kastrup et al., 2005). Functional studies in animals regarding the mechanisms underlying these behaviors have also been carried out. Many of these studies are reviewed within this volume (see chapter 13–15). We have focused on studies on the effects of the GH/IGF-1 axis on the glutamate system including the N-methyl-D-aspartyl (NMDA) receptor and its subunits (Le Grevés et al., 2002, 2005) in the rat hippocampus. We observed GH/IGF-1 induced alterations in these systems compatible with the improvements on memory and learning processes as indicated from animal studies. All these observations seen in animals are in agreement with the beneficial effect on memory and wellbeing seen during growth hormone replacement therapy (McGauley et al., 1990; Bengtsson et al., 1993; Beshyah et al., 1995; Deijen et al., 1998). The increased wellbeing seen in these patients may include the endogenous opioids, such as ␤-endorphin (Johansson et al., 1995). Evidence for a neuroprotective effect of GH and IGF-1 is also documented in the literature and this may have an impact in future treatment of brain or spinal cord trauma. A review on this topic with regard to IGF-1 is presented in chapter 15 in this book. In some preliminary studies it was previously shown that topical application of GH as well as IGF-1 may reduce the outcome of spinal cord trauma in the rat (Winkler et al., 2000; Nyberg and Sharma, 2002). Although it has been debated over a long period of time whether GH may cross the BBB or not this question has not yet been finally settled. The presence of GH in the CSF is indicative of the ability of the hormone to penetrate this barrier. All beneficial effects seen for GH during replacement therapy also suggest that the hormone may find its way from the circulatory system into the CNS. Some recent data suggest that GH may significantly diffuse into the CNS (Pan et al., 2005). In the following chapters the contribution by many investigators from various laboratories directed to research on the GH/IGF-1 axis is presented. These chapters include basic science about the biosynthesis and release of the two hormones as well as mechanisms for their actions at the receptor level. Aspects on their distribution and characteristics in the CNS are followed by reviews on their functions as assessed in cell systems and various experimental animal models. Chapters in the remaining part of this book (16–25) will highlight past and present clinical research on the somatotrophic axis in physiology and pathophysiology. These include the involvement of GH and IGF-1 in cognition, neuroprotection, degenerative and psychiatric disorders as well as acromegaly. All these contributions indicate that the functional role of the somatotrophic axis in the CNS as an important and expanding area of research both in basic and clinical sciences.

References Andersson K., Fuxe K., Eneroth P., Isaksson O., Nyberg F., Roos P. (1983). Rat growth hormone and hypothalamic catecholamine nerve terminal systems. Evidence for rapid and discrete reductions in dopamine and noradrenaline levels and turnover in the median eminence of the hypophysectomized male rat. Eur J Pharmacol. 95, 271–275. Andersson K., Fuxe K., Eneroth P., Nyberg F., Roos P. (1981). Rat prolactin and hypothalamic catecholamine nerve terminal systems. Evidence for rapid and discrete increases in dopamine and noradrenaline turnover in the hypophysectomized male rat. Eur J Pharmacol. 76, 261–265. Bengtsson B. Å., Edén S., Lönn L., Kvist H., Stokland A., Lindstedt G., Bosaeus I., Tölli J., Sjöström L., Isaksson O. G. P. (1993). Treatment of adults with growth hormone (GH) deficiency with recombinant human GH. J Clin Endocrinol Metab. 76, 309–317. Beshyah S. A., Freemantle C., Shahi M., Anyaoku V., Merson S., Lynch S., Skinner E., Sharp P., Foale R., Johnston D. G. (1995). Replacement treatment with biosynthetic human growth hormone in growth hormonedeficient hypopituitary adults. Clin Endocrinol (Oxf). 42, 73–84. Burton K. A., Kabigting E. B., Clifton D. K., Steiner R. (1992). Growth hormone receptor messenger ribonucleic acid distribution in the adult mal rat and its co-localization in hypothalamic somatostatin neurons. Endocrinology 130, 958–963. Castro J. R., Costoya J. A., Gallego R., Prieto A., Arce V. M., Senaris R. (2000). Expression of growth hormone receptor in the human brain. Neurosci Lett. 281, 147–150. Coculescu M. (1999). Blood-brain barrier for human growth hormone and insulin-like growth factor-1. J Pediatr Endocrinol Metab. 12, 113–124. Deijen J. B., de Boer H., van der Veen E. A. (1998). Cognitive changes during growth hormone replacement in adult men. Psychoneuroendocrinology 23, 45–55. Di Carlo R., Muccioli G., Lando D., Bellussi G. (1985). Further evidence for the presence of specific binding sites for prolactin in the rabbit brain. Preferential distribution in the hypothalamus and substantia nigra. Life Sci. 36, 375–382. Di Carlo R., Muccioli G., Papotti M., Bussolati G. (1992). Characterization of prolactin receptor in human brain and choroid plexus. Brain Res. 570, 341–346. Johansson J. O., Larsson G., Elmgren A., Hynsjö L., Lindahl A., Lundberg P. A., Isaksson O., Lindstedt S., Bengtsson B. Å. (1995). Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. Neuroendocrinology 61, 57–66. Kastrup Y., Le Greves M., Nyberg F., Blomqvist, A. (2005). Distribution of growth hormone receptor mRNA in the brain stem, and spinal cord of the rat. Neurosci. 130, 419–425. Lai Z. N., Emtner M., Roos P., Nyberg F. (1991). Characterization of putative growth hormone receptors in human choroid plexus. Brain Res. 546, 222–226. Lai Z., Roos P., Zhai O., Olsson Y., Fholenhag K., Larsson C., Nyberg F. (1993). Age-related reduction of human growth hormone-binding sites in the human brain. Brain Res. 621, 260–266. Le Greves M., Steensland P., Le Greves P., Nyberg F. (2002). Growth hormone induces age-dependent alteration in the expression of hippocampal growth hormone receptor and N-methyl-D-aspartate receptor subunits gene transcripts in male rats. Proc Natl Acad Sci USA. 99, 7119–7123. Le Greves M., Le Grevés P. and Nyberg F. (2005). Age-related effects of IGF-1 on the NMDA-, GH- and IGF-1-receptor mRNA transcripts in the rat hippocampus. Brain Res Bull. 65, 369–374. McGauley G. A., Cuneo R. C., Salomon F., Sonksen P. H. (1990). Psychological well-being before and after growth hormone treatment in adults with growth hormone deficiency. Horm Res. 33 Suppl 4, 52–54.

Introduction Muccioli G., Ghe C., Di Carlo R. (1991). Distribution and characterization of prolactin binding sites in the male and female rat brain: effects of hypophysectomy and ovariectomy. Neuroendocrinology 53, 47–53. Nyberg F. (1997). Aging effects on growth hormone receptor binding in the brain. Exp Gerontol. 32, 521–528. Nyberg F. (2000). Growth hormone in the brain: Characteristics of specific brain targets for the hormone and their functional significance. Front Neuroendocrinol 21, 330–348. Nyberg F., Sharma H. S. (2002). Repeated topical application of growth hormone attenuates blood-spinal cord barrier permeability and edema formation following spinal cord injury: an experimental study in the rat using Evans blue, ([125])I-sodium and lanthanum tracers. Amino Acids 23, 231–239. Pan W., Yu Y., Cain C. M., Nyberg F., Couraud P. O., Kastin A. J. (2005). Permeation of growth hormone across the blood-brain barrier. Endocrinology Aug 11; [Epub ahead of print].

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Posner B. I., van Houten M., Patel B., Walsh R. J. (1983). Characterization of lactogen binding sites in choroid plexus. Exp Brain Res. 49, 300–306. Rosen T., Wiren L., Wilhelmsen L., Wiklund I., Bengtsson B. A. (1994). Decreased psychological well-being in adult patients with growth hormone deficiency. Clin Endocrinol (Oxf). 40, 111–116. Thörnwall-LeGrevés M., Zhou Q., Lagerholm S., Huang W., Le Grevés P., Nyberg F. (2001). Morphine decreases the levels of the gene transcripts of growth hormone receptor and growth hormone binding protein in the male rat hippocampus and the spinal cord. Neurosci Lett. 304, 69–72. Winkler T., Sharma H. S., Stalberg E., Badgaiyan R. D., Westman J., Nyberg F. (2000). Growth hormone attenuates alterations in spinal cord evoked potentials and cell injury following trauma to the rat spinal cord. An experimental study using topical application of rat growth hormone. Amino Acids 19, 363–371.

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1 Regulation and Mechanism of Growth Hormone and Insulin-like Growth Factor-I Biosynthesis and Secretion C. B. CHAN, MARGARET C. L. TSE, and CHRISTOPHER H. K. CHENG Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

I. II. III. IV.

Introduction Growth Hormone Insulin-like Growth Factor-I Future Perspectives References

mass, size, and complexity of the organism. Numerous factors are involved in orchestrating this complex process in which growth hormone (GH) and insulin-like growth factor-I (IGF-I) are among the key elements mediating this vital event. The GH is a polypeptide hormone predominantly synthesized and secreted from the anterior pituitary. Since its discovery in the 1940s (Li et al., 1945), numerous investigations have been performed to study its biological functions. It is now recognized that GH is involved in multiple physiological events that regulate growth, body composition, energy metabolism, bone metabolism, cardiac functions, and immune functions, among others. It has also been demonstrated that GH not only exerts its actions on peripheral tissues, but also on the central nervous system (CNS), where it modulates appetite, cognitive functions, energy metabolism, memory, mood, neuroprotection, and sleep (reviewed in Nyberg, 2000). Early studies on the mechanisms of the growth process have revealed that GH probably does not stimulate somatic growth directly but acts through mediators called somatomedins, which are now known as insulin-like growth factors (IGFs). The presence of these mediators was first proposed in 1957 by Salmon and Daughaday, and the subsequent isolation of human IGF-I revealed that it shares significant structural homology with insulin (Rinderknecht and Humbel, 1978). IGF-I also possesses multiple functions, including the stimulation of myogenesis, inhibition of apoptosis, mediation of cell cycle progression, and modulation of immune response and sex steroid production (reviewed in Le Roith et al., 2001).

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are important regulatory factors of animal growth. Both of them are involved in multiple physiological functions ranging from controlling cell cycle progression to changing the overall metabolic status of the organism. Because of their vital roles, their biosynthesis, as well as secretion, is tightly regulated. It is now known that the expression and release of GH and IGF-I from their production sites are results of an orchestration among various factors, including hormones, metabolic status, and external stimuli. This chapter briefly reviews our current understanding of how GH and IGF-I biosynthesis and secretion are regulated. The discussion is mainly focused on GH biosynthesis and secretion in the pituitary and on IGF-I biosynthesis in the brain. The important factors involved in these processes are described as well as the mechanisms where known.

I. INTRODUCTION Growth is an important process that occurs naturally in every organism. It is a universal event resulting from a series of physiological changes in the body, including increases in

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

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Several models have been proposed to explain the participation of GH and IGF-I in growth. The “somatomedin hypothesis” was the first attempt in describing their role in stimulating somatic growth (Daughaday et al., 1972). This hypothesis suggests that GH acts mainly on the liver where it stimulates the synthesis of IGF-I, which in turn circulates to the target tissues to exert its growth-stimulating effects. This model, however, could not explain some of the findings reported in the 1980s. For example, it was found that IGF-I is not only expressed in the liver, but also in some peripheral tissues where its expression was GH independent (D’Ercole et al., 1980). Isaksson et al. (1982) reported that GH has a direct action in stimulating longitudinal bone growth, but later studies suggested that this GH-stimulated bone growth is mediated by IGF-I produced locally in the cartilage (Schlechter et al., 1986). These results led to the “dual effector hypothesis” to account for the role of GH and IGF-I on growth and differentiation (Green et al., 1985). This hypothesis suggests that, apart from having a direct action in stimulating differentiation on target cells, GH also stimulates local IGF-I production where the growth factor serves in an autocrine or paracrine fashion to enhance clonal expansion of the differentiated cells (reviewed in Ohlsson et al., 1998). Although this hypothesis is attractive, studies have questioned its completeness. In Igf-I null mice, the proliferation of the growth plate chondrocytes is normal, suggesting that IGF-I is not required for their clonal expansion (Wang et al., 1999). However, IGF-I alone is able to induce myoblast differentiation (Florini et al., 1986; Johnson and Allen, 1990). These results suggested that the relationship between GH and IGF-I in mediating growth is far more complex than our current understanding and a comprehensive model in describing the cooperative mechanism of GH and IGF-I awaits further work. Apart from stimulating growth, GH and IGF-I participate in numerous physiological events of the body, as reflected in their diverse sites of production. Studies indicated that GH is expressed in many different tissues, although it is mainly synthesized and released from the pituitary. IGF-I is expressed ubiquitously in virtually all tissues, albeit at different levels. This chapter briefly reviews the current knowledge of how GH and IGF-I biosynthesis and secretion are regulated. Discussion is mainly focused on GH expression and secretion in the pituitary and IGF-I expression in the brain.

II. GROWTH HORMONE A. Basic Mechanisms Regulating Growth Hormone Biosynthesis and Secretion 1. Transcription Factors and DNA Methylation The GH gene is mainly transcribed in the pituitary. The pituitary expression of GH is controlled by the

coordination of a number of transcription factors in which Pit-1 is probably the major trans-acting factor that accounts for the unique pituitary-specific expression of GH (Bodner and Karin, 1987; Elsholtz et al., 1990). The GH proximal promoter in both human and rat contains two Pit-1-binding sites (Catanzaro et al., 1987; Cattini et al., 1986b; Nelson et al., 1988). The expression of Pit-1 significantly enhances the activity of the GH promoter even in cells in which the GH promoter activity is extremely low (Mangalam et al., 1989). The essentiality of these Pit-1-binding sites has also been demonstrated in a transgenic mice model (Lira et al., 1993). Furthermore, studies indicate that Pit-1 binding to DNase I hypersensitive sites at 14.5 kb 5⬘ to the human GH promoter contributes to the locus control region activity in vivo (Shewchuk et al., 1999, 2002). It has been proposed that binding of Pit-1 to its response element and multimerization of the protein result in bending of the DNA molecule, which facilitates formation of the transcriptional complex (reviewed in Theill and Karin, 1993). Subsequent studies showed that Pit-1 is required during preinitiation complex assembly in gene transcription (Sharp, 1995). However, the mere presence of Pit-1-binding sites is not sufficient for efficient GH transcription, as a truncated rat GH promoter containing Pit-1-binding sites alone results in low expression of GH in transgenic mice (Lira et al., 1993). In addition, the expression of Pit-1 has been demonstrated in some extrapituitary tissues. For example, Pit-1 expression has been reported in the placenta where it is believed to regulate the expression of other members of the GH gene family (Bamberger et al., 1995). Some transcription factors are involved to cooperate with Pit-1 to mediate effective GH transcription in the pituitary. One of the factors that cooperate with Pit-1 in GH transcription is the Ets-related protein Sp1. The binding sites for Sp1 on the rat GH promoter partially overlap with the Pit-1 response elements. Although Sp1 and Pit-1 binding to the cis element is mutually exclusive, Sp1 in fact assists the loading of Pit-1 to its own binding site (Lemaigre et al., 1990; Schaufele et al., 1990). A novel member of the Cys/His zinc finger superfamily Zn-15 also cooperates with Pit-1 during GH transcription. It synergizes with Pit-1 to activate the GH promoter in cell lines in which the GH promoter minimally responds to Pit-1 alone (Lipkin et al., 1993). Other transcription factors are also involved in regulating GH expression. The cAMP response element-binding protein (CREB) has been demonstrated to play a role in the increased expression of GH (and Pit-1 as well) in response to activation of the cAMP pathway in normal and tumoral somatotrophs (Bertherat, 1997; Gaiddon et al., 1995). The activator protein AP2 can bind to two regions on the human GH promoter (Imagawa et al., 1987). However, the actual mechanism of AP2 in mediating GH transcription is not known. Similarly, an upstream stimulatory factor (USF) is able to bind to the human GH promoter at positions ⫺267 to ⫺257 but with an

1. Regulation and Mechanism of GH and IGF-I

unknown role in inducing GH transcription (Lemaigre et al., 1989). A negative regulatory element (silencer 1) is found in positions ⫺325 to ⫺280 of the rat GH promoter (Larsen et al., 1986; Pan et al., 1990). This silencer represses GH transcription by the binding of a 45-kDa protein identified in extracts isolated from cells that do not express GH (Roy et al., 1992). Again, the detailed mechanism of how this factor regulates GH transcription awaits further studies. GH transcription is also regulated by DNA methylation. It is generally known that genomic DNA methylation is associated with gene inactivation (Turker, 2002). The methyl group protrudes from the cytosine of the CpG island into the major groove of the DNA, displacing the transcription factors that bind to it (Hark et al., 2000; Kim et al., 2003). It also attracts methyl-binding proteins, thus resulting in gene silencing and chromatin compaction (Newell-Price et al., 2000). DNA hypomethylation is associated with a high level of GH production. For example, the extent of DNA methylation is less in rat GC cells that produce a large amount of GH than its variant strain GH3CDL in which the production of GH is small (Laverriere et al., 1986). Moreover, the level of GH gene methylation in human somatotrophinomas is lower than in the normal human pituitary (Huttner et al., 1994). An altered methylation pattern of specific cytosine residues in the coding region of the rat GH gene was found to be concurrent with the altered expression of GH in the pituitary during pregnancy and lactation (Kumar and Biswas, 1988). Further studies indicated that methylation of the Tha I site located 144 bp upstream of the rat GH transcription initiation site was essential for GH transcription, as DNA prepared from tissues that produce no GH was not methylated at this site (Strobl et al., 1986). However, no simple correlation could be established between the high level of rat GH promoter activity and the methylation of positive and/or negative cis-acting elements on the rat GH promoter, although an overall hypomethylation-high expression relationship was observed (Ngo et al., 1996). 2. Signaling Mechanisms of GH Secretion in the Pituitary GH secretion from somatotrophs is triggered by an elevation in the intracellular Ca2⫹ concentration (Kraicer and Spence, 1981). This cytosolic Ca2⫹ concentration elevation is brought about mainly by two signal transduction cascades: the adenylyl cyclase (AC)/protein kinase A (PKA) pathway and the phospholipase C (PLC)/protein kinase C (PKC) pathway. Agents such as growth hormone-releasing hormone (GHRH), pituitary adenylate cyclase-activating polypeptide (PACAP), and interleukin-1 (IL-1) stimulate GH secretion from the pituitary via activation of AC. In rat somatotrophs, GHRH treatment increases cAMP synthesis and cytosolic PKA activity, which in turn leads to GH release (Horvath et al., 1995; Wong et al., 1995). In rat pituitary cells, it

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has also been demonstrated that tyrosine kinase inhibitors enhance the GHRH-induced cAMP accumulation and GH secretion by inhibiting the phosphodiesterase activity (Ogiwara et al., 1997). This AC–PKA pathway activation subsequently activates both L- and T-type voltage-gated Ca2⫹ channels, resulting in an influx of Ca2⫹ ions into the somatotrophs (Chen et al., 2000). In sheep pituitary cells, the IL-1␤ stimulated GH release and gene expression depends on PKA activity, but is PKC independent (Fry et al., 1998). In porcine somatotrophs, activation of the AC–PKA pathway and extracellular Ca2⫹ entry through the L-type Ca2⫹ channel are the prevailing and requisite signals for the transduction of the stimulatory effect of both PACAP27 and PACAP38 on GH release and transcription (Hart et al., 1992; Martinez-Fuentes et al., 1998). In fact, a defect in this signal transduction pathway may underline the mechanism of growth retardation observed in dwarf rats (Brain et al., 1991; Downs and Frohman, 1991). It has also been reported that PACAP38 and GHRH evoke dual-signal transduction pathways in somatotrophs. They activate the PLC–PKC cascade in parallel with the AC–PKA pathway to mediate GH secretion in porcine pituitary (Martinez-Fuentes et al., 1998; Wu et al., 1997). However, agents such as ghrelin and orexin depend mainly on activation of the PLC–PKC pathway to stimulate GH secretion (Carreira et al., 2004; Xu et al., 2003). By activating PLC and increasing the production of inositol-1,4,5-triphosphate (IP3), they trigger an increase in intracellular Ca2⫹ concentration in two phases: a Ca2⫹ release from the endoplasmic reticulum and a Ca2⫹ influx via the L-type Ca2⫹ channel. Apart from the aforementioned signal transduction pathways, other secondary messenger systems might also be involved in mediating GH secretion. The C-type natriuretic peptide increases the intracellular cGMP level to stimulate GH secretion from rat GC cells (Shimekake et al., 1994). This GH secretion is a result of activation of the cGMPdependent protein kinase. It has also been reported that GHRH stimulates cellular guanylyl cyclase activity and increases cGMP concentration in rat somatotrophs (Kostic et al., 2001). In rat GC cells, the thyrotropin-releasing hormone (TRH) stimulates GH secretion but attenuates GH mRNA expression (Kanasaki et al., 2002). However, a contradictory result was reported that mitogen-activated protein kinase (MAPK) is involved in TRH-stimulated GH expression but not secretion, suggesting that TRH exerts dual mechanisms in mediating GH secretion and transcription.

B. Important Factors Affecting Growth Hormone Biosynthesis and Secretion in the Pituitary The major site of expression of GH is in the pituitary. However, extrapituitary tissues are also known to express GH, including other parts of the brain, as well as the peripheral nervous system (Harvey and Hull, 2003; Render et al.,

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1995), lymphoid tissues (Lantinga van Leeuwen et al., 2000; Recher et al., 2001; Wu et al., 1996), cells of the immune system (Binder et al., 1994; Kooijman et al., 1997; Rohn and Weigent, 1995; Weigent and Blalock, 1991; Wu et al., 1999), oocytes and granulosa cells (Izadyar et al., 1999), dermal fibroblasts (Palmetshofer et al., 1995), pancreatic insulinoma (Robben et al., 2002), and mammary gland tumors (Mol et al., 1995). GH produced in these extrapituitary sites probably plays a paracrine/autocrine role, while the endocrine role of circulating GH is predominantly contributed by the pituitary. However, most of the information we have to date regarding the regulation of GH biosynthesis and secretion concerns its pituitary site of production. Relatively little is known about the regulation of expression in the extrapituitary tissues. The following is an account of the important factors known to affect GH biosynthesis and secretion in the pituitary. Whether these factors play equally important roles in GH biosynthesis in the extrapituitary tissues clearly needs more work. 1. Growth Hormone-Releasing Hormone and Somatostatin GH secretion from the pituitary is pulsatile in nature. It has been proposed that this pattern of secretion is controlled by the interaction of two hypothalamic hormones in which GHRH is stimulatory whereas somatostatin (SST) is inhibitory. GHRH was first isolated from human pancreatic islet tumors (Rivier et al., 1982). Subsequent studies indicated that this polypeptide is also synthesized in the arcuate nucleus and the ventromedial nucleus of the hypothalamus (reviewed in Muller et al., 1999). It belongs to a family of brain gut peptides that include glucagon, glucagonlike peptide I, vasoactive intestinal polypeptide, secretin, gastric inhibitory peptide, and PACAP (Campbell et al., 1991). GHRH is very active in stimulating GH secretion from the pituitary. In human, GH secretion is detected within 5 min following GHRH injection (Thorner et al., 1983). In rat, GHRH has been demonstrated to be a potent GH secretion stimulator both in vitro (Badger et al., 1984) and in vivo (Wehrenberg et al., 1984b). The pivotal role of GHRH in triggering GH release is indicated by the inhibition of basal and pulsatile GH release in GHRH-immunized animals. When administered with an anti-GHRH serum, neonatal rats show drastic growth retardation (Wehrenberg et al., 1984a). This anti-GHRH serum also completely abolishes the GH release (Wehrenberg et al., 1982). Upon cessation of this GHRH immunization, the growth rate of the immunized animals returns to normal, despite the fact that the overall body mass of these immunized rats is smaller (Cella et al., 1990b). As mentioned in Section II,A,2, cAMP is the major second messenger in GHRH-stimulated GH secretion from the pituitary. GHRH activates the AC–PKA pathway, which

subsequently depolarizes the somatotrophs by opening the L-type Ca2⫹ channel. In addition to this effect on GH secretion, an increase in GH mRNA has also been demonstrated in dispersed pituitary cells as well as in cell lines (Chomczynski et al., 1988a; Gick et al., 1984). The mechanism in this GHRH-stimulated GH transcription is not entirely clear, but it has been proposed that transcription factor CREB is involved (Bertherat, 1997; Gaiddon et al., 1995). Furthermore, GHRH also increases Pit-1 transcription, which in turn elevates GH transcription indirectly (Yan et al., 2004). SST is another brain gut peptide secreted from the hypothalamus (Brazeau et al., 1973; Praydayrol et al., 1980). SST has no effect on basal GH secretion and gene transcription in both rat and bovine pituitary cells in vitro (Barinaga et al., 1985; Fukata et al., 1985; Tanner et al., 1990). Although it decreases intracellular cAMP levels, it does not seem to influence GHRH-stimulated GH transcription (Fukata et al., 1985). However, it blocks the stimulatory effects of GHRH and ghrelin on GH secretion (Bilezikjian and Vale, 1983; Law et al., 1984; Yamazaki et al., 2002). Upon binding to a specific receptor subtype in the brain, SST activates a pertussis toxin-sensitive G-protein (G␣i1⫺3), which inhibits AC activity. Indeed, SST-induced reduction in GH secretion is a result of a decreased intracellular Ca2⫹ ion concentration. This is achieved by either opening the K⫹ channels (e.g., inward-rectifier K⫹ channel), which hyperpolarize the cell and leads to the closing of the L-type Ca2⫹ channel, or by closing the L-type Ca2⫹ channel directly via the Gi protein (reviewed in Lahlou et al., 2004). Contradictory to its traditional role in inhibiting GH secretion, it was found that SST can stimulate GH release from a subpopulation of porcine somatotrophs through a cAMP-dependent pathway (Ramirez et al., 2002). This novel action of SST challenges the classic view of SST as a mere inhibitor and it has been suggested that it exerts a complex function in the control of porcine GH release. Further studies are needed to investigate if this is only a species-specific observation in this case or a more universal phenomenon. The interaction of GHRH and SST appears to be involved in controlling the pulsatile nature of GH secretion. It has been proposed that GHRH and SST are secreted tonically from the hypothalamus in which surges of each peptide are observed rhythmically. The GHRH surges induce the episodic release of GH and the rise in SST suppresses the GH release (Tannenbaum and Ling, 1984). Measurement of GHRH and SST in the rat portal blood sample supports this hypothesis (Plotsky and Vale, 1985). In sheep, however, this model cannot fully explain the episodic GH rhythm. It has been reported in sheep that SST pulses in the portal blood do not correlate with the GH troughs and are not asynchronous with the GHRH surges as well (Frohman et al., 1990). In human, control of the GH episodic release is poorly understood as portal

1. Regulation and Mechanism of GH and IGF-I

blood cannot be sampled directly. However, injection of an GHRH receptor antagonist greatly blocks the nocturnal GH pulsatility, supporting the notion that GH secretion in human is basically driven by GHRH (Jaffe et al., 1993). 2. Leptin and Ghrelin Obesity affects GH secretion from the pituitary. Obese subjects have a decreased responsiveness in GH secretion toward various GH secretagogues (reviewed in Hartman, 2000). The pulsatile secretion of GH is reduced in genetically obese Zucker rats (Tannenbaum et al., 1990). Moreover, the mRNA, as well as hormone contents of GH and GHRH, is significantly decreased in these obese animals (Ahmad et al., 1993). In human, a lowered 24-h integrated GH concentration is observed in obese men, but the circadian rhythm of GH secretion is still preserved (Veldhuis et al., 1991). However, this decreased GH production may not be the result of an increased SST function, as passive immunization against SST failed to increase the GH response to GHRH (Tannenbaum et al., 1990). Moreover, neither the mRNA nor the protein content of SST is reduced in the obese rat (Cocchi et al., 1993). As one of the characteristics of obesity is an increase in body fat mass, it has been postulated that adipose tissue exerts a role in the control of GH secretion. Leptin, a peptide hormone isolated from adipocytes (Zhang et al., 1994), is now recognized as the signal that communicates between fat tissues and the pituitary to control GH secretion. In fact, a circulating leptin level correlates well with the total body fat and is inversely related to the serum GH concentration (Fisker et al., 1997; Tuominen et al., 1997). This hormone, however, appears to exert a stimulatory action on GH secretion from the pituitary. Short-time incubation by leptin increases GH secretion and its mRNA level in rat and porcine pituitary (Baratta et al., 2002; Cocchi et al., 1999; Saleri et al., 2004). Moreover, leptin enhances GHRH and ghrelin expression but decreases SST secretion in rat (Carro et al., 1999; Cocchi et al., 1999; Toshinai et al., 2001). These results, together with the observation that administration of an anti-GHRH serum blocks leptin-induced GH release and injection of antiSST serum significantly increases GH release in these rats (Carro et al., 1999), suggest that leptin exerts a dual role in stimulating GH secretion: (1) by affecting GHRH, SST, and ghrelin expression and secretion and (2) by stimulating GH secretion from the pituitary directly. Long-time exposure of ovine pituitary cells to leptin, however, decreased GH transcription (Roh et al., 2001). When leptin was added to the culture medium for 3 days, the expression of ovine pituitary GHRH receptor was decreased, accounting for the diminished responsiveness of the pituitary toward GHRH (Chen et al., 2001). Because the leptin concentration is higher in obese subjects (Ostlund et al., 1996), this long-term exposure of the pituitary to leptin may be the main reason in accounting for the low GH level in these subjects.

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Although the signal transduction mechanism of leptin in other tissues has been well studied, the molecular mechanism of how leptin regulates GH expression and secretion in the pituitary is not understood. Nevertheless, it was found that nitric oxide production might be involved in this leptin-induced GH release (Baratta et al., 2002). Moreover, leptin-induced intracellular Ca2⫹ elevation, the prerequisite for GH secretion, was observed in porcine somatotrophs (Glavaski-Joksimovic et al., 2004). This Ca2⫹ elevation was found to depend on Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT), MAPK, and nitric oxide synthase activation. Ghrelin is another agent that links the body metabolic status to GH secretion. When first isolated from the rat stomach (Kojima et al., 1999), ghrelin was recognized as the endogenous ligand for the Gq-coupled growth hormone secretagogue receptor (GHSR) (Howard et al.,1996) found in the pituitary and hypothalamus to control GH secretion. Moreover, subsequent studies demonstrated that ghrelin is a peripheral signal to the hypothalamus when a positive energy balance is needed (Tschop et al., 2000). Ghrelin is a potent stimulator of GH secretion with multiple physiological actions (reviewed in Van der Lely et al., 2004). When bound to the GHSR located on the pituitary cell surface, it stimulates GH secretion directly by acting through the PLC–PKC pathway, which leads to an elevation of intracellular Ca2⫹ ion as mentioned in Section II,A,2. It has been shown that the AC–PKA pathway is also involved in this ghrelin-induced GH secretion in pig pituitary (Malagon et al., 2003). In fact, enormous species variation was noted in this stimulation of GH secretion by GH secretagogues. In rat somatotrophs, the PKC pathway is essential for growth hormone-releasing peptide (GHRP)-6 to induce GH secretion (Cheng et al., 1991), but Wu et al. (1997) proposed that the PKA cascade is the major signal transduction to cause GH release in ovine. This GHRPinduced GH secretion also depends partly on GHRH. Passive immunization with an anti-GHRH serum obliterated the GH response induced by GHRP-6 in rats (Tannenbaum and Bowers, 2001). Moreover, an intact GHRH system is required for the GH secretagogue function, as GHRP-6-induced GH secretion is completely blocked in patients with hypothalamopituitary disconnection (Popovic et al., 1995). In addition, a synergistic augmentation in GH secretion was observed when ghrelin and GHRH were coadministered to human (Hataya et al., 2001). This synergistic effect might be the result of receptor cross talk between GHSR and GHRH receptor in the pituitary, as coactivation of transfected human GHSR and GHRH receptors produced a cAMP response twice as much as that observed in the activation of the GHRH receptor alone (Cunha and Mayo, 2002). Controversial observations have been reported on the effects of ghrelin and GHRP on GH expression in

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pituitary. In rat somatotrophs, ghrelin and GHPR-6 increase the expression of GH, probably through the action of Pit-1, as its expression was also induced (Garcia et al., 2001). A similar observation was reported in ovine pituitary cells where Pit-1 and GH mRNAs are increased by GHRP-2 treatment (Yan et al., 2004). However, studies in rats indicated that hexarelin does not cause any GH mRNA increase in vivo (Torsello et al., 1997). However, GH mRNA expression was reported to be induced by GHRP-6 in GHRHdeprived infant rats (Locatelli et al., 1994) and by L-163,255 (a nonpeptide GH secretagogue) in uremic adult rats (Krieg et al., 2002). Despite the conventional understanding that ghrelin stimulates GH biosynthesis and/or secretion, the report that deleting ghrelin in transgenic mice does not impair their growth or appetite (Sun et al., 2003) is thought provocative. Another study further indicates that transgenic mice overexpressing des-acyl ghrelin exhibit a small phenotype (Ariyasu et al., 2005). A full understanding of how ghrelin regulates physiological functions through GH biosynthesis and secretion clearly awaits further elucidation. 3. Thyroid Hormones Apart from the transcription factors mentioned in Section II,A,1, GH transcription could be affected by hormones where the corresponding receptors are intranuclear proteins (or called ligand-activated transcription factors). In general, binding of these hormones to their specific receptors in the cytosol activates the transcriptional ability of these receptors, which are subsequently translocated into the nucleus, resulting in the activation of particular gene transcription (reviewed in Aranda and Pascual, 2001). Thyroid hormones [thyroxine (T4) and triiodothyronine (T3)] are ligands to one of these nuclear hormone receptors essential in maintaining normal growth. It has been reported that hypothyroidism impairs postnatal growth in both human and rat (Burstein et al., 1979; Chernausek and Turner, 1989). In rat pituitary, T4 stimulates GH transcription and mRNA accumulation (Dobner et al., 1981; Spindler et al., 1982). A similar observation was reported in a human pituitary tumor cell line (Chomczynski et al., 1993). This thyroid hormone-mediated GH transcription, however, is age dependent. Administration of T4 alone could not induce GH expression in the fetal rat pituitary gland on day 17 or 18 of gestation (Nogami et al., 1995). However, hypothyroidism decreases pituitary GH content in rat pups of 10 to 21 days old (De Genneraro et al., 1988), and this thyroid-dependent GH expression is sustained to adulthood, as thyroidectomy of adult rats decreases pituitary GH concentration drastically (Katakami et al., 1986). Thyroid hormones also change the pituitary response toward GHRH stimulation. Pituitary cells from rats with hypothyroidism exhibit a diminished response of GH release toward GHRH stimulation (Martin et al., 1985).

This decrease in GHRH responsiveness is possibly mediated by a reduced GHRH receptor expression in the pituitary (Tam et al., 1996). Both human and rat GH promoters contain thyroid hormone response elements (TRE) (Cattinit et al., 1986a; Lavin et al., 1988; Norman et al., 1989; Sap et al., 1990). When activated, the thyroid hormone receptor (TR) binds to the TRE on the promoter to induce GH transcription (Flug et al., 1987; Koenig et al., 1987; Tansey and Catanzaro, 1991). This thyroid hormone-induced GH transcription is independent of the action of Pit-1 in the pituitary, as deletion of the Pit-1 response element in the rat GH promoter does not abrogate T3-induced promoter activity (Suen and Chin, 1993). However, when both TR and Pit-1 are coexpressed in rat pituitary GHFT1–5 cells, synergistic induction of the rat GH promoter was observed in a ligandindependent manner (Chang et al., 1996). This result suggests that TR and Pit-1 interact with each other to drive transcription of the GH gene. Furthermore, in human monocyte U937 cells, expression of either TR or Pit-1 alone is unable to induce rat GH promoter activity. When they are coexpressed, activation of the promoter is observed (Schaufele et al., 1992). Indeed, unliganded TR is a constitutive repressor of GH transcription in which inhibition of transcription occurs at an early step during preinitiation complex assembly (Fondell et al., 1993). Upon binding with the hormone, TR is converted into an activator (Garcia-Villalba et al., 1997). Interestingly, in nonpituitary HeLa cells, the role of liganded and unliganded TR is reversed. Unliganded TR binding to the negative thyroid hormone response element (nTRE) causes a strong activation of rat GH promoter activity in this cell line, but the activity is repressed in the presence of T3. When Pit-1 is also expressed in HeLa cells, thyroid hormone produces a stimulation of GH transcription as that found in pituitary cells (Palomino et al., 1998). This mechanism is believed to be important in restricting GH expression in nonpituitary cells that express TR. Studies on the binding of TR to this nTRE in HeLa cells showed that occupancy of the promoter by TR is concomitant with the appearance of acetylated histone H3. This result suggests that histone acetylation is also involved in TR-mediated GH transcription (SanchezPacheco and Aranda, 2003). 4. GH and IGF-I GH also regulates its own secretion. The first report of this GH autoregulation appeared as early as 1966 by Krulich and McCann when exogenous administration of bovine GH was shown to reduce pituitary GH contents in rat. It is now recognized that GH is a regulator of its own secretion in both human and rat (Abrams et al., 1971; Clark et al., 1988; Leung et al., 2002). Moreover, exogenous administration of GH to human and rat blunts the release of GH stimulated by secretagogues such as GHRH (Cella et al., 1990a; Rosenthal

1. Regulation and Mechanism of GH and IGF-I

et al., 1986; Ross et al., 1987a). Because basal GH secretion as well as GHRH-induced GH release from pituitary cells is not inhibited by the presence of exogenous GH in vitro (Kraicer et al., 1988), it has been suggested that the observed GH autoregulation mainly occurs via the GHRH and SST actions. However, in bovine pituitary, both acute and chronic exposure to human GH significantly reduced GH secretion, but the same treatment has no effect on GH secretion from the rat pituitary, suggesting that there are rather large speciesspecific differences in the ability of GH to regulate its own secretion from the pituitary (Rosenthal et al., 1991). Reports that intracerebroventricular administration of human GH significantly inhibits GH secretion in rats (Abe et al., 1983) but the intracerebroventricular administration of human or bovine GH fails to alter the plasma GH concentration in sheep (Spencer, 1997) further give support to this point. In animals that show GH autoregulation, exogenous administration of GH inhibits its own secretion by reducing both GHRH secretion and expression. Hypothalamic GHRH in rat was significantly reduced after 7 days of treatment with rat GH or after inoculation of rat GH-secreting pituitary tumor cells (Bertherat et al., 1993; Cella et al., 1990a; Miki et al., 1989). In addition, GHRH mRNA is reduced by about 50% in rats bearing a GH-secreting tumor (Bertherat et al., 1993) and in rats treated with GH for 10 days (Cella et al., 1990a). Conversely, studies using hypophysectomized rats showed an increased GHRH mRNA level in the hypothalamus (De Gennaro Colonna et al., 1988; Wood et al., 1991) and this increased GHRH mRNA could be partially reversed by GH administration (Chomczynski et al., 1988a). It has been suggested that this negative feedback occurs predominantly through increased hypothalamic SST secretion (Bertherat et al., 1993; Miki et al., 1989; Ross et al., 1987b; Sato et al., 1989). Moreover, hypophysectomy decreases SST mRNA levels in the neurons of periventricular nucleus and this decrease could be restored by GH replacement (Roger et al., 1988). GH autoregulation also depends on the action of the ghrelin system. It has been found that exogenous administration of GH to rats decreases stomach ghrelin mRNA and plasma ghrelin levels (Qi et al., 2003). Moreover, hexarelininduced GH secretion is blunted in humans with prior injection of GH. This is probably mediated by a concomitant reduction in the activity of GHRH-secreting neurons (Arvat et al., 1997). However, this might also be a consequence of reduced GHSR expression in the hypothalamus and pituitary. In GH-deficient dwarf rats, GHSR in both the hypothalamus and the pituitary is higher than in normal rats and this increased GHSR expression could be decreased by exogenous administration of GH (Bennett et al., 1997; Kamegai et al., 1998). Despite the general belief that GH regulates its own biosynthesis and secretion in a negative manner, it has been observed that GH is essential to maintain its basal

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expression and secretion in the fish pituitary (Zhou et al., 2004) where GH is proposed to serve as an intrapituitary factor. Whether this observation is also applicable to other vertebrates needs further investigations. In addition to GH itself, it has been suggested that IGF-I may also be involved in regulating GH biosynthesis and secretion. In cultured rat pituitary cells, the addition of IGF-I suppresses basal GH secretion, GH mRNA level, and GHRH-stimulated GH secretion (Yamashita and Melmed, 1986, 1987). Similar findings were noted both in normal human subjects in vivo and in pituitary tumor in vitro (Bermann et al., 1994; Yamashita et al., 1986). These results indicate that IGF-I exerts a direct action on the pituitary to suppress GH secretion. Moreover, this IGF-I feedback action also depends on its influence on the levels of the hypothalamic hormones and their receptors. In hypophysectomized rat, GH replacement induces an increase in the IGF-I mRNA level in the hypothalamus, suggesting that IGF-I may serve as a hypothalamic modulator of GH secretion (Wood et al., 1991). The SST mRNA level is increased in rats infused continuously with human IGF-I (Ghigo et al., 1997), whereas in GH-deficient dwarf rats, intracerebroventricular infusion of IGF-I decreases the GHRH mRNA level (Sato and Frohman, 1993). Moreover, in a genetically IGF-Ideficient rat, expression of the GHRH receptor is higher than the control rat, indicating that IGF-I has a modulatory effect on GHRH receptor expression (Wallenius et al., 2001). Direct evidence on how IGF-I modulates the GHRH receptor level came from the work of Sugihara et al. (1999), where the level of the GHRH receptor in a rat pituitary cell culture is dose dependently reduced by IGF-I treatment. Gil Ad et al. (1996) suggested that GH and IGF-I may play a cooperative role in that both hormones stimulate SST secretion but only GH is able to inhibit GHRH release under the short-term feedback mechanism. A decrease in the pituitary GHSR level is also observed in the IGF-I-deficient rat, suggesting that GHSR expression is also affected by the IGF-I level (Wallenius et al., 2001). The mechanism of how IGF-I inhibits GH secretion in the pituitary has been studied. IGF-I signaling in the pituitary attenuates the signal cascades for GH secretion and gene expression. IGF-I inhibits both AC- and PKCmediated GH secretion (Morita et al., 1987). However, AC activation by forskolin attenuates IGF-I-induced extracellular signal-related kinase (ERK) activity (Webster et al., 1994). It has also been demonstrated that IGF-I suppression of GH transcription is independent of inositol-3-phosphate kinase and MAPK activities (Voss et al., 2000). However, this IGF-I inhibition of GH transcription does not affect the Pit-1 mRNA level, which remains constant during the IGF-I treatment (Voss et al., 2000). Further studies on IGF-Isignaling events in the pituitary connecting the cascades that stimulate GH secretion and transcription are necessary to give a clearer picture of the mechanisms involved.

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III. INSULIN-LIKE GROWTH FACTOR-I A. Insulin-like Growth Factor-I Gene Expression in the Brain Liver is the major production site of endocrine IGFs in juvenile and adult mammals. In addition, IGFs are also produced in other peripheral tissues as well as the CNS exerting autocrine and paracrine actions. IGF-I is recognized as a neuropeptide important for the growth and development of the CNS (Carlsson-Skwirut et al., 1986). Its mRNA is detected in both fetal and adult brain with the greatest abundance in the pons and cerebellum (Sandberg et al., 1988). The predominant form of the IGF-I protein in the human fetus is the truncated variant with deletion of the N-terminal tripeptide Gly-Pro-Glu by posttranslational processing (Sara et al., 1986; Sara and Carlsson-Skwirut, 1986). This truncated IGF-I in fact displays more potent biological action on brain cells than the parental IGF-I (Carlsson-Skwirut et al., 1989). Apart from this in situ production, another source of IGF-I in the brain comes from the blood circulation. Brain capillary endothelial cells express insulin and IGF-I receptors, which mediate the transport of insulin (Duffy and Pardridge, 1987; Pardridge et al., 1985) and IGF-I (Duffy et al., 1988) across the blood–brain barrier. Compared with insulin, IGF-I binds more readily to the brain capillaries, thus facilitating its crossing the blood–brain barrier into the brain parenchyma (Reinhardt and Bondy, 1994). Various transcripts of IGF-I are found in the brain. In human, formation of the IGF-I Ea subtype comes from the use of a polyadenylation site within exon 6 together with the excision of exon 5 from the primary transcript. Another transcription subtype, the Eb form, uses the polyadenylation site in exon 5 and thus exon 6 is spliced away (Rotwein et al., 1986). IGF-I Ea is the predominant form in the rat brain (Lowe et al., 1988). The major IGF-I transcripts of 7.5 and 4.4 kb and some minor bands of 10.8, 1.8, and 1.4 kb are detected in the rat fetal brain by Northern analysis (Lowe et al., 1988). The mechanism of how these transcripts are generated, however, remains unclear. IGF-I expression in the brain is believed to be regulated at the levels of transcription, RNA processing, and translation in different tissues and at different stages of development (Sussenbach et al., 1992). There is a single IGF-I gene in the human genome consisting of six exons and five introns spanning over 90 kb of chromosomal DNA. Within this single gene, four transcription initiation sites (Jansen et al., 1991) and two putative promoter regions (P1 and P2), located upstream of the two alternatively used leader exons 1 and 2, have been identified (Jansen et al., 1992; Steenbergh et al., 1993). Different promoter utilization of the human IGF-I gene is observed in which the promoter activity of P1 is higher than P2 in the neuroepithelioma cell

line (SK-N-MC), whereas the reverse is true in the ovarian carcinoma cell line (OvCar-3) (Steenbergh et al., 1993).

B. Important Factors Affecting IGF-I Biosynthesis in the Brain IGF-I exerts multiple functions on the CNS. It stimulates brain cell proliferation and maturation (TorresAleman et al., 1990) and promotes neuronal survival by inhibition of apoptosis (Tagami et al., 1997a,b). IGF-I also increases myelin ensheathment (Ye et al., 1995). Physiological concentrations of IGF-I increase the number of cultured oligodendrocytes obtained from the cerebrum of neonatal rats in serum-free medium by 60-fold (McMorris et al., 1986). It was subsequently reported that the induced expression of IGF-I in the developing oligodendroglial cells is autocrine in nature (Shinar and McMorris, 1995). Attempts to understand the regulatory mechanisms of IGF-I expression in the brain have been made. Some physiological conditions such as fasting were found to control the expression of IGF-I in the CNS. Fasting for 48 h decreases IGF-I mRNA levels in the rat brain, but this is not paralleled by changes in IGF-I binding (Lowe et al., 1989). However, an elevated cAMP level in the rat glioma C6 cell line decreases IGF-I expression at the level of transcription and mRNA stability (Wang and Adamo, 2001). The coordinate expression of the GH receptor and IGF-I in various tissues, including the brain of the developing rat, suggests that the GH receptor contributes to the tissuespecific expression of IGF-I during development (Shoba et al., 1999). Direct evidence of this relationship comes from the work of Benbassat et al. (1999) in which the IGF-I promoter responds to GH stimulation in C6 glioma cells transfected with the GH receptor. A number of transcription factors have been found to be involved in the transcriptional control of IGF-I expression in different tissues of various species. They include the insulin-responsive binding protein, CCAAT/enhancer-binding protein, hepatic nuclear factor (HNF)-1, STAT5, Sp1, and AP-1 (Kajimoto and Umayahara, 1998; Kaytor et al., 2001; Li et al., 2003; Umayahara et al., 1999; Umayahara et al., 2002; Vong et al., 2003; Zhu et al., 2000). However, in none of these studies was the importance of these transcription factors in the central expression of IGF-I addressed. Further studies in this direction are highly warranted. Nonetheless, the induction of IGF-I expression by GH and glucocorticoids, as well as during development and in brain injury, has been studied more extensively. These aspects are discussed in the following sections. 1. Developmental Regulation IGF-I expression is regulated spatially and temporally during development. A 1.1-kb IGF-I mRNA is the major

1. Regulation and Mechanism of GH and IGF-I

transcript in adult human liver (Rotwein et al., 1986), but the major IGF-I transcript in the fetal brain has a size of 7.5 kb (Sandberg et al., 1988). IGF-I mRNA is abundant in regions where neurogenesis persists after birth, including the cerebellum, olfactory bulb, and hippocampal complex (Bartlett et al., 1991). The timing of IGF-I mRNA expression appears to be temporally related to local neuronal proliferation. The expression of IGF-I in the brain is the greatest during the first 2 postnatal weeks and then declines to background levels at the end of the first month. During CNS development, the IGF-I mRNA level is the highest at embryonic day 14 (E14) and declines dramatically at birth to the level in the adult rat brain (Rotwein et al., 1988). High levels of IGF-I mRNA are found in the developing Purkinje cells of the cerebellar cortex and in the major cerebellar relay centers, including the inferior olive, medial vestibular, and lateral reticular nuclei of the brain stem and the deep cerebellar and red nuclei of the midbrain (Bondy, 1991). A high expression level of IGF-I is also found in the olfactory bulb, discrete neurons of the cranial sensory ganglia in the trigeminal region (Ayer-le et al., 1991), somatosensory system, and auditory system, including the cochlear nucleus, superior olive, lateral lemniscus, medial geniculate body, and inferior colliculus (Bondy, 1991). Bach et al. (1991) found that regional IGF-I mRNA is expressed in different developmental patterns. The olfactory bulb exhibits the highest abundance at the perinatal stage and declines dramatically by postnatal day 8 (P8). The cerebral cortex displays maximal levels of IGF-I mRNA at P8 and P13 and subsequently declines to adult levels. However, IGF-I mRNA increases from E16 up to P3 and remains high thereafter in the hypothalamus. In the brain stem and cerebellum, IGF-I mRNA and IGF-I receptor levels remain unchanged throughout E16 to P82. Another study also showed that IGF-I mRNA levels are high early in development and decrease by 4 months of age in the rat brain (Shoba et al., 1999). Unlike the situation in the liver, testis, and skeletal muscle, very little is known about the signaling events mediating IGF-I expression in the brain during development. In skeletal muscle, JAK2 and IGF-I decline coordinately. However, the pattern of JAK2 expression is discordant with IGF-I in the liver and testis. A dramatic age-dependent decrease in STAT5 and IGF-I levels is also observed in skeletal muscle and testis whereas the hepatic STAT5 level is constant. However, although the HNF-1␣ putative site is localized on the P1 promoter, no consistent change in HNF-1␣ binding to P1 in the rat brain and liver during different stages of development is observed (Shoba et al., 1999). These data indicate that the P1 promoter may not be responsible for the regulation of IGF-I expression in the brain and liver during development. Although it has been shown that members of the

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JAK/STAT proteins are expressed and indeed modulated in vivo in the rat embryonic and postnatal brain during development (De-Fraja et al., 1998), it is not clear whether their expression correlates with IGF-I expression during CNS development. 2. Brain Injury The expression of IGF-I is regulated in response to brain injury, including metabolic (Beilharz et al., 1998; Gluckman et al., 1992; Lee et al., 1996), traumatic (Li et al., 1998; Walter et al., 1999), and hypoglycemic (Cheng and Mattson, 1992) insults. Hypoxic-ischemia results in neuropathies, including ulegyria and porencephaly (Towfighi et al., 1991), changes in cerebral glucose metabolic rate and cerebral energy utilization (Vannucci et al., 1994), and altered gene expression of the IGF system (Gluckman et al., 1992; Lee et al., 1996). The expression of IGF-I, IGF-I receptor, IGF-binding protein (IGFBP)-2, and IGFBP-5 are all decreased at 1 h of recovery and these decreases are more pronounced at 24 h of recovery. At 72 h of recovery, the expression of IGF-I, IGFBP5 (Lee et al., 1996), and IGFBP3 (Gluckman et al., 1992) is activated in reactive astrocytes, whereas IGFBP2 and IGF-I receptor remain suppressed (Lee et al., 1996). The findings are further confirmed by a study done in the detection of IGF-I mRNA in the damaged regions (Beilharz et al., 1998). In these damaged regions, IGF-I mRNA is induced for 3 days following a 60-min injury and is also induced in areas of delayed, selective neuronal loss after a 15-min injury. Microglia, but not astrocytes, are responsible for IGF-I mRNA production within these regions of cell loss. Apart from the regulation of IGF expression in the ischemic brain, alteration in its gene expression also occurs during traumatic damage. Expression of IGF-I mRNA is significantly increased in the parieto-occipital lobes of both 2-week-old and adult mice following the introduction of unilateral scalpel wounds and remains elevated for 1 week after injury (Li et al., 1998). The IGF-I mRNA increases within the same injury-responsive cells after wounding but this increase is not measurable in the cerebrospinal fluid by radioimmunoassay. It was also hypothesized that the elevated IGF-I expression in the wounded rat brain acts in an autocrine/paracrine manner to regulate the cellular response in accordance with its spatial and temporal availability together with the presence of the stimulatory or inhibitory IGFBPs (Walter et al., 1997). Furthermore, the increase of IGF-I in the wounded microvasculature and its presence in serum suggest that de novo synthesis occurs in endothelial cells as well (Grant et al., 1993; Walter et al., 1997). Although IGF-I possesses neuroprotective effects toward various types of brain injury, the mechanism of how these injuries induce IGF-I expression remains unknown. Despite the fact that the signal transduction mechanisms in brain

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injury have been studied extensively, no investigation has been performed linking these mechanisms to IGF-I expression. Further studies are highly warranted to delineate the mechanisms of how IGF-I expression is induced to perform its neuroprotective effect. 3. Growth Hormone It is well known that IGF-I expression in the liver is modulated by GH (Roberts et al., 1986). This GH-stimulated IGF-I expression could also be observed in brain tissues. The brain exhibits significant increases in IGF-I mRNA levels in response to GH in dwarf mice (Mathews et al., 1986). In hypophysectomized rats, the expression of IGF-I mRNA in the brain is fourfold lower than in normal rats. This depressed IGF-I expression is recovered to 80% of normal within 4 h after the intracerebroventricular injection of human GH into the hypophysectomized rats (Hynes et al., 1987). In addition, injection of recombinant human GH fails to stimulate postnatal growth (P14 to P56) of Igf-I null mice (Liu and LeRoith, 1999). Taken together, IGF-I is the major determinant in embryonic and postnatal growth under GH modulation (Lupu et al., 2001) and it seems that its expression in the brain is also GH dependent. Although many studies have been performed on how GH affects IGF-I expression in peripheral tissues, particularly the liver, the molecular mechanism of GH-mediated IGF-I gene transcription in the brain has only been addressed using rat C6 glioma cells transiently transfected with the GH receptor and JAK2. In this cell line, GH stimulates IGF-I biosynthesis via the JAK-STAT pathway. GH binds to the GH receptor and activates JAK2, which in turn phosphorylates STAT5a and STAT5b that subsequently activates IGF-I transcription (Benbassat et al., 1999). 4. Glucocorticoids Several lines of transgenic mice that express the firefly luciferase under the control of a 11.3-kb fragment from the 5⬘ region of the rat IGF-I gene have been generated. The contiguous IGF-I gene sequence includes approximately 5.3 kb upstream of exon 1, intron 2, and the first 48 bp of exon 3 (Ye et al., 1997). It was observed that treatment by dexamethasone, a synthetic glucocorticoid, significantly reduces transgenic expression in the developing cerebral cortex, hippocampus, brain stem, and cerebellum. This result is consistent with a study performed in primary cultures of neuronal cells from rat brain (Adamo et al., 1988). Treatment of this neuronal cell culture with dexamethasone reduces IGF-I mRNA levels by 60%. It has also been suggested that this glucocorticoid-induced reduction in IGF-I production occurs at the level of transcription. Results were reported in rat C6 glioma cells where dexamethasone decreases IGF-I mRNA levels (Lowe et al., 1992). It is not clear if this reduced IGF-I expression by glucocorticoid is a result of a direct

interaction between the activated glucocorticoid receptor and the IGF-I promoter. However, results of studies in other tissues may provide some hints. In rat osteoblasts, cortisol decreases IGF-I expression via enhancing the expression of CAAT/enhancer-binding protein, which, in turn, binds to the ⫹132 to ⫹158 region of the IGF-I promoter (Delany et al., 2001). Whether the same occurs in neuronal cells remains to be elucidated.

IV. FUTURE PERSPECTIVES Not only do GH and IGF-I play important roles in the CNS, they are also produced in situ centrally. Most of our current understanding on GH biosynthesis comes from studies in the pituitary. However, the bulk of information on IGF-I biosynthesis is derived mostly from studies in the liver. Perhaps the most burning issue is whether such information obtained in these tissues would be equally applicable to the CNS. In view of the special vascularization, structure, cell types, and hormonal milieu in the CNS, it is anticipated that the situation would not be entirely the same in terms of modulatory factors encountered, promoter usage, and transcription factors involved, among other things. Novel control mechanisms peculiar to the CNS await elucidation. Research to delineate them is much needed, particularly in view of the potential application of GH and possibly IGF-I in modulating neural functions in an aging population.

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2 Ghrelin, an Endogenous Ligand for the Growth Hormone Secretagogue Receptor MASAYASU KOJIMA* and KENJI KANGAWA†,‡ *Molecular Genetics, Institute of Life Science, Kurume University, Kurume, Fukuoka, Japan of Biochemistry, National Cardiovascular Center Research Institute, Suita, Osaka, Japan and ‡Translational Research Center, Kyoto University Hospital, Kyoto, Japan

†Department

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Growth Hormone Secretagogue and Its Receptor Purification and Identification of Ghrelin Distribution of Ghrelin Physiological Functions of Ghrelin Ghrelin Knockout Mouse Ghrelin Receptor Knockout Mouse Regulation of Ghrelin Secretion Epilogue References

the bloodstream under fasting conditions, indicating that it transmits a hunger signal from the periphery to the central nervous system. Taking into account all these activities, ghrelin plays important roles for maintaining growth hormone release and energy homeostasis in vertebrates.

Small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through the GHS-R, a G-protein-coupled receptor. Using a reverse pharmacology paradigm with a stable cell line expressing GHS-R, we purified an endogenous ligand for GHS-R from rat stomach and named it “ghrelin,” after a word root (“ghre”) in Proto-Indo-European languages meaning “grow.” Ghrelin is a peptide hormone in which the third amino acid, usually a serine but in bullfrog a threonine, is modified by a fatty acid; this modification is essential for the activity of ghrelin. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by the hypothalamic GH-releasing hormone, but also by ghrelin derived from the stomach. In addition, ghrelin stimulates appetite by acting on the hypothalamic arcuate nucleus, a region known to control food intake. Ghrelin is orexigenic; it is secreted from the stomach and circulates in

Growth hormone (GH), a multifunctional hormone secreted from somatotrophs of the anterior pituitary, regulates overall body and cell growth, carbohydrate–protein– lipid metabolism, and water–electrolyte balance (Carter-Su et al., 1996). Production and release of GH are controlled tightly, but occasionally fall into imbalance; GH excess results in acromegaly and gigantism, whereas its deficiency in children results in impaired growth and short stature. GH is controlled by many factors, in particular by two hypothalamic neuropeptides: GH release is stimulated by hypothalamic growth hormone-releasing hormone (GHRH) and inhibited by somatostatin (Muller et al., 1999). However, a third independent pathway regulating GH release has been identified from studies of growth hormone secretagogues (GHSs) (Bowers, 1998; Smith et al., 1997). GHSs are synthetic compounds that are potent stimulators of GH release, working through a G-protein-coupled receptor (GPCR), the GHS receptor (GHS-R) (Howard et al., 1996). Because

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION

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Copyright 2006 Elsevier Inc. All rights reserved.

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GHSs are a group of artificial compounds and do not exist naturally, it was postulated that an endogenous ligand must exist that binds to GHS-R and carries out similar functions to GHSs in situ. We succeeded in the purification and identification of the endogenous ligand for the GHS-R from the stomach and named it “ghrelin” (Kojima et al., 1999). Ghrelin is a growth hormone-releasing and appetitestimulating peptide. This chapter reviews the structure, distribution, and physiological functions of ghrelin.

II. GROWTH HORMONE SECRETAGOGUE AND ITS RECEPTOR Figure 1 shows the structures of typical GHSs. In 1976, C. Y. Bowers found that some opioid peptide derivatives that did not exhibit any opioid activity instead had weak GH-releasing activity and were referred to as GHSs (Bowers et al., 1980). The structure of the first GHS was Tyr-D-Trp-Gly-Phe-Met-NH2, which induced GH release by directly acting on the pituitary. This synthetic peptide was a methionine enkephalin derivative, in which the second Gly was replaced with a D-Trp, and the C terminus had an amide structure. After the discovery of ghrelin, it was revealed that bulky hydrophobic side chain groups are important for its activity (Momany et al., 1981). Thus, the D-Trp in the aforementioned GHS was probably a core structure mediating its binding to the GHS receptor, which had not been yet identified at that time. The GH-releasing activity of early GHSs was very weak and was only observed in vitro. However, their discovery led to the synthesis of many peptidyl derivatives in a search for more GHSs with more potent activity. A potent GHS, GHRP-6, was synthesized in 1984 based on conformational energy calculations in conjunction with

O CH3 CH3 H N C CH2 C NH2 N

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peptide chemistry modifications and a biological activity assay (Bowers et al., 1984). A hexapeptide, GHRP-6 was shown to be active both in vitro and in vivo, which suggested its possible application for clinical use (Argente et al., 1996; Ghigo et al., 1998; Micic et al., 1999). The first nonpeptide GHS, L-692,429, was synthesized in 1993 by R. G. Smith and co-workers (Cheng et al., 1993; Smith et al., 1993). During this period, researchers investigated the mechanisms of GHS action. Whereas GH release from the pituitary was known to be stimulated by hypothalamic GHRH, exogenous GHSs were thought to induce GH release through a pathway different from that of GHRH (Akman et al., 1993; Blake and Smith, 1991; Cheng et al., 1989, 1991). GHRH acts on the GHRH receptor to increase intracellular cAMP, which serves as a second messenger. However, GHSs were found to act on a different receptor, increasing intracellular Ca2⫹ concentration via an inositol 1,4,5-trisphosphate (IP3) signal transduction pathway. In 1996, the growth hormone secretagogue receptor (GHS-R) was identified by expression cloning using a strategy based on the findings that GHSs stimulate phospholipase C, resulting in an increase in IP3 and intracellular Ca2⫹ (Howard et al., 1996). Xenopus oocytes were injected with in vitro-transcribed cRNAs derived from swine pituitary, supplemented simultaneously with various G␣ subunit mRNAs. MK0677-stimulated Ca2⫹ increase could be detected by bioluminescence of the jellyfish photoprotein aequorin, which was expressed by the Xenopus oocytes. The identified GHS-R is a typical GPCR. In situ hybridization analyses showed that GHS-R is expressed in the pituitary, hypothalamus, and hippocampus (Bennett et al., 1997; Howard et al., 1996). This receptor was for some time an example of an orphan GPCR, i.e., a GPCR with no known natural ligand. After identification of the GHS-R, a search for its endogenous ligand was actively undertaken, using the orphan receptor strategy.

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L-163,191 (MK-0677) His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 GHRP-6

His-D-2-Methyl-Trp-Ala-Trp-D-Phe-Lys-NH2 Tyr-D-Trp-Gly-Phe-Met-NH2 Hexarelin The first GHS FIGURE 1 Structures of typical growth hormone secretagogues (GHSs). GHSs are grouped into nonpeptidyl (like L-692,429 and MK-0677) and peptidyl (like GHRP-6 and hexarelin) forms. The first identified GHS was a methionine enkephalin derivative.

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2. Structure and Function of Ghrelin

III. PURIFICATION AND IDENTIFICATION OF GHRELIN A. Purification of Ghrelin Because the ligands of most GPCRs are unknown, assays for their activity generally have no positive controls. GHS-R, however, was known to bind several artificial ligands, such as GHRP-6 or hexarelin, providing a convenient positive control for constructing the assay system used to search for the endogenous ligand. A cultured cell line expressing the GHS-R was established and used to identify tissue extracts that could stimulate the GHS-R, as monitored by increases in intracellular Ca2⫹ levels. After screening several tissues, very strong activity by an endogenous ligand was unexpectedly found in stomach extracts (Kojima et al., 1999). The ligand was finally purified by reversedphase HPLC (RP-HPLC) and named as ghrelin. The name “ghrelin” is based on “ghre,” a word root in Proto-IndoEuropean languages for “grow,” in reference to its ability to stimulate GH release. Ghrelin is a 28 amino acid peptide in which the serine 3 (Ser3) is n-octanoylated and this modification is essential for the activity of ghrelin (Fig. 2). Ghrelin is the first known case of a peptide hormone modified by a fatty acid. Rat and human ghrelins differ in only two amino acid residues. There is no structural homology between ghrelin and peptide GHSs such as GHRP-6 or hexarelin. In rat stomach, a second type of ghrelin peptide has been purified and identified as des-Gln14-ghrelin (Hosoda et al., 2000b). Except for the deletion of Gln14, des-Gln14ghrelin is identical to ghrelin, even retaining the n-octanoic acid modification. Des-Gln14-ghrelin has the same potency of activities with that of ghrelin. The deletion of Gln14 in des-Gln14-ghrelin arises due to the usage of a CAG codon to encode Gln, which results in its recognition as a splicing signal. Thus, two types of active ghrelin peptide

Human NH2

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FIGURE 2 Structures of human and rat ghrelins. Both human and rat ghrelins are 28 amino acid peptides, in which Ser3 is modified by a fatty acid, primarily n-octanoic acid. This modification is essential for the activity of ghrelin.

Mammalian Human Rhesus Monkey Mouse Monglian Gerbil Rat Dog Porcine Sheep Bovine

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GSSFLSPEHQRVQQRKESKKPPAKLQPR GSSFLSPEHQRAQQRKESKKPPAKLQPR GSSFLSPEHQKAQQRKESKKPPAKLQPR GSSFLSPEHQKT QQRKESKKPPAKLQPR GSSFLSPEHQKAQQRKESKKPPAKLQPR GSSFLSPEHQKL QQRKESKKPPAKLQPR GSSFLSPEHQKVQQRKESKKPAAKLKPR GSSFLSPEHQKL Q – RKEPKKPSGRLKPR GSSFLSPEHQKL Q – RKEAKKPSGRLKPR

FIGURE 3 Sequence comparison of mammalian ghrelins. Identical amino acids are shaded. Asterisk indicates acyl-modified third amino acids. N-terminal cores with acyl-modification sites are well conserved among all ghrelins.

are produced in rat stomach: ghrelin and des-Gln14-ghrelin. However, des-Gln14-ghrelin is only present in low amounts in the stomach, indicating that ghrelin is the major active form. In addition, n-decenoyl (C10:1)-modified ghrelin exists in the stomach in small amounts.

B. Mammalian Ghrelins In mammals, ghrelin homologues have been identified in human, rhesus monkey (Angeloni et al., 2004), rat, mouse, mongolian gerbil, cow, pig, sheep, and dog (Tomasetto et al., 2001) (Fig. 3). The amino acid sequences of mammalian ghrelins are well conserved; in particular, the 10 amino acids of their N termini are identical. This structural conservation and the universal requirement for acyl modification of the third residue indicate that this N-terminal region is of central importance to the activity of the peptide.

IV. DISTRIBUTION OF GHRELIN A. Stomach and Gastrointestinal Organs In all vertebrate species, ghrelin is mainly produced in the stomach (Ariyasu et al., 2001; Kojima et al., 1999). In the stomach, ghrelin-containing cells are more abundant in the fundus than in the pylorus (Date et al., 2000a; Tomasetto et al., 2001). In situ hybridization and immunohistochemical analyses indicate that ghrelin-containing cells are a distinct endocrine cell type found in the mucosal layer of the stomach (Date et al., 2000a; Rindi et al., 2002). Four types of endocrine cells have been identified in the oxyntic mucosa with the following relative abundances: ECL, D, enterochromaffin (EC), and X/A-like cells (Capella et al., 1969; Davis, 1954; Grube and Forssmann, 1979; Solcia et al., 1975). The rat oxyntic gland contains approximately 60–70% ECL cells, 20% X/A-like cells, 2–5% D cells, and 0–2% EC cells; in the human, the corresponding percentages are 30, 20, 22, and 7%. The major products in the granules have been identified as histamine

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and uroguanylin in ECL cells, somatostatin in D cells, and serotonin in EC cells. However, the granule contents of X/A-like cells were unknown until the discovery of ghrelin. The X/A-like cells contain round, compact, electrondense granules filled with ghrelin (Date et al., 2000a; Dornonville de la Cour et al., 2001). These X/A-like cells account for about 20% of the endocrine cell population in adult oxyntic glands. However, the number of X/A-like cells in the fetal stomach is very low and increases after birth (Hayashida et al., 2002). As a result, the ghrelin concentration of fetal stomach is also very low and gradually increases after birth until 5 weeks of age. Gastric X/A-like cells can be stained by an antibody that is specific to the N-terminal, acyl-modified portion of ghrelin, indicating that ghrelin in the secretory granules of X/A-like cells has already been acyl modified. Ghrelin-immunoreactive cells are also found in the duodenum, jejunum, ileum, and colon (Date et al., 2000b; Hosoda et al., 2000a; Sakata et al., 2002). In the intestine, ghrelin concentration gradually decreases from the duodenum to the colon. As in the stomach, the main molecular forms of intestinal ghrelin are n-octanoyl ghrelin and desacyl ghrelin. The pancreas is a ghrelin-producing organ (Date et al., 2002b). Analyses combining HPLC and ghrelin-RIA revealed that ghrelin and des-acyl ghrelin both exist in the rat pancreas. However, the cell type that produces ghrelin in the pancreatic islets remains controversial, whether it be ␣ cells, ␤ cells, the newly identified islet ⑀ cells, or a unique novel islet cell type (Date et al., 2002b; Prado et al., 2004; Wierup et al., 2002, 2004). The pancreatic ghrelin profile changes dramatically during fetal development (Chanoine and Wong, 2004; Wierup et al., 2004); pancreatic ghrelin-expressing cells are numerous from midgestation to the early postnatal period, comprising 10% of all endocrine cells, and decrease in number after birth. Ghrelin mRNA expression and total ghrelin concentration are markedly elevated in the fetal pancreas, six to seven times greater than in the fetal stomach. Thus, the onset of islet ghrelin expression precedes that of gastric ghrelin. Pancreatic ghrelin expression is highest in the prenatal and neonatal periods. In contrast, gastric ghrelin levels are low during the prenatal period and increase after birth (Hayashida et al., 2002).

B. Brain and Pituitary Ghrelin has been found in the hypothalamic arcuate nucleus, an important region for controlling appetite (Kojima et al., 1999; Lu et al., 2002). In addition, a study has reported the presence of ghrelin in previously uncharacterized hypothalamic neurons adjacent to the third ventricle among dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei (Cowley et al., 2003). These ghrelin-containing neurons send

efferent fibers to neurons that contain neuropeptide Y (NPY) and agouti-related protein (AgRP) and may stimulate the release of these orexigenic peptides. These localization patterns of ghrelin suggest a role in controlling food intake. In fact, injection of ghrelin into the cerebral ventricles of rats potently stimulates food intake. GH-releasing somatotrophs in the pituitary gland are the target cells of ghrelin. In an in vivo assay, ghrelin stimulated primary pituitary cells and increased their intracellular Ca2⫹ concentration, indicating that the ghrelin receptor is expressed in pituitary cells (Bennett et al., 1997; Guan et al., 1997; McKee et al., 1997). Also, ghrelin has been found in the pituitary gland itself (Bennett et al., 1997; Guan et al., 1997; Korbonits et al., 2001a,b; McKee et al., 1997), where it may influence the release of GH in an autocrine or paracrine manner. The expression level of ghrelin in the pituitary is high after birth and declines with puberty. Pituitary tumors, such as adenomas, corticotroph tumors, and gonadotroph tumors, contain ghrelin peptides.

V. PHYSIOLOGICAL FUNCTIONS OF GHRELIN A. Growth Hormone-Releasing Activity Ghrelin acts on the ghrelin receptor, increasing intracellular Ca2⫹ concentration via IP3 to stimulate GH release. In term of both the area under the curve and mean peak GH levels, the GH-releasing activity of ghrelin is similar to that of GHRH when injected intravenously into rats (Arvat et al., 2000; Kojima et al., 1999; Peino et al., 2000; Takaya et al., 2000). However, the maximal stimulation affected by ghrelin is two to three times greater than that of GHRH (Arvat et al., 2000). Ghrelin stimulates growth hormone release both in vitro and in vivo in a dose-dependent manner (Fig. 4) (Kojima et al., 1999). An IV injection of ghrelin induces potent GH release both in rats and in humans. When anesthetized rats were injected intravenously with ghrelin, an increase in GH plasma concentration was observed [basal level: 12.0 ⫾ 5.4 ng/ml; after ghrelin injection: 129.7 ⫾ 11.3 ng/ml (SE)] (Kojima et al., 1999). GH release peaks at about 5–15 min after ghrelin injection and returns to basal levels 1 h later. A single ICV administration of ghrelin also increased rat plasma GH concentration in a dose-dependent manner, with a minimum dose of only 10 pmol (Date et al., 2000b). Thus, ICV injection appears to be a more potent route of delivery than IV administration. Ghrelin has also been shown to induce GH release in nonmammalian vertebrates, including chicken (Baudet and Harvey, 2003; Kaiya et al., 2002), fish (Kaiya et al., 2003a,b,c; Unniappan et al., 2002), and frog (Kaiya et al., 2001). Together, these in vivo assays confirmed that ghrelin is a potent GH-releasing

2. Structure and Function of Ghrelin

Concentration (ng/well/15 min)

(a)

+ Ghrelin – Ghrelin

60 50 40 30 20 10 0

GH ACTH FSH

LH

maximal level of GH release to be achieved by ghrelin administration. One possibility is transmission via the vagus nerve. When the vagus nerve is cut, the induction of GH release after IV injection of ghrelin is decreased dramatically (Date et al., 2002a), indicating that the vagus nerve pathway is required for the sufficient stimulatory effects of ghrelin. Another possibility is the involvement of GHRH in primary pituitary cells. Coadministration of ghrelin and GHRH had a synergistic effect on GH secretion, i.e., coadministration results in more GH release than either GHRH or ghrelin alone (Arvat et al., 2001; Hataya et al., 2001). This finding implies that GHRH is necessary for GH release to be maximally effective in inducing GH release.

PRL TSH

B. Appetite Regulation by Ghrelin

(b) 150 Hormone concentration in plasma (ng/ml)

29

GH ACTH FSH LH Prolactin TSH

100

50

0

0

10

20 30 40 50 60 Time (min) FIGURE 4 Effects of ghrelin on pituitary hormone secretion in vitro and in vivo. (a) Effects of a high dose (10⫺6 M) of ghrelin on hormone secretion from rat primary pituitary cells in vitro. GH, growth hormone; ACTH, adrenocorticotropin; FSH, follicle-stimulating hormone; LH, lutenizing hormone; PRL, prolactin; and TSH, thyroid-stimulating hormone. (b) Time courses of plasma hormone concentrations after IV injection of ghrelin into anesthetized male rats in vivo. Adapted from Kojima et al. (1999).

peptide. In addition, high doses of ghrelin in humans increase ACTH, prolactin, and cortisol levels (Arvat et al., 2001; Takaya et al., 2000). Ghrelin stimulates GH release from primary pituitary cells, which indicates that ghrelin can act directly on the pituitary (Fig. 4B) (Kojima et al., 1999). However, the involvement of the hypothalamus in ghrelin-mediated stimulation of GH release has been strongly suggested. Patients with organic lesions in the hypothalamic region showed insufficiency of GH release even when stimulated by ghrelin (Popovic et al., 2003). Moreover, when using primary pituitary cells, ghrelin treatment only increased GH release by two to three times above the basal level (Kaiya et al., 2001; Kojima et al., 1999), which is lower than the level of induction seen when ghrelin is peripherally administered to rats in vivo. These facts suggest that other factors are involved in vivo in order for this

1. Ghrelin Neurons in the Hypothalamic Appetite Regulatory Region Identification of appetite-regulating humoral factors reveals regulatory mechanisms not only in the central nervous system, but also mediated by factors secreted from peripheral tissues (Neary et al., 2004; Ukkola, 2004). Leptin, produced in adipose tissues, is an appetite-suppressing factor that transmits satiety signals to the brain (Friedman, 2002). Hunger signals from peripheral tissues, however, had remained unidentified until the discovery of ghrelin. Immunohistochemical analyses indicate that ghrelincontaining neurons are found in the arcuate nucleus of the hypothalamus, a region involved in appetite regulation (Kojima et al., 1999; Lu et al., 2002). This localization suggests a role of ghrelin in controlling food intake. Moreover, another report has indicated that ghrelin is also expressed in previously uncharacterized hypothalamic neurons that are adjacent to the third ventricle among the dorsal, ventral, paraventricular nucleus (PVN), and arcuate nucleus (ARC) (Cowley et al., 2003). In the ARC, these ghrelin-containing neurons send efferent fibers onto NPY- and agouti-related protein (AgRP) expressing neurons to stimulate the release of these orexigenic peptides and onto proopiomelanocortin (POMC) neurons to suppress the release of this anorexigenic peptide (Fig. 5). The neural network of ghrelin in the PVN is more complex. In the PVN, ghrelin neurons also send efferent fibers onto NPY neurons, which in turn suppress GABA release, resulting in the stimulation of corticotrophinreleasing hormone (CRH)-expressing neurons, leading to ACTH and cortisol release (Fig. 5). 2. Ghrelin Is a Potent Appetite Stimulant When ghrelin is injected into the cerebral ventricles of rats, their food intake is potently stimulated (Kamegai et al., 2001; Nakazato et al., 2001; Shintani et al., 2001; Tschop et al., 2000; Wren et al., 2001b). Among all discovered orexigenic peptide, ghrelin has been found to be

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FIGURE 5 Hypothalamic neural networks involving appetite-regulating peptides. Ghrelin-producing neurons in the arcuate nucleus (ARC) presynaptically induce NPY/AgRP neurons to release NPY/AgRP, potent orexigenic neuropeptides, thus stimulating food intake. These ghrelin-producing neurons in the ARC also increase the rate of secretion of GABA, which may postsynaptically modulate the release of POMC, an anorexigenic neuropeptide. In the PVN, ghrelin stimulates NPY release, which in turn suppresses GABA release, resulting in the simulation of CRH-expressing neurons, leading to ACTH and cortisol release.

the most powerful. Chronic ICV injection of ghrelin increases cumulative food intake and decreased energy expenditure, resulting in body weight gain. Ghrelin-treated mice also increase their fat mass, both absolutely and as a percentage of total body weight. Moreover, not only ICV injection, but also IV and subcutaneous injection of ghrelin have been shown to increase food intake (Nakazato et al., 2001; Tschop et al., 2000; Wren et al., 2001a). Ghrelin is produced primarily in gastrointestinal organs in response to hunger and starvation and circulates in the blood, serving as a peripheral signal telling the central nervous system to stimulate feeding. 3. Mechanism of Appetite Stimulation by Ghrelin The hypothalamic arcuate nucleus is the main site of the activity of ghrelin. The arcuate nucleus is also a target of leptin, an appetite-suppressing hormone produced in adipose tissues, and NPY and AgRP, which are both appetitestimulating peptides (Morton and Schwartz, 2001). NPY and AgRP are produced in the same population of neurons in the arcuate nucleus, and their appetite-stimulating effects are inhibited directly by leptin. At least part of the orexigenic effect of ghrelin is mediated by upregulating the genes encoding these potent appetite stimulants.

As suggested by the distribution of ghrelin-containing neurons in the hypothalamus, ICV injection of ghrelin induces Fos expression in NPY-expressing neurons and increases the amount of NPY mRNA in the arcuate nucleus (Chen et al., 2004; Kamegai et al., 2001; Nakazato et al., 2001; Shintani et al., 2001). Moreover, ICV ghrelin injection increases the AgRP mRNA level in the hypothalamus. The appetite-stimulating effects of ghrelin are blocked by an antagonist of NPY receptor 1. ICV injections of an AgRP inhibitor, anti-NPY IgG, and anti-AgRP IgG inhibit the appetite-stimulating effects of ghrelin. Intravenous injection of ghrelin also stimulates NPY/AgRP neurons in the hypothalamus. Immunohistochemical analysis indicated that ghrelin neuron fibers contact NPY/AgRP neurons directly (Cowley et al., 2003). These results indicate that ghrelin exerts its feeding activity by stimulating NPY/ AgRP neurons in the hypothalamus to promote the production and secretion of NPY and AgRP peptides. Ghrelin, thus, is a natural antagonist to leptin. AMP-activated protein kinase (AMPK) has been shown to be involved in hypothalamic appetite regulation (Minokoshi et al., 2004). Injection of 5-amino-4-imidazole carboxamide riboside, an activator of AMPK, increases food intake significantly. Administration of ghrelin in vivo increases AMPK activity in the hypothalamus (Andersson et al., 2004). In contrast, injection of leptin decreases hypothalamic AMPK activity. 4. Vagus Nerve and Appetite Regulation by Ghrelin Peripherally injected ghrelin stimulates hypothalamic neurons (Hewson and Dickson, 2000; Ruter et al., 2003; Wang et al., 2002) and stimulates food intake (Date et al., 2002a; Wren et al., 2001a). In general, peptides injected peripherally do not pass the blood–brain barrier. Indeed, the rate at which peripheral ghrelin passes the barrier has shown to be very low. Thus, peripheral ghrelin must activate the appropriate hypothalamic regions via an indirect pathway. The detection of ghrelin receptors on vagal afferent neurons in the rat nodose ganglion suggests that ghrelin signals from the stomach are transmitted to the brain via the vagus nerve (Date et al., 2002a; Sakata et al., 2003). Moreover, the observation that ICV administration of ghrelin induces c-Fos in the dorsomotor nucleus of the vagus and stimulates gastric-acid secretion indicates that ghrelin activates the vagus system (Date et al., 2001). In contrast, vagotomy inhibits the ability of ghrelin to stimulate food intake and GH release (Andrews and Sanger, 2002; Date et al., 2002a). A similar effect was also observed when capsaicin, a specific afferent neurotoxin, was applied to vagus nerves to induce sensory denervation. However, the basal level of ghrelin concentration is not affected after vagotomy or capsaicin treatment. Moreover, the suppression of ghrelin level by a nutrient load is not observed after vagotomy. However, fasting-induced elevation of plasma ghrelin

31

2. Structure and Function of Ghrelin

is completely abolished by subdiaphragmatic vagotomy or atropine treatment (Williams et al., 2003). These results indicate that the response of ghrelin to fasting is transmitted through vagal afferent transmission.

VI. GHRELIN KNOCKOUT MOUSE A ghrelin knockout mouse was produced, and its phenotype was examined (Sun et al., 2003; Wortley et al., 2004a,b). Ghrelin knockout mice showed normal size, growth rate, food intake, body composition, reproduction, and gross behavior, without any pathological changes. Because survival is threatened more acutely by starvation than by obesity, it may be no surprise that an orexigenic peptide-null mouse showed no change in food intake and body weight. However, the ghrelin-null mouse showed a significant reduction in respiratory quotient and a trend for lower body fat mass when the mouse was fed with a high-fat diet (Wortley et al., 2004a,b). These results indicate that ghrelin is not a critically required orexigenic factor, but may function in nutrient sensing and switching of metabolic substrates.

VII. GHRELIN RECEPTOR KNOCKOUT MOUSE Mice lacking GHS-R (Ghsr-null mice) do not show the typical increases in GH release and food intake upon ghrelin administration, indicating that GHS-R is indeed the primary biologically relevant ghrelin receptor (Sun et al., 2004). Growth and development of Ghsr-null mice are normal, and their appetite and body composition are not different from those of their wild-type littermates. Thus, ghrelin and its receptor are not critical for growth and appetite regulation. However, serum insulin-like growth factor I (IGF1) levels and body weights of Ghsr-null mice are decreased modestly compared to those of their wild-type littermates. These results suggest that ghrelin sets the IGF1 level for the maintainance of an anabolic state.

VIII. REGULATION OF GHRELIN SECRETION The most important factor for the regulation of ghrelin secretion is feeding. The plasma ghrelin concentration is increased when fasting and is decreased after food intake (Cummings et al., 2001; Tschop et al., 2001a). It is not clear what factors are involved in the regulation of ghrelin secretion. The blood glucose level may be critical: oral or intravenous administration of glucose decreases the plasma ghrelin concentration (McCowen et al., 2002; Shiiya et al.,

2002). Because gastric distention by water intake does not change the ghrelin concentration, mechanical distention of the stomach alone clearly does not induce ghrelin release. The plasma ghrelin concentration is sensitive, however, to the makeup of a meal; it is decreased by a high-lipid meal and increased by a low-protein meal (Erdmann et al., 2003; Greenman et al., 2004). Plasma ghrelin concentration showed a noctrunal increase (Dzaja et al., 2004; Yildiz et al., 2004). Ghrelin levels increased during sleep and this increase was blunted in obese subjects or by sleep deprivation. The plasma ghrelin concentration is low in obese people and high in lean people (Bellone et al., 2002; Cummings et al., 2002; Hansen et al., 2002; Haqq et al., 2003a; Rosicka et al., 2003; Shiiya et al., 2002; Tschop et al., 2001b). Related to this fact, the plasma ghrelin level is highly increased in anorexia nervosa patients and returns to normal levels upon weight gain and recovery from the disease (Ariyasu et al., 2001; Cuntz et al., 2002; Otto et al., 2001; Soriano-Guillen et al., 2004; Tanaka et al., 2003b). The ghrelin concentration is also increased in bulimia nervosa patients (Tanaka et al., 2003a). Patients with gastric bypass lose their weight and their ghrelin levels decrease (Cummings et al., 2002; Geloneze et al., 2003; Leonetti et al., 2003). Changes in ghrelin concentration associated with food intake are diminished in these patients, confirming that the stomach is the main site of ghrelin production. The plasma ghrelin concentration also decreases in patients with short bowel syndrome (Krsek et al., 2003), probably due to the loss of ghrelin-producing tissues. Exogenous treatment with somatostatin and its analogues, such as octreotide, as well as infusion of urocortin-1, a potent anorexigenic peptide, suppresses the plasma ghrelin concentration (Barkan et al., 2003; Davis et al., 2004; Haqq et al., 2003b; Norrelund et al., 2002). However, administration of leptin does not modify ghrelin levels (Chan et al., 2004). Exogenous GH decreases stomach ghrelin mRNA expression and plasma ghrelin concentration, but does not affect stomach ghrelin stores (Qi et al., 2003). These results suggest that pituitary GH exhibits a feedback regulation on stomach ghrelin production. Moreover, a relatedness between ghrelin and GH pulstility has been demonstrated, suggesting either that ghrelin participates in the pulsatile GH release or that the two hormones are coregulated simultanously (Koutkia et al., 2004).

IX. EPILOGUE In the mid-1980s, the intitial report of a GHS introduced a new regulatory pathway for GH release to accompany the known GHRH pathway. Since then, many potent GHSs have been developed, and the structure of the GHS-R has

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been identified. However, despite intensive research, identification of the endogenous ligand of the GHS-R had remained elusive until the discovery of ghrelin. This finding has launched a whole new field of research in GH and appetite regulation. Growing evidence supports the notion that GH release from the pituitary is controlled not only by GHRH from the hypothalamus, but also by ghrelin from the stomach and hypothalamus. In addition, ghrelin is a peripheral fasting signal and stimulates food intake, in contrast to the functions of leptin, a peripheral satiety signal from adipose tissues. Many interesting questions remain regarding ghrelinrelated biology and physiology. These include the identification of the biosynthetic mechanism of ghrelin production and regulation of ghrelin release from the stomach, the enzyme that catalyzes its acyl modification, as well as the continuing search for its physiological actions. Further research will answer these questions and elucidate the biochemical and physiological characteristics of this unique hormone.

Acknowledgments We express our gratitude to students, collaborators, and fellow scientists who shared their scientific interests with us and obtained the findings reviewed in this article. The studies in the authors’ laboratory are supported by grants from the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) (to M.K.), the Program for Promotion of Fundamental Studies in Health Sciences of Pharmaceuticals and Medical Devices Agency (PMDA) of Japan (to K.K.), and Grant-in-Aids for Scientific Research (B) (to M.K.) and (S) (to K.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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2. Structure and Function of Ghrelin Solcia, E., Capella, C., Vassallo, G., and Buffa, R. (1975). Endocrine cells of the gastric mucosa. Int. Rev. Cytol. 42, 223–286. Soriano-Guillen, L., Barrios, V., Campos-Barros, A., and Argente, J. (2004). Ghrelin levels in obesity and anorexia nervosa: Effect of weight reduction or recuperation. J. Pediatr. 144, 36–42. Sun, Y., Ahmed, S., and Smith, R. G. (2003). Deletion of ghrelin impairs neither growth nor appetite. Mol. Cell Biol. 23, 7973–7981. Sun, Y., Wang, P., Zheng, H., and Smith, R. G. (2004). Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc. Natl. Acad. Sci. USA 101, 4679–4684. Takaya, K., Ariyasu, H., Kanamoto, N., Iwakura, H., Yoshimoto, A., Harada, M., Mori, K., Komatsu, Y., Usui, T., Shimatsu, A., et al. (2000). Ghrelin strongly stimulates growth hormone release in humans. J. Clin. Endocrinol. Metab. 85, 4908–4911. Tanaka, M., Naruo, T., Nagai, N., Kuroki, N., Shiiya, T., Nakazato, M., Matsukura, S., and Nozoe, S. (2003a). Habitual binge/purge behavior influences circulating ghrelin levels in eating disorders. J. Psychiatr. Res. 37, 17–22. Tanaka, M., Naruo, T., Yasuhara, D., Tatebe, Y., Nagai, N., Shiiya, T., Nakazato, M., Matsukura, S., and Nozoe, S. (2003b). Fasting plasma ghrelin levels in subtypes of anorexia nervosa. Psychoneuroendocrinology 28, 829–835. Tomasetto, C., Wendling, C., Rio, M. C., and Poitras, P. (2001). Identification of cDNA encoding motilin related peptide/ghrelin precursor from dog fundus. Peptides 22, 2055–2059. Tschop, M., Smiley, D. L., and Heiman, M. L. (2000). Ghrelin induces adiposity in rodents. Nature 407, 908–913. Tschop, M., Wawarta, R., Riepl, R. L., Friedrich, S., Bidlingmaier, M., Landgraf, R., and Folwaczny, C. (2001a). Post-prandial decrease of circulating human ghrelin levels. J. Endocrinol. Invest. 24, RC19–21. Tschop, M., Weyer, C., Tataranni, P. A., Devanarayan, V., Ravussin, E., and Heiman, M. L. (2001b). Circulating ghrelin levels are decreased in human obesity. Diabetes 50, 707–709. Ukkola, O. (2004). Peripheral regulation of food intake: New insights. J. Endocrinol. Invest. 27, 96–98. Unniappan, S., Lin, X., Cervini, L., Rivier, J., Kaiya, H., Kangawa, K., and Peter, R. E. (2002). Goldfish ghrelin: Molecular characterization

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of the complementary deoxyribonucleic acid, partial gene structure and evidence for its stimulatory role in food intake. Endocrinology 143, 4143–4146. Wang, L., Saint-Pierre, D. H., and Tache, Y. (2002). Peripheral ghrelin selectively increases Fos expression in neuropeptide Y-synthesizing neurons in mouse hypothalamic arcuate nucleus. Neurosci. Lett. 325, 47–51. Wierup, N., Svensson, H., Mulder, H., and Sundler, F. (2002). The ghrelin cell: A novel developmentally regulated islet cell in the human pancreas. Regul. Pept. 107, 63–69. Wierup, N., Yang, S., McEvilly, R. J., Mulder, H., and Sundler, F. (2004). Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells. J. Histochem. Cytochem. 52, 301–310. Williams, D. L., Grill, H. J., Cummings, D. E., and Kaplan, J. M. (2003). Vagotomy dissociates short- and long-term controls of circulating ghrelin. Endocrinology 144, 5184–5187. Wortley, K. E., Anderson, K., Garcia, K., Murray, J., Malinova, L., Liu, R., Moncrieffe, M., Thabet, K., Cox, H., Yancopoulos, G. D., et al. (2004a). Deletion of ghrelin reveals no effect on food intake, but a primary role in energy balance. Obes. Res. 12, 170. Wortley, K. E., Anderson, K. D., Garcia, K., Murray, J. D., Malinova, L., Liu, R., Moncrieffe, M., Thabet, K., Cox, H. J., Yancopoulos, G. D., et al. (2004b). Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc. Natl. Acad. Sci. USA 101, 8227–8232. Wren, A. M., Seal, L. J., Cohen, M. A., Brynes, A. E., Frost, G. S., Murphy, K. G., Dhillo, W. S., Ghatei, M. A., and Bloom, S. R. (2001a). Ghrelin enhances appetite and increases food intake in humans. J. Clin. Endocrinol. Metab. 86, 5992. Wren, A. M., Small, C. J., Abbott, C. R., Dhillo, W. S., Seal, L. J., Cohen, M. A., Batterham, R. L., Taheri, S., Stanley, S. A., Ghatei, M. A., and Bloom, S. R. (2001b). Ghrelin causes hyperphagia and obesity in rats. Diabetes 50, 2540–2547. Yildiz, B. O., Suchard, M. A., Wong, M. L., McCann, S. M., and Licinio, J. (2004). Alterations in the dynamics of circulating ghrelin, adiponectin, and leptin in human obesity. Proc. Natl. Acad. Sci. USA 101, 10434–10439.

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3 Mechanisms of Signal Transduction Utilized by Growth Hormone FARHAD SHAFIEI,* ADRIAN C. HERINGTON,† and PETER E. LOBIE* *The Liggins Institute and National Research Centre for Growth and Development, The University of Auckland, Auckland, New Zealand, and †School of Life Sciences, Queensland University of Technology, Brisbane QLD 4001, Australia

I. Introduction II. The Growth Hormone Receptor III. Signal Transduction Pathways Activated by Growth Hormone IV. Conclusion References

GH receptor, potently activated JAK2, and stimulated the phosphorylation of tyrosine residues within JAK2 and the cytoplasmic domain of the GH receptor (Argetsinger et al., 1993). In the intervening decade, numerous signaling molecules have been identified that are activated in response to GH. Activation of these molecules results in various biochemical and biological outcomes, which are rapidly being elucidated and, ultimately, provide a basis for understanding GH action.

In recent years, significant progress has been made in elucidating the signaling pathways activated by the growth hormone (GH) receptor. This chapter provides a discourse on the structural and functional characteristics of the GH receptor, as well as the signaling pathways demonstrated to be utilized by GH. Activation of these pathways results in various biochemical and biological outcomes, which are rapidly being elucidated and ultimately provide a basis for understanding GH action. Accordingly, c-Src, in addition to JAK2, has emerged as a key component of GH signaling, with the ability to mediate the activation of distinct signaling pathways independently of JAK2. Together with the occurrence of both positive and negative regulators, this adds further complexity to the signal transduction mechanisms utilized by GH.

II. THE GROWTH HORMONE RECEPTOR A. The Growth Hormone Receptor Superfamily The GH receptor locus maps to the proximal short arm of human chromosome 5, region p13.1-p12 (Barton et al., 1989). The cDNA for the human GH receptor encodes a 638 amino acid protein with single extracellular, transmembrane, and cytoplasmic domains. Numerous receptors, including the GH receptor, were shown to have limited amino acid homology in a region of the extracellular domain, prompting the classification of a new cytokine receptor superfamily, of which the GH receptor was the first identified member (Cosman et al., 1990; Ihle et al., 1995). Other members of the class I cytokine receptor superfamily include prolactin, leptin, erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor

I. INTRODUCTION In 1993, the growth hormone (GH) receptor was first observed to bind to the tyrosine kinase, Janus kinase 2 (JAK2). GH increased the affinity of JAK2 for the

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(GM-CSF), neurotrophic factor (CNTF), thrombopoietin, and interleukins (IL) 2–7, IL-9, IL-11, and IL-12. A second class of more distantly related cytokine receptors has been classified and includes the interferon ␣, ␤, and ␥ receptors and the IL-10 receptor (Cosman et al., 1990; Ihle et al., 1995). Characteristics of the members of the class I receptor superfamily include the following (Bazan, 1989; Patthy, 1990): (i) presence of a single putative transmembrane domain, (ii) limited amino acid homology (14–44%) in a region spanning approximately 210 amino acids in the extracellular domain, (iii) three conserved pairs of cysteine residues in the extracellular domain and a conserved tryptophan residue adjacent to the second cysteine in the N-terminal fibronectin domain, (iv) a WSXWS (Trp, Ser, Any, Trp, Ser)-like motif in the juxta-membrane fibronectin domain (YXXFS in the mammalian GH receptor) (Waters, 1997), (v) the absence of a canonical tyrosine kinase consensus sequence, and (vi) two proline-rich motifs (termed box 1 and box 2) in the membrane-proximal region of the cytoplasmic domain (Fig. 1). A single arginine at residue 43 of the extracellular domain determines the specificity of human GH (hGH) to bind to the hGH receptor (Souza et al., 1995). Box 1 is 8 amino acid residues in length and is located within 20 residues of the transmembrane domain. Box 1 is one site

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FIGURE 1 Schematic structure of the GH receptor. Features of the GH receptor include a WSXWS-like motif, three conserved pairs of cysteine (C) residues and several N-glycosylation sites (N) in the extracellular domain, and a transmembrane domain, as well as two proline-rich boxes and numerous tyrosine (Y) residues within the intracellular domain.

of association of JAK2 with the GH receptor and is critical for many GH-stimulated cellular functions (Goujon et al., 1994; VanderKuur et al., 1994; Frank et al., 1995). Box 2 comprises a cluster of approximately 15 hydrophobic and acidic amino acid residues and is located 30 residues distal to box 1. Deletion or mutation of these boxes abrogates both JAK activation and subsequent signal transduction pathways responsible for GH-stimulated cell proliferation (Dinerstein et al., 1995; Tanner et al., 1995). The growth hormone-binding protein (GHBP) is a soluble, circulating form of the GH receptor (Herington et al., 1986). It is differentially secreted among species and is produced by two different mechanisms: by alternative splicing in mice and rats (Baumbach et al., 1989), resulting in the addition of a unique carboxyl-terminal hydrophilic “tail” sequence, and by proteolysis of the membrane-bound form of the receptor in other species (Sotiropoulos et al., 1993). At least one of the proteases involved in this cleavage is the metalloprotease “sheddase” ADAM-17 (Zhang et al., 2000), which has been shown to cleave the GH receptor at a site eight residues from the membrane in the proximal extracellular domain (Wang et al., 2002). Some C-terminally truncated, but still membrane-anchored isoforms of the GH receptor have been described in humans (Dastot et al., 1996; Ross et al., 1997) and these appear to have a greater susceptibility to juxta-membrane proteolysis. The GHBP is capable of binding GH with high affinity in the circulation and in the extracellular environment. It has no known direct biological effect on cells nor any GH signaling capacity; however, it has been shown to significantly modulate (inhibit) cellular GH action (Lim et al., 1990) via one of two presumed mechanisms: (i) direct sequestration of GH away from the cell membrane-bound GH receptor and (ii) heterodimerization with the full-length, wild-type receptor on the cell surface (Herington, 1994). As indicated in Sections II,B and III,A, dimerization of two full-length receptor molecules is required to generate normal GH receptor signaling within the cell and hence the truncated GHBP can act as a “dominant-negative” form of the receptor (Ross et al., 1997). Studies by Graichen et al. (2003) have indicated that endogenous GHBP can be actively translocated to the cell nucleus and can act as a STAT5-mediated transcriptional enhancer, thereby raising the possibility that it may play a direct transcriptional role via interaction with nuclear proteins or DNA.

B. Receptor Dimerization The use of X-ray crystallography has enabled visualization of the three-dimensional structure of the GH receptor extracellular domain and the regions that interact with GH (DeVos et al., 1992). A single GH molecule binds to two molecules of the GH receptor, leading to receptor homodimerization. GH binding to the two GH receptors

3. Mechanisms of Signal Transduction Utilized by GH

is thought to be sequential. The initial step is “high-affinity” (site 1) binding of GH to one GH receptor. A different face of GH then contacts the second GH receptor via “lower affinity” (site 2) binding. The contact with hGH at site 2 and a dimerization interface of approximately 500 Å between the two extracellular domains of the receptor stabilize the binding of the second receptor to the complex (Wells, 1996). Dimerization of the GH receptor does not produce major conformational changes in the extracellular domain (Clackson et al., 1998). At present, the structure of the cytoplasmic domain of the GH receptor is not well understood.

III. SIGNAL TRANSDUCTION PATHWAYS ACTIVATED BY GROWTH HORMONE Due to lack of intrinsic kinase activity, members of the cytokine receptor superfamily recruit and/or activate cytoplasmic tyrosine kinases to relay their cellular signals (Liu et al., 1998). This section reviews the nonreceptor tyrosine kinases demonstrated to be activated by GH and comments on their possible function in GH signal transduction.

A. JAKs The Janus family of tyrosine kinases is thought to be the predominant nonreceptor tyrosine kinases required for the initiation of GH signal transduction upon ligand binding to the receptor (Argetsinger et al., 1993; Foster et al., 1998). Members of the JAK family to date include JAK1, JAK2, JAK3, and Tyk2 (Ihle et al., 1995). They are expressed widely, except for JAK3, which is found primarily in hematopoietic cells. The predominant JAK utilized by the GH receptor is JAK2 (Argetsinger et al., 1993), although GH has been reported to induce the tyrosine phosphorylation of JAK1 (Smit et al., 1996) and JAK3 (Johnston et al., 1994) as well. The unique structural features of JAK kinases are the absence of SH2 or SH3 domains and the presence of seven conserved JH regions (JH1–JH7), of which JH1 is a functional catalytic domain and JH2 is a pseudokinase domain. The pseudokinase domain negatively regulates the activity of JAK2, presumably via interaction with the kinase domain, and is required to maintain JAK2 in an inactive state in the absence of a stimulus (Saharinen et al., 2000). Box 1 is the only cytoplasmic region conserved among all members of the cytokine receptor superfamily known to utilize JAK2 in signaling. This makes Box 1 an obvious candidate for the site of receptor–JAK2 interaction. Indeed, mutation or deletion of box 1 in the GH receptor abolishes GH receptor–JAK2 complex formation and GH-dependent activation of JAK2 (Finidori, 2000). Although box 1 is

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sufficient for activation of JAK2, maximal activation is achieved by the involvement of more distal residues, which help stabilize the interaction of JAK2 with the GH receptor. The box 1 region of the GH receptor is not a canonical proline-rich domain, and JAKs possess no SH3 domain (which typically binds proline-rich domains). Thus, interaction of the JAK–GH receptor could be mediated by an SH3 domain-containing adaptor protein (Finidori, 2000). Our current understanding of GH activation of JAK2 is that dimerization of the GH receptors brings two JAK2 molecules into close proximity so that each JAK2 can “cross-phosphorylate” the activating tyrosine residues of the other JAK2 molecule, locking JAK2 in an active conformation. The activated JAK2 in turn phosphorylates itself and the cytoplasmic domain of the GH receptor on tyrosine residues. Within the context of the surrounding amino acids, these tyrosines are thought to provide independent or unique docking sites for a variety of signaling molecules that contain SH2 or other phosphotyrosine-binding (PTB) motifs. The molecules recruited to the GH receptor following GH stimulation include Src homology 2/␣ collagenrelated (SHC) (VanderKuur et al., 1995), insulin receptor substrate (IRS) proteins (Souza et al., 1994; Yamauchi et al., 1998; Liang et al., 1999), SHP-1 (SH2 domaincontaining protein tyrosine phosphatase) (Hackett et al., 1997; Ram and Waxman, 1997; Yin et al., 1997), SHP-2 (Ram and Waxman, 1997; Kim et al., 1998), SIRP␣ (Kim et al., 1998; Stofega et al., 1998), p125FAK (Zhu et al., 1998; Takahashi et al., 1999; Ryu et al., 2000), p85 subunit of phosphoinositide-3 (PI-3) kinase (Moutoussamy et al., 1998), epidermal growth factor receptor (EGFR) (Yamauchi et al., 1997), Grb10 (Moutoussamy et al., 1998), carboxyl-terminal Src kinase (Csk) (Moutoussamy et al., 1998), SH2-B␤ (Rui et al., 1997, 2000), APS (Dhe Paganon et al., 2004), SOCS-1, SOCS-2, SOCS-3, and CIS (Hansen et al., 1999; Ram et al., 1999), PLC␥ (Moutoussamy et al., 1998), and the STAT1, STAT3, and STAT5 molecules (Smit et al., 1996; Sotiropoulos et al., 1996; Yi et al., 1996; Wood et al., 1997). Other JAK2interacting intracellular signaling proteins that undergo tyrosine phosphorylation upon cytokine/growth factor stimulation, and therefore could be potential targets downstream of JAK2 in GH signaling, include Raf-1 (Xia et al., 1996), Tec (Takahashi-Tezuka et al., 1997), signaltransducing adaptor molecule (STAM) (Takeshita et al., 1997), STAM2 (Endo et al., 2000), Vav (Shigematsu et al., 1997), and Lyn (Chin et al., 1998). The recruitment and subsequent activation of nonreceptor kinases lead to the activation of various major groups of signaling molecules. These include (1) members of the mitogen-activated protein kinase (MAPK) family, including p44/42 MAPK, p38 MAPK, and JNK/SAPK (Zhu et al., 1998a; Zhu and Lobie, 2000); (2) members of the insulin

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JAK2

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FIGURE 2 A simplified diagrammatic representation of the mechanism of GH signal transduction. GH activation of either JAK2 or c-Src leads to the activation of various receptor and nonreceptor kinases and subsequent activation of the various major signaling cascades, including MAPKs, IRSs, PI-3 kinase, small Ras-like GTPases, and STATs. (See color plate 1)

receptor substrate (IRS) group, including IRS-1, IRS-2, and IRS-3, which may act as docking proteins for further activation of signaling molecules, including PI-3 kinase (Yamauchi et al., 1998); (3) small Ras-like GTPases, including Ras, Rac, RhoA, RalA, RalB, Rap1, and Rap2 (Winston and Hunter, 1995; Ling and Lobie, 2004); and (4) STAT family members, including STAT1, STAT3, STAT5a, and STAT5b (Liu et al., 1998), which constitute one major mechanism for transcriptional regulation by GH (Fig. 2).

B. The Src Family Although almost all downstream signaling pathways activated by GH had been reported to require JAK2 activity, increasing evidence has emerged demonstrating that the Src kinase family of protein tyrosine kinases can activate and phosphorylate a series of signaling molecules in lieu of JAK kinases (Reddy et al., 2000; Zhu et al., 2002). We also observed that the inhibitor of the Src family of kinases, PP2, restored plakoglobin expression in a mammary carcinoma cell line stably transfected with the hGH gene (MCF-hGH), whereas the JAK2 inhibitor AG490 failed to alter the expression of plakoglobin (Mukhina et al., 2004). This suggests that autocrine hGH regulation of plakoglobin is JAK2 independent and predominantly requires the activation of Src kinases. The downstream substrates of c-Src include focal adhesion kinase (FAK), p130Cas (Schlaepfer and Hunter, 1998), and c-Cbl (Tanaka et al., 1996). We have demonstrated previously that GH stimulates c-Src and c-Fyn activity directly via the GH receptor, which subsequently triggers activation of a multiprotein complex centered around CrkII and p130Cas (Zhu et al., 1998a). GH induces the tyrosine phosphorylation of both p130Cas and CrkII and stimulates the assembly of multiple other tyrosine-phosphorylated proteins into the complex. These include c-Src, c-Fyn, tensin, paxillin,

IRS-1, the p85 subunit of PI-3 kinase, C3G, SHC, Grb-2, and Sos-1. c-Cbl and Nck are also tyrosine-phosphorylated and associate with the p130Cas–CrkII complex following cellular stimulation with hGH. The JNK/SAPK pathway is activated in response to hGH in accordance with formation of the p130Cas–CrkII complex (Zhu et al., 1998a). It has also been postulated that c-Src, bound through its SH2 domain to phosphorylated FAK, facilitates c-Src SH3 domain interactions with p130Cas, thereby promoting the formation of a ternary complex of c-Src-FAK-p130Cas. Thus, c-Src may be critical for the initial and JAK2-independent formation of the multiprotein signaling complex stimulated by GH (Zhu et al., 1998a). Other signaling molecules activated by c-Src independently of JAK2 have been reported and are discussed in Section III, E.

C. FAK p125FAK has been postulated to play a central role in the response of the cell to the extracellular matrix (ECM) (Guan and Chen, 1996; Cary and Guan, 1999). GH has been shown to stimulate reorganization of the actin cytoskeleton in cells with fibroblastic morphology (Goh et al., 1997) and to activate FAK. This GH-mediated activation results in tyrosine phosphorylation of two FAK-associated substrates: paxillin and tensin (Zhu et al., 1998b). GH stimulation of FAK and the subsequent changes in organization of the actin cytoskeleton have also been demonstrated, in diverse cell types, by other investigators (Takahashi et al., 1999; Ryu et al., 2000). FAK activation does require the proline-rich box 1 region of the GH receptor, indicating that FAK is downstream of JAK2 (Zhu et al., 1998b). FAK associates with JAK2 but not JAK1 after cellular stimulation with GH (Zhu et al., 1998b), and use of the JAK2-specific inhibitor AG490 prevents GH-stimulated tyrosine phosphorylation of FAK (Takahashi et al., 1999). Thus, the use of FAK by GH provides a possible mechanism for the interaction of cytokine signaling pathways and those utilized by the ECM. GH activation of FAK permits the GH signal to be propagated through multiple alternative signal transduction pathways. For example, the p85 subunit of PI-3 kinase can be tyrosine phosphorylated by FAK in vitro (Chen and Guan, 1994), suggesting that PI-3 kinase may be a substrate of FAK in vivo. The association of FAK and PI-3 kinase is direct and dependent on FAK autophosphorylation (Chen and Guan, 1994; Schlaepfer and Hunter, 1997). It is therefore possible that GH may utilize the FAK–PI-3 kinase pathway, in addition to the IRS–PI-3 kinase pathway, to increase phosphatidylinositol 3,4,5-triphosphate levels within the cell. GH has been reported to utilize IRS for activation of PI-3 kinase (Ridderstrale et al., 1995; Dominici and Turyn, 2002). Such potential utilization of two alternative pathways to activate the same kinase may permit the use of PI-3 kinase for distinct cellular purposes. For

3. Mechanisms of Signal Transduction Utilized by GH

example, the IRS-1-associated PI-3 kinase may be involved in GH stimulation of metabolic events such as lipogenesis (Ridderstrale and Tornqvist, 1994), whereas activation of PI-3 kinase via FAK may regulate GH-stimulated cytoskeletal reorganization (Goh et al., 1997). The alternative use of pathways would allow the cell to respond precisely to hormonal stimuli dependent on cell type and differentiation status. The Src family of protein tyrosine kinases may also be essential for FAK-mediated signaling events. For example, overexpression of the mutated c-Src-binding site of FAK (Phe 397) inhibits fibronectin-stimulated signaling to p44/42 MAPK (Schlaepfer and Hunter, 1998). Given that formation of the FAK–c-Src complex enhances FAK kinase activity and promotes Grb2 binding (Schlaepfer and Hunter, 1998), a potential pathway for the activation of p44/42 MAPK by GH may be through a c-Src–FAK–Grb2 complex.

D. Ras-like Small GTPases The Ras-like small GTPase family contains more than a hundred 20- to 30-kDa proteins that function as molecular switches in the regulation of diverse cellular functions, including cell proliferation/differentiation, cytoskeleton organization, and intracellular membrane trafficking (Takai et al., 2001). GH has been demonstrated to activate some of the Ras-like small GTPases. In particular, Ras is required for GH receptor JAK2-mediated activation of p44/42 MAPK (Winston and Hunter, 1995; Pandey et al., 1999). As expected from other receptor signaling pathways, GH stimulation of cells results in the assembly of a SHC–Grb2–SOS complex with resultant activation of Ras and subsequent engagement of the Raf–MEK pathway (VanderKuur et al., 1997). GH-stimulated IRS-1-associated PI-3 kinase activity has also been reported to be required for GH-stimulated Ras activation (Ling et al., 2000). GH has been reported to activate Rac, a Rho subfamily small GTPase, via a Src homology 2 (SH2) domain-containing protein, SH2-B␤ (Diakonova et al., 2002). SH2-B␤ facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating Rac effectors such as PAK (Kiosses et al., 1999; Sells et al., 1999) or Rac modulatory proteins such as DOCK 180 (Kiyokawa et al., 1998). The Ras-like small GTPases may also interact with other regulatory pathways utilized by GH. Ling and Lobie (2004) demonstrated a novel mechanism by which RhoA can regulate Stat5-mediated transcription. GH stimulates the activation of RhoA and its substrate Rho kinase (ROCK) in NIH-3T3 cells. Formation of GTP-bound RhoA requires JAK2-dependent dissociation of RhoA from its negative regulator, p190 RhoGAP. The inactivation of RhoA does not affect GH-stimulated JAK2 tyrosine phosphorylation nor p44/42 MAPK activity. However, RhoA and ROCK activities are required for GH-stimulated, Stat5-mediated

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transcription. Histone deacetylase 6 (HDAC6) activity, recruited by transcription cofactor p300, negatively regulates GH-stimulated, Stat5-mediated transcription. GH stimulates Stat5-mediated transcription by preventing HDAC6 recruitment by p300 through activation of RhoA–ROCK. The same study identified PKA as a negative regulator of GH-stimulated, Stat5-mediated transcription, and this cellular effect of PKA requires the serine residue 188 of RhoA (Ling and Lobie, 2004).

E. GH May Utilize c-Src to Activate Ras-like Small GTPases We have demonstrated that activation of the Ras-like small GTPase molecules RalA and RalB by GH is mediated by c-Src alone and does not require the activity of JAK2 per se (Zhu et al., 2002). However, full activation of RalA and RalB by GH did require the combined activity of both c-Src and JAK2, with both kinases activated by GH independently of the other. Activation of RalA by GH subsequently resulted in the activation of phospholipase D (PLD) and the formation of phosphatidic acid (PA) that was required for activation of the p44/42 MAP kinase by GH. RalA has been demonstrated to associate directly with PLD1 via its N-terminal sequence and it operates synergistically with another PLD1-interacting small GTPase, Arf, to activate PLD1 activity (Luo et al., 1998). RalA has been demonstrated previously to mediate activation of PLD in v-Src-transformed cells (Jiang et al., 1995). It is interesting to note that Ral has also been shown to regulate the activity of c-Src in response to cellular stimulation by EGF (Goi et al., 2000). It is therefore possible that Ral participates in regulating the final “output” of the GH-stimulated multiprotein signaling complex, centered around CrkII and containing c-Src, in addition to functioning in the linear pathway described here. c-Src has also been shown to be predominantly utilized by GH to activate both Rap1 and Rap2 small GTPases and thus to negatively modulate GH-stimulated p44/42 MAPK activity and subsequent Elk-1-mediated transcription. This occurs through inhibition of RalA activity (Ling et al., 2003). RalA has also been shown to be activated by c-Src (Zhu et al., 2002) and to mediate GH-stimulated p44/42 MAPK activity by phospholipase D1 (PLD1) activation and subsequent Elk-1-mediated transcription (Fig. 3). Thus, Rap1 is a GH effector molecule that provides a negative feedback mechanism for GH-stimulated p44/42 MAPK activation. GH stimulated Rap1 via activation of C3G, and Rap1 enhanced JNK/SAPK activity and subsequent c-Jun-mediated transcription in response to GH. We have therefore identified a linear JAK2-independent pathway switching GH-stimulated p44/42 MAP kinase and JNK/SAPK activities. C3G is a Rap-specific guanine nucleotide exchange factor (GEF) because it predominantly

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FIGURE 3 A proposed signal transduction model for JAK2-dependent and -independent pathways. Solid arrows indicate flow of activation, whereas dashed arrows indicate potential activation of pathways. (See color plate 2)

catalyzes the guanine nucleotide exchange reaction for Rap1 and Rap2 (Gotoh et al., 1995; Ohba et al., 2000). CrkII is the major adaptor for C3G (Matsuda et al., 1992). CrkII has been identified as a mediator of Src-dependent Rap1 activation (Xing et al., 2000), and we have demonstrated previously that CrkII is constitutively associated with C3G (Zhu et al., 1998a). Thus, CrkII-C3G possesses a pivotal role in GH signal transduction. Further support for a role of c-Src in GH signal transduction comes from the ability of Csk (Src-inactivating kinase) to inhibit GH-stimulated p44/42 MAP kinase activity (Gu et al., 2001). This observation is likely to be mediated by the Src-Ral-PLD pathway discussed here (Fig. 3). To date, Ras and Ral, the other two close relatives of Rap, together with Rac have been reported to participate in GH signal transduction. Based on our current understanding of the molecules involved in JAK2-dependent and -independent GH signaling, we have proposed a model of GH signaling pathways leading to MAPK activation (Fig. 3).

F. STATs The most studied substrates of JAKs are the STAT proteins (Liu et al., 1998). These are recruited to the proximity of the activated JAKs by binding, through their SH2 domains, to phosphotyrosine-containing motifs on the

cytoplasmic part of the GH receptor. Following tyrosine phosphorylation by JAKs and subsequent serine or threonine phosphorylation, the activated STAT molecules dissociate from the receptor as either homo- or heterodimers via a reciprocal SH2 domain–phosphotyrosine interaction and translocate to the nucleus. Here they bind to their appropriate DNA response elements within responsive gene promoters and stimulate transcription. STAT5 is the predominant STAT utilized by GH. The two forms of STAT5 (STAT5a and STAT5b) are encoded by two different genes, which exhibit ⬃90% identity in coding sequence and possess both overlapping and distinct functions in GH signal transduction (Shuai, 1999; Herrington et al., 2000). GH has been shown to utilize STAT5 for transcription of the insulin gene in insulinoma cells (Fleenor and Freemark, 2001), and a dominant-negative STAT5 mutant blocks GH-stimulated proliferation of islet ␤ cells in culture (Friedrichsen et al., 2001). STAT5b gene knockout studies in mice have defined the role of STAT5b in GH action, as disruption of this gene revealed several striking phenotypes that are visible in STAT5b-deficient males but do not manifest in STAT5bdeficient females (Udy et al., 1997). First, there was a global loss of GH-regulated, male-specific liver gene expression, including male-specific cytochrome P450 gene expression. Second, the expression of female-specific, GH-regulated liver cytochrome P450 enzymes is elevated

3. Mechanisms of Signal Transduction Utilized by GH

to near-normal female levels in STAT5-deficient male mice. Thus, the overall pattern of sexually dimorphic liver gene expression requires the presence of STAT5b and becomes feminized in its absence. This suggests that STAT5b has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute. In addition to SOCS (see Section III,G), GH may also limit the STAT response by the utilization of specific signaling molecules. Indeed, we have demonstrated that c-Cbl, a component of the multiprotein signaling complex centered around CrkII and p130Cas, is a negative regulator of STAT5-mediated transcription (Goh et al., 2002). c-Cbl overexpression in NIH-3T3 cells resulted in increased ubiquitination and proteosomal degradation of STAT5 and increased degradation of GH-stimulated, tyrosinephosphorylated STAT5 (Goh et al., 2002). Similarly, HDAC6 is recruited by transcription cofactor p300 and negatively regulates GH-stimulated, Stat5-mediated transcription (Ling and Lobie, 2004).

G. SOCS Suppressors of cytokine signaling (SOCS) are feedback inhibitors of action induced by cytokines, including interleukins and interferons, as well as cytokine hormones such as GH, prolactin, and leptin (Nicola and Greenhalgh, 2000). There are eight currently identified SOCS proteins, characterized by a variable N-terminal protein–protein interaction domain, a central SH2 domain, and a C-terminal conserved 40 amino acid motif known as the SOCS box (Hilton et al., 1998). Current models for the mechanisms of SOCS protein function include direct inhibition of JAK-STAT signaling, as well as targeting of signaling molecules for degradation via ubiquitination mechanisms involving elongin C. The SOCS box mediates the binding of SOCS to elongin C, a component of a multisubunit E3 ubiquitin ligase (Kile et al., 2002). The mechanisms of action of various SOCS proteins in GH signaling have been investigated extensively in vitro. Overexpression of SOCS1 and SOCS3, but not SOCS2, has been shown to block GH-induced transactivation of the GH-responsive serine protease inhibitor 2.1 gene promoter, with JAK2 activity being predominantly repressed in these experiments (Adams et al., 1998; Hansen et al., 1999). Similarly, complete inhibition of GH-induced STAT5bdependent transcriptional activity was observed in SOCS3 overexpression studies (Ram and Waxman, 1999). Analysis of the interaction of GH receptor/SOCS3 indicates that either Tyr487 (Hansen et al., 1999) or Tyr332 (Ram and Waxman, 1999) of the GH receptor is the binding site from which these effects are mediated. The SOCS, cytokineinducible SH2 protein (or CIS), has also been shown to be an effective inhibitor of GH-induced STAT5 activity and it is thought to interact directly with the intracellular domain

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of the GH receptor (Hansen et al., 1999; Ram and Waxman, 1999, 2000). The role of SOCS2 in GH signaling is less defined. Lower levels of SOCS2 have been shown to inhibit approximately half of all GH-induced STAT5 activity, whereas higher concentrations result in enhancement of signaling (Favre et al., 1999; Greenhalgh et al., 2002b). The mechanism by which this phenomenon occurs is unclear, but SOCS2 has been found to bind directly to phosphorylated Tyr595 on the GH receptor (Greenhalgh et al., 2002b). Interestingly, this site has also been shown to bind SHP-2 (Stofega et al., 2000). It has been proposed that the dual action of SOCS2 might reflect its concentration-dependent competition with both positive and negative regulators (perhaps including other SOCS) of GH signaling for activated receptor-binding sites, although little direct evidence has yet accumulated in support of this model. GH has been shown to induce SOCS2 expression, suppress intestinal epithelial cell proliferation, and inhibit intestinal growth (Miller et al., 2004). Gene knockout studies have provided definitive evidence that SOCS2 is a negative regulator of GH actions in growth regulation. SOCS2-deficient mice (Socs2⫺/⫺) exhibited accelerated postnatal growth that resulted in an increase in adult body weight of 40% in males and 30% in females (Metcalf et al., 2000). Socs2⫺/⫺ mice had significantly thickened dermal layers characterized by increased collagen deposition, as well as some collagen deposits found in ducts and vessels throughout the body. However, further analysis of Socs2⫺/⫺ mice has failed to find any significant alteration in GH levels to account for the excess growth. Rather, STAT5b has been shown to be a key factor in mediating a large proportion of the excess growth observed in Socs2⫺/⫺ mice (Greenhalgh et al., 2002a). SOCS2 regulation of GH action is not restricted to metabolic activities. SOCS2 expression is found in neural stem cells and neurons, and mice deficient in SOCS2 had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex. It has been suggested that SOCS2 acts to block GH inhibition of stem cell differentiation processes and thus may be an important regulator of neuronal type and number (Turnley et al., 2002). SOCS1 knockout mice have a complex pathology characterized by fatty degeneration of the liver and immune cell infiltration into many organs and tissues, usually dying within 3 weeks of birth (Starr et al., 1998). However, there is no evidence from gene knockout studies that SOCS1 is required for normal regulation of GH action in vivo (Greenhalgh and Alexander, 2004). While GH utilizes SOCS2 and potentially other SOCS proteins in a classic negative feedback mechanism, evidence has emerged that induction of SOCS expression by other stimuli may also influence GH activity. For example, IL-6 has been reported to inhibit liver GH signaling by inducing CIS and SOCS3 (Denson et al., 2003). This may

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potentially explain GH resistance in inflammatory disease. Similarly, induction of SOCS2 by estrogen has been reported to inhibit GH signaling (Leung et al., 2003), and mechanisms involving the induction of SOCS proteins have also been proposed to explain endotoxin- and TNF␣–mediated insensitivity to GH (Wang et al., 2002a; Wang et al., 2002b). These interactions raise the prospect that SOCS proteins may mediate cross talk between independent signaling pathways and provide a mechanism by which concurrent signaling processes can modulate each other.

IV. CONCLUSION Rapid progress has been made in the definition of the GH receptor signal transduction pathways and the many molecules activated by the action of GH. It is now apparent that many cytokines, including GH, share identical or similar signaling components in exerting their cellular effects. Although many of these cellular effects are pleiotropic and/or common to the cytokine receptor superfamily, some level of specificity does exist. The mechanisms by which some of the specific cellular effects of GH are achieved have been discussed, and c-Src has emerged as a key component of GH signaling, diminishing the notion that JAK2 is the predominant mediator of GH signaling.

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Sotiropoulos, A., Moutoussamy, S., Renaudie, F., Clauss, M., Kayser, C., Gouilleux, F., Kelly, P. A., and Finidori, J. (1996). Differential activation of Stat3 and Stat5 by distinct regions of the growth hormone receptor. Mol. Endocrinol. 10, 998–1009. Souza, S. C., Frick, G. P., Wang, X., Kopchick, J. K., Lobo, R. B., and Goodman. H. M. (1995). A single arginine residue determines species specificity of the human growth hormone receptor. Proc. Natl. Acad. Sci. USA 92, 959–963 Souza, S. C., Frick, G. P., Yip, R., Lobo, R. B., Tai, L. R., and Goodman, H. M. (1994). Growth hormone stimulates tyrosine phosphorylation of insulin receptor substrate-1. J. Biol. Chem. 269, 30085–30088. Starr, R., Metcalf, D., Elefanty, A. G., Brysha, M., Willson, T. A., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (1998). Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc. Natl. Acad. Sci. USA 95, 14395–14399. Stofega, M. R., Wang, H., Ullrich, A., and Carter-Su, C. (1998). Growth hormone regulation of SIRP and SHP-2 tyrosyl phosphorylation and association. J. Biol. Chem. 273, 7112–7117. Stofega, M. R., Herrington, J., Billestrup, N., and Carter-Su, C. (2000). Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol. Endocrinol. 14, 1338–1350 Takahashi, M. O., Takahashi, Y., Iida, K., Okimura, Y., Kaji, H., Abe, H., and Chihara, K. (1999). Growth hormone stimulates tyrosine phosphorylation of focal adhesion kinase (p125(FAK)) and actin stress fiber formation in human osteoblast-like cells, Saos2. Biochem. Biophys. Res. Commun. 263, 100–106. Takahashi-Tezuka, M., Hibi, M., Fujitani, Y., Fukuda, T., Yamaguchi, T., and Hirano, T. (1997). Tec tyrosine kinase links the cytokine receptors to PI-3 kinase probably through JAK. Oncogene 14, 2273–2282. Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins. Physiol. Rev. 81, 153–208. Takeshita, T., Arita, T., Higuchi, M., Asao, H., Endo, K., Kuroda, H., Tanaka, N., Murata, K., Ishii, N., and Sugamura, K. (1997). STAM, signal transducing adaptor molecule, is associated with Janus kinases and involved in signaling for cell growth and c-myc induction. Immunity 6, 449–457. Tanaka, S., Amling, M., Neff, L., Peyman, A., Uhlmann, E., Levy, J. B., and Baron, R. (1996). c-Cbl is downstream of c-Src in a signaling pathway necessary for bone resorption. Nature 383, 528–531. Tanner, J. W., Chen, W., Young, R. L., Longmore, G. D., and Shaw, A. S. (1995). The conserved box 1 motif of cytokine receptors is required for association with JAK kinases. J. Biol. Chem. 270, 6523–6530. Turnley, A. M., Faux, C. H., Rietze, R. L., Coonan, J. R., and Bartlett, P. F. (2002). Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling. Nature Neurosci. 5, 1155–1162. Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997). Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc. Natl. Acad. Sci. USA 94, 7239–7244. VanderKuur, J., Wang, X., Zhang, L-Y., Campbell, G., Billestrup, N., Norstedt, G., and Carter-Su, C. (1994). Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. J. Biol. Chem. 269, 21709–21717 VanderKuur, J. A., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C. (1995). Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor. J. Biol. Chem. 270, 7587–7593. VanderKuur, J. A., Butch, E. R., Waters, S. B., Pessin, J. E., Guan, K. L., and Carter-Su, C. (1997). Signaling molecules involved in coupling growth hormone receptor to mitogen-activated protein kinase activation. Endocrinology 138, 4301–4307.

3. Mechanisms of Signal Transduction Utilized by GH Wang, P., Li, N., and Li, J. S. (2002a). Mechanism of growth hormone insensitivity induced by endotoxin. Acta Pharmacol. Sin. 23, 16–22. Wang, P., Li, N., Li, J. S., and Li, W. Q. (2002b). The role of endotoxin, TNF-alpha, and IL-6 in inducing the state of growth hormone insensitivity. World J. Gastroenterol. 8, 531–536. Wang, X., He, K., Gerhart, M., Huang, Y., Jiang, J., Paxton, R. J., Yang, S., Lu, C., Menon, R. K., Black, R. A., Baumann, G., and Frank, S. J. (2002). Metalloprotease-mediated GH receptor proteolysis and GHBP shedding: Determination of extracellular domain stem region cleavage site. J. Biol. Chem. 277, 50510–50519. Waters, M. J. (1997) The growth hormone receptor. In “The Handbook of Physiology” (J. L. Kostyo, ed.), Vol. 5, pp. 1301–1348. Oxford Univ. Press, Oxford. Wells, J. A. (1996). Binding in the growth hormone receptor complex. Proc. Natl. Acad. Sci. USA 93, 1–6. Winston, L. A., and Hunter, T. (1995). JAK2, Ras, and Raf are required for activation of extracellular signal-regulated kinase/mitogenactivated protein kinase by growth hormone. J. Biol. Chem. 270, 30837–30840. Wood, T. J., Sliva, D., Lobie, P. E., Gouilleux, F., Mui, A. L., Groner, B., Norstedt, G., and Haldosen, L. A. (1997). Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter. Mol. Cell. Endocrinol. 130, 69–81. Xia, K., Mukhopadhyay, N. K., Inhorn, R. C., Barber, D. L., Rose, P. E., Lee, R. S., Narsimhan, R. P., D’Andrea, A. D., Griffin, J. D., and Roberts, T. M. (1996). The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner. Proc. Natl. Acad. Sci. USA 93, 11681–11686. Xing, L., Ge, C., Zeltser, R., Maskevitch, G., Mayer, B. J., and Alexandropoulos, K. (2000). c-Src signaling induced by the adapters Sin and Cas is mediated by Rap1 GTPase. Mol. Cell. Biol. 20, 7363–7377. Yamauchi, T., Kaburagi, Y., Ueki, K., Tsuji, Y., Stark, G. R., Kerr, I. M., Tsushima, T., Akanuma, Y., Komuro, I., Tobe, K., Yazaki, Y., and Kadowaki, T. (1998). Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and

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concomitantly PI3-kinase activation via JAK2 kinase. J. Biol. Chem. 273,15719–15726. Yamauchi, T., Ueki, K., Tobe, K., Tamemoto, H., Sekine, N., Wada, M., Honjo, M., Takahashi, M., Takahashi, T., Hirai, H., Tsushima, T., Akanuma, Y., Fujita, T., Komuro, I., Yazaki, Y., and Kadowaki, T. (1997). Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone. Nature 390, 91–96. Yi, W., Kim, S. O., Jiang, J., Park, S. H., Kraft, A. S., Waxman, D. J., and Frank, S. J. (1996). Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase. Mol. Endocrinol. 10, 1425–1443. Yin, T., Shen, R., Feng, G. S., and Yang, Y. C. (1997). Molecular characterization of specific interactions between SHP-2 phosphatase and JAK tyrosine kinases. J. Biol. Chem. 272, 1032–1037. Zhang, Y., Jiang, J., Black, R. A., Baumann, G., and Frank, S. J. (2000). Tumor necrosis factor-alpha converting enzyme (TACE) is a growth hormone binding protein (GHBP) sheddase: The metalloprotease TACE/ADAM-17 is critical for (PMA-induced) GH receptor proteolysis and GHBP generation. Endocrinology. 141, 4342–4348. Zhu, T., Goh, E. L., LeRoith, D., and Lobie, P. E. (1998a). Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII: Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). J. Biol. Chem. 273, 33864–33875. Zhu, T., Goh, E. L., and Lobie, P. E. (1998b). Growth hormone stimulates the tyrosine phosphorylation and association of p125 focal adhesion kinase (FAK) with JAK2: Fak is not required for Stat-mediated transcription. J. Biol. Chem. 273, 10682–10689. Zhu, T., Ling, L., and Lobie, P. E. (2002). Identification of a JAK2independent pathway regulating growth hormone (GH)-stimulated p44/42 mitogen-activated protein kinase activity: GH activation of Ral and phospholipase D is Src-dependent. J. Biol. Chem. 277, 45592–45603. Zhu, T., and Lobie, P. E. (2000). Janus kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone: Resultant transcriptional activation of ATF-2 and CHOP, cytoskeletal re-organization and mitogenesis. J. Biol. Chem. 275, 2103–2114.

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4 Insulin-like Growth Factor-I and Its Binding Proteins: Regulation of Secretion and Mechanism of Action at the Receptor Level MARK G. SLOMIANY and STEVEN A. ROSENZWEIG Department of Cell and Molecular Pharmacology and Experimental Therapeutics and Hollings Cancer Center Medical University of South Carolina Charleston, South Carolina 29425

I. INTRODUCTION AND OVERVIEW

I. Introduction and Overview II. Overview of Insulin-like Growth Factor Binding Proteins III. Summary and Future Perspective References

Since its initial description by Salmon and Daughaday (1957) as a “sulfation factor,” insulin-like growth factor-I (IGF-I) has become known as a key mediator growth, particularly during puberty (Bach et al., 1992). Subsequent studies expanded beyond cartilage sulfation to include stimulation of DNA synthesis (Daughaday et al., 1966), proteoglycan synthesis (Hall et al., 1971), and protein synthesis (Salmon et al., 1970), to name a few. A decade later, two separate groups discovered serum fraction with “nonsuppressible insulin-like activity” (NSILA) that could mimic insulin action but could not be inhibited by anti-insulin antibodies (Burgin et al., 1966; Froesch et al., 1966). In 1972, Daughaday and colleagues clarified the concept of the growth hormone (GH)-IGF axis, renaming this “sulfation factor” as “somatomedin” and recognizing its role as an effector of growth hormone or somatotrophin. Indeed, serum IGF-I, produced primarily by the liver, is regulated by GH and mediates many of its growth effects. IGF-I and IGF-2 share ⬇60% homology with each other and ⬇50% homology with insulin. IGF-I is a 70 amino acid, 7649-Da protein with three intramolecular disulfide bonds (Baxter, 1986; Raschdorf et al., 1988). The IGFs are produced by a multitude of cell types and considerable work has been amassed on their local actions and regulation, i.e., autocrine and paracrine effects (D’Ercole et al., 1984). Elevated IGF-I levels have been correlated to a wide variety of ischemic retinal disorders linked to neovascularization of the retina and iris (Meyer-Shwickerath et al., 1993). IGF-I

Since their original discovery as carrier proteins for insulinlike growth factors (IGFs), the IGF-binding protein (IGFBP) family has grown to six members, ranging in size from 216 to 289 amino acids, representing masses of 22.8 to 31.3 kDa. These proteins, which exhibit higher affinities for IGF-I and IGF-2 than the IGFI receptor (IGF-IR), have largely been portrayed as carrier proteins whose function is to block the access of their cognate ligands to the IGF-IR. Although the need for such a large array of regulatory proteins is unique to this family of growth factors, it is consistent with the constitutive release of the IGFs from numerous cell types. This is in marked contrast to the closely related ligand family member insulin, which is stored within pancreatic ␤ cells and whose secretion is tightly regulated by metabolic signals. Accordingly, the family of IGFBPs has evolved to fulfill a regulatory function at the extracellular level. On this basis, it is not surprising that dysregulation of this system in the context of disease can occur via the inappropriate proteolysis of the IGFBPs, resulting in increased levels of free IGFs and a commensurate increase in IGF-IR signaling. Evidence from a number of laboratories has begun to surface, suggesting that additional functions for the IGFBPs exist that are independent of their abilities to bind and sequester the IGFs. Thus many questions concerning the biology of the IGFBPs remain to be answered.

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has also been implicated as an autocrine and/or paracrine growth enhancer in both lung (Nakanishi et al., 1988) and breast (Quinn et al., 1996; Yee et al., 1989) cancer cells in addition to other tumors (reviewed by Macaulay, 1992). IGF-I stimulates proliferation in vitro, but it can also promote other cellular activities such as protein synthesis and secretion (Ayo et al., 1990; Moran et al., 1991; Wolf et al., 1992; Pricci et al., 1996), differentiation (Tollefen et al., 1989; Aron et al., 1992; Quinn et al., 1994), and transformation (Kaleko et al., 1990). IGF-I can prevent apoptosis (Nickerson et al., 1997; Parrizas et al., 1997) and may also act as a progression factor, allowing cells primed by other agents to enter mitosis (Stiles et al., 1979; Doi et al., 1989). All of these actions occur via activation of the IGF-I receptor (IGF-IR). IGF-I has metabolic actions similar to insulin (Froesch et al., 1985; Guler et al., 1987; Boulware et al., 1992), primarily due to common downstream signaling paradigms. High homology exists between the IGF-I receptor and the insulin receptor (IR) (Bolinder et al., 1987); both are disulfide-linked ␣2␤2 heterotetramers belonging to the tyrosine kinase family of growth factor receptors (Humbel et al., 1990). The extracellular ␣ subunits (130 kDa) contain the ligand-binding sites (Kull et al., 1986; Fujita-Yamaguchi et al., 1986), whereas the transmembrane ␤ subunits (95 kDa) have cytoplasmic tyrosine kinase domains (Ullrich et al., 1986). The IGF-IR binds IGF-I, IGF-2, and insulin with decreasing affinity (Bach et al., 1992). IGF-2 consists of 67 amino acids and has a MW of 7470 (Rinderknecht et al., 1978). Unlike IGF-I, which is more important in puberty and adult life, IGF-2 is crucial in fetal development. Also, unlike IGF-I, IGF-2 lacks regulation by GH and nutritional factors (Bach et al., 1992). IGF-2 is highly expressed by many tumors and tumor cell lines (Quinn et al., 1996; Daughaday et al., 1988; El Badry et al., 1989, 1990; Qing et al., 1996) and because IGF-2 has significant affinity for the IGF-IR, it is thought to stimulate tumor cell growth by activating the IGF-IR (Quinn et al., 1996). Indeed, most cellular actions of IGF-I and IGF-2 are mediated through the IGF-IR (Nissley et al., 1991). Although little is known about the signaling actions of the IGF-2 receptor (IGF-2R; mannose-6-phosphate receptor (Kiess et al., 1988), it is thought to be involved in tissue remodeling (Nissley et al., 1991) and compensatory hypertrophy (Hartshorn et al., 1989; Polychronakos et al., 1985). It is a ⬇274-kDa (Morgan et al., 1987) transmembrane receptor shown to stimulate calcium influx via activation of a pertussis toxin-sensitive G-protein (Nishimoto et al., 1989, 1993; Ikezu et al., 1995), leading to the generation of inositol trisphosphate and diacylglycerol (Rogers et al., 1988).

A. Signaling The type I IGF receptor (IGFR1) mediates the mitogenic signaling of both IGF-I and IGF-II (Cullen et al., 1990).

It is composed of two extracellular subunits and two transmembrane ␤ subunits. Upon ligand binding, the kinase domain of the receptor autophosphorylates tyrosine residues located in the intracellular domains of the ␤ subunit, which is an essential step in the activation cascade (Kato et al., 1993, 1994; Gronberg et al., 1993). Cellular scaffold proteins bind to the autophosphorylation sites and are phosphorylated on multiple tyrosine residues by the activated receptor kinase (Nissley et al., 1991). Most intracellular signals are assembled around the tyrosine-phosphorylated scaffold proteins, which include the IRS proteins as well as SHC, APS, and SH2B, and GAB1/2, DOC1/2, and CBL (reviewed by White, 2002). It should be noted, however, that additional molecules [IRS-2, Shc, Crk, Gab10, GrbIR/Grb10, p85, and phosphatidylinositol 3-kinase (PI3 kinase)] have all been shown to interact directly with the receptor (reviewed by Jackson et al., 1998). The IGF-IR also stimulates phosphorylation of SHPS-1, critical for SHP-2 PTPase recruitment to the IGF-IR and subsequent receptor dephosphorylation (Maile et al., 2002). IRS-1 and ⫺2 define a family of at least six multipotential signaling proteins that lack intrinsic catalytic activity but are composed of multiple interaction domains and phosphorylation motifs. IRS proteins bind multiple SH2 domain-containing signaling proteins, including Grb2, the p85 regulatory subunit of PI 3-kinase, Syp, Crk, Fyn, SHP-2, and Nck (reviewed by White et al., 1997). Two smaller members of the family include Gab1 (Grb-2associated binder-1) in mammalian cells and DOS (Daughter of Sevenless) in Drosophila. All IRS proteins are characterized by the presence of an NH2-terminal PH domain adjacent to a PTB domain, followed by a variablelength COOH-terminal tail that contains numerous tyrosine and serine phosphorylation sites in various amino acid motifs that bind distinct SH2 proteins, including p85 isoforms (PI-3 kinase), Grb-2, SHP2, nck, crk, fyn, and others (reviewed by Yenush et al., 1997). IRS proteins have several important features for receptor signaling. First, they amplify receptor signals by eliminating the stoichiometric constraints encountered by receptors that directly recruit SH2 (src-homology 2) proteins to their autophosphorylation sites. More importantly, however, IRS proteins dissociate the intracellular signaling complex from the endocytic pathways of the activated receptor. Moreover, a single receptor may engage multiple IRS proteins to expand its repertoire of accessible signaling pathways or a single IRS protein may integrate signals from multiple receptors (reviewed by White, 2002). Of particular importance, IRS proteins couple the IGF-IR to PI 3-kinase and extracellular signal-regulated kinase (ERK) cascades. PI 3-kinase is a dimer composed of a 110-kDa catalytic subunit that is associated noncovalently to a 55- or 85-kDa regulatory subunit. It becomes activated when the phosphorylated YMXM motifs in

4. Insulin-like Growth Factor-I and Its Binding Proteins

IRS protein occupy both SH2 domains in the regulatory (p85) subunit (Backer et al., 1992). Products of PI 3-kinase, including phosphatidylinositol-3,4-biphosphate and phosphatidylinositol-3,4,5-triphosphate, attract serine kinases to the plasma membrane, including the phosphoinositidedependent kinases (PDK1 and PDK2) and at least three protein kinase B (pkB) isoforms. During colocalization at the plasma membrane, PDK1 and PDK2 phosphorylate and activate protein kinase B (pkB/Akt)-1, -2, or -3. Upon activation, Akt phosphorylates proteins on serine and threonine residues. Akt substrates include GSK3, p70S6K, BAD, caspase 9, 4E-BP1/PHAS-1, IKK␣, and members of the Forkhead transcription family (FKHRL1, FKHR, AFX). BAD, caspase 9, FKHR, and FKHRL1 are death-promoting components of apoptotic regulatory pathways, and Akt, through inhibition of these targets, potentiates cell survival (reviewed by Downward, 1988). In this way, the activated protein kinase B (PKB or Akt) phosphorylates many substrates to control various biological signaling cascades, including glucose transport, protein synthesis, glycogen synthesis, cell proliferation, and cell survival (Yenush et al., 1997; Alessi et al., 1998; Brunet et al., 1999). Alternatively, activation of the serine-threonine kinase mTOR (molecular target of rapamycin) results in phosphorylation and activation of the translational regulatory proteins eIF-4E-binding protein 1 (4E-BP1) and p70 S6kinase (P70s6k) (Hara et al., 1997; Gingrass et al., 1999; Peterson et al., 1999). mTOR-stimulated phosphorylation of 4E-BP1 disrupts its inhibitory interaction with eukaryotic initiation factor 4E (eIF-4E), whereas activated p70s6k phosphorylates the 40S ribosomal protein S6 (Fukuda et al., 2002). Hence, inhibition of PI3K or FRAP/mTOR prevents growth factor- and cytokine- induced HIF-1␣ accumulation. In addition, chronic activation of mTOR by glucose in ␤ cells has been demonstrated to cause ubiquitination and a resultant decrease in IRS-2 expression and increased ␤-cell apoptosis (Briaud et al., 2005). Integrin engagement has also been shown to modulate IGF-I receptor signaling and function. Goel and colleagues (2004) demonstrated that ␤1 integrin selectively modulates IGF-IR signaling in response to IGF stimulation. Whereas ␤1A integrins form a complex with the IGF-IR and IRS-1 to promote IGF-mediated cell proliferation, ␤1C forms a complex with IGF-IR and Gab1/Shp2 to promote cell adhesion to laminin (Goel et al., 2004). Whereas insulin receptor substrates 1 and 2 are mainly involved in activation of the PI3K cascade, Shc participates in activation of the Ras/mitogen-activated protein kinase (MAPK) cascade (Caparo et al., 1995; Dey et al., 1996). The Ras/MAPK pathway is a key component in the transduction of signals, leading to growth and transformation. It consists of a linear cascade of protein kinases, Raf, MAP kinase kinase, and MAP kinase (ERKs). Similar

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to the PI 3K/Akt/mTOR pathway, the MAPK pathway has the potential to stimulate HIF-1␣ protein synthesis (reviewed by Fukuda et al., 2002).

II. OVERVIEW OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS Coincidental to IGFs came the discovery of IGFBPs in the mid-1970s. Early studies focused on binding protein purification, discrimination between “free” and IGF-bound forms, and subsequent characterization of their unique ability to bind radiolabeled IGF-I or –2. Initial reports in the late 1960s suggested the existence of proteins with the ability to bind IGFs. However, it was unclear whether these were higher molecular weight forms of the IGFs themselves or carrier proteins. This was clarified by studies in which protein extracts containing these higher molecular weight forms were acidified. The resultant lower molecular weight peptides contained IGF bioactivity, suggesting the possibility that the higher molecular weight proteins were carrier proteins (Burgi et al., 1966; Froesch et al., 1966). Subsequently, in 1975, Zapf et al. presented the first definitive report of an activity that would bind radiolabeled IGF-I. This report launched various attempts, using IGF binding assays as a readout, to purify these activities. IGFBP-1, a nonglycosylated protein of 28 kDa, was first isolated from midterm amniotic fluid (Mottola et al., 1986), a convenient source considering that IGFBP-1 is present in amniotic fluid at concentrations 100–150 times higher than serum. Purification of binding protein activity from conditioned medium of a Buffalo rat liver cell line (Moses et al., 1979) resulted in the identification of IGFBP-2, a nonglycosylated protein of 31–36 kDa found in significant amounts in both serum and cerebrospinal fluid (Shimasaki et al., 1991b). The major form of binding protein present in human circulation is IGFBP-3; its molecular mass ranges from 38 to 43 kDa depending on the number of sites glycosylated (Baxter et al., 1986). In circulation, this glycoprotein is associated with an IGF molecule and an 80-kDa acid-labile subunit (ALS) to form a 150- to 200-kDa complex (Baxter et al., 1989; Baxter, 1988, 1990). This complex consists of IGFBP-3 and IGF-I or IGF-II in an equimolar ratio, suggesting that most of the IGFBP-3 in serum is likely to be saturated. IGFBP-4, a nonglycosylated protein of 25 kDa, was first isolated from medium conditioned by human osteoscarcoma TE-89 cells (Mohan et al., 1989) and from adult rat serum (Shimonaka et al., 1989). IGFBP-5 was purified from adult rat serum, from human bone extract, and from medium conditioned by the U-2OS human osteosarcoma cell line as 29- and 23-kDa fragments (Shimasaki et al., 1991b; Bautista et al., 1991; Andress et al., 1991). This binding protein was later purified as

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a 31-kDa fragment from conditioned medium of T98G human glioblastoma cells (Camacho-Hubner et al., 1992) and a 22-kDa fragment from human cerebrospinal fluid (Roghani et al., 1989). IGFBP-6 was purified as a 34-kDa fragment (Shimasaki et al., 1991a) from human cerebrospinal fluid and from transformed human fibroblast cell cultures (Martin et al., 1990). Stemming from these analyses came limited aminoterminal amino acid sequence information for IGFBP-1 (Drop et al., 1984; Povoa et al., 1984; Koistinen et al., 1986), IGFBP-2 (Mottola et al., 1986), and IGFBP-3 (Baxter et al., 1986), which was instrumental in developing cloning strategies that were used in the late 1980s to determine the complete primary structures of these proteins (Brewer et al., 1988; Lee et al., 1988; Wood et al., 1988; Shimasaki et al., 1991; Brown et al., 1989) and cDNA sequencing and for the subsequent sequencing and expression of all six forms of IGFBPs. The development of recombinant protein expression and of large-scale purification techniques allowed for the production of significant quantities of purified proteins, enabling investigators to carry out functional studies, expanding the scope of IGFBP research. Hence, this chapter discusses the functional role of IGFBPs in relation to IGFs and their receptors.

A. IGFBP Structure The six members of the IGFBP gene family encode a family of homologous multifunctional proteins, IGFBP-1 to IGFBP-6. All six genes share a common structural organization of four conserved exons located within genes ranging from 5 kb (IGFBP-1) to more than 30 kb (IGFBP-2 and IGFBP-5) (reviewed by Baxter, 1997). Given that exon 1 of the IGFBP gene family is shared by several other genes encoding a variety of proteins, it has been proposed that they might be a member of a larger gene superfamily (Hwa et al., 1999; Vilmos et al., 2001). The secretory signal peptides of all six IGFBPs range between 20 and 39 amino acids. All six mature IGFBPs are found extracellularly, containing between 216 and 289 amino acids and core molecular masses ranging from 22.8 to 31.3 kDa. They share a highly conserved structure consisting of three domains of approximately equal size (Hwa et al., 1999). While the midregion or linker domain is highly variable (less than 30% homology) (Clemmons et al., 1993), the high homology among the six binding proteins within their amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) domains (approximately 70%) (Clemmons et al., 1993; Jones et al., 1995; Kiefer et al., 1991) has led several groups to propose that the IGFbinding domain is localized within one or both of these regions. Support for this notion comes from several studies in which N- or C-terminal fragments were shown to retain binding affinity for IGF-I and/or IGF-2. However, the

observed affinity of these fragments was reduced compared to that of the intact protein. In addition, several IGFBPrelated proteins (IGFBP-Rps) have been discovered that have slight homology with the amino-terminal domain of IGFBPs but bind the IGFs with significantly less affinity (Murphy et al., 1993). The conserved amino-terminal domain contains six disulfide bonds in all IGFBPs but IGFBP-6, which has five. Despite differences in the pairing of cysteines among IGFBP-1, -4, and ⫺6, all disulfide bonds are domain specific and do not span the midregion (Neumann et al., 1999; Chelius et al., 2001). Mutagenesis studies on IGFBP-3 and IGFBP-5 and nuclear magnetic resonance (NMR) studies on the latter (Kalus et al., 1998) have confirmed that important IGF-binding residues are found in the amino-terminal domain (Imai et al., 2000; Buckway et al., 2001; Hong et al., 2002). Interestingly, although no other major functional motifs have been identified in the amino-terminal domain, the observation that amino-terminal proteolytic fragments of IGFBP-3 cause IGF-independent inhibition of mitogenesis (Lalou et al., 1996a; Salahifar et al., 2000) has brought speculation of an additional active subdomain in this region (Firth et al., 2002). Similarly, the conserved carboxyl-terminal domain is also cysteine rich in all IGFBPs, containing three disulfide bonds by the pairing of adjacent cysteines within the domain (Forbes et al., 1998; Neumann et al., 1999; Chelius et al., 2001). IGF-binding residues in this domain have been demonstrated by the binding activity of natural carboxyl-terminal fragments of IGFBP-2 (Wang et al., 1988; Ho et al., 1997) and recombinant carboxyl-terminal IGFBP-3 fragments (Devi et al., 2000; Galanis et al., 2001) and by mutagenesis of IGFBP-5 residues (Bramani et al., 1999). Hence, the observation that residues involved in IGF binding occur in both amino- and carboxyl-terminal domains implies the existence of an IGF-binding pocket involving both domains. Headey et al. (2004) reported the NMR structure for IGFBP-6 and demonstrated that the C-terminal end of IGFBP-6 distal to the CWCV motif lacked structure (Baxter, 2000; Yao et al., 2004). On this basis it was concluded that this distal portion of the binding proteins is unlikely to participate in ligand binding, but rather the region proximal to the CWCV motif is the critical determinant for IGF binding. These findings are consistent with the work of a number of laboratories reporting that this proximal C-terminal region participates in IGF binding (Baxter, 2000; Rosenzweig, 2004). These data also serve to clarify our photoaffinitylabeling findings in which we derivatized the ␣-NH2 group of the N-terminal glycine residue of IGF-I with two different photoreactive moieties: (1) the N-hydroxysuccinimide ester of azidobenzoic acid or ab-IGF-I and (2) ([2–6-(biotinamido) 2-( p-azidobenzamido) hexanoamido]ethyl-1,39-dithiopropionoyl) or sbed-IGF-I. While ab-IGF-I labeled the C-terminal

4. Insulin-like Growth Factor-I and Its Binding Proteins

end of IGFBP-2 distal to the CWCV motif, sbed-IGF-I labeled the C-terminal end of IGFBP-2 proximal to the CWCV motif (Horney et al., 2001). Given that the Gly1 residue is not essential for IGFBP binding (Szabo et al., 1988), we speculate that it lies outside the IGFBP-binding domain. The spacer of this photoprobe is relatively small and thus reports close to the Gly1 residue, labeling the distal region of IGFBP-2. However, the spacer on sbed-IGF-I is considerably longer, making it more likely to report residues within the IGFBP-binding domain near Glu3, labeling proximal to the CWCV motif. Headey et al. (2004) also reported that the receptor-binding domain of IGF-2 contacts the C-terminal domain rather than the N-terminal domain as reported by Kalus and co-workers (1998) for IGF-I and IGFBP-5. Based on the site-specific labeling of IGF-I at each of its four amino groups with biotin moieties, we have shown that the Gly1 and Lys27 residues are in close contact with IGFBP-2 and IGFBP-3; the specific sites of interaction have not been determined (Robinson et al., 2004). Use of truncation mutants in combination with the different biotinylated species of IGF-I may enable us to determine whether the receptor-binding domain of IGF-I also contacts the C-terminal domain of IGFBP-2. Other subdomains identified within the carboxyl-terminal region of various IGFBPs include Arg-Gly-Asp (RGD) integrin-binding motifs at residues 221–223 of IGFBP-1 (Jones et al., (1993b) and residues 265–267 of IGFBP-2 (Binkert et al., 1989); functionally important 18-residue basic motifs with heparin-binding activity have been identified at residues 215–232 of IGFBP-3 and residues 201–218 of IGFBP-5—they are involved in interaction with the serum glycoprotein, acid-labile subunit (ALS) (Firth et al., 1998, 2001; Twigg et al., 1998) and other ligands such as plasminogen activator inhibitor-1 (Nam et al., 1997) and transferrin (Weinzimer et al., 2001), cell and matrix binding (Booth et al., 1995; Firth et al., 1998), and speculated nuclear transport (Schedlich et al., 2000). In contrast to amino- and carboxyl-terminal domains, the central domain of the IGFBPs lacks structural conservation among family members. Other than an intradomain bond in IGFBP-4, it contains no disulfide bonds (Chelius et al., 2001). The midregion contains various sites of posttranslational modification. IGFBP-3 resolves as four bands on SDS gels due to variable glycosylation of the three sites of N-linked glycosylation in its midregion, whereas IGFBP-5 and ⫺6 are O-glycosylated (Bach et al., 1992). IGFBP-1, -3, and ⫺5 have been shown to be serine phosphorylated in the midregion, and the remaining three IGFBPs have possible target sites for serine/threonine kinases (Coverley et al., 1997). Although the significance of IGFBP phosphorylation remains a matter of investigation, Jones and colleagues (1991) found that phosphorylation of IGFBP-1 increases its affinity for IGF-I and increases the likelihood it will be inhibitory to IGF action.

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While IGFBP-3 can be secreted as a phosphoprotein, Hoeck and Mukku (1994) demonstrated that loss of phosphorylation and glycosylation due to point mutations had no effect on its affinity for IGF-I, although it has yet to be determined whether this affects binding to the IGF-IR. IGFBPs 1 through 5 have been shown to be cell associated, which may occur by several different mechanisms. IGFBP-1 and ⫺2 contain the arginine-glycine-aspartate (RGD) sequence in the midregion (Brewer et al., 1988; Margot et al., 1989) commonly found in matrix proteins, which forms the recognition site for the integrin family of receptors. Although no evidence of functionality for the IGFBP-2 RGD has been demonstrated, Jones et al. (1993b) have shown that IGFBP-1 can bind to the ␣5␤1 integrin (the fibronectin receptor) via its RGD motif. Examination of IGFBP-3 reveals a potential cell association domain in its midregion. Finally, various sites of proteolysis in all the IGFBPs can be found in this domain as well as secondary binding sites for ALS (Fujita-Yamaguchi et al., 1986) and heparin (Ullrich et al., 1986) in the midregion of IGFBP-5.

B. Regulation of IGFBP Expression IGFBP-1 levels are influenced by various factors. Analysis of tissue mRNA levels suggests that IGFBP-1 is expressed primarily by liver, uterine deciduas, and secretory endometrium, with liver being the primary source of serum IGF-I (reviewed by Suwanichkul et al., 1990). The region immediately 5⬘ to the IGFBP-1 mRNA cap site is typical of a eukaryotic promoter, with a TATA sequence beginning 28 bp and a CCAAT promoter element beginning 72 bp upstream from this cap site. The CCAAT box region as the major cis element involved in basal IGFBP-1 promoter activity in HEP G2 cells and increased basal promoter activity is associated with the binding of at least one HEP G2 nuclear factor to the CCAAT box region (Suwanichkul et al., 1990). Cloning and characterization of a 1.18-kb fragment 5⬘ to the translation initiation codon for the rat IGFBP-3 gene revealed a transcription start site 118 bp upstream of the initiation codon, and TATA box consensus sequence located 27 bp 5⬘ to this cap site. No CAAT box is present, but a CpG island was identified. Consensus sequences for a number of putative response elements (activating protein-2, insulin, TSH/insulin-like growth factor, and GH) are present within ⫺700 bp of the cap site. Both basal and hormonally responsive (TSH and phorbol ester) promoter activities have been localized within the first 472 bases of the promoter region. Sequencing reveals 65% homology with the corresponding IGFBP-3 sequence. The region between –100 and –1 bp relative to the transcription start sites shows 85% homology (Albiston et al., 1995). IGFBP-2 has been shown to be regulated by various factors, including food deprivation, insulin, retinoic acid,

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estrogen, and T3 (reviewed by Kutoh et al., 1993). IGFBP-1 and ⫺3 both have a TATA box, and a single transcription start site, as well as a GC-rich 5⬘ end. In addition, IGFBP-1 has a CAAT box, whereas IGFBP-3 contains an upstream GC-rich element with potential Sp1-binding activity. In contrast, the putative promoter region of human, rat, and murine IGFBP-2 lacks TATA and CAAT elements. The rat IGFBP-2 promoter contains three GC-rich regions located 37, 57, and 81 nucleotides 5⬘ of the cap site. Sp1 is known to activate transcription by interacting with GC-rich elements present in a wide variety of cellular and viral promoters (reviewed by Kutoh et al., 1993). Kutoh and colleagues (1993) found that Sp1 regulates the transcription of rat IGFBP-2 (rIGFBP-2) gene and that it interacts specifically with the consensus and aberrant GC boxes on the rIGFBP-2 putative promoter. IGFBP-4 mRNA levels in several cell lines have been reported to be induced by agents that elevate intracellular cAMP levels. For example, mRNA levels of IGFBP-4 in normal bone cells and human osteosarcoma cell line TE89 were increased by treatment with dibutyryl cAMP (LaTours et al., 1990). IGFBP-4 protein levels secreted by TE89 cells were induced by treatment with forskolin (Mohan et al., 1989). In rat osteoblast-like cells (UMR 106–01), parathyroid hormone (PTH) and PTH-related peptides stimulated IGFBP-4 (Torring et al., 1991). Furthermore, IGFBP-4 mRNA levels were shown to be increased three- and twofold above basal levels by estradiol and retinoic acid, respectively, in human breast cancer cells (MCF-7) (Sheikh et al., 1992b). Analysis of the rat IGFBP-4 promoter by Gao and colleagues (1993) revealed a single transcription start site located 249 nucleotides upstream of the translational initiation ATG codon. The rat IGFBP-4 gene possesses a typical TATA box and a CAAT box. The presence of multiple potential cis elements, including three cAMP responsive elements, three AP-1-binding sites, and one progesterone receptorbinding site in the 5’ flanking region. Similar to IGFBP-4, IGFBP-5 mRNA levels were reported to be increased by forskolin in human fibroblasts. In addition, IGFBP-5 protein levels were stimulated by treatment with PTH and PTH-related peptide in culture medium of rat osteoblast-like cells (reviewed by Zhu et al., (1993a). Analysis of the rat IGFBP-5 gene by Zhu and colleagues (1993a) revealed a single transcription start site located 772 nucleotides 5’ of the ATG translational start codon. In addition to a TATA box and CAAT box, multiple putative cis-regulatory elements, including an AP-1, AP-2, and a binding site for progesterone receptor, are present in the promoter region. IGFBP-6 mRNA has been shown to be enhanced by retinoic acid in human breast cancer cells (MDA-MB-231) (Shiekh et al., 1992a). Analysis of the rat IGFBP-6 gene by Zhu and colleagues (1993b) revealed two transcriptional

start sites located 85 and 82 nucleotides upstream of the ATG translational initiation codon. The promoter region of the rat IGFBP-6 gene is devoid of a TATA or a CAAT consensus motif, but putative regulatory elements, including a Sp1, an estrogen receptor-binding site, and a retinoic acid responsive element, are present in the promoter region.

C. IGFBP Function Essential in coordinating and regulating the biological activities of the IGFs, IGFBPs act as carrier proteins, regulating IGFs in the vascular space and modulating IGF half-life and clearance. At the local level, tissue-specific release of various IGFBPs provides a means of tissue and cell typespecific action. Based on their high-affinity association with IGFs, IGFBPs have the unique ability to modulate IGF-I receptor activation through IGF-I sequestration. Finally, IGFBPs may have actions independent of IGF-I (Jones et al., 1995). The IGF binding protein gene family consists of six wellcharacterized members that encode a family of homologous multifunctional proteins, IGFBP-1 through ⫺6. Sharing a common structural organization in which four conserved exons are located within genes ranging from 5 kb (IGFBP-1) to more than 30 kb (IGFBP-2 and IGFBP-5) (Baxter, 1997), IGFBP genes, like those of IGFs themselves, are believed to have emerged early in vertebrate evolution (Upton et al., 1993). Considering that exon 1 of the IGFBP gene family is shared by several other genes encoding a variety of proteins, it has been proposed that they might be members of a larger gene superfamily (Hwa et al., 1999; Vilmos et al., 2001). In biological fluids, IGFs are normally bound to IGFbinding proteins. IGFBP-1 through -6 proteins all have higher affinities for IGFs than their respective receptors while having negligible affinity for insulin. Therefore, IGFBPs act not only as carriers of IGFs, thereby prolonging the half-life of the IGFs, but also function as modulators of IGF availability and activity (Hwa et al., 1999; Rechler et al., 1998). In this seemingly contradictory manner, IGFBPs are capable of modulating IGF-induced cell proliferation in both a positive and a negative manner (Jones et al., 1995; Rechler, 1993; De Mellows et al., 1988; Oh et al., 1993; Jones et al., 1993a). As negative regulators, IGFBPs inhibit IGF action through sequestration of the ligand from the IGF receptor (Jones et al., 1995; Rechler, 1993). Posttranslational modification may play a role in modulating this regulation. For example, whereas phosphorylated IGFBP-1 inhibits IGF actions, the nonphosphorylated form of IGFBP-1 potentiates the effect of IGF-I on DNA synthesis in porcine smooth muscle cells (Jones et al., 1995). In addition, the presence of certain IGFBPs along with IGFs may potentiate effects unique to either ligand or binding protein alone. For example, human

4. Insulin-like Growth Factor-I and Its Binding Proteins

fibroblast preincubation with IGF exhibited a growthpotentiating effect, whereas coincubation of IGF plus IGFBP-3 elicited an inhibitory effect (De Mellows et al., 1988). It has been suggested that this may be due to binding of IGFBP-3 to the cell surface, which reduces its affinity for IGF-I and results in a potentiating effect (Oh et al., 1993a). Thus the binding proteins may modulate IGF action differently, and the same binding protein may have an IGF-inhibiting or -potentiating role under different conditions. Factors influencing these differences include IGFBP phosphorylation, cell surface association, and IGFBP proteolysis. In effect, these variables may modulate IGF action in target tissues by altering the binding affinity of the IGFBPs to IGFs. A number of studies have advanced the notion that IGFBPs may have IGF-independent actions; one of the earliest studies demonstrated that murine IGFBP-3 could inhibit DNA synthesis in chick embryo fibroblasts stimulated by serum, fibroblast growth factor, or TGF␤ (Blat et al., 1989). IGFBP-3 has been shown to inhibit the proliferation of breast and prostate cancer cells by a cellular signaling pathway independent of IGFs (Oh et al., 1993b; Cohen et al., 1993). IGFBP-3 was reported to induce apoptosis of the p53-negative prostate cancer cell line PC3 through a novel pathway independent of either p53 or the IGF-IGF-I receptor-mediated cell survival pathway (Rajah et al., 1997). Lalou and colleagues (1996a) reported that plasmin-cleaved IGFBP-3 generated a 16-kDa fragment lacking IGF-I affinity that was almost as effective as intact IGFBP-3 in inhibiting IGF-I-induced DNA synthesis in chick embryo fibroblasts. Although the concept of IGF-independent effects of IGFBPs on cellular growth has gained wide acceptance in recent years, reports of a specific receptor capable of mediating the actions of IGFBPs have been variable. Oh and colleagues (1993a,b) reported the characterization of a putative cell surface receptor for IGFBP-3 in Hs578T human breast cancer cells that mediates the inhibition of cell growth by an IGF-independent mechanism (Oh 1993b). Other receptors have been proposed to mediate some IGFBP effects. For example, the type V receptor for TGF␤ has been reported to mediate IGFBP-3-induced growth inhibition (Leal et al., 1997). This receptor has been shown to be the low-density lipoprotein receptor-related protein-1 (LRP-1) (Huang et al., 2003). IGFBP-5 has also been shown to be stimulatory in a variety of cell types (Jones et al., 1993a; Mohan et al., 1995; Andress et al., 1992; Schmid et al., 1993). IGFBP-5 has been suggested to promote osteoblast proliferation via a putative cell surface-binding site (Mohan et al., 1995; Andress et al., 1992). Along with these findings have come more controversial studies providing evidence for the nuclear localization of IGFBP-3 (Li et al., 1997; Jaques et al., 1997). Although studies from a number of laboratories support the possibility that IGFBPs

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may have IGF-independent effects in certain cell types, further experimental evidence is needed to verify this mode of IGFBP action.

D. IGFBP Proteolysis Decreased affinity and release of IGFs from IGFBPs can involve binding of IGFBPs to extracellular matrix (ECM) molecules, phosphorylation of IGFBPs, and proteolytic cleavage of IGFBPs. Of these mechanisms, proteolysis has been established as the predominant mechanism for IGF release from each of the six IGFBPs. In most instances, proteolysis results in fragments with decreased affinity for the IGFs and is therefore assumed to increase the availability of IGFs to the IGF-IR (Lalou et al., 1996b; Cohen et al., 1994; Angelloz-Nicoud et al., 1995). However, IGFBP fragments have been shown to inhibit IGF-I action (Lalou et al., 1996a,b; Angelloz-Nicoud et al., 1995; Salahifar et al., 2000). In addition, IGF-I itself regulates the expression of specific IGFBPs (Camacho-Hubner et al., 1992) or their proteases (Parker et al., 1995; Salahifar et al., 1997), thus adding further complexity. A wide range of enzymes can proteolyze the IGFBPs into fragments. Three members of the metzincin class of metalloproteinases, including the matrixins or MMPs, adamalysins, and pappalysins, have been implicated in IGFBP degradation in vivo and/or in vitro (reviewed by Bunn et al., 2003). It is no coincidence that MMPs have been implicated in various IGF-mediated processes, including tumor cell invasion, cartilage and bone repair, wound healing, and angiogenesis. Fluctuations in MMP levels may significantly alter IGF bioavailability and bioactivity through their effects on IGFBP, in particular IGFBP-3, degradation (reviewed by Bunn et al., 2003). Initially identified as IGFBP-3-degrading proteases in human dermal fibroblasts (Fowlkes et al., 1994) and rat pregnancy serum (Fowlkes, 1994), MMPs contribute to the degradation of IGFBP-5 in murine osteoblast cultures (Thrailkill et al., 1995) and human fibroblast-conditioned medium (Nam et al., 1996). MMP-1, –2, and ⫺3, secreted from human dermal fibroblasts, have also been found to cleave IGFBP-3 at its midregion, thus creating biologically active aminoand carboxyl-terminal fragments (Fowlkes et al., 1994). In addition, the adamalysin class of metzincins has also been implicated in IGFBP degradation. ADAM 12-S cleaves IGFBP-3 and IGFBP-5, yielding fragments of those found in pregnancy serum (Loechel et al., 2000). The cathepsin family of lysosomal proteinases has been found to degrade IGFBP-1 through ⫺5 at low pH, leading to the proposal that cathepsins could release IGFs from IGFBPs in local acidic environments (Conover et al., 1994). This is exemplified in human prostatic cancer cells where cathepsin D has been found to degrade IGFBPs in the acidified, conditioned medium of human

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prostatic carcinoma cells (Braulke et al., 1995; Conover et al., 1995). In addition to cathepsins, a prostate-specific antigen has been established to cleave IGFBP-3 (Cohen et al., 1994). Thus, an elevated expression of IGFBP proteases may contribute to the hyperproliferative phenotype of prostatic cancer cells. Other IGFBP proteases include pregnancy-associated plasma protein A (PAPP-A) (Conover et al., 1999) and PAPP-A2 (Overgaard et al., 2001), which degrade IGFBP-4 and IGFBP-5, respectively, in an IGF-independent manner and the serine protease complement protein 1s (C1s), which is responsible for IGFBP-5 cleavage by human fibroblasts. In addition to these select examples, the tissue-specific release of IGFBP-1 through –6 and the actions of other proteases, including plasmin, thrombin, serine, and cysteine proteases, add significant complexity to IGF–IGF receptor interactions in various tissue. Adding to this complexity, certain IGFBP fragments, in particular those from IGFBP-3, appear to have distinct IGF-independent effects on cell proliferation (Baxter, 2001). A plasmin-generated 16-kDa fragment, encompassing approximately the first 95 amino acids of recombinant human IGFBP-3 and lacking affinity for IGF-I, was reported to inhibit IGF- and insulin-stimulated proliferation of chick embryo fibroblasts (Lalou et al., 1996a,b). Increasing support has grown for an IGF-independent mechanism of antimitogenesis by the 16-kDa fragment because it is capable of inhibiting fibroblast growth factor-induced mitogenesis by 80% in mouse fibroblasts lacking the type 1 IGF receptor (Zadeh et al., 1997) The function of IGFBP proteolysis thus appears to be multifaceted. The IGFBP cleavage appears to be operative in unstimulated, homeostatic conditions (PAPP-A activity in normal cell lines), yet exaggerated in conditions in which IGFBP-degrading proteinases are overproduced, hypersecreted, and/or excessively activated (MMPs in tumor growth and in pregnancy). Other IGFBP-degrading proteinases, such as the cathepsins, might function to inactivate and clear IGFs and IGFBPs from the pericellular environment. In addition, IGFBP-degrading proteinases could have secondary effects by cleaving IGFBPs into novel bioactive fragments, which have the potential to mediate cellular events distinct from those mediated via their effects on IGFs (reviewed by Bunn, 2003). Thus, modulation of IGFBP proteolysis and thus IGF action may be a useful strategy in preventing unwanted abnormal cellular growth and proliferation (e.g., tumor growth) or in promoting cellular proliferation (e.g., wound healing). 1. IGF-I and Ocular Neovascularization A number of growth factors have been implicated as regulators of retinal and choroidal angiogenesis, including GH, bFGF, IGF-I, and vascular endothelial growth factor

(VEGF). Historically, the link between IGF-I and retinal neovascularization may be traced back to early descriptions of the ameliorating effects hypophysectomy/pituitary ablation had on diabetic retinopathy (Poulsen et al., 1953). Initially, growth hormone was implicated in regulating retinal neovascularization (Merimee et al., 1978; Kimmel et al., 1985). In addition, retinal neovascularization in a diabetic patient was found to regress after pituitary infarction (Poulsen et al., 1953). Over time, attention shifted to IGF-I, as it became clear that most of the effects of GH on cell growth/proliferation are mediated by IGF-I. Significantly, IGF-I has been shown to stimulate angiogenesis in various animal models, and numerous studies have found serum and vitreous IGF-I levels to correlate in timing and degree with proliferative diabetic retinopathy associated neovascularization (Meyer-Schwickerath et al., 1993; Merimee et al., 1983; Grant et al., 1986). Meyer-Schwickerath et al. (1993) demonstrated a strong temporal correlation between vitreous IGF-I levels and diabetic retinopathy in studies showing that elevated IGF-I levels correlate with a wide variety of ischemic retinal disorders linked to neovascularization of the retina and iris (Meyer-Schwickerath et al., 1993). The role of IGF-I in neovascularization was further strengthened by Danis et al. (1995), who demonstrated that multiple injections of IGF-I into normal (nondiabetic) eyes stimulated retinal neovascularizaton in pigs. However, it was Punglia and colleagues (1997) who were the first to suggest that the angiogenic potential of IGF-I was mediated through the stimulation of VEGF expression. Subsequently, VEGF has been linked causally to neovascularization of the retina and iris (Senger et al., 1983; Keck et al., 1989; Leung et al., 1989; Adamis et al., 1996; Aiello et al., 1995). In choroidal neovascularization, a condition that develops in 10% of age-related macular degeneration (AMD) sufferers, newly formed choroidal blood vessels enter the subretinal space, where leakage and bleeding lead to retinal detachment and photoreceptor death (Mousa et al., 1999; The Macular Group, 1991; Green, 1977, 1996). As current laser treatments are ineffective, identification of the mediators of ocular angiogenesis would provide important targets for the development of selective inhibitors of choroidal neovascularization (The Macular Group, 1982; Husain et al., 1999). Of particular interest is the retinal pigment epithelium (RPE), a monolayer of highly specialized epithelial cells interposed between the retinal photoreceptors and the choroid (Zinn et al., 1979; Campochiaro et al., 1986). A major secretagogue of angiogenic (VEGF) and antiangiogenic factors, the RPE may play a central role in the modulation and progression of CNV (Tombran-Tink et al., 1991; Yi et al., 1997; Lopez et al., 1996; Wells et al., 1996;

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Amin et al., 1994; Ishibashi et al., 1997; Punglia et al., 1997). A number of animal models support a role for increased RPE VEGF in the progression of CNV, and IGF-I has been demonstrated to regulate VEGF expression in the RPE. In addition, the RPE possesses receptors for IGF-I and secretes both IGF-I and ⫺2, as well as IGFbinding proteins 3 and 6. Along with the variable expression of these components in surrounding cells, the retina contains all components of a self-contained IGF-I/IGFBP autocrine system (reviewed by Slomiany et al., 2004a). Based on a growing body of evidence demonstrating that IGF-I can induce HIF-1 activity and the secretion of VEGF and IGFBP-3 in RPE cells in vivo and in vitro, we have examined the effect of IGF-I on HIF-1␣ protein expression, VEGF and IGFBP-3 secretion, and the autocrine effects of VEGF and IGFBP-3. We have found that IGF-I stimulates an increase in HIF-1␣ in two separate human RPE cell lines (D407 and ARPE-19) as well as the apical secretion of both VEGF and IGFBP-3. We identified a unique autocrine function for VEGF in inducing the secretion of IGFBP-3 in control and IGF-I-stimulated ARPE-19 cells without affecting HIF-1 protein expression. In addition, our studies have demonstrated a negative feedback role of IGFBP-3 in sequestering and thereby attenuating IGF-I-induced VEGF secretion (Slomiany et al., 2004a,b). Together, these findings point toward the importance of the IGF system, both in modulating normal retinal function and in contributing to the pathogenesis of CNV through dysregulation. However, the creation of in vivo models demonstrating the role of IGF system dysregulation leading to CNV still need to be developed.

II. SUMMARY AND FUTURE PERSPECTIVE The molecular mechanisms by which IGFBPs modulate IGF action are complex and multifaceted. Promoter studies on IGFBPs remain vague, and mechanisms of posttranslation regulation in response to various stresses and cytokine stimulation require more detailed investigation. The role of surface association, phosphorylation, and proteolysis and their effects on the affinity of the IGFBPs for their respective ligands can clearly target cell actions. However, as few in vivo studies exist at present, such conclusions are indirect and must be made with caution. Though current in vivo data are conflicting as to whether the net effect of binding proteins is inhibitory or stimulatory, certain functions, such as the role of IGFBP-3 in serum as a potentiator of IGF action, have been delineated. Abolition of specific IGFBP release may be helpful in resolving these paradoxes in the various systems where the IGF system plays a role.

Acknowledgments This work was supported, in part, by a grant from the National Institutes of Health (CA-78887) and a Department of Defense grant to Hollings Cancer Center (N6311601MD10004) to SAR.

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5 Growth Hormone and Insulin-like Growth Factor-I in Human Cerebrospinal Fluid FRED NYBERG Department of Pharmaceutical Biosciences, Uppsala University, S-751 24 Uppsala, Sweden

I. Introduction II. Growth Hormone and Insulin-like Growth Factor-I (IGF-I) in the Cerebrospinal Fluid (CSF) Compartment III. Relations between CSF Detectable Levels of Growth Hormone and IGF-I to Various Physiological and Pathophysiological Conditions IV. Future Perspectives References

various clinics and laboratories and directed to investigation of the actual constituents of the somatotrophic axis are reviewed in brief.

I. INTRODUCTION Growth hormone (GH) or somatotrophin is a polypeptide hormone produced by somatroph cells of the anterior pituitary gland. In human it consists of a single polypeptide chain of 191 amino acids. GH is essential for body growth, but in addition the hormone is known to modulate metabolic pathways as well as neural, reproductive, immune, cardiovascular, and pulmonary functions. Several beneficial effects observed for the hormone have led to its expanded therapeutic use in both adults and children. There are increasing numbers of officially approved applications of human GH replacement therapy resulting from a large number of clinical studies on the effectiveness of the hormone as a therapeutic agent (Christ et al., 1997; Burman and Deijen, 1998; Wirén et al. 1998). GH may induce several of its action through activation of its receptor located on the surface of the target cells but many effects of the hormone are known to be mediated through the release of insulin-like growth factor-I (IGF-I). In recent years the beneficial effects of the hormone on life quality seen in studies on adult GHdeficient (GHD) patients subjected to GH replacement therapy have indicated that GH may produce profound effects also on the brain (Burman and Deijen, 1998; Wirén

This chapter reviews previous and current studies on hormones related to the somatotrophic axis in human cerebrospinal fluid (CSF). A particular focus is directed to growth hormone (GH) and its mediator insulin-like growth factor-I (IGF-I). The number of studies dealing with the assessment of CSF concentrations of GH and IGF-I and reported in the scientific literature is, to some extent, limited. One reason for this could be the fact that the influence of the somatotrophic axis on brain function has emerged from studies carried out during very recent years. Another reason may be reflected by the limited availability to CSF specimens. For instance, the possibility of collecting CSF samples at clinics is restricted in most countries for ethical reasons. Nevertheless, there are several studies examining the CSF levels of GH and IGF-I, a majority of which is focused on pathological conditions related to hyperreactivity in the GH/IGF-I axis and to hormone-producing tumors. In this review, aspects related to the characteristics of the hormone activity in the CSF and methods for the assessment of hormone levels, as well as the nature of the CSF compartment, are highlighted. Studies carried out in

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Copyright 2006 Elsevier Inc. All rights reserved.

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et al., 1998). As mentioned in Chapters 8 and 9 in this book, high densities of GH receptors (GHR) have been observed in several brain regions, including those involved in food intake, cognitive, and emotional functions (Nyberg et al., 2000, Le Grevés et al., 2002; Kastrup et al., 2005). Also, IGF-I receptors have been shown to be present in many brain areas, and this growth factor is shown to produce effects on brain function as well (e.g., Le Grevés et al., 2005). In studies of GHD patients subjected to GH replacement therapy, it has been shown that the hormone may find its way into the cerebrospinal fluid (CSF) to be detected in levels that correlate positively to the given hormone doses (Burman et al., 1996). Also, levels of IGF-I were found to increase in CSF following GH replacement therapy (Johansson et al., 1995). These observations were considered to confirm that GH, in analogy with IGF-I (Pan and Kastin et al., 2000), may cross the blood–brain barrier (BBB) in human subjects. However, data showing that growth hormone is present in CSF were reported long before the existence of GH receptors in the brain was confirmed (Schaub et al., 1977). In fact, a number of clinical studies directed to measurements of both GH and IGF-I have been published over the years. The aim of this chapter is to review some studies present in the literature dealing with GH and IGF-I in human CSF. It by no means fully covers the available literature, but it addresses some previous published possible approaches to study the GH/IGF-I axis in various neurological disorders.

II. GROWTH HORMONE AND INSULIN-LIKE GROWTH FACTOR-I (IGF-I) IN THE CEREBROSPINAL FLUID (CSF) COMPARTMENT In studies of GH and IGF-I in humans during healthy as well as pathological conditions, a large number of studies have been directed to analyses of these hormones in serum or plasma samples. In studies of GH and IGF-I in central nervous system (CNS) disorders, however, there are several limitations. First, there is the restricted availability of human CNS tissue. For ethical reasons, it is not possible to apply surgical techniques for experimental modeling like in animal experiments. Therefore, the major part of studies on GH and IGF-I in relation to human CNS physiology or pathology has been limited to brain imaging and analysis of hormone levels in CSF and plasma. In this context, the approach of measuring hormone concentrations in CSF has several advantages, as this fluid is in constant exchange with the extracellular fluid in the brain and spinal cord. Hence, alterations in hormone levels in CSF are more likely to mirror events in the CNS than changes that may be detected in the circulatory system. This chapter focuses on attempts to probe GH and IGF-I in CSF and relates their levels to various physiological and pathological conditions.

A. The CSF Compartment The CSF is characterized as a clear fluid with a comparatively low content of protein and peptide material. Various low molecular weight compounds participating in neuronal signaling are present as well. The CSF originates from the blood and is secreted from the circulation into the brain ventricles by a densely vascularized tissue called the choroid plexus. Epithelial cells of this tissue are the structural basis of the blood–CSF barrier (BCB). The blood supply to choroid plexus emerges from small branches of the internal carotid arteries. As the CSF is in direct contact with the neuronal circuits of the CNS, peptides and hormones released from central neurons and escaping proteolytic degradation should appear in this fluid. Some peptides are likely to specifically be released into the CSF to use the fluid as a medium for transport to target cells. Others may diffuse passively into the CSF. In fact, most neuroactive compounds identified in CNS tissue have also been detected in the CSF. With regard to polypeptides such as IGF-I and GH, no clear evidence that these entities are secreted into the CSF has been reported so far.

B. Source of CSF Detectable Growth Hormone and IGF-I The question about the origin of GH and IGF-I present in the CSF is not yet finally settled. Some studies suggest that GH may find its way into the CSF via the circulatory system after being secreted from the pituitary gland (Schaub et al., 1977; Johansson et al., 1995; Burman et al., 1996). Schaub and co-workers (1977) found a positive correlation between serum and CSF levels of GH in human control subjects and suggested an active transport of the hormone from the circulatory system into the CSF. However, studies also suggest that GH may be produced in brain areas apart from the pituitary (Render et al., 1995; Harvey et al., 2000). As for IGF-I, studies have shown that this growth factor may be produced in brain tissues, but strong evidence for the ability of IGF-I to cross the BCB are present in the literature (e.g., Pan and Kastin et al., 2000). Thus, it appears evident that IGF-I, following release from peripheral sources in conformity with insulin, may reach the CNS and subsequently the CSF compartment. In general the entrance of peptides into the CSF from the blood circulation is considered very restricted. Exceptions are pathological conditions connected with damage of the BBB. However, in the case of IGF-I and insulin it has been proposed that these hormones may penetrate the BCB through a carrier-mediated mechanism (Pan and Kastin, 2000; Coculescu, 2000). Some data suggest that the respective hormone receptor may account for this carrier (e.g., Duffy et al., 1988), whereas others do not (e.g., Pulford and Ishii, 2001). A receptor-mediated mechanism has been suggested for pituitary prolactin (Walsh et al., 1987), as

5. Growth Hormone and IGF-I in CSF

well as for GH (Coculescu, 2000; Nyberg, 2000). In fact, Johansson et al. (1995) showed that GH replacement therapy induced a significant increase in radioimmunoassaydetectable GH in CSF in GHD patients. Moreover, Burman and co-workers (1996) found a positive correlation between given doses of GH and the hormone level in CSF following 9 months of GH administration in patients with documented GH deficiency acquired in adult life. Also, a highly significant correlation between serum and CSF prolactin was reported earlier (Braunstein et al., 1981).

C. Characteristics of Growth Hormone and IGF-I in CSF Over the past years a number of studies directed at measuring GH and IGF-I in CSF have been published. However, due to low concentrations of the hormones in the CSF and restricted access to this fluid, no extensive characterization of GH and IGF-I has been carried out. Most studies directed at measuring of GH and IGF-I in human CSF have been confined to immunological techniques and therefore detected material should rather refer to hormone-like activity before it has been characterized chemically. Semicharacteristics of the actual hormones could be achieved by the use of chromatographic techniques. Such techniques have been used for the molecular characterization of pituitary hormones in plasma. For instance, circulating GH was reported earlier to consist of several molecular size species with different biological activity (Arosio et al., 1991). In this study, a radioimmunoassay (RIA) and immunoradiometric assay combined with gel chromatography were used to determine the size of the GH entities. The major part of prolactin immunoreactivity present in human CSF was previously characterized as an about 22-kDa entity (Nyberg et al., 1991). As for IGF-I, serum profiles indicating molecular heterogeneity have been reported (Fielders et al., 1993). No chemical characteristics regarding IGF-I in human CSF have been reported so far.

D. Methods for Measuring Growth Hormone and IGF-I in CSF As mentioned earlier, studies directed at measuring both GH and IGF-I in CSF are based on immunological techniques. The simple and predominate is the radioimmunoassay (Schaub et al., 1977; Nowak et al., 1987; Johansson et al., 1995; Burman et al., 1996). The detected level of the hormone seems to vary from one laboratory to another, and the concentration ratio from CSF to serum is around 1:6 (Schaub et al., 1997). Reported CSF concentrations of GH in human subjects range from 0 to around 2 ng/ml or from 0 to around 20 ␮U/ml (Schaub et al., 1977; Johansson et al., 1995). In patients receiving GH replacement therapy, mean CSF levels of the hormone increased

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from 13 ␮U to around 150 ␮U per milliliter (Johansson et al., 1995). In patients subjected to GH replacement therapy, a positive correlation between the given dose and the level of the hormone detected in the CSF was confirmed (Burman et al., 1996). The variation between different laboratories regarding the detected levels may be due to different antibodies or different procedures for the pretreatment of CSF samples. Moreover, an important aspect in this context is that the sampling procedure may also differ between the various clinical studies. The major approaches for the assessment of IGF-I in human CSF are also based on immunological techniques (Glick and Unterman, 1995). Some researchers have used commercially available kits (e.g., Riikonen et al., 2004), whereas others have applied an in-house-developed RIA technique (Glick and Unterman, 1995). However, the IGF-I concentration in the CSF has also been probed by binding assays using IGF-I-binding proteins (Heinze et al., 1998). In approaches using IGF-binding proteins, CSF samples need to be subjected to a preseparation procedure prior to assay. This could be accomplished by acidic gel filtration (Binoux et al., 1986) or extraction through solid-phase cartridges (Glick and Unterman, 1995). Riikonen and co-workers (1999) applied a sensitive enzyme immunoassay kit for measuring the CSF IGF-I concentration in patients with neurological disorders. The mean concentration of IGF-I in human CSF varies from 0.1 to around 1 ng/ml (Johansson et al., 1995; Riikonen et al., 2000).

III. RELATIONS BETWEEN CSF DETECTABLE LEVELS OF GROWTH HORMONE AND IGF-I TO VARIOUS PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL CONDITIONS The CSF levels of GH and IGF-I have been probed in various neurological disorders. A particular interest for detecting GH and IGF-I levels in CSF concerns patients with hypersecretion of pituitary hormones, e.g., due to pituitary adenoma or other endocrine dysfunctions. For diagnostic purposes it is essential for the practitioner to determine the normal range of GH levels in CSF.

A. Clinical Studies on Growth Hormone in CSF Braunstein and co-workers (1981), who studied patients with pituitary and parasellar tumors, found increased levels of GH in many of these subjects; however, no differentiation in GH concentration between patients with intrasellar tumors and those with pituitary tumors with suprasellar extension or primary suprasellar tumors could be observed. A similar

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observation was reported by Dogliotti and collaborators (1983), who recorded high CSF concentrations of GH and other adenohypophyseal hormones as well in patients with pituitary adenomas. No significant difference in GH was found in intra- and extrasellar tumors. High GH levels in CSF were reported in acromegaly, and a small increase was also reported after chronic administration of GH in GHD syndromes (Rolandi et al., 1982). GH was measured in serum and in CSF in 16 patients with chromophobe adenomas, in 8 with acromegaly, and in 18 subjects with neurological diseases without endocrine troubles (Roland et al., 1982). Elevated mean GH levels in serum and in CSF were found in patients with chromophobe adenomas and with acromegaly. No constant correlation was observed between serum and CSF values. The highest hormonal levels in CSF were usually observed in adenomas with suprasellar extension, but this finding was inconstant. The determination of hormonal levels in CSF does not seem to supply any reliable information about the characteristics of pituitary tumors. Assessment of GH and other adenopituitary hormones was carried out in CSF and serum samples from a group of patients with endocrine diseases during stimulation with releasing hormones in order to explore penetration of the hormone into the CSF compartment (Coculescu et al., 1985). An increase of the permeability of the BCB was frequently observed in a subgroup of patients with invasive tumors and occasionally in other tumoral or nontumoral groups. The serum/CSF ratios of gonadotropins become significantly lower than the ratios of GH and PRL in the invasive groups, and here the CSF level was correlated to increases of the serum levels during luteotropin-releasing-hormone (LRH) plus tyreotrophin-releasing-hormone (TRH) IV test only for gonadotropins. These data suggest that the specificity of BCB is not lost, but altered by tumors of the hypothalamohypophyseal region, with an increase of permeability for gonadotropins more than for GH or PRL. In a study of patients suffering from viral infections, leukemias, Hodgkin’s disease, or multiple sclerosis, GH levels in the CSF declined significantly with age. No correlation between GH levels and those of other CSF constituents belonging to the GH/IGF-I axis was found, e.g., with IGF-I and its binding protein IGFBP-3 or IGFBP2 (Heinze et al., 2004). The authors suggested that the age-related decrease of CSF levels of GH could contribute to the age-dependent decline of GH receptors in brain, which under normal function are upregulated by GH. As mentioned in Section I, CSF levels of GH have also been probed in GHD patients subjected to GH replacement therapy (Burman et al., 1996; Johansson et al., 1995). In a double-blind, placebo-controlled trial, the effect of recombinant human GH on CSF levels of GH was examined in 20 patients with GHD acquired in adult life. They were treated for 1 month with the recombinant hormone

(0.25 U/kg/week) or saline-containing vehicle. Patients participating in the study also received the appropriate thyroid, adrenal, and gonadal hormone replacement. Following treatment, the mean CSF concentration of GH increased from 13.3 ⫾ 4.4 to 149.3 ⫾ 22.2 ␮U/ml. In another double-blind, placebo-controlled 21-month study with a cross-over design, with each treatment period lasting for 9 months, the effect of long-term administration of recombinant GH on CSF concentrations of this hormone was investigated (Burman et al., 1996). The study included 24 patients with documented GHD with adult onset. Analysis of CSF collected at the end of the two treatment periods showed that the CSF concentration of GH was increased. Moreover, the level of the hormone was significantly related to the administered dose of the recombinant GH. Thus, the study suggested a transfer of GH from the circulation into the CSF, and interactions between the hormone and certain brain circuits may reflect the beneficial effect seen for GH on mood and behavior (Burman and Deijen, 1998; Wiren et al., 1998; Johansson et al., 2004).

B. Clinical Studies of IGF-I in CSF In conformity with GH its mediator of many effects, IGF-I, has also been analysed in various pituitary diseases. On the basis of the demonstration that this growth factor and its binding proteins are secreted by CNS tumors, the levels of IGF-I in the CSF were examined in patients with pituitary and other CNS tumors (Glick and Unterman, 1995). By a specific RIA, IGF-I was measured in 26 patients with tumors located adjacent to the ventricular system. CSF from patients without tumors served as controls. Prior to RIA the CSF specimens were treated with acetic acid overnight and IGF-I-binding proteins were separated from IGF-I by C-2 solid-phase cartridge extraction. A similar procedure was used to measure IGF-2, a growth factor also known to be produced within the CNS. Significantly elevated levels of IGFs were found in the CSF of patients with pituitary tumors. In nonsecreting pituitary tumors, the levels of IGF-I in the CSF were similar to normal levels, whereas IGF-2 levels were enhanced significantly. In patients with acromegaly, CSF levels of both IGF-I and -2 were elevated significantly compared to levels in normal samples and in CSF from patients with nonsecreting tumors. However, the levels of the two IGFs in CSF samples from patients with most of the primary and metastatic CNS tumors did not deviate significantly from those of normal subjects. In an earlier study (Backstrom et al., 1984), IGF (somatomedins) levels in CSF were probed in patients with acromegaly, GHD, prolactinoma, and Cushing’s disease by a RIA for IGF-I and for IGF-2 by both RIA and a radioreceptor assay (RRA) using adult human brain plasma membranes and IGF-2 as the radioligand. The mean value detected by RIA for IGF-2 (31 ⫾ 1.6 ng/ml) was about 5-fold higher than that obtained for IGF-I (5.8 ⫾ 0.3 ng/ml), but 10-fold

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higher concentrations of IGF-2 were recorded by RRA. The IGFs levels in CSF samples collected from acromegalic patients did not differ from those of GHD patients and no correlations were found between CSF levels determined by RIA or RRA when compared to circulatory levels of IGF-I or GH. However, a positive correlation between RIA assayable IGF-2 in CSF and serum IGF-I in the group of acromegalic patients was observed. All these findings suggest that IGFs in CSF consist primarily of IGF-2-like peptides, which may be produced mainly within the CNS or in the pituitary gland rather than accounting for transport from the circulation across the BCB. An attempt to correlate the CSF and serum levels of IGF-I in patients with Rett syndrome was reported by Vanhala et al. (2000). Rett syndrome is characterized as a neuro-developmental disease, including failure of somatic and brain growth. Increasing evidence shows that IGF-I plays an important role in early development of the brain, and the authors’ goal was to assess the role of IGFs in the pathogenesis of this childhood disease. They used a sensitive RIA to measure IGF-I levels in serum and cerebrospinal fluid and compared them with those in agematched controls. No difference in mean serum or CSF concentrations of IGF-I between patients and controls was found. Moreover, no significant correlation between CSF and serum levels of the IGF-I peptide in Rett syndrome was obtained. The authors suggested that this could be indicative of an independent role of IGF-I in the CNS, making serum IGF-I measurement unreliable as an indicator of disturbed function in the CNS (Vanhala et al., 2000). They concluded that their results did not support the notion that a deficit in the IGF system could explain the impaired somatic and brain growth associated with Rett syndrome. Another neurological childhood disease that has attracted search for impairments in the IGF system in the CSF is autism. This disorder represents a behaviorally defined syndrome, which is characterized by disturbances of social interaction and communication but also includes restrictions of behavior patterns and imagination. In conformity with Rett syndrome, factors involved in early brain development, such as IGFs, have attracted research in autism. A recent study was carried out in order to determine whether IGF-I levels in the CSF might be useful to probe in connection with the development of autism (Vanhala et al., 2001). The study used the RIA method to measure IGF-I levels in the CSF of 11 children with autism and compared them with the levels in 11 control subjects. The authors reporting this study found that the IGF-I levels in the CSF were statistically significantly lower in the children with autism than in the control children (Vanhala et al., 2001). They concluded that IGF-I, in addition to other neurotrophic factors, could be involved in the mechanisms behind the pathogenesis of autism. Riikonen et al. (2004) extended their studies on the IGF system to also include measurements of IGF-I in CSF from

children with acute lymphoblastic leukemia. Using RIA, they examined CSF levels of the growth factor and its binding protein IGFBP-2 to find out whether these levels correlated to any of the neurological deficits observed in children with this disease. Their result indicated that children with acute lymphoblastic leukemia had subnormal levels of IGF-I in their CSF, which, however, improved following 2 months of chemotherapy. In some patients with severe vincristine polyneuropathy, no improvement was seen throughout the chemotherapeutic period. No statistically significant correlation between the CSF level of IGF-I and any disturbed neurological function could be confirmed in this study. Studies of the IGF-I level in the CSF have also been carried out in GHD patients subjected to GH replacement therapy. Thus, the effect of recombinant human GH on CSF concentrations of IGF-I was examined in a double-blind, placebo-controlled trial (Johansson et al., 1995). Patients with GHD acquired in adult life were treated for 1 month with GH (0.25 U/kg/week) or placebo. Following treatment the IGF-I concentration in the fluid increased from 0.67 ⫾ 0.04 to 0.99 ⫾ 0.10 ng/ml, and the IGFBP-3 level also displayed an increase from 13.4 ⫾ 1.25 to 17.5 ⫾ 1.83 ng/ml. The enhanced level of IGF-I could reflect an enhancement in the transfer of the growth factor from peripheral sources over the BCB, but it could also mirror a GH-induced release of IGF-I within the CNS, as GH also appears to cross the BCB (Johansson et al., 1995).

IV. FUTURE PERSPECTIVES The increasing knowledge on functions involving GH and IGF-I in the CNS causes an enhanced need of techniques for studies of the somatotrophic axis in the brain. Therefore, the possibility of probing CSF levels of these hormones in the CSF will probably be essential for future studies. At present, immunological techniques such as RIA and enzyme-linked immunoassay, as well as RRA and radiometric assays, are the currently used methods for this purpose. However, there are several disadvantages with these assays and the development of new procedures with increased specificity, sensitivity, and reliability is desirable. Among techniques that might fulfill these desires is mass spectrometry (MS). The MS procedure has been used for studies of GH and may also discriminate between various isoforms of the hormone (Silberring et al., 1991; Zhan et al., 2005). According to the current breakthrough seen in the area of proteomics and new techniques developing in this context, it is likely that improvements in this area will also provide a basis for the development of new techniques for measurements of GH and IGF-I in the CSF. The access to such procedures will certainly respond to a great need in future approaches to probe activity in the GH/IGF-I axis in the brain.

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Acknowledgment This study was supported by the Swedish Medical Research Council (Grant 9459).

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6 Growth Hormone, Insulin, and Insulin-like Growth Factor-I: Do They Interact at the Blood–Brain Barrier? WEIHONG PAN,* YONGMEI YU,* FRED NYBERG,† and ABBA J. KASTIN* *Pennington Biomedical Research Center, LSU Systems, Baton Rouge, Louisiana, 70808 †Department of Pharmceutical Biosciences, Uppsala University, Uppsala, Sweden

I. Why There Is an Issue about Whether Growth Hormone (GH) Crosses the Blood–Brain Barrier (BBB) II. Current Knowledge about the Transport Systems for Insulin and Insulin-like Growth Factor-I (IGF-I) at the BBB III. Absence of a Saturable Transport System for GH at the BBB IV. Lack of Direct Interaction of GH and Insulin at the BBB, but IGF-I May Be an Indirect Mediator V. Conclusions References

did reduce the influx transfer constant of 125I-labeled insulin, suggesting that IGF-I may share the transport system for insulin. Thus, results indicate that GH can cross the BBB independently of the transport of insulin and IGF-I.

Growth hormone (GH) and insulin generally have opposite effects in the periphery but synergistic effects in the central nervous system. Insulin-like growth factor-I (IGF-I) mediates the effects of GH in a variety of circumstances. It has been shown that both insulin and IGF-I cross the blood– brain barrier (BBB) by saturable transport systems (Baura et al., 1993b; Banks et al., 1996; Pan and Kastin, 2000). Permeation of GH across the BBB has been suspected, but there is no direct evidence whether GH enters the brain by means of a saturable transport system. Experimental results with radioactively labeled GH show that it has significant influx from blood to the brain. This entry is not mediated by a saturable transport system at the BBB, as excess nonradioactively labeled GH had no modulatory effect on the influx transfer constant or the volume of distribution of 125I-labeled GH in the brain. Similarly, excess insulin had no effect on GH entry at the doses tested. However, IGF-I

The blood–brain barrier is a communicating interface between the cerebral microcirculation (the periphery) and the central nervous system (CNS) parenchyma. The capillary endothelial cells composing the BBB are joined by tight junctions and are lined by a continuous basement membrane. The barrier is also reinforced by astrocytic end feet and some other biochemical and structural changes of the endothelial cells and extracellular matrices. In the absence of paracellular routes, proteins that are large and not lipid soluble have limited diffusion across the lipid bilayers of endothelial cells so that if they enter the cells, they probably will be degraded by enzymes intracellularly. The principal form of growth hormone (GH) is a polypeptide of 191 amino acids (22 kDa). By the old dogma, this large size should preclude penetration into the brain. However, specific transport systems have been identified for many peptides and proteins. This growing list includes some interleukins

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. WHY THERE IS AN ISSUE ABOUT WHETHER GROWTH HORMONE (GH) CROSSES THE BLOOD–BRAIN BARRIER (BBB)

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(Banks et al., 1989, 1991, 1994a,b), tumor necrosis factor a (Gutierrez et al., 1993; Pan et al., 1997), and some other cytokines that are even larger than GH (Pan and Kastin, 1999). Speculation that GH crosses the BBB is stimulated by several lines of studies. First, evidence shows that exogenous GH used as supplementary therapy can improve human cognitive function, mood, memory, and sleep (Burman et al., 1995; Nyberg, 2000). Second, not only can GH affect CSF levels of various neuropeptides, amino acids, and monoamine metabolites (Johansson et al., 1995; Nyberg and Burman, 1996), but GH can also be recovered from the cerebrospiral fluid (CSF) after peripheral administration (Coculescu, 1999). Third, GH receptors are present in the CNS, with the highest binding density in the choroid plexus (Lai et al., 1991). GH can also cause age-dependent upregulation of its own receptor and change NMDA receptor subunit gene transcripts in the hippocampus (Le Greves et al., 2002). Thus, it seemed possible that GH could penetrate the BBB to exert its effects directly on the brain. The CNS actions of GH provide a good reason to determine whether it crosses the BBB by a transport system. For instance, GH shows neuroprotective effects in animal models of spinal cord injury (Hanci et al., 1994; Winkler et al., 2000) and hypoxic ischemia (Gustafson et al., 1999). After spinal cord injury in the rat by thoracic dorsal horn incision, there is an age-dependent increase in trauma-induced permeability to GH (Mustafa et al., 1995). If the increased entry of GH to the injured spinal cord is beneficial for neuroregeneration, delivery of therapeutic doses of GH by way of its transport system could further facilitate locomotor recovery. GH also alters appetite and feeding behavior (Wang et al., 2000). It is not known whether these effects occur in the periphery, through the circumventricular organs, or by centrally mediated alterations in other neurotransmitters. An understanding of the interactions of GH with the BBB will assist in the design of strategies to control feeding behavior. It would be very helpful, therefore, to determine whether and how GH crosses the BBB. This information would advance understanding of the physiology of GH actions and help in the design of therapeutic approaches for various CNS-driven behaviors.

II. CURRENT KNOWLEDGE ABOUT THE TRANSPORT SYSTEMS FOR INSULIN AND INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AT THE BBB Although insulin is mainly secreted by ␤ cells in the pancreas and acts on peripheral targets such as liver and muscle, it has a significant effect on brain metabolism (Henneberg

and Hoyer, 1994), sympathetic nervous system activity (Muntzel et al., 1994), and neuronal survival (Plitzko et al., 2001). In the CNS, insulin may exert different actions on blood glucose, feeding behavior, and hormonal levels in blood (Plata-Salaman, 1991; Schwartz et al., 1992; Sipols et al., 1994). Permeation of insulin across the blood–CSF barrier and the BBB was suggested by studies in human beings in 1987 (Wallum et al., 1987) and was shown in dogs several years later (Schwartz et al., 1990; Baura et al., 1993a). Banks et al. (1996, 1997) characterized the pharmacokinetics of the saturable transport system for insulin at the mouse BBB, showed its distinct regional uptake (Minors and Waterhouse, 1988; Banks et al., 1999), and illustrated its modulation in pathophysiological conditions, particularly diabetes mellitus (Banks et al., 1997). The baseline influx transfer constant for 125I-labeled insulin from various studies ranges from 0.65 to 2.55 ␮l/gmin. There is a high degree of regional difference, with the pons, medulla, and hypothalamus having the fastest rates of uptake, whereas the midbrain, thalamus, and occipital cortex have no significant uptake. Upregulation of insulin transport, shown by both in vivo multiple-time regression analysis and in situ brain perfusion studies in mice with streptozotocin-induced diabetes (Banks et al., 1997), supports the role of BBB transport of insulin in pathological states. The presence of a saturable transport system for insulin is particularly intriguing because CNS insulin can exert effects opposite from those of peripheral insulin. This is probably a mechanism to prevent “overshooting” of the feedback and feedforward loops. When peripheral insulin levels increase with hyperglycemia, more insulin will be available to penetrate the BBB. However, once insulin reaches its CNS targets, it may further elevate blood glucose concentrations. It is certainly conceivable that the actions follow different time courses to provide a finely tuned control. In a situation where dietary intake results in an acute increase in blood sugar concentrations, the resulting increase in blood insulin concentrations might overshoot; more insulin from the blood reaching the brain could then cause a compensatory increase in centrally controlled blood glucose to counteract the sudden increase in blood insulin. The CNS sensors might be able to react rapidly so as to control the glucose:insulin balance. The presence of a transport system that is saturable could prevent subsequent overstimulation of the central insulin pathways. In the mid-1980s, a receptor-mediated transport mechanism for insulin to cross bovine aortic endothelial cells (non-BBB endothelium) was indicated by a study using dual-chamber vessels (King and Johnson, 1985). In this system, the amount of 125I-labeled insulin transported from the upper to lower chamber was 15% with a confluent monolayer of aortic endothelial cells. The apical-to-basolateral flux of 125I-labeled insulin was temperature sensitive and was inhibited not only by unlabeled insulin, but also by

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6. GH, Insulin, and IGF-I

an antibody to the insulin receptor. This indicated that the insulin receptor is involved in the saturable transport. Binding sites for insulin have been found on vascular endothelial cells and may represent transporters (Frank and Pardridge, 1981; Miller et al., 1994). Although cerebral microvessel endothelial cells undergo differentiation and phenotypic changes, endocytosis of insulin at the BBB may be mediated by its receptor. IGF-I is a peptide of about 70 amino acids. After binding to the IGF-I receptor, the IGF-II receptor, the insulin receptor, or a hybrid receptor, IGF-I may mediate the effects of GH and insulin (Leroith and Blakesley, 2001). There are multiple binding proteins in blood, and the first three amino acids at the N terminus mediate a substantial amount of the binding. Thus, the pharmacokinetics of IGF-I permeation across the BBB is largely affected by the presence of binding proteins. When 125I-labeled IGF-I is injected intravenously in a bolus, most of it is associated with specific binding proteins. This probably explains why an excess dose of IGF-I can paradoxically increase the influx transfer constant of 125I-labeled IGF-I, as more 125I-labeled IGF-I becomes available for transport across the BBB. The saturable transport system for IGF-I has been shown by in situ brain perfusion studies accompanied by experiments involving HPLC and capillary depletion (Pan and Kastin, 2000).

III. ABSENCE OF A SATURABLE TRANSPORT SYSTEM FOR GH AT THE BBB The pharmacokinetics of BBB permeation by rat and human GH has been determined in ketamine-anesthetized male CD1 mice and Wistar rats with radioactively labeled GH. After an intravenous bolus injection, 125I-labeled GH was relatively stable in blood. The serum half-life of 125I-labeled GH was not affected by the presence of excess GH or insulin, consistent with the presence of growth hormone-binding proteins in the circulation. Multiple-time regression analysis (Kastin et al., 2001) showed that the influx transfer constant was about 0.44 ␮l/gmin, indicating substantial entry of 125I-labeled GH into the brain. To determine whether the permeation of 125I-labeled GH was mediated by a saturable transport system at the BBB, excess nonradioactively labeled GH was coadministered with 125I-labeled GH in the IV injection. When 1 ␮g/mouse unlabeled GH was added, there was no significant change in the influx transfer constant of 125I-labeled GH. Moreover, even as much as 200 µg/mouse unlabeled GH did not change the volume of distribution of 125I-labeled GH in the brain at 10 min. This high dose of excess GH should have replaced 125I-labeled GH from its binding proteins and competed with 125I-labeled GH for BBB transport as well.

Similarly, there was no inhibition of the permeation of rat 125I-labeled GH in rat studies. Lack of a modulatory effect of excess GH suggests that the penetration of GH across the BBB probably does not involve selective transport. To further exclude interference by serum GH-binding proteins, in situ brain perfusion was performed with serumfree buffer. Under these conditions, nonradioactively labeled excess GH still did not affect the influx transfer constant of 125I-labeled GH. Therefore, the lack of saturation was not explained by the presence of serum-binding proteins. Taken together, results suggest that the substantial entry of rat and human GH across the mouse BBB does not occur by a saturable transport system. Once GH reaches the cerebral circulation, there is only a certain amount that crosses the BBB completely. In capillary depletion studies, only about 27% of 125I-labeled GH is present in brain parenchyma, whereas 10% is trapped in the cerebral endothelium and the rest remains in the lumen of the cerebral blood vessels (Pan et al., 2005).

IV. LACK OF DIRECT INTERACTION OF GH AND INSULIN AT THE BBB, BUT IGF-I MAY BE AN INDIRECT MEDIATOR There does not seem to be evidence that GH and insulin interact directly at the BBB level. The blood-tobrain permeation of GH is not modified by excess GH or insulin. However, IGF-I mediates many of the effects of GH. We have found that IGF-I at 2 ␮g/mouse significantly decreased the influx transfer constant of 125I-labeled insulin (W. Pan et al., unpublished observations). This suggests that IGF-I and insulin share a transport system at the BBB. The saturable transport system for insulin at the BBB supplies one of the main sources of insulin from blood, as little, if any, insulin is synthesized in the brain (Banks, 2004). IGF-I is the major mediator of the indirect effects of GH, including the stimulation of body growth and actions on protein, fat, and carbohydrate metabolism. Thus, although GH has no direct modulatory action on the BBB permeation of insulin, peripheral GH may still affect the level of brain insulin by its actions on IGF-I.

V. CONCLUSIONS The interactions of GH, insulin, and IGF-I at the BBB are complex. GH by itself does not decrease the amount of insulin entering the brain, but it does stimulate the liver production of IGF-I, which in turn competes with the saturable transport of insulin. The interaction of insulin and

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IGF-I at the BBB appears to be independent of the significant BBB permeation of GH, which, in contrast to insulin and IGF-I transport, occurs by a nonsaturable system.

Acknowledgment Supported by NIH (NS45751 and NS46528 to WP, DK54880 and AA12865 to AJK).

References Banks, W. A. (2004). The source of cerebral insulin. Eur. J. Pharmacol. 490, 5–12. Banks, W. A., Jaspan, J. B., and Kastin, A. J. (1997). Effect of diabetes mellitus on the permeability of the blood-brain barrier to insulin. Peptides 18, 1577–1584. Banks, W. A., Kastin, A. J., and Durham, D. A. (1989). Bidirectional transport of interleukin-1 alpha across the blood-brain barrier. Brain Res. Bull. 23, 433–437. Banks, W. A., Kastin, A. J., and Ehrensing, C. A. (1994a). Blood-borne interleukin-1␣ is transported across the endothelial blood-spinal cord barrier in mice. J. Physiol, 479(2), 257–264. Banks, W. A., Kastin, A. J., and Gutierrez, E. G. (1994b). Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci. Lett. 179, 53–56. Banks, W. A., Kastin, A. J., Huang, W., Jaspan, J. B., and Maness, L. M. (1996). Leptin enters the brain by a saturable system independent of insulin. Peptides 17, 305–311. Banks, W. A., Kastin, A. J., and Pan, W. (1999). Uptake and degradation of blood-borne insulin by the olfactory bulb. Peptides 20, 373–378. Banks, W. A., Ortiz, L., Plotkin, S. R., and Kastin, A. J. (1991). Human interleukin (IL)1␣, murine IL-1␣ and murine IL-1␤ are transported from blood to brain in the mouse by a shared saturable mechanism. J. Pharmacol. Exp. Ther. 259, 988–996. Baura, G. D., Foster, D. M., Porte, D., Jr., Kahn, S. E., Bergman, R. N., Cobelli, C., and Schwartz, M. W. (1993a). Saturable transport of insulin from plasma into the central nervous system of dogs in vivo. J. Clin. Invest. 92, 1824–1830. Baura, G. D., Foster, D. M., Porte, D., Jr., Kahn, S. E., Bergman, R. N., Cobelli, C., and Schwartz, M. W. (1993b). Saturable transport of insulin from plasma into the central nervous system of dogs in vivo: A mechanism for regulated insulin delivery to the brain. J. Clin. Invest. 92, 1824–1830. Burman, P., Broman, J. E., Hetta, J., Wiklund, I., Erfurth, E. M., Hagg, E., and Karlsson, F. A. (1995). Quality-of-life in adults with growthhormone (GH) deficiency: Response to treatment with recombinant human GH in a placebo-controlled 21-month trial. J. Clin. Endocrinol. Metab. 80, 3585–3590. Coculescu, M. (1999). Blood-brain barrier for human growth hormone and insulin-like growth factor-I. J. Pediatr. Endocrinol. Metab. 12, 113–124. Frank, H. J., and Pardridge, W. M. (1981). A direct in vitro demonstration of insulin binding to isolated brain microvessels. Diabetes 30, 757–761. Gustafson, K., Hagberg, H., Bengtsson, B. A., Brantsing, C., and Isgaard, J. (1999). Possible protective role of growth hormone in hypoxia-ischemia in neonatal rats. Pediatr. Res. 45, 318–323. Gutierrez, E. G., Banks, W. A., and Kastin, A. J. (1993). Murine tumor necrosis factor alpha is transported from blood to brain in the mouse. J. Neuroimmunol. 47, 169–176. Hanci, M., Kuday, C., and Oguzoglu, S. A. (1994). The effects of synthetic growth hormone on spinal cord injury. J. Neurosurg. Sci. 38, 43–49.

Henneberg, N., and Hoyer, S. (1994). Short-term or long-term intracerebroventricular (icv) infusion of insulin exhibits a discrete anabolic effect on cerebral energy-metabolism in the rat. Neurosci. Lett. 175, 153–156. Johansson, J. O., Larsson, G., Elmgren, A., Hynsjö, L., Lindahl, A., Lundberg, P. A., Isaksson, O., Lindstedt, S., and Bengtsson, B. Å. (1995). Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. Neuroendocrinology 61, 57–66. Kastin, A. J., Akerstrom, V., and Pan, W. (2001). Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: Meaningful controls. Peptides 22, 2127–2136. King, G. L., and Johnson, S. M. (1985). Receptor-mediated transport of insulin across endothelial cells. Science 227, 1583–1586. Lai, Z. N., Emtner, M., Roos, P., and Nyberg, F. (1991). Characterization of putative growth-hormone receptors in human choroid-plexus. Brain Res. 546, 222–226. Le Greves, M., Steensland, P., Le Greves, P., and Nyberg, F. (2002). Growth hormone induces age-dependent alteration in the expression of hippocampal growth hormone receptor and N-methyl-D-aspartate receptor subunits gene transcripts in male rats. Proc. Natl. Acad. Sci. USA 99, 7119–7123. Leroith, D., and Blakesley, V. A. (2001). Growth factors and cytokines. In “Principles and Practice of Endocrinology and Metabolism” (K. L. Becker, ed.), pp 1588–1600. Lippincott Williams & Wilkins, Philadelphia. Miller, D. W., Keller, B. T., and Borchardt, R. T. (1994). Identification and distribution of insulin receptors on cultured bovine brain microvessel endothelial cells: Possible function in insulin processing in the bloodbrain barrier. J. Cell. Physiol. 161, 333–341. Minors, D. S., and Waterhouse, J. M. (1988). Mathematical and statistical analysis of circadian rhythms. Psychoneuroendocrinology 13, 443–464. Muntzel, M., Beltz, T., Mark, A. L., and Johnson, A. K. (1994). Anteroventral 3rd ventricle lesions abolish lumbar sympathetic responses to insulin. Hypertension 23, 1059–1062. Mustafa, A., Sharma, H. S., Olsson, Y., Gordh, T., Thoren, P., Sjoquist, P. O., Roos, P., Adem, A., and Nyberg, F. (1995). Vascular permeability to growth hormone in the rat central nervous system after focal spinal cord injury: Influence of a new antioxidant H-290/51 and age. Neurosci. Res. 23, 185–194. Nyberg, F. (2000). Growth hormone in the brain: Characteristics of specific brain targets for the hormone and their functional significance. Front. Neuroendocrinol. 21, 330–348. Nyberg, F., and Burman, P. (1996). Growth hormone and its receptors in the central nervous system: Location and functional significance. Horm. Res. 45, 18–22. Pan, W., Banks, W. A., and Kastin, A. J. (1997). Permeability of the bloodbrain and blood-spinal cord barriers to interferons. J. Neuroimmunol. 76, 105–111. Pan, W., and Kastin, A. J. (1999). Penetration of neurotrophins and cytokines across the blood-brain/blood-spinal cord barrier. Adv. Drug Deliv. Rev. 36, 291–298. Pan, W., and Kastin, A. J. (2000). Interactions of IGF-I with the bloodbrain barrier in vivo and in situ. Neuroendocrinology 72, 171–178. Pan, W., Yu, Y., Nyberg, F., and Kastin, A. J. (2005). Permeation of growth hormone across the blood-brain barrier. Submitted for publication. Plata-Salaman, C. R. (1991). Insulin in the cerebrospinal fluid. Neurosci. Biobehav. Rev. 15, 243–258. Plitzko, D., Rumpel, S., and Gottmann, K. (2001). Insulin promotes functional induction of silent synapses in differentiating rat neocortical neurons. Eur. J. Neurosci. 14, 1412–1415.

6. GH, Insulin, and IGF-I Schwartz, M. W., Figlewicz, D. P., Baskin, D. G., Woods, S. C., and Porte, D., Jr. (1992). Insulin in the brain: A hormonal regulator of energy balance. Endocr. Rev. 13, 81–108. Schwartz, M. W., Sipols, A., Kahn, S. E., Lattemann, D. F., Taborsky, G. J., Jr., Bergman, R. N., Woods, S. C., and Porte, D., Jr. (1990). Kinetics and specificity of insulin uptake from plasma into cerebrospinal fluid. Am. J. Physiol. 259, E378–E383. Sipols, A. J., Baskin, D. G., and Schwartz, M. W. (1994). Effect of intracerebroventricular insulin infusion on diabetic hyperphagia and hypothalamic neuropeptide gene expression. Diabetes 44, 147–151. Wallum, B. J., Taborsky, G. J. Jr., Porte, D., Jr., Figlewicz, D. P., Jacobson, L., Beard, J. C., Ward, W. K., and Dorsa, D. (1987). Cerebrospinal fluid

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insulin levels increase during intravenous insulin infusion in man. J. Clin. Endocrinol. Metab. 64, 190–194. Wang, X., Day, J. R., Zhou, Y., Beard, J. L., and Vasilatos-Younken, R. (2000). Evidence of a role for neuropeptide Y and monoamines in mediating the appetite-suppressive effect of GH. J. Endocrinol. 166, 621–630. Winkler, T., Sharma, H. S., Stalberg, E., Badgaiyan, R. D., Westman, J., and Nyberg, F. (2000). Growth hormone attenuates alterations in spinal cord evoked potentials and cell injury following trauma to the rat spinal cord: An experimental study using topical application of rat growth hormone. Amino Acids 19, 363–371.

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7 Growth Hormone and Insulin-like Growth Factors in the Central Nervous System: Localization in Mammalian Species VINCENZO C. RUSSO and GEORGE A. WERTHER Centre for Hormone Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Department of Paediatrics, University of Melbourne, Parkville, 3052 Victoria, Australia

I. Expression of the Growth Hormone System in the Mammalian Brain II. Expression of the Insulin-like Growth Factor System in the Brain III. Concluding Remarks References

human brain. It is, therefore, of critical importance to determine the sites of expression and action of these two key neuroendocrine systems in the mammalian brain. This chapter presents a comprehensive overview on the localization of the GH and IGF system components in the CNS and suggests likely mechanisms of action.

In addition to the well-known physiological mechanisms activated by the growth hormone (GH)/insulin-like growth factor (IGF) somatotrophic endocrine axis, increasing evidence for the expression of components of the GH and IGF system within the mammalian brain clearly points to the presence of physiologically important paracrine or autocrine GH/IGF neuroendocrine circuits in the central nervous system (CNS). A critical role for the GH and IGF system in fetal and postnatal brain development is strongly supported by a number of studies employing transgenic and knockout animal models for either GH or IGF system components. Alteration of either the GH or IGF system results in similar alteration of the size and morphology of the CNS during development, thus suggesting that both the GH and/or IGF system, alone or in combination, modulate these events. Furthermore, both GH and IGF-I have been utilized successfully as neuroprotective agents in animals for a variety of brain diseases and injury models, thus warranting their potential use as alternative neuroprotective and neurogenic therapies for diseases affecting the

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. EXPRESSION OF THE GROWTH HORMONE SYSTEM IN THE MAMMALIAN BRAIN A. Growth Hormone Sites of Action in the Developing Mammalian Brain Growth hormone (GH) gene expression occurs predominantly in the pituitary gland, although it also occurs in many extrapituitary sites, including the brain (GarciaAragon et al., 1992; Edmondson et al., 1995; Harvey et al., 1998) with brain GH immunoreactivity not affected by hypophysectomy (Ajo et al., 2003). The cellular location and ontogeny of neural GH production is, however, not well documented. Limited data on GH mRNA expression in the brain, determined by in situ hybridization, provide some support for local expression of GH (Garcia-Aragon et al., 1992; Edmondson et al., 1995; Harvey et al., 1998; Lobie et al., 2000).

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Early studies by Garcia-Aragon et al. (1992), followed by Edmondson et al. (1995), have clearly demonstrated expression (mRNA) and synthesis (immunoreactivity) of the GH receptor/binding protein (BP) during embryonic and fetal development of the rat brain. In these studies, immunohistochemical and molecular analyses revealed GH receptor/binding protein expression in all major organ systems of the E18 rat fetus, including the brain (GarciaAragon et al., 1992; Edmondson et al., 1995). A further detailed postnatal ontogenic study demonstrated widespread but discrete GH receptor/BP expression in many regions of the rat and rabbit brains (Lobie et al., 1993; Lincoln et al., 1994). Regions with high expression included the cerebral cortex, neuronal-rich formations of the thalamus and hypothalamus, cerebellum (Purkinje cells), neuronal cell in the brain stem, and retinal ganglion cells (Lobie et al., 1993; Lincoln et al., 1994). Some expression was also associated with glial cells and astrocytes and in other nonneuronal structures, including the ependyma of the choroid plexus, ventricular lining, and pia mater (Lobie et al., 1993). More discrete was the localization/immunoreactivity of GH receptor/BP in the neonatal rabbit, with neuronal formation in the cerebral cortex, pyramidal cells of the hippocampus, brain stem reticular formation, dorsal thalamus, and hypothalamus being the major areas. Although the presence of GHR-immunoreactive protein in the brain suggests target sites for GH action, this, however, could potentially reflect the presence of GH-binding proteins rather than authentic receptors. A different approach to unequivocally identifying sites of GH action/ GR receptor activity in brain has been described by Harvey and co-workers (2002) in a nonmammalian species, birds. They localized sites of GH action in the brain by looking at the expression of specific GH-responsive genes, including the novel GH-responsive gene GHRG-1. This gene, not expressed in GH-resistant dwarfs with dysfunctional GH receptor has been identified as an intracellular marker of GH action (i.e.,) upregulated by exogenous GH). These studies by Harvey et al. (2002) clearly showed the expression of a GH-responsive gene in neural tissues. GHRG-1 mRNA was abundantly and widespread expressed in the brain of normal chickens, suggesting that GH might act in an autocrine or paracrine fashion within the central nervous system (CNS) (Harvey et al., 2002). In human brain, GH and its receptor are seen in a similar distribution (i.e., choroid plexus, hippocampus, hypothalamus, pituitary, putamen, and thalamus) to that described in the rat brain (Nyberg and Burman 1996; Nyberg, 2000). Binding studies from Lai et al. (1991) for human GH (hGH) in the human brain clearly demonstrated the highest density of human GH binding in the choroid plexus. Other brain regions with dense binding of labeled hGH were the hippocampus and the hypothalamus formation and

the pituitary gland (Lai et al., 1991). This pattern of hGH binding was observed in brain tissue from both sexes (Lai et al., 1991). However, brain tissue from females shows relatively higher binding than male tissue, suggesting that binding of hGH to the prolactin receptor might account for this apparently higher binding of hGH in female brain tissue (Lai et al., 1991; 1992). This sexdependent difference in binding is not observed in rats when rat-GH is used, whereas a sex difference was observed in rat when hGH was employed (Mustafa et al., 1997), once again explained by the known cross-reactivity of hGH with the lactogenic receptor. Whether GH receptors in the brain are activated by circulating GH synthesized in the pituitary gland or locally expressed GH remains unclear, especially since data on local expression are limited.

B. Growth Hormone and Insulin-like Growth Factor-I in the Nervous System Although insulin-like growth factor (IGF-I) acts as a paracrine factor for multiple GH actions and is also found in the cerebral hemispheres, where it exerts neurotrophic effects, only a few studies have attempted to demonstrate coexpression of the GH and IGF systems during fetal and postnatal mammalian brain development. Early work from Edmondson et al. (1995) and later studies from Lobie and co-workers (2000) have demonstrated the sites of expression of growth hormone receptor in the rodent brain and compared these to the sites of IGF-I expression (Lobie et al., 2000). From these studies it appears that while some IGF-I and GH receptor/BP expression sites colocalized macroscopically to the same brain region, at a cellular level, GH receptor/BP and IGF-I expression sites are distinct (Edmondson et al., 1995; Lobie et al., 2000). GH receptor/BP mRNA is expressed in tissues adjacent to those expressing IGF-I mRNA (Edmondson et al., 1995) as detected in the rat brain olfactory bulb with GHR/BP mRNA highly expressed in the internal granular layer (Fig. 1), distal from the IGF-I mRNA localized to the outer mitral and glomerular layers (Edmondson et al., 1995) (Fig. 1). It is likely therefore that, in vivo, GH action in the brain is not mediated via local IGF-I expression. Several experimental in vivo models support a key and IGF-independent role for GH in brain development. The Snell dwarf mouse (Pit1) and the GHRH receptor-deficient little mouse exhibit microcephalic cerebra with hypomyelination, retarded neuronal growth with poor synaptogenesis. Postnatal GH administration normalizes neuronal growth in these mice (Sugisaki et al., 1985; Noguchi et al., 1984). In vivo studies by Scheepens et al. (1999, 2000, 2001) have demonstrated that GH is involved in neuroprotection

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7. GH and IGF in the CNS Section A-A A

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FIGURE 1 GH and IGF systems in the rat brain olfactory bulb. GH and IGF systems are expressed and synthesized in distinct areas of the rat brain (i.e., olfactory bulb, shown). This provides strong evidence for autocrine and paracrine actions of both GH and IGFs in the nervous system. IGF action is modulated by locally expressed insulin-like growth factor-binding protein (IGFBPs). Olfactory nerve layer (ONL); glomerular cell layer (GL); external plexiform cell layer (EPL); mitral cell layer (MI); granular cell layer (GRL); and growth hormone receptor/binding protein (GH-R/BP). Section A-A shows H/E staining of an olfactory bulb. Area in the square is enlarged and represented in the schematic. Section A-A and rat brain insert reprinted from Brain Maps: Structure of the brain, 2nd Edition, Swanson, L. W., 1998–1999, Elsevier, Amsterdam. (See color plate 3)

during hypoxic/ischemic brain injury. These studies showed GH-like immunoreactivity on injured brain cells and demonstrated that GH administered intracerebroventricularly is capable of preventing brain cell loss (Scheepens et al., 1999, 2000, 2001). Whether the neurogenic/ protective effects of GH involve IGF-I induction is not clear. However, while an in vitro study by Ajo et al. (2003) demonstrated IGF-mediated GH action in fetal cerebral cortical cells, most in vivo data (Garcia-Aragon et al., 1992; Edmondson et al., 1995; Harrey et al., 1998; Lobie et al., 2000; Sugisaki et al., 1985; Noguchi et al., 1984; Scheepens et al., 1999, 2000, 2001) strongly suggest a direct and IGF-independent action for GH in the CNS. In summary, it is now reasonably well established that growth hormone gene expression is not restricted to the

pituitary gland and occurs in many extrapituitary tissues, including the central and peripheral nervous systems (Harvey and Hull, 2003) with GH-immunoreactive proteins abundant in the brain, spinal cord, and peripheral nerves. GH- and GHR-like proteins are relatively abundant in neural tissues during fetal and postnatal development and are independent of changes in pituitary GH secretion (Harvey et al., 1998; Harvey and Hull, 2003). The localization of GH and GHR in these tissues, prior to the appearance of endocrine circulating GH and distant from sites of IGF-I expression, suggests direct autocrine or paracrine roles for GH in the brain during early embryogenesis, pointing to a potential initial role for GH as a neuropeptide, rather than as an endocrine hormone (Harvey et al., 1998; Harvey and Hull, 2003).

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II. EXPRESSION OF THE IGF SYSTEM IN THE BRAIN A. IGF Expression in the Nervous System Mammalian CNS development begins in the embryo with the formation and closure of the neural tube followed by the rapid division of pluripotential cells (stem cells), which then migrate to the periphery of the neural tube and differentiate into neural or glial cells. These cells form special structures such as nuclei, ganglia, and cerebral cortical layers and develop a network with their cytoplasmic extensions, neurites. These complex processes are regulated by a number of growth factors, including the IGFs (Richard-Parpaillon et al., 2002; Pera et al., 2001). IGF-I plays a key role in the development of the nervous system, with demonstrated effects on many stages of brain development, including cell proliferation, cell differentiation, and cell survival (Richard-Parpaillon et al., 2002; Pera et al., 2001; D’Ercole et al., 1996; Leventhal et al., 1999; Cheng et al., 2001; Bondy and Cheng, 2002). Although reports have demonstrated, in rats, that postnatal circulating IGF-I might exert neurogenenic/survival activity (Trejo et al., 2001, 2002; Carro et al., 2001, 2002; Busiguina et al., 2000; Popken et al., 2004; Pulford and Ishii, 2001), systemic IGF-I is not readily transported through the blood–brain barrier and therefore local production of IGF-I is considered the primary source of the ligand (autocrine and paracrine action) for brain cells. During embryogenesis, IGF-I mRNA expression is detectable in many regions of the rat brain (Han et al., 1987a,b, 1988; Rotwein et al., 1988; Andersson et al., 1988; Lee et al., 1992; Bondy et al., 1992; Ayer-le Lievre et al., 1991; Ajo et al., 2003). IGF-I gene expression is particularly high in neuronally rich regions such as the spinal cord, midbrain, cerebral cortex, hippocampus, and olfactory bulb (Edmondson et al., 1995; Rotwein et al., 1988; Garcia-Segura et al., 1991). More precisely, IGF-I is expressed in neuronal cells with large soma and complex dendritic formations (Bondy and Lee, 1993; Cheng et al., 2003), including sensory and projecting neurons, such as the Purkinje cells of the rat cerebellum (Bondy et al., 1992). In most neurons, IGF-I transcription decreases significantly postnatally, a decrease that correlates with the degree of cell maturation and reaches very low levels in the adult (Rotwein et al., 1988; Andersson et al., 1988). However, in rodents, exceptions are the mitral and tufted cells of the olfactory bulb (Fig. 1), which undergo constant cell renewal/turnover; in these cells, IGF-I expression persists at a high level throughout life (Graziadei and Monti-Graziadei, 1978). IGF-II mRNA is expressed abundantly in the embryonic rat CNS; however, IGF-II expression in cells of neuroepithelial origin is controversial (Hirvonen et al., 1989;

Bondy et al., 1990; Caelers et al., 2003). IGF-II is the most abundantly expressed IGF in the adult CNS, with the highest level of expression found in myelin sheaths, but also in leptomeninges, microvasculature, and the choroid plexus, all nonneuronal structures that enable diffusion of growth factors to their sites of activity (Bondy et al., 1990, 1992; Ayer-le Lievre et al., 1991; Logan et al., 1994; Couce et al., 1992; Stylianopoulou et al., 1988; Aberg et al., 2003).

B. Type I and II IGF Receptor Expression in the Nervous System IGF receptors are widely expressed throughout the mammalian (rat and mouse) central nervous system with high levels of expression found in specific regions and located to specific cell types (D’Ercole et al., 1996; Bondy et al., 1992; Bondy and Cheng, 2004; Werther et al., 1989, 1990, 1998a,b; Hawkes and Kar, 2003; Wilczak et al., 2000). Given that IGF receptors are expressed from early stages of embryogenesis and throughout life and that their ligands also show a similar “temporal-spatial” pattern of expression, it is evident that the local brain IGF circuits are crucial modulators of the processes activated during brain development. Type I IGF receptors (IGF-IR) are expressed throughout the rat CNS (Garcia-Segura et al., 1991; Werther et al., 1989; Gammeltoft et al., 1985, 1988; Bondy and Lee, 1993) with high levels of expression detected in the developing cerebellum, midbrain, and olfactory bulb (Fig. 1) and in the ventral floorplate of the hindbrain (Rotwein et al., 1988; Bondy et al., 1990; Werther et al., 1989). The level of IGF-IR decreases to adult levels soon after birth (Baron-Van Evercooren et al., 1991; Kar et al., 1993), but remains relatively high in the choroid plexus, meninges, and vascular sheaths (Bondy et al., 1992; Werther et al., 1989). It is thus not surprising that knockout of the IGF-I receptor gene (Liu et al., 1993; Baker et al., 1993; Beck et al., 1995) produced, in addition to in utero growth retardation, a clear brain phenotype, namely a small brain. The type II IGF receptor, which has low affinity for IGF-I, but a high affinity for IGF-II and also binds mannose-6-phosphate, is selectively expressed in all major brain regions (Couce et al., 1992; Hawkes and Kar, 2003, 2004; Wilczak et al., 2000; Valentino et al., 1990). It is highly expressed in the pyramidal cell layers of the hippocampus, the granule layer of the dentate gyrus, olfactory bulb, the choroid plexus, and in the cerebral vasculature, ependymal cells, retina, pituitary, brain stem, and spinal cord (Couce et al., 1992; Hawkes and Kar, 2003; Wilczak et al., 2000; Valentino et al., 1990). However, very little is known about the physiological significance of the type II IGF receptor in the functioning of the central nervous system (Hawkes and Kar, 2004).

7. GH and IGF in the CNS

C. IGF-Binding Proteins: Expression and Function in Brain The mRNA expression profiles and location of the most abundant IGF-binding proteins (IGFBPs) 2, 4, and 5 in the normal developing and adult CNS are well defined (Lee et al., 1992, 1993; Bondy and Lee, 1993; Ocrant, 1991, 1993; Russo et al., 1994; Han et al., 1996). IGFBP-3 (Russo et al., 1994; Han et al., 1996; Ocrant et al., 1990) and IGFBP-6 (Naeve et al., 2000) are also expressed discretely in the CNS, but at lower levels, and therefore data on their mRNA expression distribution are limited. IGFBP-1 is not expressed in the CNS. 1. IGFBP-2 In the rat, IGFBP-2 is expressed early in embryogenesis (Wood et al., 1992) and by embryonic day 10 is highly expressed in neuroectoderm structures, including the neural tube and neuroepithelium (Wood et al., 1992). During development the most prominent sites of IGFBP-2 expression in the rodent CNS comprise cells with non-neuronal phenotypes, including the epithelium of the choroid plexus, the floor plate, and the infundibulum (Wood et al., 1992). Later in development, IGFBP-2 mRNA is detectable throughout the rat brain (Lee et al., 1993; Liu et al., 1994), particularly in brain regions undergoing continuous remodeling, such as the olfactory bulb (Fig. 1), the cerebellum, and the hippocampus (D’Ercole et al., 1996; Lee et al., 1993; Russo et al., 1994; Liu et al., 1994). IGFBP-2 expression correlates with and complements that of IGF-II (IGFBP-2 preferential ligand), and both IGFBP-2 and IGF-II protein are highly abundant in the CSF and choroid plexus (D’Ercole et al., 1996). It is also known that IGFBP-2 binds IGF-II with a moderate preferential affinity over IGF-I (Firth and Baxter, 2002). IGFBP-2 associates to cell surface proteoglycans in rat brain tissue (Russo et al., 1997) and neuronal cells (Russo et al., 1999), and IGF-I/IGFBP-2/proteoglycan complexes have been identified in rat brain tissue (Russo et al., 2004), but the role of these cell membrane complexes is not completely understood. However, it has been suggested that differential localization of the IGFBPs in the pericellular and extracellular space, involving components of the extracellular matrix (Firth and Baxier, 2002; Russo et al., 1997, 1999; Jones and Clemmons, 1995; Parker et al., 1996, 1998; Rees and Clemmons, 1998) might regulate levels of diffusible/free IGFs. We have shown that IGF-I complexes with IGFBP-2 to promote neurogenesis in adult stem cells (Brooker et al., 2000) and further demonstrated that neurogenesis was inhibited by the IGFBP-2 antibody blockade (Brooker et al., 2000), thus suggesting a key role for IGFBP-2 in this process. Further evidence in support of an IGF-facilitating role for IGFBP-2 in the brain comes from colocalization of injected IGF-I with IGFBP-2 (Guan et al.,

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2000) and subsequent neuroprotection following hypoxic ischemic injury, an effect not seen with an IGF variant that does not bind IGFBP-2 (Guan et al., 1996). It is therefore possible that brain IGFBP-2 regulates IGF-I cellular responses by facilitating receptor targeting of IGF-I or modulating IGF-I bioavilability in the pericellular space in vivo (Firth and Baxter, 2002; Jones and Clemmons, 1995). Cell-associated IGFBP-2 may therefore act as a “linker” molecule, allowing pericellular storage of IGF. In vivo, this IGF-I storage might be affected by the presence of IGFBP-2 proteases (Ishikawa et al., 1995), which generate IGFBP-2 fragments (Ishikawa et al., 1995) that have a markedly reduced binding affinity for the IGFs (Firth and Baxter, 2002; Russo et al., 1999, 2004). An IGFBP-2 fragment was identified on the cell surface of human neuronal cells (Russo et al., 1999). The presence of this proteolytic fragment of IGFBP-2, capable of binding IGF-I while simultaneously being bound to the neuronal cell surface, might point to a process whereby the proteolysis of membrane-bound IGFBP-2 provides a mechanism for creating perireceptor low-affinity IGF-I-binding sites (Russo et al., 1999). Although it has been demonstrated that IGFBP-2 proteolysis occurs during the differentiation of human neuronal cells (Russo et al., 2004), the precise physiological significance of IGFBP-2 proteolysis in the mammalian nervous system remains to be determined. An example of a physiological change in local brain levels of IGFBP-2 and how this might affect IGF action has been reported by Cardona-Gomez et al. (2000) in a specialized group of glial cells of the third ventricle called tanycytes. These cells have the ability to accumulate IGF-I and thus regulate IGF-I availability (Fernandez-Galaz et al., 1996; Garcia-Segura et al., 1994; Duenas et al., 1994). It was shown that estradiol and progesterone regulate local levels of IGFBP-2, including “peri-IGF-I-receptor” IGFBP-2 (Cardona-Gomez et al., 2000) and that these changes affect the accumulation of IGF-I in tanycytes. This accumulation process might involve the interaction of IGF-I with cell surface-associated IGFBP-2 prior to IGF-I being translocated intracellularly (Cardona-Gomez et al., 2000). Unexpectedly, ablation of the IGFBP-2 gene (Wood et al., 1993) generated a phenotype less dramatic than that initially predicted. Selective alterations were reported for spleen and liver size, while the level of other circulating IGFBPs was found to be increased in the adult animals (Wood et al., 2000). The absence of a “brain phenotype” in the IGFBP-2⫺/⫺ mouse (Wood et al., 1993) suggests functional redundancy in the IGFBP family during development of the CNS (Pintar et al., 1995). However, the IGFBP-2 transgenic mouse model developed by the Hoeflich group suggests that IGFBP-2 may, under certain conditions, be a negative regulator of postnatal growth, including brain growth, in rodents (Hoeflich et al., 1999), potentially by reducing the bioavailability of IGF-I.

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2. IGFBP-3 IGFBP-3 is normally expressed at a low level in the mammalian CNS, mainly in nonneuronal structures, including epithelial cells (Leventhal et al., 1999; Russo et al., 1994; Lee and Bondy, 1993; Beilharz et al., 1998) (Fig. 1), and the effects of IGFBP-3 gene deletion on CNS are either unknown or have not been reported. Furthermore, no change in brain growth or phenotype has been reported in IGFBP-3 Tg mice (Murphy et al., 1995). Nevertheless, in a in vitro study by Ajo et al. (2003), IGFBP-3 was found to be upregulated in rat cerebral cortical cells following GH stimulation. Ajo et al. (2003) showed that GH promotes the proliferation of neural precursors, neurogenesis, and gliogenesis and that these responses are mediated by locally produced IGF-I and its modulator IGFBP-3. Conversely, a study by Rensink et al. (2002), investigating the mechanisms of amyloid-␤ (Abeta) deposition in cerebral blood vessel walls and Abeta-induced toxicity in Alzheimer’s disease (AD), proposed that IGFBP-3 might contribute to neuronal degeneration in AD. It is therefore possible that, as seen in other systems, IGFBP-3 might promote either enhancement or inhibition of IGF-I action in brain cells in vitro depending on the experimental conditions. Its role in the brain in vivo remains unclear. 3. IGFBP-4 IGFBP-4 is normally expressed at a very low level in the mammalian CNS where its mRNA is found in a variety of brain cell types, including meningeal cells, astrocytes, and fetal neuronal cells (Chernausek et al., 1993). During early brain development, IGFBP-4 expression is increased and its mRNA is easily detectable in regions such as the choroid plexus, meninges, and basal ganglia (Brar and Chernausek, 1993). Postnatally, IGFBP-4 mRNA is found in the meningeal cell layer surrounding the developing cerebellum in the hippocampal formation and olfactory bulb (Fig. 1) (Russo et al., 1994; Chernausek et al., 1993). In some of these IGFBP-4-expressing brain regions, which maintain a degree of tissue remodeling/plasticity (Werther et al., 1998a), local expression for IGF-I and its receptor is also seen, thus suggesting that IGFBP-4 may play a role as a local modulator of IGF action (Chernausek et al., 1993). Overexpression of IGFBP-4 gene has been investigated only recently in smooth muscle cell-rich tissue, and therefore the effects on the nervous tissue are unknown. Whether ablation of the IGFBP-4 gene (Pintar et al., 1995) affects the CNS of these mice also remains unknown. 4. IGFBP-5 IGFBP5 gene expression is highly abundant during rat brain development (Bondy and Lee, 1993). The early expression of IGFBP-5 at embryonic day 10.5 suggests a

key role of this IGFBP during embryogenesis (Green et al., 1994). This hypothesis is further supported by a study from Pera and co-workers (2001), which shows that IGFBP-5, as well as IGFs expressed in early embryos, promoted anterior development by increasing the head region in a nonmammalian species, namely Xenopus embryos. Thus, active IGF signals, including IGFBP-5, appear to be required for anterior neural induction in Xenopus (Pera et al., 2001). Whether IGFBP-5 has similar functions in early mammalian neural development is not known. In rodents, IGFBP-5 appears to be coexpressed with IGF-I in principal neurons of sensory relay systems, cerebellar cortex, hippocampal formation, and many other neuron-rich regions (Bondy and Lee, 1993; Cheng et al., 1996), including the olfactory bulb (Fig. 1) (Russo et al., 1994). These data point to the presence of potential autocrine and paracrine interactions between IGFBP-5 and IGF-I in specific brain regions, where IGFBP5 may act as a modulator or determinant of IGF action (Roschier et al., 2001). Further to this spatiotemporal coexpression of IGFBP-5 and IGF-I, it is now becoming clear that IGF-I specifically regulates IGFBP-5. Using two IGF-I transgenic mice lines, Ye and D’Ercole (1998) demonstrated that IGF-I upregulates IGFBP-5 expression in vivo. This increase is specific for IGFBP-5 mRNA, as the level of expression of IGFBP-2 and IGFBP-4 mRNAs in these mice was found unchanged (Ye and D’Ercole, 1998). The effects of IGFBP-5 gene ablation in the brain have not been reported. 5. IGFBP-6 IGFBP-6 is poorly expressed in the nervous system, and information regarding its mRNA expression distribution, in both developing and adult nervous systems, is limited. The unique property of IGFBP-6 of preferential binding to the IGF-II ligand (Bach, 1999), coupled with the fact that this ligand is the most abundantly expressed IGF in the adult CNS, suggests that the IGFBP-6/IGF-II complex has a unique role in modulating IGF-II function in the adult brain (Naeve et al., 2000). During CNS embryogenesis, IGFBP-6 expression is tightly restricted to trigeminal ganglia and, relative to the rest of the embryo, this structure has the highest expression (Naeve et al., 2000). The expression in the forebrain and cerebellum does not occur until after postnatal day 21 and then is primarily associated with GABAergic interneurons (Naeve et al., 2000). The highest levels of expression in the adult animal are in the hindbrain, spinal cord, cranial ganglia, and dorsal root ganglia (Naeve et al., 2000). These nuclei in the hindbrain and periphery that express IGFBP-6 are all associated with the coordination of sensorimotor function in the cerebellum, which suggests an important role for the IGFBP-6/IGF-II complex in the function and maintenance of these systems. IGFBP-6 binds IGF-II preferentially and is regarded as a relatively specific inhibitor of IGF-II actions (Bach, 1999).

7. GH and IGF in the CNS

IGFBP-6 is often expressed in nonproliferative cells and its expression is associated with inhibition of growth of tumor cells in vitro (Russo et al., 2004; Bach, 1999; Babajko et al., 1997) and in vivo (Bach, 1999). These findings are also supported by overexpression of IGFBP-6 in vivo, a model developed by Bienvenu et al. (2004) with strong expression of IGFBP-6 in GFAP-positive cells. Preliminary analysis of the IGFBP-6 Tg mouse shows reduced cerebellum size and weight combined to altered/reduced differentiation of astrocytes (Bienvenu et al., 2004). Abnormalities in the hypothalamus and pituitary were also reported (Bienvenu et al., 2004). The effects on the brain of IGFBP-6 gene ablation remain unknown.

III. CONCLUDING REMARKS In addition to the well-known GH/IGF somatotrophic endocrine axis, paracrine or autocrine GH/IGF neuroendocrine circuits might also be present in the mammalian CNS. These two systems are critical for fetal and postnatal brain development, and alteration of either the GH or the IGF system results in dramatic neuroanatomical deficits of the developing brain. Although precise localization of the sites of expression and synthesis for components of either the GH or the IGF system in the CNS suggests potential sites of action of these two systems, the detailed mechanisms involved in GH and/or IGF action are still poorly understood. It is likely that some of the effects of GH are mediated via the IGF system, but it is also possible that other “secondary mediators” might be involved. Their actions are likely to include either developmental, neurotrophic and remodeling functions, as well as neuroprotective roles. Many questions remain unanswered, including the role of circulating versus locally produced GH and IGF forms, the potential mechanism by which circulating GH and IGF find their ways across the blood–brain barrier into the CNS, possibly transported via their receptors in the choroid plexus, and the nature of the relationship between GH and IGF systems in the brain.

Acknowledgments This work was supported by grants to VCR and GAW from the National Health & Medical Research Council of Australia (#209067 and #149219), Juvenile Diabetes Research Foundation International (#1-2004-501), Diabetes Australia DART Grant, and Murdoch Childrens Research Institute.

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8 Purification of Growth Hormone Receptors from Human Brain Tissues ZHENNAN LAI* and FRED NYBERG† *Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, and †Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden

I. Introduction II. Methods for Purification III. Characteristics of Highly and Partly Purified Growth Hormone Receptors IV. Conclusion References

(h ⫽ human) and further characterized using analytical sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With this electrophoresis, combined with autoradiography, it was found that the receptor preparation from the choroid plexus migrated in conformity with a protein of molecular mass around 50,000 kDa. A similar result was obtained for GHR in the hippocampus, whereas those of the pituitary and hypothalamus behaved as molecular entities of around 60,000 kDa. The association constant for GH to its receptors in the choroids plexus was 0.83 nM. Ultimate purification of GHR was achieved by affinity chromatography (with antibodies against GH coupled to the gel matrix) and preparative zone electrophoresis in columns of agarose suspension.

The effects that growth hormone (GH) may exert on brain function have received particular attention during the past decade. The hormone was shown to induce several psychological improvements following GH replacement therapy in GH-deficient patients, thereby increasing life quality among these subjects. In animal models, GH was shown to improve cognitive capabilities. The mechanism by which GH exerts its effects on the brain has been explored in our laboratory during the past decade. One important step in this research has been to characterize the GH receptor (GHR) located on target cells in the brain. This article reviews some of the approaches to recover and purify GHRs in human brain tissues for further biochemical characterization. In particular we have focused on GHR in the choroid plexus but studies have also been directed to GHR recovered from other brain tissues, such as the hippocampus, hypothalamus, and pituitary. Thus, GHR was prepared from the human choroid plexus by extraction followed by molecular sieve chromatography before affinity chromatography and subsequently zone electrophoresis. From other brain tissues GHR was recovered by extractions followed by consecutive centrifugations. Highly purified, as well as partially purified GHRs, were covalently cross-linked to 125I-hGH

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION Growth hormone (GH), a 22-kDa polypeptide secreted from the anterior pituitary, is known to exert profound effects in a great variety of peripheral tissues but also in the brain (Nyberg, 2000). Receptors for this protein hormone are consequently widely distributed and a number of studies on their properties in tissues of both animal and human origin have been carried out. Several studies have confirmed the presence of specific GH receptors (GHR) in the central nervous system (CNS) (for review, see Nyberg, and Burman, 1996; Nyberg, 2000). Studies using receptor assays have verified the high

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density of GH binding in the choroid plexus, pituitary, hypothalamus, and hippocampus, as well as in the spinal cord (Di Carlo et al., 1984; Walsh et al., 1990; Lai et al., 1991, 1993; Zhai et al., 1994). In human, an apparent sex difference in GH binding was observed (Lai et al., 1993). However, the addition of excessive prolactin abolished this difference, suggesting that the higher level of GH binding in women was due to a higher density of lactogenic receptors known to be recognized by GH. The distribution in the rat brain of GH receptors is similar to that found in human (Zhai et al., 1994). Binding studies using radioactive iodine-labeled rat GH confirmed that the rat brain also has the highest content of GHR in the choroid plexus (Mustafa et al., 1994a,b; Zhai et al., 1994). Sex-dependent GH binding in the rat was observed previously using the human variant of the hormone (Mustafa et al., 1994a). The binding of labeled rGH, which does not recognize lactogenic sites, did not indicate any sex differences (Mustafa et al., 1994a; Zhai et al., 1994). Characterization of GH-binding sites in the various brain areas has revealed that these protein entities display molecular heterogeneity. For instance, in the human brain the calculated molecular mass of the GHR in the hypothalamus is about 10 kDa higher than that of GHR in the choroid plexus (Lai et al., 1993). Also, in the rat, the molecular weight of GHR in different regions varies (Zhai et al., 1994). To explore this further, attempts to identify any difference in the structure of the gene transcripts have been carried out. Studies on the expression of the gene transcript of GHR have confirmed its presence in a variety of brain areas in both human and rat. A study on the distribution of the GHR message in the rat brain carried out by in situ hybridization has been described (Burton et al., 1992; Kastrup et al., 2005). Similar studies were also reported from other groups (Hasegawa et al., 1993; Lobie et al., 1993). Areas with high expression of GHR mRNA-containing cells are the hypothalamus, thalamus septal region, hippocampus, dentate gyrus, and amygdala. Moreover, the presence of a 4.4-kb GHR transcript in an ovine choroid plexus cell line has been reported (Thornwall et al., 1995). The gene for an extracellular domain of the GH receptor in human choroid plexus has been cloned (Nyberg, 2000). The predicted amino acid sequence of this part of the choroid plexus receptor was found to be homologous with the GH receptor in human liver (Leung et al., 1987). By Northen blot analysis, no difference between sex and expression of the GHR transcript in the choroid plexus was found. The expression of GHR in the human brain has also been studied in other laboratories (Castro et al., 2000). The nucleotide sequence of the transcripts of GHR in the rat hippocampus and spinal cord has been reported (Thornwall-LeGrevés et al., 2001). The nucleotide sequence of GHR in other areas of the rat brain is described in Chapter 9. As written in Chapter 9, cloned rat brain GHRs exhibit nucleotide sequences, which are identical to that of the liver variant of the receptor. Therefore, the size heterogeneity among GHR receptor proteins may be

due to posttranslational events, rather than to differences in the nucleotide sequences of the gene transcripts. In order to examine the cause and nature of the observed size heterogeneity, we have attempted to purify GHR receptors from various brain tissues. For that purpose we have focused on brain areas with a high density of receptor binding, e.g., the choroid plexus. The choroid plexus is the region in the human brain that contains not only the highest amount of GHR, but also of prolactin (PRL) receptors (Lai et al., 1992). We have carried out studies on GHR in the choroid plexus of human origin and outlined a procedure for its purification (Lai et al., 2005). We also for the first time describe the complete separation of GHR from prolactin receptors in the CNS. After extraction and homogenization, GHR was purified further using molecular sieve chromatography and subsequently attained by affinity chromatography followed by zone electrophoresis in agarose suspension columns, utilizing a novel multibuffer system with initially generated zone sharpening by displacement electrophoresis.

II. METHODS FOR PURIFICATION A. Tissue Material Human brain tissues were collected in connection with autopsy where dissections occurred 15–48 h postmortem. All collected material was immediately frozen and kept stored at ⫺70 °C before further processing. Tissues (choroid plexus, hippocampus, hypothalamus, pituitary, putamen, and thalamus) were collected from adult females and males (39–90 years old). No brain pathology was found in any of these cases (Lai et al., 1992, 1993).

B. Receptor Preparation All procedures were performed at 4 °C. Prior to processing the frozen tissues were thawed, minced, and subsequently homogenized in 0.3 M sucrose (5 ml/g) (Lai et al., 1991, 1993). The homogenate was centrifuged at 150,000 g for 90 min, and the pellet (containing the membrane-bound receptors) was collected and suspended in phosphatebuffered saline (PBS: 137 mM NaCl, 2 mM KCl, 8.4 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4; 0.5 ml buffer per g starting tissue) and was used immediately or stored at ⫺20 °C. To prepare solubilized receptors from the choroid plexus, the tissues was homogenized (5 ml/g) in a suitable buffer (e.g., 50 mM Tris–HCl buffer, pH 7.8) for 2 h at 4 °C with continuous stirring. Homogenization was followed by centrifugation for 25–30 min at 20,000 g. The insoluble precipitate was suspended in the aforementioned Tris–HCl buffer, containing 1% Triton X-100, and incubated overnight at 4 °C. Centrifugation for 25 min at 20,000 g gave a supernatant that was the source of solubilized receptors.

8. Purification of GHR from Human Brain Tissues

C. Hormone Iodination Highly purified GH (Roos et al., 1963) was labeled with using the iodogen method (Paus et al., 1982) to a specific activity of 70–100 ␮gCi/␮g. In GH-binding studies, 50,000–100,000 cpm was used as described earlier (Lai et al., 1991, 2005). In the case of prolactin (Roos et al., 1979), the hormone was iodinated using the chloramine T method (Silberring et al., 1982). Both hormones were recovered from excessive iodine and other reaction constituents by gel filtration. 125I

D. Preparative Chromatography and Electrophoresis 1. Molecular Sieve Chromatography The solubilized (Triton X-100) receptor active material from the various brain tissues was applied onto a Sepharose CL-6B column and equilibrated in Tris/MgCl2 buffer (25 mM Tris–HCl, 10 mM MgCl2, pH 7.4) containing 0.1% (v/v) Triton X-100 (see Lai et al., 1992). The column was eluted with this buffer and assayed for protein content and receptor activity. The column was calibrated with standard proteins (ferritin, catalase, and aldolase) as molecular weight markers. 2. Affinity Chromatography Affinity chromatography was used to separate the GHR from impurities, including PRL receptors. Highly purified human GH or PRL (3 mg) was coupled at pH 8.0 to activated CH-Sepharose 4B powder (Lai et al., 1992, 2005) according to the instructions provided by the manufacturer (Pharmacia). The freeze-dried Sepharose powder (for GH-gel, 6 g; for hPRL-gel, 1 g) was suspended in 1 mM HCl (200 ml/g) on a sintered glass filter and mixed with the ligand (hGH or PRL) dissolved in coupling buffer (0.1 M NaHCO3, pH 8.0, containing 0.5 M NaCl) for 1–2 h at room temperature or for 4 h at 4 °C. Excess hormones, which did not attach to the gel matrix, were eliminated by repeated washings on the filter with the coupling buffer. Subsequently, any remaining active groups were blocked by treatment with 0.1 M Tris–HCl buffer, pH 8.0. The coupled Sepharose gel was also washed alternately (four times) with buffer solutions of low and high pH (0.1 M acetate buffer containing 0.5 M NaCl, pH 4.0, followed by 0.1 M Tris–HCl buffer, pH 0.8, containing 0.5 M NaCl). The gel was equilibrated with 25 mM Tris–HCl buffer, pH 7.4, containing 0.1% Triton X-100 and mixed with solubilized receptor with continuous slow rocking for 16 h at 4 °C. The receptors were dialyzed earlier against this buffer. Following affinity binding the gel was centrifuged at 1000 g for 10 min at 4 °C. The pellet was suspended in the aforementioned buffer and poured into the columns (for hGH, 1.5 ⫻ 10 cm, for hPRL, 1.0 ⫻ 2.0 cm), which were subsequently eluted with this buffer. Consecutively 0.1 M Tris–HCl buffer, pH 7.4, 0.1 M glycine–NaOH buffer, pH 8.8, and

93

0.1 M glycine–NaOH buffer, pH 8.8/5 M MgCl were used as eluents. All buffers contained 0.1% Triton X-100. Fractions were collected and analyzed by the radio receptor assay. The protein content in the various eluates was recorded by measuring the absorbance at 280 nm. 3. Preparative Zone Electrophoresis in a Novel Multibuffer System Additional or final separation of the affinity-purified GH or PRL receptors was accomplished by applying preparative column electrophoresis in agarose suspension (Lai et al., 2005). This step was initiated by a prior zone-sharpening created (in the same column) by displacement electrophoresis. The technique was based on the development of a previously described procedure (Hjertén, 1990) using a novel multibuffer system for carrier-free electrophoresis in capillaries. The composition of the buffer system was such that initially the sample solutes were concentrated (stacked) by displacement electrophoresis. Subsequently, by increasing the ionic strength the conditions for this type of electrophoresis were no longer fulfilled and the solutes were destacked and separated by zone electrophoresis. Runs were carried out as described (Lai et al., 2005) utilizing equipment described earlier for preparative isoelectric focusing in agarose suspension (Roos, 1987). Following completed separation, fractions were collected and their protein content was recorded as the difference in absorbance at 280 and 310 nm. The binding activities in the collected fractions were assessed by receptor assays using both 125I-labeled GH and 125I-labeled PRL as radioligands (Lai et al., 2005).

E. Analytical Techniques 1. Assays for the Assessment of Receptor Activities The various chromatographic and electrophoresis steps during receptor purification were guided by binding assays. Collected fractions from the described purification steps were screened for binding affinity using the protocols described earlier (Lai et al., 1991, 1992). Briefly, samples (100 ␮l) from the various separation steps were incubated with 125I-labeled hGH (50,000 cpm) or 125I-labeled hPRL (60,000 cpm) at room temperature for 16 h in 0.025 M Tris–HCl buffer, pH 7.4, containing 0.01 M MgCl2, and the carrier human ␥-globulin (0.016%). Following completed incubation, 1.0 ml of an ice-cold water solution (18.75%) of polyethylene glycol 6000 (PEG) was added to each incubation vial. After 20 min the precipitates that formed were collected by centrifugation (10,000 g) at 4 °C for 10 min and taken for radioactivity counting. Specific binding was calculated by subtraction of nonspecific binding determined in the presence of excessive amounts of unlabeled hormones (10 ␮g) from total binding. All determinations were carried out in triplicate. Protein concentrations were obtained by measuring the absorbance at 280 nm.

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Zhennan Lai and Fred Nyberg

2. Covalent Cross-Linking of Purified Receptors to Labeled and Unlabeled Ligands Cross-linking of the iodinated hormones to the purified receptors was performed using disuccinimidyl suberate (DSS, ICN Biomedicals Inc., Planriew, NY) as described (Lai et al, 1991, 1992). Briefly, the purified receptor material was incubated with iodine-labeled hormones overnight at room temperature in PBS (137 mM NaCI, 3 mM Cl, 8.4 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4) containing MgCl2 (10 mM). The following day, DSS (2.5 mM, freshly dissolved in dimethyl sulfoxide) was added and the tubes were subsequently kept at 0 °C for 15 min. The addition of buffer (2 M Tris–HCl buffer, pH 7.5, containing 1 mM EDTA) terminated the reaction. The same procedure was used for cross-linking to unlabeled hormones. Details are given in previously published studies (Lai et al., 1991, 1992, 1993). 3. Sodium Dodecyl Sulfate (SDS)–Polyacrylamide Gel Electrophoresis SDS electrophoresis was performed in a polyacrylamide gel (T ⫽ 10%, w/v, C ⫽ 2.7%, w/v) according to the procedure of Laemmli (1970) as described earlier (Lai et al., 1992, 1993). Samples of cross-linked complexes or proteins collected directly after the purification were mixed with a “sample cocktail” [75 ␮l of 0.3 M Tris–HCl buffer, pH 8.8, containing 14% (w/v) SDS, 10 mM dithiothreitol, 70 ␮l of 50% Trasylol (Fluka Chemica A. G., Switzerland), and 36% sucrose (w/v)] and boiled for 5 min. Following alkylation with iodoacetamide (60 mM) for 15 min at 20 °C, aliquots (40 ␮l) were subjected to electrophoresis for 45 min at 200 V using a Mini-Protean II apparatus (BioRad Laboratories, Richmond, CA). In experiments with labeled complexes the gels were stained with Coomassie brilliant blue R prior to autoradiography (Kodak X-AR 5, Eastman Kodak, Rochester, NY) to locate molecular weight markers. If unlabeled complexes were applied, all proteins were visualized using the silver stain method (Tunant and Johansson, 1984).

III. CHARACTERISTICS OF HIGHLY AND PARTLY PURIFIED GROWTH HORMONE (GH) RECEPTORS A. Binding Constants for GH Receptors in Various Brain Regions The association constants calculated for GH binding in the different brain regions are shown in Table I. Data are based on studies of receptor material present in crude receptor homogenates (Lai et al., 1993). As shown in Table I, the highest affinity for the hormone was found in the pituitary, while the highest binding capacity was in the choroid plexus. It is obvious that the latter tissue is rich

TABLE I 125I-hGH Bound to Membrane Receptors from Human Brain Regions Displaced by Unlabeled hGHa Association constant Ka (nM⫺1)

Binding capacity (pM)

Choroid plexus

0.83 ⫾ 0.12

350 ⫾ 40

Hippocampus

4.85 ⫾ 0.10

18 ⫾ 7

Hypothalamus

7.36 ⫾ 0.14

12 ⫾ 5

Pituitary

9.87 ⫾ 0.40

30 ⫾ 16

Brain tissue

a

See also Lai et al. (1991, 1993).

in GH receptors and these sites have been suggested to be involved in a transport mechanism for the hormone across the blood–brain barrier (Nyberg, 2000). In all these tissues, GH binding was found to decrease with increasing age. A similar observation was also seen in studies of male rats (Zhai et al., 1994). Thus, it seems that the well-known decline in the level of circulating GH seen to occur with aging also concerns its receptors in the brain. The binding of GH to the various areas of the human brain also appeared to be sex dependent (Lai et al., 1993). Significant higher specific binding of GH was recorded in almost all tissues originating from female brains. However, as mentioned earlier, in the presence of excessive prolactin this difference was depleted, suggesting that the increased binding in female tissue is due to an increased density of lactogenic-binding sites. Human GH is also shown to display binding affinity for lactogenic sites.

B. Molecular Sizes of GH Receptors from Molecular Sieve Chromatography The GH receptors retained their ability to bind the hormone also after solubilization and gel filtration chromatography. However, the molecular sizes recorded after this chromatographic step highly exceed those predicted from the nucleotide sequence of the corresponding gene transcript (Lai et al., 1991). The explanation of the appearance of this large complex may be that the receptors form aggregates that may bind the hormone. Similar observations have been made for receptors from other sources (see Waters and Friesen, 1979).

C. Molecular Weights of GH Receptors in Some Brain Regions Established from Sodium Dodecyl Sulfate Electrophoresis of Receptors Covalently Cross-Linked to Labeled GH Using SDS electrophoresis of cross-linked hormone– receptor complexes, it was possible to determine the molecular weights of GHR from various brain regions in both crude and purified receptor preparations (Lai et al., 1991,

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8. Purification of GHR from Human Brain Tissues

TABLE II Mr Values for Hormone-Binding Units Calculated from Data Obtained by SDS–Polyacrylamide Gel Electrophoresis of Cross-Linked Hormone Receptor Complexesa

Brain tissue

Mr for hGH-binding unit

Binding capacity (pM) 350 ⫾ 40

Choroid plexus

51,000 ⫾ 1600

Hippocampus

48,000 ⫾ 1800

18 ⫾ 7

Hypothalamus

58,000 ⫾ 1500

12 ⫾ 5

59,000 ⫾ 1200

30 ⫾ 16

Pituitary Liver b

130,000

a Values are means from three different experiments (see Lai et al., 1991, 1993). b Predicted from the DNA sequence of the human liver receptor (Leung et al., 1987).

1992, 1993, 2005). Calculated molecular sizes for GHR in the human choroid plexus, hippocampus, hypothalamus, and pituitary are given in Table II. As shown in Table II, values for the choroid plexus and hippocampus are around 10 kDa lower than those estimated for the hypothalamus and the pituitary. Moreover, the molecular size of GHR predicted from the nucleotide sequence of the liver gene transcript of GHR is significantly higher than those of the brain regions (Table II). Also, the size of the GHR receptor protein in the liver deviates from those of the brain (Hoquette et al., 1989). This was considered to indicate that the origin of brain congeners of GHR results from alterative splicing of the original receptor gene transcript. However, as it appears that the message of GHR in

the human choroid plexus (Nyberg, 2000) and those of the choroid plexus, hippocampus, hypothalamus, and spinal cord in the rat (see Chapter 9) exhibit identical sequences to the corresponding GHR gene transcript in the liver, it is suggested that the difference in molecular sizes is due to posttranslational truncations (Nyberg, 2000).

D. Affinity Chromatography and Preparative Zone Electrophoresis The existence of GHR as a separate entity distinct from the PRL receptor in the brain has been debated. However, we demonstrated that these two receptors in the choroid plexus may be separated from each other by affinity chromatography using a gel matrix to which GH antibodies (ab) have been attached (Lai et al., 1992, 2005). We found that the GH-ab affinity gel was suitable for an ultimate separation of GHR and the PRL receptor. Moreover, we also observed that all PRL receptor activity can be removed from the GHR fraction by affinity chromatography with PRL antibodies coupled to the gel matrix (Lai et al., 2005). The distribution and recovery of GH- and PRL-binding activities from these affinity chromatography experiments are summarized in Fig. 1. As shown in Fig. 1, affinity chromatography on the GH-ab column resolved the active binding material in three separate fractions. GHR-binding activity eluting from the GH-ab column appeared in a major fraction with negligible amounts of PRL-binding activity. The main part of PRL-binding activity emerged from the column in a fraction eluting ahead of the GHR-like material. This fraction contained minor amounts of GH-binding activity but meant a significant purification of the PRL receptor material. By this

(a)

(b)

250

160 140

200

120 100

150 hGH-binding hPRL-binding

100

80

hGH-binding hPRL-binding

60 40

50

20 0

0

Buffer 1 Buffer 2 Buffer 3 Buffer 4

Buffer Buffer Buffer Buffer 1 2 3 4

FIGURE 1 Recovery of GHR and PRL receptors following affinity chromatography with highly purified hGH and hPRL coupled to activated CH-Sepharose 4B. The solubilized proteins were loaded onto an affinity column (1.5 ⫻ 10 cm) eluted consecutively with four different buffers (1–4, see text 3 Lai et al., 2005) at a flow rate of 10 ml/h. Fraction volumes of 1.3 ml were collected. The specific binding in receptor assays with both 1251-hPRL and 1251-hGH are given. (a) Affinity chromatography on CH-Sepharose 4B with GH coupled to the gel matrix. (b) Affinity chromatography on CH-Sepharose 4B with PRL coupled to the gel matrix.

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Zhennan Lai and Fred Nyberg

separation step (on protein basis) the GHR activity also displayed significant purification. The majority of inert proteins appeared in the break-through volume. Using the PRL affinity column, a complete separation of the GH respectively PRL-binding activity was achieved (see Fig. 1; Lai et al, 2005). The GHR-like activity was collected together with inert proteins in the void volume, whereas the PRL binder was recovered highly purified in a fraction free from any GHR-like material. It is obvious that the GH- and PRL-ab affinity chromatography steps provide a powerful purification of both GH and PRL receptors. The application of preparative zone electrophoresis provides an additional powerful tool for the purification of the GHR. We developed a new technique based on electrophoretic runs in columns of agarose suspension (Lai et al., 2005). Using this technique, it was possible to obtain the GHR protein in a single peak associated with binding activity. The purity of this material was confirmed by SDS electrophoresis, where the GH binder was silver stained as a single band (Lai et al., 2005). In a separate experiment we were able remove the GHR-contaminating material in the PRL receptor fraction collected from the GH-ab affinity column (A:GH-ab II, see Fig. 1). Thus, the PRL receptor protein recovered in a separate peak also stained as a single band following SDS electrophoresis with silver staining (Lai et al., 2005). On the basis of specific binding per milligram protein, the GHR protein recovered by zone electrophoresis displayed more than 100-fold purification (purification factor 115) relative to the activity in the crude homogenate. The corresponding purification factor for the PRL receptor was estimated to be 160.

appears desirable to outline a purification procedure fast enough to avoid freezing and storage of partially purified fractions, as this is often connected with dropped activity and loss of material. However, it is obvious that it is possible to find suitable separation techniques that allow high resolution and recovery within a relatively short time. Both the affinity chromatographic steps and the preparative zone electrophoresis mentioned in this chapter represent methods that fulfill these criteria. The affinity techniques appear very selective and remove large amounts of contaminating proteins and are connected with comparatively high yields. The electrophoresis equipment represents not only a rapid technique, but due to the displacement step prior to zone electrophoresis, the protein mixture to be separated is highly concentrated, allowing sharp zones and recovery of the active material in very narrow peaks, which could be collected in small volumes (Lai et al., 2005). These properties of the actual techniques appeared essential to obtain enough protein for subsequent analysis by various analytical approaches. Thus, working with relatively small amounts of brain material, the affinity chromatography and the preparative zone electrophoresis in agarose suspension seem to represent methods of choice for purification and recovery. We also believe that these kinds of techniques may be of importance in the future in many other projects in biochemical research directed at the characterization of cell surface receptors.

IV. CONCLUSION

References

Most data regarding the structure and properties of receptors for hormones produced in the anterior pituitary have emerged from molecular cloning of these receptors and subsequent studies following expression of these entities in various cells. Before these techniques came into frequent use in the various laboratories, many attempts to purify and characterize protein hormone receptors using conventional biochemical approaches have been carried out (e.g., Water and Friesen, 1979; Hoquette et al., 1989). These approaches were connected with great difficulties for several reasons. First they require very specific and reliable assays for screening the activity of the actual receptor. Second, as the actual hormone receptors are located in the cell membrane and hardly soluble in water, it is necessary to include suitable detergents to prevent loss of material due to aggregation and precipitation. Furthermore, during progressive purification the conditions for optimal binding may alter as important cofactors may be removed. It also

Burton, K. A., Kabigting, E. B., Clifton, D. K., and Steiner, R. (1992). Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat and its colocalization in hypothalamic somatostatin neurons. Endocrinology 130, 958–963. Castro, J. R., Costoya, J. A., Gallego, R., Prieto, A., Arce, V. M., and Senaris, R. (2000). Expression of growth hormone receptor in the human brain. Neurosci. Lett. 281, 147–150. DiCarlo, R., Muccioli, G., Bellussi, G., Pagnini, G., Papotti, M., and Bussolati, O. (1984). Lactogenic binding sites in the brain: Regional distribution, species variation and characterization. Adv. Biosci. 48, 303–308. Hasegawa, O., Minami, S., Sugihara, H., and Wakabayashi, I. (1993). Developmental expression of the growth hormone receptor gene in the rat hypothalamus. Dev. Brain Res. 74, 287–290. Hjertén, S. (1963). Zone electrophoresis in columns of agarose suspensions. J. Chrom. 12, 510–526. Hjertén, S. (1990). Zone broadening in electrophoresis with special reference to high–performance electrophoresis in capillaries: An interplay between theory and practice. Electrophoresis 11, 665–690. Hocquette, J. F., Postel-Vinary, M. C., Dijiane, J., Tar, A., and Kelly, P. A. (1989). Human liver growth hormone receptor and plasma binding protein: Characterization and partial purification. Endocrinology 127, 1665–1672.

Acknowledgment This study was supported by the Swedish Medical Research Council (Grant 9459).

8. Purification of GHR from Human Brain Tissues Hughes, J. P., Elsholtz, H. P., and Friesen, H. G. (1985). Growth hormone and prolactin receptors. In “Polypeptide Hormone Receptors” (B.I. Posner, ed.), pp. 157–199. Dekker, New York. Kastrup, Y., Le Greves, Nyberg, F., and Blomqvist, A. (2005). Distribution of growth hormone receptor mRNA in the brain stem, and spinal cord of the rat. Neuroscience 130, 419–425. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 277, 680–685. Lai, Z., Emtner, M., Roos, P., and Nyberg, F. (1991). Characterisation of putative growth hormone receptors in human choroid plexus. Brain Res. 546, 222–226. Lai, Z., Roos, P., Olsson, Y., Larsson, C., and Nyberg, F. (1992). Characterisation of prolactin receptors in human choroid plexus. Neuroendocrinology 56, 225–233. Lai, Z., Roos, P., Zhai, Q.-Z., Olsson, Y., Fhölenhag, K., Larsson, C., and Nyberg, F. (1993) Age-related reduction of growth hormone-binding sites in the human brain. Brain Res. 621, 250–266. Lai, Z., Roos, P., and Nyberg, F. (2005). Affinity chromatography and zone electrophoresis for the separation of growth hormone and prolactin receptors from human choroid plexus. Submitted for publication. Leung, D. W., Spencer, S. A., Cachianes, O., Hammonds, R. C., Collins, C., Henzel, W. J., Barnard, R., Waters, M. J., and Wood, W. I. (1987). Growth hormone receptor and serum binding protein: Purification, cloning and expression. Nature 330, 537–543. Lobie, P., García-Aragón, J., Lincoln, P., Barnard, R., Wilcox, J. N., and Waters, M. J. (1993). Localization and ontogeny of growth hormone receptor gene expression in the central nervous system. Dev. Brain Res. 74, 225–233. Mustafa, A., Adem, A., Roos, P., and Nyberg, F. (1994a). Sex differences in binding of human growth hormone to rat brain. Neurosci. Res. 19, 93–99. Mustafa, A., Nyberg, F., Bogdanovic, N., Islam, A., Roos, P., and Adem, A. (1994b). Somatogenic and lactogenic binding sites in rat brain and liver: Quantitative autoradiographic localization. Neurosci. Res. 20, 257–263.

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Nyberg, F., and Burman, P. (1996). Growth hormone and its receptors in the central nervous system: Location and functional significance. Horm. Res. 45, 18–22. Nyberg, F. (1997). Ageing effects on growth hormone receptors binding in the brain. Exp. Gerontol. 32, 521–528. Nyberg, F. (2000). Growth hormone in the brain: Characteristics of specific brain targets for the hormone and their functional significance. Front. Neuroendocrinol. 21, 330–348. Posner, B. I., Houten, M., Patel, B., and Walsh, R. J. (1983). Characterization of lactogen binding sites in choroid plexus. Exp. Brain Res. 49, 300–306. Roos, P. (1987). Preparative isoelectric focusing in agarose suspension columns. J. Biochem. Biophys. Methods 14(Supp. 7), 57–58. Roos, P., Fevold, H. R., and Gemzell, C. A. (1963). Preparation of human growth hormone by gel fiItration. Biochem. Biophys. Acta. 74, 525–531. Roos, P., Nyberg, F., and Wide, L. (1979). Isolation of human pituitary prolactin. Biochem. Biophys. Acta. 588, 368–379. Silberring, J., Golda, W., and Szybinski, Z. (1982). A universal and simple chloramine T version for hormone iodination. Int. J. Appl. Radiat. Isot. 33, 117–119. Thörnwall, M., Chhajlani, V., Le Grevés, P., and Nyberg, F. (1995). Detection of growth hormone receptor mRNA in an ovine choroid plexus epithelium cell line. Biochem. Biophys. Res. Commun. 217, 349–353. Thörnwall-LeGrevés, M., Zhou, Q., Lagerholm, S., Huang, W., Le Grevés, P., and Nyberg, F. (2001). Morphine decreases the levels of the gene transcript of grwoth hormone receptor and growth hormone binding protein in the male rat hippocampus and spinal cord. Neurosci. Lett. 304, 69–72. Tunon, P., and Johansson, K. E. (1984). Yet another improved silver staining method for the detection of proteins in polyacrylamide gels. J. Biochem. Biophys. Methods 9, 171–179. Waters, M. J., and Friesen, H. G. (1979). Purification of and partial characterization of a nonårimate growth hormone receptor. J. Biol. Chem. 254, 6815–6825. Zhai, Q. Z., Lai, Z., Roos, P., and Nyberg, F. (1994). Characterisation of growth hormone binding sites in the rat brain. Acta Paedr. 406, 92–95.

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9 Growth Hormone Receptor Message in the Rat and Human Central Nervous System: Structure and Function MADELEINE LE GREVES Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, SE-751 24 Uppsala, Sweden

I. II. III. IV. V.

I. INTRODUCTION

Introduction Cloning of the Growth Hormone Receptor (GHR) Growth Hormone-Binding Protein GHR mRNA in the Central Nervous System Regulation of GHR Gene Expression References

In the past decade, it has become more apparent that the growth hormone (GH) has profound effects in the central nervous system (CNS) (Harvey et al., 1993; Nyberg, 2000). Studies have shown the involvement of GH in the regulation of emotion (Burman and Deijen, 1998; Gibney et al., 1999; McGauley et al., 1996), cognitive functions and memory (Deijen et al., 1996; Rollero et al., 1998), appetite (Stoving et al., 1999), neuroprotection (Scheepens et al., 1999), and sleep (Drucker-Colin et al., 1975; Frieboes et al., 1999; Matsuno et al., 1998; Perras et al., 1999). The physiological mechanisms behind this are not clear, but it suggests the presence of specific binding sites for GH in the CNS. Originally, growth hormone receptor (GHR) expression was considered to be restricted to the liver, but Mathews and co-workers (1989) demonstrated the presence of both GH-binding sites and GHR mRNA throughout the body, including the CNS. This was later confirmed by several reports (Fraser et al., 1990; Hull and Harvey, 1998; Kastrup et al., 2004; Lai et al., 1991; Le Grevès et al., 2002, 2005; Lobie et al., 1993; Zhai et al., 1994). In addition to the single chain transmembrane GHR belonging to the cytokine receptor superfamily, there is also a GH-binding protein (GHBP) present in the circulation (Bazan, 1990). These proteins have been cloned and characterized in a number of species and depending on the species, the

The growth hormone (GH) influences a variety of central nervous system (CNS)-controlled functions such as appetite, memory, mood, and sleep. This has led to an increased interest in the biological role of the hormone and its receptor, the GHR, in the brain. Many studies using different techniques have demonstrated a widespread distribution of GHR in the brain, brain stem, and spinal cord. After the pioneer work of cloning and sequencing cDNA encoding for the GHR in the rabbit liver, GHR has subsequently been isolated and characterized in many species, including human and rat. It has been shown that the GHR protein present in the CNS is a low-molecular variant of that existing in the liver. After cloning and sequencing, fully or partially, cDNA in the hippocampus, hypothalamus, choroid plexus, and spinal cord, no tissue-specific differences were found, i.e., GHR present in the CNS is encoded by the same DNA sequence as the previously cloned receptor existing in the liver. However, regulation of the expression of the GHR gene seems to be tissue specific and is influenced by a large number of factors, such as ontogeny, hormones, and nutritional status.

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

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Copyright 2006 Elsevier Inc. All rights reserved.

Transmembrane domain

2 4 6

2 4 7

2 4

2 4

2 4

2 4

3 5 0

3 5 0

3 5 0

3 4 9

84%

69%

Rat GHBP

1 8 0

Rat GHR

2 4 6

Rabbit GHR

Extracellular/ hormone-binding domain

Human GHR3-

Madeleine Le Greves

Human GHR

100

2 4 5 1 7

Intracellular domain

FIGURE 1 The central dogma of molecular genetics. DNA is amplified through replication. The synthesis of mRNA complementary to the DNA is referred to as transcription. The conversion of the genetic code into an amino acid sequence of proteins is called translation.

GHR consists of ⬃620 amino acids with a molecular mass between 100 and 130 kDa (Edens and Talamantes, 1998), which is derived from a mRNA transcript of ⬃4.5 kb. The GHBP is identical to the extracellular domain of the GHR and is produced by either alternative splicing of the GHR gene or proteolytic cleavage (Baumbach et al., 1989; Schantl et al., 2004; Smith et al., 1989). Following binding of GH to its receptor, a cascade of events has been identified. Upon binding, a dimerization of the GHR is induced (Cunningham et al., 1991) and an association with janus Kinase 2 and mitogen-activated protein kinase is initiated whereby the kinases and the receptor itself become phosphorylated (Goujon et al., 1994) and activation of a variety of genes occurs, leading to the effects mentioned earlier. The relationship among DNA, RNA, and protein is described in Fig. 1 (Crick, 1970). A single DNA chain serves as a template for either complementary DNA molecules, in the process of DNA replication, or complementary mRNA, in the process of transcription. The mRNA molecule serves as the template that organizes the amino acids within the protein chain during the process of translation.

FIGURE 2 Schematic structure of the GHR and GHBP protein in human, rabbit, and rat. White boxes represent the hormone-binding extracellular domain. Light gray boxes represent the transmembrane domain, and the dark gray represents the signaling intracellular domain. The hydrophilic tail of rat GHBP is shown as a striped region. Numbers in each region indicate the amount of amino acids present in the corresponding domain. Percentage homology with the human GHR sequence is indicated below each receptor. From Edens and Talamantes, (1998). (See color plate 4)

cDNA library with a restriction fragment probe from one of the rabbit clones. After sequencing it was revealed that both rabbit and human GHR clones contain an open reading frame of 638 amino acids (Fig. 2). This pioneer work paved the way for further elaboration of the amino acid sequences in many species, including monkey (Martini et al., 1997), ovine (Adams et al., 1990), porcine (Cioffi et al., 1990), bovine (Hauser et al., 1990), chicken (Burnside et al., 1991), mouse (Smith et al., 1989), and rat (Baumbach et al., 1989).

A. The Human GHR Transcript II. CLONING OF THE GROWTH HORMONE RECEPTOR (GHR) In 1987, Leung and co-workers reported the cloning of the first GHR cDNA from rabbit and subsequently in human liver. The cDNA encoding for the rabbit GHR was isolated by screening liver cDNA libraries with oligonucleotide probes based on a 19 residue amino acid sequence of a tryptic fragment of the purified receptor protein. The positive clones were mapped and sequenced, and the translated DNA sequence matched exactly one of the oligonucleotide probe used for screening. By using these obtained clones, the same libraries were rescreened and clones of the entire coding and 3⬘-untranlated regions were isolated. The human GHR was then cloned by screening a human liver

The human GHR gene has been located to chromosome 5p13.1-p12 (Barton et al., 1989). After the alternate first exon of the GHR, there are nine coding exons. Exon 2 encodes 11 bp of the 5⬘-UTR and the signal peptide, exons 3 to 7 encode the extracellular domain (246 amino acids), exon 8 encodes the transmembrane domain (24 amino acids), and the cytoplasmic domain is encoded by exons 9 and 10 (350 amino acids) (Fig. 3) (Godowski et al., 1989; Zou et al., 1997). Exon 10 also encodes a 2-kb 3⬘-UTR of the transcript. The importance of 3⬘-UTR in the GHR is poorly investigated but, in general, it is thought that these 3⬘-UTR affect the rate of degeneration of the mRNAs and initiate translation of the GHR protein (Decker and Parker, 1995; Jacobson and Peltz, 1996). Only one single mRNA transcript encoding the GHR, sized 4.7 kb, has been identified in human.

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5′UTRs

Transmembrane domain Intracellular domain Extracellular hormone binding domain and 3′UTR

Signal peptide

2

3

4

5

6

7

8

9

10

FIGURE 3 Structure of the genomic DNA encoding the human GHR located on chromosome 5p13.1-p12 (Barton et al., 1989). Numbered boxes represent exons, and introns are drawn as lines. Exon 1 encodes the 5⬘-UTR (V1–V9), exon 2 the signal peptide, exons 3–7 the extracellular domain, exon 8 the transmembrane domain, and exons 9 and 10 the intracellular part and the 3⬘-UTR. (See color plate 5)

B. The mRNA Transcript for GHR in the Rat The rat GHR was first cloned from the liver and revealed two cDNAs (Fig. 3) (Baumbach et al., 1989): one cDNA encoding a membrane-bound protein, the GHR, and one cDNA encoding a soluble secreted protein that contained the same putative GH-binding region but lacking the transmembrane and intracellular domains, the GHBP. The open reading frame in the cloned rat GHR contained 638 amino acids where amino acids 1 to 17 predicted the signal sequence and amino acids 266 to 289 the transmembrane domain. The homologies between the rat and the previously cloned rabbit and human GHRs were 74 and 69% identity, respectively. As determined with Northern blot analysis, the major mRNA transcript that encodes the full-length receptor is 4.6 kb in length. A smaller transcript of 1.2 kb was also detected in the rat liver tissue and this smaller mRNA is considered to code for the GHBP. Similar to the human GHR gene there are several 5⬘-UTR variants (V1–V5) of the corresponding gene in rat. The expression of these variants exhibited a tissuespecific pattern, e.g., V2 is expressed exclusively in the liver while V4 is the only variant expressed in the brain (Domene et al., 1995).

III. GROWTH HORMONE-BINDING PROTEIN GHBP corresponds to the extracellular part of the GHR (Fig. 2), essential for GH binding, with a short hydrophilic tail instead of the transmembrane and the intracellular domains of the receptor. The GHBP is found in the circulation and is believed to act as a reservoir for GH. The generation of GHBP is species specific. In rat and mouse an alternative splicing of the GHR transcript has been proven (Baumbach et al., 1989); the GHBP is produced from a mRNA of 1.2–1.4 kb. In the rat, the transcripts for GHR and GHBP are expressed in a tissue-specific manner, where the GHBP mRNA is predominant in the liver and the GHR mRNA is expressed predominantly in the brain (Domene et al., 1995).

In human and rabbit it is postulated that soluble GHBP results from proteolytic cleavage of the membrane-bound receptor (Bick et al., 1996; Dastot et al., 1996; Sotiropoulos et al., 1993). The human GHBP is generated by a transmembrane metalloprotease called tumour necrosis factor␣–converting enzyme (Schantl et al., 2004). In the Rhesus macaque, however, Martini and co-workers (1997) suggested that these two mechanisms coexist and that the monkey GHBP is generated partly by alternative splicing and partly by proteolytic cleavage. GHBP prolongs the half-life of GH by protecting it from degradation, which may enhance its bioactivity in vivo (Baumann et al., 1987; Clark et al., 1996).

IV. GHR mRNA IN THE CENTRAL NERVOUS SYSTEM (CNS) Fraser and co-workers (1990) first identified the presence of mRNA encoding the GHR in the CNS, namely in brains from chicken and rabbit. RNA extracted from hypothalamic and extrahypothalamic tissues contained mRNA that hybridized to a cDNA probe for the rabbit liver GHR. The size of the transcript was similar to the major GHR mRNA transcript found in rabbit liver. Futhermore, the gene expression for GHR was found to be age related; levels were higher in adult compared to neonatal animals (Hasegawa et al., 1993). In the rat CNS, the GHR mRNA transcript has been localized using techniques such as in situ hybridization and Northern blot (Edmondson et al., 1995; Kastrup et al., 2004; Le Grevès et al., 2002; Lobie et al., 1993; Thörnwall-Le Grevès et al., 2001). Mapping the neuroanatomical distribution by in situ hybridization revealed that cells in brain regions (Fig. 4) such as the thalamus, septal region, hippocampus, dentate gyrus, amygdala, and hypothalamus contain the GHR mRNA (Table I) (Bennett et al., 1996; Burton et al., 1992). Burton et al. (1992) showed that GHR mRNA and somatostatin mRNA are coexpressed in perventricular and paraventricular nuclei of the hypothalamus, suggesting that GH acts directly on

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Cortex CA1

Cerebellum

DG CA3 Hippocampus

Midbrain

Thalamus Pons Amygdala

Medulla oblongata

Spinal cord

Hypothalamus Pituitary

FIGURE 4 A section of the rat brain cut in a sagittal plane. (See color plate 6)

TABLE I Distribution of GHR Expression in Rat CNS Structures Braina Lateral septum Bed nucleus of the stria terminalis Diagonal band-horizontal limb Thalamus, Reticular thalamic nucleus Pararential thalamic nucleus Amygdala Hippocampusb Dentate gyrus Hypothalamus, Paraventricular nucleus Periventricular nucleus Arcuate nucleusb Dorsomedial nucleus Ponsc Dorsal nucleus raphe Parabrachial nucleus, superior lateral part Nucleus of the trapezoid body Dorsal tegmental nucleus Motor nucleus of the trigeminal nerve Nucleus incertus Locus coeruleus Medulla oblongatac Facial nucleus Paragigantocellular reticular nucleus, dorsal part Hypoglossal nucleus Nucleus of the solitary tract, commissural part Area postrema Spinal cordc Dorsal horn, superficial layers Ventral horn a

From Burton et al. (1992). From Bennett et al. (1995). c From Kastrup et al. (2004).

b

hypothalamic neurons and participates in the regulation of its own secretion. The generation of GH is regulated by two hypothalamic peptides: somatostatin that inhibits production and growth hormone-releasing hormone that stimulates production. In a rigorous publication (Kastrup et al., 2004), the presence and distribution of GHR mRNA in the brain stem and spinal cord of the rat were investigated (Table I). Dense concentrations were seen in the arcuate nucleus of the hypothalmus, the locus coeruleus, the area postrema, and the commissural part of the nucleus of the solitary tract while a number of other structures were labeled with less intensity. The presence of GHR mRNA in these areas supports a direct action of GH on brain regions that are involved in widely differing aspects of which most physiological functions are poorly understood. However, the GHR mRNA detected in the arcuate nucleus of the hypothalamus is believed to be highly involved in the feedback regulation of GH secretion from the pituitary, whereas the GHR mRNA in the commissural part of the nucleus of the solitary tract, the superior lateral parabrachial nucleus, and the area postrema has been suggested to be implicated in the control of food intake and/or sleep pattern (Perras et al., 1999; Phifer and Berthoud, 1998; Rinaman et al., 1998; Stoving et al., 1999). The action of GH on the spinal level has been proposed to be involved in pain-processing mechanisms (Bennett, 1998; Kastrup et al., 2004), whereas GH action in the hippocampus is thought to be involved in learning and memory functions (Le Grevès et al., 2000; M. Le Grevès et al., unpublished data). Northern blot analysis of CNS tissues of the hippocampus, hypothalamus, choroid plexus, and spinal cord revealed two RNA transcripts: one of 4.5 kb encoding for

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GHR and one of 1.2 kb encoding for GHBP (Hasegawa et al., 1993; Le Grevès et al., 2002; Minami et al., 1993; Thörnwall-Le Grevès et al., 2001). The molecular mass of the GHR in the rat choroid plexus was estimated to be 60 kDa (Lai et al., 1991), indicating the existence of a smaller form of GHR in this tissue compared to that in the liver (130 kDa). It was speculated that the difference in size might be due to a specific role for GHR as a mediator in the transport of the hormone over the blood–brain barrier. To clarify the presence of this smaller variant of GHR in the brain, its cDNA in different CNS tissues was cloned. The amino acid sequence of the cloned GHR cDNA derived from rat spinal cord (Thörnwall-Le Grevès et al., 2001) was found to be identical to the already cloned liver GHR (Baumbach et al., 1989). The spine GHR was cloned using the reversed transcriptase polymerase chain reaction (RTPCR) technique where the primers were designed according to the liver sequence and therefore only variants of the known GHR could be matter for this cloning. Using the same technique, the cDNA encoding the GHR was also cloned, partly or fully, in the rat hypothalamus (59%), hippocampus (84%), and choroid plexus (100%) (Fig. 5). These clones predicted the same amino acid sequence as the previously cloned liver receptor. In the human CNS, localization and distribution studies on mRNA encoding GHR are almost absent; only two reports have been published so far. In one of them (Mercado et al., 1994) the existence of GHR mRNA in the brain stem was reported, and in a more recent report (Castro et al., 2000) the presence of GHR mRNA in the frontoparietal and

temporal cortex, as well as in a human glioblastoma cell line, was demonstrated.

A. Variants of GHR mRNA A number of variants of the mRNA encoding the human GHR in peripheral tissues have been identified: nine 5⬘-UTR variants (V1–V9) (Edens and Talamantes, 1998; Goodyer et al., 2001; Pekhletsky et al., 1992) and several GHR gene variants that are partially and fully lacking an exon, resulting in an unfunctional receptor (Dastot et al., 1996; Ross et al., 1997; Urbanek et al., 1992). The 5⬘-UTR variants are alternatively spliced forms of the transcript and have been suggested to be tissue specific (Ballesteros et al., 2000; Mercado et al., 1994) and of importance for the regulation of GH response, where the truncated forms may act as inhibitors (Ballesteros et al., 2000). Laron and co-workers (1966) first reported the GH insensitivity syndrome (GHIS). Laron patients are characterized by a short stature and normal to high GH levels but low serum insulin-like growth factor I concentrations. This syndrome is, in almost all documented cases, caused by a defect in the GHR gene, resulting in failure of ligand binding (Rosenbloom et al., 1997), receptor dimerization (Duquesnoy et al., 1994), and/or receptor signaling (Baumbach et al., 1997; Rosenfeld et al., 1994; Sobrier et al., 1997). Examples are the GHR-(1-279) and GHR(1-277) that were first identified in human liver (Dastot et al., 1996; Ross et al., 1997). Both these alternative

Transmembrane domain 1

Extracellular domain

746 818

Intracellular domain

5′-ATG Liver1 Choroid plexus 2

Spinal cord 3 Hippocampus 3

Hypothalamus 2

FIGURE 5 Schematic of the advancement in cloning and sequencing the GHR in different CNS tissues of the rat. Mathews et al. (1989) cloned the liver sequence in 1989. By using the RT-PCR technique with primers designed after the liver sequence the GHR in the rat choroid plexus (100%), spinal cord (100%), hippocampus (84%), and hypothalamus (59%) were sequenced. (See color plate 7) 1 Baumbach et al., 1989. 2 M. Le Grevès et al., unpublished data. 3 Thörnwall-Le Grevès et al., 2001.

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TAG-3′

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B. GHR mRNA Variants in the CNS The abundance and distribution of GHR mRNA variants in different areas of the human CNS are poorly studied, and there is only one report by Mercado and co-workers (1994) that demonstrates the existence of both GHR and GHR3- in the brain stem. The proportion of GHR3- was relatively high (93%) compared to that in the liver (21%), suggesting the expression of GHR to be tissue specific. The exon 3-deleted variant of GHR has been considered to be species specific and has not so far been reported to exist in any other species than human (Pantel et al., 2000). However, it was demonstrated that the GHR3- isoform is also present in the sheep choroid plexus (M. Le Grevès et al., unpublished data). A full-length cDNA was generated from ovine choroid plexus tissue and a sheep choroid plexus cell line (SCP) by RT-PCR using specific primers. The cDNAs were cloned and, when sequenced, an isoform of the GHR mRNA lacking exon 3 was identified. Choroid plexus tissues showed a heterozygous GHR expression, both GHR and GHR3-, whereas SCP cells showed a homozygous expression pattern of GHR3-. The mechanism for the generation of ovine GHR3- has not yet been revealed.

1998). In liver, conflicting results were reported concerning the regulation of GHR by GH: downregulated, upregulated, or unchanged (Butler et al., 1996; Frick et al., 1990; Hull and Harvey, 1998; Maiter et al., 1988; Mathews et al., 1989). In studies on GHR-expressing cell lines, GH has been shown to downregulate the receptor in fibroblasts and IM-9 lymphocytes (Lesniak and Roth, 1976; Murphy and Lazarus, 1984). In the brain, the ontogeny of GHR gene expression was studied in the rat hypothalamus from embryonic day 15 until 56 days of age, and both RNA species were detected during this period of life. The level of 1.2- and 4.5-kb transcripts was low in embryonic animals but increased between days 7 and 35 of age, thereafter the levels declined. No sex differences were shown in the levels of the two transcripts of GHR in the hypothalamic tissue (Hasegawa et al., 1993). Studies on the transcriptional level showed that central infusions of GH in normal rats decreased GHR gene expression in the hypothalamus (Bennett et al., 1995). However, a single injection of GH resulted in an increase of GHR and GHBP mRNA in the brain, whereas in the liver, only the GHBP transcript was increased, and it was suggested that GHR gene transcription is autoregulated in a tissue-specific manner (Hull and Harvey, 1998). Furthermore, chronic sc administration of GH or IGF-I enhanced the GHR mRNA expression in the hippocampus of young adult but not in older rats. In the same study the GHBP mRNA expression was unaffected by treatment with GH but declined in the elderly group treated with IGF-I (Fig. 6) (Le Grevès et al., 2002). Administration of opiates influences the GHR protein present in certain areas of the brain. It was demonstrated that exogenous opiate administration decreases the level of GHR **

2.0 mRNA (arbitrary units)

spliced mRNA derive from a frameshift and a premature stop codon occurs, resulting in a receptor protein that has intact extracellular and transmembrane domains but lacks the signaling intracellular part. The GHR3- variant lacks 66 bp, exactly corresponding to exon 3, and was first isolated in human placenta (Urbanek et al., 1992). Individuals expressing the GHR3- have a full functional receptor and do not have any GHIS symptoms. The generation of human GHR3- seems to be a result of polymorphism in the GHR gene (Stallings-Mann et al., 1996) and it was suggested that it is produced by a retrovirus-mediated alternative splice mimicry (Pantel et al., 2000). The physiological importance of the retention or deletion of exon 3 is still not clarified. Many studies comparing binding of GH and related proteins to GHR and GHR3- have been performed without revealing any differences (Sobrier et al., 1993; Urbanek et al., 1993). However, it has been shown that short-statue children, carrying at least one allele encoding the GHR3-isoform, show a 1.7–2 times higher response to GH therapy (Dos Santos et al., 2004).

*

1.0 0 GHBP 2.0 *

1.0

V. REGULATION OF GHR GENE EXPRESSION A variety of factors, including ontogeny, hormonal and nutritional status, and tissue/cell-specific control, regulate GHR gene expression. These factors can take place at the gene or protein level (Schwartzbauer and Menon,

Control GH IGF-1

0

11 week old rats

14–16 month old rats

FIGURE 6 Effects of daily sc injections of saline (control), GH (1 mg/kg), or IGF-I (0.3 mg/kg) on GHR and GHBP mRNAs in the hippocampus of 11-week-old and 14- to 16-month-old rats. *P ⬍ 0.05; **P ⬍ 0.01 compared to control animals (unpaired Student’s t test) (Le Grevès et al., 2002, 2005). (See color plate 8)

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mRNA level (% of control)

9. GHR mRNA in the Rat and Human CNS Control GH GHBP

150 Spinal cord 100

100 *

*

*

50 0

References

150 Hippocampus

*

50

30 min

4h

24 h

0

30 min

4h

24 h

FIGURE 7 Effects of saline (control) or morphine (10 mg/kg) on GHR and GHBP mRNA in rat spinal cord and hippocampus at 30 min, 4 h, or 24 h after a single injection. *P ⬍ 0.05, ANOVA followed by Fisher PLSD (Thörnwall-Le Grevès et al., 2001). (See color plate 9)

GHR-binding sites in the choroid plexus and the hypothalamus during its acute phase (Zhai et al., 1995). When tolerance to morphine was developed, the level of GH binding was restored to the control level. In the hippocampus and spinal cord of rats treated with a single dose of morphine, a decrease in the levels of GHR and GHBP transcripts was seen (Fig. 7) (Thörnwall-Le Grevès et al., 2001). However, in the isolated human lymphoblast cell line IM-9, it was shown that morphine increases both the number of binding sites and the expression of GHR mRNA in a naloxonereversible manner (Henrohn et al., 1997). Bennett and co-workers (1996) showed that dexamethasone treatment of rats reduced hepatic GHR mRNA expression considerably but had no effect on GHR expression in either the arcuate nucleus or the hippocampus. The opposite situation was seen when rats were treated with estradiol, which upregulated hepatic GHR expression and decreased arcuate nucleus and hippocampal GHR mRNA levels significantly (Bennett et al., 1996). In pregnant mice, it was shown that both GHR and GHBP mRNA increased dramatically in the liver with fetal age while the levels of mRNA of these genes did not alter in the CNS (Ilkbahar et al., 1995). Finally, rats that were subjected to acute stress by restraint stress in the water (RSW) have an increased serum level of corticosterone and a decreased level of GH. In parallel were the mRNA levels of GHR measured in the hippocampus. Half an hour after RSW the GHR mRNA level in the dentate gyrus of the hippocampus was downregulated by 14%, while the transcript was upregulated by 38% 4 h after RSW (Fujikawa et al., 2000). Seven hours after the induction of stress the level of GHR mRNA in the dentate gyrus returned to normal. Taking these studies together, the GHR transcript is highly regulated in a tissue-specific, developmentally, and gestational manner.

Acknowledgment The Swedish Medical Research Council (Grant 9459) supported the studies conducted in our laboratory.

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10 Interaction of Growth Hormone and Prolactin in Brain Circuits DAVID R. GRATTAN* and TANJA A. E. MÖDERSCHEIM† *Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin, New Zealand †Liggins Institute and National Research Centre for Growth and Development, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand

I. INTRODUCTION

I. Introduction II. Common Features of the Somatotrophic and Lactotrophic Systems in the Central Nervous System III. Differential Actions of Growth Hormone and Prolactin in the Brain IV. Potential Interaction of the Two Hormones in Brain Function V. Conclusion References

Growth hormone (GH) and prolactin are phylogenically old hormones and exert a wide range of adaptive functions in vertebrates (Forsyth and Wallis, 2002). The genes for prolactin and growth hormone are structurally similar, as they originate from a common ancestral gene (Soares, 2004). They are expressed in the anterior pituitary gland in a manner that is highly conserved across mammalian species. Typically, they act as hormones, traveling in the blood to bind to and activate specific receptors on target organs. Both proteins, however, have been reported to be produced in extrapituitary sites, including the brain, and may exert local paracrine actions (Ben-Jonathan et al., 1996). Both hormones require a similar mode of action to influence brain circuits. As large polypeptide hormones, prolactin and growth hormone would be expected to be excluded from the brain by the blood/brain barrier (Nilsson et al., 1992; Smith et al., 2004). Hence, they must be actively transported into the brain, through a yet to be well-elucidated mechanism. There are also a number of similarities in their signal transduction mechanisms. Their receptors are closely related molecules, being members of the cytokine type 1 family of receptors. Signal transduction is predominantly through a JAK /STAT pathway. Both hormones have been reported to activate a range of STAT proteins, although STAT5a and STAT5b are the major intracellular signaling molecules (Goffin and Kelly, 1996; Postel-Vinay and Kelly, 1996; Bole-Feysot et al., 1998).

Growth hormone and prolactin are closely related molecules, acting through similar mechanisms. The goal of this chapter is to summarize the common features of the somatotrophic and lactotrophic systems in the central nervous system, including transport mechanisms into the brain, distribution of hormone receptors, similarities in signal transduction pathways, and extrapituitary sources of the hormones. Differences between the systems are also highlighted, in particular focusing on differences in hormonal feedback mechanisms and neuronal functions that appear to be specific to each hormone. Finally, a number of examples are given where there is evidence for interactions between the hormones either through common mechanisms or through independent convergent pathways. While there has been little direct investigation of interactions between these molecules in the regulation of brain circuits, there are a number of functions that the hormones appear to have in common and several potential pathways whereby they may interact. Recognition of these potential interactions will be important in interpreting effects of either hormone in the central nervous system.

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Despite the similarities just described, the two hormones exert relatively distinct neuroendocrine functions. Arguably the most important functions are the feedback regulation of their own secretion: GH, acting predominantly through specific GH receptors located on somatostatin neurons in the periventricular nucleus to increase somatostatin secretion into the portal blood and indirectly suppress growth hormone-releasing hormone secretion from the arcuate nucleus (Bluet-Pajot et al., 1998; Veldhuis et al., 2001), and prolactin, acting through specific prolactin receptors expressed on tuberoinfundibular dopaminergic (TIDA) neurons in the arcuate nucleus, regulating dopamine secretion into the portal blood (Freeman et al., 2000). Their receptors, however, are expressed in a number of common regions, potentially providing the opportunity for interaction in the regulation of brain function. Moreover, because of the close relationship between the molecules, in some species the hormones can exert actions on receptors for the other molecule. In particular, human growth hormone is also lactogenic, suggesting that growth hormone could significantly influence prolactin-sensitive circuits in the human brain. There has been relatively little research directed at evaluating the interactions between these closely related neuroendocrine hormones. Hence, the goal of this chapter is to summarize common features of the lactotrophic and somatotrophic systems in the central nervous system (CNS), to review the distribution of GH and prolactin receptors in the brain, and to identify potential sites of interaction between these hormones. Specific examples of interaction between the hormones are provided, together with some speculation as to other neuronal circuits that may be concurrently regulated by the two hormones.

II. COMMON FEATURES OF THE SOMATOTROPHIC AND LACTOTROPHIC SYSTEMS IN THE CENTRAL NERVOUS SYSTEM A. Transport of GH and Prolactin into the Brain The access of polypeptides to the brain is limited by the specialized tight junctions between vascular endothelial cells in the brain that form the so-called “blood/brain barrier” (Nilsson et al., 1992; Smith et al., 2004). For GH and prolactin of peripheral origin to exert actions in the brain, they must either act in regions of the brain, such as the circumventricular organs, that have an incomplete blood/brain barrier or be transported across the blood/brain barrier. There is now considerable evidence that GH and prolactin gain access to the cerebrospinal fluid (CSF) by means of a saturable, carrier-mediated transport system located predominantly in the choroid plexus (Walsh et al., 1987). Once in CSF, hormones could exert actions throughout the brain

(Smith et al., 2004). The choroid plexus contains the highest density of both GH (Lai et al., 1993; Lobie et al., 1993; Zhai et al., 1994, 1995) and prolactin (Silverman et al., 1986; Brooks et al., 1992; Di Carlo et al., 1992; Lai et al., 1992; Chiu and Wise, 1994; Pi and Grattan, 1998a; Augustine et al., 2003) receptors in the brain. These are distinct molecules (Lai et al., 1992; Zhai et al., 1994), suggesting specific and separate uptake and transport of the respective hormones. The high level of receptor expression, coupled with evidence for carrier-mediated transport of the hormones, has led to the suggestion that receptors actually function as hormone transporters. The precise mechanism whereby a cytokine receptor could mediate translocation of prolactin or GH across this epithelial layer into the CSF, however, has not been determined. Extensive evidence shows that both GH and prolactin receptors undergo a ligand-dependent internalization through an endocytotic mechanism (Roupas and Herington, 1989; Vincent et al., 1997; Govers et al., 1999; Lobie et al., 1999; Lu et al., 2002). There has been considerable investigation of this pathway as a mechanism of downregulating functional receptor expression on the cell membrane. In addition, for both GH and prolactin, evidence shows that internalization may be associated with nuclear translocation of the hormone/receptor complex, resulting in transcriptional regulation (Rao et al., 1993, 1995; Lobie et al., 1994; Rycyzyn et al., 2000; Rycyzyn and Clevenger, 2002; Mertani et al., 2003). Internalization occurs independent of transcriptional activation through the membrane-associated JAK/STAT pathway (see later) (Allevato et al., 1995; Harding et al., 1996; Mertani et al., 2003). Despite much research, however, the function of the internalized hormone/ receptor complex is still not well understood. Following internalization, the internalized hormone is distributed to various subcellular departments, including the Golgi apparatus (Roupas and Herington, 1989), and exocytotic release of GH from internalized stores has been reported (Ilondo et al., 1992). Hence, it is possible that in polarized epithelial cells, such as the choroid plexus, this intracellular trafficking system could be utilized for receptor-mediated transcellular transport of the ligand. Such a mechanism, however, has not been directly demonstrated. Hence, the evidence for GH and prolactin receptors in the choroid plexus functioning as transporters remains circumstantial. The concept has been supported, however, by investigations into leptin access into the brain. This is an analogous system involving a protein hormone that is produced peripherally and is actively transported into the brain to exert actions on hypothalamic circuits. Like GH and prolactin receptors, leptin receptors are members of the cytokine type-I receptor superfamily, are highly expressed on choroid plexus cells, and provide a high-affinity uptake system concentrating leptin in epithelial cells (Karonen et al., 1998; Zlokovic et al., 2000; Thomas et al., 2001).

10. Interaction of GH and PRL in the Brain

Direct evidence that the leptin receptor can act as a transporter has been provided by a study using the MDCK epithelial cell line. Monolayers of these cells are incapable of transporting leptin. Transfection of the cells with the short form of the leptin receptor, however, confers an ability to transport leptin intact across the epithelial monolayer (Hileman et al., 2000). Hence, evidence is mounting suggesting that these cytokine receptors can function as epithelial protein hormone transporters in the choroid plexus, providing a conduit to allow CNS actions. Interestingly, because the different isoforms of the prolactin receptor may have differential internalization properties (Vincent et al., 1997; Lu et al., 2002), it is possible that only certain isoforms will function as a transporter. The presence of a transport system in the choroid plexus also provides a potential point of regulation of hormone action in the brain. Conceivably, suppression of transport could limit hormone action in the brain, as has been suggested in some forms of leptin resistance (Banks, 2001). Decreases in levels of receptor proteins or increased expression of factors that alter the availability of hormones to the transporter systems could reduce the transport of peripheral hormones into the brain. For both GH and prolactin, soluble forms of the hormone receptor have been reported (Postel-Vinay, 1996; Kline and Clevenger, 2001), produced either by proteolytic cleavage of the receptor molecule or by alternative splicing of the mRNA. These proteins are thought to bind to the hormone in plasma, thus limiting biological activity of the hormones (Ross et al., 1997). Such binding would certainly be expected to reduce the availability of GH and prolactin to transporter receptors in the choroids plexus, thereby limiting access to the brain. Interestingly, because of the ability of human GH to bind to prolactin receptors, serum prolactin-binding proteins may also influence biologically available GH (Dannies, 2001). In contrast to the mechanisms reducing access to the brain, discussed earlier, it might also be possible to enhance hormone action in the brain through increased transport into the CSF. For example, hyperprolactinemia (Mangurian et al., 1992; Kvitnitskaya-Ryzhova et al., 1994) and conditions such as pregnancy and lactation that elevate serum prolactin levels (Pi and Grattan, 1999; Augustine et al., 2003) are known to induce an increased expression of prolactin receptors in the choroid plexus. These changes are often associated with enhanced prolactin action in the brain (Grattan, 2002), presumably due to the increased transport of prolactin across the blood–brain barrier.

B. Distribution of GH and Prolactin Receptors in the Brain Several studies have documented the expression of GH and prolactin receptors in the brain. In addition to the prevalence of receptors for both hormones in the choroid

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plexus, discussed previously, there are a number of similarities in the distribution of receptors for the two hormones in the hypothalamus. Both mRNA and protein for GH and prolactin receptors have been identified in the mediobasal hypothalamus, in particular in arcuate and periventricular nuclei, and in the paraventricular nucleus (Burton et al., 1992; Lobie et al., 1993; Chiu and Wise, 1994; Bakowska and Morrell, 1997; Pi and Grattan, 1998a; Bakowska and Morrell, 2003). In situ hybridization studies have identified mRNA for both receptors in the bed nucleus of the stria terminalis and in the medial amygdala, although this has not yet been confirmed with studies of protein distribution (Burton et al., 1992; Chiu and Wise, 1994; Bakowska and Morrell, 1997, 2003). There are also some regions of differential expression, notably the hippocampus, where GH but not prolactin receptors are found, and the slightly more widespread hypothalamic distribution of prolactin receptors, including medial preoptic, supraoptic, and ventromedial hypothalamic nuclei. The general pattern of expression of the receptors for the two hormones is summarized in Fig. 1. The overall similarity in the distribution of receptors may reflect the fact that the hormones have related functions. Alternatively, it is possible that the similar distribution is merely a consequence of the common mode of access to the CNS, resulting in limited regions of the brain that can be affected by these hormones.

C. Signal Transduction Pathway Both prolactin and GH receptors are members of the class 1 cytokine receptor superfamily (Goffin and Kelly, 1996; Postel-Vinay and Kelly, 1996; Bole-Feysot et al., 1998), characterized by an extracellular domain, a single transmembrane domain, and an intracellular domain that couples with various intracellular kinase pathways. There are at least two isoforms of the prolactin receptor molecule, a long form and a short form, produced by alternative splicing of the prolactin receptor gene (Bole-Feysot et al., 1998). The GH receptor also occurs as two isoforms: a long form analogous to the long form of the prolactin receptor and a soluble form that corresponds to the extracellular domain of the receptor molecule and functions as a GH-binding protein (Talamantes and Ortiz, 2002). The different isoforms of the receptor protein have not been specifically identified within brain tissue, but several studies have examined the distribution of the two forms of prolactin receptor mRNA (Bakowska and Morrell, 1997, 2003; Pi and Grattan, 1998b). Both forms are highly expressed in the choroid plexus, but the long form is predominant in the hypothalamus (Augustine et al., 2003). The two forms of the prolactin receptor differ in their ability to activate intracellular signaling pathways. Binding of prolactin to its receptor induces dimerization of the receptor molecules and then activation of multiple intracellular

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FIGURE 1 Approximate distribution of growth hormone (left) and prolactin (right) receptors in the rat brain. Diagrams represent coronal sections at three levels of the brain, based on the atlas of Paxinos and Watson (1997). Major regions where both mRNA and protein have been identified by in situ hybridization and immunohistochemistry, respectively, are indicated with a star (✭). Regions where mRNA but not protein has been detected are indicated with a filled circle (●). For growth hormone receptor, data are based predominantly on Burton et al. (1992) and Lobie et al. (1993). For prolactin receptor, data are based predominantly on Bakowska and Morrell (1997) and Pi and Grattan (1998a).

signaling proteins (Bole-Feysot et al., 1998; Freeman et al., 2000). Ligand interaction with the long form of the receptor leads to phosphorylation of the receptor-associated janus tyrosine kinase 2 (JAK2). JAK2 recruits and phosphorylates a family of latent cytoplasmic proteins known as signal tranducers and activator of transcription (STATs). Phosphorylated STAT molecules form homo- or heterodimers, translocate to the nucleus, and bind to specific promoter sequences of target genes (Leonard and O’Shea, 1998). In various tissues, several STAT molecules have been reported to be involved in prolactin signal transduction, including STAT1, STAT3, STAT5a, and STAT5b

(Bole-Feysot et al., 1998). In TIDA neurons, prolactin treatment induces nuclear translocation of STAT5 (Lerant et al., 2001), but the role of other STAT molecules has not been examined. Furthermore, STAT5b-deficient mice have extraordinarily high levels of serum prolactin, which appear to arise from a failure of prolactin signaling in the TIDA neurons (Grattan et al., 2001). These data suggest that STAT5b is required for the negative feedback action of prolactin on TIDA neurons. As only the long form of the receptor can activate the JAK–STAT pathway, involvement of STAT5b in the prolactin regulation of TIDA strongly implicates the long form of the receptor as the critical protein-mediating prolactin signal, at least in this population of neuroendocrine neurons. While the short form of the receptor cannot activate the JAK–STAT pathway, it can mediate some actions of prolactin through the mitogen-activated protein kinase (MAPK) pathway (Das and Vonderhaar, 1995). Furthermore, in vitro experiments have suggested that the short form of the receptor might act as a competitive inhibitor to suppress prolactin signal transduction by forming inactive heterodimer receptor complexes with the long form and preventing activation of the JAK/STAT pathway (Perrot-Applanat et al. 1997). Hence, the short form of the receptor, which is expressed in the hypothalamus (Bakowska and Morrell, 2003), may also play a role in regulating neuronal function. Growth hormone binding to its receptor also acts predominantly through activation of JAK2/STAT5b (Thomas, 1998), although there has been little examination of this pathway in neurons. Nevertheless, in neurons expressing both prolactin and GH receptors, the intracellular response might be expected to be similar. However, it is possible that exposure to one hormone might alter the responsiveness to the other. In pancreatic islet cells, which express both GH and prolactin receptors, the two hormones activate STAT5b over a slightly different time course, and prior exposure to GH can suppress the subsequent response to prolactin or GH (Brelje et al., 2004). This “desensitization” of the receptors may be mediated by a family of proteins known as suppressors of cytokine signaling (SOCS) (Greenhalgh and Alexander, 2004). SOCS proteins are activated by cytokines and hormones (including GH and prolactin) and function to provide an intracellular negative feedback loop by suppressing further activation of the JAK/STAT pathway. Overexpression of these proteins leads to suppression of the GH (Greenhalgh and Alexander, 2004) and prolactin (Pezet et al., 1999; Tomic et al., 1999) signaling pathways. In mice overexpressing bovine GH, there is a desensitization of the JAK2/STAT5 signaling pathway due, at least in part, to chronic activation of SOCS proteins (Miquet et al., 2004). During conditions of hyperprolactinemia, such as lactation, differential activation of SOCS results in loss of responsiveness to prolactin in specific tissues (Tam et al., 2001). At least two of these SOCS proteins (CIS and SOCS3) are induced in hypothalamic

10. Interaction of GH and PRL in the Brain

FIGURE 2 Diagrammatic representation of the JAK/STAT intracellular signal transduction pathway, which is the major pathway mediating both GH and prolactin action. Because both hormones activate essentially identical signaling pathways, there is significant potential for interaction. In particular, one mechanism depicted in the diagram involves hormoneinduced expression of SOCS proteins. In neurons expressing both GH and prolactin receptors, exposure to one hormone might alter the responsiveness to the other through the activation of SOCS proteins, resulting in a feedback suppression of further activation of the JAK/STAT pathway.

neuronal populations in response to GH (Kasagi et al., 2004) or prolactin (Anderson and Grattan, unpublished result). The physiological roles of these proteins in regulating brain actions of GH and prolactin have yet to be elucidated, but their presence provides a mechanism by which signaling pathways for the two hormones may interact. A summary of these signaling pathways is depicted in Fig. 2. Using primary cultures of hypothalamic neurons, we have demonstrated that, in addition to activation of the JAK2/STAT5b pathway in neurons, prolactin can acutely alter tyrosine hydroxylase activity through a variety of protein kinase-dependent pathways, including Protein Kinase C (PKC) and Mitogen Activated Protein Kinase (MAPK) (Ma et al., 2005). Growth hormone has also been reported to activate these kinase pathways in a number of tissues (Carter-Su et al., 1996), although it has not yet been examined in neurons.

D. Brain Production of GH and Prolactin In addition to hormones of peripheral origin being transported into the central nervous system, evidence shows that both GH and prolactin are produced within the brain, and hence may function as neuropeptides to influence brain function. GH has been detected in the brain by radioimmunoassay (Hojvat et al., 1982), and levels were not suppressed by hypophysectomy. In addition, GH mRNA has been detected using reverse transcriptase polymerase chain reaction (RT-PCR) (Render et al., 1995), in situ hybridization (Gossard et al., 1987), and in situ RT-PCR (Yoshizato

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et al., 1998). The distribution of brain GH mRNA is not clear, however, with the earlier study reporting quite widespread distribution in the hippocampus, caudate putamen, and cerebral cortex (Gossard et al., 1987), while the more recent study found that GH mRNA was restricted to the lateral hypothalamus (Yoshizato et al., 1998). The reasons for this discrepancy are not clear. Immunoreactive prolactin has also been identified in the brain in intact and hypophysectomized animals (Emanuele et al., 1986, 1987; DeVito, 1988). Prolactin mRNA has been identified in the brain by RT-PCR (DeVito et al., 1992b; Wilson et al., 1992), and its expression appears to be regulated independently of pituitary prolactin (Emanuele et al., 1992). A range of studies using immunohistochemistry have produced somewhat inconsistent descriptions of the distribution of prolactin protein, with cell bodies expressing prolactin identified in the mediobasal hypothalamus (Harlan et al., 1989), periventricular hypothalamus (Siaud et al., 1989), or lateral hypothalamus (Paut-Pagano et al., 1993), but more recent research suggests that magnocellular hypothalamic nuclei might represent a major source of prolactin (Clapp et al., 1994; Torner et al., 1999) with local release presumably contributing to the regulation of prolactin-sensitive circuits (Mejia et al., 1997; Torner and Neumann, 2002; Torner et al., 2002; Mejia et al., 2003; Torner et al., 2004). The physiological role of brain GH and prolactin is not yet clear. It is possible that the systems are only activated under certain conditions. For example, upregulation of “neuropeptide” GH has been reported following brain tissue damage (Scheepens et al., 2001) (discussed later). Similarly, increases in brain prolactin occur during lactation (Torner et al., 2002).

III. DIFFERENTIAL ACTIONS OF GROWTH HORMONE AND PROLACTIN IN THE BRAIN Although there are a number of similarities in the somatotrophic and lactotrophic systems in the brain, most of the functions of these hormone systems reported to date have been examined in isolation. Hence, there are a number of functions that appear to be distinct and specific to the individual hormone. Where examined, the receptors appear to be on different neuronal populations (see Table I). This section summarizes some of the major specific functions of each hormone in the brain.

A. Feedback Regulation of Hormone Secretion Growth hormone is secreted in a pulsatile pattern driven by alternating secretion of two hypothalamic factors: GH-releasing hormone (GRH) and somatostatin, which suppresses GH release from the pituitary gland. GH exerts a short loop negative feedback control over its own secretion

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TABLE I Phenotype of GH and Prolactin (PRL) Receptor-Containing Neurons (Where Identified) Brain region

GH receptors

PRL receptors

Arcuate nucleus

NPY neuronsa

TH neuronsb

Supraoptic nucleus

Not presentc

Oxytocin and vasopressin neuronsd

Paraventricular nucleus

Present, neurons unidentifiedc

Periventricular nucleus

Somatostatin

neuronsc

Oxytocin and vasopressin neuronsd Present, neurons unidentifiede

a

From Chan et al. (1996) and Kamegai et al. (1996). From Lerant and Freeman (1998) and Grattan (2001). c From Mejia et al. (2003). d From Burton et al. (1992). e From Pi and Grattan (1998a). b

mediated predominantly through GH receptors on somatostatin neurons in the periventricular nucleus (Pellegrini et al., 1996). There is also some evidence of GH stimulation of GRH neurons (Chomczynski et al., 1988), although it is not clear that GRH neurons express functional GH receptors (Burton et al., 1992). Effects on GRH are likely to be mediated indirectly through actions of somatostatin on GRH neurons. The majority of GH receptor-containing neurons in the arcuate nucleus appear to be NPY neurons (Chan et al., 1996; Kamegai et al., 1996), and there is also evidence that NPY may contribute to suppression of GH secretion. In peripheral tissues, many of the actions of GH are mediated by either local or liver production of IGF-I. IGF receptors have been reported in the hypothalamus (Ocrant et al., 1988), but evidence suggests that IGF has little or no central action to suppress GH secretion (Minami et al., 1997). Prolactin secretion is tonically inhibited by the hypothalamus by means of dopamine released from three populations of neuroendocrine dopaminergic neurons located within the periventricular and arcuate nuclei of the hypothalamus (Freeman et al., 2000). Dopamine acts in the anterior pituitary to suppress prolactin secretion. Prolactin acts in the hypothalamus to stimulate the activity of all three populations of dopaminergic neurons (DeMaria et al., 1999), thereby inhibiting its own secretion by short loop negative feedback. Prolactin appears to act directly on hypothalamic dopamine neurons, as these neurons express prolactin receptors (Arbogast and Voogt, 1997; Lerant and Freeman, 1998; Grattan, 2001). Double-label in situ hybridization studies suggest that dopamine neurons make up the vast majority of neurons expressing the prolactin receptor in the arcuate nucleus, although there is evidence that additional neurons may express the prolactin receptor during lactation (Kokay and Grattan, unpublished results). Hence, both GH and prolactin are regulated by a short loop feedback system, and for this to be functionally significant, a hormone of peripheral origin (rather than a centrally produced hormone) must be the primary factor influencing brain hormone receptors. Despite the clear

similarities in mode of feedback, the neuronal pathways involved appear to be quite distinct. Thus, receptors for GH are expressed on somatostatin neurons in the periventricular nucleus and neuropeptide Y neurons in the arcuate nucleus, whereas prolactin receptors are predominantly on dopamine neurons in these same nuclei (see Table I).

B. Maternal Behavior and Neuroendocrine Adaptation to Pregnancy and Lactation Apart from regulation of its own secretion, the major function ascribed to prolactin in the brain is the neurobiological adaptation to pregnancy and lactation (Grattan, 2002). Prolactin receptor-deficient mice exhibit extreme deficits in maternal behavior (Lucas et al., 1998), clearly demonstrating a functional role for prolactin in this process. Administration of exogenous prolactin to suitably hormoneprimed nonpregnant animals can advance the expression of maternal behavior (Bridges et al., 1990), whereas blockade of prolactin signaling in the brain can significantly delay the onset of maternal behavior (Bridges et al., 2001). In addition to these behavioral actions, prolactin has been implicated in regulating a range of other adaptations that occur in the brain of the mother during lactation, including increasing appetite and food intake, attenuating the stress response and suppressing fertility (for review, see Grattan, 2002). These effects appear to be specific to prolactin, mediated through prolactin receptors, and there are very little data implicating GH in these processes. Exogenous GH has been reported to mimic actions of prolactin on maternal behavior (Bridges and Millard, 1988), but this may be mediated through cross-reactivity with prolactin receptors. GH is elevated during pregnancy and lactation, including the production of placental GH-like hormones in some species (Soares, 2004), although high levels of GH-binding protein during pregnancy (Ilkbahar et al., 1995) may limit access to the brain. Nevertheless, it is possible that somatotrophic hormones of maternal or placental origin may contribute to the regulation of brain function during pregnancy and lactation,

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at the very least through known interactions with lactogenic receptors in the brain.

C. Cognitive Function The GH receptor, but not the prolactin receptor, is expressed in the hippocampus (Burton et al., 1992; Lai et al., 1993; Lobie et al., 1993; Zhai et al., 1994) (see Fig. 1), suggesting a specific role for GH in the regulation of hippocampal function. There is extensive interest in potential cognitive actions of GH, given clinical observations that GH replacement results in cognitive improvement in GH-deficient children and in aging (Burman and Deijen, 1998; Deijen et al., 1998; Creyghton et al., 2004; Oertel et al., 2004). In animal studies, GH has been reported to improve long-term memory in young but not old rats (Schneider-Rivas et al., 1995). Chronic GH treatment alters the expression of NMDA receptor subunits in the hippocampus and increases hippocampal GH receptor expression in young but not old rats (Le Greves et al., 2002). Hence, GH may enhance cognitive function through receptor-mediated actions on glutamatergic neurotransmission. There is little or no evidence of prolactin receptor expression in the hippocampus under normal conditions (Bakowska and Morrell, 1997, 2003; Pi and Grattan, 1998a).

IV. POTENTIAL INTERACTION OF THE TWO HORMONES IN BRAIN FUNCTION Interactions between somatotrophic and lactotrophic axes in the brain could occur through coexpression of receptors on the same neuronal populations and, hence, interactions of signaling pathways, as described earlier. While colocalization of GH and prolactin receptors has been described in some tissues, there is no direct evidence of this in the brain at present (see Table I). Nevertheless, functional evidence shows that both GH and prolactin influence the development of the tuberoinfundibular dopamine system, so this may be an example of this type of interaction (discussed later). Alternatively, interactions could occur through independent regulation of pathways with common functions. A number of functions that appear to be regulated by both GH and prolactin, either independently or through shared pathways, are also discussed.

A. Tuberoinfundibular Development A significant body of work by Phelps and colleagues has demonstrated that the development of TIDA neurons, while initially independent of prolactin feedback, requires neurotrophic support from prolactin during a critical postnatal period (Romero and Phelps, 1995; Phelps and Hurley, 1999;

Phelps and Horseman, 2000; Phelps et al., 2003; Phelps, 2004). Thus, in Ames and Snell dwarf mice, which due to mutations impairing pituitary development lack prolactin and GH, there is a postnatal loss of TIDA neurons that can be rescued by treatment with prolactin from postnatal day 12 but not at later times (reviewed in Phelps and Hurley, 1999). These observations are consistent with the established feedback role of prolactin on these neurons (discussed earlier), and neurotrophic effects are presumably mediated through prolactin receptors that are expressed on those cells. Interestingly, however, transgenic mice that are specifically lacking prolactin have normal development of the TIDA neurons (Phelps and Horseman, 2000), and preliminary evidence suggests that prolactin receptor-deficient mice also have normal numbers of TIDA neurons (Phelps, 2004). These observations suggest that GH may be able to compensate for the postnatal neurotrophic action of prolactin on these neurons through an action independent of the prolactin receptor. Interestingly, STAT5b-deficient mice, which are likely to have impaired signal transduction of both GH and prolactin, have markedly suppressed activity of TIDA neurons (Grattan et al., 2001), although the number of TIDA neurons present has not been quantified. The compensatory action of GH on TIDA development provides functional evidence that these neurons may express GH receptors. Consistent with this concept, mice lacking GH receptors are hyperprolactinemic (Chandrashekar et al., 1999), suggesting that under normal circumstances, the somatotrophic axis may contribute to the suppression of prolactin secretion. In contrast, however, mice overexpressing bovine GH show an increase, rather than a decrease, in prolactin secretion (Steger et al., 1991). Hence, the overall role of GH in regulating TIDA activity requires further investigation.

B. Food Intake The role of GH in regulating appetite and food intake is complex. Clinical evidence shows that GH induces food intake in GH-deficient patients (Snel et al., 1995; Blissett et al., 2000), but the mechanism is not clear. Much recent research has focused on the interactions of GH with the endogenous GH secretagogue ghrelin, which, in addition to its ability to stimulate GH secretion, is a potent appetitestimulating hormone (Konturek et al., 2004). In addition, many of the peripheral actions of GH result in metabolic changes that can influence appetite (Scacchi et al., 2003). Nevertheless, there is some evidence that GH can act directly on hypothalamic appetite regulatory centres. It has been demonstrated that chronic overexpression of bovine GH in the brain in mice induced an increase in hypothalamic expression of AGRP and NPY, associated with obesity and hyperphagia (Bohlooly et al., 2005). Acute icv administration of GH also stimulated food intake in mice (Bohlooly et al., 2005).

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Prolactin has also been reported to induce hyperphagia. Hyperprolactinemia is accompanied by weight gain and obesity (Creemers et al., 1991; Greenman et al., 1998). Prolactin receptors are expressed in the paraventricular, ventromedial, and arcuate nuclei (see Fig. 1), all intimately involved in the regulation of food intake (Elmquist et al., 1999). Feeding and appetite are stimulated in response to prolactin (GerardoGettens et al., 1989a,b; Noel and Woodside, 1993; Sauvé and Woodside, 1996, 2000; Heil, 1999), an effect that is mediated within the hypothalamus (Sauvé and Woodside, 2000). Prolactin is also implicated in the hyperphagia that occurs during lactation (Moore et al., 1986). Prolactin receptor-deficient mice exhibit lower body weight and reduced fat mass compared with wild-type controls, consistent with the hypothesis that prolactin has a long-term role in the regulation of body weight (Freemark et al., 2001). While GH and prolactin have both been implicated in the regulation of food intake, postulated mechanisms appear to be dissimilar. This difference is highlighted by effects of these hormones on orexigenic NPY neurons in the mediobasal hypothalamus. GH clearly directly regulates the arcuate NPY neurons involved in the stimulation of appetite (Chan et al., 1996; Kamegai et al., 1996). In contrast, best evidence suggests that prolactin acts in the paraventricular nucleus to enhance food intake (Sauvé and Woodside, 2000). Prolactin stimulates NPY neurons in the DMH, but does not appear to regulate arcuate NPY neurons (Li et al., 1999; Chen and Smith, 2004). Hence, the two hormones interact using apparently different mechanisms to influence appetite and food intake.

C. Neurotrophic Actions, Neurogenesis and Neuroprotection In recent years, there has been extensive interest in hormonal factors that might influence neuronal development and/or confer protection to neuronal systems against aging or pathological insult. The GH/IGF axis has received considerable attention in this area. It is well established that the GH/IGF axis is active in the developing brain (Duenas et al., 1994), and IGF appears to function as a neural stem cell differentiation factor (Arsenijevic and Weiss, 1998; Arsenijevic et al., 2001). Both GH and GH receptors are present in the developing brain (Hojvat et al., 1982; Lobie et al., 1993), and a variety of dwarf mice models that lack GH exhibit neuronal development deficits (Noguchi, 1996). In cultures of fetal cerebral cortical tissue, GH induced increased cell division of both neurons and glia and stimulated differentiation (Ajo et al., 2003). These latter actions appeared to be mediated by a GH-induced activation of IGF production. In contrast with a stimulatory action through IGF, GH appears to directly inhibit the differentiation of neuronal progenitor cells (Turnley et al., 2002) and hence have an inhibitory effect on neurogenesis (Ransome et al., 2004). These

inhibitory actions of GH are blocked by SOCS2 (Turnley et al., 2002), suggesting that they are mediated directly through the JAK/STAT pathway downstream of the GH receptor. Despite reported inhibitory actions, neurogenesis promoting actions of GH are currently being investigated (Weiss and Shingo, World Intellectual Property Organisation Patent Application, WO 03/024471 A2). In addition to neurogenesis and developmental roles, GH and IGF may have neuroprotective functions during the cellular response to brain injury. GH (Scheepens et al., 2001), GH receptor (Scheepens et al., 1999), and IGF (Beilharz et al., 1998) are upregulated in the brain following hypoxia/ischemia injury. Exogenous treatment with either GH (Scheepens et al., 2001) or IGF (Gluckman et al., 1993; Guan et al., 1993) has proven to provide some protection against apoptotic cell loss following such brain injury. Interestingly, the spatial neuroprotection mediated by GH treatment does not correlate with the neuroprotection found following IGF-I treatment, suggesting that neuroprotection through GH is independent from IGF-I action (Scheepens et al., 2001). The possible dissociation of brain GH and IGF-I is confirmed in studies of Ames dwarf mice that have normal IGF-I mRNA levels and elevated IGF-I protein levels in the hippocampus, despite lower GH mRNA levels (Sun et al., 2004). In addition, it has been demonstrated that an absence of GH signaling does not impair IGF-I expression in the mouse brain (Lupu et al., 2001; Sun et al., 2004). Prolactin has also been implicated in the regulation of neurogenesis and the response to neuronal damage. In addition to its neurotrophic effect on developing hypothalamic dopamine neurons, discussed earlier, prolactin is mitogenic for astrocytes in culture (DeVito et al., 1992a, 1993; Mangoura et al., 2000) and induces the expression of a variety of cytokines (DeVito et al., 1995b; DeVito and Stone, 1999). Moreover, neuronal tissue damage has been shown to upregulate prolactin expression in the hypothalamus (DeVito et al., 1995b) and telencephalon (Möderscheim, Kokay, Grattan, Williams, and Scheepens, unpublished result), suggesting that prolactin may be involved in the brain response to injury. Prolactin has also been implicated in the pregnancy-induced increase in neurogenesis in the brain (Shingo et al., 2003), promoting neuronal cell division in the adult subventricular zone, and enhanced integration of new neurons into the olfactory bulb. These observations suggest that in addition to developmental effects and responses to injury, prolactin may play a physiological neurotrophic role in the adult brain. Although their expression is similarly increased at the wound site following brain injury (Scheepens et al., 2001; Möderscheim et al., unpublished results), the specific roles of GH and prolactin may differ. In the hypoxicischemic rat brain, neuronal rescue can be achieved through GH treatment (Scheepens et al., 2001) but not through PRL treatment at concentrations previously found to be effective in the rat brain (Möderscheim et al., unpublished results). Furthermore,

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using primary neuronal cultures, we have found that prolactin had limited neuroprotective potency as compared to GH (Möderscheim et al., unpublished result). As hypoxic ischemic injury is associated with an increased neuronal progenitor proliferation in the subgranular zone (Arvidsson et al., 2001), the SVZ (Jin et al., 2001; Zhang et al., 2001), and the cerebral cortex (Gu et al., 2000) and prolactin has been reported to induce neurogenesis and migration of progenitor cells during pregnancy (Shingo et al., 2003), it seems possible that prolactin may function to stimulate the generation and migration of stem cells/progenitors following injury. Alternative roles for prolactin might lie in the mediation of trophic support through the induction of astrogliosis. Astrocytic support is important for the spontaneous recovery of ischemic neurons (Zoli et al., 1997). Astrogliosis restores the integrity of the blood–brain barrier and seals off the wound site (Herx and Yong, 2001). Prolactin may influence astrogliosis and induce a rapid expression of inflammatory cytokines, such as Interleukin-1␤, Tumor necrosis factor ␣ and Transforming growth factor, factors that are important in regulating the response to injury (DeVito et al., 1995a,b; 1997, 2000). This is confirmed by the finding that a local increase in prolactin synthesis mediates the regulation of the neuroimmune response to hypothalamic injury (DeVito et al., 1995a). Although prolactin and GH are likely to have numerous functions following brain injury, GH seems to be involved in immediate neuroprotection and in the processes of postinjury plasticity and neurogenesis, whereas prolactin appears to promote long-term tissue repair, adaptation, and functional recovery.

V. CONCLUSION There is very little direct evidence of interactions between the somoatotrophic axis and the lactogenic axis within the central nervous system. Both GH and prolactin have a wide variety of roles in the brain, the majority of which appear to be adaptive, helping the organism adjust to changing physiological conditions or to pathological damage. Given the similarities in function, distribution of receptors within the brain, mode of hormone access to the brain, and intracellular mechanism of action, however, it seems likely that significant interaction between the two hormones could occur. Such interactions, however, remain largely hypothetical. The prevailing literature documents a range of independent functions in which the role of the other hormone has not been evaluated. In humans, however, GH is able to activate the prolactin receptor, providing a direct mechanism of interaction between the two signaling pathways. To fully understand the role of either hormone in the CNS, therefore, it will become increasing important to be cogniscent of potential actions through the parallel hormone pathway.

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10. Interaction of GH and PRL in the Brain Scheepens, A., Sirimanne, E., Beilharz, E., Breier, B. H., Waters, M. J., Gluckman, P. D., and Williams, C. E. (1999). Alterations in the neural growth hormone axis following hypoxic-ischemic brain injury. Mol. Brain Res. 68, 88–100. Scheepens, A., Sirimanne, E. S., Breier, B. H., Clark, R. G., Gluckman, P. D., and Williams, C. E. (2001). Growth hormone as a neuronal rescue factor during recovery from CNS injury. Neuroscience 104, 677–687. Schneider-Rivas, S., Rivas-Arancibia, S., Vazquez-Pereyra, F., VazquezSandoval, R., and Borgonio-Perez, G. (1995). Modulation of long-term memory and extinction responses induced by growth hormone (GH) and growth hormone releasing hormone (GHRH) in rats. Life Sci. 56, PL433–441. Shingo, T., Gregg, C., Enwere, E., Fujikawa, H., Hassam, R., Geary, C., Cross, J. C., and Weiss, S. (2003). Pregnancy-stimulated neurogenesis in the adult female forebrain mediated by prolactin. Science 299, 117–120. Siaud, P., Manzoni, O., Balmefrezol, M., Barbanel, G., Assenmacher, I., and Alonso, G. (1989). The organization of prolactin-like-immunoreactive neurons in the rat central nervous system: Light- and electron-microscopic immunocytochemical studies. Cell Tissue Res. 255, 107–115. Silverman, W. F., Walsh, R. J., and Posner, B. I. (1986). The ontogeny of specific prolactin binding sites in the rat choroid plexus. Brain Res. 389, 11–19. Smith, D. E., Johanson, C. E., and Keep, R. F. (2004). Peptide and peptide analog transport systems at the blood-CSF barrier. Adv. Drug Deliv. Rev. 56, 1765–1791. Snel, Y. E., Brummer, R. J., Doerga, M. E., Zelissen, P. M., and Koppeschaar, H. P. (1995). Energy and macronutrient intake in growth hormone-deficient adults: The effect of growth hormone replacement. Eur. J. Clin. Nutr. 49, 492–500. Soares, M. J. (2004). The prolactin and growth hormone families: Pregnancy-specific hormones/cytokines at the maternal-fetal interface. Reprod. Biol. Endocrinol. 2, 51. Steger, R. W., Bartke, A., Parkening, T. A., Collins, T., Buonomo, F. C., Tang, K. C., Wagner, T. E., and Yun, J. S. (1991). Effects of heterologous growth hormones on hypothalamic and pituitary function in transgenic mice. Neuroendocrinology 53, 365–372. Sun, L. Y., Evans, M. S., Hsieh, J., Panici, J., and Bartke, A. (2004). Increased neurogenesis in dentate gyrus of long-lived Ames dwarf mice. Endocrinology. Talamantes, F., and Ortiz, R. (2002). Structure and regulation of expression of the mouse GH receptor. J. Endocrinol. 175, 55–59. Tam, S. P., Lau, P., Djiane, J., Hilton, D. J., and Waters, M. J. (2001). Tissue-specific induction of SOCS gene expression by PRL. Endocrinology 142, 5015–5026. Thomas, M. J. (1998). The molecular basis of growth hormone action. Growth Horm. IGF Res. 8, 3–11. Thomas, S. A., Preston, J. E., Wilson, M. R., Farrell, C. L., and Segal, M. B. (2001). Leptin transport at the blood–cerebrospinal fluid barrier using the perfused sheep choroid plexus model. Brain Res. 895, 283–290. Tomic, S., Chughtai, N., and Ali, S. (1999). SOCS-1, -2, -3: Selective targets and functions downstream of the prolactin receptor. Mol. Cell. Endocrinol. 158, 45–54. Torner, L., and Neumann, I. D. (2002). The brain prolactin system: Involvement in stress response adaptations in lactation. Stress 5, 249–257.

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11 Growth Hormone and Insulin-like Growth Factor-I and Cellular Regeneration in the Adult Brain N. DAVID ÅBERG,* MARIA A. I. ÅBERG,† and PETER S. ERIKSSON† *Research Centre of Endocrinology and Metabolism, Institute of Internal Medicine and †Arvid Carlsson Institute for Neuroscience, Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden

I. II. III. IV.

Introduction Neuroprotective Actions of the Somatotrophic Axis Models and Techniques Adult Cell Genesis by Insulin-like Growth Factor-I and Growth Hormone V. Physiological and Functional Correlates to Adult Cell Genesis References

recruitment as well as increasing newborn cells with the endothelial phenotype. The increase of IGF-I on the endothelial cell phenotype may explain the increase in cerebral arteriole density observed after GH treatment. The functional consequences of newborn cells of different lineages in the adult brain are reviewed especially with reference to exercise, enriched environment, learning, aging, and disease. The findings summarized in this chapter extend the role of IGF-I as being a putative regenerative agent in the adult CNS. GH, being less studied in these aspects, may have similar effects, especially as it is the main stimulus of IGF-I in vivo. It will be interesting and exciting to see if these agents can be used in clinical trials in the years ahead.

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) have various effects in both the developing and the adult central nervous system (CNS). Some of these effects derive from local IGF-I, whereas other effects origin from circulating IGF-I. Both GH and IGF-I affect the adult brain functionally and in cognition. Several neurobiological correlates have been found to these effects, including effects on adult cell genesis. This chapter tries to summarize the significance of GH and IGF-I in adult CNS progenitor cell regulation and cellular regeneration and possible clinical and functional aspects. Neurogenesis in the adult brain occurs in the hippocampal dentate gyrus and the subventricular zone. In addition, astrocytes and oligodendrocytes are also formed from these brain regions. Apart from the well-established neuroprotective effects of IGF-I (in the acute phase of various injuries to the CNS), peripheral infusion of and circulating IGF-I increase progenitor cell proliferation and neurogenesis in the dentate gyrus of the hippocampus. It appears that the MAPK signaling pathway is required for IGF-I-stimulated proliferation in vitro. In the hippocampus, IGF-I treatment seems to enhance oligodendrocyte

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION A. Adult Neurogenesis: History Up until the early 1990s it was thought that cell genesis in the adult mammalian brain was restricted to pathological situations such as the “glial scar” and to endothelial cell proliferation. Physiological adult cell proliferation forming oligodendrocytes, astrocytes, and neurons was simply not taking place. Animal and human data presented in the past decade confirm, however, that the brain retains its capacity of cell renewal throughout life. The old concept that the adult brain cannot produce new neurons has now been rejected.

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In the first half of the 20th century, there were occasional reports of postnatal neurogenesis in mammals (Kershman, 1938; Levi, 1898; Schaper, 1897). These scattered reports, which raised the possibility of adult mammalian neurogenesis, were largely ignored. Presumably, this was because of opposition to the idea of authorities in the neuroscience field and the inadequacy of the available methods both for detecting cell division and for distinguishing glia from small neurons. An important advance in the study of neurogenesis came in the late 1950s with the introduction of [3H]thymidine autoradiography. [3H]Thymidine is incorporated into the DNA of dividing cells. Therefore, the progeny of cells that had just divided could be labeled, and their time and place of birth determined. Starting in the early 1960s, Joseph Altman began publishing a series of papers in which he reported thymidine autoradiographic evidence of new neurons in various structures in the young and adult rat, including the neocortex, dentate gyrus, and olfactory bulb (Altman, 1962, 1966, 1969; Altman and Das, 1965, 1966). These papers were taken with skepticism because they used morphological classification of newborn neurons. The newborn neurons were small and could be mistaken for other cells (e.g., astrocytes). Fifteen years after Altman’s first report, Kaplan and co-workers reported autoradiographic and further ultrastructural evidence for new neurons in the adult rat dentate gyrus, olfactory bulb, and cerebral cortex (Kaplan, 1985; Kaplan and Hinds, 1977; Kaplan et al., 1985). Again, this was still not sufficient for the general acceptance of newborn neurons in the adult brain. However, a few years later, there were three factors that changed this view. The first was a series of studies showing neurogenesis in adult birds (Goldman and Nottebohm, 1983; Paton and Nottebohm, 1984; Paton et al., 1985). The second factor was the introduction of new methods for labeling new cells and for distinguishing neurons from glia. Finally, the demonstration that adult neurogenesis could be up- and downregulated by important stimuli such as stress and learning, as well as hormones, raised the possibility that adult hippocampal neurogenesis could be important for cognition in higher animals. The decline in belief in the stability of the neuronal population seems to be part of a more general paradigm shift that has extended the concept of plasticity in the adult brain to include structural modulation in terms of adding new cells.

B. Definition of a Neural Stem Cell Neurogenesis exists in the adult nervous system in rodents (Altman and Das, 1965; Bayer, 1985; Stanfield and Trice, 1988) and primates (Gould et al., 1998) and in the human brain as well (Eriksson et al., 1998; Roy et al., 2000). A commonly accepted definition of the term “neural stem cell” relies on three hallmark criteria: a neural stem cell (1) can generate neural tissue or that it is derived

from the nervous system, (2) has the capacity for selfrenewal, and (3) can give rise to at least two cell types other than stem cells through asymmetric cell division. However, the detection of continuous neurogenesis is not evidence for the presence of neural stem cells as it would have to be proven that one particular cell (the stem cell) is capable of giving rise to both neurons and glia. This was shown by Palmer et al. (1997) by genetic marking of adult-derived hippocampal progenitors (AHPs). Moreover, the same progenitors have been transplanted back to the adult hippocampus, where they retain their capacity to become new granule cell layer (GCL) neurons (Gage et al., 1995a). To evaluate the plasticity of AHPs within the optic retina, retrovirally engineered AHPs were grafted into the vitreous cavity in the eye of adult and newborn rats (Takahashi et al., 1998). Within the adult eye, AHPs formed a uniform nondisruptive lamina in intimate contact with the inner limiting membrane. Four weeks after grafting to the developing eye, the AHPs were well integrated into the retina and adopted the morphologies and positions of Müller, amacrine, bipolar, horizontal, photoreceptor, and astroglial cells. This suggests that adult-derived stem cells can adapt to a variety of heterologous environments such as in the eye and the hippocampus. A schematic presentation of potential stem cells with neural capability is shown in Fig. 1. Whereas embryonic stem cells in the early embryo are transient during development, adult stem cells appear to reside permanently in some tissues, including the brain, bone marrow, skin, and intestinal mucosa. Adult stem cells were initially thought to be limited in their developmental potential to the regeneration of cell types comprising their tissues of origin, e.g., cells derived from the neuroectoderm were destined to become neurons or glial cells. However, neuroectodermal stem cells from the adult brain (Björnson et al., 1999) have been shown to regenerate the entire hematopoietic system in lethally irradiated adult mice and, in fact, can give rise to most tissues when injected into mouse or chick blastula (Clarke et al., 2000). Also, reports suggest that stem cells of, for example, mesenchymal origin — adult marrow stroma cells — can be induced to overcome their mesenchymal commitment and become neurons under certain conditions (Mezey et al., 2000; Sanchez-Ramos et al., 2000). Although received with enthusiasm, the concept of transdifferentiation is, however, still controversial and under debate (Wells, 2002). The origin of adult stem cells is not known. They may be residual undifferentiated embryonic cells or, as recent evidence suggests, committed/differentiated adult cells that require multipotency in response to environmental signals. In the latter case, most of the neural stem cells lie dormant, waiting for an activating stimulus. The group of Arturo Alvarez-Buylla has shown both in the subventricular zone (SVZ) (Doetsch et al., 1999b) and in the subgranular zone (for the hippocampus) (Seri et al., 2001, 2004) that the cells

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FIGURE 1 Schematic drawing of multipotent stem cells and their relation to IGF-I (in the hippocampus). IGF-I presumably acts at several levels, stimulating both stem cell renewal and asymmetric cell division in committed cells into neural precursor cells. Nonneuronal cells are also formed under IGF-I stimulation, e.g., oligodendrocytes, as discussed in the text.

that have stem cell properties are also GFAP positive. These cells, which should not be termed “astrocytes,” have been suggested to originate from radial glia, which originate from the early ventricular zone. Whatever their origin, their presence in the central nervous system (CNS) throughout life suggests new and intriguing possibilities for recovery and repair after damage to the CNS.

C. Neurogenic Areas in Adult Brain and Regulation 1. Two Regions with Neurogenesis During the early 1990s the concept of adult neurogenesis was consolidated by the discoveries of self-renewing cells

with multilineage potential in the adult mammalian brain (Reynolds and Weiss, 1992; Richards et al., 1992), the human brain included (Eriksson et al., 1998; Roy et al., 2000). There are two sites with a high density of proliferating progenitor cells in the adult brain: the subgranular zone (SGZ) of the dentate gyrus of the hippocampal formation (Altman and Das, 1965; Kuhn et al., 1996; Roy et al., 2000) and the subventricular zone (Chiasson et al., 1999; Doetsch et al., 1999a; Johansson et al., 1999). In the dentate gyrus, progenitor cells migrate into the granule cell layer where they differentiate into granule cell neurons (Fig. 2). In the SVZ, the progenitors migrate through the rostral migratory stream to the olfactory bulb where they differentiate into interneurons. In addition to neurons, cells of the glial lineages are also formed in both of these two sites.

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et al., 1999b) and living in an enriched environment increase neurogenesis in the hippocampus (Kempermann et al., 1998; Nilsson et al., 1999). Conversely, animal models for chronic stress decrease hippocampal neurogenesis (Westenbroek et al., 2004). Substances that may be biological correlates of these conditions have been shown to affect adult cell genesis as well, e.g., brain-derived neurotrophic factor (BDNF) and serotonin (Mattson et al., 2004; Sairanen et al., 2005), ciliary neurotrophic factor (CNTF) (Emsley and Hagg, 2003; Åberg et al., 2001), glucocorticoids (Kim et al., 2004), and blocking the opioid system (Persson et al., 2004). It is interesting that insulin-like growth factor I (IGF-I) and growth hormone (GH) either affect or are affected by several of these conditions.

D. Somatotrophs and Cell Genesis in Adult Brain

FIGURE 2 The adult dentate gyrus within the hippocampus contains dividing cells. (A) Diagram of the hippocampal anatomy showing the granule cell layer (GCL) located in the dentate gyrus. The hilus region is located within the dentate gyrus. (B) Diagram showing the proliferative zone, often designated the subgranular zone of GCL. The sites of migration and differentiation are also shown. (See color plate 10)

It was evident quite early that the GH–IGF-I system affected the brain. Already by 1968 a study indicated that GH had neural trophic effects, such as it increased the thickness of diencephalic structures as anterior and posterior commisure in the growing postnatal brain (Diamond, 1968). Therefore, it was plausible that somatotrophic hormones could affect cell genesis as well as cellular size and arborization and possibly extracellular dimensions.

2. The Cerebral Cortex: Cell Genesis but No Neurogenesis Under physiological conditions, the newly generated daughter cells in the SVZ migrate through the rostral migratory pathway to the olfactory bulb. Normally this process restricts their dispersion and migration into adjacent brain structures. An exception to this was observed in adult macaques, in which migration of the proliferating cells from the SVZ toward the cortex was suggested as a source of newly generated neurons (Gould et al., 1999b). However, the first reports claiming neurogenesis in the cerebral cortex (Gould et al., 1999b) have been convincingly contradicted by other studies (Kornack and Rakic, 2001; Magavi et al., 2000) showing cell genesis and differentiation toward other lineages. Taken together, in mammalian brains of most species, adult cell genesis without neurogenesis can be observed under physiological conditions in the cerebral cortex, whereas neurogenesis occurs in the dentate gyrus of hippocampus and in the olfactory bulb. However, neurogenesis appears to occur in other brain regions after brain injury. Newborn cerebrocortical neurons have been identified in studies of adult rodents subjected to different types of ischemic lesions (Arvidsson et al., 2002; Gu et al., 2000).

1. Insulin-like Growth Factor-I The significance of IGF-I for cell genesis in the CNS has been shown in vivo during development as well as in adulthood. In addition, mechanisms of IGF-I on specific cell genesis have been further elucidated in vitro using several CNS culture paradigms. Using combinations of in vivo and in vitro models, the role of local and peripheral IGF-I on cell genesis has been demonstrated. In vivo, studies on IGF-I knockout mice have shown decreased numbers of granule cells in the hippocampus and reduced neuron and oligodendrocyte numbers within the olfactory bulb (Beck et al., 1995; Cheng et al., 1998). Conversely, studies on IGF-I overexpression show increased neuron number and growth in medullary nuclei of the mouse (Dentremont et al., 1999) and promotion of neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development (O’Kusky et al., 2000). These findings have parallels to different in vitro situations. For example, IGF-I is a mitogen for embryonic progenitors (DiCicco Bloom and Black, 1988; Ye et al., 1996) and is necessary for the establishment of neurospheres derived from fetal CNS (Arsenijevic et al., 2001) and neuroblast cultures (Bozyczko Coyne et al., 1993). These studies have focused mainly on the effects of IGF-I defiency on cell genesis during development and young ages. However, IGF-I is also known to affect cell genesis in the adult brain, as discussed in Section IV.

3. Conditions Affecting Neurogenesis Adult cell genesis can be affected by a number of conditions and substances. For example, exercise (van Praag

2. Growth Hormone In the aspect of CNS cell proliferation, GH has been less studied than IGF-I. In terms of direct effects of GH on adult

11. GH and IGF-I and Cell Genesis in Adult Brain

cell proliferation, there are no studies available. However, there is reason to believe that GH affects proliferation in CNS for several reasons. First, systemic GH has similar effects as IGF-I, as systemic IGF-I is mostly produced in response to systemic GH stimulation but probably also locally within the brain (Frago et al., 2002; Lopezfernandez et al., 1996; Ye et al., 1997). Second, it appears that GH per se affects cell proliferation during development. This was shown by two reports of a suppressor of a cytokine signaling-2 (SOCS-2) knockout model. These mice show altered cell proliferation with altered cell composition in the CNS (Ransome et al., 2004; Turnley et al., 2002). SOCS-2 inhibits GH signaling through the JAK-STAT pathway, which indirectly reveals effects of GH. GH increased astrocyte numbers and decreased neuron number in vitro via decreasing neurogenin-1. This could be overcome by SOCS-2 overexpression in vitro (Turnley et al., 2002). Similarly, GHR ⫺/⫺ mice show more neurons and fewer astrocytes in several brain regions in vivo (Ransome et al., 2004). In addition, dendritic branching decreased in pyramidal cells and neuronal somas were also decreased in size (Ransome et al., 2004). As IGF-I predominantly signals via other cellular pathways than JAK-STAT, these studies favor the view that GH has IGF-I-independent effects on cellular proliferation in the CNS, at least in development. 3. Peripheral Administration of IGF-I and Adult Cell Genesis With the discovery of adult neurogenesis extending to human brain, conditions affecting this phenomenon have gained increased interest. In this context, the extracerebral administration of GH and IGF-I into adult animals is of special interest as they are part of a physiological response to exercise and also because GH or IGF-I may be used as exogenous agents for enhancing cell genesis in the CNS. The investigation of proliferation and differentiation of progenitor cells in the adult brain has been performed using hypophysectomized (hx) rats (Åberg et al., 2000a), liverinduced IGF-I-deficient mice treated with peripherally administered IGF-I (Lopez-Lopez et al., 2004), or blocking peripheral IGF-I with antibodies (Trejo et al., 2001). In these paradigms, bromodeoxyuridine (BrdU) has been used to monitor proliferation and double/triple immunofluorescence for cell-specific markers for the evaluation of cell fate differentiation. Details of these data are described in Section IV.

II. NEUROPROTECTIVE ACTIONS OF THE SOMATOTROPHIC AXIS A. Neuroprotection and Neuroregeneration The research on acute treatment of stroke has largely focused on various neuroprotective agents, sparing the

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neurons in the penumbra zone of an ischemic lesion. Several such compounds, including IGF-I and GH, have been successful in reducing the damage after ischemic lesions in experimental animal models. No substance so far has been successful in clinical studies on humans. After the acute phase in an ischemic lesion, which may extend to at least days in humans, there is a recovery phase where patients usually recover some lost abilities and skills. The recovery is often enhanced with active programs for rehabilitation. Interestingly, this may have a parallel to the situation where several plasticity mechanisms, including neurogenesis, are observed in rats and mice living in an enriched environment (Kempermann et al., 1998; Nilsson et al., 1999), learning tasks (Gould et al., 1999a), and physical activity (van Praag et al., 1999b). Formerly, the recovery phase was believed to depend only on subcellular and functional reorganization of the CNS. However, the discovery of extended neuroand gliogenesis has added yet another mechanism for how this rehabilitation may occur, namely the cellular regeneration in the adult CNS. The concepts of neuroprotection and neuroregeneration include different aspects of beneficial effects for the brain, and it is not uncommon that a particular substance may act both in a protective and in a regenerative manner. Although this chapter is focused on regenerative effects of GH and IGF-I, a short overview of neuroprotective effects is given.

B. Neuroprotective Effects of IGF-I Since the early 1990s, studies have shown neuroprotective effects of IGF-I in different models of injury and insults to the brain. IGF-I has been administered through different routes. For example, IGF-I has been applied directly through intracerebroventricular administration (Brywe et al., 2005; Guan et al., 1993). However, similar effects were found after peripheral administration of IGF-I (Fernandez et al., 1999; Tagami et al., 1997). Interestingly, it has also been shown that elevated IGF-I after exercise may mediate neuroprotective effects against hypoxic ischemia in the adult rat (Carro et al., 2001). Initial studies were focusing mainly on the protective aspects of IGF-I. These effects can largely be linked to antiapoptotic mechanisms (Brywe et al., 2005; Guan et al., 2003). Also, the neuroprotective effect of IGF-I has been shown to synergize with erythropoietin in (N-methyl-Daspartate) NMDA-induced insult (Digicaylioglu et al., 2004). However, in addition to protective effects of IGF-I, there are other effects of IGF-I on the CNS. Most likely IGF-I acts simultaneously at several levels of cell turnover, including both protective (cell survival) and regenerative (cell genesis). As cell turnover can be detected in both rodent (Åberg et al., 2000a) and human brain (Eriksson et al., 1998), both actions of IGF-I may be important and beneficial after lesions to the CNS (Fig. 3).

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FIGURE 3 Potential cell proliferative or protective effects of a neuroactive agent. So far, the concept of neuroprotection has been examined in a number of studies. In an acute situation, protecting CNS cells from death is of course the most important issue. Later, in the recovery phase, it may additionally be of interest to enhance cellular regeneration. The concept of neuroprotection mainly deals with how many cells that are saved in a pathological situation that causes cell death, either by apoptosis or by necrosis. However, the concept of cellular regeneration by cell genesis (proliferation) may be added in studies of positive effects of a putative agent to the CNS. Likely, both concepts may act simultaneously at various degrees in different situations and conditions. (a–c) Three situations showing how the number of BrdU-positive cells varies after an initial BrdU injection time point, depending on the proliferation rate and cell survival (roughly equaling neuroprotective effects of a substance). (a) A putative neuroactive agent that increases proliferation (thin line) compared to a control situation (bold line). As a proportion of newborn cells generally go into apoptosis, the BrdU-positive cells will decrease in numbers. However, the relatively increased proportion of cells surviving will be conserved if boosted in the beginning. To stringently examine such a phenomenon, the treatment with the particular agent should be terminated after the time of BrdU injections. (b) A putative neuroactive agent that increases cell survival (thin line) compared to a control situation (bold line). The proportion of cells surviving will be relatively increased with time. Stringent examination of this phenomenon would start the particular treatment after the time of BrdU injections. (c) The most likely biological effect of a neuroactive compound that promotes recovery is a combination of increasing cell survival and enhancing cell proliferation (thin line, and control situation bold line).

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C. Growth Hormone In the 1990s, a number of studies showed similar neuroprotective effects of GH given ICV as well as in the periphery to that of IGF-I (Fernandez et al., 1999; Guan et al., 1993; Gustafson et al., 1999; Scheepens et al., 1999, 2001; Tagami et al., 1997). In treatment paradigms where GH is administered close to the time of injury (within hours), the usual rise in IGF-I levels (Scheepens et al., 2001) cannot account for the neuroprotective effects of GH. Therefore, it seems that GH on its own has protective effects on the CNS. However, in studies using extended periods of GH treatment, i.e., more than at least 12–24 h, the interpretation of effects is more uncertain, and the effect of GH may be due to the rise of IGF-I (Scheepens et al., 1999). In recent years, apart from neuroprotective effects of GH and IGF-I, neuroprotective data have also been presented for GH secretagogues (Frago et al., 2002). Some of these effects may be due to activation of IGF-I expression, but GH secretagogues may also have intrinsic protective effects per se. Although numerous studies have been performed regarding protective effects of IGF-I and later GH, no study has so far examined the long-term cell regenerative effects of GH and IGF-I after injury. This, of course, largely depends on the difficulties of interpreting the results of long-term cell genesis after injuries. First, injuries vary largely between animals (requiring large numbers of animals). Second, it may be difficult to correlate and interpret data in terms of functional benefits.

III. MODELS AND TECHNIQUES A. Animal Models and Hormonal Treatment To study the effects of hormonal manipulation of the somatotrophic axis in the adult brain at a given time point, there are a few options available, all requiring different animal models. Using standard transgenic animals may be less favorable because the effects of the transgene would be expected to induce secondary adjustments, making the interpretation complicated. Also, it may be difficult to discern adult and developmental effects of the transgene. However, in recent years additional transgenic constructs have become available (e.g., Turnley et al., 2002) that are partly compatible with studies on adult cellular regeneration. However, the use of conditional adult transgenic animals would greatly enhance the interpretation of specific adult cell genesis. Treating normal animals with GH or IGF-I for extended periods of time (i.e., more than about 24 h) poses problems in interpretation, as endogenous GH from the pituitary is decreased due to feedback inhibition.

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Nevertheless, such studies are important if they can demonstrate an effect in a more normal situation. We have used female Sprague–Dawley rats that were hypophysectomized at 50 days of age (becoming almost totally GH and IGF-I deficient) to maximize the effect of peripherally administered GH and IGF-I. Hormonal treatment started 7 to 10 days after hypophysectomy. All hx rats received subcutaneous injections of cortisol phosphate and L-thyroxine (Sjöberg et al., 1994; Åberg et al., 2000a). Recombinant human IGF-I, however, was given as a continuous subcutaneous infusion using osmotic minipumps (Åberg et al., 2000a). GH may be administered as injections, daily or more, whereas IGF-I has to be administered evenly in pumps to avoid its sometimes lethal hypoglycemic effects. Hormonal treatment continued for 6 or 20 days. During the first 5 days of each treatment period, all animals received a daily intraperitoneal injection of bromodeoxyuridine at 50 mg/kg body weight. In some cases, a normal group of rats was kept as an additional control group. The normal group should not be used for a direct comparison of GH/IGF-I effects, but rather as a control of the physiological levels of cell genesis. Other possible models include IGF-I null mice and blocking of IGF-I by administering antibodies into the circulation of the animals. The problem with IGF-I null mice is obvious: no postnatal survival of animals. Blocking antibodies have several inherent problems concerning specificity and duration of effect, especially in the context of the blood–brain barrier. To study the significance of GH and IGF-I in the adult (and aging) animal, conditionally transgenic models would be needed. Although an adult liver-specific-inducible IGF-I knockout is available (Lopez-Lopez et al., 2004; Sjögren et al., 1999), more such models would be desirable. Generally, cell genesis is monitored by incorporation of the thymidine analog BrdU into DNA synthesis, which occurs almost exclusively at the S phase of cell division. In the brain, usually a 1- to 8-day-long daily treatment of BrdU coinciding with initiation of the particular treatment is used as a measure of proliferative capacity (Fig. 4), sacrificing the animals shortly after the last BrdU injection (Cameron and McKay, 2001). A few weeks after a specific BrdU injection, many of the divided cells have died or migrated and matured into different phenotypes. Analysis of cell survival (equaling to total number of remaining BrdU-positive cells) and phenotype (double labeling of BrdU and cell-specific markers) is therefore often performed 2–4 weeks after the last BrdU injection.

B. Immunohistochemistry Standard immunohistochemistry for BrdU immunoreactivity and specific cellular antigens favorably quantifies cell survival of newborn cells and their cell progeny

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FIGURE 4 Experimental paradigms used for evaluating effects on cell genesis by IGF-I in the adult CNS. Adult female rats hypophysectomized at about 50 days of age are treated for 6 days with IGF-I and with BrdU injections for the last 5 days before sacrifice. Analyzed with immunohistochemistry, this gives a good estimate of the proliferation rate. After a total of 20 days, the rest of the animals are sacrificed and analyzed (both regarding remaining BrdU-positive cells and their phenotype). Biological data obtained are a combination of originally proliferated cells, survival effect, and phenotypic differentiation of the cells (see discussion in the text). Oligodendrocyte is abbreviated oligodendr.

(Åberg et al., 2000a). However, in addition to standard immunofluorescence microscopy, confocal z-series laser scanning of the cell of interest using cell-specific markers should be performed to evaluate the phenotype of newborn cells (e.g., one focal plane each 0.5 ␮m through the entire cell). Confocal scanning with z-series detects the three-dimensional relationships between BrdU-positive cells and cell-specific markers for neurons, astrocytes, and oligodendrocytes. This technique has shown that neurogenesis in cerebral cortex of the macaque brain was an artifact (Kornack and Rakic, 2001), formerly being reported present with less detailed analysis (Gould et al., 1999a). The section thickness of 40 ␮m (microtome setting) is often used in the dissector estimation of volume. The number of BrdU-positive cells should then be counted by ignoring the cells in the uppermost focal plane and focusing through the thickness of the section to avoid errors caused by oversampling (for a discussion of the optical dissector principle, see Coggeshall and Lekan, 1996; Gundersen et al., 1988; West, 1993). When comparing similarly prepared sections of the brain, this technique yields accurate relative numbers of cells. However, when calculating absolute cell numbers from brain sections (e.g., for comparing to other studies), different techniques of fixation of tissue yield different shrinkages of sections and therefore may give a significant error in absolute cell number estimates (Skoglund et al., 1996). Differences in use of the optical dissector principle and in differential packing of cells due to different shrinkages of sections are two major explanations for different cell numbers of

the same brain region in different studies (when elsewhere comparable).

C. BrdU Incorporation into DNA and Detection of Cellular Proliferation The incorporation of BrdU into SVZ cells reflects the number of dividing progenitor cells at the time of administration (Kuhn et al., 1996; del Rio and Soriano, 1989). The BrdU bioavailability after injection lasts at most for 2 h, and BrdU labels DNA only during the S phase, which has been estimated to last approximately 8 h (Nowakowski et al., 1989; Takahashi et al., 1992). Animals killed the day after the last BrdU injection provide information about proliferation, whereas the population that is allowed to survive for 3 weeks provide data on the ultimate survival and phenotypes of those cells. Cells that have undergone multiple divisions (after several weeks) may pose the problem of dilution of BrdU. Such cell progeny may be negative for BrdU and may affect the interpretation of the results in terms of altered cell survival. To our knowledge there is no study that addresses the exact nature of BrdU dilution and multiple divisions. BrdU detection is anyhow usually considered to be reliable for at least several cell divisions. One could argue that DNA repair might contribute to BrdU labeling observed in vivo. Palmer and colleagues (2000) evaluated this and concluded that the BrdU methodology and concentration (50 mg/kg) often used were not sensitive enough to detect radiation-induced DNA repair in cultured fibroblasts. The repair processes that possibly occur in the CNS should therefore not contribute significantly to

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the measurements of mitosis in this paradigm. This is in line with a study by Cameron and McKay (2001) showing that BrdU is a specific and nontoxic marker of S phase cells in the adult dentate gyrus (Cameron et al., 1998). In that study, and in a later study (Cameron and McKay, 2001), it was shown that 50 mg/kg of BrdU does not label all cells in S phase, implying that the optimal dose for BrdU is higher. Another concern of BrdU administration is that cells entering the apoptotic process may take up BrdU, without dividing, before they disappear in apoptosis (Kuan et al., 2004). This would pose a substantial problem to short-term studies going on for time ranges of hours (where the preapoptotic cells may constitute a substantial fraction of all BrdU-positive cells) but not for long-term studies (days), as the preapoptotic BrdU-positive cells mostly would then have disappeared. Finally, the use of tritiated thymidine for labeling dividing cells should be mentioned. Tritiated thymidine also incorporates into DNA in the S phase of the cell cycle and can be used both in vivo and in vitro. This type of labeling is of course radioactive and uses photographic films for detection in vivo or a scintillation counter for in vitro assays. The technique is difficult [although possible (Cameron et al., 1993)] to combine with immunofluorescent labeling of cell-specific markers of cell progeny in vivo. However, as BrdU labeling does this more easily in vivo, the tritiated thymidine technique is less used today. However, in vitro the technique is widely used for quantifying the amount of cells dividing in cultures. However, also for in vitro use, BrdU detection is preferred when analyzing the phenotype of the cell progeny.

D. Differentiation Two to 4 weeks after the last BrdU injections, cell progeny may be analyzed, as in that time window the newborn cells will have migrated and differentiated or gone into apoptosis. Differentiation of cell progeny is made by colocalization of BrdU with cell-specific markers (Fig. 5) and as described earlier. Genetic labeling may also preferably do analysis of differentiation. For example, a retroviral vector expressing green fluorescent protein that only labels dividing cells has been used for tracing cell progeny and electrophysiological examination of newborn cells in live hippocampal slices (van Praag et al., 2002). However, when studying surviving progeny of proliferating cells in a particular (sub-)region of the brain, one also needs to consider the migration of cells away from the place of genesis. Genetic labeling of specific cells has the advantage in that it may track the route of migration as well as differentiation.

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FIGURE 5 Colocalization of BrdU (green) with neuronal markers MAP2 (red) and calbindin (blue) in a section of the dentate gyrus of the hippocampus. Cells are found in the subgranular zone of the dentate gyrus. Migration will occur outward to the right. The darker section to the left is the hilus and the more red section to the right is the molecular layer. (Insert) Magnification of BrdU-positive cells. Adapted and reprinted with permission from the Journal of Neuroscience 2000©. (See color plate 11)

E. In Vitro Models: Adult Hippocampal Progenitors Progenitor cells may be isolated from various parts of the CNS, including the striatum (Gritti et al., 1996; Palmer et al., 1995), the hippocampus (Gage et al., 1995a; Palmer et al., 1997), the subventricular zone (Zhang et al., 2003), the adult mouse thoracic spinal cord (Weiss et al., 1996), and the hypothalamus (Markakis et al., 2004). The two most common adult in vitro progenitor cell types are, however, derived from the two major neurogenic areas, i.e., the hippocampal subgranular zone (Gage et al., 1995a; Palmer et al., 1997) and the subventricular zone of the lateral ventricle (Zhang et al., 2003). Proliferative multipotent progenitors from adult SVZ or hippocampal tissues may be expanded in vitro with different growth factors (Gage et al., 1995b; Temple and Qian, 1996). These progenitor cells or stem cells derived from the hippocampus, striatum, or SVZ or are thought to divide asymmetrically to produce both replacement progenitors and cells destined to form neurons or glia (Craig et al., 1996; Palmer et al., 1997; Reynolds and Weiss, 1992). Although to some extent similar in appearance, the two pools of progenitor cells may not be regarded synonymous (Kuhn et al., 1997) and they are usually propagated differently (Craig et al., 1996; Palmer et al., 1995). In our studies, the isolation of AHPs was performed with a clonal population that was used between passages 5 and 15 postcloning showing stable progenitor cell properties (Palmer et al., 1997). For proliferating conditions, clonal progenitor cells were cultured in Dulbecco’s modified Eagles medium/Hams’s F12 (DMEM/F12) containing N2 supplement, which is a defined medium (instead of fetal calf serum) composed for maintaining brain cultures. This

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defined medium contains a comparatively high concentration of insulin (5 ␮g/ml), which at this high concentration would cross-bind to the IGF-I receptor. Therefore, in experiments with IGF-I, a special N2 supplement was constructed using a low concentration of insulin (100 ng/ml) (Åberg et al., 2003a). Twenty nanograms per milliliter of hFGF-2 is used to maintain the progenitor state of the cells. FGF-2 should be withdrawn in all differentiation experiments (as it strongly favors the undifferentiated state through maintaining cells proliferating). After 7–10 days in vitro, the cells were fixed and stained with antibodies against cell-specific markers. The effects of FGF-2 may additionally be explained by the fact that it seems to upregulate telomerase activity in neural progenitor cells, making them stable through passages (Haik et al., 2000). Their differentiation into either neurons or glial cells leads to downregulation of telomerase activity.

IV. ADULT CELL GENESIS BY INSULIN-LIKE FACTOR-I AND GROWTH HORMONE A. IGF-I Affects Cell Genesis in Adult Brain Studies from Little mice that are deficient in GH and IGF-I have shown that these substances were important for cell proliferation and for oligodendrocyte parameters in the developing brain (Morisawa et al., 1989). Similarly, treatment with GH antiserum caused a decrease in proliferation and myelination in growing rats (Pelton et al., 1977). Conversely, GH overexpression seems to increase brain weights and the astrocyte intermediate filament glial fibrillary protein (GFAp) (Miller et al., 1995). IGF-I overexpression shows more pronounced effects with increased (⫹55%) brain size in vivo and the myelin content increasing 130% at 55 days of age, while oligodendrocyte cell density was not increased (Carson et al., 1993). Studies of IGF-I overexpression in hippocampal areas show a promotion of neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development (O’Kusky et al., 2000). Conversely, in IGF-I knockout mice, hypomyelination, reduction of granule cells in the hippocampus, and reduction of brain total size are found (Beck et al., 1995). Essentially, these studies indicate the importance of GH and IGF-I in cell genesis during development, although the exact significance of this during adulthood remains to be established. However, in the last few years, studies of adult transgenic animals (Ransome et al., 2004; Turnley et al., 2002) and adult treatment experiments [data from our groups and Turnley et al. (2002)] have added data suggesting that some of the aforementioned effects persist into adulthood in a brain region-specific manner, acting on proliferation, cell survival, and differentiation.

1. The Hippocampus a. Proliferation Peripheral administration of IGF-I in young adult rats affects both cellular proliferation in the dentate subgranular proliferative zone and the subsequent migration and differentiation of progenitor cells within the GCL (Lopez-Lopez et al., 2004; Åberg et al., 2000a). IGF-I increases BrdUpositive cells by about 80% in GCL after 6 days of administration (Fig. 6A). After 20 days of IGF-I treatment, almost 50% of the numbers of BrdU-positive cells remained (Fig. 6B). This likely reflects both apoptosis and cellular migration away from the site of genesis (see also Section III). b. Differentiation: Increase of New Neurons and Oligodendrocytes Peripheral rhIGF-I treatment for 20 days in hypophysectomized rats increases both BrdU-positive cells (Fig. 6A) and the fraction of newly generated neurons in the GCL (Fig. 6B), as evaluated by the neuronal markers calbindin D28K, MAP2, and NeuN (Åberg et al., 2000a). IGF-I does not appear to affect the fraction of newly generated astrocytes (Åberg et al., 2000a). A positive correlation between weight gain (by IGF-I treatment) and the number of BrdUpositive cells in the GCL was observed, which demonstrates that a stronger peripheral biological activity of IGF-I also has a greater effect on hippocampal neurogenesis. Combining GCL volume data with BrdU–calbindin D28K staining, we found an overall increase of 78 ⫾ 17% of new neurons after 20 days in IGF-I-treated animals (Åberg et al., 2000a). Peripheral IGF-I induced by exercise shows similar effects on hippocampal neurogenesis (Trejo et al., 2001). In addition to neurogenesis, peripheral IGF-I treatment increases BrdU-positive cells positive for lectin (an endothelial marker), which indicates an angiogenetic effect of IGF-I in the hippocampus (Lopez-Lopez et al., 2004). In addition to peripheral IGF-I, it seems that local IGF-I also has significance for hippocampal neurogenesis because there are both increased local IGF-I and increased neurogenesis in GH-deficient long-lived Ames dwarf mice, despite low levels of circulating IGF-I (Sun et al., 2005). Finally, it has been shown that IGF-I has an instructive role in inducing oligodendrocyte cell fate from adult hippocampal progenitor cells in vitro and that IGF-I increases oligodendrocyte markers in the hilus region of hippocampus in vivo (Hsieh et al., 2004). It appears that IGF-I preserves its capacity to induce adult hippocampal proliferation of cells after ischemic injury (Dempsey et al., 2003). Whether this is of functional benefit has not been examined. The action of IGF-I on hippocampal neurogenesis may require permissive conditions acted by other agents such as the estradiol receptor system. This is because the estrogen receptor antagonist ICI 182,780 blocked intracerebroventricular administration of IGF-I-induced neurogenesis in the

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FIGURE 6 rhIGF-I affects cellular proliferation and differentiation in the adult hippocampus. (a) Density of BrdUpositive cells in the GCL and hilus of the dentate gyrus. rhIGF-I d was given for 6 or 20 days to hypophysectomized rats [all rats given L-thyroxine, cortisol, as described in Åberg et al. (2000a)]. (b) Rats that underwent the 20-day IGF-I were analyzed phenotypically by confocal z-series laser scanning of immunohistochemical cell-specific markers. The relative abundance of calbindin–BrdU-positive cells (neuronal phenotype) and glial fibrillary acidic protein (GFAp)–BrdU-positive cells (astrocytes phenotype) is shown *P ⬍ 0.05, **P ⬍ 0.01. Error bars are standard error of the mean (SEM). Adapted and reprinted with permission from the Journal of Neuroscience 2000©.

dentate gyrus, whereas coadministration of subcutaneous estradiol and intracerebroventricular IGF-I enhanced neurogenesis (Perez-Martin et al., 2003). Estrogen receptors of both ␣ and ␤ types are widely expressed in the hippocampus and elsewhere in the brain of both sexes (for review, see Shughrue and Merchenthaler, 2000). IGF-I and estradiol also have permissive and additive effects for other processes in the brain, e.g., synpatogenesis and neuroprotection (for review, see Shughrue and Merchenthaler, 2000).

distribution after 10 days of IGF-I treatment in vitro showed that the fraction of newly generated neurons increased significantly. These results suggest that the proliferative effects of IGF-I on adult hippocampal progenitor cells found in vivo are also found in vitro and that the proliferative effect is mediated via the MAPK signaling pathway.

c. In Vitro Adult hippocampal progenitor cells thought to correspond to progenitor cells in the adult subgranular zone express IGF-I receptor, IGFBP-2, and IGFBP-4 (Åberg et al., 2003b). IGF-I treatment increases the amount of IGFBPs, whereas FGF-2 pretreatment increases the amount of IGF-I receptor protein. IGF-I-treated cultures showed a dose-dependent increase in DNA synthesis, total number of cells, and number of cells entering the mitosis (M) phase, demonstrating a robust proliferative effect of IGF-I. Using inhibitors and dominant-negative constructs, it appears that the MAPK signaling pathway is required for IGF-I-stimulated proliferation in adult progenitor cells (Åberg et al., 2003b). The proliferative effect is well separated from the effects mediated by insulin, as shown in dose–response experiments with high or low concentrations of insulin. The proliferative effect of IGF-I is additive to the proliferative effect of FGF-2. Furthermore, analysis of the phenotypic

The SVZ is the other main site of dividing cells that persist throughout adulthood. Migration of cells occurs mainly through the rostral migratory stream (RMS) to the olfactory bulb. In the olfactory bulb, the cells mature into interneurons. Although not well understood, data support an additional migration of cells from the SVZ into the cerebral cortex (Bernier et al., 2002; Gould et al., 1999b; Levison et al., 1999). As yet, no study has examined the effects of IGF-I on adult proliferation in the SVZ. However, in the development of the olfactory bulb, IGF-I knockout animals show an abnormal formation of the olfactory bulb mitral cell layer and altered radial glia morphology (Vicario-Abejón et al., 2003). As SVZ is the source of olfactory neurons, also in development, it appears that IGF-I has the potential to affect SVZ proliferation. This is supported by the fact that IGF-I (Bartlett et al., 1992) and the IGF-I receptor (Aguado et al., 1993) are expressed in these cell layers .

B. The Subventricular Zone

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C. The Olfactory Bulb Neurogenesis occurs in the olfactory epithelium, which is actually part of the peripheral nervous system (Huard and Schwob, 1995), and within the olfactory bulb (part of the CNS). The adult olfactory bulb is a target for most cells migrating from the SVZ. It appears that type A cells within the SVZ are the source of migrating neuroblasts (Doetsch, 2003), which arrive to the olfactory bulb to mature locally (Carleton et al., 2003). Although their origin is from the SVZ, it also seems that some precursor cells continue to divide in situ within the olfactory bulb (Gritti et al., 2002). IGF-I clearly affects the development of the olfactory bulb in that knockout animals show abnormal formation of the olfactory bulb mitral cell layer and altered radial glia morphology (Vicario-Abejón et al., 2003). Furthermore, in adult glial olfactory cultures, IGF-I stimulates proliferation (Yan et al., 2001). Although it seems plausible that peripheral IGF-I would affect adult cell genesis in the olfactory system, there is, however, no data available on that issue.

D. GH and Adult Proliferation and Differentiation 1. Distinguishing between GH and IGF-I Effects Clearly, IGF-I has been studied more extensively than GH in terms of adult brain cell proliferation and cellular differentiation. The most important reason for this is that whenever GH is given systemically, IGF-I will also be produced both into the circulation from the liver (Mathews et al., 1986) and locally in the brain (Frago et al., 2002; Lopezfernandez et al., 1996; Mathews et al., 1986). Therefore, there are a number of different approaches to study the effects of GH on the brain (see also Section III). One alternative is to study short-term effects of GH where IGF-I would not have had time to be induced. However, that reduces the time window in studying acute effects. A second alternative would be to study GH (also inducing mostly liver IGF-I systemically) and IGF-I (slightly decreasing systemic GH due to feedback inhibition) treatments and compare their relative effects (Chen et al., 1997). If this is done in hypophysectomized rats, IGF-I treatment will not cause a feedback inhibition of systemic GH (as it is already depleted) (Åberg et al., 2000b), and in that way a more distinct IGF-I response can be compared to the combined effect of IGF-I and GH, which is the effect of GH treatment. The third way to study effects of GH would be use antibodies against IGF-I. A fourth approach would be to use transgenic constructs, disrupting liver-IGF-I, brain IGF-I, or both. Liver IGF-I knockout mice are available, but to our knowledge no one has used them for assessing GH effects. A fifth approach are transgenic constructs with disrupted GH signaling, e.g., GHR disruption (Ransome et al., 2004) or SOCS-2 disruption (Ransome et al., 2004; Turnley et al., 2002). It would be desirable if there were

conditional transgenic animals using disruption of GH, GHR, and SOCS-2 at adult time points. At present, there is, however, one liver–IGF-I conditional adult or young adult knockout animal model (Lopez-Lopez et al., 2004; Sjögren et al., 1999). 2. Proliferation Effects in the Developing CNS Data in the developing animal indicate that GH affects both neuron and astrocyte proliferation. SOCS-2 is held to be an inhibitor of GH signaling. Therefore, in SOCS-2-disrupted animals, the effect of GH is hyperresponsive (Ransome et al., 2004). In these mice, having grown to a young adult age, there are fewer neurons and more astrocytes in several layers of the cerebral cortex and striatum, although the hypothalamus was unaffected (Ransome et al., 2004). Conversely, the neuron:glia ratio increased in GHR –/– animals (Ransome et al., 2004). This was also shown in embryonic progenitor cultures taken from the SVZ, where GH decreased neuronal differentiation (Turnley et al., 2002). In addition, it seems that GH overexpression increases GFAp content in the brain (Miller et al., 1995), indicating an effect on astrocytes. Surprisingly, GH increases both neuronal and glial relative cell numbers in embryonic cultures from the cerebral cortex (Ajo et al., 2003). These phenomena were indicated already in 1968, when it was shown that GH had neural trophic effects, such as increasing the thickness of diencephalic structures as anterior and posterior commissures in the growing postnatal brain (Diamond, 1968). However, these effects may be transient for the embryonic period. 3. IGF-I Independent Action of GH in the Adult CNS? The studies mentioned earlier assessed the effect of GH during development and the effects could be to a secondary rise in IGF-I levels, either in the circulation or locally within the brain. However, it appears from a number of studies that GH has some effects independent of IGF-I in the postnatal brain. For example, ICV administration of GH has partly different patterns of neuroprotection to hypoxic ischemia as compared to IGF-I (Scheepens et al., 2001). Still, it would be possible for GH to act via local IGF-I synthesis, which could mediate the action of GH. Similarly, peripheral administration of GH in hypophysectomized rats induces increased expression of the gap junction protein connexin43, whereas peripheral administration of IGF-I does not (Åberg et al., 2000b). Interestingly, it was only IGF-I that increased connexin43 in glial cell cultures (Åberg et al., 2003b), which does not exlude direct effects of GH [as the cell cultures expressed only low levels of GH receptors (Åberg et al., 2003b)] but it may support the notion that the effect of GH in vivo may be mediated by IGF-I synthesis within the brain. Another example is that motor neuron size is larger in postnatal GH transgenic animals than in IGF-I transgenic animals [which instead exhibit a larger spinal cord (Chen et al., 1997)]. Altogether,

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there is support for IGF-I-independent action within the adult CNS, but it remains to be investigated whether GH has IGF-I-independent (in terms of local IGF-I synthesis) proliferative and differentiative effects in the adult CNS.

V. PHYSIOLOGICAL AND FUNCTIONAL CORRELATES TO ADULT CELL GENESIS A. Physiological Endogenous Regulation of Neurogenesis It is now well established that cell genesis and neurogenesis occur in the adult brain, but it is even more intriguing that it can be affected (see also Section I). Conditions such as exercise (Trejo et al., 2001; van Praag et al., 1999b) and living in an enriched environment (Kempermann et al., 1997; Nilsson et al., 1999) increase hippocampal neurogenesis, whereas models for depression decrease it (Westenbroek et al., 2004). As IGF-I and GH are increased by as little as 10 min of moderate exercise (for review, see Jenkins, 1999), there is the potential that they are involved in several of these conditions. Indeed, IGF-I has been shown to mediate effects of exercise upon hippocampal neurogenesis (Trejo et al., 2001). This section reviews some functional aspects of adult cell genesis.

B. Functionality of Newborn Cells? 1. Neurons Adult neurogenesis is a new research field and most researchers have concentrated their attention to characterization, regulation, and the effects of different growth factors but fewer studies have focused on the functional aspects. However, data indicate that the newly formed GCL neurons indeed are functional. It has been shown that the new adult-generated neurons develop dendritic and axonal processes and receive synaptic contacts on their cell bodies (Markakis and Gage, 1999; Stanfield and Trice, 1988), expressing hypothalamic neuropeptides (Markakis et al., 2004). Neurons colabeled for BrdU and early neuronal markers, PSA-NCAM (Seki, 2003) and TOAD-64, are all found at the inner edge of the granule cell layer and these neurons are characterized by one or two main dendrites with compact dendritic branches (Gould et al., 1999a; Scott et al., 1998). In young adult rats, many such neurons are present, as they are produced rapidly within the first few months, with an average new cell production of about 9000/day of which almost half mature into a neuronal phenotype (Cameron and McKay, 2001). In adult animals, neurons located in the deep aspects of the granule cell layer, near the proliferative zone, have different properties than those located in the superficial layers. In contrast to

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superficial and mature neurons, which never produce LTP when GABAA inhibition is intact, neurons located near the proliferative zone, tentatively designated as young, are characterized by their ability to produce LTP unaffected by GABAA inhibition (Wang et al., 2000). In the last years, direct evidence has gathered supporting that newborn adult neurons are functionally integrated. In the hippocampus, newborn neurons, targeted via GFP labeling of dividing subgranular zone progenitor cells, have been prospectively followed during migration and they show normal electrophysiological patterns in acutely isolated tissue slices (van Praag et al., 2002). Conversely, specific ablation by the injection of methylazoxymethanol (MAM) of dividing progenitor cells in the dentate gyrus alters hippocampus-specific memory imprinting (Shors et al., 2001). Still, the exact significance of hippocampal neurogenesis for memory formation is uncertain, as Morris water maze testing, considered specific for spatial memory formation located in the hippocampus, has not been affected by the aforementioned ablation of subgranular zone progenitor cells of the dentate gyrus (Shors et al., 2002). Similarly, in the olfactory bulb, live EGFP-labeled neurons have been traced from the subventricular zone (Carleton et al., 2003), with tangentially migrating neurons expressing extrasynaptic GABAA receptors, later AMPA receptors, before the appearance of NMDA receptors. Electrophysiological characterization showed spontaneous synaptic activity, which emerged soon after migration was complete, whereas spiking activity appeared later (Carleton et al., 2003). In summary, there is growing support for actual functional integration of adult newborn neurons. 2. Oligodendrocytes and Astrocytes Gliogenesis (i.e., genesis of astrocytes and oligodendrocytes) in the adult CNS per se may potentially harm or benefit the functions of CNS. The classical glial scar that occurs after injury may be an example of harmful proliferation and differentiation toward massive amounts of astrocytes that hinder the functional regeneration of axons (as discussed in Kinouchi et al., 2003). However, studies have shown that directed gliogenesis may be linked to a positive functional outcome. The formation of new oligodendrocytes and increased myelin staining has been shown to have a link to positive functional outcome in different situations. First, in mice with experimental autoimmune encephalomyelitis (a model for multiple sclerosis), erythropoeitin was shown to enhance NG2-positive (a marker for more immature oligodendrocytes) cells double positive for BrdU in the striatum and corpus callosum. The myelin content was also increased, and the observations coincided with a significantly improved clinical outcome after 30 days of treatment (Zhang et al., 2005a). It has also been shown how neurogenin-2-transfected neural stem cells transplanted to a site of thoracic spinal

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cord injury are associated with increased new oligodendrocytes, increased myelin, and fewer astrocytes compared to control (without neurogenin-2 transfection) transplantation (Hofstetter et al., 2005). Most importantly, neurogenin-2transfected neural stem cell transplantation improved motor and sensory function. Astroglial proliferation may be regarded from two different aspects. The first aspect is the normal physiological astrocyte function, where low-level astrocyte proliferation may be functionally beneficial. An example of this may be the increased proportion of astrocytes (and astrocyte processes) observed in the visual cortex of rats living in an enriched environment (Jones and Greenough, 1996), as well as in the hippocampus (Gomez-Pinilla et al., 1998). The second aspect of astrocyte proliferation is the massive reactive gliosis that often takes place after injuries to the CNS, which is unfortunately linked to a negative functional outcome. However, it appears that the negative consequences of reactive astrocytosis may be overcome. In mice in whose astrocytes are deficient of GFAp and vimentin (astrocyte intermediate filaments), neural transplants to the retina integrated successfully with normal axonal extensions (Kinouchi et al., 2003). Altogether, it appears that directed astrocyte proliferation may be associated with a positive functional outcome.

administration of IGF-I, has been shown to have neuroprotective actions after injury (Fernandez et al., 1999; Pulford et al., 1999). In addition, circulating IGF-I, increased after exercise, has been shown to mediate the neuroprotective effects against various types of damage to the CNS (Carro et al., 2001). To settle the effect of IGF-I on cell survival, one would have to quantify the rate of apoptosis at different time points. In the hippocampus, perhaps the proliferative effect of IGF-I is evident, as the relative proportion of cells surviving until day 20 is unchanged (Fig. 6A). This could be favorable in a situation where increased cell genesis (including neuro- and gliogenesis) is preferable, as after ischemic events.

D. Roles of IGF-I-Mediated Neurogenesis 1. Aging

C. Proliferation or Cell Survival?

The activity of the GH–IGF-I axis decreases with age. Both spontaneous GH secretion and IGF-I levels are frequently low in the elderly (especially above 60 years of age), overlapping with those usually recorded in younger GH-deficient patients (Savine and Sonksen, 2000). These age-related perturbations also occur with the IGF-I levels in the hippocampus (Lai et al., 2000; Sonntag et al., 1999). Translational deficiencies or deficits in the transport of IGF-I through the blood–brain barrier contribute to the decline in brain IGF-I with age. Studies have demonstrated that the proliferation of rodent granule cell progenitors, and ultimately the production of new granule cells, is dependent on the levels of circulating adrenal steroids. Glucocorticoids have been shown to inhibit adult rat neural progenitor cell proliferation, which has been proposed to be mediated through an NMDA receptor-mediated excitatory pathway (for review, see Gould and Tanapat, 1999). Aging is characterized by increased levels of glucocorticoids and decreased neurogenesis (Cameron and McKay, 1999; Lupien et al., 1994; Sapolsky and Altmann, 1991). As downregulation of IGF-I mRNA by glucocorticoids has been shown in several cell systems, including the brain, it is reasonable to postulate that the decrease in neurogenesis induced by glucocorticoids may be at least partly mediated via its inhibition of IGF-I synthesis and secretion (Adamo et al., 1988; Bitar, 2000; Lowe et al., 1992). The observation that IGF-I declines in the aging brain and that intracerebroventricular administration of IGF-I reverses aspects of functional and cognitive deficiencies with age further shows the importance of IGF-I in adult brain function (Markowska et al., 1998).

Parallel to the proliferative or regenerative action of IGF-I (Hsieh et al., 2004; Åberg et al., 2000a, 2003a) there is also a neuroprotective effect of IGF-I (Brywe et al., 2005; Carro et al., 2001). In fact, not only intracerebroventricular administration of IGF-I, but also peripheral

2. Stress and Depression Indirect evidence supports the notion that stress and depression inhibit neurogenesis in the adult hippocampus. There seems to be a complex relationship during depression,

3. Angiogenesis Generally, there is an overlap between neurogenesis and angiogenesis. For example, it has been shown that dividing cells in the subgranular zone of the dentate gyrus in the hippocampus are found in dense clusters and are associated with the vasculature (Palmer et al., 2000). Overlap between neurogenesis and angiogenesis also extends to several growth factors. For example, neural progenitors of several origins will proliferate in response to several “angiogenic” factors, including bFGF (Cameron et al., 1998), TGF␣ and ␤ (Cameron et al., 1998), PDGF (Wolswijk et al., 1991), VEGF (Jin et al., 2002), and testosterone in the adult songbird brain (Louissaint et al., 2002). Data have now established that IGF-I (Lopez-Lopez et al., 2004) and GH (Sonntag et al., 1997) have significance for different aspects of angiogenesis in the adult brain. Interestingly, cerebrocortical angiogenesis has been shown to be associated with improved clinical outcome after embolic stroke in aged rats (Zhang et al., 2005b).

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with increased glucocorticoids, decreased serotonin, and subsequent decreased hippocampal neurogenesis (Jacobs, 2002). The inhibitory effects of depression on granule cell production may be prevented by 5HT1A receptor agonists (Gould, 1999; Malberg et al., 2000). Depression also decreases GH secretion, measured during the night (Sakkas et al., 1998), and a serotonin antagonist could mimic the effects, giving a significant decline in serum growth hormone concentration (Kuhn et al., 1978). Moreover, serotonin has been shown to stimulate IGF-I release in different nonneural cell systems (Lambert and Lauder, 1999; Schaeffer and Sirotkin, 1997). A decreased serum level of GH and increased glucocorticoids as in depression both decrease IGF-I levels. Whether it is decreased serotonin levels per se or an effect of serotonin/glucocorticoids on the GH/IGF-I axis that is mediating depression or if ameliorated neurogenesis is a major factor in pathology remains to be proven. In the last years, however, evidence have been presented showing a distinct role of serotonin on hippocampal neurogenesis (Banasr et al., 2004; Brezun and Daszuta, 1999; Malberg and Duman, 2003). Although the definite causality of hippocampal neurogenesis is unclear, present data suggest an intimate coupling between hippocampal neurogenesis and depression, and perhaps also with the burnout syndrome (Eriksson and Wallin, 2004). 3. Exercise Exercise gives increased serum levels of GH and subsequent increased IGF-I levels in serum and also in the brain (Carro et al., 2000; Eliakim et al., 2000). Exercise has been shown to increase neurogenesis (van Praag et al., 1999a,b). In fact, circulating IGF-I has been shown to mediate such an effect (Trejo et al., 2001). Just running in a wheel enhances the number of BrdU-labeled cells in the subgranular zone of the dentate gyrus in the hippocampus. This effect is probably not due to environmental stimulation, as that mainly affects the survival of newborn cells and not proliferation (Kempermann et al., 1997; Nilsson et al., 1999). Exercise is associated with a sensation of wellbeing, and this subjective state has been quantified objectively with psychometric, cardiovascular, and neurophysiological data. Cortisol and ␤-endorphin levels increase transiently after exercise, although the cortisol level has been shown to be below baseline hours after the exercise, which may indicate a lower stress level (Heitkamp et al., 1996). In line with this, blocking opioid receptors during exercise partly decreases the positive effect of exercise on hippocampal neurogenesis (Persson et al., 2004). Exercise alone is also known to increase angiogenic activity in the adult rat cerebellum and hippocampus (Isaacs et al., 1992; Lopez-Lopez et al., 2004). It may be that exercise by running influences neurogenesis by a vascular intermediate (separable from within CNS-specific signaling). Then, the initial proliferative response observed in the SGZ may

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be caused by exercise-elevated peripheral factors, e.g., vascular endothelial growth factor-A (VEGF-A) or IGF-I (Breen et al., 1996; Carro et al., 2000; Gustafsson et al., 1999; Åberg et al., 2000a). As mentioned earlier, circulating IGF-I has been shown to mediate an exercise-induced increase in hippocampal neurogenesis (Trejo et al., 2001). Another factor that increases in the hippocampus after exercise is FGF-2, which is a well-known mitogen of adult neural progenitors (Gomez-Pinilla et al., 1998). There are important interactions between IGF-I and FGF-2, as FGF-2 has been shown to produce an increase in IGF-I receptors and IGF-binding proteins (Drago et al., 1991; Pons and Torres Aleman, 1992) and, in vitro, IGF-I potentiated the effect of FGF-2 on progenitor proliferation (Åberg et al., 2003a). 4. Learning The benefits of exercise are becoming increasingly apparent, including enhanced cardiovascular fitness, retention of muscle mass and strength, increased bone density (Position Stand, 1998), and increased adult neurogenesis, at least in rats. It is suggested that increased neurogenesis in the runners contributes to learning. Indeed, several factors that elevate production of new neurons are also associated with enhanced learning. Both running (through increased cell proliferation) and living in an enriched environment (through increased survival of newborn cells) enhance the number of newborn cells and improve the performance in a learning task (Kempermann et al., 1998; van Praag et al., 1999a). Running was also shown to selectively enhance dentate gyrus LTP. In line with this, estrogen increases progenitor cell proliferation (Tanapat et al., 1999) and improves memory (Luine et al., 1998). In contrast, factors that reduce neurogenesis, e.g., glucocorticoids, are associated with diminished performance in spatial learning tasks (Krugers et al., 1997; McEwen, 1999). 5. Why Increased Neurogenesis? Why does exercise, via IGF-I, induce an increase in neurogenesis? When would wild rodents have a larger need of neurogenesis? There is a seasonal variation in the access to food. During periods when it is difficult to find food, the rodents have to run long distances to find food and then find their way back to the nest. As a result, when they are increasing their territory size in order to seek food, they need enhanced spatial memory and the increased physical activity would increase IGF-I levels and thereby neurogenesis and spatial memory. This assumption is supported by a study of wild meadow voles, where female animals captured during the nonbreeding season when they were seeking food had higher rates of cell proliferation in the granule cell layer (GCL) than animals captured during the breeding season (Galea and McEwen, 1999). In contrast, males increased their home range during the breeding season and

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the rate of cell proliferation increased. Barnea and Nottebohm (1994) also found a seasonal change in the number of neurons produced in the hippocampus of canaries that was related to territory size. Thus, it is likely that increased exercise in rats during food seeking would increase hippocampal IGF-I levels and subsequently increase neurogenesis and learning in a dynamic manner.

E. IGF-I and Adult Human Brain 1. Structural Plasticity Related to Learning and Memory in Humans? It is indeed intriguing that IGF-I affects adult cell genesis in various parts of the brain. However, would this apply to human adult neurogenesis and learning? Potentially, there are progenitor cells for IGF-I to act on. It was shown that neurons were born in the adult human hippocampus (Eriksson et al., 1998) and somewhat later that these cells could be maintained in culture (Roy et al., 2000). One important role of the hippocampus is to facilitate spatial memory in the form of navigation, and one population with extensive navigation experience is taxi drivers in London. A structural magnetic resonance image study of the brains of taxi drivers compared to matched controls showed that the posterior hippocampus, which is believed to store a spatial representation of the environment, was significantly larger in the taxi drivers (Maguire et al., 2000). Hippocampal volume correlated with the amount of time spent as a taxi driver (positively in the posterior and negatively in the anterior hippocampus). It appears that adult cell genesis would account for at least part of these macrostructural trophic increases in hippocampal volume. In contrast, hippocampal atrophy is found in humans with (i) Cushing syndrome, which is characterized by a pathological oversecretion of glucocorticoids; (ii) episodes of repeated and severe major depression, which is often associated with hypersecretion of glucocorticoids; and (iii) posttraumatic stress disorder (for reviews, see Sapolsky, 2000, 2001). It might thus be that, as in the rodent, prolonged stress or prolonged exposure to glucocorticoids has adverse effects on the human hippocampus and probably depresses the memory capacity (Eriksson and Wallin, 2004). To our knowledge there are no data available on primate adult cellular proliferation after physiological conditions such as learning, exercise, or depression. In summary, there are indications but no definite data that memory and learning could be correlated to the rate and quantity of neurogenesis in adult humans. 2. IGF-I and Old-Aged Human Brains It appears that peripheral IGF-I, passing the blood–brain barrier, plays an important role in neurotrophic signaling in the young adult rat brain. However, if IGF-I is eligible for exogenous treatment of human brain injury, it would mostly have to be used in older ages. At least in the rodent brain,

it seems that IGF-I has positive behavioral/cognitive effects in rats 32 months old (Markowska et al., 1998). Similarly, bGH increases surface vascular density in 30-month-old rats (Sonntag et al., 1997). Also, it appears that GH (and thus probably also IGF-I) has positive effects in humans (in normal, uninjured healthy brains), as evaluated by the quality of life in humans suffering from hypopituitarism with GH–IGF-I deficiency aged above 65 years (Feldt-Rasmussen et al., 2004). Of course, this cannot be extrapolated to a situation where one would administer IGF-I after an injury to enhance cell genesis in the recovery phase. However, IGF-I or GH administration certainly appears to have a potential for such use, even in humans.

F. Final Remark Exogenous administration of IGF-I to the adult brain affects both proliferation and cellular differentiation in various regions of the brain. Most importantly, IGF-I enhances adult neurogenesis in the hippocampus and increases oligodendrocyte recruitment of newborn cells in the hippocampus. For a number of years, the neuroprotective role of IGF-I has been known and accepted. The findings summarized in this chapter extend the role of IGF-I as being a putative regenerative agent in the adult CNS. GH, being less studied in these aspects, may have similar effects, especially as it is the main stimulus of IGF-I in vivo. It will be interesting and exciting to see if these agents can be used in clinical trials as treatment for human brain insults in the years ahead.

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12 Growth Hormone and Insulin-like Growth Factor-I and Their Effects on Astroglial Gap Junctions N. DAVID ÅBERG Research Center for Endocrinology and Metabolism, Institute of Internal Medicine, Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden

I. Gap Junctions and Connnexins in the Central Nervous System (CNS) II. Astrocytes in the CNS III. Growth Hormone (GH) and Insulin-like Growth Factor-I (IGF-I) and Astrocytes and Connexin43 – Gap Junctions IV. Methodological Aspects V. Significance of Regulation of Connexin43 and Gap Junctional Coupling VI. Regulation of Connexin43 by GH and IGF-I in the Context of Other Effects VII. Gap Junctional Plasticity and Cortical Functions References

Systemic bGH but not rhIGF-I increased the expression of cx43 mRNA and protein in the cerebral cortex and hypothalamus of the brain. However, in vitro, only rhIGF-I increased GJC and cx43 abundance. The astroglial cells secreted IGBFPs in vitro, and a high abundance of these were associated with a low response to rhIGF-I. These results indicate that the effect of GH on cx43 expression in vivo may be mediated locally by IGF-I and interactions with insulin-like growth factor binding protein. Altogether, results show that cx43 and astroglial gap junctions are under dynamic regulation by GH and IGF-I. This may be of significance by enhancing the spatial buffering capacity of astrocytes needed to sustain extended neurotransmission, as well as after insults to the nervous system.

Connexins form gap junctions that are aqueous pores, allowing low-molecular (⬍1000 Da) compounds to pass from cell to cell. In astroglial cells, connexin43 (cx43) is the dominant connexin making up gap junctions. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) have been shown to affect levels of neurotransmitters in the brain, myelination, and cognition. As glial cells express receptors for GH and IGF-I, it was hypothesized that these hormones could affect astrocytic gap junctions and cx43. The effects of bovine GH (bGH) and recombinant human IGF-I (rhIGF-I) on cx43 expression and gap junctional coupling (GJC) were studied in vivo and in vitro. In vivo, hypophysectomized adult female rats were substituted with bGH (1 mg/kg) and rhIGF-I (0.85 mg/kg). In vitro, bGH and rhIGF-I were added to primary astroglial cell cultures.

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. GAP JUNCTIONS AND CONNEXINS IN THE CENTRAL NERVOUS SYSTEM (CNS) A. Introduction Higher brain function is thought to involve processing of information. For this to occur, the information needs to be transmitted—communicated—from one cell to another. The intercellular communication from cell to cell occurs by two principal ways (Fig. 1). These are chemical transmission via synapses occurring from neuron to neuron and transmission via gap junctions that are electrically coupled occurring in

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FIGURE 1 Definitions and semantics of different types of intercellular communication. Intercellular communication in the brain is often defined as chemical and electrical. Chemical transmission is what is referred to when a neurotransmitter leaves a presynaptic nerve terminal into the small extracellular space in the synaptic cleft to exert effects in the postsynaptic nerve terminal (vertical in figure). This is a very rapid process compared with electrical transmission, which occurs via gap junctions (horizontal in figure). Interestingly, the substance being transmitted via synapses and gap junctions may be the same substance (designated y), e.g., glutamate. Finally, extracellular release of substances (hormones, neurotransmitters etc., designated x) outside the synaptic cleft may be referred to as indirect intercellular communication, called autocrine, paracrine, and endocrine.

both astrocytes and neurons. In the adult brain, the electrical coupling via gap junctions is low in neurons, whereas astroglial cells are highly electrically coupled by gap junctions. Gap junctions are intercellular pores that allow lowmolecular mass compounds ⬍1000 Da to pass almost freely between cells and that form an electrically coupled syncytium. Gap junctions are built up of connexin proteins. The principal and ubiquitous gap junction protein of astroglial cells is connexin43 (cx43), whereas neurons in the adult CNS indeed are coupled by gap junctional coupling (GJC) but to a lower degree than astrocytes and by other connexins. Furthermore, it seems that neuronal gap junctions do not couple to astrocytes (Rash et al., 2001). In recent years, another connexin, namely connexin 30, has been shown to be relatively abundant in astrocytes in certain subregions of the brain, especially in gray matter (Nagy et al., 1999). This chapter focuses on regulation of the gap junction protein connexin43 and its functional correlate in gap junctional coupling by brain region heterogeneity and by growth hormone (GH) and insulin-like growth factor I (IGF-I).

B. Connexin Expression in the CNS Many cell types in the body, including astrocytes, are coupled to each other via membrane channels called gap

junctions. This was shown for the first time in 1964 by Kanno and Loewenstein. In 1986 Paul cloned the gene for the 27-kDa rat liver gap junctional protein. The following year, Beyer and co-workers (1987b) cloned the 3.0-kb transcript of the gene for the gap junction 43-kDa protein found in the heart, being cx43. From then on, several additional connexins have been demonstrated in the brain or neural cultures, e.g., cx26 in developing neurons (Nadarajah et al., 1997), cx30 in mature gray matter astrocytes (Nagy et al., 1999), cx32 in oligodendrocytes (Kunzelmann et al., 1997), cx33 in hippocampal progenitor cells (Rozental et al., 1998), cx36 in neurons (Condorelli et al., 1998), cx40 in hippocampal progenitor cells (Rozental et al., 1998), and cx45 in oligodendrocytes (Kunzelmann et al., 1997). In the brain, cx43 is more or less restricted to astrocytes, although ependymal and meningeal cells also express cx43 (Yamamoto et al., 1990a). However, endothelial cells do not express cx43 within neural tissue (Simard et al., 2003), although endothelial cells have been reported to express cx43 elsewhere. Instead, cx43 seems to be confined to the astrocyte end feet, which are in close apposition to endothelial cells at the gliovascular interface (Simard et al., 2003). Neurons do not express cx43 at detectable levels in normal neocortex in the adult brain (Rash et al., 1997, 2000; Theis et al., 2003b). Instead the principal connexin of neurons seem to be connexin36 (Rash et al., 2000). However, subgroups of neurons have been reported to express cx43 in other brain regions, e.g., in the olfactory bulb (Miragall et al., 1996), and early in development when neuronal circuit formation occurs (Bittman et al., 1997; Miragall et al., 1997; Rozental et al., 2000). Altogether, in the adult rat brain, cx43 expression may be regarded as being nonneuronal and expressed predominantly by astrocytes. The astrocyte GJC is widespread and forms large networks of coupled cells. For example, entire subdivisions of the hippocampus are coupled, as studied from slices (Konietzko and Muller, 1994). Additionally, fetal cx43-knockout astrocyte cultures from mice lose about 95% of their intercellular coupling (Naus et al., 1997), supporting the fact that cx43 is the predominant connexin in astrocytes. Adult conditional astrocyte-specific cx43-knockout experiments have revealed similar reduction in gap junctional communication in vitro, whereas in vivo the reduction was about 50% in slices of the hippocampus (Theis et al., 2003a). Probably the smaller the reduction in vivo was due to an upregulation of cx30. Interestingly, it is evident that cx43 and astrocytes gap junctions have significance for higher brain function, as adult conditional astrocyte-specific cx43 knockout mice show altered learning patterns and impaired motor capacities (Frisch et al., 2003). Astroglial cells may therefore transmit messages via the astroglial syncytium over large distances independently and separated from neuronal cells and it seems that these functions have significance for cognitive processes.

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C. Connexin43 and Gap Junction Coupling

N-terminal and C-terminals inside the cell [Fig. 2, for review, see Beyer (1993)]. After synthesis, the protein is transported to the endoplasmic reticulum, where it is thought to be phosphorylated to become a 41-kDa species. After this event, the cx43 peptide is believed to be assembled into hexameres, called connexons, in the Golgi compartment (Jordan et al., 1999). Additional phosphorylation to 42–44 kDa is believed to take place distal to the Golgi compartment (Puranam et al., 1993). These connexons may be stored for sometime and may thus be a pool that can be recruited rapidly (minutes) to form functional gap junctions in the cell membrane. The channels of connexons are found in clusters of hundreds to thousands, and these clustered assemblies were originally called gap junctions when observed in electron microscopes.

Gap junction coupling or permeability is subject to complex regulation at several levels, including transcription, translation, posttranslational phosphorylation, and dephosphorylation, which is important for gap junction assembly, permeability, and degradation. 1. Synthesis The newly synthesized 382 amino acid cx43 protein has a calculated molecular weight of 43,036 (Beyer et al., 1987a), but is believed to be represented by a 40- to 41-kDa band in SDS–PAGE Western blots (Puranam et al., 1993). Connexins have four membrane-spanning regions and form two extracellular loops and one intracellular loop with both

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FIGURE 2 Cx43 from transcription to gap junction. (a) The cx43 gene spans over two exons interrupted by one 8.5-kbp intron. The primary transcript is spliced to a transcript of approximately 2.8 kbp. Part of this transcript is translated to the final cx43 protein. The untranslated regions are probably important for regulating mRNA stability. The sizes are not scaled exactly. (b) The cx43 protein is 382 amino acids long and has four membrane-spanning segments. The two extracellular loops have six cysteine residues thought to be important for attaching the connexins of an opposing cell. Amino acids 252–270 are often used to make antibodies. (c) Six cx43 (red) molecules oligomerize into a hexamere called a connexon. Such a connexon is transported to the cell membrane, where it joins to another connexon of another cell. When that happens, an aqueous pore is formed and a gap junction channel is established (as indicated). (See color plate 12)

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Today, such a cluster is designated as a gap junction plaque as opposed to a single gap junction channel. However, sometimes these terms are still used intermingingly. 2. Gap Junction Coupling When gap junction channels (usually in clusters of channels) are established, the cells become coupled intercellularly, and the coupling is then subject to several types of regulation. The intercellular coupling degree may be divided semantically according to how it affects the channel status, i.e., the readiness of a channel to be permeated (permeability) and the number of channels (Fig. 3). These two factors determine the degree of GJC between a pair of cells. In practice, these conceptions—gap junctional permeability and number of gap junction channels—are often substituted for one another, as a change in GJC often involves changes in both factors. This section attempts to describe different timescales of GJC regulation involving both true permeability changes and changes in gap junction channel number. The number of gap junction channels is determined by the efficacy of transcription and translation, whereas permeability is determined by factors at the cell membrane. First there is the ultrashort (⬍10 ms) regulation of permeability called channel gating, which is dependent on H⫹ ions (pH dependent or chemical gating) and transjunctional voltage (“voltage gating”) (Bukauskas and Peracchia, 1997). A low intra- and extracellular pH closes the channels by an unknown mechanism. Carbon dioxide (CO2) (Bukauskas and Peracchia, 1997) and lactic acid have also been reported to decrease gap junction permeability, perhaps due to lowering of pH (Anders, 1988). A high transjunctional voltage REGULATION Short-term

Long-term

Permeability of single channels

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FIGURE 3 The regulation of gap junction coupling (GJC) strength can principally be referred to as short-term and long-term acting. Short-term regulation occurs when a substance (or a condition) acts at the permeability of single channels (left). This process occurs at the cell membrane, directly affecting the channel to open or to close, which is designated as “gating” of the channel. Long-term regulation (right) involves changing the numbers of channels. This is affected by the efficacy of transcription, translation, posttranslational events, and the rate of removal of channels from the cell membrane. Sometimes a single substance may act both short term (acting directly at the permeability of single channels) and long term via altering the transcription and translation of connexins.

also affects the permeability in that it keeps the gap junction channels open, despite high CO2 (Bukauskas and Peracchia, 1997). High extracellular K⫹ increases astroglial gap junctional permeability, possibly via membrane depolarization (Enkvist and McCarthy, 1994). Furthermore, a high sustained intracellular (Deleze and Loewenstein, 1976) or extracellular (Pfahnl and Dahl, 1999) Ca2⫹ closes the gap junction channels. The second type of regulation of gap junction channel permeability is thought to involve different types of phosphorylations and dephosphorylations and generally occurs on a timescale of seconds to minutes (Godwin et al., 1993). These phosphorylations should be distinguished from the ones needed for the assembly of connexins into hexameres. It seems that phosphorylation of a third or possibly fourth type is required for optimal permeability. This is reflected in higher molecular weights in migration on Western blots designated as phosphorylation 1 (P1) and phosphorylation 2 (P2) sites as opposed to nonphosphorylated (NP) (Guan et al., 1995). Disruption of P2 bands seems to coincide with disruption of gap junction permeability (Guan et al., 1995). Often the P1, P2, and further P bands are almost indistinguishable and may blur into each other, and therefore it may be difficult to interpret the exact state of phosphorylation on Western blots. Furthermore, live dephosphorylation with phosphatases inhibits GJC by cx43 in granulosa cells (Godwin et al., 1993). The regulation by phosphorylation and dephosphorylation is complex and sometimes contradictory (Cruciani and Mikalsen, 2002; Hervé and Sarrouilhe, 2002). The third possibility, occurring on a timescale of hours and which should be referred to as “long term,” involves an effect on connexin abundance, i.e., a certain compound exerts effects on the abundance of cx43 by influencing the translation (and/or transcription), thereby affecting the number of gap junction channels. For example, in myometrial cells of the uterus, the cx43 gene (Fig. 2A) responds to estrogen treatment after 2–3 h and peaks after 4–6 h, whereas Fos and Jun increase and peak by 2 h (Piersanti and Lye, 1995). Sometimes the process of increasing cx43 abundance is linked simultaneously to posttranslational events (i.e., phosphorylation). An example of this is transforming growth factor ␤1 (TGF-␤1), which increases phosphorylation, cx43 protein abundance, and GJC in bovine endothelial cells and in rat astroglial primary cultures (Larson et al., 1997; Robe et al., 2000). Other known compounds foremost affecting cx43 protein abundance (but sometimes phosphorylation as well) in different culture systems and species include estrogen [increase (Yu et al., 1994)], progesterone [decrease (Orsino et al., 1996)], basic fibroblast growth factor (bFGF) [increase or decrease depending on maturation (Nadarajah et al., 1998; Reuss et al., 1998)], platelet-derived growth factor [PDGF, decrease (Hossain et al., 1999)], thyroxine [increase (Stock et al., 1998)], hepatocyte growth

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factor (HGF) [decrease (Moorby et al., 1995)], and TGF-␤1 [increase in astroglial cells, decrease in glioblastoma cells (Robe et al., 2000)]. The gap junction channel blockers form a functional fourth group of regulation as they involve a quick (less than minutes) closure of the gap junction channels. Biochemically, these can be divided into cell membrane interacting and connexin interacting, which can partly be assigned to the former examples mentioned as well. Cell membrane-interacting molecules include alcohols such as octanol (Li et al., 1996) or lipids such as oleamide (Guan et al., 1997), arachidonic acid and polyunsaturated fatty acids (Hii et al., 1995), and halothane (Finkbeiner, 1992). Connexin-interacting inhibitors of gap junction coupling include 18␣-glycyrrhetinic acid 3␤-o-hemisuccinate (18-␣-GA) and its synthetic derivative carbenoxolene (Bani-Yaghoub et al., 1999). It can be argued that membrane-interacting molecules are not completely specific, whereas 18-␣-GA and carbenoxolene are considered to be more specific. In addition to the phosphorylation events that seem to be required for the assembly of gap junctions, evidence shows that other types of phosphorylations take place in order to close and inactivate the gap junction channels. These events most probably precede the transport of inactivated connexons away from the cell membrane to lysosomal vesicles, where they are finally degraded (Jordan et al., 1999; Laing et al., 1997). Connexins in general are subject to a rapid turnover, which is also the case for cx43. The half-life of cx43 has been reported to be in the range of hours (Hertzberg et al., 2000).

D. Gap Junctional Function 1. Relation to Astrocyte Function In its ability to redistribute low-molecular weight compounds, the astroglial syncytium is believed to contribute to several important functional features in the adult brain. Extracellular levels of potassium are elevated after axonal propagation of action potentials, as well as after the synaptic release of neurotransmitters. Astrocytes take up excess potassium and redistribute the ion via the so-called spatial redistribution of potassium, considered to be mediated by gap junctions (Gardner Medwin, 1983a; Odette and Newman, 1988; Reichenbach, 1991). Intracellular increases of calcium in astrocytes affect K⫹ channels so that the uptake of K⫹ into astroglial cells may be increased (Cooper, 1995; Quandt and MacVicar, 1986). In vitro, the calcium waves, traveling at approximately 20 ␮m/s (Finkbeiner, 1992), are able to affect neuronal cytosolic calcium and excitability (Hassinger et al., 1995). Also, calcium waves are believed to be closely associated with the spreading depression phenomenon, which is a slowly propagating wave of neuronal depolarization after focal ischemia or focal epileptic activity in the brain.

Interestingly, functional blockade of the gap junctions with inhibitors (see earlier discussion) abolishes the spreading depression (Nedergaard et al., 1995) and reduces the size of the lesion after cerebral ischemia (Rawanduzy et al., 1997). 2. The Astroglial Calcium Wave The astroglial calcium wave has been considered to be mediated through gap junctions built by connexins as described earlier (Fig. 2). However, in addition to cx43 gap junctions, there are clearly other factors affecting the propagation of calcium waves. Altogether, the most common view is that the astroglial calcium wave requires mediators, which pass through open gap junction channels and subsequently activate the release of intracellular calcium stores in the endoplasmic reticulum. When the astroglial calcium wave was first described, it was thought that calcium itself (Cornell Bell et al., 1990; Saez et al., 1989) propagated through the gap junctions and was able to release calcium via ryanodine receptors in the endoplasmic reticulum (Peracchia, 1990) or via extracellular inflow of calcium (Cornell Bell et al., 1990). It was later shown that phospholipase C (PLC) and its second messenger inositol 1,3,4-trisphosphate (IP3) was a more significant mediator of the intracellular release of calcium in the endoplasmic reticulum (Venance et al., 1997). It has also been proposed that the release of extracellular ATP, after reentering neighboring cells, mediated the release of calcium via purinergic receptors (Cotrina et al., 1998). Furthermore, nitric oxide (NO), via the cyclic guanosine monophosphote pathway, has been shown to be able to mediate calcium waves in glial cells (Willmott et al., 2000), as well as the release of extracellular glutamate (Innocenti et al., 2000). Together, these data show that the glial calcium wave is subject to complex regulation by several systems—perhaps active to different degrees during various steps of maturation and differentiation. However, there is a considerable amount of data supporting the notion that one mechanism mediating the calcium wave is direct (and not only indirect via extracellular release of ATP or glutamate) mediation by intact gap junctions (Venance et al., 1997, 1998).

II. ASTROCYTES IN THE CNS A. Astrocytes As cx43 is mainly expressed in astrocytes, the function of cx43 and gap junctional communication is linked to other properties of astrocytes. 1. Types of Astroglial Cells and Astrocytes The major cell types of the brain are neurons and glial cells. Of these two, astrocytes are the most abundant, although astrocytes are slightly outnumbered by neurons in

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Central Nervous System (CNS)

Neuroectodermal cells

Other cells

Mesenchymal cells

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Astroglial cells

Ependymocytes

Microglial cells

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Oligodendrocytes

Astrocytes

In vivo

Protoplasmic

In vitro

Fibrous

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FIGURE 4 Nomenclature of neural cells. Neural tissues consist of a number of cells that are derived from different dermal layers of the embryo and different stages and lineages of differentiation. However, this is not always reflected in the naming of the cells. The relationships between subtypes of glial cells and neuronal cells and their source of origin are shown.

the rodent cerebral cortex (Bass et al., 1971; Young, 1991). However, in larger brains, as in human and dolphin cerebral cortices, astrocytes outnumber neurons (Young, 1991). Glial cells also constitute the bulk of the volume in the brain, and in the cerebral cortex of mammals they have been estimated to constitute about 20% of the total volume (Pope, 1978). Astrocytes are star-shaped cells that belong to the glia cell family (Fig. 4). The astrocytes were formerly considered to be the inactive connective tissue of the brain. However, in the last decades, astroglial cells have come into focus as having active functional relevance to the CNS. The processes of the astrocytes contact virtually all other cell types in the brain. The processes extend and cover neuronal cell bodies and axons, other astrocytes, synapses, the brain surface (pia mater), and ependymal cells (Fig. 5). The so-called end feet of astrocytes also cover endothelial cells and they form the blood–brain barrier together with the tight junctions of endothelial cells. There are morphologically distinct types of glial cells whose terminology may sometimes be confusing (Fig. 4). The astroglia, derived from astro ⫽ star (Latin) and glia ⫽ glue (Greek), are divided into several subtypes of astrocytes.

The oligodendrocytes that ensheath axons of neurons and ependymocytes covering the ventricular surface may be regarded as glial cells, although they are not considered astroglial. The astrocytes are further divided into the protoplasmic astrocytes, which are found predominantly in the

FIGURE 5 Schematic structural relationships between astrocytes and other cell types of the CNS. Astrocyte processes extend their end feet to cover other astrocytes, neuronal cell bodies, axons, endothelial cells, synapses, and pia mater (the latter not shown here). Red boxes symbolize gap junctions, through which low-molecular substances (⬍1000 Da) may pass for relatively long distances between astrocytes. Note the high abundance of gap junctions in proximity to neurons and endothelial cells. (See color plate 13)

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gray matter, and the fibrous astrocytes, mainly found in white matter. Furthermore, there are specialized glial cells, e.g., Bergmann glia in the cerebellum, Müller cells of the retina, tanycytes of the median eminence (hypothalamus), and pituicytes of the pituitary gland. In vitro, two main morphological classes of astrocytes are distinguishable: type 1 and type 2. These were originally found in cultures of the optic nerve and it is uncertain whether they are correlates to the protoplasmic and fibrous astrocytes in vivo. However, the protoplasmic astrocyte (in vivo) and the type 1 astrocyte (in vitro) have much larger cell bodies and have coarser but shorter processes. The fibrous astrocyte (in vivo) and the type 2 astrocyte (in vitro) have substantially smaller cell bodies but longer and thinner processes that can be numerous. In addition to the morphological classification, astrocytes in the adult brain have been shown to be highly functionally heterogeneous in terms of, for example, synthesis of neuroactive compounds and receptor expression. 2. Astrocyte Function The function of astrocytes can be looked at from several different angles. All such terminology may preferably be viewed from the standpoint of a functional unit of neuron–astrocyte interaction (Hyden, 1962). In this paradigm, astrocytes are affected by neurons and astrocytes affect neurons, and the concerted output determines the action. In this respect astrocytes actively participate and modulate many processes of brain physiology. a. Functions in Common to Astrocytes and Neurons Many of the attributes that were assigned to neurons have now been shown to be properties of astrocytes as well. For example, astrocytes can directly release several neurotransmitters, including glutamate (Araque et al., 2000), aspartate, and taurine (O’Connor and Kimelberg, 1993). In addition, astrocytes have receptors for most neurotransmitters, including 5-hydroxytryptamine (5-HT), glutamate, GABA, opioids, noradrenaline, purines, histamine, acetylcholine (muscarinic), and somatostatin (for reviews, see Hansson and Rönnbäck, 1995; Porter and McCarthy, 1997). Especially uptake and clearance of glutamate from synapses, for which hippocampal astrocytes have been shown to be principally responsible (Bergles and Jahr, 1998), are important in synaptic neurotransmission. Glutamate that is not taken up at the synapse affects neighboring synapses (Mitchell and Silver, 2000). An increase in glial glutamate uptake is also accompanied by enhanced glucose utilization and lactate release (VoutsinosPorche et al., 2003), which can be utlilized by neurons demanding more energy. b. Functions: Astrocyte Specific There are certain functions that are astrocyte specific in the brain. For example, in cell energy metabolism, astrocyte-

specific functions are demonstrated in gluconeogenesis, storing glycogen, breaking down glycogen to lactate, and utilizing glycogen phosphorylase (for review, see Hamprecht and Dringen, 1995). Furthermore, astrocytes release glutamine, which may be used as a precursor amino acid for glutamate (Martinez Hernandez et al., 1977), GABA (Peng et al., 1993), and arginine [which is a precursor for nitric oxide in neurons (Grima et al., 1997)]. Astrocytes also protect against oxidative damage, as they contain glutathione, which scavenges free radicals (for review, see Dringen, 2000). c. Astrocytes and Structural Events at the Synapse It seems that astrocyte function is needed for neurotransmissional plasticity. Blocking of astrocyte function, which inhibits neurotransmission in hippocampal slices (Keyser and Pellmar, 1994), shows that astrocyte function is a prerequisite for neuronal function. To a large degree that probably depends on their close apposition to neuronal synapses and neurons. In that aspect it is interesting that astrocytes, under certain conditions, are highly adaptable in their morphological appearance. For example, in the hypothalamus, astrocytes may withdraw or extend their processes in response to different physiological stimuli (for review, see Theodosis and MacVicar, 1996). In addition, living in an enriched environment leads to both increased astrocyte cell number (Diamond et al., 1966) and increased astroglial wrapping of synapses in the visual cortex of rats (Jones and Greenough, 1996). Altered astrocyte wrapping of synapses especially brings the focus to gap junctions, as the processes of astrocytes are rich in cx43 (Nagy and Rash, 2000). Thus astrocyte function and morphology are both important in many highly regulated plastic processes in the brain. In light of this, the role of intercellular signaling in astroglial cells becomes interesting.

III. GROWTH HORMONE (GH) AND INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AND ASTROCYTES AND CONNEXIN43 – GAP JUNCTIONS A. General 1. Structul, Cognitive, and Neuroprotective Effects Growth hormone releases hepatic IGF-I, and both these hormones have numerous effects on the growth and metabolism of the body. However, it has become evident that both hormones are also important for structural (biochemical), cognitive, and neuroprotective parameters of the brain. In rats with growth hormone deficiency, there is hypomyelination and reduced brain size in adulthood (Morisawa et al., 1989; Noguchi et al., 1982; Pelton et al., 1977). IGF-I has been shown to have partly similar and even

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stronger effects, with hypomyelinated brains and reduced weights in IGF-I knockout mice (Beck et al., 1995). Furthermore, GH treatment improves the quality of life in GH-deficient human adults (for review, see Hull and Harvey, 2003) and improves memory and learning in rodents (Drago et al., 1996; Schneider Rivas et al., 1995), and short-term memory and iconic memory have been shown to be improved in GH-deficient humans after GH replacement (Deijen et al., 1998). It seems that IGF-I has similar mental effects (Markowska et al., 1998), although IGF-I has been less studied concerning this aspect. Moreover, early neuroprotective treatment with both GH (Gustafson et al., 1999; Scheepens et al., 2001) and more robustly IGF-I (Gluckman et al., 1998; Tagami et al., 1997) has been implicated with reducing the damaged area after ischemia. 2. GH or IGF-I? When discussing the effects of GH and IGF-I, one has to keep in mind that GH induces IGF-I synthesis not only in the liver (inducing an increase of circulating bloodborne IGF-I), but also in the brain (Frago et al., 2002; Hynes et al., 1987; Johansson et al., 1995; Lopezfernandez et al., 1996; Wood et al., 1991; Ye et al., 1997). The time course of the induction within the brain seems to be relatively fast, as IGF-I mRNA was already maximal by 4 h after an intraperitoneal injection of GH in hypophysectomized rats (Hynes et al., 1987). There are clearly both systemic and local effects exerted by GH and IGF-I, and a study of the relative effects of GH and IGF-I in vitro in comparison with the in vivo situation may serve as a model to distinguish between these effects. For example, overexpression of GH in transgenic mice affects astrocytes in that glial fibrillary acidic protein (GFAp) increases, but it cannot be excluded that this is actually an effect of IGF-I (Miller et al., 1995).

B. GH and Cellular Signaling In the brain, both glial and neuronal cells express the GH receptor (GHR) (Lobie et al., 1993; Nyberg, 2000). The rat GHR is a 620 amino acid transmembrane receptor belonging to the cytokine receptor family (for review, see (Edens and Talamantes, 1998). GH is thought to bind one receptor molecule, which dimerizes and exerts effects primarily via the JAK-STAT pathway (Carter-Su and Smit, 1998). However, GH signaling occurs through a number of other pathways as well (Carter-Su and Smit, 1998). For instance, PLC, insulin receptor substrate 1 (IRS-1), and phosphatidyl inositol 3 kinase (PI3K) have been reported to be activated after GH receptor stimulation (Carter-Su and Smit, 1998). The GHR has not been previously reported to be present in astroglial cells in vitro, but we have shown

GHR immunoreactivity in astrocyte primary cultures (Åberg et al., 2003b) and GH has been shown to exert effects on astroglial cultures (Almazan et al., 1985). In vivo, GHR have been demonstrated in neurons in wide parts of the brain (Lobie et al., 1993) and in glial cells along the ventricular lining, pia mater, and choroid plexus (for review, see Harvey, 1995). Furthermore, GH is expressed endogenously, although at low levels compared to the pituitary in wide parts of the brain (Hojvat et al., 1982; Martinoli et al., 1991). There may be variants of the GH peptide in the brain, although only one transcript has been found (for review, see Harvey, 1995). It was initially believed that GH exerted its effect primarily only via liverderived somatomedin, now referred to as IGF-I. However, accumulating data indicate that GH acts on an organ both by intrinsic action and by stimulating local synthesis of IGF-I, which has similar or, in some cases, differential effects (for review, see Ohlsson et al., 1998). In this aspect, there is a link between GH and local brain IGF-I, as it is known that administration of systemic GH may increase IGF-I synthesis in the brain (Frago et al., 2002; Hynes et al., 1987; Johansson et al., 1995; Lopezfernandez et al., 1996; Wood et al., 1991; Ye et al., 1997).

C. IGF-I and Cellular Signaling IGF-I is also expressed within the adult brain under normal circumstances mostly in neurons (D’Ercole et al., 1996), but also in astrocytes (Duenas et al., 1994). IGF-I is especially upregulated in astrocytes in response to injury (Garcia-Estrada et al., 1992). In vitro, astrocytes secrete substantial amounts of IGF-I (Chernausek, 1993). The IGF-I receptor is expressed in both neurons and processes of astrocytes and by oligodendrocytes, as studied in the hypohthalamus and cerebellum (Garcia-Segura et al., 1997). IGFBPs are expressed in astroglial cell cultures and in C6 glioma cell lines (Bradshaw et al., 1993a,b) and are differentially upregulated in vivo in both astrocytes and neurons after hypoxic-ishemic injury (Beilharz et al., 1998). The IGF-I receptor (IGF-IR) is built up of two ␤ and two ␣ subunits and does not belong to the cytokine receptor family (as GHR). Instead, it shares homology with the insulin receptor, and insulin and IGF-I may cross bind to the other receptor with low affinity. The IGF-IR is phosphorylated upon ligand binding and signals via several pathways, the two most important ones being PI3K (via IRS-1 and -2) and mitogen-activated protein kinase (MAPK) via Shc proteins (for review, see Baserga and Morrione, 1999). These intracellular signaling cascades show complex interactions that direct the outcome of IGF-I binding to its receptor. The effects of IGF-I and IGF-IR may be divided into (i) proliferative, (ii) differentiative, (iii) transformative, and (iv) antiapoptotic effects, as suggested by Valentinis and co-workers (1999).

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D. Regulation of Connexin43/Gap Junctional Coupling by GH and IGF-I GH and IGF-I are known to affect brain biochemistry as well as cognitive functions. As evidence shows that astrocytic intercellular communication by gap junctions is part of these processes, we hypothesized that these hormones could affect long-term regulation of gap junctions by cx43 abundance in astrocytes. 1. In Vivo cx43 mRNA and protein abundance in four brain regions of hypophysectomized female rats substituted with cortisol (400 ␮g/kg) and thyroxine (10 ␮g/kg). Bovine GH (1 mg/kg) given for 6 and 19 days increased the relative cx43 mRNA abundance in homogenates of the cerebral cortex and hypothalamus (Fig. 6) (Åberg et al., 2000b). In both the cerebral cortex and the hypothalamus the increase was somewhat more pronounced after the 19-day treatment, indicating foremost a long-term effect of bGH treatment. In addition, cx43 protein abundance was increased after 19 days of bGH treatment (Fig. 6). In the brain stem and hippocampus, no increases in cx43 mRNA or protein were found. Because

(a)

(b) Cortex, 6d 250 cx43 mRNA (%)

cx43 mRNA (%)

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Different signal cascades affect different functions. For example, IGF-I has been shown to exert proliferative effects by the MAPK pathway, while the IRS-1/PI3K pathway may mediate effects on cell adhesion and cell–cell interaction (Baserga and Morrione, 1999). However, different cell types utilize different signaling pathways for the same function, e.g., proliferation. In addition, under some circumstances there may be a cross-activation of receptor signaling by GH and IGF-I. It has been reported that a direct physical interaction exists between the GH and IGF-I signaling cascades in preadipocytes, as GHR forms a complex between JAK2 and the IGF-I receptor and that IGF-I stabilizes this complex (Huang et al., 2004). Insulin-like growth factor binding proteins (IGBFPs) interact with IGF-I and affect the bioactivity of IGF-I. There are six known IGF-I high-affinity-binding IGBFPs, designated IGFBP1–6 (Clemmons, 1997). These bind IGF-I in the interstitium and in the cell membrane and may thereby prolong IGF-I half-life and stability as well as preventing IGF-I from reaching its receptors. The complexity of the IGF-I system is further substantiated by the fact that IGBFPs do not always inhibit the effects of IGF-I but under certain circumstances enhance them. For instance, if IGFBP-3 is preincubated in cell medium, the effect of IGF-I is enhanced (for review, see Clemmons, 1997). Both GH and IGF-I have been reported to have bellshaped effect curves for certain of their effects. In the case of GH, this phenomenon is thought to depend on the fact that excess GH prevents GH receptor dimerization, which is required for intracellular signal transduction, whereas for IGF-I the mechanism is not clearly resolved.

**

200 150 100 50 0

–

+

GH

GH

– – – + + +

– – – + + +

FIGURE 6 Effect of bGH treatment on cx43 mRNA and protein abundance in the cerebral cortex and hypothalamus of T4/C-treated hypophysectomized rats (6 and 19 days). (a–f) Effect of bGH-treatment (⫹) on cx43 mRNA levels in the cerebral cortex (Cortex) (a ⫹ c ⫹ e) and hypothalamus (b ⫹ d ⫹ f) of T4/C-treated hypophysectomized rats (⫺). The amount of cx43 transcript was determined by a solution hybridization assay. (a⫺d). Effect of bGHtreatment (⫹) on cx43 protein levels in the cerebral cortex (Cortex) (e) and hypothalamus (f) of T4/C-treated hypophysectomized rats (⫺). The amount of cx43 protein was determined by the densitometry of Western blots. Representative samples of cx43 in the Western blot are shown. Data are presented as mean ⫾ SEM. *P ⬍ 0.05, **P ⬍ 0.01. The mean of the bGH treatment group is expressed as a percentage of the mean of the T4/C-treated hx rats (⫽100). (a–f) Reprinted and modified with permission by Åberg et al. (2000b) and holder of copyright 2000, The Endocrine Society.

IGF-I is produced in response to GH treatment, we examined if circulating IGF-I alone could contribute to the increase in cx43 found in the brain. However, rhIGF-I (0.85 mg/kg) did not have the corresponding effect on cx43 abundance in any of the brain regions examined (Åberg et al., 2000b), despite that the circulating IGF-I levels were maximal (157 ⫾ 17 ng/ml) (Åberg et al., 2003b) with respect to uptake and affecting the brain (Armstrong et al., 2000). To explore

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whether GH affected astrocytes in general to a corresponding degree, the abundance of GFAp protein was examined. However, neither bGH nor rhIGF-I treatment affected GFAp abundance as cx43 was affected in the cerebral cortex (Åberg et al., 2000b). 2. In Vitro In vivo experiments indicated that it was primarily circulating GH that was able to affect cx43 in the brain (Åberg et al., 2000b). However, it had been reported that GH stimulated local IGF-I production in the brain (Johansson et al., 1995; Lopezfernandez et al., 1996; Ye et al., 1997) that could mediate the effects of systemic GH. Therefore, the possibility of local IGF-I stimulation of astroglial cx43 and GJC in vitro was examined (Åberg et al., 2003b). GH receptor immunoreactivity was present in astroglial cell cultures derived from the cerebral cortices of newborn rats as well as abundant IGF-I receptor immunoreactivity (Åberg et al., 2003b). The cultures were supplemented with bGH and rhIGF-I, and cx43 and GJC were assessed. In these cultures, rhIGF-I increased intercellular GJC (⫹25%) as evaluated by dye transfer of Lucifer Yellow (Fig. 7) and cx43 protein abundance (⫹45%) (Åberg et al., 2003b). Bovine GH, however, did not affect GJC. In the aspect that IGF-I increases cx43 and GJC, IGF-I contrasts to most substances hitherto examined for longterm exposure, which mostly decrease GJC (for review, see Rouach et al., 2002). IGF-I has previously been reported to (a)

(b)

decrease GJC in oocyte cultures in a shorter time window, possibly via a direct interaction on the connexon channels (Homma et al., 1998). Therefore, we examined rhIGF-I effects after 30 min of stimulation. However, we saw no effects of rhIGF-I on either GJC decrease or increase in the primary cultures (Åberg et al., 2003b). During the course of the experiments the effect of IGF-I was found to vary. As high proliferation rates have been reported to affect cx43 abundance negatively (Bradshaw et al., 1993a,b), we monitored this factor by adding [3H]thymidine to the medium. The highest concentration of rhIGF-I (150 ng/ml) increased proliferation in the cells by 54%. Interestingly, this concentration of rhIGF-I did not increase GJC by Lucifer Yellow dye transfer or cx43 protein (Åberg et al., 2003b). IGFBP abundance is well known to interact with IGF-I access and efficacy of signaling through the IGF-I receptor. To explore the possibility of IGFBP regulation of the effect of rhIGF-I, IGFBP abundance was analyzed in conditioned media of the cultures using radiolabeled IGF ligand blotting. These assays showed that in conditioned media of cultures with low responses to rhIGF-I there was a higher abundance of the different IGBFPs present in the culture media (Åberg et al., 2003b). As IGFBPs adhere to cell membranes, likely some bovine IGFBP was left from the fetal calf serum (FCS) supplement prior to the experiments. Controls with FCS and Western blots indeed showed that both bovine and rat IGFBPs were present in the culture media. Using Western blots and an antibody being specific for rat IGFBP-2, we found that rat endogenous IGFBP-2 in conditioned media of the cultures was related to a low response to rhIGF-I in terms of dye spreading (Åberg et al., 2003b). The definite contribution of the other bands could not definitely be determined and was probably a partial remainder from FCS. However, the abundance of these bands showed the same inverse pattern to the GJC response to rhIGF-I. Altogether, these results support the notion that it is primarily GH that affects brain cx43 from the periphery, but that local IGF-I, being produced as a result of systemic GH stimulation of the brain, may mediate the effect of GH in concert with the abundance of IGBFPs.

IV. METHODOLOGICAL ASPECTS FIGURE 7 Scrape-loading and dye-transfer experiments under serumfree conditions as indicated, without (a) or with rhIGF-I (b) at 30 ng/ml given for 24 h before the experiment. In the lower panel, the figures were analyzed three-dimensionally by Scion Image software and drawn as a three-dimensional graph showing Lucifer Yellow spreading as “mountains” next to the central scrape. (a and b). Reprinted and modified with permission by Åberg et al. (2003b) and holder of copyright 2003 WileyLiss, Inc (http://www.interscience.Wiley.com/). (See color plate 14)

A. In Vivo Experimental Protocol and Hypophysectomy Female Sprague–Dawley rats (Møllegaard Breeding Center Ltd., Ejby, Denmark) were hypophysectomized at 50 days of age, which corresponds to adoloscence, and experiments were started about 10–15 days later.

12. GH and IGF-I and Astroglial Gap Junctions

When supplementing a certain hormone to study longterm (⬎1–2 days) hormonal effects in animals, there is always a risk of feedback inhibition of the endogenous secretion of that particular hormone. Both exogenous GH (Tannenbaum, 1980) and IGF-I (Aguila et al., 1993) have been shown to inhibit GH secretion from the pituitary, thereby blurring the study of long-term effects. To eliminate such effects, hypophysectomized animals, being deficient of all hormones from the anterior pituitary, are used. The animals are often substituted with cortisol and thyroxine (as described earlier), in order to normalize water/fluid homeostasis (Friedman et al., 1966) and growth (Jansson et al., 1982; Thorngren and Hansson, 1973). Additionally, thyroxine and glucocorticoids are important for normal central neural plasticity and function and it may therefore be important to normalize the environment in which GH or IGF-I acts (for reviews, see McEwen, 1996; Sinha et al., 1994). However, the doses mentioned do not completely match normal hormonal serum concentrations, and there are other hormones not being substituted at all, such as adrenocorticotrophic hormone, prolactin, follicle-stimulating hormone and luteinizing hormone, and their subsequent adrendal/gonadal steroid hormones. The main disadvantage of partial substitution of hypophysectomized rats is that a hormonal effect may remain undetected due to a lack of permissive actions from other pituitary-dependent hormones, such as steroid (i.e., the unsubstituted) hormones. The main advantage is that the risk of studying a secondary hormonal effect is minimized, which is not the case when intact rats are studied. Corticosterone is the principal glucocorticoid in the rat. However, the substitution of cortisol or other glucocorticoids (dexamethasone/cortisone/triamcinolone, etc.) to rats or other animals is probably mainly a matter of glucocorticoid potency, i.e., binding affinity to the glucocorticoid receptor. Thus, the corticosterone glucocorticoid potency is about 30% of that of cortisol in terms of affinity to the glucocorticoid receptor. Bovine GH and recombinant human IGF-I were administered at 1 and 0.85 mg/kg/day, respectively, per 24 h. These concentrations were both shown to increase serum IGF-I and body growth, although GH exerted a greater effect. The concentration of IGF-I was probably near maximal with respect to the brain, as the IGF-I is taken up into the brain via a carrier-mediated mechanism, which has been shown to be saturated at a serum concentration of approximately 150 ng/ml (Armstrong et al., 2000).

B. In Vitro 1. Cell Cultures Primary brain cultures have been used as a model system to study astroglial and neuronal function for about 30 years (Booher and Sensenbrenner, 1972). In our experiments, the

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cultures were enriched in astroglial cells (without neuronal cells) from 1- to 2-day-old Sprague–Dawley (Hansson, 1984). In primary cultures, there are many factors that can be controlled, e.g., pH, O2 partial pressure, growth factor supplementation, and, to a large extent, the composition of cells. However, other factors are lost in the primary cultures. First they are derived from immature rats, and second the local environment with regard to both extracellular milieu and cytoarchitecture is lost upon cultivating. To preserve a three-dimensional (3D) environment for the cells, aggregate or matrix cultures may be used instead of using monolayer cultures. In cultured hepatocytes, this technique of culturing has significance as it enhances the effect of GH as compared to classical monolayer cultures (Guzelian et al., 1988). An alternative to monolayer or 3D culture is to obtain fresh slices of brain tissue, either for direct (acute) use or for long-term (organotypic) culture. The greatest advantage is that with preserved cytoarchitecture the cellular networks are largely intact. However, these techniques still cause an abrogation of the local environment that may change, for example, synaptic density (Kirov et al., 1999). An advantage in using astroglial cell cultures (without neurons) is they are more homogeneous and monitored more easily. However, the presence of neurons in a primary culture affects the astroglial cells in that they express different ion channels and receptors (Barres et al., 1990; Corvalan et al., 1990), and using mixed (neuron-containing) cultures may have been more similar to the in vivo situation, as in the case ion channels (Barres et al., 1990). Indeed, the presence of neurons increases astroglial GJC (Fischer and Kettenmann, 1985). Therefore, using mixed cell cultures may be a suitable tool for future studies with regard to GH and IGF-I effects on GJC.

C. Scrape Loading/Dye Transfer There are several techniques available for evaluating gap junctional coupling. All of them use dyes that are small enough to penetrate gap junctions that can be detected using a microscope. Lucifer Yellow (MW 521.6) is nontoxic at the particular concentration used and does not interfere with plasma membranes (Stewart, 1981). GJC was determined using the scrape-loading/dye transfer technique (Giaume et al., 1991; Venance et al., 1995; el Fouly et al., 1987) (Fig. 7). When spreading out from the initial scrape, there is a risk of underestimating the actual area of spread due to dilution of the dye after long-distance diffusion (e.g., an actual increase threefold difference could be measured as less than threefold). This may be exemplified by the fact that a threefold increase in the number of cells being coupled (as measured by microinjections of Lucifer Yellow) corresponded to only a twofold increase in dye transfer areas as measured by scrape loading (Pepper and Meda, 1992). This is a problem common to all techniques

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evaluating GJC. Another problem with scrape loading is the mechanical trauma induced by damaging the cells in the scrape. This risk is reduced in other techniques assessing GJC, e.g., the microinjection techniques (Meda, 2000) and especially fluorescence recovery after photobleach (Trosko et al., 2000). The greatest advantage of the scrape-loading technique, as compared to the other two mentioned, is that it is much faster, thereby eliminating time-biased differences after a certain treatment or experiment.

D. In Vivo versus In Vitro 1. Cellular Heterogeneity When studying homogenates of a brain region, an effect or quantification will be evened out due to the fact that not all cells respond. A heterogeneous cell distribution is exemplified by the observation that the granule cell layer in the dentate gyrus has abundant IGF-I receptor as analyzed by in situ hybridization of mRNA (Aguado et al., 1993). However, when quantifying IGF-I receptor mRNA from homogenates of hippocampus with RNase protection assay, only moderate amounts of the IGF-I receptor were found (Rotwein et al., 1988). This phenomenon may indeed explain why cx43 in homogenates of the hippocampus and brain stem was not affected by GH treatment (Åberg et al., 2000b), although in certain subgroups of cells or nuclei they might have been. In cultures, these problems are, to a large extent, avoided, as culture conditions can be chosen to favor more homogeneous cell populations. 2. Extracellular Compartment Brain tissue is composed of many cell types and an extracellular compartment, which is approximately 25% [as extrapolated from corpus callosum (Chvatal et al., 1997)] of the total brain volume. However, in the local environment the extracellular space may be larger or smaller. For example, the extracellular space between two neurons and adjacent astrocytes covering a synaptic cleft may be small enough for a transmitter such as glutamate to reach high concentrations without having the option to diffuse easily into the surrounding extracellular compartment, at least on a short timescale. This is of course very different from the vast “extracellular” space found in culture medium. This is important to keep in mind because to a large extent culture medium eliminates concentration peaks after release of a certain substance. Finally, application of a substance to culture medium only tests a situation with a long and steady biological effect. In vivo, an oscillatory or long-term variation of release of a substance is more likely. 3. Cellular Localization When performing studies on in vivo specimens, astrocytes and neurons are often closely packed together.

Astrocyte processes may especially be mistaken for being of neuronal origin when light microscopic techniques are used. Previously, cx43 was identified as being present in neurons using light microscopy with a resolution of 0.2–0.4 ␮m, which is larger than the thickness of some (or even most) astrocyte processes. However, these observations have been contradicted with high-resolution electron microscopy, showing a strict localization of cx43 to astrocytes (Rash et al., 1997). Such problems are largely eliminated with monolayer cultures. Similar explanations may account for the cellular identity of the IGF-I receptor. The IGF-I receptor was previously only found in cultured astrocytes (Masters et al., 1991), as also found by us (Åberg et al., 2003b), whereas in vivo the IGF-I receptor seemed to be confined to neurons (Folli et al., 1994). However, a later report using electron microscopy showed comparatively abundant localization of the IGF-I receptor in astrocytes, as shown in the hypothalamus and cerebellum (Garcia-Segura et al., 1997).

V. SIGNIFICANCE OF REGULATION OF CONNEXIN43 AND GAP JUNCTIONAL COUPLING A. Connexin43 Transcription and Translation Gap junctional coupling is regulated at several levels, i.e., gap junction permeability, the number of established connexons, and the translation efficiency and transcription rate (Fig. 3). A regulatory event may affect one or two of these levels (Darrow et al., 1996) or, less commonly, all steps simultaneously (Larson et al., 1997). One study showed that a sixfold increase in cx43 mRNA was followed by a twofold increase in cx43 protein (Pepper and Meda, 1992). These results are in line with our results, as the increase in cx43 mRNA was larger than the increase in cx43 protein after GH being given in vivo (Åberg et al., 2000b). Hypothetically, the assumption that the translation rate is linear to that of cx43 mRNA abundance would imply that the cx43 protein would increase to an equal amount as cx43 mRNA. Therefore, these findings could indicate that the turnover rate of cx43 protein was increased, which in itself would be an interesting observation. An increased turnover with a decreased half-life of cx43 protein might indicate a need to fine-tune the level of cx43 in short timescales.

B. Other Connexins and Gap Junctions There are several isoforms of connexins. Since 1987, when the first connexin was cloned, at least 12 types of connexins have been reported in the rat nervous system

12. GH and IGF-I and Astroglial Gap Junctions

(for review, see Rouach et al., 2002). However, in adulthood, mostly glial cells express connexins. Furthermore, of glial cells, astrocytes are the cell type that expresses cx43 and cx30 (Naus and Bani-Yaghoub, 1998). In astroglial cell cultures, the astrocytes seem mostly to express cx43, whereas cx30 is not found until prolonged times in culture (Kunzelmann et al., 1999). In agreement with these data, no cx30-IR was detected in 16-day-old primary astroglial cultures derived from cerebral cortices of newborn rats (data from our laboratory, not shown). However, in vivo, there is still a possibility for the gap junctions to be formed of several connexins, both within a cell (heteromeric gap junctions with both cx30 and cx43 in the same connexon, or heterotypic gap junctions with cx30 and cx43 in different connexons) and between different types of cells (heterologous gap junctions). Of course, such terminology offers multiple theoretical combinations of connexins. However, many combinations have been shown to be biochemically incompatible or at least dysfunctional. For example, the heterotypic gap junctions between cx40 and cx43 have been shown to be biochemically incompatible with gap junction permeability (Haubrich et al., 1996). Of all combinations reviewed by White and Bruzzone (1996), cx43 only forms functional channels with cx30.3 and cx37, whereas cx26, cx31, cx31.1, cx32, cx33, and cx40 were reported to be incompatible with cx43. These connexins are not significantly expressed by astrocytes. However, the possibility still remains for cx30 to form gap junctions with cx43, as that alternative seems not to have been investigated. Anyhow, in most cases, homomeric, homotypic, and homologous gap junctions can be considered the standard, and other combinations may be exceptions to the rule. Altogether, these data indicate that connexins other than cx43 have a limited abundance in astrocytes in vitro and that some of the potential interactions of cx43 with other connexins in vivo are biochemically incompatible with permeability. This is substantiated further by the finding that no heterocellular contact was found between neuronal and astroglial gap junctions in the cerebral cortex (Rash et al., 2001).

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The propagation of astroglial calcium waves depends on gap junction permeability but probably involves more mechanisms than just permeability. Calcium itself (discussed in Giaume and Venance, 1998), IP3 (Venance et al., 1997), ATP (Cotrina et al., 1998), and glutamate (Innocenti et al., 2000) have been proposed to mediate the propagating intracellular calcium increments called “calcium waves.” Of these, ATP and glutamate have been proposed to be released extracellularly (Cotrina et al., 1998; Innocenti et al., 2000). Probably this does not exclude the possibility that either glutamate (glutamic acid MW 147.1) or ATP (free acid ATP MW 507.2) may pass the gap junction channels, as they are small enough to diffuse within such a channel. The increase in astroglial calcium wave extent may further make the astroglial cells able to transmit relatively fast signals that may activate signal transduction pathways and even gene expression in other astroglial cells (reviewed in Verkhratsky et al., 1998) or by signaling calcium increments to neurons [in vitro (Hassinger et al., 1995) or in situ (Bezzi et al., 1998)], affecting their gene expression (for review, see Bito, 1998). Glutamate and the IGF-I system have further links in other aspects than affecting gap junctional regulation of neurotransmission. It is interesting that glutamate increases the permeability of the gap junctions in cultures of the cerebral cortex and in hippocampal cultures, which are two brain regions important for memory and cognition (Blomstrand et al., 1999). Extended ongoing glutamate neurotransmission probably produces an excess of both extracellular glutamate (as the neurons cannot reuptake all of the glutamate) and K⫹. The extracellular glutamate may stimulate astrocytes to increase gap junction permeability, which may redistribute (“buffer”) excess K⫹ distant from the site of increased neurotransmission. In this aspect it is interesting that IGF-I has been found to increase glutamate transport as well as the glutamate transporter glutamate/aspartate transporter in cultured astrocytes (Suzuki et al., 2001).

D. Connexin43 and Proliferation C. Gap Junctional Communication, Calcium Waves, and Neurotransmission The regulation of gap junction permeability is often referred to as short-term regulation (Fig. 3). However, a compound may sometimes act directly on a channel (⫽short term) as well as recruiting new channels (⫽long term), either from intracellular stores or via de novo translation of connexin protein, e.g., by bFGF in endothelial cell cultures (Pepper and Meda, 1992). We showed that rhIGF-I exerts no evident short-term effects after 30 min of stimulation on gap junctional communication in astroglial cells in vitro (Åberg et al., 2003b). Therefore, the effects of GH and IGF-I may be regarded as predominantly long-term regulation.

In addition to making up gap junctions, connexins and cx43 may have other functions as well. One such effect has been suggested to be an antiproliferative effect of connexins (Bradshaw et al., 1993a,b). However, this observed effect of cx43 transfections in glioblastoma cells may be a side effect of the establishment of gap junctions that mediate antiproliferative effects directly via the gap junction channels or the gap junctions may form an anchor for receptors that mediate antiproliferative signaling between the cells. Nevertheless, results from our group support that idea, as higher proliferative rates by rhIGF-I were associated with an absence of cx43 and GJC increases (Åberg et al., 2003b). Another possibility is that the establishment of connexin–gap junctions

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signals to the cells that differentiation should occur, which in turn should induce the cells automatically to slow down their mitogenic activity. A third possibility is that connexin (without forming gap junctions) actually itself inhibits the mitogenic action of other signals on the cell. Either the connexin protein or its mRNA (or derivatives of both) may bind directly to transcription factors or even the promoter region itself to inhibit uncontrolled mitogenic action of the cells. However, the last two suggestions (direct connexin inhibition of growth factor signaling) have not been examined (Trosko and Ruch, 1998). In summary, although there is no definite evidence concerning the underlying mechanisms (Trosko and Ruch, 1998), there seems to be a link between high cx43 and low proliferation (Bradshaw et al., 1993a,b; Naus et al., 1997), most likely mediating antiproliferative signals through communicating gap junctions (Trosko and Ruch, 1998).

Cx43 gap junction

The rat brain: N/A IGF-I-R

III Local IGF-I

II GH-R

Unknown(s) I The pituitary GH The blood-stream

GH IGF-I Effector organs (liver)

VI. REGULATION OF CONNEXIN43 BY GH AND IGF-I IN THE CONTEXT OF OTHER EFFECTS A. Systemic versus Local Pathways GH and IGF-I have many effects in common while some effects are dominated by one of the hormones. To distinguish between the hormones, one needs to keep in mind that GH induces IGF-I expression locally in many tissues. In addition, IGF-I is increased by GH in the circulation, mainly coming from the liver (Gronowski and Rotwein, 1995), making IGF-I able to affect various tissues systemically (for a summary on the routes of GH and IGF-I action on the brain, see Fig. 8). 1. GH Increases Brain IGF-I Once having accessed the brain, GH may act differentially on the different cell types the GH receptor is present. As the GHR is primarily expressed on neurons but also on astrocytes to a lesser extent (Lobie et al., 1993), GH has several potential ways to act on the brain. Furthermore, GH may act by increasing other substances, presumably IGF-I, peripherally or locally in the brain, as reported in several studies (Hynes et al., 1987; Johansson et al., 1995; Lopezfernandez et al., 1996; Wood et al., 1991; Ye et al., 1997). However, although peripheral rhIGF-I does not increase cx43 in the brain as bGH does (for summary, see Table IA), it exerts other effects on, for example, hippocampal neurogenesis (Åberg et al., 2000a). There are many potential explanations for this involving cellular (neuronal/astroglial or other) localization of the GH receptor, IGF-I receptor, binding proteins for GH and IGF-I, and, of course, synthesis of IGF-I in the brain (Table IB).

FIGURE 8 Hypothetical routes of action for GH and IGF-I to induce cx43 gap junction formation in the brain. The first possibility (I) is that GH itself (or bioactive fragments thereof) directly reenters the brain via the BBB and acts directly via the GH receptor to induce cx43 gap junctions. The second possibility (II) is that GH reenters the brain via the BBB and induces local brain IGF-I, which in turn induces cx43 formation. Our studies does not address the question of other unknown intermediates for the stimulation of IGF-I by GH (as indicated). The third possibility (III) is that circulating IGF-I, known to be stimulated by GH in, for example, the liver, may reenter the brain to stimulate the IGF-I receptor [being of astrocytic (A) or neuronal (N) type].

2. Local IGF-I Concentrations One plausible explanation for why peripheral IGF-I acts on adult hippocampal progenitor proliferation but not astrocytes is that IGF-I affects the two cell types at different concentration intervals. This may be explained by a different abundance of subtypes of the IGF-I receptor, requiring more IGF-I to affect astrocytes than hippocampal progenitors. Indeed, a neuronal and astrocytic subtype of the IGF-I receptor has been reported (Burgess et al., 1987). In addition, in the hippocampus, IGF-I receptors are very abundant in the granule cell layer of the dentate gyrus as compared to the hilus, where astrocytes are mostly situated (Ayer le Lievre et al., 1991). This is in agreement with in vitro data showing a much stronger IGF-I receptor immunoreactivity in hippocampal progenitor cells compared with astrocytes [visually comparing (Åberg et al., 2003a,b)]. Proliferation in adult hippocampal progenitor cells was already increased at 0.3 ng/ml, as compared to 30 ng/ml for astrocytes. It therefore seems that IGF-I has to be additionally synthesized locally in the brain to reach substantially higher concentrations in the local microenvironment than the concentration in cerebrospiral fluid (CSF) reported to be

161

12. GH and IGF-I and Astroglial Gap Junctions

Other effects

Connexin

TABLE IA Effects of GH and IGF-I in the Four Brain Regions Stated or in Cultures of These Different Brain Regions

冦

冦

Treatment

Hypothalmus

GH in vivo

⫹

Cx43

Cortex

Hippocampus

Brain stem

⫹

⫺

⫺

Cx43

Cx43

Cx43

GH treatment in humans ➔ CSF 150 ␮U/liter (⫽57 ng/ml)

Concentration ⫺

IGF-I in vivo

⫺

Cx43

Concentration

⫺

Cx43

Åberg et al. (2000a) Johansson et al. (1995)

⫺

Cx43

Reference

Cx43

Rat CSF ➔ 0.7 ng/ml (peak of 3 ng/ml)

Åberg et al. (2000a) Armstrong et al. (2000)

GH in vitro

⫺

Concentration

10–100 ng/ml

Åberg et al. (2003b)

IGF-I in vitro

⫹

Åberg et al. (2003b)

Concentration

30 ng/ml ⫹

GH in vivo

⫹

c-fos, feedback

Cx43 Cx43

Åberg et al. (2003b) p NE, 5-HIAA

⫹

RNA-synth.

Concentration

As above ⫹

IGF-I in vivo

⫹

Feedback

Proliferation Progenitor

Niblock et al. (2000); Åberg et al. (2000a)

As above

In vitro GH

Minami et al. (1992); Berti Mattera et al. (1983); Stern et al. (1975) Johansson et al. (1995)

⫹

Dendrites Neuron

Concentration

Armstrong et al. (2000)

⫹ embryonic day 15 telencephalon, MBP q

Almazan et al. (1985)

1–100 ng/ml dose dependent

Almazan et al. (1985)

Concentration

IGF-IR p Neuron Astrocyte

In vitro IGF-I

⫹

Concentration

1 nM ⫽ 7.5 ng/ml

⫹/⫺Additional

Åberg et al. (2003b)

⫹

qGlycogen Astrocyte

⫹

Proliferation Progenitor

⬎0.5 nM ⫽ ⬎3.5 ng/ml

Dringen and Hamprecht (1992); Pons and Torres Aleman (1992)

⬎0.3 ng/ml

Dringen and Hamprecht (1992); Pons and Torres Aleman (1992)

data on brain region-specific effects is provided in “Other effects”.

TABLE IB Presence of GH, GH Receptors, IGF-I, IGF-I Receptors, GH-Binding Proteins, and IGBFPs in Four Brain Regions Factor

Hypothalmus

Cortex

Hippocampus

Brain stem

Reference

Brain GH mRNA

⫹⫹

Neuron

⫹⫹

?

⫹

?

⫹

Marks et al. (1991); Gossard et al. (1987)

Brain GH

⫹⫹

?

⫹

?

⫹

?

GH rec. mRNA

⫹⫹

Neuron

⫹

?

⫹⫹

?

GH rec. protein

⫹⫹ ⫹⫹

Neuron Ventr. Astr.

⫹⫹ ⫺

Neuron Astrocyte

⫹ ⫹⫹

Neuron Ventr. Astr.

⫹

?

⫹⫹

Neuron

IGF-I mRNA

⫹

?

IGF-I

⫹⫹

Astrocyte

IGF-I rec. mRNA

⫹⫹ ⫹

Neuron Astrocyte

⫹⫹

Neuron ?

⫹⫹

?

⫹⫹

?

IGF-I rec. (ligand binding)

?

Hojvat et al. (1982) Burton et al. (1992) ⫹

?

Lobie et al. (1993) Ayer le Lievre et al. (1991) Duenas et al. (1994)

⫹⫹ ⫹

GCL Hilus

⫹

?

Garcia–Segura et al. (1997); Aguado et al. (1993)

⫹⫹ ⫹

CA3, GCL Hilus, etc.

⫹/⫺

?

Marks et al. (1991); Lesniak et al. (1988)

GHBP

All cell types in the brain show GHBP IR, no detailed data

IGBFPs (1–6)

Complex regulation and interaction with IGFs, synthesized by both neurons and astrocytes

Lobie et al. (1992)

a Examples of GH and IGF-I effects are shown and should not be taken as exhaustive. For example, neuroprotective effects of GH and IGF-I are left out. ⫹/⫺, positive or negative effect. ?, cell type is unknown. Ventr. Astr., ventricular astrocytes.

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0.5 to 3 ng/ml after the systemic administration of rhIGF-I (Armstrong et al., 2000) to affect astrocytes and cx43. In the literature, there is evidence of higher concentrations of IGF-I in the brain tissue (Lopezfernandez et al., 1996) than can possibly have been added by serum or diffusion from the CSF. In the young adult rat, the concentration of IGF-I is about 2 ng/mg of brain total protein (Lopezfernandez et al., 1996), which would correspond to at least several tens of nanograms per milliliter of wet weight brain tissue. This implies that IGF-I is produced at high levels within the brain tissue, comparable to the levels used in vitro (Åberg et al., 2003b). 3. Neurons: The Cellular Route? As GH receptors seem to be found predominantly in neurons (Burton et al., 1992) and as neurons seem to be the chief producer of IGF-I under normal circumstances with no astrogliosis (Ayer le Lievre et al., 1991), this may be the cellular route by which GH induces astroglial cx43 and gap junctions in the brain. This could be achieved possibly by comparatively low concentrations of Des-IGF-I, which at least in humans is the form of IGF-I in brain (CarlssonSkwirut et al., 1986). Des-IGF-I could be substantially more effective in exerting effects due to its lower binding affinity to IGBFPs. Indeed, IGFBPs seem to be important in the context of IGF-I affecting cx43 and GJC. Especially IGFBP-2 in the cell medium was correlated to a lack of effect of rhIGF-I on GJC (Åberg et al., 2003b). 4. Astrocytes: The Cellular Route? However, as GH receptors are found in astrocytes, there is still a possibility that GH directly affects astroglial cx43 in vivo. The lack of effect of GH on monolayer astroglial cultures does not exclude effects of GH on astroglia in vivo, as suggested that monolayer culturing conditions with hepatocytes substantially reduces biological effects of GH (Guzelian et al., 1988). However, we would not have been able to assess gap junctional communication by other means than using monolayer cultures. This is, however, a challenging opportunity for future studies using single cell microinjections in tissue slices where the spatial environment is essentially preserved (Meda, 2000). 5. Conclusion Our results show that GH affects the brain directly and others have shown that IGF-I is increased locally after GH treatment in the brain, implying that IGF-I could mediate the effect on cx43. The fact that rhIGF-I was able to increase cx43 in astroglial cell cultures supports the notion that IGF-I may mediate the effect of GH in vivo. The effect of GH on cx43 found in vivo would then be mediated by an effect on primarily neuronal cells producing local IGF-I, which in turn increases neighboring astroglial expression of cx43.

B. Other Possible Mediators/Pathways 1. GH Affects Thyroxine That May Affect Connexin43 There may be other pathways of GH and IGF-I action both systemically and in the local microenvironment. Systemically, apart from affecting IGF-I, GH induces increases in thyroxine (T4), triiodothyronine (T3) (Geelhoed Duijvestijn et al., 1992), and insulin [at least in humans (Johansson et al., 1996)], whereas in rats the latter is uncertain (Daugaard et al., 1999). T4 has been shown to increase cx43 in other cell types (Stock et al., 1998). However, at least in humans, T4 levels are decreased in CSF after GH treatment (Burman et al., 1996). In neural systems, however, these compounds have not been examined on GJC or cx43. 2. TGF-␤1 and bFGF: A Link to the IGF-I System and Connexin43? Compounds that have been shown to regulate the translation (and/or transcription) of cx43 in neural systems include bFGF [increase in cortical progenitor cells, being mainly astroglial cells (Nadarajah et al., 1998), decrease in postnatal astroglial cells (Reuss et al., 1998)] and TGF-␤1 [increase in astroglial cells, decrease in glioblastoma cells (Robe et al., 2000)]. These have been shown to have links to the IGF-I system. Basic FGF has been reported to release IGF-I from hypothalamic cultures (Pons and Torres Aleman, 1992), whereas TGF-␤1 is reported to increase IGFBP-3, which binds free IGF-I (Rajah et al., 1997). As bFGF increases GJC (dye coupling) and cx43 in a similar way, as found in vitro (Nadarajah et al., 1998), the increase in cx43 and GJC may actually be at least partly mediated by IGF-I. However, under other conditions, bFGF downregulates cx43 and GJC (Reuss et al., 1998). If the effect of bFGF is mediated by IGF-I, these contradictory data may be explained by our finding of different effects on gap junction cellular coupling in selective concentration windows of rhIGF-I and especially its interaction with IGFBPs. In our experiments, a high abundance of IGFBPs (especially IGFBP-2) made rhIGF-I unable to increase GJC, whereas a lower abundance of IGFBPs was associated with a robust increase in GJC (Åberg et al., 2003b). A difference in the abundance of IGFBPs between the experiments of Reuss and co-workers and Nadarajah and co-workers could therefore explain the opposite effects of bFGF on cx43 and GJC (Nadarajah et al., 1998; Reuss et al., 1998). Conversely to being the mediator of GH, IGF-I may regulate other paracrine factors that increase cx43 and/or gap junctional coupling. However, there are little data on IGF-I-induced synthesis of different compounds, especially in the brain. Anyhow, IGF-I has been reported to induce steroidogenesis in human luteal cells (Apa et al., 1996). If the same mechanisms are present in the brain, it may be of

12. GH and IGF-I and Astroglial Gap Junctions

interest because estrogen binds the cx43 promoter and induces transcription (Yu et al., 1994). Another compound being induced by IGF-I in endothelial cells is nitric oxide (NO) (Tsukahara et al., 1994). That would also be a candidate for affecting gap junctional communication (and perhaps cx43), as NO is reportedly expressed in astroglial cells and disrupts gap junctional communication (Bolanos and Medina, 1996). However, in order to affect cx43 and gap junctional communication by NO, IGF-I would have to affect NO release in astrocytes negatively.

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ischemia (Gluckman et al., 1998; Gustafson et al., 1999; Scheepens et al., 2001; Tagami et al., 1997) and inhibit neuronal apoptosis (Russo et al., 2004). This idea—that cx43 may be part of the regenerative process after ischemia—is supported by the finding that cx43 is upregulated in the penumbra zone after induced hypoxia (Hossain et al., 1994). Furthermore, the process of glucose uptake into astroglial cells seems to involve an interaction with IGFBP abundance, as the addition of IGFBP-2 has been reported to enhance the glucose uptake effect of IGF-I in astroglial cells (Raizada, 1991).

C. The IGF-I System and Connexin43/Gap Junctions The IGF-I system is complex and involves interactions among insulin, IGF-I, IGF-II, the IGF-I receptor (IGF-IR), and the six IGBFPs. We have demonstrated the presence of the IGF-IR and examined the effects of rhIGF-I and insulin on cx43 and GJC (Åberg et al., 2003b). Furthermore, a high abundance of IGBFPs reduced the GJC increase found after giving rhIGF-I. There are some issues important to have in mind when discussing the effects of IGF-I. 1. Proliferation First, IGF-I has robust dose-dependent proliferative effects (Åberg et al., 2003b). Abundant expression of several IGBFPs in astroglial cell cultures and in C6 glioma cell lines has been reported before (Bradshaw et al., 1993a,b). Some of the IGBFPs have been linked to high or low rates of proliferation (Bradshaw et al., 1993a,b). Interestingly, cx43 seems to be involved in these processes. For example, cx43 transfection downregulates proliferation rates in C6 glioma cells and alters the binding protein profile from IGFBP-3 to IGFPBP-4 and IGFBP-2 (Bradshaw et al., 1993a,b). As discussed previously, a high proliferative rate, including induction by IGF-I, seems to be coupled to a low expression of connexins, as we have also found. 2. Glucose Metabolism In astroglial cell cultures, IGF-I increases the glucose transporter abundance (Werner et al., 1989) and glucose uptake into astroglial cultures (Raizada, 1991), as well as glycogen formation (Dringen and Hamprecht, 1992). The effect of IGF-I on glucose transporter abundance is already found at 1 ng/ml (Werner et al., 1989), a concentration where pilot experiments showed no effect of rhIGF-I on GJC in our system. Interestingly, glucose (MW 180.2) is small enough to permeate gap junctions. Therefore, a concerted effect of increased GJC by cx43 and glucose uptake might synergistically enhance the ability of the astroglial syncytium to sustain an increased metabolic demand as would be required by extended neural activity. This could indeed be part of the mechanism by which GH and IGF-I reduce the area damaged after

VII. GAP JUNCTIONAL PLASTICITY AND CORTICAL FUNCTIONS A. Astrocytes and Learning/Enriched Environment Learning and memory are believed to involve many aspects of plasticity in the brain. An enriched environment, which is associated with an enhanced learning ability (Falkenberg et al., 1992; Liljequist et al., 1993; Nilsson et al., 1999), affects a number of parameters in the brain. Morphological changes are increased synaptic densities, including enlarged dendritic trees with more spines (Camel et al., 1986), increased astrocytic ensheathment of synaptic elements (Jones and Greenough, 1996), increased vasculature (Isaacs et al., 1992), and increased newborn cells [e.g., neurons (Nilsson et al., 1999) and astrocytes (GomezPinilla et al., 1998)] in the hippocampus, as well as astrocytes in the visual cortex (Diamond et al., 1966). In addition, functional changes in synaptic connectivity as studied by long-term potentiation and kindling seem to be associated with learning. Furthermore, there are data on molecular changes associated with an enriched environment or learning conditions, as, for example, increases in bFGF (Gomez-Pinilla et al., 1998) and brain derived neurotropic factor (BDNF) (Falkenberg et al., 1992). Evidently, in addition to neuronal engagement, some of these processes may involve astrocytes. In terms of learning and memory, astrocytes have been proposed to affect neurons in several different ways (Smith, 1994) (Table II). It appears that astroglial function is at least involved in consolidating memory formation, as, for example, shown by the fact that astrocyte glycogenolysis is required (O’Dowd et al., 1994), as well as for securing a continuous glutamine supply to the neurons to form memory (Gibbs et al., 1996). In addition, data suggest that astroglial uptake of K⫹ is required for memory formation (for review, see Gibbs, 1991). The question that then arises is, do these processes have a link to gap junctional communication via connexins?

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N. David Åberg

TABLE II Proposed and Shown Mechanisms for Astrocyte Participation in Functional Plasticity in the Brain with Relevance to Memory Formation and Learning

Astrocyte function affecting neurons

Ref.

Gap junctions involved?

Astroglial calcium waves and Ca2⫹-sensitive K⫹ channels affect neuronal excitability by modulating K⫹ in narrow extracellular spaces

Smith (1994)

Astrocytic ion fluxes drive slow electrical currents in the neuropil (also known as slow field potentials)

Relation to learning and memory?

Ref.

How?

Ref.

Yes, directly

GJs mediate calcium waves

Blomstrand et al. (1999)

Unknown

—

Laming (1989)

Yes, possibly

GJs may mediate these (evoked pot. ➔ p in cx32 mutation)

Bahr et al. (1999)

Association with evoked potentials

Lickey and Fox (1966)

Ca2⫹ may be directly regulated by astrocytes in synaptic clefts

Eriksson et al. (1993)

Yes, indirectly

GJs may redistribute Ca2⫹

Ca2⫹ ⬍1000 Da

Unknown

—

Neuroactive agents from astrocytes

Smith (1994)

Yes, indirectly

Via redistribution

Molecules ⬍1000 Da

Unknown

—

Astrocytic modulation of extracellular dimensions

Smith (1994)

Yes, indirectly

Astrocyte processes are rich in GJs

Yamamoto et al. (1990a)

Enriched environment ➔ q learning ➔ q astrocyte

Jones and Greenough (1996); Nilsson et al. (1999)

Synaptic transmission may be modulated by uptake of neurotransmitters

Smith (1994)

Yes, indirectly

Via redistribution

Molecules ⬍1000 Da

Unknown

—

Astrocyte modulation of cerebrovascular blood flow

Smith (1994)

Yes, indirectly

GJs are abundant in end-feet

Yamamoto et al. (1990a)

Blood flow is changed under learning conditions

Isaacs et al. (1992)

Astrocytic regulation of carbohydrate access

Pearce et al. (1988); Smith (1994)

Yes, indirectly

Via redistribution

Carbo-hydrates ⬍1000 Da

Yes, astrocyte glycogen ➔ lactate (neonate chicks)

O’Dowd et al. (1994)

Potassium redistribution

Gardner-Medwin, (1983b)

Yes, directly

Redistribution

Gardner-Medwin (1983b)

Na/K ATPase inhibition in chicks

Gibbs (1991)

Supply of glutamine to neurons

Martinez Hernandez et al. (1977)

Yes, indirectly

Via redistribution

Glutamine ⬍100 Da

Yes, astrocyte glutamine synthetase

Gibbs et al. (1996)

Astroglial Ca2⫹ oscillations (relation to calcium waves)

Pasti et al. (1995)

Yes, directly

Memory of oscillatory patterns

—

Unknown – may affect gene expression

—

Astroglial GJC

—

Yes, directly

Connexin43 Frisch et al. knockout ➔ (2003); Theis GJC reduced 50% et al. (2003a)

q exploratory behaviour, p motor capacity, changes in brain acetylcholine

Frisch et al. (2003); Theis et al. (2003a)

B. Significance of Astroglial Gap Junctions for Learning and Memory There are both strong indirect and recently more direct evidence for gap junctional involvement in terms of learning and memory (Table II).

1. Indirect Evidence a. Connexin43 and Maturation First, it seems that astroglial gap junctions increase dramatically with maturation of the CNS. Both cx43 (being most abundant) and cx30 increase their expression

12. GH and IGF-I and Astroglial Gap Junctions

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postnatally and toward adulthood (Nagy et al., 1999), which implies that gap junctions are important for the function of the adult CNS. Second, connexins (and cx43) have an especially high expression in brain regions with a high degree of plasticity, e.g., the hippocampus and hypothalamus in vivo and in vitro ((Blomstrand et al., 1999; Micevych and Abelson, 1991; Åberg et al., 1999)).

conditions where there is a need for robust and indiscriminate neurotransmission. This is indeed the case in the supraoptic nucleus (SON) in the hypothalamus, where astrocytes withdraw processes covering SON neurons and neuronal elements during dehydration (Tweedle and Hatton, 1976), which is associated with markedly increased neuronal activity in releasing vasopressin and oxytocin.

b. Connexin43 and K⫹ Redistribution It is well known that the thicker the brain gets in various species, the higher the astroglia:neuron index (Reichenbach, 1989). It is interesting to note that even the rat hippocampus shows a comparatively high astroglia:neuron index of approximately 10 (Walsh, 1981), indicating that more astrocytes are required in a brain region intimately coupled to learning and memory imprinting . There has been speculation on the reason for this phenomenon, and the most widely held hypothesis is that redistribution of excess K⫹ away from sites of active neurotransmission requires more astrocytes. As astroglial cell-to-cell conductance (largely attributed to gap junctions) is thought to be essential in this (GardnerMedwin, 1983c; Odette and Newman, 1988; Reichenbach, 1989); a high number of astrocytes may be regarded as a measure of brain regions with a high degree of activity and plasticity. In line with this, cx43 is highly expressed in the globus pallidus, a region known for extended tonic neuronal activity (discussed in Yamamoto et al., 1990b).

d. Connexin43 and Extracellular Matrix In addition to astroglial ensheathing of neuronal synaptic elements, astroglia secrete proteins that form the extracellular matrix (ECM). The ECM proteins form nets called perineuronal nets (Blumcke et al., 1995). Together with astroglial processes (rich in gap junctions and cx43), these perineuronal nets are thought to form barriers to growth factors and may buffer cations (Bruckner et al., 1993) in the local microenvironment. This may allow substances to reach substantially higher concentrations locally. As astroglial processes probably secrete a substantial part of these proteins (Blumcke et al., 1995), there is an additional role for astrocytes to regulate both the size and the quality of the extracellular space in the local microenvironment.

c. Connexin43 and Astrocyte Enwrapment of Neuronal Synapses Furthermore, astroglial processes extend their end feet to cover synapses, dendrites, and neuronal somata. As cx43 and gap junctions are abundant in these subcellular compartments of astroglial cells (Yamamoto et al., 1990a,b), it is likely that they are regulated in the same fashion as when astroglial processes are withdrawn or extended in response to certain physiological stimuli or requirements. It is interesting in this context to note that astrocytes increase the proportion of astrocytic processes to ensheath neuronal synaptic elements in the visual cortex of rats that have lived in an enriched environment (Jones and Greenough, 1996), whereas synaptic density is unchanged (Turner and Greenough, 1985). It should be mentioned that, upon enrichment of the environment, the overall thickness of this brain region is increased, and although neuron number is not increased (Diamond et al., 1966), dendrites and synapses per neuron do increase (Turner and Greenough, 1985). This may reflect an increased ability of these networks to sustain extended neurotransmission. The astroglial process ensheathment of synaptic elements may also reflect an increased participation of astrocytes in selecting, for example, which neurons or, more specifically, which synapses in a specific dendritic tree should be allowed to transmit information (via neurotransmission). In line with this idea, astrocytes could withdraw their ensheathing processes in

2. Direct Evidence: Adult Connexin43 Conditional Astrocyte Knockout An astrocyte-specific adult cx43 knockout model has been established (Frisch et al., 2003; Theis et al., 2003a). Earlier, global cx43 knockout models were incompatible with cognitive experiments due to the postnatal death of these mice. However, conditional adult astrocyte-specific knockout experiments have revealed increased exploratory behavior, impaired motor capacities, and changes in brain acetylcholine levels (Frisch et al., 2003; Theis et al., 2003a). These cognitive changes are associated with functional biochemical alterations in that hippocampal slice GJC was reduced by about 50%. In addition, a physiological phenomenon thought to be a functional correlate to calcium wave spreading, called spreading depression, was increased by 20% in stratum radiatum in the hippocampus (Theis et al., 2003a). Interestingly, the spreading depression phenonenon is also thought to have links to diverse brain pathologies such as migraine (Ramadan, 2003), epileptic activity (Martins-Ferreira et al., 2000), and ischemic damage in the penumbra zone (Anderson et al., 2003). Taken together, these facts support the notion that astroglial processes, which are rich in cx43 gap junctions, are involved in enabling and regulating the selectivity of neurotransmission.

C. Conditions Where the GH/IGF-I Axis Affects Cognitive/Mental Function GH and, to a certain extent, IGF-I have been reported to affect memory formation positively in rodents (Drago et al., 1996; Schneider Rivas et al., 1995), and these findings may

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be an analogue of positive psychological effects of GH treatment in GH-deficient humans (Hull and Harvey, 2003). It is interesting in this context that a number of conditions affect GH and IGF-I levels, which subsequently may impact the brain. 1. Exercise Short-term effects of exercise increase serum GH (Coiro et al., 1994; Eliakim et al., 1999), as well as IGF-I levels in serum (Bang et al., 1990). In fact, IGF-I has been shown to be taken up through the blood–brain barrier more efficiently within hours after training (Carro et al., 2000). Interestingly, long-term effects of exercise (14 days) increase GHBP (Roelen et al., 1997), which may prolong GH half-life in serum. In addition to exercise, GH secretion may be elevated by excitatory anticipation, as serum GH levels were increased markedly before performing parachute jumps as compared to a control situation and control subjects (Nelson, 2000). 2. Aging and Depression Conversely, GH secretion and IGF-I levels are decreased in elderly people (Ghigo et al., 1996). Furthermore, the GH secretory pattern is altered in depressed patients, with a higher daytime secretion of GH (Linkowski et al., 1987). In addition, the peak in GH after exercise is blunted if subjects are mentally depressed (Harro et al., 1999). Patients that are deficient in GH are depressed to a greater extent than controls (Deijen et al., 1996). In analogy, GH secretion is altered (decreased) in rat pups exposed to maternal deprivation (Evoniuk et al., 1979), which decreases ornithine decarboxylase activity in various tissues, including the brain (Evoniuk et al., 1979). Interestingly, this condition could be reversed, incuding GH secretion, by allowing tactile stimulation of the rats (Evoniuk et al., 1979). These phenomena are similar to the symptoms that occur in children with psychosocial dwarfism (Nelson, 2000). 3. Conclusion In summary, different conditions affect both the pattern of and the total secretion of GH and, secondary to GH secretion, IGF-I levels. Of these, at least exercise may be an option where GH may exert positive effects on the brain by enhancing the astrocyte gap junctional communication capacity by cx43 to cope with increased neurotransmission. Moreover, GH treatment has been reported to have neuroprotective effects (Gustafson et al., 1999; Scheepens et al., 2001), and upregulation of cx43 may be part of that effect, as cx43 (Hossain et al., 1994) and perhaps gap junctional communication are upregulated after induced ischemia. This may represent a repair response in the brain tissue. An appropriate moderate physical activity causing a rise in endogenous GH secretion and cx43 might therefore potentially mediate restorative effects after brain insult.

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13 Rodent Models for the Study of the Role of Growth Hormone–Insulin-like Growth Factor-I or the Insulin Axis in Aging and Longevity: Special Reference to a Transgenic Dwarf Rat Strain and Calorie Restriction ISAO SHIMOKAWA Department of Pathology and Gerontology, Nagasaki University Graduate School of Biomedical Sciences, 12-4 Sakamoto 1-chome, Nagasaki City 852–8523 Japan

I. Introduction II. Longevity Models for the Growth Hormone–Insulin-like Growth Factor-I/Insulin Axis III. The Glucose–Insulin System IV. Stress Response V. Conclusion References

lowered while insulin sensitivity is increased; (3) stress resistance is augmented; and (4) the traits in 1,2, and 3 are similar, if not completely identical, to those in CR animals. Although data on aging-related alterations of brain functions in these animal models are presently limited, the models should provide insights into a role for the GH–IGF-I/insulin axis in brain aging and help in protective strategies against it.

Aging is a major risk factor for neurodegenerative and cardiovascular diseases in the brain; thus, long-lived rodent models can be useful tools for the study of agerelated impairments in brain functions. Calorie restriction (CR) is an experimentally reproducible intervention that delays many aging processes and extends the life span in laboratory animals. However, current research has revealed that loss-of- or reduction-of-function mutations of single genes can also extend the life span in rodents as well as invertebrates. Many longevity genes are clustered into the growth hormone (GH)–insulin-like growth factor (IGF)-I or the insulin axis. Reviewing both published data and that obtained from a transgenic dwarf rat strain compared to CR rats in our laboratory, this chapter describes some aspects of the glucose–insulin system and the stress response of these longevity models. In most of the models, (1) GH–IGF-I signaling is attenuated; (2) concomitantly, the plasma insulin level is

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION Aging is a major risk factor for neurodegenerative and cardiovascular diseases in the brain (Thal et al., 2004). Animal models that extend the life span can be utilized to analyze the pathogenesis of those disorders and give insights into possible protection against them. Long-term restriction of calorie intake with essential nutrients, referred to as calorie restriction (CR), has been the only intervention that has been reliably shown to retard the aging process and increase survival rates in aging laboratory rodents (Masoro, 2003). The antiaging and life-prolonging effects of CR have been demonstrated in diverse organisms, including yeasts (Lin et al., 2000), invertebrates (Klass, 1977; Pletcher et al., 2002),

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and dogs (Kealy et al., 2002). The effects are also expected in primates (Mattison et al., 2003). These findings suggest the presence of a universal, evolutionarily conserved, mechanism(s) that regulates aging and life span in animals. However, our knowledge of the mechanisms, particularly in mammals, is incomplete because of physiological complexity. In contrast, in simple organisms such as nematodes and yeasts, a number of single gene mutations, which assure longevity, have been identified (Hekimi and Guarente, 2003) since the late 1980s when a reduction-of-function mutation in a gene called age-1 in nematodes was reported to extend the life span (Friedman and Johnson, 1988). In 1996, a single gene mutation was reported to cause life span extension in rodents (Brown-Borg et al., 1996). Ames mice bearing a loss-of-function mutation in prop-1 gene and thus showing a developmental defect of growth hormone (GH)-, prolactin (PRL)-, and thyroid-stimulating hormone (TSH)-secreting cells in the pituitary gland (Sornson et al., 1996) live longer than wild-type mice. Since then, a number of long-lived rodent models, in which a single gene is spontaneously mutated or genetically engineered, have been reported (Miskin and Masos, 1997; Migliaccio et al., 1999; Coschigano et al., 2000; Flurkey et al., 2001; Mitsui et al., 2002; Shimokawa et al., 2002; Bluher et al., 2003; Holzenberger et al., 2003); many are related to a reduction of GH–IGF-I and/or insulin signaling (Liang et al., 2003; Table I). Longevity models for the

GH–IGF-I/insulin axis share some phenotypes with CR rodents (Shimokawa et al., 2003), e.g., reduced body size and a modified glucose–insulin system. This chapter describes several aspects of the glucose– insulin system and the stress response in a transgenic dwarf rat strain that has been reported as a longevity model by the author’s laboratory (Shimokawa et al., 2002), in comparison with other longevity models for the GH–IGFI/insulin axis and CR. Studies using those models for analysis of aging-related impairments in brain functions are still limited in number (Kinney et al., 2001a,b). However, longevity models could serve as tools to elucidate answers to the controversy about a role for the GH–IGF-I/insulin axis in impaired brain functions in aged animals (Kinney–Forshee et al., 2004).

II. LONGEVITY MODELS FOR THE GROWTH HORMONE–INSULIN-LIKE GROWTH FACTOR-I/INSULIN AXIS When life span extension was reported in Ames mice, a role for the GH–IGF-I axis in the life-prolonging effect in the animal model was obscure because PRL and TSH were also deficient in Ames mice. The phenotype did not exclude a potential effect of life span extension of PRL,

TABLE 1 Rodent Longevity Models

Mutant rodents

Mutated or modified gene

Ames mice

Life spana

Phenotype

Reference

Prop-1

F, ⫹ 68% (718 ⫾ 45) M, ⫹ 49% (723 ⫾ 54)

Defect of pituitary GH, PRL, and TSH secreting cells/dwarf

Brown–Borg et al. (1996)

Snell mice

Pit-1

F, ⫹ 42% (811 ⫾ 20) M, ⫹ 26% (822 ⫾ 34)

Defect of pituitary GH, PRL, and TSH secreting cells/dwarf

Flurkey et al. (2002)

Ghrhr lit/lit mice

Ghrhr

F, ⫹ 25% (857 ⫾ 169) M, ⫹ 23% (886 ⫾ 148)

Defect of GH-releasing hormone signaling/dwarf

Flurkey et al. (2001)

GHR/BP⫺/⫺ mice

GHR/BP

F, ⫹ 38% (749 ⫾ 41) M, ⫹ 55% (629 ⫾ 72)

Loss of GH signaling/dwarf/ susceptible to paraquat

Coschigano et al. (2000)

Igf1r ⫹/⫺ mice

Igf1r

F, ⫹ 33% (568 ⫾ 49) M, ⫹ 16% (585)

Reduction of igf-I signaling/almost normal body size/resistant to paraquat

Holzenberger et al. (2003)

FIRKO mice

Insulin receptor (fat specific)

M ⫹ F, ⫹ 18% (753)

Fat-specific loss of insulin receptor/ reduced fat tissue/dwarf

Bluher et al. (2003)

p66shc⫺/⫺ mice

p66shc

M ⫹ F, ⫹ 30% (761 ⫾ 191)

Loss of an adaptor protein between insulin receptor and downstream molecules/normal body size/ resistant to paraquate

Migliaccio et al. (1999)

anti-GH (tg/⫺) rats

Antisense GH

M, ⫹ 10% (882)

Moderate reduction in the GH–IGF-I axis/dwarf/ resistant to endotoxin

Shimokawa et al. (2002)

a

Percentage increase in mutant animals (mean life span ⫾ SE in control wild animals).

13. Rodent Longevity Models

TSH, or their combination. Indeed, experimental hypothyroidic rats have been reported to live longer (Ooka et al., 1983). However, it was thought that inhibition of the GH–IGF-I axis exhibited the principal effect on life span extension because GH overexpression mice show premature aging phenotypes, such as a shorter life span, earlier loss of fertility, and altered metabolism of hypothalamic neurotransmitters (Steger et al., 1993). In 2000, mice whose GH receptor/binding protein (GHR/BP) gene was disrupted were reported to live longer than their wild counterparts (Coschigano et al., 2000). In these mice, the plasma concentration of IGF-I is severely suppressed because of the disruption of GH signaling. In 2001, “little” mice with a mutation of the GH-releasing hormone receptor gene were shown to also undergo life span extension when fed a low-fat diet (Flurkey et al., 2001). These models suggest that selective suppression of the GH–IGF-I axis increases the life span in mammals. Life span extension of heterozygous knockout mice for the IGF type 1 receptor (Igf1r) gene (Holzenberger et al., 2003) also supports the importance of the GH–IGF-I axis in the regulation of aging and life span. The binding of IGF-I or insulin to its receptor on the cell membrane leads to activation of the receptor tyrosine kinase that phosphorylates insulin receptor substrate (IRS) proteins (Fig. 1; Rhodes and White, 2002). Tyrosinephosphorylated IRS proteins then activate phosphatidylinositol 3-kinase (PI3K) and its downstream kinases, including protein kinase B (PKB/Akt). This pathway mediates mainly

IGF-1

Insulin

GH

JAK2

PI3K

IRS

JAK2

Shc Grb2

SOS

PDK-1

Ras Raf-1

PKB/Akt

MEK FoxO

Metabolic effects

MAPK

Mitogenic effects FIGURE 1 IGF-I, insulin, or GH receptor signaling in mammalian cells. Binding of IGF-I or insulin to its receptor elicits two major signaling pathways: IRS-PI3K and Shc-MAPK. These pathways mediate metabolic and mitogenic effects of IGF-I and insulin, respectively. The GH signaling pathway also activates these two pathways (Herrington and Carter-Su, 2001; Rhodes and White, 2002).

175

the metabolic effects of insulin or IGF-I. The binding of the ligand also leads to autophosphorylation of the receptor, which generates binding sites for Shc proteins that have a Src homology 2 (SH2) domain. The association of Shc with the receptor activates the RAS mitogen-activated protein (MAP) kinase pathway, which mediates mitogenic effects of insulin or IGF-I. Also notable are the IRS-PI3K and the Shc-MAPK pathways initiated by the binding of GH to its receptor (Herrington and Carter-Su, 2001). The receptor–IRS-PI3K pathway in mammals is considered intriguing for the regulation of life span because it became apparent that the Daf-2-Age-1 pathway, which has been studied extensively for a longevity signal in nematodes, is homologous to the mammalian receptor–IRS-PI3K pathway (Hsieh et al., 2002). Daf-2 and Age-1 are homologues of the insulin or IGF-I receptor (Kimura et al. 1997) and PI3K (Morris et al., 1996), respectively. In fruit flies (Drosophila melanogaster), a mutation of the chico gene, which is an analogue of mammalian IRS, also results in a longer life span (Clancy et al., 2001). Evidence suggests that the IGF-I/ insulin receptor–IRS-PI3K pathway has been conserved during evolution, i.e., a common pathway controls life span and aging in diverse organisms. Three Shc genes, ShcA, ShcB, and ShcC, have been found in mammals (Luzi et al., 2000). The ShcA gene encodes two mRNA species, p66shc and p46/p52shc; the latter encodes two proteins because of its alternative translation start sites. Targeted mutation of the mouse p66shc gene induces stress resistance and also prolongs the life span in mice (Migliaccio et al., 1999). The finding suggests the presence of a pathway different from that of IRS-PI3K in insulin/ IGF-I signaling that controls life span in mammals; however, p66shc is not involved in activation of the RAS-MAPK pathway (Migliaccio et al., 1997). Although IGF-I and insulin signaling pathways are not separated in nematodes as in mammals, the reduced GH–IGF-I signaling was thought a principal component of life span extension, and thus retardation of aging in mammals, because direct introduction of loss-offunction mutations into insulin signaling shortens the life span because of metabolic disorders such as diabetes (Accili et al., 1996). However, in long-lived rodent models, plasma insulin and glucose concentrations are also reduced (Table II). Mice whose insulin receptor gene is knocked out exclusively in fat tissues (FIRKO) are also reported to show a longer life span (Bluher et al., 2003), although insulin receptor knockout in other tissues induces insulin resistance and /or metabolic disorders (Bruning et al., 1998; Kulkarni et al., 1999; Bruning et al., 2000; Michael et al., 2000). In FIRKO mice, the plasma IGF-I concentration does not differ from that in wild type (Bluher et al., 2002). Although IGF-I and insulin signalings may converge into a common pathway in the cell, the exact mechanism(s) underlying the life span extension

176

Isao Shimokawa

TABLE II Glucose–Insulin System in Longevity Modelsa Blood glucose (mg/dl)

Plasma insulin (ng/ml) Glucose tolerance

Insulin tolerance

⫺42%

Impaired glucose clearance

Intensified hypoglycemia

Dominici et al. (2002)

n/a

n/a

n/a

Hsieh et al. (2002)

Fed

Fasting

Fed

Fasting

Ames mice

n/ab

⫺27%

n/a

Snell mice

⫺57%

n/a

⫺98%

Reference

GHR/BP-KO mice

⫺25–30%

⫺14–35%

⫺66%

⬍⫺83%

n/a

n/a

Coschigano et al. (2003)

lit/lit mice

n/a

n/a

NSc

n/a

n/a

n/a

Donahue and Beamer (1993)

Igflr⫹/⫺ mice

M, ⫹ 12%; F, ⫺4%d

NS

NS

n/a

Impaired in male; normal in female

n/a

Holzenberger et al. (2003)

FIRKO mice

NS

NS

NS

⫺42%

Normal; inhibit aging-related impairment

Normal; inhibit aging-related insulin resistance

Bluher et al. (2002)

Anti-GH (tg/⫺) rats

⫺16%

NS

⫺79%

⫺49%

Normal

Intensified

Yamaza et al. (2004)

Control (⫺/⫺)-CR rats

⫺15%

NS

⫺84%

⫺40%e

Slightly improved glucose clearance

Intensified

Yamaza et al. (2004)

a

Values are relative to those in wild-type animals. No data found in the literature. c Statistically insignificant. d Statistically unmentioned. e p ⬍ 0.10. b

remains unknown. FIRKO mice might use different mechanisms to extend the life span from those in other longevity models with a reduction in the GH–IGF-I axis. We started a longevity study in 1997 using a transgenic strain of male Wistar rats in which GH synthesis and secretion were selectively reduced by overexpression of the antisense GH transgene (Matsumoto et al., 1993). In this project, we tested a hypothesis that isolated suppression of GH and its downstream pathways retard aging processes and extend the life span in animals. During preliminary

studies for the project, we noticed that the transgenic strain of rats shares several phenotypes with CR rats when they are fed ad libitum. Comparative studies between transgenic and CR rats were also conducted to evaluate a potential role for the GH–IGF-I axis in the antiaging effect of CR. The not-fasting plasma concentrations of GH, IGF-I, and insulin in anti-GH (tg/⫺) rats at 6 months of age are demonstrated in Table III. Part of the findings have been published elsewhere (Shimokawa et al., 2002, 2003; Yamaza et al., 2004).

TABLE III Not-Fasting Plasma Concentration of GH, IGF-I, Insulin, and Glucosea GH (ng/ml)

IGF-I (ng/ml)

Insulin (ng/ml)

Glucose (mg/dl)

AL

172.1 ⫾ 44.1

626.5 ⫾ 89.6

21.6 ⫾ 17.7

105.5 ⫾ 18.0

CR

129.6 ⫾ 35.9

345.6 ⫾ 39.8

23.5 ⫾ 26.0

90.2 ⫾ 13.1

AL

157.3 ⫾ 55.0

1058 ⫾ 127.2

101.8 ⫾ 48.8

126.1 ⫾ 33.9

CR

178.3 ⫾ 26.7

818 ⫾ 82.3

16.0 ⫾ 9.3

107.1 ⫾ 10.2

Anti-GH (tg/⫺)

Control (⫺/⫺)

a

Values represent the mean ⫾ SD of 5–12 rats. Results of 2-f ANOVA are as follows. (1) GH: Tg effect, CR effect, ns; (2) IGF-I: Tg effect, p ⬍ 0.0001; CR effect, p ⬍ 0.0001; (3) insulin: Tg effect, p ⬍ 0.05; C Tg ⫻ CR, p ⬍ 0.05; (4) glucose: Tg effect, p ⬍ 0.05; CR effect, p ⬍ 0.05.

177

13. Rodent Longevity Models

III. THE GLUCOSE–INSULIN SYSTEM

B. The Glucose–Insulin System in Anti-GH (tg/⫺) Rats in Comparison with CR Rats

A. The Glucose–Insulin System in Longevity Models

To characterize the glucose–insulin system in anti-GH (tg/⫺) rats in comparison with CR rats, glucose and insulin tolerance tests (GTT and ITT, respectively) were performed in anti-GH (tg/⫺) male rats and control (⫺/⫺) rats at 6 to 8 months of age (young age group) and at 24 to 25 months of age (old age group). The experimental rats, either (tg/⫺) or (⫺/⫺) rats, were fed ad libitum throughout their life span (AL group) or were fed a 30% calorie-restricted diet (CR group) from 6 weeks of age (Shimokawa et al., 2003). Although not-fasting blood glucose concentrations were reduced slightly in anti-GH (tg/⫺)-AL rats and the CR group at the young age, overnight-fasting blood glucose levels did not differ between anti-GH (tg/⫺) and control (⫺/⫺) rats (Fig. 2a); there was also no difference between AL and CR groups or between age groups. At a young age, the fasting plasma insulin concentration was reduced in anti-GH (tg / ⫺) rats, while there was no statistical difference between AL and CR groups (Fig. 2b). The fasting plasma insulin level fluctuated significantly but increased in old age in all the rat groups. These findings suggest that these animals remained in an euglycemic state, while aging-related insulin resistance had already occurred in all the rat groups. Further analysis will be needed to elucidate whether the occurrence of insulin resistance is delayed in anti-GH (tg/⫺) rats during the period of the experiment. Glucose clearance from blood in GTT was normal or slightly facilitated in anti-GH (tg/⫺)-AL, anti-GH (tg/⫺)-CR, and (⫺/⫺)-CR rats as compared with control (⫺/⫺)-AL rats at a young age (Fig. 3a). Glucose tolerance was impaired at

Insulin resistance and glucose intolerance in the aging process induce hyperinsulinemia and hyperglycemia, both of which promote aging-related pathologies as the process of diabetic complications (Facchini et al., 2000; Anisimov, 2003). In contrast, CR causes a life-long decrease in plasma glucose and insulin levels (Masoro et al., 1992). Therefore, proper modulations of the glucose-insulin system could favor a long life span in mammals. Table II summarizes traits of the glucose-insulin system in longevity models. The plasma insulin concentration has been reduced mostly when animals are either fed or fasted; there is no case with an increased insulin level. The blood glucose level is, in most circumstances, reduced, except for male igf1r⫹/⫺ mice that show a slight elevation of the plasma glucose level (Holzenberger et al., 2003). Increased sensitivity of insulin, in a notion based on intensified hypoglycemia in the insulin tolerance test, characterizes the longevity models (Bluher et al., 2002; Dominici et al., 2002; Yamaza et al., 2004), although glucose tolerance is not always improved. Ames mice and male igf1r⫹/⫺ mice show glucose intolerance (Dominici et al., 2002; Holzenberger et al., 2003). FIRKO mice exhibit normal glucose tolerance and insulin sensitivity (Bluher et al., 2002). However, aging-related glucose intolerance and insulin resistance, which are shown in control mice at 10 months of age, are inhibited in FIRKO mice (Bluher et al., 2002). (a)

(b) 100

120

Insulin (ng/ml)

Glucose (mg/dl)

150

90 60 30

75 50 AL

25

CR 0

0 Y

O (–/–)

Y

O (tg/–)

Y

O (–/–)

Y

O (tg/–)

FIGURE 2 Fasting blood glucose and plasma insulin concentrations in anti-GH (tg/⫺) rats: the effect of aging and calorie restriction. Blood samples were obtained from the tail vein after overnight fasting. Data were analyzed by three-factor ANOVA for effects of the antisense GH transgene [(tg/⫺) versus (⫺/⫺), age (Y, young, 6–7 months of age versus O, old, 24–25 months of age), and diet (AL, ad libitum feeding versus CR, calorie restriction)]. Interaction between or among the effects is noted when it is statistically significant. (a) Blood glucose concentrations. Each bar and line represent the mean ⫾ SD of four to five rats. 3-f ANOVA: Tg effect, not significant; age effect, not significant; diet effect, not significant. (b) Plasma insulin concentrations. 3-f ANOVA: Tg effect, p ⬍ 0.05; age effect, p ⬍ 0.0001; diet effect, not significant. Each bar and line represent the mean ⫾ SD of four to five rats.

178

Isao Shimokawa (a)

(b) 280 Young 240 200 160 120 80

0

15

30 60 90 Time (min)

Blood glucose (mg/dl)

Blood glucose (mg/dl)

280

Old 240 (tg/–)-AL

200

(tg/–)-CR

160

(–/–)-AL 120 80

120

(–/–)-CR 0

15

30 60 90 Time (min)

120

FIGURE 3 Blood glucose concentration during glucose tolerance testing. (a) Young age (6–7 months of age). Each bar and line represent the mean ⫾ SE of five to seven rats. 3-f ANOVA: Tg effect ⬍0.05; diet effect, p ⬍ 0.001; time effect, p ⬍ 0.0001. (b) Old age (24 months of age). Each bar and line represent the mean ⫾ SE of four to seven rats. 3-f ANOVA: Tg effect, not significant; diet effect, p ⬍ 0.0001; time effect, p ⬍ 0.0001; diet ⫻ time, p ⬍ 0.001; Tg ⫻ diet ⫻ time, p ⬍ 0.05. In the (tg/⫺) or (⫺/⫺) rat group, 3-f ANOVA was also performed for the effects of diet, age, and time, and a significant age effect was confirmed in each rat group.

(Fig. 4b). The aging-related fluctuation and increase in plasma insulin were minimized in the anti-GH (tg/⫺)-AL and CR groups of (tg/⫺) and (⫺/⫺) rats (Fig. 4b). ITT demonstrated that hypoglycemia was slightly facilitated in the CR group at a young age (Fig. 5a), but there was no difference between (tg/⫺) and (⫺/⫺) rats. In old age, insulin-induced hypoglycemia was intensified in (⫺/⫺)-AL, (⫺/⫺)-CR, and (tg/⫺)-CR rats as compared to the respective young age groups (Fig. 5b). Generally, statements of enhanced insulin sensitivity are based on findings that hypoglycemia is

old age in (⫺/⫺)-AL rats (Fig. 3b). Aging-related glucose intolerance was almost inhibited in the CR group of anti-GH (tg/⫺) and (⫺/⫺) rats (Fig. 3b). It is of interest that there was no insulin surge after glucose load in anti-GH (tg/⫺)-AL and -CR rats and (⫺/⫺)-CR rats, although the plasma insulin concentration was transiently elevated at 15 min in (⫺/⫺)-AL rats at a young age (Fig. 4a). In old age, the plasma insulin level fluctuated greatly in (⫺/⫺)-AL rats without any peak value being detectable, although the levels were generally increased (a)

(b) 200

200

Old

160

Plasma insulin (ng/ml)

Plasma insulin (ng/ml)

Young

120

80

40

0

160

120 (tg/–)-AL (tg/–)-CR

80

(–/–)-AL (–/–)-CR

40

0

15 30 Time (min)

60

0

0

30 15 Time (min)

60

FIGURE 4 Plasma insulin concentration during glucose tolerance testing. (a) Young age (6–7 months of age). Data represent the means ⫾ SE of four to seven rats. 3-f ANOVA: Tg effect, p ⬍ 0.0001; diet effect, p ⬍ 0.001; time effect, p ⬍ 0.01; Tg ⫻ diet, p ⬍ 0.01; diet ⫻ time, p ⬍ 0.01; Tg ⫻ diet ⫻ time, p ⬍ 0.001. (b) Old age (24 months of age). Data represent the means ⫾ SE of four to seven rats. 3-f ANOVA: Tg effect, p ⬍ 0.01; diet effect, p ⬍ 0.05; time effect, not significant. In the (tg/⫺) or (⫺/⫺) rat group, 3-f ANOVA was also performed for the effects of diet, age, and time, and a significant age effect was confirmed in each rat group.

179

13. Rodent Longevity Models (a)

(b) 120

120 Old

100

100

80

80

% Glucose

% Glucose

Young

60 40

(tg/–)-AL

60

(tg/–)-CR

40

(–/–)-AL (–/–)-CR

20

20 0

0

15 30 Time (min)

60

0

0

15 30 Time (min)

60

FIGURE 5 Blood glucose concentration during insulin tolerance testing. Data are normalized by values at 0 min. (a) Young age (7 months of age). Data represent the means ⫾ SD of five to six rats. 3-f ANOVA: Tg effect, not significant; diet effect, p ⬍ 0.05; time effect, p ⬍ 0.0001. (b) Old age (25 months of age). Data represent the means ⫾ SD of five to six rats. 3-f ANOVA: Tg effect, not significant; diet effect, p ⬍ 0.0001; time effect, p ⬍ 0.0001; Tg ⫻ diet, p ⬍ 0.01; diet ⫻ time, p ⬍ 0.05. In the (tg/⫺) or (⫺/⫺) rat group, 3-f ANOVA was also performed for the effects of diet, age, and time, and a significant age effect was confirmed in each rat group.

intensified in ITT (Bluher et al., 2002; Dominici et al., 2002). In the experiment given earlier, insulin sensitivity could be increased in the CR group at a young age, but not in anti-GH (tg/⫺)-AL rats. However, in old age, hypoglycemia was facilitated in the rat groups except for (tg/⫺)-AL rats, although insulin resistance was observed in all rat groups. In summary, fed and fasting blood glucose and insulin concentrations at a young age suggest that a reduced GH–IGF-I axis lowers blood and insulin levels as with CR. Glucose clearance is normal in anti-GH (tg/⫺) rats and is achieved without a significant surge of plasma insulin. Insulin tolerance testing indicates that a reduced GH–IGF-I axis does not affect insulin sensitivity, while CR slightly sensitizes the action of insulin. These findings suggest the presence of an insulin-independent mechanism for glucose clearance in anti-GH (tg/⫺) and CR rats, although the insulin-dependent mechanism seems to be enhanced in CR rats, i.e., longevity animals have some mechanisms to efficiently metabolize glucose without insulin, and thus these animals do not have to increase the plasma concentration of insulin during the feeding cycle. Insulin resistance and glucose intolerance apparently occur in aged rats. Plasma insulin does not respond properly to glucose load in GTT. Hypoglycemia after exogenous insulin administration in ITT is also intensified in aged rats. This finding might not simply indicate an increased insulin sensitivity in aged rats because the basal level of insulin is increased, suggesting insulin resistance. Mechanisms counterbalancing the insulin-induced hypoglycemia might be dysfunctional in aged rats. Taken together data from anti-GH (tg/⫺) and CR rats, the longevity model results indicate that insulin signaling seems to be more important for the extension of life span than

lowered blood glucose. Although hyperglycemia leads to a Millard reaction in cellular molecules and induces some pathology (Monnier and Cerami, 1981), it may not be essential for longevity in mammals.

C. Insulin Signaling in Longevity Models To characterize insulin signaling in Ames mice, a high dose of insulin was injected into the portal vein, and insulin signaling molecules were analyzed in liver and skeletal muscle tissues (Dominici et al., 2002, 2003). In the liver, insulin-induced phosphorylation levels of insulin receptor (IR), IRS-1, and IRS-2 are reported to increase as compared with those in control rat. The abundance of the p85 subunit of PI3K associated with IRS-1, but not that with IRS 2, is also increased. Insulin-stimulated, phosphotyrosinederived PI3K activity is, however, similar between Ames and control mice. Nonetheless, the level of phospholylated Akt is increased 41% in Ames mice. Similar findings are also reported in GHR/BP-KO mice liver (Dominici et al., 2000). These findings support that the finding that insulin sensitivity is increased in long-lived mice liver. In contrast, in the muscle of Ames mice, most phosphorylation of the signaling molecules and PI3K activity are decreased, indicating that the response to a high dose of insulin is diminished in skeletal muscle in dwarf mice (Dominici et al., 2003). This finding may be linked to glucose intolerance in this mice strain because glucose clearance from blood is greatly dependent on glucose uptake in skeletal muscle (Saltiel and Kahn, 2001). This experimental setting of a high-dose insulin administration may not reflect the physiology of insulin signaling in

180

Isao Shimokawa

the longevity models because the plasma insulin concentration is expected to be lower in the longevity models during most of a feeding cycle as already described. In a basal not-fasting condition in the liver of Snell dwarf mice, the protein abundance of IR is reported to be similar to that in control mice, and the autophosphorylation of IR-␤ is also almost the same, although the proportion is increased in old age in Snell mice (Hsieh et al., 2002). IRS 2 protein in the liver is increased twofold in Snell mice, whereas PI3K activity associated with IRS-1 does not differ between Snell mice and control mice at a young age. Data on insulin signaling in longevity models are limited. Tissue-specific studies for the insulin signal pathway are needed because, as described previously, the fat-specific disruption of the insulin receptor gene affects glucose–insulin signaling and extends the life span in mice (Bluher et al., 2002, 2003).

D. Selected Parameters for Insulin Resistance The aging-related reduction in plasma GH causes an increase in fat tissue and a decrease in lean body mass in

(a)

aged animals (Toogood, 2003). One may predict that the reduced GH signaling facilitates aging-related accretion of fat mass and insulin resistance. However, Ames mice show a reduction of body fat relative to body weight (Heiman et al., 2003); this reduction is correlated to increased insulin sensitivity and thus to life span extension in those mice. In contrast, lit/lit and GHR/BP-KO mice exhibit a greater proportion of fat mass relative to body weight (Flurkey et al., 2001; Berryman et al., 2004). At a young age, the body fat content in anti-GH (tg/⫺)-AL rats did not differ from that in (⫺/⫺)-AL rats when normalized by body weight (Fig. 6a). CR also did not affect fat content at a young age. However, it was increased significantly in old age in both (tg/⫺) and (⫺/⫺) rats when fed ad libitum. Body fat content stayed constant with aging in the CR group of both (tg/⫺) and (⫺/⫺) rats. These data suggest that the moderate reduction of the GH–IGF-I axis in the present study does not affect body fat content. CR results in inhibition of an aging-related increment in fat content and thus maintains leanness in rodents. The plasma leptin level is known to reflect body fat content (Niswender and Schwartz, 2003). Changes by CR and

(b) 120 Leptin (ng/ml)

Fat (g/100 g BW)

10 8 6 4

80

40

2 0

Y

O

Y

(–/–)

0

O

Y

O

Y

(–/–)

(tg/–)

O (tg/–)

(c)

Adiponectin (µg/ml)

15 12 9 6

AL

3

CR

0

Y

O (–/–)

Y

O (tg/–)

FIGURE 6 Selected parameters for insulin resistance in anti-GH (tg/⫺) rats: Effects of aging and calorie restriction. (a) Body fat content represents the total wet weight of perirenal and epididymal fat normalized by body weight [g/100 g body weight (BW)]. Each bar and line represent the mean ⫾ SD of 9–17 rats. Three-factor ANOVA: Tg effect, not significant; age effect, p ⬍ 0.001; diet effect, p ⬍ 0.0001; age ⫻ diet, p ⬍ 0.0001. (b) Plasma leptin concentration. Each bar and line represent the mean ⫾ SD of 4–5 rats. Three-factor ANOVA: Tg effect, not significant; age effect, not significant; diet effect, p ⬍ 0.0001; age ⫻ diet, p ⬍ 0.01. (c) Plasma adiponectin concentration. Each bar and line represent the mean ⫾ SD of 5–14 rats. Three-factor ANOVA: Tg effect, p ⬍ 0.0001; age effect, not significant; diet effect, p ⬍ 0.0001; Tg ⫻ diet, p ⬍ 0.001.

181

13. Rodent Longevity Models

including neurons. Overexpression of the GH gene affects memory or learning ability in mice, probably through elevated reactive oxygen species (ROS) in the brain (Lemon et al., 2003). Therefore, stress resistance can be correlated with retardation of brain aging and extension of life span. Indeed, CR rodents exhibit resistance to many types of stressors, including toxic substances, inflammatory stimuli, ambient temperature, and surgery (Masoro, 1996). Efficient induction of stress proteins in response to cellular insults could be one of the mechanisms underlying the effect of CR (Heydari et al., 1993). p66shc⫺/⫺ mice and igf1r⫹/⫺ mice also show enhanced resistance to oxidative stress in vivo. The survival rate after intraperitoneal injection of paraquat, a ROS-generating herbicide, is increased in mice models (Migliaccio et al., 1999; Holzenberger et al., 2003). We also evaluated whether transgenic dwarf rats exhibit stress resistance by their response to lipopolysaccharide (LPS)-induced inflammatory challenge, which causes cellular injury by generation of ROS and reactive nitrogen species (RNS) (Van Amersfoort et al., 2003). Anti-GH (tg/) and control (⫺/⫺) rats at 6 months of age, fed ad libitum or 30% calorie-restricted from 6 weeks of age, were administered LPS (1.6 mg/kg body weight) from the tail vein and were sacrificed at 0, 1, 4, and 8 h after LPS administration. This dose of LPS induced foci of coagulation necrosis in the liver at 8 h in 50% of (⫺/⫺)-AL rats. Serum asparate aminotransferase levels were measured as an index of tissue damage caused by LPS. The experiment clearly demonstrated that (tg/-) rats were resistant to LPS-induced inflammatory challenge (Fig. 7). Interestingly, (tg/⫺) rats seemed to be more resistant to LPS than CR rats in this experimental setting. The molecular mechanisms involved in the stress response in longevity models remain to be elucidated. In vitro experiments using mouse embryo fibroblasts (MEFs) have also demonstrated that MEFs prepared from p66shc⫺/⫺ and

(–/–) Serum AST (IU/L)

aging in plasma leptin concentration in (tg/⫺) and (⫺/⫺) rats are correlated to alterations in body fat content (Fig. 6b); the leptin level appeared lower in (⫺/⫺)-CR rats at a young age when compared with (⫺/⫺)-AL rats, as previously confirmed the reduction of plasma leptin in young F344 rats (Shimokawa and Higami, 1999). Adiponectin is exclusively expressed in adipocytes and secreted into the circulation (Wolf, 2003). Of interest is that in obese and diabetic subjects, its expression in adipose tissues and the plasma level are reduced; adiponectin increases insulin sensitivity with activation of insulin signaling and glucose uptake. Administration of full-length adiponectin lowers plasma glucose concentrations by hepatic gluconeogenesis in obese and diabetic mice. FIRKO mice with increased insulin sensitivity also exhibit significantly greater levels of serum adiponectin (Bluher et al., 2002). The plasma concentration of adiponectin was elevated in anti-GH (tg/⫺) and CR rats (Fig. 6c), although the effect of CR was greater in the former, and aging did not significantly affect plasma adiponectin levels. These findings suggest that (1) the reduced GH–IGF-I axis increases the plasma adiponectin level, and this effect decouples from the body fat content; (2) the increased insulin sensitivity in (tg/⫺) and the CR group rats at a young age results partly from the upregulated level of plasma adiponectin; and (3) agingrelated insulin resistance might not be explainable by the plasma adiponectin level. Adipose tissues are now known to secrete proinflammatory cytokines, growth factors, and hormones, some of which affect insulin action and contribute to insulin resistance (Fasshauer and Paschke, 2003). For example, tumor necrosis factor-␣ suppresses tyrosine kinase phosphorylation of the insulin receptor, resulting in defects in insulin signaling and impaired glucose tolerance (Hotamisligil et al., 1993). Direct effects of leptin on insulin sensitivity in insulin-responsive tissues such as liver, muscle, and fat are contradictory (Fasshauer and Paschke, 2003). However, there is general agreement that leptin expression and secretion are increased in obesity and that a strong correlation exists between body fat stores and leptin plasma concentrations; thus leptin may have some indirect effects on insulin sensitivity. Characterization of plasma levels of adipocytokines and their roles in the glucose–insulin system in longevity models remain to be determined.

104 103

Aging is driven by oxidative stresses, and CR is hypothesized to retard aging by modulating oxidative stresses (Yu, 1996). Accumulation of oxidative damage in mitochondria insults cellular functions, the impairments of which are more critical in tissues composed of postmitotic cells,

AL CR

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IV. STRESS RESPONSE

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FIGURE 7 Stress response to lipopolysaccharide (LPS)-induced inflammation in anti-GH (tg/⫺) rats: The effect of calorie restriction. Rats were sacrificed 0, 1, 4, and 8 h after intravenous injection of a dose (1.6 mg/kg BW) of LPS. The serum asparate aminotransferase (AST) level was measured as an index of tissue injury after LPS injection. Each bar and line represent the mean ⫾ SD of four to seven rats. Three-factor ANOVA: Tg effect, p ⬍ 0.001; diet effect, p ⬍ 0.0001; time effect, p ⬍ 0.0001; Tg ⫻ time, p ⬍ 0.001.

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igf1r⫹/⫺ mice are resistant to ultraviolet- or H2O2-induced apoptosis (Migliaccio et al., 1999; Holzenberger et al., 2003). H2O2 induces oxidative damage by increasing intracellular levels of ROS. A series of experiments indicate that serine 36 phosphorylation in p66shc is critical for the activation of apoptosis in cells exposed to oxidative damage (Migliaccio et al., 1999). Activated p66shc causes phosphorylation and thus inactivation of FKHRL1, a transcription factor of the FoxO family, probably mediated by Akt activity (Nemoto and Finkel, 2002). Because FoxO family transcription factors upregulate gene expression levels of free radical scavenger enzymes (Van Der Heide et al., 2004), a lack or reduction of p66shc serine phosphorylation, which is observed in MEFs from p66shc⫺/⫺ or igf1r⫹/⫺ mice, can be linked to the enhanced resistance of cells to oxidative damage-induced apoptosis. Fibroblasts cell cultured from tail skin of Snell dwarf mice also show increased resistance to ultraviolet, heat, paraquat, H2O2, and cadmium, a toxic metal (Murakami et al., 2003). Of interest, the effect required an interval (⬃27 h) of culture in serum-free medium. Serum starvation increases intracellular oxidative stress (Nemoto and Finkel, 2002), suggesting the possibility that some protective mechanisms are induced efficiently in fibroblasts prepared from Snell mice skin during the period of serum starvation. However, the reduced rate of apoptosis in cell culture after oxidative damage is not simply correlated with increased longevity in animals, as efficient removal of damaged cells by apoptosis is also thought to be one of the mechanisms by which CR reduces the occurrence of neoplasms (James and Muskhelishvili, 1994). It has been shown that GHR/BP-KO male mice are more susceptible to paraquat toxicity in vivo (Hauck et al., 2002). Some studies have demonstrated that stress resistance can be decoupled with longevity in nematodes (Liang et al., 2003), although most long-lived mutants are stress resistant. Therefore, studies are needed to evaluate the role of stress response machinery in life span extension in longevity models.

V. CONCLUSION This chapter described some aspects of rodent longevity models in respect to the involvement of the GH–IGF-I/ insulin axis. Six of the eight models focused on here exhibited a reduction in plasma IGF-I or IGF-I signaling. However, in FIRKO and p66shc⫺/⫺ mice, there is no evidence of a reduction of the GH–IGF-I axis. Most of the models show a reduction in plasma insulin levels during the feeding cycle, although insulin sensitivity seems to be augmented. Some of the models are resistant to stresses, including oxidation, although one model, GHR/BP-KO mice, is susceptible to oxidative stress. At present, we cannot

determine whether the IGF-I or the insulin pathway or their cross talk is more essential for life span regulation. However, if mammals have obtained multiple and more complex signal systems for sensing nutritional status and eliciting adaptive responses during evolution, it is reasonable that many signaling molecules and related pathways would be involved in the regulation of aging and life span in mammals. In this sense, hypothalamic arcuate nucleus neurons, which sense nutritional status and eliciting neuroendocrine adaptive responses, may play a crucial role in the regulation of aging because they have receptors for insulin, IGF-I, GH, leptin, and ghrelin, which are considered molecules involved in informing details of nutritional states to the hypothalamus (de Graaf et al., 2004). It is hypothesized that CR induces alterations of the hormones specifically modulated by reduced energy intake. Therefore, the effect of CR could be greater than just modulation of a single molecule, as observed in the anti-GH (tg/⫺) rats (Shimokawa et al., 2003). Genetic models for these nutritional sensing molecules could serve as tools to dissect the molecular mechanisms underlying the antiaging effect of CR, and thus aging-related impairments in brain function.

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14 Growth Hormone and Insulin-like Growth Factor-I and Their Interactions with Brain Circuits Involved in Cognitive Function MELINDA RAMSEY and WILLIAM E. SONNTAG Department of Physiology and Pharmacology, Roena Kulynych Center for Memory and Cognition Research Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157

I. Introduction II. The Growth Hormone/Insulin-like Growth Factor-I (IGF-I) Axis III. Actions of Growth Hormone and IGF-I in the Mammalian Brain IV. Growth Hormone/IGF-I Axis and Brain Aging V. Summary References

age-related decreases in serum growth hormone and IGF-I and potential mechanisms that may influence cognitive function in the elderly population, but interventional studies are needed to establish a more definite link between the these hormones and function of the aging brain. In rodents, long-term growth hormone/IGF-I replacement improves hippocampal-dependent learning and memory in aged rats. While the exact mechanism underlying these cognitive improvements is unknown, growth hormone and IGF-I replacement to aged animals increased neurogenesis, vascular density, and glucose utilization and altered NMDA receptor subunit composition in brain areas implicated in learning and memory. Furthermore, IGF-I may regulate brain levels of A␤ peptide associated with neurodegenerative diseases such as Alzheimer’s disease. While these observations offer valuable insight into the influence of growth hormone and IGF-I on neuronal events in the aged mammal, additional functional studies are required to link these changes to cognitive improvements.

A vast literature supports the requirement of growth hormone and insulin-like growth factor-I (IGF-I) for normal development of both the mammalian body and brain. These hormones promote long bone and tissue growth and cardiovascular maturation and function and interact with other hormonal systems to regulate blood glucose levels and reproduction. Furthermore, IGF-I crosses the blood–brain barrier and promotes neurogenesis and synaptogenesis throughout development and during adulthood. The hormone protects neurons from chemical toxicity and cell death resulting from hypoxic-ischemic injury and reduces cell loss when administered postinsult. IGF-I is essential for normal dendritic development, and several groups have reported that IGF-I regulates excitatory synaptic transmission in a regionally specific manner. Nevertheless, studies of growth hormone treatment in children and adults with growth hormone receptor deficiency have yielded inconsistent results related to the benefits of this therapy to intelligence, learning, or memory. In recent years, much attention has focused on

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION Growth hormone and its anabolic mediator, insulin-like growth factor-I (IGF-I), have long been recognized for their critical roles in mammalian growth and development.

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However, these hormones affect numerous organ systems and participate in diverse processes such as wound healing and glucose homeostasis. Numerous studies have focused on the potential roles of growth hormone and IGF-I in brain development, neurogenesis, and neuroprotection. Only recently, however, have scientists explored the possible benefits of growth hormone and IGF-I to the aging brain. This chapter focuses on the potential mechanisms by which upregulation of the growth hormone/IGF-I axis improves learning and memory in aged rodents and humans.

II. THE GROWTH HORMONE/INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AXIS Pure bovine growth hormone was first isolated from the pituitary gland by Li et al. (1945) and was subsequently shown to stimulate fatty acid metabolism and amino acid uptake, as well as DNA, RNA, and protein synthesis (Corpas et al., 1993; Finkelstein et al., 1972; Steiger et al., 1992). These actions contribute to its regulatory role in cell division and tissue growth and, with the exception of fatty acid metabolism, occur via the anabolic mediator insulinlike growth factor-I. In humans, growth hormone is released in pulsatile bursts from the pituitary gland with the majority of secretion occurring nocturnally in association with slow-wave sleep (Corpas et al., 1993; Born et al., 1988; Tannenbaum and Martin, 1976). Similar pulses are observed in rodents, except that high-amplitude secretory pulses occur every 3.5 h in males (Tannenbaum and Martin, 1976) and hourly in females (Clark et al., 1987; Saunders et al., 1976). Regulation of these pulses involves at least two hormones released by the hypothalamus: growth hormonereleasing hormone (GHRH), which increases growth hormone release (Rivier et al., 1982; Brazeau et al., 1973, 1981), and somatostatin, which inhibits its release (Lanzi et al., 1994). It is generally believed that somatostatin tone is dominant during trough periods, whereas when somatostatin is suppressed, growth hormone is released in response to the secretion of GHRH (Tannenbaum and Martin, 1976). The dynamic interactions between these hormones are responsible for high-amplitude, pulsatile growth hormone secretion. Although the precise function of this ultradian pattern remains unknown, the pulsatile nature of growth hormone release has been confirmed in every species examined to date and appears to be essential to optimize biological potency of the hormone. Both growth hormone and IGF-I inhibit growth hormone release in a typical negative feedback manner either at the level of the pituitary or indirectly via stimulation of somatostatin and inhibition of GHRH release from

the hypothalamus (Berelowitz et al., 1981; Wallenius et al., 2001; Chomczynski et al., 1988; Bertherat et al., 1993; Kamegai et al., 1998). Extrahypothalamic regulation of growth hormone release can be achieved by growth hormone secretagogues (GHS), which act at GHS receptors in the hypothalamus and anterior pituitary to stimulate growth hormone release (Bona and Bellone, 2003). Ghrelin, a peptide secreted by the stomach endothelium, has been identified as an endogenous ligand for the GHS receptor and specifically stimulates growth hormone release from the anterior pituitary in vitro and in vivo (Kojima et al., 1999). Upon release from the anterior pituitary, growth hormone binds with high affinity to the growth hormone receptor found in tissues throughout the body (Fig. 1). Plasma growth hormone is carried by a growth hormone-binding protein, which is homologous to the cleaved extracellular domain of the growth hormone receptor (Zhou et al., 1997). The growth hormone molecule exhibits two binding sites for the growth hormone receptor, resulting in dimerization,

FIGURE 1 Growth hormone (GH), produced in the anterior pituitary, is modulated by two hypothalamic hormones: growth hormone-releasing hormone (GHRH), which stimulates both the synthesis and secretion of GH, and somatostatin (SS), which inhibits GH release in response to GHRH. GH also feeds back to inhibit GHRH secretion and probably has a direct inhibitory effect on secretion from the somatotroph (GH-producing cells). In mammals, GH is secreted in pulsatile bursts from the anterior pituitary gland, a pattern that is necessary to achieve full biological activity. GH binds with high affinity to its receptor, found in tissues throughout the body, and activation of this receptor stimulates the synthesis and secretion of insulin-like growth factor-I (IGF-I). Although 90% of circulating IGF-I is synthesized and secreted by the liver, many types of cells, including some found in the brain and vasculature, are capable of IGF-I production. Binding of the hormone to the IGF-I receptor causes potent mitogenic effects, including increases in DNA, RNA, and protein synthesis. Although heterogeneity exists in the processing of IGF-I mRNA, these transcripts appear to produce a single peptide that is homologous to the structure of proinsulin. Blood and tissue levels, as well as activity of the peptide, are regulated by IGF-I-binding proteins (IGFBP). Although it was initially proposed that all of the actions of GH were mediated through IGF-I, data from several studies support direct roles for GH in the regulation of lipolysis and insulin sensitivity that are independent of IGF-I. Adapted from Carter et al. (2002). (See color plate 15)

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a step that is required for biological activity of the hormone. Activation of the growth hormone receptor initiates the JAK-STAT signal transduction pathway commonly utilized by cytokine receptors. The stimulated receptor associates with the JAK2 tyrosine kinase with subsequent phosphorylation of both proteins (Roupas and Herington, 1989). Many other intracellular proteins are subsequently phosphorylated, including protein kinase C (PKC), mitogenactivated protein kinase (MAPK), insulin receptor substrate (IRS) proteins, and the signal transducers and activators of transcription (STAT) proteins (Gent et al., 2003). The result of growth hormone receptor activation is an increase in c-fos, c-jun, serine phosphatase inhibitor-1, IGF-I gene expression, and finally IGF-I synthesis and release (Xu and Sonntag, 1996). It should be noted that although early hypotheses proposed that all of the actions of growth hormone were mediated through IGF-I, several studies have provided relatively convincing data that growth hormone synergizes with IGF-I produced in tissue and/or has direct effects on specific tissues (Isaksson et al., 1988). For instance, growth hormone decreases insulin sensitivity, glucose uptake, and adiposity and increases lipolysis via an IGF-I-independent mechanism (Russell-Jones et al., 1993a,b; Dietz and Schwartz, 1991). IGF-I is a small peptide (about 7.5 kDa) structurally related to proinsulin. Most of the circulating IGF-I is derived from liver, but activation of the growth hormone receptor in tissues throughout the body promotes paracrine, or local synthesis, secretion, and action of IGF-I (Murphy and Friesen, 1988; Yamamoto and Murphy, 1995). For example, mice deficient in liver-derived IGF-I (LID mice) grow to normal size, presumably due to an increased synthesis of local IGF-I acting in a paracrine and/or autocrine manner. The absence of feedback inhibition by IGF-I in this model results in high levels of endogenous growth hormone that stimulates local IGF-I production and postnatal growth (Liu et al., 2000). About 1% of plasma IGF-I circulates in the free form (t1/2 is about 15–20 min), while the remainder is bound to specific binding proteins that prolong the half-life of the peptide (Janssen and Lamberts, 1999). Presently, six tissue-specific IGF-I binding proteins (IGFBP) have been identified and constitute an elaborate transport and regulatory system for IGF-I. IGFBPs are generally believed to decrease the availability of IGF-I to its receptor in target tissues (Daughaday and Rotwein, 1989), with specific proteases required to cleave the IGF-I peptide from its binding protein complex. For instance, IGFBP-3 is the major IGF-I binding protein in the blood, where it binds about 80% of the protein in a ternary complex that also includes acidlabile subunit (ALS). IGFBP-3 must be cleaved by specific proteases, which decrease affinity of the binding protein for IGF-I and cause release of the free hormone (Lalou et al.,

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1996; Angelloz-Nicoud and Binoux, 1995). In contrast, some reports indicate that IGFBPs can enhance IGF-I activity. IGFBP-5, for example, enhances IGF-I-mediated DNA synthesis and cell proliferation in vascular smooth muscle cells, whereas IGFBP-4 inhibits IGF-I activity in these cells (Duan and Clemmons, 1998). Furthermore, dephosphorylated IGFBP-1 is believed to promote IGF-I activity, while the phosphorylated form inhibits effects of IGF-I in a variety of cell types (Busby et al., 1988; Elgin et al., 1987; Yu et al., 1998). IGFBPs-2 and -6 are generally believed to inhibit IGF-I action, but are associated primarily with inhibition of IGF-II activity (Schneider et al., 2002; Sandhu et al., 2002; Firth et al., 2002). Free IGF-I binds the IGF-I receptor, the product of a gene found in nearly every mammalian tissue and cell type (Bondy et al., 1990). The IGF-I receptor is structurally similar to the insulin receptor and consists of two extracellular ␣ subunits, which form binding sites for IGF-I, and two transmembrane ␤ subunits, which contain tyrosine kinase and signal transduction domains (Kato et al., 1994). IGF-I binding initiates autophosphorylation of the IGF-I receptor as well as phosphorylation of other tyrosinecontaining peptides, including insulin receptor substrate-1 (IRS-1). Following the phosphorylation of IRS-1, the IGF-I signaling cascade is believed to activate two separate pathways: phosphatidylinositol-3 kinase (PI3-K) and ras/MAPK. Phosphorylated IRS-1 activates PI3-K, which in turn activates Akt kinase. Phosphorylated IRS-1 can also activate Shc, Grb, and Sos complex and culminates in the activation of ras, ␬-raf-1, MAPK kinase (MEK), mitogenactivated protein kinase (MAPK), and Erk-1 and -2 (Qiang et al., 2002; Scrimgeour et al., 1997; Kim et al., 1998).

III. ACTIONS OF GROWTH HORMONE AND IGF-I IN THE MAMMALIAN BRAIN While the peripheral actions of growth hormone and IGF-I have been explored extensively, the contributions of these hormones to brain development, neuronal differentiation, and cognitive function are less completely understood. However, current literature suggests that IGF-I is essential for normal brain development and promotes neurogenesis. Furthermore, in a number of in vitro and whole animal studies, the peptide has been reported to possess prosurvival and antiapoptotic actions by regulating the activity and expression of proteins involved in cell survival and apoptosis. Neuroprotective effects and neuronal rescue have also been demonstrated in vitro utilizing chemical insults, as well as in vivo models of hypoxic-ischemic (HI) injury. Electrophysiological studies have also revealed the ability of IGF-I to acutely regulate synaptic transmission.

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A. Endocrine, Paracrine, and Autocrine Sources of Growth Hormone and IGF-I Act on the Brain Although the brain, with the exception of the hypothalamus, has not typically been considered a target for growth hormone action, autoradiography has demonstrated the expression of growth hormone receptors in the choroid plexus, hippocampus, pituitary, and spinal cord in rodents, with reduced receptor density observed in the cortex (Zhai et al., 1994). Growth hormone receptors have also been observed in human choroid plexus (Lai et al., 1991), and growth hormone receptor mRNA has been observed in human brain stem (Mercado et al., 1994) and astrocytic tumors (Castro et al., 2000). While the main source of growth hormone is the anterior pituitary gland, brain tissues appear to produce small amounts of the hormone that can act at receptors in local tissues. Therefore, paracrine growth hormone action coexists with pituitary-derived circulating growth hormone, which may be transported across the blood–brain barrier via receptor-mediated transcytosis (Coculescu, 1999). Using coronary artery perfusion techniques in rats, Reinhardt and Bondy (1994) demonstrated that liver-derived circulating IGF-I crosses the blood–brain barrier and can then bind IGF-I receptors believed to be expressed throughout the brain, although concentrated in the superficial and deep cortical layers, olfactory bulb, amygdala, thalamic nuclei, and hippocampus (Araujo et al., 1989; Reinhardt and Bondy, 1994; Lesniak et al., 1988; Dore et al., 1997b; Breese et al., 1991; Bondy et al., 1992). Substantial evidence also shows that brain tissue produces IGF-I, which can also act in a paracrine or autocrine fashion (Schneider et al., 2003). Brain expression of growth hormone and IGF-I, as well as their receptors, is modulated by a variety of factors outside the growth hormone/IGF-I axis. For instance, Fujikawa et al. (2000) demonstrated that, in response to acute stress, growth hormone receptor mRNA expression paralleled increases in plasma glucocorticoid levels and mRNA levels of glucocorticoid and mineralocorticoid receptors in rat dentate gyrus (Fujikawa et al., 2000). Furthermore, transgenic mice overexpressing the cytokine tumor necrosis factor (TNF-␣) demonstrate decreased abundance of IGF-I in the cerebellum, cerebral cortex, and diencephalon. Increased expression of IGFBP-3 and decreased levels of IGFBP-5 were also observed in cerebellar tissue of these animals when compared to wild type (Ye et al., 2003). Several investigators have hypothesized that the developmental expression of some IGFBPs may affect IGF-I synthesis in the brain. For instance, spatiotemporal correlations in the expression of IGFBP-2 and -5 mRNA with IGF-I mRNA in the developing rat brain have been reported (Lee et al., 1992; Bondy and Lee, 1993). In summary, the growth hormone/IGF-I axis is important to structures throughout the brain, and

expression of its components is regulated by one another and regulates a number of other endocrine and nonendocrine factors.

B. IGF-I Promotes Neurogenesis from Early Stages of Development 1. Perinatal Studies Growth hormone has been identified via immunoassay in fetal rat brain as early as day 10 of gestation (Hojvat et al., 1982), and Ajo et al. (2003) reported the expression of mRNA for growth hormone and IGF-I receptors as well as that encoding the IGF-I peptide in cerebral cortical cultures at embryonic day 14 (E14). As part of the same study, it was reported that incubation of E17 cells with growth hormone increased cell proliferation as determined by total cell number, [3H]thymidine incorporation, levels of proliferating cell nuclear antigen (PCNA), and bromodeoxyuridine (BrdU) staining. Growth hormone incubation also induced neuronal differentiation as determined by the quantification of ␤-tubulin/BrdU-positive cells and ␤-tubulin protein expression and by the development of astrocytes as determined by the quantification of glial fibrillary acidic protein (GFAP)/BrdU-positive cells and GFAP expression. These effects were blocked by IGF-I antisera, suggesting that growth hormone-induced changes in neuronal proliferation and differentiation were mediated by actions of IGF-I (Ajo et al., 2003). 2. Transgenic Studies Results obtained from transgenic studies further support an essential role for growth hormone and IGF-I in mammalian brain development. For example, Carson et al. (1993) utilized transgenic mice overexpressing IGF-I to demonstrate that at postnatal day 55, transgenic brains were 55% larger than controls and exhibited increases in both cell size and number. Transgenic brain tissue also exhibited an average of 130% increase in total myelin content (Carson et al., 1993). Conversely, 2-month-old IGF-I-/- mic demonstrated reduced volume of white matter structures due to decreased numbers of axons and oligodendrocytes. IGF-I null mice also possessed a greater reduction in myelinated axons than unmyelinated axons (Beck et al., 1995). More recent studies in transgenic mice carrying an IGF-II promoter-driven IGF-I transgene (IGF-II/I Tg mice) highly expressed in postnatal brain also suggested an important role for IGF-I in developmental neurogenesis and synaptogenesis. Stereological analyses of the dentate gyrus were performed in these animals and compared to nontransgenic littermates from postnatal days 7 to 130. During this early postnatal period, granule cell layer and molecular layer volumes, total neuron number, and synapse-to-neuron ratio were increased significantly in the dentate gyrus of

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transgenic mice as compared to nontransgenic controls (O’Kusky et al., 2000). Mice carrying the IGF-II/IGF-I transgene highly expressed in cerebellum exhibited significant increases in cerebellar weight (90%) and DNA content (143%) by 50 days of age. These transgenic mice also demonstrated significant increases in total number of cerebellar granule cell progenitors, cerebellar granule cells, Purkinje cells, external granular layer cells, and BrdUlabeled external granular cells compared to nontransgenic littermates (Ye et al., 1996). Similarly, mice expressing high levels of IGF-I in medulla exhibit increased total medullar volume at all ages studied, and morphometric analyses of the medulla revealed significant increases in volume and neuron number in the nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV), but only increased volume in the hypoglossal nucleus (HN) and facial nucleus (FN) of transgenic mice. Motor neurons in the DMV, HN, and FN of transgenic mice also exhibited morphological changes consistent with increased neuritic outgrowth (Dentremont et al., 1999). Clearly, transgenic technology has contributed substantial evidence for the necessity of IGF-I action in normal brain growth. 3. Exercise and IGF-I IGF-I has also been shown to exert neurogenic and synaptogenic effects in adult rodents and may be responsible for beneficial effects associated with exercise (Anderson et al., 2002). Aberg et al. (2000) administered IGF-I to adult hypophysectomized rats via subcutaneous injection for 6 days and observed an increase in the proliferation of progenitor cells in the dentate granule cell layer (GCL) of the hippocampus. Furthermore, a 20-day IGF-I treatment resulted in a 78 ⫾ 17% increase in newly generated GCL neurons (Aberg et al., 2000). Additional reports suggest that physical activity in rodents increases the expression of neurotrophic factors and early response genes, improves spatial memory, and increases neurogenesis and long term potentiation (LTP) in the dentate gyrus of hippocampus (Gomez-Pinilla et al., 2002; van Praag et al., 1999). Carro et al. (2000) postulated that circulating IGF-I modulates these responses to exercise and reported that 1 h of running stimulates uptake of circulating IGF-I by neurons in the cortex, hippocampus, striatum, and a variety of other areas of rat brain. The increased accumulation of IGF-I was accompanied by an increased spontaneous firing and a prolonged increase in sensitivity to afferent stimulation. The authors also used an antiserum to IGF-I and an IGF-I receptor blocker in combination to prevent IGF-I uptake during exercise. This intervention also attenuated the exerciseinduced increase in c-Fos protein abundance throughout the brain (Carro et al., 2000). The same group later found that a subcutaneous administration of IGF-I to sedentary adult rats

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for 7 days increased hippocampal neuron number, and use of an IGF-I antiserum showed that an uptake of circulating IGF-I is required for the exercise-induced increase in BrdU⫹ hippocampal granule cells (Trejo et al., 2001). In summary, these reports emphasize the importance of growth hormone and IGF-I to neural development and healthy adult brain function. Published studies indicate that these hormones begin exerting neurogenic actions during embryonic development, and studies in vitro confirm that IGF-I promotes the growth of neurons and astrocytes. Furthermore, transgenic mice overexpressing IGF-I demonstrate increased brain mass, whereas IGF-I-null mice have deficits in myelination. Evidence also supports a role for IGF-I in adult neurogenesis, particularly in the granule layer of the dentate gyrus, and evidence suggests that an increased uptake of IGF-I may mediate the increased neurogenesis and cognitive benefits associated with exercise.

C. IGF-I Exerts Prosurvival/Antiapoptotic Actions via Several Mechanisms 1. Studies in Vitro In addition to supporting neurogenesis, IGF-I acts to promote survival and block apoptosis in both developing and adult brains. For instance, organotypic cortical slices obtained from rats at postnatal day 4 and incubated in IGF-I (200 ng/ml) for 24 h demonstrated significantly decreased average cell death index (number of TUNEL-positive neurons divided by total number of cells in a counting frame) across cortical layers II/III, IV, V, and IV (Niblock et al., 2001). Wilkins et al. (2001) demonstrated that oligodendrocyte precursors (OPCs) and differentiated oligodendrocytes promote the survival of cultured cortical neurons via contact-mediated and soluble mechanisms and used IGF-I antisera to show that IGF-I contributes to the trophic actions of these support cells. These results are consistent with earlier findings, including the report that the addition of IGF-I to serum-free cultures of embryonic cerebellar neurons increased the number of neurite-bearing cells in a dose-dependent manner. Neuronal survival was increased by about 100% after incubation in 10⫺9 M IGF-I, an effect that was attenuated by the addition of an antibody to the IGF-I receptor (Torres-Aleman et al., 1994). The addition of IGF-I (25 ng/ml) to culture media also prevented the induction of apoptosis that normally occurs in cerebellar granule neurons switched from media containing high to lower levels of potassium (D’Mello et al., 1993). The aforementioned studies offer substantial evidence of the trophic effects that IGF-I exerts on neuronal cells in culture. Studies published from the mid-1990s to the present have explored possible signaling mechanisms behind the prosurvival and antiapoptotic actions of IGF-I and other growth factors. In a pivotal study, Dudek et al. (1997)

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observed that IGF-I induced activation of PI3-K via binding of PI3-K to IRS-1 in embryonic (E6–E8) cerebellarcultured neurons. IGF-I also induced the activation of Akt, an event that was blocked by wortmannin, and therefore required PI3-K. The same cells were transfected with wild-type and mutant forms of Akt in order to demonstrate the necessity of Akt for growth factor-dependent survival. Furthermore, investigation of a possible role for another PI3-K target, p70 ribosomal protein S6 kinase (p70s6K), revealed that this protein is not involved in PI3-K dependent survival. It was also reported that neither insulin nor IGF-I activates MAPK in cerebellar cultures, and the authors concluded that activation of the ras/MAPK pathway is not required for the survival-promoting effects of IGF-I (Dudek et al., 1997). These findings are consistent with those of Miller et al. (1997), who demonstrated that PI3-K activity is essential for the survival-promoting effects of IGF-I in serum-free cultures of cerebellar granule cells obtained from 7-day-old Sprague–Dawley rats (Miller et al., 1997). Additionally, Yamada et al. (2001) reported that a 5-min incubation of embryonic cerebral cortical neurons in IGF-I (100 ng/ml) induced a threefold increase in PI3-K activity, as well as a significant increase in Akt activation, an effect that was dependent on PI3-K activity. The survival-promoting effects of IGF-I were partially attenuated by the coaddition of LY294002, an inhibitor of PI3-K, to the culture medium (Yamada et al., 2001). The contribution of PI3-K/Akt signaling to the regulation of cell survival has been confirmed in a number of other neuronal and PC-12 cell models (Zheng et al., 2002; Kumari et al., 2001; Datta et al., 1997; Ryu et al., 1999). Datta et al. (1997) also used transfection techniques in vitro to demonstrate that IGF-I induces phosphorylation of the proapoptotic protein Bad, preventing Bad-induced apoptosis in cerebellar granule cell cultures (Datta et al., 1997). Similar methods were used to demonstrate that Akt activation is required for the inactivation of Bad, a finding supported by several other reports (Peruzzi et al., 1999; Cardone et al., 1998). Another study utilizing the same cells demonstrated that rat cerebellar granule neurons incubated in serum-free media with IGF-I demonstrated inhibition of caspase-3 and caspase-9 activation via PI3-K, preventing the release and redistribution of cytochrome C from the mitochondria. IGF-I also blocked induction of the Bcl-2-interacting member of cell death (Bim) via a signaling mechanism that included the activation of PI3-K but not the inhibition of c-Jun. Consistent with other reports, IGF-I also induced a sustained increase in Akt activity and prevented the dephosphorylation and nuclear translocation of the proapoptotic transcription factor FKHRL1 (Zheng et al., 2002). Previous findings have demonstrated that FKHRL1 regulates the expression of Bim (Dijkers et al., 2000), which led Linseman et al. (2002) to conclude that IGF-I causes activation of PI3-K and Akt and Akt prevents

transcription modulation by FKHRL1, which subsequently suppresses the induction of Bim and promotes cell survival (Linseman et al., 2002). These findings are consistent with those of Brunet et al. (1999), who used cerebellar granule neurons to demonstrate that, when allowed to translocate from cytoplasm to nucleus, FKHRL1 can cause induction of the Fas ligand gene, another gene with central involvement in apoptotic cell death. The aforementioned reports suggest that IGF-I may promote neuronal survival and inhibit apoptosis through a number of mechanisms. While a network of links between some seemingly independent mechanisms may exist, it is clear that the PI3K/Akt branch of the IGF-I signaling cascade is involved and that the regulation of downstream elements, including the Bcl-2 family of proteins, the transcription factor FKHRL1, and enzymes including the proapoptotic caspase-3, is critical to the antiapoptotic properties of the hormone. 2. In Vivo Studies Investigators are currently examining signaling elements downstream of PI3-K and Akt, specifically the BCL-2 family of proteins, for possible involvement in survivalpromoting effects of growth factors. The BCL-2 family includes both prosurvival (e.g., Bcl-2 and BAG-1) and proapoptotic proteins (e.g., Bad and BAX). The results of many studies have led to the conclusion that the addition of growth factors to neuronal cultures promotes survival and inhibits apoptosis by altering the balance in expression or activation of prosurvival and proapoptotic signaling elements. For instance, Frago et al. (2002) demonstrated that administration of growth hormone or GHRP-6 to rats for 1 week caused significant increases in IGF-I mRNA expression in the hypothalamus, hippocampus, and cerebellum. Parallel increases in Akt activation, inactivation of the proapoptotic protein Bad, and increased abundance of the antiapoptotic protein Bcl-2 further supported the hypothesis that shifts in the expression and activity of a number of proteins mediate the survival-promoting effects of growth hormone, IGF-I, and other growth factors (Frago et al., 2002). Transgenic technology has also aided investigation into the signaling mechanisms by which IGF-I promotes cell survival. Transgenic mice overexpressing IGF-I in the central nervous system exhibit significantly fewer apoptotic cerebellar neurons at postnatal day 7 (P7) than wild-type mice, with similar trends observed at P14 and P21. At P7, P14, and P21, Western immunoblotting revealed that transgenic cerebella exhibited decreased expression of procaspase-3 and caspase-3, as well as poly(adP-ribose) (PARP), a cleavage fragment and indicator of caspase activity. Cerebella from transgenic mice also showed increased expression of the antiapoptotic protein Bcl-XL at P7 and decreased expression of both Bax and Bad proteins at P14 and P21 (Chrysis et al., 2001). Baker et al. (1999) used immunohistochemistry to demonstrate that Bcl-2 protein

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levels are increased in cerebellar Purkinje cells obtained from 3-day-old transgenic mice overexpressing IGF-I in brain. The same authors also reported a converse relationship in which newborn transgenic mice overexpressing Bcl-2 protein in mitral cells of the olfactory bulb demonstrate greater expression of IGF-I and increased survival in serum-free culture compared to those obtained from wild-type mice (Baker et al., 1999).

D. IGF-I Imparts Neuroprotective Effects Pre- and Postinjury 1. In Vitro Studies In addition to establishing prosurvival and antiapoptotic actions of IGF-I on neuronal cells in serum-free media, cell culture systems have been utilized in the study of the neuroprotective effects of IGF-I in response to chemical toxicity. For instance, Leski et al. (2000) demonstrated that the addition of IGF-I (5–100 ng/ml) to serum-free media prevents the development of kainate sensitivity in cerebellar granule cell cultures that generally results 5–6 days after serum withdrawal. Wortmannin and LY294002, as well as rapamycin, were used to demonstrate that the neuroprotective effects of IGF-I were dependent on PI3-K and p70S6k but not MAPK (Leski et al., 2000). Furthermore, glutamate-mediated apoptosis was attenuated for at least 48 h when immature prooligodendroblasts (pro-OLs) were coincubated with IGF-I (10 ng/ml). Western blots revealed that the prolonged protective effects of IGF-I were associated with sustained IGF-I receptor and Akt activation, as well as inhibition of caspase-3 activation (Ness and Wood, 2002). Similarly, Takadera et al. (1999) reported that IGF-I (50 ng/ml) treatment prevented MK-801-induced apoptosis in primary cerebral cortical cells, an effect that coincided with attenuation of caspase-3 activation. These results indicate that the same antiapoptotic signaling that allows IGF-I to protect neurons from death induced by serum-free media also plays an important role in the neuroprotective actions of IGF-I against chemical insults. 2. Hypoxic-Ischemic Injury Many investigators have also focused on a role for the growth hormone/IGF-I axis in attenuating neuronal damage that occurs in HI injury. Guan et al. (1996) reported that HI injury can enhance the transport of IGF-I from the cerebrospinal fluid (CSF) to the cerebrum via perivascular spaces and white matter tracts, including the corpus callosum and external capsule. The same group studied the potential protective effects of IGF-I during perinatal asphyxia in near-term fetal sheep. IGF-I was infused icv (3 ␮g over 1 h) 90 min after 30 min of carotid artery occlusion, and the animals were allowed to recover for 4 days. Tissue sections from IGF-I-treated animals exhibited a

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significantly reduced loss of striatal neurons compared to those obtained from vehicle-treated controls, and immunostaining techniques revealed that the majority of rescued neurons were associated with cholinergic and GABAergic systems (Guan et al., 2000a). It was later reported that a single dose of IGF-I (50 ␮g) delivered icv to rats 2 h after HI injury reduced somatosensory deficits up to 20 days postinjury compared to vehicle-treated animals. IGF-Itreated animals also exhibited significantly reduced neuronal loss 20 days postinjury (Guan et al., 2001). Guan et al. (2000b) also reported that recovery from HI injury in a cool environment (23 °C) extended the time window for effective IGF-I-mediated (50 ␮g) rescue of cortical neurons from 2 h at 31 °C to 6 h. Guan et al. (1996, 2000, 2001) have thus established the benefits of IGF-I treatment in neuronal survival post-HI injury. However, mechanisms by which IGF-I accomplishes the observed neuroprotection are not as clear. In an effort to elucidate the intracellular signaling that must occur in order for IGF-I to exert neuroprotective effects, Chavez and LaManna (2002) studied the interaction between IGF-I and hypoxia-inducible factor-1 (HIF-1), a transcription factor believed to influence adaptive responses to hypoxic conditions. Northern and Western blotting, as well as immunostaining methods, were used to determine that 7-day icv (0.25 ␮g/h) or systemic administration of IGF-I (a dose sufficient to increase plasma IGF-I levels twofold) was able to induce HIF-1␣ and HIF-1 target genes in rat cerebral cortex. The IGF-I-mediated increases in HIF-1␣ mimicked those achieved by a 4-h exposure to hypoxic conditions (8% O2), as well as those observed 4 days following cardiac arrest and resuscitation. It was also reported that IGF-I protein levels were increased nearly four-fold 2–7 days following cardiac arrest and resuscitation and that icv infusion of the IGF-I receptor antagonist JB-1 during the 4 days following the injury attenuated HIF-1␣ accumulation. The authors concluded that HIF-1 activation may mediate some neuroprotective actions of IGF-I in response to ischemia (Chavez and LaManna, 2002). Sizonenko et al. (2001) hypothesized that the N-terminal tripeptide of IGF-I (GPE) mediates some of the neuroprotective effects of IGF-I. Unilateral HI injury was induced in 21-day-old rats via carotid artery ligation and exposure to 8% O2, followed by infusion of GPE into the injured lateral ventricle (30 ␮g) or peritoneal cavity (ip 300 ␮g). Animals that received GPE via either route exhibited significantly increased neuronal survival in the CA1 region of hippocampus, while only ip injection protected neurons of the lateral cortex. Furthermore, icv injection of [3H]GPE revealed binding mostly in glial cells, as opposed to neurons, leading the authors to postulate that the neuroprotective effects of GPE may be mediated by glial cells (Sizonenko et al., 2001). It is not clear whether des(1–3)-IGF-I, which lacks the GPE peptide, provides neuroprotective benefits.

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Consistent with hypothesized involvement of glial cells in IGF-I-mediated neuroprotection, Cao et al. (2003) determined that icv infusion of IGF-I (3 ␮g) to near-term fetal sheep 90 min after ischemic injury prevents loss of oligodendrocytes due, in part, to decreased caspase-3 activation in these cells. Immunohistochemical analysis of cortical tissue obtained from IGF-I-treated animals revealed increases in proliferation of oligodendrocytes, astrocytes, and microglia, leading the authors to conclude that both antiapoptotic and proliferative actions of IGF-I on these support cells may mediate the protective effects of the hormone on white matter (Cao et al., 2003). Additional authors have reported induction of IGF-I and IGFBPs in microglia and astrocytes surrounding brain lesions in response to HI and other injuries (O’Donnell et al., 2002; Beilharz et al., 1998). 3. Neuronal Rescue While the aforementioned studies focused on the neuroprotective properties of IGF-I, many experiments have been designed to determine whether IGF-I plays a role in neuronal rescue postinsult. Fernandez et al. (1997) used 3acetylpyridine to inflict partial deafferentation of the cerebellar cortex and employed a peptide antagonist to the IGF-I receptor and an antisense oligonucleotide to IGF-I to demonstrate that IGF-I receptor activation is required for injury-induced increases in neuronal c-fos protein expression. An increase in reactive gliosis was also observed when IGF-I signaling was disrupted (Fernandez et al., 1997). The same group later determined that subcutaneous or intraventricular administration of IGF-I rescued neurons of the inferior olive and resulted in full recovery of motor function in rats rendered ataxic by the 3-acetylpyridine approach (Fernandez et al., 1998). The ability of IGF-I to rescue injured neurons and reduce cell death also extends to tissue damaged by HI events. Liu et al. (2001) reported that intranasal administration of IGF-I to rats after onset of a 2-h middle cerebral artery occlusion and at 24 and 48 h postocclusion significantly reduced infarct volume and improved performance in neurologic tests requiring motor, sensory, reflex, and vestibulomotor functions. Additionally, Scheepens et al. (2001) reported increases in growth hormone immunoreactivity in injured neurons and glial cells after moderate and severe HI injury. Furthermore, growth hormone (20 ␮g) administered icv 2 h following HI injury reduced neuronal loss in areas of the cortex, hippocampus, and hypothalamus, a pattern of neuroprotection that correlates well with the distribution of the growth hormone receptor in the rat brain. However, areas afforded the most protection by growth hormone treatment were not the same areas protected by IGF-I infusion, suggesting that growth hormone may have neuroprotective actions that do not involve IGF-I (Scheepens et al., 2001).

4. Transgenic Studies One transgenic model has also been used in the investigation of the abilities of IGF-I to protect and rescue neurons from nutritional deficits. Ye et al. (2000) used transgenic mice that overexpress IGF-I in brain beginning shortly after birth to demonstrate that IGF-I protects myelin from postnatal undernourishment. Brain weights of transgenic mice undernourished from postnatal day 1 (P1) to P20 were comparable to well-fed control animals and significantly increased compared to undernourished control mice. Undernourished transgenic animals exhibited significant increases in mRNA for myelin basic protein (MBP) and proteolipid protein (PLP) compared to undernourished control mice, a finding that was consistent with subsequent Western blotting analysis. The authors also used immunostaining to demonstrate significantly increased numbers of oligodendrocytes and their precursors in the cerebral cortex and corpus callosum of undernourished transgenic mice as compared to undernourished control animals. Overall cell numbers were similar between the two groups, and the authors concluded that IGF-I protects against neuronal insult, in part, via the maintenance of myelination (Ye et al., 2000). 5. Behavioral Studies Behavioral assays have also been utilized to assess the extent of functional neuroprotection afforded by IGF-I. Carro et al. (2001) hypothesized that IGF-I mediates the neuroprotective effects of exercise and subjected adult rodents to neuronal insults of the hippocampus (domoic acid in mice) and brain stem (3-acetylpyridine in rats). The pcd mouse model of inherited cerebellar Purkinje cell degeneration was also utilized. Brain-damaged animals were divided into a sedentary or exercising group, which ran 1 km/day on a treadmill 15 days before, 4–5 weeks after, or before and after brain insult. The hippocampallesioned animals were subsequently evaluated in a water maze procedure designed to assess spatial memory, whereas the brain stem-lesioned and pcd mice were evaluated in a rotarod test for motor coordination once every week. Exercise improved spatial memory acquisition and retention in hippocampal-lesioned mice and attenuated the loss of motor coordination in hippocampal- and brain stemlesioned animals, respectively. Five weeks of treadmill exercise following brain injury also induced the recovery of motor coordination to control levels in brain stem-lesioned rats, whereas the motor performance of pcd rats was comparable to normal rats after only 1 week of exercise. Chronic subcutaneous administration of a blocking antiIGF-I antibody to exercising animals abrogated the protective effects of exercise on cognitive and motor performance. It was later determined that antibody-treated exercising animals exhibited neuronal loss comparable to that observed

14. Growth Hormone, IGF-I, and Cognitive Function

in sedentary animals as determined by calbindin staining in the brain stem and Purkinje cells and by Nissl staining in the hippocampus. The authors suggested that the antibody blocked the increased brain uptake of IGF-I associated with exercise. Chronic IGF-I treatment also caused increases in glucose uptake in the hippocampi of domoic acid-lesioned mice similar to the increases in glucose uptake that occur with exercise (Carro et al. 2001). These results support earlier conclusions by the same group (Carro et al., 2000) and establish IGF-I as a mediator of some effects of physical activity on the brain. The reports by Carro et al. (2000, 2001) discussed earlier are also consistent with the Saatman et al. (1997) report that IGF-I administered via subcutaneous injection (1 mg/kg) at 12-h intervals for 14 days following moderate fluid percussion (FP) brain injury attenuated loss of motor function in rats. Rats implanted with subcutaneous pumps delivering IGF-I (4 mg/kg/day) for 2 weeks post-FP injury exhibited improved coordinated motor function compared to vehicle-treated rats. IGF-I-treated animals also demonstrated improvements in spatial memory as assessed by a reduced latency to reach an escape platform and improved probe trial performance in the Morris water maze (Saatman et al., 1997). The aforementioned studies of the effects of IGF-I treatment on cultured cells exposed to chemical toxins, as well as whole animal models of HI and other injuries, offer substantial support for neuroprotective properties of this hormone, both pre- and postinsult. Findings indicate that the protective effects of this peptide in the brain may depend on inhibition of caspase-3 and Bad, as well as activation of HIF-1 and NF-␬B. Reports also suggest a role for nonneuronal support cells in the neuroprotective processes and indicate that protection of myelination and increased glucose uptake may lead to increased neuronal survival with IGF-I treatment. Future studies may identify the signaling pathways that allow growth hormone to impart neuroprotective effects independent of IGF-I induction.

E. IGF-I Affects Synaptic Structure, Function, and Strength 1. Developmental Effects In addition to neuroprotective and neurogenic actions, many investigators have reported effects of IGF-I on elements that affect synaptic structure and function, particularly in the developing brain. IGF-I gene expression was examined in developing rats utilizing in situ hybridization, and results indicate that IGF-I mRNA is abundant in long axon projection neurons throughout the brain, but appears during the later stages of development, during a period that accompanies the maturation of dendrites and synapse formation (Bondy, 1991). Functional studies confirm a role

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for IGF-I in synaptic development. For example, Kelsch et al. (2001) utilized whole cell patch clamp recordings in cultured embryonic hippocampal neurons to determine that the maturational transition to outward Cl⫺ transport via the KCC2 transporter is accelerated by the stimulation of IGF-I receptors. Furthermore, the KCC2 transporter can be activated rapidly by conjoint application of IGF-I and Src kinase, an effect that is blocked by tyrosine kinase inhibitors. The authors concluded that IGF-I works in conjuction with cytosolic tyroine kinase activity to promote the transition to neuronal K⫹/Cl⫺ cotransport via KCC2. This transition is required for the maintenance of low intracellular Cl⫺ concentrations, allowing the hyperpolarizing influx of Cl⫺ mediated by the GABAA receptor in the postnatal brain (Kelsch et al., 2001). 2. Dendritic Structure Investigators have also examined the actions of IGF-I on dendritic architecture in both cortical and cerebellar structures. For example, Niblock et al. (2000) used organotypic slices of rat primary somatosensory cortex to demonstrate that a 24-h incubation in IGF-I (200 ng/ml) increases branching and total length of both apical and basal dendrites of pyramidal cells (Fig. 2). The authors emphasize that IGF-I treatment did not result in increased somal size and, therefore, results are not due to overall stimulation of growth. The specific regulation of dendritic elaboration by IGF-I may indicate an important role for the hormone in the formation of cortical connections during development (Niblock et al., 2000). Cheng et al. (2003) studied somatic and dendritic growth in early postnatal IGF-I-null mice and reported an increase in pyramidal neuron density and a decrease in soma size in the frontoparietal cortex. Reductions in dendritic length, dendritic spine density, and overall dendritic arbor complexity were reported for cortical layers II and III of the mutant as compared to wildtype mice. Cortical levels of CDC42, a protein involved in neurite growth, were also reduced in mutant mice. A 30% reduction in total brain synaptotagmin levels in the brains of IGF-I-null mice supported the hypothesis of a reduction in synapses in IGF-I-null mice. Biochemical analyses revealed a 10% reduction in both cholesterol and phospholipids levels, as well as significant reductions in lipogenic enzymes in the IGF-I-null brain. The authors concluded that alterations in lipid metabolism may contribute to impaired neuronal somatic and dendritic growth in this transgenic model (Cheng et al., 2003). In a study that utilized adult rats, Nieto-Bona et al. (1997) reported that injection of IGF-I antisense into the inferior olive nucleus resulted in significant reductions in the size and density of dendritic spines in the same region, without affecting density of synapses or climbing or parallel fiber terminals. The authors suggest that IGF-I derived from the inferior olive is important to the well-documented

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FIGURE 2 Quantitative effects of growth factors on dendritic branching and total dendritic length. (A) IGF-I elicited a 75% increase in the mean number of branches in the apical dendritic arbor (*p ⬍ 0.05, SNK). (B) IGF-I produced a 50% increase in the mean total length of the apical arbor ( p ⬍ 0.05, SNK). (C) Plotting the total apical dendritic length for each control and IGF-I-treated neuron against its rank (within the treatment group) revealed the wide range in measured values and suggested that even the largest layer 2 neurons responded to IGF-I treatment with an increase in apical dendritic length. (D) Each of the growth factors tested elicited an increase in the mean number of branches within the basal dendritic arbor ( p ⬍ 0.05, SNK). (E) It appeared that each factor also produced an increase in total basal dendritic length. When all neurons in each group were compared, however, differences failed to reach statistical significance ( p ⬎ 0.05, ANOVA). (F) Plotting the total length for each neuron against its rank indicated that there was an upper limit on basal length at which the values for control and treated neurons converged. After eliminating the two greatest values in each group (e.g., neurons that were presumably already near the limit before treatment; values in box), a second ANOVA indicated a significant effect of treatment, and post hoc tests demonstrated significant increases in mean length in response to each factor ( p ⬍ 0.05, SNK). From Niblock et al. (2000).

14. Growth Hormone, IGF-I, and Cognitive Function

transynaptic trophic action of climbing fibers on Purkinje cells (Nieto-Bona et al., 1997). A more recent study of developing Purkinje cells supports this conclusion. Kakizawa et al. (2003) reported that delivery of the mouse IGF-I peptide (100 pg/mg cerebellar tissue) or human recombinant IGF-I (100 ng/mg cerebellar tissue) to cerebellar tissue of neonatal mice in vivo promotes the maintenance of multiple innervation of Purkinje cells by climbing fiber afferents from the inferior olive nucleus. However, application of antisera to IGF-I or its receptor decreased the degree of multiple climbing fiber innervation, suggesting that the absence of IGF-I action promotes their elimination. These findings indicate that IGF-I influences the normal transition from multiple to monoinnervation of Purkinje cells that occurs during development, promoting the survival of climbing fiber synapses (Kakizawa et al., 2003). Taken together, the aforementioned reports indicate that IGF-I is important for normal dendritic development and maintenance. 3. Calcium Signaling The aforementioned effects of IGF-I on synaptic activity may be due, in part, to changes in calcium signaling. Blair and Marshall (1997) demonstrated that IGF-I application to cultured cerebellar granule neurons results in a rapid potentiation of calcium channel currents. This effect was dependent on activation of PI3 kinase, as demonstrated by the application of PI3-K inhibitors and transfection of neurons with cDNAs encoding inactive regulatory subunits of the kinase. Furthermore, the authors isolated calcium channel subtypes pharmacologically and observed IGF-Imediated potentiation of calcium currents through only N and L channels. Activities of these channel subtypes were affected by IGF-I in a manner dependent on membrane potential: N currents increased at potentials from 0 to 40 mV, whereas L currents increased at potentials from ⫺20 to ⫺40 mV (Blair and Marshall, 1997). These results not only indicate that N- and L-type calcium channels are downstream targets of IGF-I, but also suggest that acute actions of IGF-I include effects on calcium-mediated events. 4. Synaptic Transmission Many of the actions of IGF-I on the structure and function of synapses in the mammalian brain are consistent with those reported for other growth factors, including neurotrophins and transforming growth factors (Schuman, 1999). Due in part to the similarities in signaling between IGF-I and growth factors such as BDNF, investigators have begun to focus on a role for IGF-I in the modulation of synaptic strength and membrane excitability. For instance, external application of IGF-I induced an attenuation of AMPA receptor-mediated currents, had no effect on NMDA receptor-mediated currents (Wang and Linden, 2000), and potentiated kainate receptor-mediated currents in cultured

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cerebellar neurons (Gonzalez et al., 2001). Additionally, Nunez et al. (2003) reported that systemic administration of IGF-I elicited a prolonged increase in the excitability of dorsal column nuclei (DCN) cells in vivo. The same group observed that, in a slice preparation, IGF-I induced a sustained depolarization of 2–5 mV in 81% of DCN neurons and increased evoked excitatory post-synaptic potential (EPSP) peak amplitude and rising slope by what appeared to be a presynaptic facilitatory process. The increased excitability evoked by IGF-I in DCN cells in vitro and in vivo depended on activation of MAPK (Nunez et al., 2003). Taken together, this body of evidence suggests that IGF-I affects excitatory neurotransmission in a manner that may differ between brain regions. Although the effects of acute application of IGF-I to hippocampal slices have not been investigated, studies in LID mice have provided valuable insights related to the electrophysiological and effects of chronic alterations in IGF-I signaling. Trejo et al. (2003) reported that the perforant path granule cell synapse in hippocampal slices obtained from LID mice exhibits an absence of LTP, a form of synaptoplasticity involved in learning and memory. Indeed, LID mice demonstrate learning impairments in the Morris water maze by 2 months of age, but show substantial recovery of learning scores and hippocampal LTP after 14 days of IGF-I treatment (Trejo et al., 2002). These observations suggest that liver-derived IGF-I is critical for hippocampal synaptic plasticity and spatial learning. 5. Plasticity Castro-Alamancos and Torres-Aleman (1993) have focused on the possible involvement of IGF-I in cerebellar plasticity and discovered that coadministration of glutamate and IGF-I to the cerebellar cortex inhibits glutamateinduced GABA release from Purkinje cells in a long-lasting and dose-dependent manner. Furthermore, stimulation of the inferior olivary complex coupled with glutamate administration resulted in decreased GABA release from cerebellar cells in response to subsequent glutamate pulses, an effect similar to that reported with conjoint treatment with glutamate and IGF-I. The authors suggested that IGF-I may be involved in plastic processes of cerebellar neurons, including long-term synaptic depression (LTD) (Castro-Alamancos and Torres-Aleman, 1993). Subsequent studies utilized pharmacological manipulation of the protein kinase C and nitric oxide signaling pathways, for which important roles in cerebellar plasticity have been postulated. These experiments revealed that the long-lasting inhibition of glutamate-induced GABA release observed with IGF-I administration occurs, at least in part, via the protein kinase C and nitric oxide signaling cascades (Castro-Alamancos et al., 1996). The authors continued these studies in an experiment that utilized an IGF-I antisense oligonucleotide injected into the inferior olive of

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male Wistar rats. This procedure was reported to inhibit learning of a conditioned eye-blink response, but not retention of the conditioned response, further emphasizing a role for IGF-I in cerebellar plasticity and motor function (Castro-Alamancos and Torres-Aleman, 1994).

F. Actions of Estrogen and IGF-I Interact in Neural Tissue and Are Often Interdependent There have been numerous reports suggesting interaction and often interdependence of estrogen and IGF-I in the brain (Cardona-Gomez et al., 2000b). Most neurons expressing the IGF-I receptor also express estrogen receptors, and expression of receptors for IGF-I and estrogen is cross-regulated, as blockade at either receptor decreases the expression of the other (Cardona-Gomez et al., 2000a, 2002). Furthermore, systemic estrogen administration to female rats was followed by upregulation of 125I-labeledIGF-I binding in the pituitary gland that reached a peak during the proestrous phase (Michels et al., 1993). Studies by Quesada and Etgen (2001) demonstrated that IGF-I enhances the activity of ␣(1B)-adrenoceptors in the preoptic area and hypothalamus only after estradiol administration. Furthermore, estrogen-induced increases in the expression of preoptic and hypothalamic ␣(1B)-adrenoceptors are mediated by IGF-I, and blockade of the IGF-I receptor inhibits the estrogen-induced release of luteienizing hormone and inhibited hormone-dependent reproductive behavior (Quesada and Etgen, 2002). In primary hypothalamic cultures, antagonism of the estrogen receptor or treatment with an antisense oligonucleotide to IGF-I receptor mRNA blocks the trophic effects of both estradiol and IGF-I (Duenas et al., 1996). Similarly, Azcoitia et al. (1999) examined the in vivo neuroprotective effects of estradiol and IGF-I on hippocampal neurons. The neuroprotective effects of each hormone against kainic acid insult were blocked by antagonizing the receptor for the other, suggesting interdependence of estradiol and IGF-I receptor signaling in neuroprotection (Azcoitia et al., 1999). Cross talk between estrogen and IGF-I receptor signaling has been emphasized by biochemical investigations into the mechanisms by which these hormones promote growth and survival. Numerous reports indicate that estrogen and IGF-I receptors share signaling elements, including Akt/PKB (Duenas et al., 1996; Honda et al., 2001; Singh, 2001; Zhang et al., 2001; Cardona-Gomez et al., 2002). Estrogen- or IGF-induced activation of Akt/PKB leads to phosphorylation of the proapoptotic protein Bad, thereby preventing Bad-induced cell death. Furthermore, induction of the prosurvival protein Bcl-2 in the hypothalamus via estrogen-induced activation of Akt/PKB requires IGF-I receptor activity (Cardona-Gomez et al., 2001; Honda et al., 2001). Future investigations may expand what is known of the mechanisms underlying the

cooperative trophic actions of estrogen and IGF-I in the mammalian brain.

G. Studies of Humans with Growth Hormone Deficiency Indicate a Possible Cognitive Role for Growth Hormone/IGF-I There have been multiple investigations related to the effects of growth hormone deficiency on cognitive function in humans (Sytze and Aleman, 2004). Studies of cognitive function in children with abnormalities of the growth hormone/IGF-I axis are numerous, but often contradictory. For instance, a study performed in Ecuador by Rosenbloom et al. (1990) found that IGF-I deficiency resulting from growth hormone receptor deficiency was associated with exceptional performance in school. However, Kranzler et al. (1998) reported that intelligence measures in 18 growth hormone receptor-deficient Ecuadorian children did not differ from family members or community control subjects, regardless of whether they were homozygous or heterozygous for the mutated growth hormone receptor gene. Furthermore, a cohort of Israeli children with mutated growth hormone receptors and resultant IGF-I deficiency exhibited inferior intelligence (Rosenbloom et al., 1992). It should be noted that interpretation of these studies is difficult due to psychological effects of growth retardation that can affect measures of intelligence (Stabler et al., 1991; Siegel et al., 1991). Several other studies focused on adults with childhoodonset growth hormone deficiency, and results have suggested that growth hormone administration may impart benefits on memory and intelligence in these patients (Almqvist et al., 1986; Sartorio et al., 1995). For instance, impaired iconic, short-term, and long-term memory was reported in 17 men with childhood-onset isolated growth hormone deficiency compared to age-matched healthy men (Deijen et al., 1996). The same group randomized 48 hypopituitary men aged 19–37 to placebo or one of three doses of growth hormone replacement and used several tasks to compare memory to age-matched healthy men. All hypopituitary subjects received adequate replacement therapy for pituitary insufficiency other than growth hormone. After 6 months of growth hormone treatment, only subjects demonstrating supraphysiological serum IGFI concentrations (n ⫽ 13) exhibited significant improvements in short- and long-term memory assessed in the associate learning task and associate recognition task, respectively. However, the 17 patients with IGF-I levels in the normal range also achieved significant improvements in memory after 1 year, and growth hormone-induced normalization of iconic, short-term, and long-term memory persisted after 2 years of treatment (Deijen et al., 1998). Considered together, these reports provide compelling evidence that growth hormone replacement provides cognitive

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IV. GROWTH HORMONE/IGF-I AXIS AND BRAIN AGING A. Growth Hormone/IGF-I Axis Exhibits Profound Changes with Age In the mid-1980s, it was revealed that elderly individuals experience a decline in the ability to secrete growth hormone in response to several stimuli, including insulininduced hypoglycemia and arginine administration (Laron et al., 1970). Subsequent studies revealed loss of the nocturnal surges of growth hormone (Carlson et al., 1972; Finkelstein et al., 1972) and a decrease in plasma IGF-I that paralleled the decline in growth hormone pulses (Johanson and Blizzard, 1981; Rudman et al., 1981; Corpas et al., 1993). These early studies in humans have been extended to rodents (Sonntag et al., 1980; Florini et al., 1981) and nonhuman primates; today, declines in high-amplitude growth hormone secretion and plasma IGF-I concentrations are some of the most robust and well-characterized endocrine alterations that occur with age (Fig. 3). Furthermore, these changes appear to be an important component of agerelated changes in tissue function. Aside from the established age-related changes in plasma levels of growth hormone and IGF-I, studies have revealed additional age-related changes in the brain that involve additional elements of the growth hormone/IGF-I axis. Several reports suggest age-related reductions in the density of growth hormone receptors throughout the brain (Lai et al., 1991, 1993; Nyberg, 1997). A more recent study revealed increased densities of IGF-I receptors in the pyramidal cells of cortical layers II and III and V and VI as well as in the CA3 area of hippocampus in aged rats (Chung et al., 2002). However, other investigators have reported age-related decreases in hippocampal (Sonntag

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benefits to patients with childhood-onset growth hormone deficiency. Published observations focused on the cognitive effects of growth hormone replacement in patients with adult-onset growth hormone deficiency are less consistent with one another. Oertel et al. (2004) studied 18 hypopituitary patients with adult-onset growth hormone deficiency who received replacement therapy sufficient to normalize and stabilize adrenal, thyroid, and gonadal parameters. Improved attentional performance was noted in those assigned to growth hormone treatment for at least 3 months, but neither verbal memory nor nonverbal intelligence differed from subjects receiving placebo (Oertel et al., 2004). Baum et al. (1998) studied 40 men with adult-onset growth hormone deficiency and reported reduced verbal learning and visual memory scores. However, a specific relationship between growth hormone deficiency and cognitive deficits could not be established, as no attenuation of these cognitive deficits was achieved by those randomized to 18-month growth hormone treatment versus placebo (Baum et al., 1998). Furthermore, adult hypopituitary women with probable growth hormone deficiency demonstrated attenuated scores on measures of verbal knowledge, perceptual-motor speed, reaction time, and spatial learning (Bulow et al., 2002). In conclusion, the age of onset as well as the etiology of growth hormone deficiency may affect the degree to which cognitive benefits are manifest after growth hormone replacement. Furthermore, interpretation of these studies should be conducted with caution, as measures of memory, intelligence, and other end points differ between studies, making comparisons of outcomes difficult. Finally, the utility of growth hormone replacement therapy may be limited by the risks inherent in the administration of anabolic hormones. These risks may be particularly relevant in the aged population, which is considered in the next section.

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et al., 1999) and cortical IGF-I receptor density (D’Costa et al., 1993). Furthermore, Paneda et al. (2003) reported that mRNA encoding the IGF-I peptide is decreased in the cerebellum of old rats compared to young animals. This decrease was associated with increased cell death and activation of proapoptotic enzymes caspase-3 and -9. However, when aged rats were treated with GHRP-6, a synthetic peptide that binds the ghrelin receptor, the authors observed increases in IGF-I mRNA expression, decreased cell death, and decreased activation of caspases (Paneda et al., 2003). While controversies still exist regarding age-related changes in specific elements of the growth hormone/IGF-I system, these reports support the hypothesized links among the growth hormone, IGF-I, and brain aging.

B. Decreased Plasma Levels of IGF-I May Be Associated with Age-Related Cognitive Deficits 1. Human Studies In an effort to address whether the age-related downregulation of growth hormone and IGF-I activity has physiological consequences, investigators have administered growth hormone to aged animals and humans. These studies revealed that age-related decreases in lean muscle mass, bone mass, immune function, and skin thickness are ameliorated by growth hormone administration (Rudman et al., 1990; Andersen et al., 2000; Sugimoto et al., 2002; Sonntag et al., 1985; Davila et al., 1987). Khansari and Gustad (1991) reported an increased life span in mice administered growth hormone from 17 months of age (Khansari and Gustad, 1991). However, mice heterozygous for deletion of the IGF-I receptor allele (Igf1r⫹/⫺) live an average of 26% longer than wild-type mice (Holzenberger et al., 2003). The implications of growth hormone and IGFI deficiency on the aging brain are also incompletely understood and often controversial. Numerous reports have addressed a possible relationship between serum IGF-I levels and human cognitive function. Aleman et al. (1999) observed statistically significant associations between both perceptual-motor performance and information processing speed and IGF-I levels in a sample of 25 subjects 65–76 years of age (Aleman et al., 1999). The same subject group demonstrated a significant association between serum IGF-I and fluid intelligence, a cognitive measure that is sensitive to aging (Aleman et al., 2001). In a 2-year prospective study involving 186 participants between 55 and 80 years of age, high total serum IGF-I concentrations and high IGF-I/IGFBP-3 ratios were significantly correlated with decreased age-adjusted cognitive decline determined by scores on the 30-point MMSE (Kalmijn et al., 2000). In 19 healthy centenarians, a significant correlation was reported between IGF-I/IGFBP-3 molar ratios, which have been postulated to reflect the

amount of biologically available IGF-I, and MMSE scores (Paolisso et al., 1997; Juul et al., 1995). Rollero et al. (1998) documented a direct correlation between serum IGF-I concentrations and MMSE scores in 22 subjects between 65 and 86 years of age. Morley et al. (1997) reported a significant correlation between IGF-I/growth hormone ratio and both visual and auditory learning in men 20–84 years of age. In a 3-year longitudinal study of cognitive decline in 1318 subjects 65–88 years of age, Dik et al. (2003) observed an association between low serum levels of IGF-I and deficits in information processing speed. However, Papadakis et al. (1995) reported no association between serum IGF-I levels and age-adjusted cognitive status in 104 healthy older men, and others observed no correlation in IGF-I levels and attention, fluid intelligence, or memory (Aleman et al., 1999, 2000). While these correlational observations have offered valuable information related to connections between growth hormone/IGF-I axis and human cognitive function, intervention studies offer a more realistic picture of the therapeutic potential of growth hormone or IGF-I replacement. However, very few such investigations have been performed to date. Friedlander et al. (2001) reported that, in women over 60 years of age, 1-year IGF-I treatment sufficient to increase plasma IGF-I to that found in younger subjects had no effect on memory in name-face and word-list recall tasks. Nevertheless, 6-month growth hormone treatment in men of mean age 75 resulted in significant improvements on the Trails B test compared to those receiving placebo (Papadakis et al., 1996). Dissention among investigators regarding the success of growth hormone and IGF-I replacement for the attenuation of agerelated cognitive impairment in humans necessitates additional studies. 2. Rodent Studies Several investigations employing growth hormone or IGF-I replacement in aged rats may help resolve the controversy, as confounding variables inherent in human populations are markedly reduced in rodent models (Fig. 4). Studies utilizing the Morris water maze have demonstrated that treatment with GHRH from 9 to 30 months of age prevents age-related cognitive decline in aged brown Norway ⫻ F344 rats (Thornton et al., 2000). Furthermore, a 28-eight-day icv infusion of IGF-I attenuated age-related deficits in working and reference memory assessed in the Morris water maze and object recognition tasks (Markowska et al., 1998). While the mechanism by which upregulation of the growth hormone/IGF-I axis benefits learning and memory in aged rodents is unknown, administration of growth hormone for 28 days was found to increase microvascular density in aged animals (Sonntag et al., 2000b). Subsequent analyses indicated that the cerebral vasculature may be an important paracrine source of

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IGF-I for the brain. Furthermore, a 28-day icv infusion of IGF-I to aged brown Norway ⫻ F344 rats increases hippocampal NMDAR2A and R2B subunit expression (Sonntag et al., 2000a) (Fig. 5), a finding made especially important by a report by Clayton et al. (2002) that ablation of the NMDAR2B subunit function abolishes hippocampal LTP and impairs spatial learning in young animals (Clayton et al., 2002). Le Greves et al. (2002) have subsequently reported that a 10-day treatment with growth hormone in vivo resulted in increased expression of NMDA receptor subunits R1 and R2A in aged hippocampal tissue. Shortterm IGF-I treatment also increased rates of local cerebral glucose utilization, a function believed to be correlated with neuronal activity, in the anterior cingulate of the cortex (14.2%) and the CA1 region of the hippocampus (11.0%) in aged rats (Lynch et al., 2001). Furthermore, administration of IGF-I to aged brown Norway ⫻ Fisher rats ameliorates the decline in hippocampal neurogenesis associated with aging (Lichtenwalner et al., 2001). One report focused on the relationship between the growth hormone/IGF-I axis and the function of aging cortical tissue in vitro. Shan et al. (2003) used whole cell electrophysiological techniques in vitro and reported that IGF-I increased the amplitude of Ca2⫹ currents through P/Q- and N-type calcium channels in the cortical region that controls hindlimb muscle function of senescent rats. This effect occurred without changing the voltage dependence of Ca2⫹ currents, and enhancement of Ca2⫹ current flow with IGF-I application was similar to that observed in slices obtained from young and adult rats. The authors concluded that despite the decline in synthesis of IGF-I that occurs with age, voltage-gated calcium channels are able to respond to the peptide (Shan et al., 2003). In recent years, significant progress has been made toward a more complete understanding of growth hormone and IGF-I activity in the aging brain. The goal of most related investigations is to explore the implications for this hormonal axis in cognitive function. While large-scale human studies have been correlational

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FIGURE 5 Analysis of changes in NMDA R2 receptor subtype protein expression in the hippocampus assessed by Western analysis. NMDAR2A and NMDAR2B indicated a significant decline between 21 and 30 months of age that was reversed by treatment with IGF-I for 28 days. No alterations in protein expression of NMDA R2C were observed with age or treatment with IGF-I (*p ⬍ 0.05 compared to young or middle-aged, **p ⬍ 0.01 compared to old saline). From Sonntag et al. (2000a).

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FIGURE 6 Immunohistochemical identification of newborn cells in the dentate gyrus. BrdU-labeled cells are evident in representative DIC photomicrographs of the dentate gyrus (insets) in (A) young, (B) middle-aged, (C) old, and (D) old IGF-I-treated animals in the short-term survival group. Arrows within the insets indicate areas shown in the higher magnification micrographs. Whereas few BrdU-labeled cells are evident in middle-aged and old saline-infused rats (B and C, respectively), many BrdU-positive cells are apparent in young saline-infused and old IGF-I-treated animals, particularly in the PZ (A and D, respectively). Scale bar: 50 m (inset scale bar: 200 m). For presentation purposes, adjustments were made to brightness and contrast. From Lichtenwalner et al. (2001).

designs, several intervention studies in rodents have revealed that systemic GHRH treatment or icv administration of IGF-I imparts improvements in learning and memory. These cognitive benefits may be the result of concurrent increases in neurogenesis (Fig. 6), vascular density, or glucose utilization. Changes in the subunit composition of NMDA receptors or alterations in calcium signaling may also contribute to the cognitive improvements associated with GHRH or IGF-I treatment in aged rats. Advanced techniques, including imaging technology, may allow the eventual extension of similar studies to humans.

C. Age-Related Decreases in Plasma IGF-I Have Been Implicated in the Development of Neurodegenerative Diseases 1. In Vitro Studies Multiple in vitro and whole animal models have also been employed in efforts to investigate the ability of IGF-I to protect against the neurodegeneration associated with aging in humans. Offen et al. (2001), for example, reported that the addition of IGF-I to rat cerebellar neurons in culture attenuated cell death induced by high concentrations of dopamine, which have been implicated in the neuronal loss associated with Parkinson’s disease (Offen et al., 2001). In a whole animal study utilizing a model of Huntington’s disease, GPE administered directly into the striatum for 7 days following quinolic acid lesion protected against the loss of GABAergic projection neurons of the calbindin phenotype. Similar protection

was observed in cholinergic interneurons identified by immunocytochemical and histochemical staining (Alexi et al., 1999). Several groups have also focused on possible benefits of IGF-I to patients with Alzheimer’s disease (AD) (Gasparini and Xu, 2003). Hashimoto et al. (2001) used trypan blue exclusion and calcein flurescence assays to demonstrate that IGF-I protects primary cultured neurons from A␤1–43, a ␤-amyloid peptide involved in the neurotoxicity of AD (Hashimoto et al., 2001). Dore et al. (1997a) demonstrated that simultaneous incubation with IGF-I dose dependently protects rat primary hippocampal neurons exposed to the A␤ peptide fragment A␤25–35 or human amylin. IGF-I was also able to rescue primary hippocampal neurons when applied within 4 days of amyloidogenic peptide exposure (Dore et al., 1997a). Additionally, the same group found that IGF-I protected cultured primary hippocampal neurons from toxicity induced by both A␤25–35 and A␤1–42. Treatment of the same cell type with IGF-I up to 5 days after ␤-amyloid peptide exposure also provided significant neuronal rescue as determined by the thiazoyl blue colorimetric assay, an indicator of mitochondrial activity (Dore et al., 1999). In a different approach to examining the neuroprotective actions of IGF-I against ␤-amyloid peptides associated with AD, Heck et al. (1999) applied H2O2, an oxidative stressor and mediator of A␤ neurotoxicity, to rat primary cerebellar neurons and GT1–7 hypothalamic cells. H2O2 treatment resulted in reduced cell viability as determined by MTT 3-(4,5 dimethyl triazol-2-yl)-2,5-diphenyl-tetrazolium bromide and trypan blue exclusion assays. However, a 24-h

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incubation in IGF-I prior to the addition of H2O2 increased cell viability in both cell types in a dose-dependent manner. The authors also used electrophoretic mobility shift assays and transfection technology to demonstrate that IGF-I treatment caused specific increases in DNA binding and transcriptional activity of NF-␬B, a transcription factor previously found to possess neuroprotective actions. Western blot analysis of nuclear extracts obtained from GT1–7 cells revealed that IGF-I treatment caused concurrent increases in nuclear translocation of the NF-␬B subunit p65 and degradation of I␬B␣, an inhibitory protein believed to cause cytoplasmic sequestration of NF-␬B. Furthermore, overexpression of the NF-␬B subunit c-Rel protected GT1–7 cells from H2O2-mediated death, whereas transfection with a mutant form of I␬B␣ decreased survival after oxidative insult. The aforementioned effects of IGF-I on cell survival and NF-␬B activation were inhibited by LY294002 and wortmannin and were therefore dependent on the PI3-K pathway (Heck et al., 1999). Hong and Lee (1997) reported that IGF-I influences tau phosphorylation, a step in the formation of neurofibrillary tangles associated with AD. In human neuronal cultures, IGF-I reduced tau phosphorylation and facilitated tau binding to microtubules (Hong and Lee, 1997). These in vitro studies represent significant progress toward the understanding of mechanisms by which IGF-I affects processes associated with neurodegenerative conditions. 2. In Vivo Studies Investigations in vivo have also offered insights into the relationship between IGF-I and pathophysiological changes associated with AD. A study by Carro et al. (2002) complements in vitro studies that indicate a role for IGF-I in the regulation of brain ␤-amyloid protein levels, which may contribute to age-related cognitive deficits. The authors used immunoblotting techniques to demonstrate that aged rats possess increased levels of A␤1–40 protein tissues of the hippocampus and choroid plexus compared to young rats. IGF-I administration decreased A␤1–40 protein levels in both brain regions of aged rats to levels comparable to young rats. IGF-I treatment also attenuated the reactive gliosis characteristic of aged frontal cortical tissue as determined by GFAP and vimentin immunoreactivity. Furthermore, systemic treatment with IGF-I prior to intraparenchymal injection of the biotinylated A␤1–40 protein resulted in decreased accumulation of A␤1–40 protein in cortical tissue and increased the presence in the CSF as compared to saline-injected rats. In a complementary study, the same group found that young liver-IGF-I deficient (LID) mice demonstrate decreased circulating levels of IGF-I and increased concentration of A␤1–40 protein in the hippocampus compared to wild-type littermates. Similar effects on A␤1–40 protein expression and A␤ protein-associated gliosis were observed in cortical and

hippocampal tissues when transgenic mice that overexpress a mutant form of human amyloid precursor protein (APP695) and demonstrate high levels of A␤ proteins were treated with IGF-I. Carro et al. (2002) also demonstrated that IGF-I increases the permeability of the brain–CSF barrier to albumin, an A␤ carrier protein, and also increases blood and CSF binding of A␤ proteins to albumin. Similar findings were reported with regard to transthyretin, another A␤ carrier protein, levels of which were increased in the cortex, choroid plexus, and CSF with systemic IGF-I treatment. This study provides evidence that serum IGF-I modulates the expression of A␤ proteins associated with AD in the mammalian brain. The authors also suggested that IGF-I modulates the availability of A␤ protein carriers, which can increase the ratio of A␤ protein in the CSF to that in brain tissue (Carro et al., 2002). Additional research is required to determine the therapeutic potential of IGF-I in the treatment of AD, but a reduction in A␤ protein levels in the brain may account for some of the improvements in learning and memory reported in aged animals treated with GHRH and IGF-I.

D. Conclusions While the age-related declines in plasma levels of growth hormone and IGF-I are well documented, changes in other elements of the growth hormone/IGF-I system are controversial. The benefits of growth hormone or IGF-I replacement relative to cognitive function in the elderly are uncertain due to the lack of interventional studies performed to date. However, long-term administration of GHRH or IGF-I was associated with improvements in learning and memory in aged rats. The mechanisms underlying the cognitive benefits of upregulation in growth hormone/IGF-I signaling include increases in hippocampal neurogenesis and glucose utilization and altered composition of NMDA receptors. Several groups have also provided support for the hypothesis that IGF-I inhibits processes associated with the development of neurodegenerative diseases, including AD. For example, studies utilizing neuronal cultures and transgenic animals suggest that IGF-I protects neurons from A␤ toxicity, regulates tau phosphorylation, and facilitates A␤ clearance. In summary, IGF-I is linked with AD, and the decreased plasma levels of IGF-I associated with age may contribute to the progression of the disease.

V. SUMMARY The studies summarized here represent a vast literature that supports the requirement of growth hormone and IGF-I for normal development of the mammalian body and brain.

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These hormones promote long bone and tissue growth, cardiovascular maturation and function, and work with other hormonal systems to regulate blood glucose levels and reproduction. Furthermore, IGF-I crosses the blood–brain barrier and promotes neurogenesis and synaptogenesis throughout development and during adulthood. The hormone protects neurons from chemical toxicity and cell death resulting from hypoxic-ischemic injury and reduces cell loss when administered postinsult. IGF-I is essential for normal dendritic development, and several groups have reported that IGF-I regulates excitatory synaptic transmission in a regionally specific manner. This hormone also acts synergistically with estrogen to mediate trophic and neuroprotective effects in brain tissue, and there is evidence of overlap between IGF-I and estrogen receptor signaling pathways. While investigators have reported numerous actions of growth hormone and IGF-I in various brain areas, studies of the functional contributions of growth hormone and IGF-I are less abundant. Indeed, studies of growth hormone treatment in children and adults with growth hormone receptor deficiency have yielded inconsistent results related to the benefits of this therapy to intelligence, learning, or memory. In recent years, much attention has focused on agerelated decreases in serum growth hormone and IGF-I and on potential mechanisms that may influence cognitive function in the elderly population. Several groups have reported positive correlations between serum IGF-I levels and cognitive abilities in older humans, but interventional studies are needed to establish a more definite link between these hormones and function of the aging brain. Such interventions have been performed in rodents and have demonstrated that long-term growth hormone/IGF-I replacement improves hippocampal-dependent learning and memory in aged rats. While the exact mechanism underlying these cognitive improvements is unknown, growth hormone and IGF-I replacement to aged animals increased neurogenesis, vascular density, and glucose utilization and altered NMDA receptor subunit composition in brain areas implicated in learning and memory. One group has also suggested that voltage-gated calcium channels in aged neurons are able to respond to IGF-I. Furthermore, IGF-I may regulate brain levels of A␤ peptide associated with neurodegenerative diseases such as AD. While these observations offer valuable insight into the influence of growth hormone and IGF-I on neuronal events in the aged mammal, additional functional studies should be performed to link these changes to cognitive improvements.

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15 Insulin-like Growth Factor-I and Neuroprotection E. CARRO, J. L. TREJO, S. FERNANDEZ, A. M. FERNANDEZ, and I. TORRES-ALEMAN Laboratory of Neuroendocrinology, Cajal Institute, 28002 Madrid, Spain

I. INTRODUCTION

I. Introduction II. Neuroprotective Signaling III. Insulin-like Growth Factor-I (IGF-I) Regulates New Brain Cell Populations IV. IGF-I Maintains Brain Cell Function V. Mechanisms of Neuroprotection by IGF-I VI. Loss of IGF-I Function Contributes to Brain Aging and Disease VII. Perspectives References

Due to its intense metabolic activity and low cell turnover, the brain is probably more susceptible to damaging metabolic by-products and cell loss than other organs. To counteract this greater vulnerability, brain cells rely on a neuroprotective network whose chief function is to keep not only neurons, but all types of brain cells, healthy and functional (Torres-Aleman, 2000). However, although the concept of neuroprotection is used increasingly in neuroscience research, it is ill-defined. This chapter refers to neuroprotection as encompassing all types of brain mechanisms displaying self-repair characteristics. Many include activitydependent processes whose protective traits are not usually acknowledged. Examples of these are increased synaptic contacts underlying learning-related events (Zhou and Poo, 2004), which in turn maintain neuronal function by diverse mechanisms such as increased uptake of neurotrophic factors (Carro et al., 2000). Recruitment of new neurons in response to environmental and hormonal cues is a fascinating new aspect of brain physiology that most likely participates in neuroprotection; for instance, exercise-induced neurogenesis allows increased resilience to insults (Carro et al., 2001). Based on these and many other examples, we conclude that neuroprotection is an intrinsic brain mechanism that probably evolved to cope with the specially demanding conditions of brain function. An indirect but convincing evidence of the existence of a physiological neuroprotective network is that during the aging process many, if not all, of its putative components

Neuroprotection is a relatively new concept in neuroscience research coined to encompass a great variety of mechanisms aimed at preserving brain function. These include humoral growth factors such as insulin-like growth factor-I (IGF-I), which are integrated within a network of protective signaling between different brain cell types and the periphery. IGF-I displays a wide array of neuroprotective activities, ranging from those addressing basic cell needs such as energy balance to brain-specific requirements such as modulation of neuronal plasticity. Recent developments emphasize the potential therapeutic utility of IGF-I and its downstream targets in brain diseases and underscore the important role played by glial cells in IGF-I neuroprotection. While novel findings indicate that blood-borne IGF-I is an important trophic source for brain cells, the precise role played by brain IGF-I is still not clear. Similarly, molecular pathways of IGF-I neuroprotection remain largely uncharted. Understanding the role played by IGF-I in the adult brain will surely illuminate essential aspects of neuroprotective mechanisms.

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

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decay, which is probably the reason of a greater incidence of neurodegenerative diseases in old age (Mayeux, 2003) Therefore, research into the mechanisms of brain aging constitutes an appropriate physiological setting to analyze neuroprotective mechanisms. Although neuroprotection operates during normal physiological processes, it is also activated in response to injury. As with other homeostatic mechanisms (i.e., immune surveillance), in some cases its reactivity may prove counterproductive. For instance, a double-sided effect of the glial reaction to injury has long been discussed (Liberto et al., 2004; McGraw et al., 2001). Glial plasticity is associated to normal synaptic function, but may prove detrimental to axonal regeneration, while at the same time it is important to promote the survival of damaged neurons. However, the overall role (either beneficial or detrimental) played by glial reactivity in response to injury remains to be established (McGraw et al., 2001). Greater insight into the processes underlying neuroprotection will allow the design of new therapeutic strategies in neurological diseases based on the potentiation and/or repair of endogenous neuroprotective pathways. In addition, they may prove useful for therapeutic intervention in brain aging.

II. NEUROPROTECTIVE SIGNALING An ample set of molecules and mechanisms are involved in neuroprotective networks. Among these, humoral cellto-cell messengers are well positioned to constitute a primary arm of neuroprotection, as during brain development they exert neurotrophic activity. However, their role in the adult brain is not yet well defined. Building on the reported actions of neurotrophic molecules in adult tissues other than brain, as well as their much better described activities in the developing brain, we are slowly understanding the important role played by neurotrophic signals in the adult. This chapter documents the role of a prototypical neuroprotective signal, i.e., insulin-like growth factor I (IGF-I), a peptide growth factor of the insulin family. Using IGF-I as a paradigm, we can illustrate different aspects of neuroprotection. Similar to other neurotrophic signals within the neuroprotective network [i.e., brain-derived neurotrophic factor (BDNF), neurosteroids, etc.], IGF-I is a pleiotropic peptide with a wide array of biological activities, most of them better characterized in tissues other than the brain. Because IGF-I belongs to the insulin family of peptide hormones and is under the control of growth hormone, its physiological role in endocrine physiology has been studied intensely (Yakar et al., 2002). IGF-I is one among many neurotrophic factors involved in brain development promoting proliferation, survival, and differentiation of brain cell populations (D’Ercole et al., 1996a). The presence of relatively high

levels of IGF-I protein in the adult brain — albeit brain IGF-I mRNA levels are relatively low as compared to the developmental period or to other organs such as liver (Murphy et al., 1987)—has prompted interest on its possible role in the adult period. Taking into account its wellknown growth-promoting actions in other tissues, IGF-I has been considered as a promoter of tissue growth also in the adult brain. Naturally, because brain tissue lacks the regenerative potential of other tissues, IGF-I adapts its growthpromoting activities to the particularities of this organ.

III. INSULIN-LIKE GROWTH FACTOR-I (IGF-I) REGULATES NEW BRAIN CELL POPULATIONS Classical views stated that the adult brain produces very little, if any, new neurons, but recent findings indicate the existence of neurogenic areas in the adult mammalian brain (Gross, 2000). The control of new neuron formation is currently under intense scrutiny because of its impact in brain physiology and its therapeutic potential. Abundant literature illustrates the fact that neurogenesis is regulated by both intrinsic and extrinsic cues; most prominently physical and mental activity, stress, disease, and hormones (Gould et al., 2000). Among the latter, IGF-I is probably one of the most important modulators of new neuron formation (Anderson et al., 2002), very likely participating in the effects of physical exercise and depressive illness on adult neurogenesis (Carro et al., 2003). However, the cellular and molecular pathways involved in neurogenesis in general and in particular in response to IGF-I are not well defined yet. Because the actions of IGF-I on proliferation, survival, and differentiation of new neurons during development are better known (D’Ercole et al., 1996a), it is assumed that similar processes may participate in the adult. For instance, the protein kinase Akt, a known mediator of prosurvival actions of IGF-I in developing cells (Dudek et al., 1997), appears also important in adult neurogenesis (Sinor and Lillien, 2004). Nevertheless, these pathways remain to be established. Other brain cells, including glia, the major cellular component of brain tissue, fibroblasts, epithelial cells, or endothelial cells, have not been studied in detail in this regard, except for the latter. Although glial cell proliferation is very modest in the adult brain, except when confronted to injury, it is probable that IGF-I plays a role in proliferation and recruitment of new glial cells, including macrophages from the periphery. However, evidence indicates that brain endothelial proliferation depends on IGF-I (Lopez-Lopez et al., 2004) and that maintainance of vessel density depends on appropriate IGF-I input (Sonntag et al., 1997). Therefore, in those cell types where the actions of IGF-I have been analyzed in detail, it has been found that their growth is modulated by IGF-I.

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As for cellular pathways involved, while in general it is considered that growth factors act locally to control cell growth, evidence in the case of IGF-I strongly indicates that blood-borne IGF-I is critical for new neuron formation during adulthood (Trejo et al., 2001). Novel findings point to the blood–cerebrospinal fluid (CSF) interface in the choroid plexus as the route of passage of serum IGF-I into the brain (Carro et al., 2000). This specialized epithelium is particularly rich in IGF-I receptors and IGF-binding proteins (Lee et al., 1993; Marks et al., 1991) and translocates molecules from the bloodstream into the CSF through as yet poorly understood transcytotic processes, although the best known role of choroid plexus is the production of CSF (Chodobski and Szmydynger-Chodobska, 2001). At any rate, the ability of serum IGF-I to modulate new cell formation in the adult brain provides an unpredicted way to link peripheral signals with brain function.

IV. IGF-I MAINTAINS BRAIN CELL FUNCTION Due to its modest capacity to produce new neurons, the brain relies on tight regulation of neuron performance to maintain its function. Neurons require not only nutrient and oxygen supply as any other cell type, but sustained interactions with local and distant partners are important in keeping their functional status. This specialized characteristic, together with the enormous variety of brain cell phenotypes, probably contributed to development of a complex network of dedicated protective mechanisms that include humoral signals such as IGF-I. An intriguing feature is that growth factors appear to play roles in the brain not previously described in other target organs. This apparent gain of function is very well exemplified by IGF-I. While this growth factor is a universal prosurvival signal, as well as stimulating proliferation and differentiation of many types of cells throughout the body, IGF-I in the brain also participates in the control of neuronal membrane excitability, carbohydrate metabolism, cell-to-cell contacts, or clearance of potential hazardous compounds. It is true that in most instances, the molecular underpinnings of this striking variety of actions are not well understood. However, those that are slowly being unveiled are giving important cues to the physiological role of IGF-I in the adult brain. A common characteristic is that the reported actions of IGF-I on mature neurons are aimed to preserve their functionality. This is illustrated with probably the most unpredictable action of IGF-I on mature neurons: its ability to modulate membrane ion channels directly contributing to neuronal excitability. The first observation is that IGF-I interacts with many types of ion channels, including K⫹, Cl⫺, or Ca2⫹ channels, and neurotransmitter-associated conductances (Table I). Because the contribution of these channels

TABLE I Ion Channels Modulated by IGF-I in Neuronal Membranes Channel

IGF-I action

Reference

K

Inhibits IA currents

Nunez et al. (2003)

Cl

Activates K/Cl cotransporter

Kelsch et al. (2001)

Ca

Activates N and L channels

Blair and Marshall (1997)

Translocates GRC channels to the cell membranea

Kanzaki et al. (1999)

Translocates voltage-gated channels to the membranea

Viard et al. (2004)

AMPA

Translocates receptors to the cytosolb

Man et al. (2000)

Kainate

Stimulates receptor currents

Gonzalez, V et al. (2001)

a Results b Results

in channel activation. in receptor run-down.

to membrane excitability varies widely, in many occasions, even in opposite ways, it is not possible even to hypothesize the functional role of these modulatory actions, although for at least specific neuronal phenotypes, the final effect found is that IGF-I increases its excitability (Carro et al., 2000; Liou et al., 2003; Nunez et al., 2003). A second observation in this regard is that IGF-I usually modulates the membrane availability of ion channels by regulating its membrane trafficking through pathways not fully clarified. Usually these involve translocation of the target protein from cytosolic stores (the Golgi network, etc.) to the membrane (Kanzaki et al., 1999; Man et al., 2000; Russo et al., 2004; Viard et al., 2004). Regulation of cytosolic and membrane compartmentalization of different membrane proteins is probably a major characteristic of IGF-I action on target cells, which appears of particular importance for brain function (Fig. 1). Among these, IGF-I-regulated trafficking of AMPA receptors in neuronal membranes or of amyloid

IGF-I

Cytosol

Membrane

Membrane protein

Membrane protein

FIGURE 1

Membrane protein trafficking and IGF-I. A major characteristic of IGF-I actions on brain cells is its ability to modulate traffic of membrane proteins (neurotransmitter receptors, ion channels, cargo proteins, etc.) in and out of the membrane compartment. In many cases this is achieved by phosphorylation of either the target protein or associated proteins. Therefore, IGF-I modulates cell–environment interactions. (See color plate 16)

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carriers in choroid plexus epithelial cells (Carro et al., 2002, 2003; Carroll et al., 2001; Man et al., 2000; Wang and Linden, 2000), presently the best studied, constitutes a new intriguing aspect of neuroprotection.

V. MECHANISMS OF NEUROPROTECTION BY IGF-I Surprisingly little detailed information is available on the cellular and molecular pathways involved in IGF-I neuroprotection. A bias in neurotrophic research tends to assume that the actions of neurotrophic factors in the adult brain are similar to those seen during development. However, while there is no firm evidence to either reject or confirm this notion, it is increasingly apparent that new pathways appear in adulthood, at least in the case of IGF-I. One such new pathway is provided by the blood–CSF route that is established postnatally, whereby serum IGF-I accesses the brain (Carro et al., 2000). Strong evidence indicates that this is probably the main source of IGF-I in the adult brain rather than locally produced IGF-I. Low serum IGF-I levels produce more deficits in brain than when brain IGF-I is low, and these deficits are rescued by systemic treatment with IGF-I (unpublished observations). Therefore, a peripheral source of IGF-I should be taken into account when analyzing the actions of this growth peptide on the brain. This means that classical endocrine regulation of the growth hormone (GH)–IGF-I axis is impinging on brain physiology, which may help explain brain disturbances seen in GH-deficient patients (Deijen et al., 1996). Probably the only relatively well-defined intracellular pathway of IGF-I in neurons is that linking IGF-I receptordependent activation of the kinase Akt with cell survival by inhibiting proapoptotic pathways involving the cell nucleus and mitochondria (Dudek et al., 1997). In this regard, regulation of the forkhead transcription factor FoxO3 by IGFI-stimulated Akt is currently the subject of intense analysis, as FoxO3 appears pivotal in the cell response to stress and antiapoptotic signals (Brunet et al., 2004). As a matter of fact, this transcription factor, together with its upstream regulators, including the IGF signaling cascade and the deacetylase Sir2, is center stage in the genetic regulation of aging (Brunet et al., 2004; Daitoku et al., 2004; Kaeberlein et al., 2004). However, IGF-I activates multiple intracellular cascades recruiting kinases such as Mitogen activated protein kinases (MAPKs), Protien Kinase C (PKCs), phosphatases, or even G-coupled proteins (LeRoith, 2000; LeRoith et al., 1995). While many of these are known to be activated by IGF-I also in brain cells, their biological significance remains to be established. Moreover, most work has focused on neurons as target cells, whereas recent work indicates that glial cells are major mediators of the protective actions of IGF-I. In this regard, we have found that IGF-I stimulates

calcineurin and Phosphatase and TEnsion homolog deleted from chromosome 10 (PTEN)-dependent pathways in astrocytes, whereas in neurons, these phosphatases are not affected by IGF-I (unpublished observations). As indicated in Fig. 2, these two novel pathways are intimately involved in the tissue response to the generation of reactive oxygen species (ROS) and proinflammatory mediators. Therefore, a glial-to-neuron signaling mediates important neuroprotective actions of IGF-I that require further work to be defined.

VI. LOSS OF IGF-I FUNCTION CONTRIBUTES TO BRAIN AGING AND DISEASE An apparently paradoxical role of IGF-I in aging and disease is currently under debate (Bartke et al., 2003; Carter et al., 2002; Trejo et al., 2004). While reduced IGF-I input is reportedly beneficial in reducing cancer risk and prolonging life (Giovannucci, 2003; Kenyon, 2001), an increased incidence of highly debilitating diseases such as age-associated cognitive loss, cardiopathy, or type 2 diabetes is expected to develop in states of IGF-I deficiency (Boger et al., 2003; Carro and Torres-Aleman, 2004; Dunger et al., 2003). This controversy has not yet been resolved, probably because it is difficult to define a simple conceptual framework for the biological significance of IGF-I, a growth factor with an enormous variety of actions. However, mounting evidence supports the notion that a state of IGF-I deficiency, due to low IGF-I levels, development of resistance to IGF-I, or both (Trejo et al., 2003), underlies many of the brain deficits commonly associated with old age, as well as helps explain the development of diseases such as age-associated Alzheimer’s disease (Carro and Torres-Aleman, 2004). As indicated in Fig. 3, reduced IGF-I input may explain the characteristic features of the aging brain, including cognitive deterioration, tissue atrophy, vascular impairment, and deposits of metabolic byproducts. As described by Carro et al., (2005), reduced IGF-I results in decreased ␤ amyloid clearance and, consequently, brain amyloidosis, which associates to other characteristic traits of Alzheimer’s disease, including cognitive loss. Mutant mice with premature IGF-I deficiency show increased vulnerability to insult together with other brain deficits characteristic of old age, such as reduced neurogenesis and memory impairments (Trejo et al., 2003, 2005). Importantly, these deficits are rescued when exogenous IGF-I is administered, pointing to low IGF-I as the underlying cause of these disturbances. Whether potentiation of IGF-I signaling or its downstream pathways will hinder brain aging is therefore a promising new target of research into aging. A compelling argument favoring an important role of IGF-I in brain disease is the observation that widely different detrimental situations, such as inflammation, excitotoxicity, or cytotoxicity, that take place during brain trauma,

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FIGURE 2

Pathways in IGF-I neuroprotection. While the IRS/PI3K/Akt prosurvival cascade following IGF-I receptor activation is well established in neurons, several other pathways recruited by IGF-I in other cells, such as MAPK or PKC, remain ill-defined. Activation of Akt is also involved in other actions of IGF-I in neurons, including enhancement of Ca2⫹ currents. In glial cells such as astrocytes, the role of Akt and other kinases downstream of the IGF-I receptor is not known. Evidence indicates that novel pathways include stimulation of phosphatases such as calcineurin or PTEN and that these routes are involved in anti-inflammatory and antioxidative actions of IGF-I that protect neurons against these type of insults. (See color plate 17)

Age-associated low IGF-I input leads to Age-associated brain deficits Altered synaptic plasticity Cell loss and/or impairment

Impaired cognition

Diminished reparative capacity Reduced neurogenesis Altered glucose metabolism

Tissue atrophy

Impaired angiogenesis Inflammation

Vascular dysfunction

Disturbed metabolic homeostasis

Accumulation of deleterious substances

ischemia, amyloidosis, epilepsy, or drug-related insults, to mention a few, share a common pathological effect, i.e., they all interfere with IGF-I signaling on neurons and/or glial cells (Garcia-Galloway et al., 2003; Venters et al., 1999; Xie et al., 2002; Zhang et al., 1998). This led us to propose that interruption of IGF-I trophic support to brain cells is a characteristic feature of brain disease (Trejo et al., 2003). In other words, pathological challenges rely in part in their ability to counteract the protective actions of IGF-I. This notion also requires further study.

VII. PERSPECTIVES FIGURE 3

Brain aging and IGF-I. An association between lowering IGF-I input during aging and age-associated brain deficits can be established by analyzing the actions played by IGF-I on brain cells. All the major age-associated changes in brain function can be explained by specific deficits resulting from lower IGF-I input. Direct proof of this relationship is provided by observations linking recovery of function after IGF-I treatment in old laboratory animals.

As with good stories, the study of neuroprotective actions of IGF-I leaves open more questions than it answers. An important one is the biological significance of locally produced IGF-I. While neurotrophic actions of IGF-I in the adult brain have been implicitly ascribed to brain-derived IGF-I,

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probably because local IGF-I levels are high and are clearly involved in brain growth during development (D’Ercole et al., 1996b), it is not at all apparent that this is the case. Rather, most actions of IGF-I in the adult brain are better explained by accounting for a peripheral source of IGF-I (TorresAleman, 2000). For instance, work with a neuron-specific IGF-I knockout mouse further substantiates this notion, as no gross brain changes are seen in the absence of neuron-derived IGF-I (unpublished observations). Coupled to the observation that local synthesis of IGF-I is restricted to a few areas and that IGF-I mRNA levels are very low in the adult brain, the conclusion is that brain IGF-I in the adult is likely involved in very specific local actions still to be determined. However, because old notions are hard to overcome, current neurotrophic research widely assumes that brain growth factors are local modulators, leaving the abundant observations on the brain actions of peripherally derived brain neurotrophic factors mostly ignored. Other important questions remain. Is the comprehensive nature of the biological actions of IGF-I on brain tissue specific to this growth factor? Do other bona fide factors such as BDNF behave similarly? Are they effectors of the protective role of IGF-I? What is the functional relationship between them? At any rate, why is IGF-I acting on all cell types in the brain and controlling so many different mechanisms? Are these observations mere experimental artifacts or are we unveiling an important characteristic of brain function? It is clear that much more work is needed to answer these points.

Acknowledgment Work in the authors’ laboratory is funded by grants from CAM, FIS, and DGICYT.

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16 Quality of Life in Adult Growth Hormone Deficiency ROBERT D. MURRAY* and STEPHEN M. SHALET † *Department

of Endocrinology, Leeds Teaching Hospitals NHS Trust, St James’s Hospital, Leeds, and of Endocrinology, Christie Hospital, Manchester, United Kingdom

†Department

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV.

Introduction Why Is Quality of Life (QoL) Important? What Do We Mean by “Quality of Life?” Measurement of Quality of Life Effect of Untreated Growth Hormone (GH) Deficiency (GHD) on Quality of Life Is GH Causative in the Impaired QoL of Hypopituitary Adults? QoL Following Discontinuation of Childhood GH Therapy Natural History of QoL in Adults with Untreated GHD Effects of GH Replacement on QoL in GHD Hypopituitary Adults Specific Patient Groups Effect of Long-Term GH Replacement on QoL Predictors of Improvement in QoL with GH Replacement Potential Biases in Open Studies of QoL Summary References

have centered around the indications to initiate replacement therapy. In the absence of an absolute indication for treatment, such as prevention of excess mortality or fractures, QoL has constituted the area in which patients perceive the most benefit and where evidence for a beneficial effect of GH replacement therapy is the most robust. Evidence for impaired QoL in GHD adults and improvements following GH replacement in both placebo-controlled and open studies support the use of GH replacement in GHD adults with impaired QoL. With our increasing knowledge there has been a slow evolution in the way studies of the effect of GH replacement on the QoL of GHD hypopituitary adults are conducted in terms of dosing regimens and patient selection. As a result, we still may not have undertaken the study that will finally answer the question of whether GH replacement has a significant effect on QoL in severely GHD adults. While awaiting the results of these placebo-controlled, double-blinded studies in GHD adults with impaired QoL who receive GH replacement by a low-dose titration regimen, GHD adults should continue to receive GH replacement, as the current evidence is supportive in terms of risk–benefit ratio.

I. INTRODUCTION II. WHY IS QUALITY OF LIFE (QoL) IMPORTANT? Since the realization of the beneficial effects of growth hormone (GH) replacement on body composition, bone mass, surrogate markers of cardiovascular risk, and quality of life (QoL) of GH-deficient hypopituitary adults, discussions

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

The impact of GH deficiency on the hypopituitary adult has been well characterized since the late 1980s (Jorgensen et al., 1989; Salomon et al., 1989). The salient

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features encompass a decrease in total body and truncal fat mass (Hoffman et al., 1995; Rosen et al., 1993), increased lean body mass (Hoffman et al., 1995), reduced bone mass (de Boer et al., 1994), reduced strength and exercise tolerance (Cuneo et al., 1990), insulin resistance (Johansson et al., 1995), adverse surrogate markers of cardiovascular risk (Abdu et al., 2001; Johansson et al., 1994), and impaired quality of life (McGauley, 1989; Rosen et al., 1994). In addition to the aforementioned morbidity, hypopituitary adults on conventional hormone replacement, but not GH replacement, have an approximately twofold increase in relative risk of mortality. It is intuitive to hypothesize that GH deficiency may contribute at least part of the excess mortality given the presence of insulin resistance, excess levels of procoagulant factors, an adverse lipid profile, and truncal adiposity in these patients. Following GH replacement, there are improvements in the lipid profile, procoagulant factors, and body composition; however, insulin resistance may be worsened and the independent cardiovascular risk factor lipoprotein(a) frequently increases. The overall effect of GH replacement on life expectancy is therefore difficult to predict and the impact of GH replacement on mortality rates has yet to be demonstrated. Until this information is available, cardiovascular risk factors alone do not constitute an absolute indication for treatment of all GHD adults with GH. This leaves the dilemma of which GHD adults should receive GH? Selection of patients must therefore be based on certain specific areas of morbidity. The symptoms of GHD recognized throughout the literature are nonspecific, as is frequently observed in endocrine practice. Reduced quality of life, in particular lack of energy, although not specific for GHD, is the symptom patients with GHD are likely to complain of and to detect benefit in if GH replacement is commenced. If the patient is to accept that daily injections will be required, possibly for life, then compliance will be increased if they perceive benefit. Current medical literature consistently documents reduced QoL in adult GH-deficient patients. Improvements in QoL following initiation of GH, however, are not seen in all studies and possible reasons for this are discussed. In the United Kingdom, the National Institute of Clinical Excellence (NICE) evaluates evidence supporting the use of new medical therapies and medical devices for efficacy and cost-effectiveness and provides guidance on clinical use of these therapies. The NICE review of the use of GH replacement therapy in GH-deficient adults has recommended its use only for patients with significant impairment in QoL. Evidence for a beneficial effect of GH replacement in patients with osteopenia or to reduce vascular mortality was deemed inadequate to recommend more widespread use of GH replacement in hypopituitary adults.

III. WHAT DO WE MEAN BY “QUALITY OF LIFE?” Despite the term “quality of life” being well used and accepted in research and clinical practice, there is little consensus as to its definition. In this context, studies examining QoL in adult GH deficiency do not define exactly what they are studying. Patients, clinicians, and researchers are likely to interpret this term differently depending on which aspects of QoL they consider most important. QoL is a sum of the effect of symptoms, function, and sense of wellbeing on the patient. The effect of each of these on an individual’s QoL is variable and further complicated by the process of adaptation. Adaptation is the process by which a person with a disability maintains a good QoL by taking up pursuits that are within their capabilities. Alterations in QoL should reflect the overall effect of an illness and its treatment on a patient. It must be remembered that different disease states affect different aspects of QoL (i.e., vitality, anxiety, physical mobility) and that the patient is the best judge of their QoL.

IV. MEASUREMENT OF QUALITY OF LIFE Quality of life can be assessed either qualitatively or quantitatively. Qualitative assessment depends on a clinical interview, and the degree of distress experienced by the patient is assessed by the interviewer or observer. Lynch et al. (1994) interviewed 41 GHD adults and, using standardized psychiatric ratings, identified 46% of the cohort as definite psychiatric cases. Only 24% of an age- and sex-matched group with diabetes mellitus fitted a psychiatric diagnosis (Lynch et al., 1994). Holmes et al. (1995) interviewed 35 adult GHD patients and reported their major concerns: 94% disliked their body image, 91% had low energy, 83% had poor concentration and memory, and 71% felt irritable (Holmes et al., 1995). These trials have been invaluable not only in documenting that GHD adults experience a marked reduction in QoL, but in determining the areas of QoL most commonly affected. However, as qualitative assessments do not provide a numerical value from which the actual degree of impairment of QoL can be assessed and compared with other disease states and from which health care managers can determine cost–benefit analyses, the majority of studies have employed quantitative measures of QoL. Quantitative measures of QoL are obtained from questionnaires completed by the patient to provide a numerical value that reflects QoL (self-rating questionnaires). Questionnaires completed by the patient’s partner also have value in assessing a patient’s QoL and may be less

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subjective, but have the disadvantage that not all patients have a partner. Quantitative measures have the advantage of allowing comparison of QoL between patient groups and/or control subjects. Multiple self-rating questionnaires have been developed to quantify QoL in illness. These can generally be divided into those aimed at determining QoL between different disease states, generic questionnaires, and those designed for a specific disease state, disease-specific questionnaires. The more frequently used generic inventories include the Nottingham Health Profile (NHP-I and II), Psychological General Well-Being Schedule (PGWB), Rand 36 Item Health Survey (SF-36), Sickness Impact Profile (SIP), and the General Health Questionnaire (GHQ). Several of these inventories (NHP-I, PGWB, SF-36) are divided into subsections allowing particular areas of distress (domains) to be studied. To date there are few adult GHD-specific questionnaires. Several have relied on changing items within more established generic questionnaires (Wallymahmed et al., 1996). The Adult Growth Hormone Deficiency Assessment (AGHDA) is a disease-specific questionnaire that has been derived from unstructured interviews with GH-deficient adults. The major concerns of these patients were documented and used to construct the questionnaire (Holmes et al., 1995; Hunt, 1994). The format consists of 25 statements to which a “yes” or “no” answer is requested. The score range for the AGHDA is 0–25, with a score of 25 representing the greatest morbidity. Given that the AGHDA reflects the physical and physiological complaints expressed by GHD adults, this questionnaire should be sensitive to changes in the specific areas of QoL impaired in these adults. Concerns have been raised over the specificity of the AGHDA. Barkan (2001) administered the AGHDA to patients with GHD, acromegaly, and healthy controls. Scores in the controls (3.3 ⫾ 0.7) were significantly different from those in hypopituitary patients with unsubstituted GHD (10.6 ⫾ 1.5) and active acromegaly (11.6 ⫾ 1.6); however, the AGHDA was unable to discriminate between the latter two groups. It was suggested therefore that the ability of the AGHDA to detect changes in QoL with GH replacement be viewed with scepticism (Barkan, 2001). The results of this study are, however, not surprising, as the two major concerns expressed by GHD adults at interview were dislike of their body image and low energy levels, both of which are also concerns of patients with active acromegaly. The AGHDA should therefore more correctly be termed a disease-generated questionnaire rather than a disease-specific questionnaire. Whether the AGDHA is more sensitive to changes in QoL of GHD adults than generic questionnaires is debatable given the high degree of correlation between AGHDA and generic questionnaires such as the PGWB (Fig. 1) (Murray et al., 1999a).

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FIGURE 1 The relationship between generic (PGWB) and diseasespecific (AGHDA) quality of life questionnaires in untreated severe GHD adults.

V. EFFECT OF UNTREATED GROWTH HORMONE (GH) DEFICIENCY (GHD) ON QUALITY OF LIFE Studies aimed at documenting QoL in GHD adults have consistently found these patients to perceive significant impairment in a number of areas of their health status and psychological well-being. The vast majority of studies have used self-rating questionnaires to allow direct comparison of QoL between GHD adults and healthy control subjects. One of the earliest studies compared QoL scores derived from the NHP-I, PGWB, and GHQ of 24 GHD adults with 21 control subjects matched for age, gender, ethnic origin, and area of residence (McGauley, 1989; McGauley et al., 1990). The overall scores of the patients for all three questionnaires reflected significantly greater levels of psychological distress. Subsection analysis of the NHP-I revealed GHD adults to perceive themselves as being less energetic, more emotionally labile, and more socially isolated, whereas subsection analysis of the PGWB showed GHD patients to perceive themselves as having less vitality, less self-control, more anxiety, and poorer general health (McGauley, 1989; McGauley et al., 1990). A larger study comprising 86 GHD adults and 86 healthy controls matched for age, sex, marital status, and socioeconomic class, using the NHP, confirmed GHD adults to have impaired QoL from the overall score, with the areas of greatest psychological distress being reduced energy, social isolation, and emotional lability (Rosen et al., 1994). The patients additionally completed part 2 of the NHP, which showed significantly greater health-related problems among the patients concerning their sex life. A trend toward greater

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than expected numbers of patients with disablement pensions was also observed among the patients (19 vs 12,4, P ⫽ 0.09). By studying a control group with short stature (⬍3rd percentile), in addition to normal height controls and GHD adults, Rikken et al. (1995) were able to study the impact of both GHD and short stature on social status. The questionnaire completed by the subjects covered the areas of education, profession, income, living situation, marital status, and children (Rikken et al., 1995). No differences were found between normal height and short control subjects, suggesting that height does not impact significantly on QoL. GHD adults did not differ from controls on education, but scored lower on the professional scale, had a lower income, less often had a partner, and if they had a partner, they less frequently had children. More of the patients lived with their parents. No differences were detected between GHD adults with isolated GHD and those with multiple pituitary hormone deficits, suggesting that the cause of the lower scores on the social parameters was GHD itself (Rikken et al., 1995). In confirmation of this latter finding, no differences in QoL scores, using the AGHDA and PGWB, have been detected between patients with isolated GHD and multiple pituitary hormone deficits in a further study of GHD adults (Murray et al., 1999b). Patients who were found to have GHD during childhood (childhood onset, CO) have a number of phenotypic differences compared with patients who acquire GHD after completion of growth and puberty (adult onset, AO) (Attanasio et al., 1997). Relative to adult-onset GHD patients, childhood-onset patients are reported to have lower IGF-I levels (Attanasio et al., 1997; Lissett et al., 2002), lower bone mass (Holmes et al., 1994; Kaufman et al., 1992), and reduced lean body mass (Attanasio et al., 1997). Adultonset patients have a higher waist–hip ratio and lower HDL-cholesterol levels (Attanasio et al., 1997). With respect to QoL, Attanasio et al. (1997) reported that both AO and CO GHD adults have impaired QoL relative to age- and sex-matched healthy control subjects. The level of psychological distress was, however, greater in the AO group in all domains of the NHP. This reached significance for the domains of energy and physical mobility (Attanasio et al., 1997). A further study aimed at determining the subgroups of GHD adults with the greatest psychological distress, before GH replacement therapy, confirmed AO patients to have significantly more problems than CO patients on both the PGWB and the AGHDA questionnaires (Murray et al., 1999b). In contrast to these two studies, when QoL was assessed in patients enrolled in a large GH replacement postmarketing surveillance program (KIMS), no differences in baseline AGHDA scores were observed between AO and CO patients (Abs et al., 1999). It is possible that this latter finding is the consequence of selection bias, as it has been shown that patients who enter a trial of GH replacement therapy are those with greater impairment

of their baseline QoL (Holmes and Shalet, 1995a). Thus the lack of difference in baseline QoL between AO and CO patients in the postmarketing surveillance study may result from a selection bias in that CO patients with a “normal” quality of life do not request a trial of GH replacement. To explain the difference in self-reported QoL between AO and CO GHD patients in the former studies, it has been proposed that CO GHD patients who were born with or developed GHD early in life had grown up with any problems it created and therefore had little experience with which to contrast their current feelings (Hunt, 1994). Selfrating questionnaires may thus underestimate the true impairment of QoL in CO GHD adults.

VI. IS GH CAUSATIVE IN THE IMPAIRED QoL OF HYPOPITUITARY ADULTS? There are, however, a number of problems in assuming the impaired QoL observed in hypopituitary adults is a consequence of GHD. The majority of studies in the literature compare QoL in hypopituitary adults with that in healthy control subjects. Hypopituitary adults have generally undergone pituitary surgery and/or radiotherapy, the vast majority of patients have additional pituitary hormone deficits and are therefore required to take regular medication on a daily basis, and most will also be under hospital follow-up and will be attending for optimization of their replacement therapy and to monitor whether there has been a recurrence of the primary pathology. Given this degree of involvement with health professionals, it would be intuitive to expect these individuals to have impaired QoL even in the absence of GHD or active pituitary disease. The obvious answer to this dilemma is to use a control group of patients with normal GH status who have undergone pituitary surgery, radiotherapy, and are on additional pituitary hormone replacement for which they regularly attend hospital clinics. Unfortunately, the GH axis is the most frequently affected by pituitary surgery and radiotherapy, and thus patients who have additional pituitary hormone deficits are also likely to be GHD (Toogood et al., 1994). Comparison of QoL in hypopituitary patients with and without GHD is therefore unlikely to be possible. A further option is to compare QoL of GHD hypopituitary adults with that in patients diagnosed with another chronic disease. Lynch et al. (1994) compared QoL in GHD hypopituitary adults with that in adults with diabetes mellitus. The authors concluded that there was a significantly higher prevalence of psychiatric disturbance in hypopituitary patients than could be attributable solely to the presence of a chronic disease (Lynch et al., 1994). A further study compared QoL in hypopituitary patients with patients whom had undergone mastoid surgery and were followed up at

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least annually (Page et al., 1997). No differences were detected in QoL scores between the two groups. A possible explanation is that a proportion of the hypopituitary patients did not have GHD; however, when patients with at least two anterior pituitary hormone deficits were compared with mastoid patients, no difference in QoL scores was detected. Further analysis of data showed the hypopituitary patients who had received pituitary radiotherapy had impaired QoL compared to the mastoid patients, particularly depression and emotional lability (Page et al., 1997). This latter finding could be interpreted as suggesting pituitary radiotherapy is the cause of impaired QoL in hypopituitary adults instead of GHD.

VII. QoL FOLLOWING DISCONTINUATION OF CHILDHOOD GH THERAPY There are few studies examining the impact of discontinuation of childhood GH therapy after attainment of final height on QoL. Wiren et al. (2001) assessed the psychological well-being of 40 adults treated with GH during childhood. Baseline was considered to be the time at which GH was discontinued. GH status was reassessed after discontinuation of GH and on the basis of this the patients GH status was defined as GHD or GH replete. The patients were followed up for 2 years. At baseline, patients defined as GHD showed reduced well-being on the PGWB overall score as well as within the domains of depression and general health. After 2 years of follow-up there were no significant changes in PGWB scores of the GH replete patients; however, the PGWB overall score improved in the GHD cohort (Wiren et al., 2001). The only significant difference in the PGWB domains of the deficient and replete patients after 2 years was of greater anxiety in the GHD adults. Given current data concerning QoL in untreated GHD adults, combined with that following discontinuation of childhood GH, it is not possible to say without a doubt that the impaired QoL in hypopituitary adults is a consequence of GHD and does not relate purely to the presence of a chronic disease. Thus the only way to establish if GHD is causative, at least in part, to the impaired QoL of hypopituitary adults is to determine whether beneficial effects on psychological wellbeing and physical health occur during replacement therapy.

VIII. NATURAL HISTORY OF QoL IN ADULTS WITH UNTREATED GHD Limited data exist as to the long-term natural history of QoL in hypopituitary adults. Badia et al. (1998) administered the AGHDA to 356 GHD hypopituitary adults on

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conventional hormone replacement but not GH at baseline and 12 months. Although AGHDA scores worsened over the observation period, the difference was not statistically significant (Badia et al., 1998). In a cohort of elderly GHD hypopituitary adults, QoL was assessed at baseline and after 2 years. Similar assessments were made in an age- and sex-matched control group. Although QoL in the GHD elderly was observed to deteriorate in a number of domains of the multiple questionnaires used, similar deteriorations were observed in the control group (Li Voon Chong et al., 2002). As part of a long-term follow-up study of patients previously enrolled in a 6-month placebo-controlled study of GH replacement, Gilchrist et al. (2002) reviewed the changes in the NHP and PGWB after 9 years in patients who discontinued GH at the end of the initial placebocontrolled study. In these patients a decrease in the physical mobility subsection of the NHP was observed, but no significant change occurred in any of the domains of the PGWB (Gilchrist et al., 2002). In a similar study, Gibney et al. (1999) reassessed QoL after 10 years, using the NHP, in 11 patients who did not wish to continue GH replacement following an initial placebo-controlled study. No changes in the overall score or any of the subsection scores were observed (Gibney et al., 1999). Data, therefore, support the notion that in the absence of GH replacement therapy, QoL is reduced in individuals with GHD and impaired QoL does not improve significantly in the absence of GH replacement.

IX. EFFECTS OF GH REPLACEMENT ON QoL IN GHD HYPOPITUITARY ADULTS A. Double-Blind Placebo-Controlled Studies A number of double-blind placebo-controlled studies of GH replacement have assessed QoL as an end point. In nearly all of these studies the duration of GH replacement under placebo-controlled conditions has been 6 months, which has then frequently been followed by a period of open treatment with GH. This section considers only the findings of the initial double-blinded phase. The first placebo-controlled study of the effects of GH replacement on QoL in GHD hypopituitary adults involved 23 patients randomized to placebo (n ⫽ 12) or GH at a dose of 0.163 mg/kg body weight/week (⬃1.63 mg/day in a 70-kg man, n ⫽ 11) (McGauley, 1989; McGauley et al., 1990). No differences were observed in total or subsection scores of the placebo and GH-treated groups at 1 month. At 6 months there was a significant improvement in the overall score of the NHP, as well as the subsection for energy; no change in the PGWB overall score was

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noted; however, there was a significant improvement in the subsection for mood: the 60 item general health questionnaire showed a significant reduction in psychological distress. Six of the patients on GH replacement experienced side effects of edema and joint pain. Wallymahmed et al. (1997) performed a similar study in 30 GHD adults, with GH-treated patients receiving 0.083 mg/kg body weight/ week (⬃0.83 mg/day in a 70-kg man, n ⫽ 17). QoL was assessed using the NHP, Hospital Anxiety and Depression score (HAD), the Self-Esteem scale (SE), the Mental Fatigue Questionnaire (MFQ), and the Life Fulfilment and Impact scales. Eleven of the patients on active treatment reported side effects of arthralgia or edema warranting a dose reduction. Although there was an improvement in the energy subsection of the NHP within the GH-treated group at 6 months, this was not significantly different from the placebo group. There were no significant improvements in the HAD score, SE scale, or MFQ with GH therapy. The Life Fulfilment scale showed a reduction in material satisfaction at 6 months with GH therapy, whereas the Impact scale showed a reduction in the perceived impact of GHD with GH therapy; however, no differences from the placebo group were shown (Wallymahmed et al., 1997). In two small 6-month double-blinded crossover studies, Whitehead et al. (1992), in 14 GHD adults, failed to demonstrate an effect of GH on either the NHP or the PGWB, whereas Bengtsson et al. (1993), in 10 GHD adults, documented improvements in the Comprehensive Psychological Rating scale (CPRS) of 7 patients, but no change in the Symptom Check List-90 (SCL-90). It is unclear if improvements in the CPRS of the latter study reached significance. The maintenance GH dose used in both these studies was 0.083 mg/kg body weight/week, and significant side effects were documented in 5 and 7 patients, respectively. In 40 patients with GHD adults randomized to 6 months GH (0.093 mg/kg body weight/week) or placebo, Beshyah et al. (1995), during clinical interview, documented that 55% of patients on GH and 20% of patients on placebo reported improvements in well-being (P ⬍ 0.01). When assessed quantitatively using the GHQ and CPRS, however, no significant improvement in QoL could be demonstrated (Beshyah et al., 1995). In one of the largest double-blinded studies to date, no improvement in the NHP or NHP subscales was observed after 6 months on GH (0.083 mg/kg body weight/week) (Cuneo et al., 1998). Analyzing “treatment ⫻ time” interactions, an improvement in the overall and pain subscale scores was observed. In addition to the NHP, no improvement in the GHDQ or subscales was observed (Cuneo et al., 1998). In contrast to the aforementioned studies, which used weight-based dosing regimens extrapolated from the use of GH therapy in children, Baum et al. (1998) performed a randomized double-blind placebo-controlled study of 18 months duration in which GH was started at a dose of 0.07 mg/kg

body weight/week and the dose adjusted to normalize the serum IGF-I level (Baum et al., 1998). The mean GH maintenance dose during the study was 0.028 mg/kg body weight/week. QoL was assessed using the NHP, PGWB, GHQ, and Minnesota Multiphasic Personality Inventory-2 (MMPI-2). Cognition was assessed with the Wechsler Adult Intelligence Scale-Revised, Peabody Picture Volcabulary Test-Revised, Raven’s Standard Progressive Matrices, and the Controlled Oral Word Association Test. During the 18 months of the study there were no notable improvements in any of these measures with GH compared with the placebo group (Baum et al., 1998). The findings of the aforementioned double-blind placebo-controlled studies have thus been mixed. The majority show minimal improvements in QoL scores when GH replacement is compared with placebo-treated subjects. Most of the documented improvement occurs within selfrating questionnaire subscales rather than in overall quality of life. A number of reservations, however, must be made as to the appropriateness of studies undertaken to date to assess the impact of “physiological” GH replacement on QoL. First, the vast majority of these studies used weightbased dosing regimens extrapolated from pediatric practice, which have resulted in supraphysiological insulin-like growth factor I (IGF-I) levels, side effects, and a high proportion of patient withdrawals from the studies. It is likely that these side effects have detracted from any beneficial effect of GH on QoL. Furthermore, the occurrence of side effects in a high proportion of the GH-treated patients effectively unblinds the study, leading to doubt as to whether the study was truly blinded to the patient or the observer. Second, many of the patient cohorts were unselected, including patients with normal and impaired QoL. In 166 GHD adults entered into a study of GH replacement where QoL was assessed using the NHP, Cuneo et al. (1998) reported 77, 70.5, 59.5, 45.5, 43.5, and 36.5% of patients to score “0” (no impairment) in the subscales for pain, social isolation, physical mobility, emotional reaction, sleep, and energy, respectively. Thus in unselected cohorts with a large proportion of patients with normal psychological and physical well-being at baseline, any beneficial effect observed in the additional severely affected patients will be diluted and thus difficult to detect. At first glance, the study of Baum et al. (1998) looks to have provided the answer to whether GH has a beneficial effect on QoL in that it was conducted as a double-blind placebo-controlled study and the GH dose was adjusted to normalize the serum IGF-I level (Baum et al., 1998). Despite titration of the GH dose to the IGF-I level, patients were commenced on a GH dose of 0.07 mg/kg body weight/week (0.7 mg/day in a 70-kg man), which likely resulted in supraphysiological IGF-I levels and side effects in a number of patients given that the maintenance dose was less than half of the initial dose. Therefore, as in other

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16. Quality of Life Issues in Adult GHD 110 sGHD patients

Controls 100

(a) Holmes et al (1995) 92.0 (b) Holmes et al (1995) 83.0 Wiren et al (1998) 81.4

90 80 .

Page et al (1997) 75.0 Burman et al (1995) 70.0 McGauley (1989) 69.6

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Murray et al (1998) 59.7

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88.9 McGauley (1989) 83.5 Sacramento, community 82.0 Page et al (1997) 80.3 NHANES 76.5 Nashville, community

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FIGURE 2 The psychological well-being schedule (PGWB). Overall scores from community surveys and studies of severely GHD adults (sGHD): (a) those patients who entered a study of GH replacement and (b) those who were invited, but declined. Adapted from Murray and Shalet (1999).

studies, this would lead to unblinding of the treatment and detract from any beneficial effects of GH on QoL. Also the patients entering the study were not selected on the basis of impaired QoL and, in fact, had mean QoL scores on the PGWB at baseline (GH-treated group, 83; placebo-treated group, 85) similar to normal control individuals and random community samples (Fig. 2). Given that QoL of subjects in this study was normal at baseline, it is unreasonable to expect that further improvement to a QoL above that observed in normal health would be observed with GH replacement.

B. Open Studies of GH Replacement A number of 6-month double-blind placebo-controlled studies continued as open treatment studies (Beshyah et al., 1995; Wallymahmed et al., 1997). Not all patients continued in the open phase and a number of patients discontinued GH therapy due to side effects. The significant reduction in numbers of patients in turn resulting in a lack of power to successfully show a beneficial change in QoL. It is not surprising therefore that these studies failed to show a significant change in QoL at later time points (Beshyah et al., 1995; Wallymahmed et al., 1997). In a larger study of 99 adult-onset and 74 childhood-onset GHD adults, GH was continued for 12 months as an open study, following the initial 6-month double-blind phase. No improvement in QoL was observed in childhood-onset patients at any stage; however, QoL measured using the NHP improved in the adult-onset cohort by the end of the double-blind phase and remained unchanged thereafter until the end of the study (Attanasio et al., 1997). Improvements were observed in the energy and physical mobility subsections.

The majority of open treatment studies of GH replacement in unselected hypopituitary adults have demonstrated improvements in QoL. In 71 GHD adults of predominantly adult onset treated with GH at a dose of 0.084 mg/kg body weight/week, changes in QoL were assessed using the NHP-I and II and PGWB (Wiren et al., 1998). The overall NHP score improved at 6 months and this improvement was maintained at 20–50 months of follow-up. There were significant improvements in three of the six NHP subsections—energy, emotional reaction, and physical mobility. All areas of the NHP-II also improved, with the most marked improvements occurring in the areas of sex life, paid employment, housework, and hobbies. Total PGWB scores improved by around 7.5 points, with improvements in five of the six subsections (Wiren et al., 1998). Importantly in this study, the patients QoL was assessed at enrollment into the study and then at 6 months time, the point at which GH replacement was commenced. No change in QoL was observed during this 6-month run-in period, suggesting that changes in QoL were not due purely to enrollment into a study. In a smaller study of eight patients treated with 0.175 mg/kg body weight/week, six patients demonstrated significant improvements in QoL, particularly reduced fatigue (Binnerts et al., 1992). Many of the later studies of GH replacement used a lowdose GH titration regimen, which entails starting GH at a low dose, usually in the region of 0.2–0.3 mg/day, and slowly titrating the GH dose to normalize the serum IGF-I level as is undertaken in clinical practice (Drake et al., 1998; Murray et al., 1999a). In addition to using a low-dose titration regimen, Murray et al. (1999) selected a cohort of GHD hypopituitary adults with subjectively impaired QoL and assessed changes in the AGHDA and PGWB during 8 months of GH treatment. Objective assessment of QoL

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X. SPECIFIC PATIENT GROUPS A. The Elderly As GH secretion declines by around 14% per decade of adult life, it has been suggested that phenotypic differences between normal and GHD elderly would be less than that observed in younger adults, and replacement therapy may therefore have less to offer in older individuals. At baseline, elderly GHD adults show impaired QoL compared with age- and sex-matched control subjects. Using the NHP the patients express greater distress with respect to energy, mobility, emotional reactivity, and social isolation (Li Voon Chong et al., 2000) and, on the SF-36, they reported impaired social functioning, mental health, and general health (Li Voon Chong et al., 2000). Follow-up of these patients, untreated with GH, for 2 years showed a deterioration in energy, depression, and pain subscales; however, follow-up of a control group of healthy age- and sex-matched elderly also showed significant deteriorations in energy, vitality, self-esteem, depression, and mental health scores. At 2 years the only detectable difference between patients and controls was greater depression in the patients (Li Voon Chong et al., 2002). The authors therefore suggested that these results raised doubts over the potential beneficial effect of GH in the elderly. Further studies are clearly needed before the impact of GH replacement in elderly GHD hypopituitary patients can be fully assessed.

B. Long-Term Survivors of Childhood Cancer Survival rates for some childhood malignancies have increased dramatically since the mid-1980s, leading to 5-year survival rates in excess of 70% in many common childhood malignancies. As a consequence of the complex treatment regimens employed, significant detrimental effects on the endocrine system, growth, and fertility are recognized. GH secretion is recognized to be the most vulnerable of the hypothalamo–pituitary axes to radiation-induced damage. Young adult survivors of cancer who received cranial irradiation are frequently GHD and demonstrate symptoms and signs compatible with GHD. The relative contribution of GHD to these abnormalities has not been disentangled from the direct effects of the primary pathology, irradiation, chemotherapy, high-dose corticosteroids, and insufficient exercise. In an attempt to answer this question, QoL in 27 GHD survivors of childhood cancer was compared with age- and sex-matched controls both before and after 12 months of low-dose GH replacement therapy. At baseline, QoL was significantly impaired relative to the controls on both the AGHDA and the PGWB (Murray et al., 2002). Following GH replacement, QoL improved on both questionnaires within 3 months and was maintained at 12 months (Fig. 3), suggesting that GHD contributes significantly toward the impaired QoL in these patients. In a further study, QoL of GHD cancer survivors was compared with that of GHD adults of pituitary etiology before and after 6–13 and 24–77 months of GH replacement (Mukherjee, 2005). QoL as assessed by the AGHDA and PGWB was similarly impaired at baseline, and ranking of the PGWB domains was similar between the groups, with vitality the most impaired in both groups. All QoL variables improved with treatment, with the greatest improvement in the PGWB seen in the vitality domain for both GHD cancer survivors and GHD adults of pituitary etiology. No differences in response were detected between GHD cancer survivors and pituitary GHD adults. The authors concluded that QoL impairment in childhood

70 60 50 Change in PGWB

confirmed the patients to have severe impairment of QoL at baseline. Following GH replacement the overall PGWB score improved by around 14 points and there was also a significant improvement in the AGHDA score of 5.5 points (Murray et al., 1999a). These values represent some of the greatest improvements in QoL observed with GH replacement in hypopituitary adults. All PGWB subscales showed significant improvement, although the change in the vitality subsection was of the greatest magnitude. Two similar studies using low-dose titration regimens reported significant improvements in the AGHDA score of 7.0 and 3.3 points in 50 GHD adults treated for up to 12 months (Drake et al., 1998) and 46 GHD adults treated for 3 months, respectively (Ahmad et al., 2001). Additional information regarding the effect of GH replacement on QoL is available from analysis of data collected in large postlicensing surveillance programs from multiple centers. In an analysis of data from one of these surveillance programs, the effect of GH on the AGHDA of 665 GHD adults after 6 and 12 months of therapy showed improvements of 2.2 and 2.8 points, respectively (Bengtsson et al., 1999).

40 30 20 10 0 0 –10

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–20

FIGURE 3 The change in PGWB following 3 months of GH replacement compared with the PGWB score before commencing GH.

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16. Quality of Life Issues in Adult GHD

cancer survivors appeared to relate to GHD rather than cancer diagnosis and treatment.

the NHP and the vitality subsection of the PGWB were present after 9 years of follow-up.

XI. EFFECT OF LONG-TERM GH REPLACEMENT ON QoL

XII. PREDICTORS OF IMPROVEMENTS IN QoL WITH GH REPLACEMENT

Two studies have assessed QoL in patients at entry to a 6-month double-blind study of GH replacement and then attempted to reassess these patients’ QoL around a decade later. Gibney et al. (1999) reviewed NHP scores of 10 patients who continued GH after the initial doubleblinded study and 11 who did not. No significant change in NHP scores was observed in the untreated group; however, those patients still receiving GH showed improvements in the energy and emotional reaction subsection scores as well as in the overall NHP score (Gibney et al., 1999). Gilchrist et al. (2002) assessed QoL using the NHP and PGWB in a similar long-term study. Those patients who discontinued GH replacement after the initial 6-month double-blind study showed deterioration in the physical mobility domain of the NHP, but no change in PGWB subsection scores (Gilchrist et al., 2002). In those who remained on GH therapy, long-term improvements in the energy subsection of

A number of studies have tried to determine which patients are most likely to respond to GH replacement with a significant improvement in QoL. No effect of gender (Murray et al., 1999b; Wiren et al., 1998), age (Wiren et al., 1998), duration of GHD (Wiren et al., 1998), severity of GHD (Wiren et al., 1998), number of additional anterior pituitary hormone deficits (Bengtsson et al., 1999; Murray et al., 1999b; Rikken et al., 1995), concurrent changes in body composition (Ahmad et al., 2001; Murray et al., 1999a), or IGF-I (Ahmad et al., 2001; Bengtsson et al., 1999; Burman et al., 1995; Murray et al., 1999a) has been found to predict QoL changes in the majority of studies. To date, the only factor consistently able to predict the degree of improvement in QoL with GH replacement has been baseline QoL. Murray et al. (1999) found a significant correlation between baseline PGWB and AGHDA scores and the magnitude of improvement in the respective scale following GH replacement (Fig. 4)

(a)

(b) 25 100 20 80

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AGHDA

15

10

60

40 5 20 0 0 Controls

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3 months 12 months

Controls

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3 months 12 months

FIGURE 4 Box and whisker plots representing quality of life as represented by adult growth hormone deficiency assessment (AGHDA, range 0–25, high values represent greater morbidity) (a) and the psychological general wellbeing schedule (PGWB, range 0–110, low values represent greater morbidity) (b) in GH-deficient survivors of childhood cancer during 12 months of GH treatment and in healthy control subjects. The lower boundary of the box indicates the 25th percentile, a line within the box marks the median, and the upper boundary of the box indicates the 75th percentile. Error bars above and below the box indicate 90th and 10th percentiles, respectively.

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(Murray et al., 1999a). Females have been reported to show a greater improvement in QoL with GH replacement (Ahmad et al., 2001; Bengtsson et al., 1999); however, this has not been reported consistently. Following the study by Attanasio et al. (1997) showing improvements in the NHP with GH therapy in adult-onset patients but not childhood-onset patients, there has been considerable debate as to the effect of GH on QoL of childhood-onset patients (Attanasio et al., 1997). However, when childhood-onset patients with subjectively impaired QoL are specifically selected, these patients show at least equal improvements in QoL to adult-onset patients also selected for poor QoL (Murray et al., 1999b).

XIII. POTENTIAL BIASES IN OPEN STUDIES OF QoL Despite the fairly consistent improvements in QoL documented in open studies of GH replacement in GHD adults, one must first comprehend the potential biases that could influence the findings of these studies. The first potential bias is in patient selection. This problem was exemplified nicely by Holmes et al. (1995), who invited 98 unselected hypopituitary patients to enter a 12-month study of GH replacement. Sixty-five patients agreed to participate and 33 declined. Comparison of patients who entered the trial and those who declined entry revealed no differences in gender, age, BMI, peak stimulated GH value, duration of GHD, etiology of GHD, timing of onset of GHD, employment, or marital status. In contrast, QoL at baseline was significantly more impaired in the patients who entered the study, as shown by greater impairment overall, and in the energy and emotional reaction subsections of the NHP, as well as in the vitality subsection of the PGWB (Holmes and Shalet, 1995a). In addition to selection of patients with poorer QoL at baseline, those patients wishing to continue GH replacement as an open treatment study following an initial double-blind phase also have greater psychological distress at baseline (Gilchrist et al., 2002; Holmes and Shalet, 1995b) and greater improvements in well-being at the end of the double-blinded stage (Holmes and Shalet, 1995b; Wallymahmed et al., 1997). Together these findings suggest that patients entering a study of GH are those with the worst QoL, whereas those continuing treatment long term are those who feel benefit early during treatment. One of the problems occurring as a result of this self-“selected” population when studying QoL is the potential for regression toward the mean, leading to an improvement in QoL with time whatever treatment was administered. In the study by Murray et al. (1999) where improvements in the PGWB of 14 points were observed, regression to the mean was calculated to account for 35 to 70% of this improvement (Murray et al., 1999a). Additionally, as those patients

most likely to continue GH replacement are those whom have gained the most benefit early in the study, it is possible that those patients who leave the study are those with no notable change in their QoL. As a consequence this would leave only patients who feel a beneficial effect of GH in the latter stages of open studies when not analyzed on an intention to treat basis. Finally, there is an inevitable placebo effect, which has been documented in a number of placebocontrolled studies, although it has generally not reached significance. The mean improvement in the overall PGWB score of placebo-treated patients ranged from 1.0 to 1.9 points (Baum et al., 1998; Burman et al., 1995). There are therefore a number of problems inherent in open treatment studies when assessing QoL as an end point, and there can be no substitute for a properly conducted double-blind placebo-controlled study. To finally answer the question as to whether GH replacement has a therapeutic role in improving the quality of life of GHD hypopituitary adults, what is required is a double-blind placebo-controlled study of the effect of low-dose, titrated GH replacement on GHD adults with impaired QoL. Until this study is undertaken, we will be left with an endocrine community that is divided regarding the impact of GH replacement therapy on QoL in the adult.

XIV. SUMMARY Although GHD hypopituitary adults are consistently found to have impaired QoL and it would be simple to think of GH as causative, it is likely that a number of additional factors contribute to the overall psychological and physical well-being of these patients. Early treatment studies, which include the vast majority of double-blind placebo-controlled studies, used weight-based dosing regimens extrapolated from pediatric practice that resulted in a high proportion of side effects and withdrawals. Collectively these studies showed only minor improvements in QoL within self-rating questionnaire subscales rather than in overall scores. This may, in part, be the consequence of side effects detracting from the potential benefit of GH therapy. Later studies have generally been undertaken in the form of open treatment studies. Open studies have mostly documented a beneficial effect of GH on QoL but suffer from a placebo effect, regression to the mean, and patient selection biases. Long-term studies have been important in showing us that the beneficial effect of GH is likely maintained, although patient selection biases may have influenced the findings of these open studies. GHD long-term survivors of childhood cancer seem to benefit from GH replacement to a similar extent as hypopituitary patients, whereas the benefit to elderly GHD adults remains unclear.

16. Quality of Life Issues in Adult GHD

Along with the earlier double-blind placebo-controlled studies, open treatment studies have provided us with important information on how a definitive double-blind placebo-controlled study should be designed and conducted.

References Abdu, T. A., Neary, R., Elhadd, T. A., Akber, M., and Clayton, R. N. (2001). Coronary risk in growth hormone deficient hypopituitary adults: Increased predicted risk is due largely to lipid profile abnormalities. Clin. Endocrinol. (Oxf.) 55, 209–216. Abs, R., Bengtsson, B.-A., Hernberg-Stahl, E., Monson, J. P., Tauber, J., Wilton, P., and Wuster, C. (1999). GH replacement in 1034 growth hormone deficient hypopituitary adults: Demographic and clinical characteristics, dosing and safety. Clin. Endocrinol. 50, 703–13. Ahmad, A. M., Hopkins, M. T., Thomas, J., Ibrahim, H., Fraser, W. D., and Vora, J. P. (2001). Body composition and quality of life in adults with growth hormone deficiency; effects of low-dose growth hormone replacement. Clin. Endocrinol. (Oxf.) 54, 709–717. Attanasio, A. F., Lamberts, S. W., Matranga, A. M., Birkett, M. A., Bates, P. C., Valk, N. K., Hilsted, J., Bengtsson, B. A., and Strasburger, C. J. (1997). 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. Endocrin. Metab. 82, 82–88. Badia, X., Lucas, A., Sanmarti, A., and Ulied, A. (1998). One year followup of quality of life in adults with untreated growth hormone deficiency. Clin. Endocrinol. 49, 765–771. Barkan, A. L. (2001) The “quality of life-assessment of growth hormone deficiency in adults” questionnaire: Can it be used to assess quality of life in hypopituitarism? J. Clin. Endocrinol. Metab. 86, 1905–1907. Baum, H. B. A., Katznelson, L., Sherman, J. C., Biller, B. M. K., Hayden, D. L., Schoenfeld, D. A., Cannistraro, K. E., and Klibanski, A. (1998). Effects of physiological growth hormone (GH) therapy on cognition and quality of life in patients with adult-onset GH deficiency. J. Clin. Endocrinol. Metab. 83, 3184–3189. Bengtsson, B. A., Abs, R., Bennmarker, H., Monson, J. P., FeldtRasmussen, U., Hernberg-Stahl, E., Westberg, B., Wilton, P., and Wuster, C. (1999). The effects of treatment and the individual responsiveness to growth hormone (GH) replacement therapy in 665 GH-deficient adults: KIMS Study Group and the KIMS International Board. J. Clin. Endocrinol. Metab. 84, 3929–3935. Bengtsson, B. A., Eden, S., Lonn, L., Kvist, H., Stokland, A., Lindstedt, G., Bosaeus, I., Tolli, J., Sjostrom, L., and Isaksson, O. G. (1993). Treatment of adults with growth hormone (GH) deficiency with recombinant human GH. J. Clin. Endocrinol. Metab. 76, 309–317. Beshyah, S. A., Freemantle, C., Shahi, M., Anyaoku, V., Merson, S., Lynch, S., Skinner, E., Sharp, P., Foale, R., and Johnston, D. G. (1995). Replacement treatment with biosynthetic human growth hormone in growth hormone-deficient hypopituitary adults. Clin. Endocrinol. 42, 73–84. Binnerts, A., Swart, G. R., Wilson, J. H., Hoogerbrugge, N., Pols, H. A., Birkenhager, J. C., and Lamberts, S. W. (1992). The effect of growth hormone administration in growth hormone deficient adults on bone, protein, carbohydrate and lipid homeostasis, as well as on body composition. Clin. Endocrinol. 37, 79–87. Burman, P., Broman, J. E., Hetta, J., Wiklund, I., Erfurth, E. M., Hagg, E., and Karlsson, F. A. (1995). Quality of life in adults with growth hormone (GH) deficiency: Response to treatment with recombinant human GH in a placebo-controlled 21-month trial. J. Clin. Endocrinol. Metab. 80, 3585–3590. Cuneo, R. C., Judd, S., Wallace, J. D., Perry Keene, D., Burger, H., Lim Tio, S., Strauss, B., Stockigt, J., Topliss, D., Alford, F., Hew, L.,

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lipid profiles and quality of life after 2 years compared to controls. Clin. Endocrinol. (Oxf.) 56, 175–181. Lissett, C. A., Murray, R. D., and Shalet, S. M. (2002). Timing of onset of growth hormone deficiency is a major influence on insulin-like growth factor-I status in adult life. Clin. Endocrinol. (Oxf.) 57, 35–40. Lynch, S., Merson, S., Beshyah, S. A., Skinner, E., Sharp, P., Priest, R. G., and Johnston, D. G. (1994). Psychiatric morbidity in adults with hypopituitarism. J. R. Soc. Med. 87, 445–447. McGauley, G. A. (1989). Quality of life assessment before and after growth hormone treatment in adults with growth hormone deficiency. Acta Paediatr. Scand. Suppl. 356, 70–72; discussion 73–74. McGauley, G. A., Cuneo, R. C., Salomon, F., and Sonksen, P. H. (1990). Psychological well-being before and after growth hormone treatment in adults with growth hormone deficiency. Horm. Res. 33(Suppl. 4), 52–54. Murray, R. D., Darzy, K. H., Gleeson, H. K., and Shalet, S. M. (2002). GH-deficient survivors of childhood cancer: GH replacement during adult life. J. Clin. Endocrinol. Metab. 87, 129–135. Murray, R. D., and Shalet, S. M. (1999). The use of self-rating questionnaires as a quantitative measure of quality of life in adult growth hormone deficiency. J. Endocrinol. Invest. 22, 118–126. Murray, R. D., Skillicorn, C. J., Howell, S. J., Lissett, C. A., Rahim, A., and Shalet, S. M. (1999a). Dose titration and patient selection increases the efficacy of GH replacement in GHD adults. Clin. Endocrinol. 50, 749–757. Murray, R. D., Skillicorn, C. J., Howell, S. J., Lissett, C. A., Rahim, A., Smethurst, L. E., and Shalet, S. M. (1999b). Influences on quality of life in GH deficient adults and their effect on response to treatment. Clin. Endocrinol. 51, 565–573. Page, R. C., Hammersley, M. S., Burke, C. W., and Wass, J. A. (1997). An account of the quality of life of patients after treatment for non-functioning pituitary tumours. Clin. Endocrinol. 46, 401–406. Rikken, B., van Busschbach, J., le Cessie, S., Manten, W., Spermon, T., Grobbee, R., and Wit, J. M. (1995). Impaired social status of growth hormone deficient adults as compared to controls with short or normal stature: Dutch Growth Hormone Working Group. Clin. Endocrinol. 43, 205–211.

Rosen, T., Bosaeus, I., Tolli, J., Lindstedt, G., and Bengtsson, B. A. (1993). Increased body fat mass and decreased extracellular fluid volume in adults with growth hormone deficiency. Clin. Endocrinol. 38, 63–71. Rosen, T., Wiren, L., Wilhelmsen, L., Wiklund, I., and Bengtsson, B. A. (1994). Decreased psychological well-being in adult patients with growth hormone deficiency. Clin. Endocrinol. 40, 111–116. Salomon, F., Cuneo, R. C., Hesp, R., and Sonksen, P. H. (1989). The effects of treatment with recombinant human growth hormone on body composition and metabolism in adults with growth hormone deficiency. N. Engl. J. Med. 321, 1797–1803. Toogood, A. A., Beardwell, C. G., and Shalet, S. M. (1994). The severity of growth hormone deficiency in adults with pituitary disease is related to the degree of hypopituitarism. Clin. Endocrinol. 41, 511–516. Wallymahmed, M. E., Baker, G. A., Humphris, G., Dewey, M., and MacFarlane, I. A. (1996). The development, reliability and validity of a disease specific quality of life model for adults with growth hormone deficiency. Clin. Endocrinol. 44, 403–411. Wallymahmed, M. E., Foy, P., Shaw, D., Hutcheon, R., Edwards, R. H., and MacFarlane, I. A. (1997). Quality of life, body composition and muscle strength in adult growth hormone deficiency: The influence of growth hormone replacement therapy for up to 3 years. Clin. Endocrinol. 47, 439–446. Whitehead, H. M., Boreham, C., McIlrath, E. M., Sheridan, B., Kennedy, L., Atkinson, A. B., and Hadden, D. R. (1992). Growth hormone treatment of adults with growth hormone deficiency: Results of a 13-month placebo controlled cross-over study. Clin. Endocrinol. 36, 45–52. Wiren, L., Bengtsson, B. A., and Johannsson, G. (1998). Beneficial effects of long term GH replacement therapy on quality of life in adults with GH deficiency. Clin. Endocrinol. 48, 613–620. Wiren, L., Johannsson, G., and Bengtsson, B. A. (2001). A prospective investigation of quality of life and psychological well-being after the discontinuation of GH treatment in adolescent patients who had GH deficiency during childhood. J. Clin. Endocrinol. Metab. 86, 3494–3498.

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17 The Somatotropic Axis in Psychologic Functioning: Effects on Sleep and Psychopathology HARALD JÖRN SCHNEIDER and GÜNTER KARL STALLA Internal Medicine, Endocrinology and Clinical Chemistry Max Planck Institute of Psychiatry, 80804 Munich, Germany

both GH and IGF-I may pass the blood–brain barrier suggest important clinical effects of the somatotropic system on the brain (Schneider et al., 2003). Several clinical studies have revealed interesting findings on GH, IGF-I, and growth hormone-relasing hormone (GHRH) actions in the central nervous system. Deficiency of GH secretion and low IGF-I levels may cause typical changes in brain function, whereas certain pathological brain affections can lead to changes in GH secretion. The classical reasons for GH deficiency are intrapituitary masses such as pituitary adenomas (Fig. 1, left). Studies have shown that brain damage such as traumatic brain injury (Fig. 1, right) or subarachnoid hemorrhage may often lead to hypopituitarism and cause GH deficiency in 10 to 20% of all affected patients (Schneider et al., 2004). Here, generally, it is difficult to clinically distinguish whether brain injury, GH deficiency, or both cause brain dysfunction. It is still not clear if GH substitution might lead to improvement in these conditions, as clinical studies on this field are still lacking. This chapter focuses mainly on GH deficiency and its substitution and their effects on sleep and psychopathology, as other aspects, such as cognitive function and the effects of GH excess, are reviewed in different chapters.

I. Introduction II. Sleep III. Mood and Psychiatric Symptoms References Mutual interactions of the somatotropic system with behavioral functions such as sleep and mood regulation are well documented. Growth hormone (GH) secretion is closely associated to slow wave sleep. Growth hormone-releasing hormone and GH secretagogues have been found to have sleep-promoting effects, whereas somatostatin may suppress sleep. In depressed patients, a blunted GH response after stimulation of the somatotropic axis and a disturbed pattern of GH secretion have been found. Patients with GH deficiency report deficits in mood and well-being, which have been shown to be improved by GH replacement in some but not all studies.

I. INTRODUCTION The presence of growth hormone (GH) and insulin-like growth factor-I (IGF-I) receptors in many areas in the brain (Nyberg et al., 2001) and the strong evidence that

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FIGURE 1 Typical causes of GH deficiency. (Left) Macroadenoma of the pituitary (sagital MRI scan). (Right) Traumatic brain injury (initial CT scan at ICU showing scull fractures, frontal bleedings, and traumatic subarachnoid hemorrhage). (See color plate 18)

II. SLEEP There are well-documented bidirectional interactions between human sleep and the somatotropic system (reviewed by Steiger and Holsboer, 1997; van Cauter et al., 1998; Steiger, 2003). There is a close association among slow wave sleep (SWS), sleep stages III and IV, and the nocturnal growth hormone surge in humans (Quabbe et al., 1966; Steiger et al., 1987; Mullington et al., 1996). In young men the GH peak at sleep onset is normally the most reproducible peak. In middle-aged and elderly men this is often the only interval during which measurable GH secretion takes place. In premenopausal women the GH peak at sleep onset also occurs but does not normally constitute the main part of 24-h secretion. In shifts of sleep onset the GH peak still normally occurs at sleep onset (van Cauter et al., 2000), but in about 25% of young men a GH peak occurs before sleep onset (Steiger et al., 1989; Mullington et al., 1996). With increasing age, a parallel exponential decline in SWS duration and nocturnal GH secretion occurs (van Cauter et al., 1998). Therefore, it has been hypothesized that there is a common cause for SWS and the regulation of pulsatile GH secretion (Steiger and Holsboer, 1997). Manipulations of the sleep–wake cycle result mostly in changes of GH secretion: for example, in a blunted GH release during sleep deprivation (Beck et al., 1975), although not in young men (Mullington et al., 1996), a delay of the GH peak in parallel to an increased sleep latency (Takahashi et al., 1968).

Preclinical, human, and clinical studies have shown that all components of the somatotropic system are involved in the physiology of sleep regulation: GHRH (Krueger and Obál, 1993; Steiger and Holsboer, 1997), somatostatin (Danguir, 1986; Frieboes et al., 1997; Ziegenbein et al., 2004), GH (Stern et al., 1975; Mendelson et al., 1980, Aström et al., 1990; Kern et al., 1993; Obál et al., 1997), IGF-I (Obál et al., 1998), synthetic GH secretagogues (Frieboes et al., 1995; Copinschi et al., 1997), and the natural GH secretagogue ghrelin (Weikel et al., 2003). In healthy individuals, GH has been found to reduce SWS and increase rapid eye movement (REM) duration (Mendelson et al., 1980) or to exert no changes in the sleep pattern (Kern et al., 1993). When GHRH is applied in a pulsatile fashion during the first half of the night, an increase in SWS and GH secretion and a suppression of cortisol can be seen (Steiger et al., 1992). Other authors found enhanced REM sleep after a bolus injection of GHRH but an increase of SWS only after sleep deprivation or when GHRH was given in the third REM sleep period (Kerkhofs et al., 1993). When comparing pulsatile GHRH injections to continuous GHRH infusions, a more pronounced effect of pulsatile GHRH administration on sleep promotion and GH secretion was found (Marshall et al., 1996). During aging the response of GH and SWS to GHRH declines (Guldner et al., 1997). Somatostatin decreases SWS in elderly subjects (Frieboes et al., 1997), whereas in young individuals, no effect of somatostatin on sleep electroencephalography (EEG) can be

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found (Steiger et al., 1992, Kupfer et al., 1992). REM sleep is also stimulated by GHRH in normal but not in hypophysectomized rats (Obal et al., 1996). Thus non-REM sleep appears to be directly stimulated by GHRH, whereas REM sleep seems to be mediated by GH but not by GHRH. The fact that GHRH stimulates non-REM sleep in both normal and hypophysectomized rats implies that GHRH directly stimulates non-REM sleep. However, both GHRH and GH increase REM sleep and GH reduces SWS, which implies that REM sleep is stimulated by GH (reviewed by Steiger and Holsboer, 1997) and that the decrease in SWS by GH is induced by a negative feedback mechanism on GHRH. Pulsatile administration of ghrelin, the natural GH secretagogue receptor ligand, leads to enhanced SWS and stimulation of GH and cortisol secretion (Weikel et al., 2003). A single dose of GHRP-6, a synthetic GH secretagogue, induces an increase in stage II sleep (Frieboes et al., 1995, 1999). Oral administration of MK-677, another GH secretagogue, over 1 week led to an increase in REM and stage IV sleep (Copinschi et al., 1997). In some respects the adrenocorticotropic system appears to act as an antagonist of the somatotropic system. Corticotropinreleasing hormone (CRH) reduces nocturnal GH secretion (Holsboer et al., 1988), and CRH, adrenocorticotropic hormone (ACTH), and cortisol decrease REM sleep duration (Born et al., 1989): SWS duration is reduced by CRH (holsboer et al., 1988; Tsuchiyama et al., 1995) and increased by cortisol (Born et al., 1989, 1991). This implies a central inhibition of SWS by CRH and an inhibition of REM sleep by cortisol. During normal aging and in depression sleep becomes shallow, SWS and the activity of the somatotropic system decrease, and the activity of the adrenocorticotropic system increases so that the balance between the two systems shifts toward the adrenocorticotropic system (Steiger and Holsboer, 1997). Only few studies have investigated the sleep EEG in patients with pathological changes of GH secretion so far. Patients with acromegaly who suffer from GH hypersecretion have been reported to have reduced SWS and REM sleep (Aström et al., 1991). In children with growth hormone deficiency (GHD), a reduction in REM sleep and partial normalization after GH substitution have been described (Wu and Thorpy, 1988; Hayashi et al., 1992). Data regarding effects of GH substition in adult GH-deficient patients are somewhat controversial. Whereas Aström et al. (1990) found a partial normalization of prolonged total sleep time and reduced SWS by GH replacement in 9 patients mainly with isolated childhoodonset (Co) GHD, no significant effects of 6 months of GH substitution were found in 17 patients with adulthood-onset (Ao) GHD, having mostly multiple pituitary hormone deficiencies (Schneider et al., 2005). Moreover, sleep EEG at baseline was comparable to healthy controls from the literature, and assessment of daytime sleep propensity by

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a multiple sleep latency test also revealed no effect of GH substitution (Schneider et al., 2005). Differences in the enrolled collectives might explain these diverging findings. In patients with traumatic brain injury, a reduction of nocturnal GH secretion and stage II sleep has been found, a sleep pattern similar to patients with remitted depression (Frieboes et al., 1999). However, here it is not clear if impairment of GH secretion is causally related or an accompanying sequel of brain injury. In conclusion, it has been shown that all parts of the somatotropic system interact with sleep. It has been shown that GHRH and GH secretagogues have strong sleep-promoting effects, whereas diverging findings on the effects of GH substitution in GH-deficient subjects have been reported.

III. MOOD AND PSYCHIATRIC SYMPTOMS Several studies have attempted to determine the possible effects of GH on the brain, including cognition, quality of life, and well-being (reviewed by Schneider et al., 2003). Changes of somatotropic secretion have been found in mood disorders; however, data are still scarce and not quite conclusive. Other studies have investigated the changes in psychological functioning caused by GH deficiency, depending on the time of its onset and the effects of GH replacement therapy. Unfortunately, many studies do not make the distinction between patients with isolated growth hormone deficiency and those with multiple pituitary hormone deficiencies, where the lack of GH is accompanied by additional deficiencies in other pituitary hormones. Healthy young men with high depression and anxiety scores show a reduced or no increase in GH secretion after physical exercise (Harro et al., 1999). In depressive patients, a reduced 24-h GH secretion has been found that has been attributed mainly to decreased nocturnal GH levels (Voderholzer et al., 1993; Fiasche et al., 1999). This has been confirmed in another study measuring only nocturnal GH secretion (Sakkas et al., 1998). Other authors have found unchanged daytime secretion (Deuschle et al., 1997) or even increased 24-h GH secretion due to elevated diurnal levels (Mendlewicz et al., 1985; Linkowski et al., 1987). The GH response to stimulation testing with GHRH (Voderholzer et al., 1993) and clonidine (Mokrani et al., 2000) is reduced, but elevated levels of IGF-I have been observed (Deuschle et al., 1997, Franz et al., 1999). In depressed children, a decreased GH response to insulininduced hypoglycemia (Ryan et al., 1994) and GHRH administration (Dahl et al., 2000) has also been found. In addition, in children and adolescents who are not depressive but have a high genetic risk due to a family history of depression, a reduced increase of GH to GHRH stimulation testing was found (Birmaher et al., 2000). Taken

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together, most studies report a disturbance of GH secretion in depression. Even though data are somewhat conflicting, evidence points toward a blunted GH response after stimulation in depressed patients. As this also has been observed in nondepressed family members of depressive patients, it is possible that blunted GH secretion may be a trait marker for subjects at high gentic risk for depression. The pattern of GH secretion in depression seems to be changed with a shift toward elevated daytime and reduced nocturnal levels. Investigations have shown that GHD in adulthood is associated with lack of vitality, low mood, decreased quality of life, and increased social isolation, as well as an impaired sense of well-being relative to the normal population (Rosen et al., 1994). Adults with CoGHD who were treated with GH during childhood had similar school achievements as their peers, but were more likely to be unemployed or underemployed and were often unmarried. Depression, particularly anxiety disorders and social phobia, was observed in these patients as well (Deijen et al., 1996; Stabler, 2001). It has been found that patients with AoGHD have more difficulties in working and reduced enjoyment from social occasions than patients with CoGHD (Attanasio et al., 1997; Hunt et al., 1993). This has been attributed to the fact that CoGHD patients grow up with the problems associated with GHD, whereas patients with AoGHD are aware of the difference between before and after the development of GHD. Several studies have attempted to assess the effects of GH replacement on mood and well-being. Some authors have found significant improvement of psychological wellbeing and mood after GH replacement (Bengtsson et al., 1993; Burman et al., 1995; Attanasio et al., 1997; Wiren et al., 1998). The effect was shown to be dose dependent and more pronounced in adults with acquired GHD than in those with CoGHD (Attanasio et al., 1997; Bengtsson et al., 1999). Other authors have reported no beneficial effect of GH replacement on mood and well-being (Degerblad et al., 1990; Whitehead et al., 1992; Baum et al., 1998; Deijen et al., 1998). Possible explanations for these diverging findings might be differences in methodology of psychological assessment, dosing regimens, and patient populations. Only few studies have tried to assess the prevalence of psychiatric diseases in GHD. Korali et al. (2003) assessed the lifetime occurence of mental disorders in 93 patients with pituitary adenomas compared to a matched control group with a standardized psychopathological assessment. They found no increased prevelance of mental disorders in this sample. However, these results should be interpreted carefully with regard to GHD as the study population was not selected for GHD and only 53% of the patients in this sample were GH deficient. Bulow et al. (2002) studied 33 women with GHD and found an increased incidence of mental disorders compared to healthy controls with more symptoms of

somatization, anxiety, depression, obsession–compulsion, hostility–irritability, phobic, and psychotic symptoms (Bulow et al., 2002). In summary, clinical studies show that mood and GH secretion are related. Changes of GH secretion and IGF-I levels are found in depression. However, GH deficiency causes typical complaints of decreased well-being, low mood, and lack of vitality. These symptoms have been shown to be improved by GH substitution. There is some evidence that the prevalence of mental disorders is increased in GH-deficient subjects; however, data on this topic are still scarce and further studies are needed.

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Takahashi, Y., Kipnis, D. M., and Daughaday, W. H. (1968). Growth hormone secretion during sleep. J. Clin. Invest. 47, 2079–2090. Tsuchiyama, Y., Uchimura, N., Sakamoto, T., Maeda, H., and Kotorii, T. (1995). Effects of hCRH on sleep and body temperature rhythms. Psychiatr. Clin. Neurosciences 49, 299–304. Van Cauter, E., and Copinschi, G. (2000). Interrelationships between growth hormone and sleep. Growth Horm. IGF Res. 10(Suppl. B), S57–S62. Van Cauter, E., Plat, L., and Copinschi, G. (1998). Interrelations between sleep and the somatotropic axis. Sleep 21, 553–566. Van Coevorden, A., Mockel, J., Laurent, E., Kerkhofs, M., L’HermiteBalériaux, M., Decoster, C., Neve, P., and Van Cauter, E. (1991). Neuroendocrine rhythms and sleep in aging men. Am. J. Physiol. 260, E651–E661. Voderholzer, U., Laakmann, G., Wittmann, R., Daffner-Bujia, C., Hinz, A., Haag, C., et al. (1993). Profiles of spontaneous 24-hour and stimulated growth hormone secretion in male patients with endogenous depression. Psychiatr. Res. 47, 215–227. Weikel, J. C., Wichniak, A., Ising, M., Brunner, H., Friess, E., Held, K., Mathias, S., Schmid, D. A., Uhr, M., and Steiger, A. (2003). Ghrelin promotes slow-wave sleep in humans. Am. J. Physiol. Endocrinol. Metabol. 284, E407–E415. Whitehead, H. M., Boreham, C., McIlrath, E. M., Sheridan, B., Kennedy, L., Atkinson, A. B., Hadden, D. R. (1992). Growth hormone treatment of adults with growth hormone deficiency: Results of a 13-month placebo controlled cross-over study. Clin. Endocrinol. 36, 45–52. Wiren, L., Bengtsson, B. A., and Johannsson, G. (1998). Beneficial effects of long-term GH replacement therapy on quality of life in adults with GH deficiency. Clin. Endocrinol. 48, 613–620. Wu, R. H., and Thorpy, M. J. (1988). Effect of growth hormone treatment on sleep EEGs in growth hormone-deficient children. Sleep 11, 425–429. Ziegenbein, M., Held, K., Kuenzel, H. E., Murck, H., Antonijevic, I. A., and Steiger, A. (2004). The somatostatin analogue octreotide impairs sleep and decreases EEG sigma power in young male subjects. Neuropsychopharmacology 29, 146–151.

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18 Growth Hormone Replacement Therapy in Adults: Responsiveness Related to Life Quality ÅSE KROGH RASMUSSEN AND ULLA FELDT-RASMUSSEN Medical Department of Endocrinology Rigshospitalet, University of Copenhagen DK-2100 Copenhagen, Denmark

I. II. III. IV.

Introduction Quality of Life (QoL) Measurements QoL in Growth Hormone (GH)-Deficient Adults Effects of GH Deficiency and GH Therapy on QoL in Specific Conditions V. Therapeutic Mechanisms of GH Replacement VI. Cost–Benefit Considerations VII. Conclusion References

of QoL in GH-deficient patients and an improvement or normalization after GH replacement exists.

I. INTRODUCTION Growth hormone (GH) exerts pleiotropic effects on all organ systems and may influence quality of life (QoL) via neurological mechanisms or secondary to GH effects on metabolic, cardiovascular, immune, reproductive, bone, and/or muscular functions. The typical phenotype of GH deficiency has as part of the syndrome a reduction in many classical QoL measures (Table I). QoL has been defined by the World Health Organization as “the state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity” (WHO, 1947). QoL is complex and is a difficult concept to quantify and measure across disease states. Prior to the existence of recombinant GH, GH was only used therapeutically to correct height deficit in children. The access to recombinant GH opened up the possibility of also treating adults with GH deficiency. However, GH therapy remains expensive, and it has been suspected to potentially worsen or induce cancer by being a growth stimulator (Hankinson et al., 1998; Shaneyfelt et al., 2000). It is therefore mandatory to collect documentation both for safety and for efficacy of therapy.

Growth hormone (GH) exerts pleiotropic effects on all organ systems and may influence quality of life (QoL). The importance of measuring health-related QoL as one of several valid indicators of whether treatment with GH is beneficial is stressed. There are two main types of health-related QoL measures—disease specific and generic—and a combination is generally advocated, as each provides complementary information. However, it is mandatory that the questionnaires are validated correctly and that tests in the relevant language with a sufficient population-based reference group are available. Former published clinical studies of effects of GH replacement therapy on QoL in adults are scrutinized, and the therapeutic mechanisms of GH replacement are discussed. Although many of the studies performed to assess QoL and GH replacement are not long enough, not controlled, or use too high GH dose or the wrong QoL instruments, a growing body of evidence for an impairment

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TABLE I Symptoms and Signs of Adult Growth Hormone Deficiency Symptoms of growth hormone deficiency Decreased psychological well-being Reduced energy and vitality Poor general health Impaired self control Disturbed emotional reactions Lack of positive well-being Depressed mood Increased anxiety Increased social isolation Increased abdominal adiposity Reduced strength and physical endurance Thin dry skin Signs of growth hormone deficiency Truncal obesity Increased waist–hip ratio Thin dry skin Abnormal body composition Decreased psychological well-being Reduced exercise performance Abnormal cardiac structure and function Cardiovascular risk factors Decreased bone density Disturbed renal function Lowered basal metabolic rate Decreased sweat secretion Increased insulin resistance

The advisability of GH replacement therapy for GHdeficient adults is still controversial. Although a change in body composition to a more favorable state has been highly reproducible in all studies on the effect of GH replacement in patients with hypopituitarism, other variables, such as lipid profile, blood pressure, morbidity, and mortality, have been more controversial. The importance of measuring health-related QoL as one of several valid indicators of whether treatment with GH is beneficial has thus been highly acknowledged and is commonly noted in consensus statements of official bodies. The American Association of Clinical Endocrinologists (AACE) states that “Replacement therapy should be monitored carefully . . . and special emphasis should be placed on perceived and objectively measured benefits and adverse effects” (AACE Growth Hormone Task Force, 2003). Also, in the technology appraisal for GH replacement therapy in adults published by National Institute for Clinical Excellence (NICE) (2003), decreased QoL, as determined by the diseasespecific questionnaire on quality of life assessment of growth hormone deficiency of adults (AGHDA) (Mc Kenna et al., 1999), is prerequisite for starting GH replacement, and evidence of improvement of AGHDA scores should be provided after 9 months in the individual patient (3 months of titration followed by 6 months of stable therapy).

This underlines the importance of having access to reliable routine measures of QoL in these patients, with a sufficient accuracy, precision, sensitivity, and specificity of the assessment.

II. QUALITY OF LIFE (QoL) MEASUREMENTS With the broad definition of WHO in mind, the assessment of health-related quality of life is an attempt to determine how variables related to a condition or its treatment relate to particular domains of life considered important to people in general or to people who have a specific disease condition (Guyatt et al., 1993; Wilson and Cleary, 1995). Most concepts of health-related QoL emphasize the effects of illness on physical, psychological, and social functioning (Ware, 1995). The importance of measuring health-related QoL in clinical research has been acknowledged, and many patient-reported measures are becoming increasingly accepted as valid indicators of whether a medical treatment is beneficial (Spilker, 1996; Testa and Simonsen, 1996). Thus, patient-reported health-related QoL measures are becoming increasingly accepted also in the assessment of patients with GH deficiency, and a number of different questionnaires have been used to quantify the effect of GH therapy on QoL in adults (reviewed in Hull and Harvey, 2003). There are two main types of health-related QoL measures: disease specific and generic (Table II). Older studies have usually relied on generic tests measuring the total burden of disease, such as the Short Form 36 (SF-36) Health Survey (consisting of 36 items that assess eight health dimensions: physical functioning, role limitations due to physical health, bodily pain, general health perceptions, vitality, social functioning, role limitations due to emotional problems, and mental health), the Nottingham Health Profile (NHP) (consisting of 38 items that assess six dimensions of health problems: physical mobility, pain, social isolation, emotional reactions, energy level, and sleep), or the General Health Questionnaire (GHQ). However, in theory, disease-specific measures demonstrate greater sensitivity than generic measures. Questions in the disease-specific health survey refer directly to typical symptoms of the GHdeficient condition and address problems that are specific to or common among patients with GH deficiency. The first GH deficiency-specific questionnaire was the AGHDA (Mc Kenna et al., 1999). The QoL-AGHDA was developed through in-depth interviews with hypopituitary adults, validated, and adobted for cross-cultural use (Holmes et al., 1995). It is a needs-based questionnaire with item questions such as “It is difficult for me to make friends,” “I often forget what people have said to me,” “I find it difficult to plan ahead,” “I have difficulty in controlling my emotions,” and “I often feel too tired to do the things I ought to do.”

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TABLE II Tests Used to Assess Quality of Lifea General tests AS – Apathy Evaluation Scale BDI – Beck Depression Inventory BSI – Brief Symptom Inventory CIS – Clinical Interview Scale CPRS – Comprehensive Psychological Rating Scale DSQ – Disease Specific Questionnaire GHQ – General Health Questionnaire GWBS – General Well-Being Schedule HDS – Hamilton Depression Scale HSCL – Hopkins Symptoms Check-List HADS – Hospital Anxiety and Depression Scale KSQ – Kellner Symptom Questionnaire LFS – Life Fulfilment Scale LSC – List of Somatic Complaints MFQ – Mental Fatigue Questionnaire MFS – Mental Fatigue Scale MMPI-2 – Minnesota Multiphasic Personality Inventory-2 MACL – Mood Adjective Check List NHP – Nottingham Health Profile PAS – Personality Assessment Schedule POMS – Profile of Mood States PGWB – Psychological and General Well-Being Schedule SES – Self-Esteem Scale SF-36 – Short Form 36 SAS – Social Adjustment Scale SRS – Social Relationship Scale SCL-90 – Symptom Check-List-90 Disease-specific tests QLS(M)-H – Questions on Life Satisfaction Modules-Hypopituitarism DSQ – Disease Specific Questionnaire AGHDA – Adult Growth Hormone Deficiency Assessment GHD-LFS – Modified Life Fulfilment Scale GHD-IS – Modified Impact Scale GHDQ – Growth Hormone Deficiency Questionnaire a

Modified from Hull and Harvey (2003), with permission from the authors.

A combination of disease-specific and generic measures is generally advocated because each provides complementary information (Guyatt et al., 1993), and it should possibly be acknowledged that no single QoL instrument will provide absolute discrimination between all patients and a control population. Individual clinical assessment remains paramount (Monson, 2000). A major problem in comparing data from various studies is the fact that not all of the questionnaires have been validated correctly and tested in all languages, and very rarely is a sufficient population-based reference group available by each country. This is true for QoL measures of both generic- and disease-specific types. Also, GH-deficient populations treated with GH replacement may differ very much by a different approach and physician attitude toward whether the criteria set down by the Growth

Hormone Research Society (GRS) should be followed (Growth Hormone Research Society, 1998). Furthermore, different official criteria such as those by NICE in the United Kingdom may create the basis for large population differences, as only very severe and long-lasting GH deficiency can be treated in the United Kingdom, whereas an appropriately performed GH stimulation test according to GRS criteria is sufficient in Denmark and Sweden. In other countries it is even allowed to treat patients with partial deficiency and not only with very severe GH deficiency. In some countries a low QoL can no longer be demonstrated in the majority of the patients because patients are now treated as soon as they develop GH deficiency (e.g., after pituitary surgery) and therefore do not develop the full GH deficiency phenotype. When assessing effects of GH replacement, it also has to be taken into account that earlier studies used supraphysiological doses of GH, rendering the patients biochemically acromegalic, while the treatment regimens of today aim at a titrated GH dose to keep insulinlike growth factor-I (IGF-I) Z scores between 0 and ⫹ 2SDS. This treatment monitoring modality then depends on the quality of the reference population for IGF-I measurements for each laboratory in calculating Z scores. All these factors have to be taken into account when assessing results of the various studies in the literature from previous and present treatment regimens.

III. QoL IN GROWTH HORMONE (GH)-DEFICIENT ADULTS A. At Baseline Clinical studies have, in most cases, demonstrated that symptoms such as increased emotional stress, mental fatigue, social isolation and decreased energy, life satisfaction, and self-esteem are more prevalent in the GH-deficient population (reviewed in Hull and Harvey, 2003), consistent with the typical features of signs and symptoms in patients with severe GH deficiency (Table I). Some studies, however, have failed to detect any difference in QoL between GH-deficient individuals and the normal population (Page et al., 1997; Baum et al., 1998; Zenker et al., 2002). These three studies all used generic tests for measurement of QoL. Zenker et al., (2002) compared outcomes of the questionnaire answers with the level of IGF-I and the GH response from either GHreleasing hormone test or insulin tolerance. They concluded from their study that the absence of any relationship with the severity of GHD and the level of apathy/depression/psychosomatic complaints suggested that the impairments are not specific for GH deficiency. They suggested that symptomatic improvement might be secondary to somatic improvements from GH replacement. Finally, they found that the indication

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for GH substitution therapy should not be based on psychological impairments alone without the presence of somatic symptoms of GH deficiency. Their results and conclusions are in contrast to a number of other studies, e.g., McMillan et al. (2003) who performed a double-blind placebo-controlled trial in which GH replacement in 12 patients with severe GH deficiency was discontinued for 3 months (placebo) while the other group (n ⫽ 9) continued GH replacement. They found a significant difference between the groups in scores for SF-36, HDQoL, and, by interview, most patients identified correctly whether they had received GH or placebo. These authors concluded that withdrawal of GH treatment from adults with severe GH deficiency had detrimental psychological effects. One of the reasons for such contrasts in conclusions might be that the patient populations in the various studies were very heterogeneous, as were also the comparative control groups or populations, and the methods for measurement of QoL (Table III). Often both childhood-onset (CO) and adultonset (AO) GH deficiency patients are included. Adult-onset GH deficiency is often associated with other pituitary hormone deficiencies, which may interfere in the scores of QoL in these patients compared to isolated GH deficiency in childhood-onset GH deficiency, whereas childhood-onset patients may have a series of other psychological problems related to the disease in childhood, including a low height. The importance of GH deficiency may be evidenced by studies demonstrating that even though replacement of all hormones except GH to pituitary-insufficient adults was done, QoL was not normalized (Rosen et al., 1994; Zenker et al., 2002). QoL was different in patients with GH deficiency among different countries (unpublished data from the KIMS database). A specific reason for that is not quite clear, as the same questionnaire (AGHDA) was used in all countries. This questionnaire had been validated in GH-deficient patients from different countries and across languages. It is, however, possible that an assessment of QoL by any questionnaire should be investigated in the normal population in each country in order to correct for country differences (KoltowskaHäggström et al., 2004). It was shown by KoltowskaHäggström et al. (2004) that among approximately 1000 members of a general population in England and Wales, the mean AGHDA score for females was 7.0 and 6.3 for men. In a population of patients with GH deficiency from the same area the scores were approximately 7 higher (poorer QoL) and significantly different from the healthy general population mean. A similar study in Spain (Casanueva et al., 2004) also found a significant difference between healthy members of the general population and Spanish patients with GH deficiency — the scores for both groups were, however, much lower (i.e., better QoL) than those from England and Wales. The same was the case for German and Swedish patients (Wiren et al., 2000). Similar differences among countries with the United Kingdom displaying the poorest QoL in both controls and patients with GH deficiency were also shown by

use of a different disease-specific questionnaire (QLS-H) (Blum et al., 2003).

B. Effects of GH Replacement Therapy on QoL in Adults Both generic and GH deficiency-specific QoL measurement tools in most clinical studies indicate that GH replacement therapy enhances QoL (Table IV) (reviewed in Hull and Harvey, 2003 — fully or partly placebo controlled: McGauley, 1989; Bengtsson et al., 1990; Mardh et al., 1994; Beshyah et al., 1995; Burman et al., 1995; Attanasio et al., 1997; Caroll et al., 1997; Verhelst et al., 1997; Wallymahmed et al., 1997; Cuneo et al., 1998; Florkowski et al., 1998; Soares et al., 1999; nonplacebo controlled: Bjork et al., 1989; Wirén et al., 1998; Drake et al., 1998; Gibney et al., 1999; Murray et al., 1999; Ahmad et al., 2001; Hernberg-Stahl et al., 2001; Gilchrist et al., 2002). However, some studies (fully or partly placebo controlled: McGauley, 1989; Degerblad et al., 1990; Whitehead et al., 1992; Bengtsson et al., 1993; Baum et al., 1998; Giusti et al., 1998; Deijen et al., 1998; Cuneo et al., 1998) failed in at least one of the QoL scales to show any effect of GH supplementation on QoL and demonstrated normal QoL parameters compared to control population. The studies were very heterogeneous and generally reported very few patients (Table IV). Only a few studies have applied a fully placebocontrolled double-blinded approach — all of them of rather short duration (Baum et al., 1998; Burman et al., 1995; McGauley et al., 1989; Soares et al., 1999); 13 of the studies were a combination of a placebo-controlled approach followed by open treatment (Table IV), whereas another 9 had only an open trial approach (Table IV). The dosages used were also extremely variable and largely depended on when the study was performed: from gross overtreatment in the early days of GH replacement therapy and treatment based on weight and/or surface to now a titrated dose in each individual patient according to the IGF-I level (Ahmad et al., 2001; Drake et al., 1998; Murray et al., 1999; Rosilio et al., 2004). Among the open trials, some need a special comment. Two studies looked into very long-term effects (Gibney et al., 1999; Gilchrist et al., 2002), and two others were part of the long-term pharmacoepidemiological survey databases from Pfizer (KIMS ⫽ Pfizer International Metabolic Database) and Eli Lilly (HypoCCS ⫽ Hypopituitary Control and Complications Study), each incorporating a large number of patients mainly for safety, but in a number of situations, valuable efficacy data have also come out (Hernberg-Stahl et al., 2001; Rosilio et al., 2004). These four long-term trials showed an increase in QoL with time during GH replacement. The very variable designs of all the controlled trials almost without any common denominator unfortunately also precludes the performance of a meta-analysis of the results.

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TABLE III Quality of Life in Growth Hormone-Deficient (GHD) Adultsa

GHD onset AO ⫹ CO

n 1034

AO

356

AO

41

AO ⫹ CO

957

Controls

Tests used to quantify QoL b

Change in QoL in GHD adults

Reference

Normal population

AGHDA

p QoL

Abs et al. (1999)

963 normals matched for age, sex, and education

AGHDA

p QoL

Badia et al. (1998)

Normal population

NHP PGWB GHQ MMP1-2

⫽ QoL

Baum et al. (1998)

Normal populations corrected for sex, age, and country

QLS-H

p QoL

Blum et al. (2003)

CO

23

47 normals

NHP PGWB

q Sleep problems q Social isolation q Physical mobility problems

Björk et al. (1989)

CO ⫹ AO

36

Normals

HSCL NHP

p QoL proportional to duration of GHD

Burman et al. (1995)

31 MPHD 17 IGHD

48

41 normals matched for age

HSCL POMS Cognitive tests

p IQ, memory, vigor q anxiety (MPHD)

Deijen et al. (1996)

CO ⫹ AO

41

41 diabetics

CIS PAS CPRS

q Depression q Personality disorders

Lynch et al. (1994)

Mostly AO

24

Normals matched for sex, age, ethnicity, class, and residence

NHP PGWB

p QoL (NHP) q psychological distress (PGWB)

McGauley (1989)

AO (hypopituitary tumors)

48

Mastoid surgery patients

GWBS SF-36

⫽ QoL

Page et al. (1997)

Mostly AO

86

Normals matched for sex, age, marital status, and class

NHP

p QoL, energy, sex life q Emotional lability, social isolation

Rosen et al. (1994)

Same-sex siblings

SF-36 SRS SAS BSI Interview

p General health ⫽ Emotional and mental health

Sandberg et al. (1998)

CHO

140

MPHD

25

Normals matched for sex, age, height, and class

Stress-reactivity, psychometric testing

p Openess q Assertiveness

Stabler et al. (1992)

AO

57

Age-matched diabetics

HADS SES MFQ LFS

p QoL, self-esteem p Life fulfillment q Depression, fatigue q Anxiety

Wallymahmed et al. (1999)

111

1448 normal population

AGHDA

p QoL

Wiren et al. (2000)

CO

21

GH-sufficient CO-GHD

NHP MACL PGWB

q Anxiety ⫽ Cognition

Wiren et al. (2001)

Mostly AO

98

Normal population

BDI AS LSC

⫽ QoL

Zenker et al. (2002)

AO ⫹ CO

a QoL, quality of life; GHD, growth hormone deficiency; CO, childhood onset; AO, adult onset; IGHD, isolated GHD; MPHD, multiple pituitary hormone deficiencies; n, number of subjects with GHD; q, ⫽, p, change in QoL parameter compared to appropriate controls. Modified and updated from Harvey and Hull (2003), with permission from the authors. b Abbreviations given in Table II.

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TABLE IV Effects of Growth Hormone Replacement Therapy on Quality of Life in Adults in Published Trialsa GHD onset

N

AO

40

2–6 ␮g/kg

18

PCDB

NHP PGWB GHQ MMPI-2 Cognition tests

⫽ Cognition, QoL

Baum et al. (1998)

Mostly AO

21

2–4 U

9

PCDB

NHP PGWB HSCL Spousal report

qQoL placebo ⫹ GH groups (HSCL) qQoL GH group (NHP, spousal report)

Burman et al. (1995)

Mostly AO

24

0.07 U/kg

6

PCDB

NHP PGWB GHQ

qSubjective well-being qQoL (NHP) qQoL (PGWB)

McGauley et al. (1989)

Not stated

9

0.035 U/kg

6

PCDB

HDS BDI Cognitive tests

qQoL, cognition

Soares et al. (1999)

AO ⫹ CO

173

12.5 ␮g/kg

18

6 months PCDB 12 months open

NHP

⫽ Mobility, energy (6 months) qMobility, energy (12 months)

Attanasio et al. (1997)

AO ⫹ CO

40

0.04 U/kg

18

6 months PCDB 12 months open

CPRS GHQ

qQoL 12 months (CPRS) qQoL 6 months placebo (GHQ)

Beshyah et al. (1995)

Not stated

42

0.024 (6 months)–0.012 (6 months) ␮g/kg

12

6 months PCDB 6 months open

NHP PGWB

qQoL on both scales qNHP score in placebo

Caroll et al. (1997)

Not stated

12–18

6 months PCDB 6–12 months open

NHP PGWB

qQoL (NHP) qWell-being

Mardh et al. (1994)

AO

124

Dosage per day

Duration (months)

Controls

Tests used to quantify QoLb

Change in QoL in GHD adults

Reference

Mostly AO

32

0.018 (1 month) 0.035 (5 months) U/kg

12

6 months PCDB 6 months open

GHD-LFS GHD-IS NHP HADS SES MFS

qSelf-esteem qEnergy and emotional reaction (transient)

Wallymahmed et al. (1997)

AO

10

13–26 ␮g/kg

6

PCDB Crossover

CPRS SCL-90

qQoL (CPRS) ⫽ QoL (SCL-90)

Bengtsson et al. (1993)

AO

6

0.07–0.09 U/kg

3

PCDB Crossover

⫽ Mood, cognition qVitality, mental alertness

Degerblad et al. (1990)

0.07 U/kg

6

PCDB Crossover

Mood questionnaires Psychometric Testing PGWB

⫽ QoL, but no q IGF-I

Whitehead et al. (1992)

0.018 (1 month) 0.036 (11 months) U/kg

12

6 months PC 6 months open

NHP GHDQ Social history

qQoL 12 months (NHP) ⫽ QoL (GHDQ)

Cuneo et al. (1998)

AO ⫹ CO Mostly AO

14 166

CO (men)

48

1–3 U/m2

24

PC

Psychological Testing

⫽ Well-being qMemory

Deijen et al. (1998)

AO ⫹ CO

20

0.035 U/kg

3

Randomized

DSQ

qQoL placebo ⫹ GH

Florkowski et al. (1998)

PC, crossover

SCL-90

groups

SAS AO Mostly AO

25 148

0.5–1 U 0.035 U/kg

6 24

Randomized

HDS

qQoL (HDS)

PC

KSQ

⫽ KSQ

6 months PC

NHP

qQoL placebo ⫹ GH

18 months open

Social history

pSick leave, hospitalization

Giusti et al. (1998) Verhelst et al. (1997)

Continued

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18. Growth Hormone Replacement Therapy in Adults

TABLE IV Effects of Growth Hormone Replacement Therapy on Quality of Life in Adults in Published Trialsa—cont’d GHD onset

N

AO

46

Normal IGF-I

AO

50

Normal IGF-I

AO ⫹ CO

11

AO ⫹ CO

61

AO

304

AO ⫹ CO

65

Dosage per day

Duration (months)

Controls

Tests used to quantify QoLb

Change in QoL in GHD adults

3

Open

AGHDA

qQoL after 1 and 3 months

Ahmad et al. (2001)

6

Open

AGHDA

qQoL after 3 and 6 months

Drake et al. (1998)

0.025 U/kg

120

Control

NHP

qQoL (NHP), energy, emotional reaction

Gibney et al. (1999)

Not stated

108

No GH

NHP

qEnergy (NHP)

Gilchrist et al. (2002)

PGWB

qVitality (PGWB)

0.125–0.25 U/kg

12

Open

AGHDA

qQoL after 1 months, higher after 3 months

Hernberg-Stahl et al. (2001)

Normal IGF-I

8

Open

PGWB

qQoL (large)

Murray et al. (1999)

qQoL (large, 3 months)

Murray et al. (2001)

Reference

AGHDA CO (cancer)

27

Normal IGF-I

18

Open

PGWB AGHDA

AO ⫹ CO

576

AO ⫹ CO

71

Normal IGF-I

12

Open

QLS-H

qQoL

Rosilio et al. (2004)

6–12 ␮g/kg

20–50

Open

NHP

qQoL

Wiren et al. (1998)

PGWB a QoL, quality of life; GHD, growth hormone deficiency; AO, adult onset; CO, childhood onset; n, number of subjects; PC, placebo controlled; PCDB, placebo controlled, double blind; open, open label; q, ⫽, p, change in QoL parameter in GH-treated patients compared to controls. Modified and updated from Hull and Harvey (2003), with permission from the authors. b Abbreviations given in Table II.

In some studies, the reported improvements in QoL measurements were seen as soon as after 1 or 3 months of GH replacement (Ahmad et al., 2001; Hernberg-Stahl et al., 2001; Murray et al., 2001), but in some studies, 12–24 months of GH replacement treatment were needed for an effect to be demonstrated (Beshyah et al., 1995). Sustained improvements in QoL have been reported after 2 and 3 years of GH replacement (Burman et al., 1995; Wirén et al., 1998; Gibney et al., 1999; Gilchrist et al., 2002; Svensson et al., 2004; Rosilio et al., 2004). These studies were in complete contrast to a report that compared QoL scores of a group of patients receiving GH replacement therapy for at least 1 year (mean 3 years of treatment) with scores from age- and sex-matched control subjects without GH deficiency from the same geographical area. The GH-treated group still had significant impairments in QoL scoring compared to the control population (Malik et al., 2003).

IV. EFFECTS OF GH DEFICIENCY AND GH THERAPY ON QoL IN SPECIFIC CONDITIONS Growth hormone production is declining with age after puberty, and the existence of a human somatopause has been discussed (Toogood et al., 1996). In an elegant study demonstrated by Toogood et al. (1998), a properly

performed stimulation test for GH deficiency was able to distinguish between the GH response in healthy elderly persons above 65 years and that of hypopituitary patients with assumed GH deficiency. Toogood (2003) later demonstrated that these elderly GH-deficient hypopituitary patients had indeed also a significantly unfavorable body composition and bone mineral content compared to healthy age-matched counterparts. It was later demonstrated that these patients ⬎65 years of age improved their QoL as assessed by AGHDA scores to the same extent as young GH-deficient patients when receiving GH replacement (Monson et al., 2000; FeldtRasmussen et al., 2004). Li Voon Chong et al. (2002) studied 27 elderly (mean age 73 years) patients with severe GH deficiency for 2 years without GH replacement and compared their QoL with an age-matched group of elderly healthy individuals. They were investigated by SF-36, NHP, LFS, HADS, and MFQ, and although the QoL of the patients with GH deficiency did decrease during the 2 years, it also decreased in the healthy control group. Other measured variables, such as body composition and lipids, did not differ either. Their results have therefore raised some doubt about benefits of GH replacement in elderly people with GH deficiency. Most of the aforementioned studies were performed in small heterogeneous groups of patients of different etiologies. The larger surveillance databases, such as KIMS and

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HypoCCS, have provided a possibility to look into QoL in subgroups of patients. It has thus been possible to investigate if different causes of GH deficiency show differences in QoL both at baseline and in response to GH replacement. By such subdivisions it has been demonstrated in 665 GH-deficient adults that patients with AO GH deficiency had a better responsiveness to GH replacement than CO GH-deficient patients (Bengtsson et al., 1999). They did, however, have a poorer QoL at baseline, thus indicating that the poorer the measure at baseline, the better the potential for improvement by replacement. From the KIMS database, 268 patients with craniopharyngioma and severe GH deficiency were extracted. They had a mean AGHDA score of approximately 11, equal in men and women, and largely similar to that of other GHdeficient adults with other etiologies. Their improvement was approximately 4 AGHDA scores after 2 years of GH replacement (Verhelst and Kendall-Taylor, 2004). It is noteworthy that this would not be sufficient to meet the aforementioned NICE guidelines for GH replacement therapy. Similarly, equal responsive effects from GH replacement on QoL have been reported in irradiated GH-deficient patients compared to nonirradiated ones (Maiter, 2004) and in Sheehan’s syndrome (Kelstimur, 2004), which is still a prevalent cause of GH deficiency in some countries. The effect of growth hormone on the brain and thereby QoL is probably two phased, where both too much (acromegaly or overtreatment with GH) and too little (GH deficiency) are detrimental. The adverse effect of GH overproduction as in acromegaly has been assessed by a number of QoL measures (Pantanetti et al., 2002; Biermasz et al., 2004), but the interpretation is often difficult because GH may influence the QoL through other effects than directly on the brain, such as on the sleep pattern (Astrom et al., 1990), e.g., by an interaction with melatonin (Sinisi et al., 1997) or by low selfesteem due to disfiguring of the patients with long-standing disease (Pantanetti et al., 2002). It has been demonstrated that 40 previously acromegalic patients with GH deficiency due to treatment had a similar reduction in QoL by AGHDA, as had 1392 patients with GH deficiency for other reasons (mainly nonfunctioning adenomas) (Feldt-Rasmussen et al., 2002). These data were derived from the KIMS database and also demonstrated that the acromegalic patients had a similar beneficial effect from GH replacement as other GH-deficient patients.

V. THERAPEUTIC MECHANISMS OF GH REPLACEMENT How GH works to improve QoL is not very clear. Some of the effects mentioned later have been reviewed (Hull and Harvey, 2004). A direct effect on the brain is one possibility, which is dealt with in other chapters. GH does cross

the blood barrier and treatment increases the concentration in the spinal fluid of both GH and neurotransmitters (Johansson et al., 1995). The demonstration of growth hormone receptors in the brain increases the likelihood of a direct effect. It is also possible that GH has an influence on brain growth and development, as well as on neural repair. The brain is considered now to be a site of GH production and action, and it therefore seems likely that GH deficiency has detrimental effects on cognition and mood and consequently on QoL. GH deficiency has a number of well-documented somatic effects on metabolism, cardiovascular function, pulmonary function, muscle strength, body composition, sweat secretion, immune function, and bones, all of which are more or less completely reverted after GH replacement. Thus, a number of the items in the QoL questionnaires that have to do with energy and physical mobility would have an impact on the QoL scores. It is also likely that the changes in body composition with more fat mass in GH deficiency can contribute to a reduced self-perceived QoL, as may also a constant change in fluid homeostasis with a resulting reduction of blood and plasma volume. Decreased sweating results in impaired heat dissipation (Juul et al., 1993), which, together with low muscle strength, can impair the person’s self-esteem due to performance difficulties and reduced exercise capacity. Therefore, the impact of GH status on clinical indices such as height, body composition, metabolism, cardiovascular, and bone health may be responsible for some of the GH-dependent QoL indices. The responses to GH replacement are heterogeneous as to the somatic changes and might therefore also reflect the heterogeneity of QoL responses. Finally, an interaction with other hormones may be in play. For instance, it is well documented that GH increases the peripheral conversion of thyroxine to triiodothyronine (T3) (Burman et al., 1996; Jorgensen et al., 1994). A low T3, being the active hormone, in GH deficiency may contribute to a low activity level and thus a poor QoL, which should be reverted by GH replacement. A correlation between T3 concentration and QoL has, however, not been proven.

VI. COST–BENEFIT CONSIDERATIONS Calculating the cost of GH replacement treatment is rather complicated, as the direct cost of GH is only a small part of the equation. Furthermore, most patients with GH deficiency, whether started in childhood or in adulthood, have other disease-related conditions such as other pituitary hormone deficiencies or consequences of a treated disease such as Cushing’s disease or irradiated intracranial childhood cancer. In the other extreme, some adult patients have childhood-onset idiopathic-isolated GH deficiency. The estimated

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18. Growth Hormone Replacement Therapy in Adults

morbidity of patients with GH deficiency is therefore very heterogeneous and highly dependent on the etiology of the disease. A few earlier studies have estimated the increased cost of health care consumption in patients with untreated GH deficiency (Ehrnborg et al., 2000; Hakkaart-van Roijen et al., 1998; Jonsson and Nilsson, 2000; Sanmarti et al., 1999). Only a few studies have attempted to calculate the costs of GH replacement (reviewed in Radcliffe et al., 2004). In one study (Hernberg-Stahl et al., 2001) using patients from the KIMS database, a health-related QoL questionnaire was used (KIMS Patient Life Situation Form), along with a needsbased questionnaire (AGHDA). Before receiving GH replacement, approximately 20% of the group of 304 patients with GH deficiency received a disability pension. QoL in these patients was more impaired than in those working or those retiring normally. During GH replacement therapy the use of specific health care resources decreased. Those reported were days of sick leave, visits to the doctor, hospital days, and assistance with daily activities. At the same time the QoL increased, as evidenced by increased AGHDA scores. This was followed by publication of similar calculations from a single country (Sweden) (Svensson et al., 2004). They studied 237 Swedish patients enrolled into KIMS and treated for 3 years with GH. A reduction was seen in days of sick leave, days in hospital, and visits to the doctor. At the same time, leisure-time physical activity and satisfaction with physical activity increased, as did AGHDA scores. After 3 years the patients had the same number of days of sick leave as before start of treatment, but all the other items remained improved. Findings from the heterogeneous group of GH-deficient patients from different countries (Hernberg-Stahl et al., 2001) could thus be confirmed in a homogeneous patient population from Sweden (Svensson et al., 2004).

VII. CONCLUSION Although many of the studies performed to assess QoL and GH replacement are not long enough, not controlled, or use too high GH dose or the wrong QoL instruments, there is a growing body of evidence for an impairment of QoL in GH-deficient patients and a sustained improvement or normalization after GH replacement. GH may improve QoL via neurological mechanisms. However, the beneficial effect of GH replacement in hypopituitary GH-deficient patients on cardiovascular, reproductive, metabolic, and immune functions may have an additional impact on the improved QoL. GH therapy does not improve QoL in every GH-deficient individual. Dosage of GH is important, as no improvement in QoL may be expected if the GH dosage is suboptimal. However, it may be difficult to determine the optimal dose based on weight, surface, gender, and correct titration according to

the plasma IGF-I concentration. Degree of impairment of QoL is also important because no improvement can be expected in a GH-deficient patient that has not developed the full phenotype, e.g., shortly after surgery. However, many clinical guidelines rely on QoL measures for the indication for GH replacement in the individual patient and also treatment follow-up. A combination of diseasesensitive and generic instruments must be advised, and a proper registration in each country of the QoL score by each particular questionnaire in a comparative healthy control population is mandatory. It will also be important in the future to focus on subgroups of patients that may benefit more from GH replacement than others. With proper instruments, GH replacement does seem to decrease health care consumption in parallel with improvements in QoL. This needs to be translated into a full calculation of cost-effectiveness of GH replacement in each country, not only considering the price of GH but also the health care-related costs, such as visits to the doctor, hospitalization, and daily assistance from others.

References AACE Growth hormone Task Force (2003). America association of clinical endocrinologists medical guidelines for clinical practice for growth hormone use in adults and children: 2003 update. Endocr. Pract. 9, 65–76. Abs, R., Bengtsson, B.-Å., Hernberg-Ståhl, E., Monson, J. P., Tauber, J. P., Wilton, P., and Wüster, C. (1999). GH replacement in 1034 growth hormone deficient hypopituitary adults: Demographic and clinical characteristics, dosing and safety. Clin. Endocrinol. 50, 703–713. Ahmad, A. M., Hopkins, M. T., Thomas, J., Ibrahim, H., Fraser, W. D., and Vora, J. P. (2001). Body composition and quality of life in adults with growth hormone deficiency: Effects of low dose growth hormone replacement. Clin. Endocrinol. 54, 709–717. Astrom, C., and Lindholm, J. (1990). Growth hormone deficient young adults have decreased deep sleep. Neuroendocrinology 33, 495–500. Attanasio, A. T., Lamberts, S. W., Matranga, A. M., Birkett, M. A., Bates, P. C., Valk, N. K., Hilsted, J., Bengtsson, B.-Å., and Strasburger, C. J. (1997). 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. 82, 82–88. Badia, X., Lucas, A., Sanmarti, A., Roset, M, and Ulied, A. (1998). One-year follow-up of quality of life in adults with untreated growth hormone deficiency. Clin. Endocrinol. 49, 765–771. Baum, H. B., Katznelson, L., Sherman, J. C., Biller, B. M., Hayden, D. L., Schoenfeld, D. A., Kannistraro, K. E., and Klibansky, A. (1998). Effects of physiological growth hormone (GH) therapy on cognition and quality of life in patients with adult-onset GH deficiency. J. Clin. Endocrinol. Metab. 83, 3184–3189. Bengtsson, B.-Å., Eden, S., Lonn, L., Kvist, H., Stokland, A.,Lindstedt, G., Bosaeus, I., Tolli, J., Sjostrom, L., and Isaksson, O. G. (1993). Treatment of adults with growth hormone (GH) deficiency with recombinant human GH. J. Clin. Endocrinol. Metab. 76, 309–317. Bengtsson, B.-Å., Abs, R., Bennmarker, H., Monson, J. P., FeldtRasmussen, U., Hernberg-Stahl, E., Westberg, B., Wilton, P., and Wüster, C. (1999). The effects of treatment and the individual responsiveness to growth (GH) hormone replacement therapy in 665 GH-deficient adults. J. Clin. Endocrinol. Metab. 84, 3929–3935.

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Beshyah, S. A., Freemantle, C., Shahi, M., Anyaoku, V., Merson, S. Lynch, S., Skinner, E., Sharp, P., Foale, R., and Johnston, D. G. (1995). Replacement treatment with biosynthetic human growth hormone in growth hormone deficient hypopituitary adults. Clin. Endocrinol. 42, 73–84. Biermasz, N. R., van Thiel, S. W., Pereira, A. M., Hoftijzer, H. C., van Hemert, A. M., Smit, J. W., Romijn, J. A., and Roelfsema, F. (2004). Decreased quality of life in patients with acromegaly despite long-term cure of growth hormone excess. J. Clin. Endocrinol. Metab. 89, 5369–5376. Björk, S., Jonsson, B., Westphal, O., and Levin, J. E. (1989). Quality of life of adults with growth hormone deficiency: A controlled study. Acta Paediatr. Scand. Suppl. 356, 55–59. Blum, W. F., Shavrikova, E. P., Edwards, D. J., Rosilio, M., Hartman, M. L., Marin, F., Valle, D., van der Lely, A. J., Attanasio, A. F., Strasburger, C. J., Heinrich, G., and Herschbach, P. (2003). Decreased quality of life in adult patients with growth hormone deficiency compared with general populations using the new, validated, self-weighted questionnaire, questions on life satisfaction hypopituitarism module. J. Clin. Endocrinol. Metab. 88, 4158–4167. Burman, P., Broman, J. E., Hetta, J., Wiklund, I., Erfurth, E. M., Hagg, E., and Karlsson, F. A. (1995). Quality of life in adults with growth hormone (GH) deficiency: Response to treatment with recombinant human GH in a placebo-controlled 21-month trial. J. Clin. Endocrinol. Metab. 80, 3585–3590. Burman, P., Hetta, J., Wide, L., Mansson, J.-E., Ekman, R., and Karlsson, A. (1996). Growth hormone treatment affects brain neurotransmitters and thyroxine. Clin. Endocrinol. 44, 319–324. Caroll, P. V., Littlewood, R., Weissberger, A. J., Bogalho, P., McGauley, G., Sonksen, P. H., and Russel-Jones, D. L. (1997). The effects of two doses of replacement growth hormone on the biochemical, body composition and psychological profiles of growth hormone-deficient adults. Eur. J. Endocrinol. 137, 146–153. Casanueva, F., Mattson, A., Espadero, R., Badia, X., Sanmarti, A., Kind, P., and Koltowska-Häggström, K. (2004). Quality of life (QoL) in adults with growth hormone deficiency (GHD) measured by QoL-assessment for growth hormone deficiency in adults (QoL-AGHDA); to what extent are Spanish results comparable to those from England and Wales. In “International Congress of Endocrinology,” Lisbon, Portugal. [Abstract.] Cuneo, R. C., Judd, S., Wallace, J. D., Perry-Keene, D., Burger, H., LimTio, S., Strauss, B., Stockigt, J., Topliss, D., and Alford, F. (1998). The Australian Multicenter trial of growth hormone (GH) treatment in GHdeficient adults. J. Endocrinol. Metab. 83, 107–116. Degerblad, M., Almquist, O., Grunditz, R., Hall, K., Kaijser, L., Knutsson, E., Ringetz, H., and Thoren, M. (1990). Physical and psychological capabilities during substitution therapy wuth recombinant growth hormone in adults wuth growth hormone deficiency. Acta Endocrinol. 123, 185–193. Deijen, J. B., de Boer, H., Blok, G. J, and van der Veen, E. A. (1996). Cognitive impairments and mood disturbances in growth hormone deficient men. Psychoneuroendocrinology 21, 313–322. Deijen, J. B., de Boer, H., and van der Veen, E. A. (1998). Cognitive changes during during growth hormone replacement in adult men. Psychoneuroendocrinology 23, 45–55. Drake, W. M., Coyte, D., Camacho-Hubner, C., Jivanji, N. M., Kaltsas, G., Wood, D. F., Trainer, P. J., Grossman, A. B., Besser, G. M., and Monson, J. P. (1998). Optimizing growth replacement therapy by dose titration in hypopituitary adults. J. Clin. Endocrinol. Metab. 83, 3913–3919. Ehrnborg, G., Hakkaart-van Roijen, L., Jonsson, B., Rutten, F. F. H., Bengtsson, B.-Å., and Rosen, T. (2000). Cost of illness in adult patients with growth hormone deficiency. Pharmacoeconomics 17, 621–628. Feldt-Rasmussen, U., Abs, R., Bengtsson, B-Å., Bennmarker, H., Bramnert, M., Hernberg-Ståhl, E., Monson, J. P., Westberg, B., Wilton, P., and Wüster, C. (2002). Growth hormone deficiency and replacement in hypopituitary patients previously treated for acromegaly or Cushing’s disease. Eur. J. Endocrinol. 146, 67–74.

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18. Growth Hormone Replacement Therapy in Adults Juul, A., Behrenscheer, A., Tims, T., Nielsen, B., Halkjaer-Kristensen, J., and Skakkebaek, N. E. (1993). Impaired thermoregulation in adults with growth hormone deficiency during heat exposure and exercise. Clin. Endocrinol. 38, 237–244. Kelestimur, F. (2004). Sheehan’s syndrome. In “Growth Hormone Deficiency in Adults: 10 Years of KIMS” (R. Abs and U. Feldt-Rasmussen, eds.), pp. 277–287. PharmaGenesis Ltd., Oxford. Koltowska-Häggström, M., Hennessy, S., and Kind, P. (2004). Quality of life deficit in hypopituitary adults with growth hormone deficiency (GHD): Comparison of age-sex matched patient data and a UK population survey. In “2nd International Congress on GH-IGF-I Research,” Cairns, Australia. [Abstract]. Li Voon Chong, J. S., Groves, T., Foy, P., Wallynahmed, M. E., and Macfarlane, I. A. (2002). Elderly people with hypothalamic-pituitary disease and untreated GH deficiency: Clinical outcome, body composition, lipid profile and quality of life after 2 years compared to controls. Clin. Endocrinol. 56, 175–181. Lynch, S., Merson, S., Beshyah, S. A., Skinner, E., Sharp, P., Priest, R. G., and Johnston, D. G. (1994). Psychiatric morbidity in adults with hypopituitarism. J. R. Soc. Med. 87, 445–447. Maiter, D. (2004). Radiotherapy for the treatment of pituitary tumours and growth hormone deficiency in irradiated patients. In “Growth Hormone Deficiency in Adults: 10 Years of KIMS” (R. Abs and U. Feldt-Rasmussen, eds.), pp. 253–267. PharmaGenesis Ltd., Oxford. Malik, I. A., Foy, P., Wallymahmed, M., Wilding, J. P. H., and MacFarlane, I. A. (2003). Assessment of quality of life in adults receiving long-term growth hormone replacement compared to control subjects. Clin. Endocrinol. 59, 75–81. Mardh, G., Lundin, K., Borg, G., Jonsson, B., and Lindeberg, A. (1994). Growth hormone replacement therapy in adult hypopituitary patients with growth hormone deficiency: Combined data from 12 European placebo-controlled trials. Endocrinol. Metab. 1, 43–49. McGauley, G. A. (1989). Quality of life assessment before and after growth hormone treatment in adults with growth hormone deficiency. Acta Pediatr. Scand. Suppl. 356, 70–72. Mc Kenna, S., P., Doward, L. C., Alonso, J., Kohlman, T., Niero, M., Prieto, L., and Wiren, L. (1999). The QoL-AGHDA: An instrument for the assessment of quality of life in adults with growth hormone deficiency? In “Growth Hormone Therapy” (J. P. Monson, ed.), pp. 160–176. Blackwell Science, London. McMillan, C. V., Bradley, C., Gibney, J., Healy, M. L., Russel-Jones, D. L., and Sonksen, P. H. (2003). Psychological effects of withdrawal of growth hormone therapy from adults with growth hormone deficiency. Clin. Endocrinol. 59, 467–475. Monson, J. P. (2000) Application of a disease-specific, quality-of-life measure (QoL-AGHDA) in growth hormone-deficient adults and a random population sample in Sweden: Validation of the measure by Rasch analysis. Commentary. Clin. Endocrinol. 52, 141–142. Monson, J. P., Abs, R., Bengtsson, B.-Å., Bennmarker, H., FeldtRasmussen, U., Hernberg-Stahl, E., Thoren, M., Westberg, B., Wilton, P., and Wüster, C. (2000). Growth hormone deficiency and replacement in elderly hypopituitary adults. Clin. Endocrinol. 53, 281–289. Murray, R. D., Darzy, K. H., Gleeson, H. K., and Shalet, S. M. (2002). GH-deficient survivors of childhood cancer: GH replacement during adult life. J. Clin. Endocrinol. Metab. 87, 129–135. Murray, R. D., Skillicorn, C. J., Howell, S. J., Lissett, C. A., Rahim, A., Smethurst, L. E., and Shalet, S. M. (1999). Influences of quality of life in GH deficient adults and their effect on response to treatment. Clin. Endocrinol. 51, 565–573. National Institute for Clinical Excellence (2003). Human growth hormone (somatropin) in adults with growth hormone deficiency. Technology Appraisal 64. www.nice.org.uk Page, R. C., Hammersley, M. S., Burke, C. W., and Wass, J. A. (1997). An account of the quality of life of patients after treatment for nonfunctioning pituitary tumours. Clin. Endocrinol. 46, 401–406.

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19 Psychological Importance to the Child of Growth Hormone Replacement JOHN ERIC CHAPLIN, JOVANNA DAHLGREN, BERIT KRISTRÖM, and KERSTIN ALBERTSSON WIKLAND Göteborg Paediatric Growth Research Centre Sahlgrenska Academy at Göteborg University Institute for Women’s and Children’s Health Department of Paediatrics University of Göteborg, Sweden

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Modes of Action of Growth Hormone (GH) Location of Action of GH GH Secretion Versus Sensitivity Typical Cognitive Development and Critical Windows of Development Effect of GH on Cognitive and Academic Performance Effect of GH on Psychological Well-Being Methodological Difficulties in Measurement Future Directions References

Finally, we look at directions for future research in the area and the need for methodological standardization.

I. INTRODUCTION Growth hormone (GH) treatment is recommended for children with proven clinical diagnosis of GH deficiency (GHD). Treatment with GH has a pleiotropic, and often unforeseen, effect on nearly every organ system and therefore its effects are not restricted only to growth. Indeed the NICE Committee (2002), having reviewed the evidence on both the clinical effectiveness and the cost effectiveness of GH treatment, concluded that the utility gain from the height gain in treatment of GH deficiency was “a worthwhile gain for the resource, given its lifelong value and the psychological importance to the child.” The “psychological importance to the child” is a broad concept that can be defined in many ways. This chapter reviews what is known and what is concluded about the effects of GH on the brain, cognition, and finally the wellbeing of GHD children. It also examines evidence for an effect or otherwise of GH treatment and discusses some of the methodological problems arising from drawing firm conclusions about the psychological effects of this treatment.

The life-long psychological importance of growth hormone (GH) treatment in childhood has been proposed as an indicator of the greater utility value over costs of treatment. This chapter reviews the various studies that have examined the psychological importance of GH treatment in the child. The measurement of cognitive and intellectual effects is put into the perspective of cognitive theory and is categorized in terms of intelligence quotient, school achievement, and specific cognitive skills, including attention, visual motor skills, and memory. Evidence for the psychological well-being of the GH-deficient child is also reviewed together with quality of life issues. This is followed by an examination of the methodological difficulties of examining the long-term psychological benefits of GH in children.

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II. MODES OF ACTION OF GROWTH HORMONE (GH) There are three possible modes of action of GH; neurological, endocrinological, and psychological. First, we know that GH gene expression occurs in the central and peripheral nervous systems (Harvey and Hull, 2003) and that following subcutaneous GH injection, GH reaches the cerebrospinal fluid (CSF) (Jorgensen et al., 1990). Binding sites for GH have been identified in the human brain, particularly in the chorioid plexus (Lai et al., 1991). In a double-blind, crossover design study, Burman et al. (1996) demonstrated that GH administered to adults (adult-onset GHD) entered into the CSF. Moreover, it has been observed that a decrease in homovanillic acid (HVA), and a decrease in serum levels of free T4 concentration in the CSF may be linked to improved well-being following treatment (Burman et al., 1993, 1996; Johansson et al., 1995). These findings may be of particular significance for cognitive functions. Therefore, consideration must be given to potential effects of GH on the central nervous system (CNS). Second, we know that GH stimulates longitudinal bone growth of the body as well as organ development, increasing morphology (body size), muscle strength, and bone structure (Isaksson et al., 1987). These physical growth factors affect the child’s experience, and these experiences in turn influence the course of the child’s cognitive and social development (Bee and Mitchell, 1986). Third, psychosocial functioning is affected by appearance (Erling et al., 2002). It is observed that not only is the GHD child of short stature, but the child also has immature facial features, which will lead to them being treated as if they are younger than their chronological age. They are, therefore, more likely than children of idiopathic short stature (ISS) to miss developmentally relevant social interactions with family, friends, and adults (Clopper et al., 1986; Rotnem, 1986). Children with GHD have a more negative view of their appearance than children of normal height (Erling et al., 2002). Improving self-confidence due to increased satisfaction with height and appearance can lead to reductions in stress and greater involvement in school and social activities, which ultimately affect well-being and ability to incorporate new information and experiences, thus improving cognitive abilities. Therefore, all three forms of action may be of psychological importance to the child.

III. LOCATION OF ACTION OF GH Growth hormone and insulin-like growth factor-I (IGF-I) receptors can be found in several areas of the brain (Clayton and Cowell, 2000; D’Ercole et al., 2002),

suggesting a major influence of the somatotropic axis on the brain (Nyberg, 2000). GH receptors are mainly found in the choroid plexus, thalamus, hypothalamus, pituitary, putamen, and hippocampus, whereas IGF-I receptors are mainly concentrated in the hippocampus and parahippocampal areas (Lai, 1991). In early life, GH and IGF-I have an important role in the development and differentiation of the CNS (Bunn et al., 2005). In the more developed CNS, GH and IGF-I are thought to have a variety of functions, including various cognitive functions. In children with GHD, improvement of cognitive functions is expected to be observed after the administration of GH; however, the exact action of the hormone and likely cognitive outcomes from effects of different receptor sites are as yet unknown.

IV. GH SECRETION VERSUS SENSITIVITY The effect of GH replacement therapy can only occur if the receptor sites are sensitive to the GH. When the patient does not respond to treatment in terms of growth, we can speculate whether the problem lies in the sensitivity of the organs to GH. Diminished responsiveness is known to be a reason for nongrowth following GH treatment. It is therefore possible that a similar action can be found in the brain. If diagnosed correctly, GHD children should have low or absent levels of GH, whereas their responsiveness to GH should be normal. Children who have ISS should have normal or high levels of GH in their bloodstream but are normal or nonresponsive to GH, resulting in growth failure.

V. TYPICAL COGNITIVE DEVELOPMENT AND CRITICAL WINDOWS OF DEVELOPMENT The period of childhood is characterized by the development of the child both physically and cognitively. It has been proposed by Piaget (1929) and others that cognitive development occurs in universal, discontinuous stages that characterize structural reorganizations common to every child. Piaget posited that all children progress through four stages in the same order (see Fig. 1). Piaget called his theoretical framework “genetic epistemology” because he was interested primarily in how knowledge develops in humans. The concept of a cognitive structure is central to his theory. Cognitive structures are patterns of physical or mental actions that underlie specific acts of intelligence and correspond to stages of child development. Each stage is a mental framework within which the child has the ability to understand and remember information at different levels of sophistication. The possibility to

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19. Psychological Importance to the Child Stage

Age

Major Features

Sensorimotor

Birth to 2 years

Pre-Operational Thought

2 to 6/7 years

Concrete operational

6/7 to 11/12 years

Formal operational

11/12 ⫹ years

The child learns to generalise its activities to a wider range of situations and coordinate them into increasingly lengthy chains of behaviour. The child learns to represent objects through mental imagery symbols and there is rapid language growth The child learns to represent the thoughts of others and understands transformations from one state to another. The child learns to think logically and abstractly about problems.

FIGURE 1 Piaget’s primary cognitive structures.

move to the next stage is achieved by the maturity of the brain and happens naturally as part of a continual progress of cognitive development. The stage theory has been challenged in recent years. The stages that children go through have been shown to vary (Snow, 1989), and progress through the stages may have a typical or atypical character (Wishart and Duffy, 1990). An argument that is often seen as an alternative view to the hypothesis of cognitive stages is that cognitive development is a continuous process, with all children showing individual developmental patterns as a function of their interaction with the environment and learning opportunities. In this view, there is no uniform, incremental sequence of stages that the child passes through. Evidence suggests that children do not move through each stage evenly and that children can be in two cognitive levels at one and the same time. Piaget has been criticized for using concepts and objects unfamiliar to children; evidence now suggests that children can perform beyond Piaget’s levels when using familiar objects (Sutherland, 1992). There is also evidence to suggest, for example, that children with certain learning difficulties may regress to a lower level than previously attained if the skills are not maintained (Wishart and Duffy 1990). A resolution of this argument between structural and functional approaches is a framework that combines both organism and environment, a collaborative model based on stages and individual differences. This idea can be seen as being presented in the concepts of developmental plasticity and life span development. This theory suggests that development varies in response to environmental circumstances within a range of capability, determined by the maturity of the organism. In this view, the concept of stages becomes less significant but there is still the notion of incremental knowledge-building appropriate at different developmental levels. The large number of independent factors influencing the individual (e.g., genetics, neuroanatomy, physiology, behavior, environment, social group effects, cultural organization) means that any cognitive development is probabilistic and that variation at one stage will have an effect on future developmental expression (Gollin, 1981; Scarr and McCartney, 1983). Nevertheless, under typical circum-

stances, the cognitive capability of a child increases with age as the structures in the brain develop. When there are structural abnormalities, the structures have not fully developed, or the neural structures function inadequately, an atypical cognitive development will occur. In order to identify context-sensitive variations within the structural framework, the concept of performance as opposed to capability has been proposed (Chomsky, 1965; Overton and Newman, 1982). This idea proposes that capability is determined by the organism but that performance can vary widely within the possible range. Illustration of this approach is the literature on intelligence testing, where capability is defined as the best score the child can obtain, whereas the child’s performance can vary (see Fig. 2). Growth hormone deficiency may result in cognitive capacity below normal at any point in time. If this is so, then the next question is whether this is a reduced capacity or delayed development that can be compensated for through increased educational opportunities (of course this involves extra resources. The child’s cognitive capability at any point defines the developmental stage, and apparent variations are due to performance factors. Most cognitive tests emphasize the importance of achieving the best possible performance of the individual in order to identify the capability. In other words, all tests presume to indicate capacity through the measuring of performance. In order to achieve this, the child must be prepared to perform to the highest level of their capability. With endocrinological treatment, it is proposed that the capability of the mental structures will be increased. However, the problem of measuring capability via performance still persists. Highest capability possible Highest performance level

Total possible range of cognitive capacity Age of the child

FIGURE 2 Cognitive capacity versus performance.

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VI. EFFECT OF GH ON COGNITIVE AND ACADEMIC PERFORMANCE There is evidence both for and against a possible treatment effect of GH on cognitive function. In recent years, several studies examining the effects of GH treatment on cognitive function in GHD patients have been published. Many researchers have concluded that a substantial minority of children with GHD have problems in cognitive and academic performance. Siegel (1990) pointed out that poor academic achievement is explained in the literature by three conflicting theories. 1. The low ability theory: that there are structural or developmental deficits leading to poor performance. If a cognitive benefit of GH treatment is to be found then it should be identifiable through a decrease in the difference between the IQ levels of GHD and ISS children. 2. The cognitive underfunctioning theory: that because of psychosocial and environmental factors leading to low self-esteem, the child does not demonstrate the intellectual performance they are capable of achieving. If a cognitive benefit of GH treatment is to be found then it should be similar for GHD and ISS children. 3. The cognitive deficit theory: that poor intellectual achievement is due to specific cognitive, attentional, and visual-spatial deficits. If a cognitive benefit of GH treatment is to be found then it should be identifiable in the improvement of specific deficits. This section examines evidence for a cognitive effect in relation to the elements of all three theories. A brief description of some of the tests used to measure intelligence and school achievement is given in Table I.

A. Intelligence Quotient The low ability theory would predict that we should find lower IQ levels for children with GHD and that if GH treatment has a beneficial cognitive effect, it should lead to an increase in IQ levels. It has been noted that there is often a significant difference between the intelligence levels of short children and control groups but this was not a consistent finding. However IQ scores above the normal were also found. Despite this intelligence levels were still within the normal range (Wheeler et al., 2004). From adult studies, we know that support for a relationship between GHD and impaired cognitive functioning is given by studies of IGF-I plasma level and intellectual functioning. Serum IGF-I concentrations in GHD males have been shown to be correlated positively with IQ score and education level (Deijen et al., 1996). Cognitive function has been shown to improve following GH treatment using the digit span subset

and digit symbol test of the Wechsler Adult Intelligence Scale (Lasaité et al., 2004), and total IQ has improved significantly in children born short for gestational age (SGA) (van Pareren, 2004). The GH/IGF-I axis may, therefore, play a role in the level of cognitive functioning in healthy persons and GHD patients (Stouthart et al., 2003). However, even in adult studies, data are too limited to allow conclusions concerning the effects of GH treatment on cognition (Arwert et al., 2005).

B. School Achievement (SA) The cognitive underfunctioning theory would suggest that the psychosocial effects of short stature would lead to decreased school achievement, which should be true for all children who are of short stature. GH treatment, if it has a cognitive effect, should lead to an improvement in school achievement for both GHD and ISS children. School achievement is a partial indicator of IQ and is not equivalent to IQ. School achievement includes social and behavioral aspects that have an influence on the measurement of SA. However, SA has often been used as an outcome variable, partly because this is a better indicator than IQ of future adult achievement, e.g., job success (Hunter and Hunter, 1984), and as a measure it requires fewer resources to collect data. Wheeler et al. (2004) examined the school achievement of short children, which included growth hormone deficient (GHD), multiple hormone deficient (MDH), ideopathic short stature (ISS), and constitutional growth delay (CGD) children. They found almost as many studies showing no difference in SA as there were showing an effect. Short stature, whether it was due to GHD or ISS, could not be identified as being significantly related to lower school achievement. In line with Wheeler’s review, it has been pointed out that problems of academic achievement in children with GHD have not been found to be related to height (Sandberg et al., 1998); however, Björk et al. (1989) pointed out that lower educational advancement is found in child-onset growth hormone deficient (COGHD) adults when compared to a control group. This suggests that GHD may have a long-term role to play in cognitive abilities, which is not shown in Wheeler’s review. Stabler et al. (1998) compared 109 GHD children (mean age 11.1) at start and after 3 years of GH treatment with a control of 86 ISS children for achievement using the Wide Range Achievement Test (WRAT-R). They found no difference between GHD and ISS at baseline and these scores did not change significantly during treatment.

C. Specific Cognitive, Attentional, and Visual-Spatial Deficits Cognitive deficit theory identifies poor intellectual performance as reliant upon specific cognitive, attentional, and visual-spatial deficits. The theory is that children with GHD

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TABLE I Measures of IQ and School Achievement Tests

Description

Study

Wechsler Adult Intelligence Scale-Revised (WAIS-R). Designed for age 17 and above. Wechsler Intelligence Scale for Children, Third Edition (WISC-III). Designed for children age 6 to 17 years. Wechsler Preschool and Primary Scale of Intelligence (WPPSI) Designed for children age 4–6 1/2 years.

The Wechsler Intelligence Scales are a series of standardized tests used to evaluate cognitive abilities and intellectual abilities in children and adults. This test is divided into six verbal and five performance subtests. The complete test takes 60–90 min to administer. Verbal and performance IQs are scored based on the results of the testing and then a composite Full Scale IQ score is computed.

Sartorio et al. (1995)

Slosson Intelligence Test— Revised (SIT-R3)

Measures various cognitive areas: vocabulary, general information, similarities and differences, comprehension, quantitative, and auditory memory, Age range 4 years upward. Takes 10–20 min to administer. Correlates highly with the WISC-R, verbal VIQ.

Kaufman Brief Intelligence Test K-BIT

Individual screener of verbal and nonverbal intelligence. Age range 4 years and upward. Takes 15–30 min to complete.

Kaufman Test of Education Achievements (KTEA)

School achievement: Individual measure of processing and cognitive abilities. Age range 3 to 18 years. Scales include sequential processing/short-term memory, simultaneous processing/ visual processing, learning ability/long-term storage. In addition, a nonverbal option allows assessment of a child whose verbal skills are severely limited.

Wide Range Achievement Test—Revised (WRAT-R)

School achievement: A brief school achievement test measuring reading recognition, spelling, and arithmetic computation. There are two levels: level I is normed for children ages 5.0 to 11.11; level II is normed for children aged 12 through adults aged 64.

Suffolk reading scales

A comprehensive measure of pupils’ reading ability, which gives standard age scores and reading ages.

British Ability Scales (BAS)

Ability (intelligence test) revised and updated 1996, similar in structure to the WISC. Covers the age range 2.6 to 17.11 through two batteries. The design is hierarchical, leading from specific scales to domain (clusters) and to a general conceptual ability (GCA). As with the WISC, core skills reflect interrelated cognitive processes linked to the three most significant information processing systems in the Horn-Cattell model: verbal (Gc), visual/spatial (Gv), and nonverbal (Gf).

Peabody Picture Vocabulary Test revised (PPV-T)

An individually administered, norm-referenced test of hearing vocabulary containing items arranged in order of increasing difficulty. Each item has four simple, black-and-white illustrations arranged in a multiple-choice format. The subject selects the picture that best illustrates the meaning of a stimulus word presented orally. Age range 2.5 to 40 years.

Peabody Individual Achievement Test (PIAT)

An individual measure of academic achievement: reading, mathematics, and spelling. Multiple-choice format that requires only a pointing response for most items. It is ideal for assessing individuals with limited expressive abilities. Age range 5.0 to 22.11.

Stabler et al. (1998) (GHD / ISS children)

Brown et al. (2004)

Kranzler et al. (2000) (ISS children)

Chang et al. (2002) (stunted children)

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will demonstrate certain discrete deficiencies that may be related to developmental delay and that may lead to an overall poor performance or atypical variation in cognitive abilities. Improvements in “cognitive functioning” following GH therapy have been reported in several studies; however, where an effect on cognitive function has been found, the effects have been heterogeneous. It is therefore important to identify how cognitive functioning is being measured. A brief description of some of the tests used to measure attention and memory is given in Tables II and III.

1. Verbal and Performance IQ Discrepancy The common measurement of IQ is based on a composite of two parts: verbal (VIQ) and nonverbal or performance IQ (PIQ). Taken together, they complete the full IQ (FIQ). According to the WISC manual (Wechsler, 1991), a significant difference between VIQ and PIQ can give rise to concern that the child is developing cognitively in an atypical pattern. An abnormal discrepancy between VIQ and PIQ is found in school-aged children born preterm (Gabrielson et al., 2002). It is also found in children with congenital

TABLE II Measures of Attention, Performance, and Impulsivity Tests

Description

Study

Digit cancellation test

Search for a letter (d or p) with dashes above or below within a text. The test variable is the number of correct letters cancelled within 1 min.

Oertel et al. (2004) (hypopituitarism in adults)

Trail-making test

Connect numbers from 1 to 90 in an ascending order. The test variable is the number of correct connections within 1 min.

Oertel et al. (2004) (adults) Baum et al. (1998) (AOGHD) Soares et al. (1999) (GHD adults)

Evocated response potential recorded while completing a selective attention task.

Rapid (750–950 ms) pseudorandom sequences of four different square-wave patterns in the center of a visual field. The test variable is based on the frequency and orientation of the patterns. Subjects respond with a button press.

Lijffijt et 2002 (COGHD)

Digit backwards

A serial task of mental control derived from the WISC.

Soares et al. (1999) (GHD adults)

Stroop color test

Selective attention and response inhibition. The subject reads aloud color names that are printed in a different colors. The level of interference that this causes gives a measure of cognitive ability.

TABLE III Measures of Memory and Learning Tests

Description

Study

Face recognition task

Recognition of faces within a multiple presentation. This task is completed faster for developmentally normal children.

Almqvist et al. (1986) (GHD adults)

Associate learning; Associate recognition tasks

Memory tasks based on free recall of presented material.

Deijen et al. (1998) (GHD adults)

Associate learning task

Nine word pairs consisting of a name and an occupation are displayed on a computer screen at a constant rate of one pair per 3 seconds to learn as many paired names and occupations as possible. By means of recognition short-term memory is measured. An associate learning test after one hour, tests long-term memory.

Stouthart et al. (2003) (GHD young adults)

15 word immediate and delayed recall test (LTM)

A version of the Rey auditory verbal learning task where 15 words are recalled immediately after presentation and 15 min later following another nonassociated task.

van Dam et al. (2005) (COGHD)

Digit span (STM)

Part of the WISC and WAIS — progressively longer sequences of numbers are presented. In task 1, recall is in the order presented, and in subjects have 2, recall is in reverse order.

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hypopituitarism, where verbal IQ is within the normal range and not significantly different to sibling controls but where performance IQ is significantly lower than the control group (Brown et al., 2005). Brown et al. (2004) used the WISC-III to evaluate IQ, which revealed that of the four index measures, the only significant difference was found in perceptual organisation, with no difference in processing speed, verbal comprehension, or freedom from distractibility. Tasks comprising perceptual organization are those concerning organization of visual information: identifying missing elements from pictures, arranging pictures in logical and chronological order, constructing abstract patterns, and picture assembly. The consequences of this discrepancy might be referred to as nonverbal learning disorder (NVLD). NVLD was first identified in the early 1970s (Myklebust, 1975) but remains little understood. Looking at GHD adults with childhood onset, Sartorio et al. (1995) found that, following 6 months of GH treatment, there were improvements on the nonverbal scales of the WAIS, i.e., the symbol–number–association test, which is a measure of cognitive and procedural speed. 2. Attention, Perception, and Impulsivity Research concerning GH effects on cognition was started as early as 1985 (Smith et al., 1985) when eight children with GHD were studied in a counter-balanced, placebo study with GH treatment. This study showed that after only 4 weeks, the attentional abilities of these children could be seen to respond to the treatment and the attentional ability correlated with the blood levels of GH replacement. GHD has also been associated with patients’ inability to concentrate. However, this is also reported in children with ISS (Skuse et al., 1994). Stabler and colleagues (1994) administered a battery of psychological tests to 166 children referred for GH treatment, 86 of whom had GHD. Following 3 years of GH treatment, there were improvements in attention and larger effects were noted in the GHD group than in the ISS group (Stabler et al., 1998). Soares et al. (1999) reported enhanced performance in attentional parameters after at least 6 months of GH therapy. In a closed label study using adult subjects, Oertel et al. (2004) found that compared to baseline, even after 3 months of GH treatment, a significant improvement in the attention performance of adult patients with hypopituitarism was found in the GH group but not in a placebo group. Using the digit cancellation test (DCT) and the traitmaking test (TMT), attention scores were significantly different from the non-GH group after 6 months. Lijffijt et al. (2003) reported an EEG study that showed lower event-related potentials (N2b) during selective-attention tasks in patients with low levels of GH and IGF-I (COGHD). Patients were presented with a series of four visual patterns, one of which had been previously indicated to be the target pattern for them to identify. Target detection was significantly impaired in comparison to a control group, although the

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reaction time did not show a difference. Relevant information is therefore selected less adequately. Lijffijt et al. (2003) reported observations from two EEG scalp sites over the medial-frontal/anterior area (Fz) and over the posterior occipital visual cortex (Oz). These have been shown previously to indicate cortical correlates of selective attention. The only significant group effect found reflected attention-related negativity (N2bs) at Fz for controls. A smaller attention-related brain potential (N2b) in COGHD patients suggests poor integrated processing of stimulus characteristics (Kenemans et al., 2002). Lijiffijt et al. (2003) suggested that this indicates that GH and IGF-I have a specific effect on elements of cognition rather than a general effect. The mechanism(s) explaining the possible relationship between attention and GH/IGF-I requires much further investigation. However, it might be due to cognitive immaturity caused by developmental delay. One mechanism that could potentially be investigated further is the action of myelination. We know that myelination is partly controlled by growth factors and hormones (van der Pal et al., 1988; Besnard et al., 1989) and that increasing evidence suggests the importance of IGF-I in this process (D’Ercole et al., 2002). A delay in brain myelination has also been implicated as a potential explanation for attention deficit hyperactivity disorder (ADHD) (Mattes, 1980; Sieg et al., 1995). The frontal lobe and the prefrontal area are among the last areas of the brain to be myelinated (at around 16 or 17 years), and it is these areas that are concluded to exercise restraint over impulsive behavior (Asahi et al., 2004; Beckman, 2004). We might therefore also expect to find increased impulsivity in children with GHD. 3. Visual-Motor Skills Siegel and Hopwood (1986) noted a significant delay in the visual-motor skills of children with GHD or multiple pituitory hormone deficiency (MPHD): 26% of children with GHD scored less than the 16th percentile, which is equal to or greater than level 4 brain injury. Siegel and Hopwood (1986) used the Bender visual motor test as a brief test of visual-motor integration that may provide interpretive information about a child’s (from 3 years) or adult’s development and psychological functioning. The standard test consists of nine figures presented one at a time, which the child is asked to copy. The results are scored based on accuracy and organization. 4. Memory and Learning Although some evidence shows that memory deficits in adults with COGHD are ameliorated by treatment with GH (van Dam, 2005), there are no equivalent studies on children. A specific role for GDH has been observed for memory deficits related to both short and long-term memory in adults (Chrisoulidou et al., 1998). Looking at the adult studies, however, we learn that Almqvist et al. (1986) found that five adult subjects had

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improved memory function for face recognition after 8 weeks of GH treatment without changes in other cognitive parameters. Deijen et al. (1998) also found an improvement in memory in patients with COGHD. Changes in memory performance were positively correlated to the GH-induced changes in serum IGF-I concentration. Deijen et al. (1998) also observed that memory performance normalized during GH many years after the completion of brain development, which strongly suggests that memory impairments in GHD are mainly caused by a reversible disturbance in neural cell metabolism. Memory improvements following GH treatment may be due to the presence of GH in the hippocampus, an area known to be important for memory and other cognitive functions (Nyberg and Burman, 1996). Functions mediated by the GH receptors identified in the hippocampus are not yet known but it has been suggested that they may be involved in the action of the hormone on memory and cognitive functions (Nyberg, 2000; Le Greves et al., 2002). IGF-I has also been positively correlated to vigor and memory (Stouthart et al., 2003). However, research on adult patients put some doubt on the effects of GH on memory. Long-term memory (LTM) could not be shown to improve compared to baseline during GH therapy, and short-term memory (STM) improved equally in both a GH and placebo group after 3 and 6 months. The improvement in STM was considered a placebo or practice effect (Oertel et al., 2004). Memory was assessed using a text reproduction task (Wechsler,

1987) where details of a story are recalled immediately after presentation (STM) and after 24 h (LTM). Sleep is also an important element to consider. It is known that sleep is needed for the consolidation of memory for complex tasks and that elements of the GH axis are important in the regulation of sleep. The GH axis also up-regulates protein synthesis, which is required for memory consolidation (Rollero et al., 1998). Therefore, the relationship between GH treatment and improved memory could be mediated through improved sleep patterns. Evidence that the decline in GH/IGF-I in older adults is correlated with learning and memory capacities (Aleman et al., 1999; Kalmijn et al., 2000) suggests that normal learning functioning may depend on somatotropic functioning.

VII. EFFECT OF GH ON PSYCHOLOGICAL WELL-BEING Well-being can depend on many aspects of the psychological status of the individual. Patients who lack GH demonstrate symptoms of a lack of energy, tiredness, and irritability (Deijen et al., 1996; Bengtsson et al., 1993). Many studies identify specific aspects of well-being as their outcome measures. A brief description of some of the tests used to measure psychological well-being are given in Tables IV to VII.

TABLE IV Measures of Self-esteem and Self-perception Tests

Description

Study

I think I am

Self-assessment likert type response to a series of questions concerning family, self-esteem, and self-worth. A Swedish test with two age versions.

Erling et al. (2002) (ISS)

Silhouette apperception test

Self-assessment of height in relation to five different-sized silhouettes.

Child visual analogue scale for growth (CvasG)

VAS scale set of paired adjectives selected on the basis of factor-analyzed responses of children with short stature.

Machover, Draw a person

Projective test. Subject draws two “persons” and answers a set of standard questions concerning the lives and opinions of the drawn people.

Youth self-report (YSR)

Self-ratings for 20 competence and problem items paralleling those of the child behaviour checklist (CBCL)/ages 6–18. The YSR also includes open-ended responses to items covering physical problems, concerns, and strengths.

Self-esteem inventory

Respondents state whether a set of generally favorable or unfavourable aspects of a person are “like me” or “not like me.”

Self-perception profile for children (SPPC)

Measures six domains: academic, social, athletic, physical, behavioral, and global self-perceptions, as well as an individual’s perceptions of global self-worth. It also includes the degree of importance an individual gives the various self-perception domains.

Lagrou et al. (1998) (Turner)

van der ReijdenLakeman (1996) (intraoterine growth retordation children) Ross et al. (2004) (ISS children)

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TABLE V Measures of Behavior Problems and Psychological Immaturity Tests

Description

Study

Child Behaviour Checklist (CBCL) parent version

Parent provides information on 20 competence items and rates the child on 118 problem items using a 0-1-2 scale on behavior in the past 6 months. Separate scoring for boys and girls, age 4–11 and 12–18.

Lagrou et al. (1998) (Turner girls) Steinhausen et al. (2002) (ISS and Turner)

CBCL teacher version

Teacher-rated behavior problems questionnaire.

Parent rating of the behaviour assessment system ⫹ teacher rating scales Behaviour Assessment System for Children (BASC)

Adaptive and problem behaviours of children 4–18 years in school, home, and community

Stabler et al. (1998) (GHD children) Kranzler et al. (2000) (ISS children)

McGarth “how are you?”

Simple measure of emotional stability — choice of nine smiley faces.

Ross et al. (2004) (ISS children)

Hamburg Neuroticism Extraversion Scale (HANES)

Measuring sociability, social activity, and neuroticism.

Vineland

Daily living skills: A semistructured interview and classroom questionnaire for measuring personal and social skills used for everyday living. This assessment provides critical data for the diagnosis or evaluation of a wide range of disabilities, including mental retardation, developmental delays, functional skills impairment, and speech/language impairment.

Soares et al. (1999) (GHD adults)

Connors 93-item parents questionnaire

Connors scales rate several areas of functioning, including hyperactivity, conduct problems, emotional over-indulgence, anxiouspassive, asocial, and daydream-attendance problems.

Scarth et al. (1994) (ISS children)

Susan Harter’s SelfPerception Profile (SPP)

A self-report estimation of a child’s sense of general self-worth and self-competence in the domain of academic achievement. It describes five specific domains of self-concept as well as global self-worth.

Children’s Depression Inventory (CDI)

A brief self-report test that helps assess cognitive, affective, and behavioral signs of depression in children and adolescents 6 to 17 years old. It consists of 27 questions and is adapted from the Beck Depression Inventory.

Revised Child Manifest Anxiety Scale (RCMAS)

A self-report inventory used to measure anxiety in children for clinical purposes (diagnosis and treatment evaluation), educational settings, and research purposes. The RCMAS consists of 28 anxiety items and 9 lie (social desirability) items. Each item is related to feeling or action that reflects an aspect of anxiety

Norwicki–Strickland Locus of Control Scale

A measure of personality characteristics within predetermined situations.

Rutter Teacher questionnaire

Contains 20 pro-social behavioral items. Teachers are asked to compare each item to the child’s behavior over the previous 3 months.

A. Self-esteem Erling et al. (2002) examined the relationship between GH and psychological functioning, especially self-perception and well-being, in 60 prepubertal boys of short stature with a wide range of GH levels.

Scarth et al. (1994) (ISS children) Chang et al. (2002) (stunted children)

Psychological functioning was measured using a growthrelated measure of alertness, mood, self-esteem, inhibition, stability, and litheness (Wiklund et al., 1994); Well-being was assessed using the Swedish “I think I am” questionnaire coving aspects of self-perception, physical

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TABLE VI Measures of Motivation, Mood, and Depression Tests

Description

Study

Profile of Mood States (POMS)⫹ Vigor-anxiety subscale

A standard method of assessing mood state. A self-report scale. It contains adjectives relating to feelings, which in the original version are divided into seven subscales but shortened versions have been developed.

Stouthart et al. (2003) (GHD young adults)

Hospital Anxiety and Depression Scale (HAD)

The HAD scale comprises statements that the patient rates based on their experience over the past week. The 14 statements are relevant to either generalized anxiety (7 statements) or “depression”.

State-Trait Anxiety Inventory

A self-evaluation measure of anxiety (trait) and level of tension (state). Child and adult versions may be used with emotionally disturbed individuals, as well as the general population.

Hopkins Symptom Checklist (SCL-90)

Psychological, somatic complaints including anxiety and obsessivecompulsive behavior

Stouthart et al. (2003) (GHD adults) Bengtsson et al. (1993) (GHD adults)

Hamilton Depression scale (HAM-D)

Provides an indication of depression.

Soares et al. (1999) (GHD adults)

Beck Depression Inventory (BDI)

A 21-item test presented in multiple choice format that measure-presence and degree of depression in adolescents and adults.

TABLE VII Growth-Related Measures of Quality of Life in Children Tests

Description

Study

TNO-AZL Children’s Quality of Life (TACQOL)

Offers respondents the possibility of differentiating between how they function now and their feelings about this level of functioning. It has 56 items covering seven domains: physical complaints, motor, cognitive, social functioning, autonomy, and positive and negative emotions. Age range 6–15 years.

Theunissen et al. (2202) (ISS children)

Idiopathic Short Stature Quality of Life (ISSQOL-CF)

An 8 item ISS-specific scale measuring vitality between 0 and 100

Dutch Children’s AZL/TNO Quality of Life (DUCATQOL)

A 25 item generic self-report questionnaire. Age range 5–16 years. Four domains plus a total HRQoL score using abstract smiley faces.

Global HRQoL of the child

Judgement of the pediatrician. Three questions: seriousness of situation, distress caused by condition, and distress of participating in research.

appearance, skills and talents, psychological well-being, family relationships, and non-family relationships (OuvinenBirgerstam, 1985). A comparison was made of the wellbeing and self-perception of children with GH insufficiency, children with ISS, a normative sample, and healthy boys with normal stature. They found that the lower the endogenous levels of GH, the more inhibited were the boys of short stature, as perceived both by themselves and by their parents. Boys with GHD had a more negative perception of their physical appearance than

the normative sample, leading to the conclusion that low levels of GH lead to inhibition and social withdrawal.

B. Behavior Problems and Psychological Immaturity Stabler and colleagues (1994) reported six times higher than expected rates of behavior problems among GHD and ISS children, reporting that the total behavior problem scores as assessed by the Child Behaviour Check List

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(CBCL) were significantly higher ( p ⬍ 0.001) at baseline than those of a control group. Psychological immaturity has been identified in children with GHD (Hayashi et al., 1992; Sartorio et al., 1996; Rotnem et al., 1977, 1979). Short stature alone was found not to be related with psychological problems or selfconcept, but a trend toward an improvement of behavior problems was measured by the CBCL in the GH treatment group in years 3 and 4. Steinhausen et al. (2002) showed that there was a significant but clinically small decline of behavioral abnormalities (as measured by the parental CBCL) over time. Parents themselves reported being more satisfied. Results were independent of diagnostic category, gender, height velocity, puberty, and age. Stabler et al. (1998) studied 195 children with short stature (109 had GHD) aged between 5 and 16 years. They discussed the possibility that observed improvement in behavior problems following GH treatment might be related to indirect effects of the GH on the brain, the positive social value associated with increased height, or effects related to the expectations of parents. Stabler et al. (1998), who used the parent-completed CBCL, observed that behavioral problems decreased following GH treatment. Ross et al. (2004) also found that GH treatment was associated with a trend toward improvement in problem behavior as measured by parental questionnaires (CBCL).

significant relationship between height and feelings of guilt using the Draw A Person test. Again, there are no child GHD studies where anxiety and depression are the outcome measures. However, from adult studies we find that in the first 6 months of treatment with GH, depression decreased (Feldt-Rasmussen, 2004). Depression levels continued to decrease over the 12 months of the study period. IGF-I levels were found to be negatively correlated to depression, fatigue, tension, and anxiety and positively correlated to vigor (Feldt-Rasmussen, 2004; Lasaité et al., 2004). There is a remarkable intercorrelation between mood and IGF-I. Thus, the IGF-I response seems a most valuable predictor of the impact of GH (Stouthart et al., 2003). We can anticipate that an effect on mood might be found because we know that GH secretion capacity is reduced during depression (Schneider et al., 2003), and Burman et al. (1996) demonstrated the passage of GH into the CSF. They stated that the “changes in homovanillic acid and free T4 are similar to those reported after successful treatment of depressive illness with antidepressant drugs.” They concluded that “this may reflect a beneficial affect of GH on mood and behaviour.” It has also been shown that GH secretion capacity is reduced during depression and sleep (Schneider et al., 2003), which would seem to lend support to the importance of this mechanism in the area of mood.

C. Motivation, Mood, Vigor/Vitality, Energy, and Depression

D. Sleep

We know from clinical observations that children with untreated GHD appear to lack physical and mental initiative. There are a number of terms used in the literature that could refer to this factor: motivation, mood, vigor, energy, and depression. It is in this area that clinicians and parents expect to see the greatest psychological changes take place. From adult studies, we know that GHD leads to tiredness, decreased vitality, problems with depressed mood, and difficulties in coping with stressful situations (Björk et al., 1989; McGauley et al., 1990; Rosén et al., 1994; Holmes and Shalet, 1995). It has been shown that neuroendocrine centers modulate mood and are functionally related to GH secretion by the pituitary (Abelson et al., 1992). Therefore, we might also expect to find an effect of GH on mood. Stouthart et al. (2003) also showed that IGF-I was positively correlated to vigor. Vigor is measured as a dimension within the Profile of Mood States (POMS). Kesleman et al. (2000) found that depression, social isolation, and dependent lifestyle were evident in GHD patients. This was measured by juvenilization, which was a common complaint. Molinari et al. (2002) showed that children with GHD had higher levels on a total anxiety index. They found a

Sleep disturbance is also indicated in GHD. GHD patients complain of reduced vitality, general fatigue, lack of concentration, irritability, and reduced alertness during daytime. It is unclear whether these symptoms are due primarily to GHD or secondary to GHD-related sleep impairments. Animal studies show that the GH-releasing hormone (GHRH) is implicated in the regulation of sleep–wake activity (Obal et al 2001), and we know that the natural cycle of GH secretion is that it occurs during sleep. Schneider (2003) showed that sleep is affected by GH secretion. We can therefore conclude that sleep will be affected and that this is likely to have an effect on other psychological variables.

E. Anxiety Anxiety has been identified as a factor in GHD. One study that looked specifically at anxiety and compared GHD with non-short controls was Stabler et al. (1994). A semistructured clinical psychiatric interview (SNID-NP) was conducted and participants completed a series of measures of anxiety, including fear of negative evaluation, FNE; social avoidance and distress scale, SAD; fear questionnaire (social and agoraphobia subscales); depression inventory; and tridimensional personality questionnaire TPQ. Results

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showed that young adults with GHD were more likely to be at risk for anxiety and depression than age- and gendermatched control groups. From the literature, Stabler and colleagues (1998) were able to identify significant improvements in mental health factors (i.e., withdrawal; somatic complications; anxiety/ depression) following 3 years of GH treatment in children (mean age 11.2 years). Using the parent-completed CBCL, they found that behavioural scores improved significantly ( p ⬍ 0.001) over the 3-year study period. There were also improvements in the GHD group on the subscales of social and thought problems following treatment.

F. Stress Kosten et al. (1984) demonstrated that the GH secretion rate correlates to the neuroendocrine response to stress. Those individuals with low levels of GH may have a weak or ineffective psychological defense pattern, thus exposing them to distress. It is interesting to speculate what effect this could have on a child. Would they show greater effects of family distress than a non-GHD child? As yet there are no direct studies examining stress as an outcome in GHD children treated with GH. However, many symptoms have been found that could be described as nonspecific stress responses (Gilmour and Skuse, 1999). Adult studies also show that depression and psychological complaints increase during the first 6 months of discontinuation of GH (Stouthart et al., 2003). This supports the hypothesis that decreased psychological well-being may be related to GHD per se.

G. Appetite It has been proposed in the literature that a possible connection exists between appetite and GHD. A common observation is that GH treatment is associated with an increase in appetite. Children with severe GHD often lack appetite, which may be interpreted as a lack of mental energy and depression. Therapy has been observed to increase appetite and improve eating habits (Blissett et al., 2000; Mericq et al., 2003).

H. Quality of Life Following GH treatment, we expect that the child will increase in body composition, strength, and exercise capacity. In line with the aforementioned analysis, it is also expected that there will be improvements in psychological well-being, improved mood state, less anxiety, and so on. All these factors lead to an anticipated improvement in quality of life (QoL). Review studies often conclude that QoL studies have been inconclusive in the identification of GH treatment effects (e.g., McGauley, 2000; Radcliffe

et al., 2004; South Thames Drug Information Service, 1998). However, assessment of QoL is complicated by the multifaceted nature of the concept and disagreement on what is to be included within an objective, multidomain measurement or how to measure QoL as a subjective concept. One possible reason that the evidence is inconclusive is that the instruments used are often generic and not relevant to the condition of GHD. A number of generic instruments have been used with populations of short stature adults and children but there are only a few measurement instruments specifically designed for children with growth problems (Radcliffe et al., 2004). Two instruments designed for children with growth disorders are the parentcompleted “Short Stature in Children Questionnaire” by Haverkamp and Noeker (1998) and the “Child Visual Analogue Scale for Growth” by Wiklund et al. (1994). There are very few studies on the QoL effects of GH treatment on GHD children. There are, however, several studies on ISS children. These reports, sometimes with control groups, do not show a clear improvement of healthrelated QoL (HRQoL). These studies indicate no change in HRQoL after treatment (Voss, 2000; Kranzler et al., 2000; Sandberg, 2000; Downie et al., 1996; Voss et al., 1994; Boulton et al., 1991). Evidence for a decrease in QoL has been found in only one study (Theunissen et al., 2002) with the use of the TACQOL-CF (a quality of life questionnaire developed by the TNO research institute in the Netherlands) at the second year follow-up. This finding needs to be investigated further; however, Theunissen et al. (2002) pointed out that in this group of ISS children, HRQoL and self-esteem were not lower than in a norm population, therefore it might be unreasonable to expect an increase above the norm. It was also pointed out that satisfaction with height was no different between control and treatment groups and that satisfaction was related positively to improvements in HRQoL. That children in the treatment group had a worsening HRQoL could be due to their higher level of psychosocial expectations that could not be met with gains in centimeters at the 2-year follow-up. A longer term follow-up may be required to determine the true benefit.

VIII. METHODOLOGICAL DIFFICULTIES IN MEASUREMENT It has long been recognized that many issues need to be considered in the measurement of response to GH treatment and that the identification of the best method is not straightforward (Wiklund et al., 1991). Many factors have on impact on the child’s cognitive development that are simultaneous with any hormone treatment. For example, Erling (1999) showed that there were greater psychological benefits from

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GH for taller GHD boys who benefited more than shorter GHD boys. It is speculated that a lesser, but still positive, treatment effect might be due to the shorter boys having to cope with both GHD and extreme short stature (Erling, 1999). Hunt et al. (2000) suggested that it was not the actual height but the personal satisfaction with the height that was more closely associated with psychological outcomes. Boys who perceived themselves to be short were more likely to be bullied (Erling, 2004).

A. Developmental Issues The developmental picture of advanced reasoning abilities in children is incomplete due to its complexity and the methodological difficulties of studying these processes with sufficient range and validity. We need measures of cognitive development that represent valid cultural and linguistic factors, which must take into account the developmental age of the child. The developmental age of a child is, however, complicated by the plasticity of the development process. Each child will develop cognitively at their own pace, dependent on their abilities, past knowledge, and educational surroundings. The situation is less complicated with the assessment of cognitive abilities of adults whose cognitive development no longer develops at the same speed as the child’s. The child’s cognitive development is also characterized by large leaps or paradigm shifts in understanding, which make testing procedures more complicated. The child may also be at different cognitive levels at the same time, which also makes assessment more complicated. Where the respondent may have learning difficulties or behavior problems, the measurement of cognitive capacity and change-over time is further complicated. Any improvement in cognitive capabilities may be due to normal developmental changes and not due to hormone replacement therapy. The longitudinal assessment of child cognitive development is thus complicated by the individual nature of the child’s development, making the choice of a matching sample or control group difficult.

B. Structural Issues (Primary or Secondary Effects) The difficulty in isolating specific psychological findings is that GH treatment has both primary and secondary effects in this area. The primary effect is on the GH receptors in the brain and metabolic changes in the body leading to physical maturation. The secondary effect is the consequence of social interaction leading from changes in appearance and changes in interpretation of subjective experience.

C. Bias in Referring to Growth Units There are also confounding variables that may give rise to an apparent effect of GH. Parents may refer their child for

reasons other than height. It has been shown that children with emotional/behavioural problems, who are also short, are more likely to be referred to a growth clinic (Voss and Sandberg, 2004). The referral may also be more to do with the parental fear that “something is going wrong” than with there actually being a problem. This fear leads to anxiety, which can be passed on to the child, in whom it may be expressed in a range of behaviors from excessive dependency to behavioral deviancy. Once treatment has started, the parents may feel that they have done everything it is possible to do and feel assured that they are in fact “good parents.” The anxiety in the parent–child relationship then declines and the child’s behavior normalizes.

D. Placebo Effect It has been suggested that placebo effects can contribute as much as 50–70% of the initial effect of a treatment but that this effect declines over time (Wiklund, 1993). However, Ross et al. (2004), looking specifically at the effects of GH, found a persistent placebo effect at follow-up after 4 years. They showed that there was an equal decline in behavioral problems between ISS children treated with GH and ISS children treated with a placebo, despite increased physical height in the GH-treated group (0.50 SDS; 3.7 cm). They concluded that this shows that there are no negative effects of GH treatment; however, it can also be interpreted as a placebo effect. The notion that the height problems are being taken seriously may have a positive effect on behavior. It could also be hypothesized that less time worrying about height problems may leave more time for developing cognitively. The new experiences of participating in a growth study will include increased experiential exposure and thereby improved self-confidence, leading to benefits in learning and attention. Parents and teachers expectations may also increase due to expectations of treatment success and thus stimulate the child to achieve more. The fact that the child is having the height issue dealt with “professionally” may give rise to feelings of support and security, which, over time, reassure the child. Of course, in order to be able to determine a placebo effect in GHD, it is necessary to carry out the type of placebo-controlled trial performed by Ross et al. (2004) with ISS children. However, this is morally and ethically undesirable in a population for whom a delay in treatment may lead to long-term or irreversible effects.

E. Cognitive Maturity Needed to Recognize Height Differences The use of child self-report questionnaires, assumes that the child is mature enough to recognize and categorize relevant differences between themselves and others. Young children may not have the cognitive maturity to recognize

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that there are height differences between themselves and other children (Radcliffe et al., 2004) or they may deny differences as a coping strategy (Siegel et al., 1991; Erling et al., 1994).

Another problem arises in that what we are measuring is not clearly defined. Psychological importance, as stated earlier, can be interpreted in a number of ways. Unlike height, there is not a standard, universally agreed measure of psychological importance.

F. Adaptation In addition to the aforementioned considerations, the measurement of psychological importance is also complicated by the child adapting to their short stature. Height is just another issue to deal with during teenage years; children cope by joining a social group that accepts them in exchange for them accepting the other members. The child may have learnt to cope with the “problem” by associating with younger children who are shorter so that age-related short stature is less apparent. At this point of the equation we also have to consider individual differences of the children in dealing with the issue of short stature. As has been pointed out, temperament, family support, and coping strategies will all effect the problem expression (Erling, 2004) and every child will deal with the problem differently. The expression of psychological importance of GH treatment is therefore very difficult to disentangle from the psychosocial interactions taking place. This may be the reason why it is concluded that the psychological benefits of GH are more obvious in patients with adult-onset GHD (Almqvist et al., 1986; Attanasio et al., 1997; Burman et al., 1995; Degerblad et al., 1990; Mårdh et al., 1994; McGauley et al., 1990; Whitehead et al., 1992). It can be argued that CO-GHD has a more profound effect on the individual, perhaps because of the prolonged time with GHD and especially because of GHD at a critical period of psychological development. However, it might also be argued that the child has learnt to cope and adapt to its short stature and therefore the beneficial effects are less obvious.

G. Tools: Measurement Instruments Given the argument that GH has profound effects on the body and brain in excess of height, along with the frequent clinical observation that GH treatment has effects on the child’s behavior, it seems likely that a number of psychologically important changes are taking place. It is therefore surprising that these are sometimes difficult to identify and measure. However, the measurement instruments we are using are not sufficiently sensitive to indicate change. Often the instruments have been designed for the measurement of psychological effects in other conditions and it has been argued that it is necessary to develop instruments that are condition specific in order to improve their sensitivity (McGauley, 1989). Adult psychological instruments have been developed, and Wiklund and Erling (1994) have developed an instrument specifically for GHD children. However, specific self-report instruments for the child are lacking.

IX. FUTURE DIRECTIONS Short stature, as well as the lack of GH, is, in certain vulnerable children, likely to result in mental health and cognitive problems that continue into adulthood. It is therefore vital that we understand fully the psychological importance of GHD and how the child’s mental development is affected by GH treatment. In order to achieve this, we need to agree on the definition of what is meant by psychological importance. Once we have established this, we will be better able to identify those psychological and cognitive instruments that will be needed to measure changes in this model. Ideally, it would be beneficial to identify differences among metabolic, neurological, and social effects, which may be possible as we learn more about the specific actions of GH on the brain. A consistent picture of the cognitive profile of children with GHD and the effects of GH has not been achieved thus far in the literature. In order to achieve this, it will be necessary to have cooperative, national or international studies that allow the identification of confounding factors that may be obscuring the cognitive effects of GH treatment. Larger studies with agreed standardized tests are needed to be able to establish the cognitive effects of GH treatment. In the area of growth deficiency, it is advised to identify the problem as early as possible so that early treatment can be instigated. It seems likely that similar demands should be applied to the cognitive and psychological well-being of the child. Therefore, we need to develop instruments sensitive enough to measure changes in young children so that we are able to instigate treatment as early as possible, ideally prior to the child experiencing stress due to the condition, in order to prevent the possible negative cognitive complications. New methods of looking at attention issues need to be found and comparative data for different methods need to be established. One method that is currently being tested is to use computer-assisted attention tests developed for ADHD that could be normed for GHD children. Such a study is being carried out at the Gothenburg Paediatric Growth Research Centre. There is a great deal of contradictory evidence in the literature and much of this is difficult to evaluate as a result of different methods being used and poor study designs. Due to the often small sample sizes of studies in this area, it is important that similar standardized methods are used in order to be able to pool results so that firm conclusions can be reached.

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20 Acromegaly and Brain Function: Effects on the Human Brain during Conditions with Increased Growth Hormone Concentrations SIGBRITT WERNER Department of Medicine, Karolinsko University Hospital Huddinge, Stockholm, Sweden

ghrelinoma causing notably high levels of ghrelin in serum, 2100 ␮g/liter (reference level ⬍5 ␮g/liter), could not document signs or symptoms of chronical hypersecretion of GH. The patient’s insulin-like growth factor I (IGF-I) and GH levels in serum were normal. The syndrome acromegaly was first described by the French physician Pierre Marie. Its association with disturbed GH production was gradually discovered; in 1921 Evans and Long reported that anterior lobe extracts stimulated growth. In 1944 Li and Evans isolated bovine GH, which proved inactive in the human. In 1957 Raben and in 1963 Roos et al. reported on improvements of the purification of human growth hormone, which has since been given to GH-deficient individuals. In 1969 Li and co-workers revealed the protein structure of GH. During this decade, radioimmunoassays for the determination of GH were also developed and GH-producing tumors could be characterized according to the patients’ GH profiles in serum; in acromegaly, GH was detectable throughout the day and night on a high or low level without the repetitive nadirs down to nondetectable levels and without the repetitive peaks seen in healthy subjects (Fig. 1). The knowledge of IGF-I and the radioimmunoassays used for the determination of IGF-I in serum developed later. Only since the 1990s has IGF-I been established and spread for wide clinical use. Today, determination of serum IGF-I is the best long-term marker of growth hormone production per time. So far there has been no report on a patient with a GH-producing tumor in whom IGF-I in serum has not been increased (Fig. 2). The diagnosis of GH-producing tumours is generally confirmed on an outpatient basis with a series of GH

I. Brain Effects during Growth Hormone Hypersecretion II. Treatment of Growth Hormone-Producing Tumors References Chronical surplus of endogenous and exogenous growth hormone (GH) will, to various degrees, affect all cell systems in the human body. However, no one has yet been able to document beneficial effects on the central nervous system, such as improved cognition, learning capacity, quality of life, or memory. Possible anabolic effects of growth hormone have also been studied in patients with Turner’s syndrome who show diminished height and a spectrum of characteristic cognitive disturbances. Long-term GH administration was not associated with abbreviations of these urocognitive deficits nor does long-term growth hormone administration induce improved mental development in patients with Down’s syndrome. Growth hormone (GH) hypersecretion causing the clinical syndrome of gigantism in the young, still growing individual, and acromegaly in the adult is usually caused by a growth hormone-producing pituitary adenoma. Another rare cause of endogenous hypersecretion with clinical signs of GH hypersecretion is the ectopic production of growth hormone-releasing hormone from tumors, e.g., of the lung, pancreas, thymus, mammary gland, and ovary. An even more rare cause of endogenous GH hypersecretion is ectopic production of the growth hormone (Ezzat et al., 1993). The 28 amino acid peptide ghrelin is a potent GH stimulator that acts on the GH-producing pituitary cell via the receptor GHS-␣. Interestingly, Tsolakis et al. (2004) in a report on a patient with a malignant gastric

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FIGURE 1 GH levels in serum during 24 h in two patients with acromegaly compared with a healthy individual. Patient A has a stable, moderate hypersecretion throughout the 24 h without periods of nondetectable GH levels.

determinations, e.g., from 8 to 10 h followed by an oral glucose test, which, in acromegaly, fails to inhibit GH production to ⬍0.1 ␮g/liter. Concomitantly, blood is drawn on the first sample occasion to assess possible insufficient secretion of adrenocorticotropic (ACTH), thyroid stimulating hormone (TSH), or gonadotropins and possible hypersecretion of prolactin and TSH. The incidence of GH-producing tumors is approximately 4 cases per million per year. The prevalence of gigantism/acromegaly is approximately 70 patients per million inhabitants. The disease is equally represented in women and in men. Most patients are diagnosed in their forties but the tumors can be found in children as well as in the very old (Sheppard, 2003). Approximately 30% of the tumours cosecrete prolactin (Barkan, 1989). Previously, acromegaly was diagnosed late; patients came to diagnosis due to local symptoms from a large tumor—headache, pituitary insufficiencies, visual field defects, atrophy of the

optical nerves, severe pains from degenerative arthrosis, diabetes, and hypertension. Nowadays, with the clinical practice widely used analysis of IGF-I in serum, the sensitive marker of chronically increased GH production, patients are diagnosed early via symptoms of water retention, such as sweating, snoring, and soft tissue swelling in hands. Simplistically, one can state that the clinical effects of GH on any tissue can be summarized in its stimulating effect on protein synthesis (anabolism), its lipolytic effect, and its stimulation of water retention both intra- and extracellularly. The only metabolic effect of GH that does not require adequate nutrition is the lipolytic effect, which is active and increased during starvation. Somatically healthy individuals taking GH to increase lean body mass and to diminish fat mass (doping) counteract the “side effect” of water retention with diuretics. However, the majority of patients with acromegaly seek a physician due to symptoms of growth, e.g., of hands, feet, nose, cheek, and jaw. Amazingly, many are diagnosed by friends and physicians macroscopically en passant.

I. BRAIN EFFECTS DURING GROWTH HORMONE HYPERSECRETION One might hypothesize that during the early phase of tumor growth and GH hypersecretion, while the tumor is not yet visible on magnetic resonance imaging or only some

20. Acromegaly and Brain Function

millimeters in diameter, there would be a true anabolic, positive effect from GH on brain function in analogy with GH transgenic mice, which solve various tasks more quickly than control mice (Rollo et al., 2002) without any of the side effects known from growth hormone surplus. However, the patient would not seek a doctor but would consider himself a healthy person, continue to work, and possibly rejoice over any mental or somatical improvement. An individual seeks a doctor when signs and symptoms of illness and disease come. Improvements in any cerebral function in gigantism or in acromegaly due to chronical GH hypersecretion have not been reported so far. However, the first evidencebased, disease-specific, internationally scrutinized rating scale for estimating quality of life in acromegaly was published as late as in 2002 (Webb et al., 2002). It is documented that psychiatric morbidity and depression are not increased in patients with newly diagnosed untreated acromegaly (Abed et al., 1987). Quality of life in acromegaly is decreased both due to somatical stigmata such as disfiguring features, pains, and hampered motility and due to mental stigmata such as tiredness and lack of energy (Richert et al., 1987).

II. TREATMENT OF GROWTH HORMONE-PRODUCING TUMORS For many years, surgery has been the most commonly chosen first treatment in acromegaly. There is still consensus internationally that surgery should be the first chosen treatment for an intrasellar, well-delineated tumour. There is also a widely accepted consensus that criteria for successful surgery or “cure/adequate control” are postoperatively normalized IGF-I and GH levels ⬍1 ␮g/liter basally or after oral glucose administration (Guistina et al., 2000). Untreated patients with acromegaly or patients with postoperative growth hormone levels above 2.5 ␮g/liter will continue to show increased morbidity and mortality with a decreased life span of about 10 years (Sheppard, 2003). In contrast, patients cured according to the consensus criteria show mortality and morbidity comparable with a control population (Sheppard, 2003). Even after successful surgery, the patients show persistently decreased quality of life (Biermasz et al., 2004). Patients not controlled adequately by surgery and therefore treated further with conventional external photon irradiation generally via three fields and with a total dose of 40–50 Gy showed significantly lower quality of life scores than those patients treated with surgery only. The further impaired quality of life could not be explained by larger, more invasively growing tumors, longer disease duration, or higher age in the group of patients receiving radiotherapy (Biermasz et al., 2004).

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Long-acting somatostatin analogues constitute a third alternative in treating patients with acromegaly/gigantism. Successful treatments during which the patients show lowered GH levels and normalized serum IGF-I were not associated with a significantly improved quality of life (Biermasz et al., 2004). A fourth manner used to treat patients with GH-producing adenomas is to administer the genetically engineered GH receptor antagonist pegvisomant/trovert, which inhibits GH action on cells. This GH receptor antagonist is remarkably effective, controlling disease activity and normalizing IGF-I levels in serum in over 90% of patients. The treatment is accompanied by unchanged or moderately upregulated growth hormone levels in serum. So far there are no reports on alterations in brain parameters before and during treatment with the GH receptor antagonist. Long-term follow-up studies on possible brain effects or brain side effects are also lacking; the drug was registered for treatment of acromegaly only recently (Trainer et al., 2000; Van der Lely et al., 2001). The central and peripheral nervous systems were studied in patients with untreated acromegaly by recording the somatosensory-evoked potentials and brain stem auditorial-evoked potential concomitantly with distal motor latency, nerve conduction velocity, and other variables. The 10 patients studied showed abnormalities in the peripheral nervous system but no changes in the central nervous system compared with controls (Ozata et al., 1997). One confounding factor in patient materials when evaluating the effects of GH- or IGF-I hypersecretion on cerebral and cognitive functions is the concomitant hypersecretion of prolactin seen in approximately 30% of patients with GH-secreting pituitary adenomas. The hyperprolactinemia causes hypogonadism, loss of sexual desire, impotence, and difficulties of mentally concentrating and memorizing. The hypogonadism is also accompanied by diminished production of the anabolic steroids: estradiol, androstendion, androsterone, and testosterone. In particular, testosterone is a permissive factor for optimizing GH effects on cell systems (Chowen et al., 2004). Concomitant thyroid and/or cortisol insufficiencies where the patients risk oversubstitution as well as undersubstitution with thyroxin and cortisol/cortisone will also obscure possible GH or IGF-I- and IGF-II-mediated effects on the brain in acromegaly. To study possible anabolic brain effects of GH in girls with Turner’s syndrome, a long-term, double-blind, placebocontrolled study was performed. The ages of the patients were from 5 to 12 years and the treatment duration was from 1 to 7 years. Growth hormone was given three times per week subcutaneously, ensuring pharmacologically increased growth hormone concentrations and increased IGF-I and IGF-II in serum. Intelligence quotient, verbal memory, language reception and expression, spatial memory, spatial perception, visual-motor function, and aural affect recognition were measured. These characteristic nonverbal neurocognitive deficits

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Sigbritt Werner headache

enlarged nose enlarged tongue

enlarged ears

enlarged lips

coarse features

protruding lower jaw

cheeks lacking fat goiter

sleep apnoea

cardiac enlargement

galactorrhea

cardiac myopathy

hypertension liver enlargement

spleen enlargement

kidney enlargement

carpal tunnel syndrome

enlarged hands

colon polyps widened and prolonged gastro intestinal tract

spinal stenosis arthrosis

diabetes mellitus

broadened feet

FIGURE 3 Some signs in a patient with advanced acromegaly due to a GH-producing pituitary tumor.

were not altered with growth hormone treatment (Ross et al., 1997). Possible mental and neurological benefits from growth hormone treatment were also studied in young children with Down’s syndrome; 15 patients, 6–9 months old with GH levels within the normal range were treated with GH for 3 years. Growth velocity normalized but GH had no effect on head circumference, mental development, or gross motor development. There was some (p ⬍ 0.01) improvement in fine-motor development (Annerén et al., 2000). In conclusion, in the human individual, it has so far been difficult to document possible beneficial effects on the central nervous system during endogenous or exogenous growth hormone surplus. The side effects illustrated by all cell systems of the body disguise the possible benefits from GH surplus (Fig. 3).

Referencess Abed, R. T., Clark, J., Elbadawy, M. H., and Cliffe, M. J. (1987). Psychiatric morbidity in acromegaly. Acta Psychiatr. Scand. 75, 635–639. Annerén, G., Tuvemo, T., and Gustafsson, J. (2000). Growth hormone therapy in young children with Down syndrome and clinical comparison of Down and Prader-Willi syndromes. Growth Horm. IGF Res. Suppl. B, S87–S91.

Barkan, A. L. (1989). Acromegaly: Diagnose and treatment. Endocrinol. Metab. Clin. North Am. 18, 277–310. Biermasz, N. R., van Thiel, S. W., Pereira, A. M., et al. (2004). Decreased quality of life in patients with acromegaly despite long-term cure of growth hormone excess. J. Clin. Endocrinol. Metab. 89, 5369–5376. Chowen, J. A., Frago, L. M., and Argente, J. (2004). The regulation of GH secretion by sex steroids. Eur. J. Endocrinol. 151, Suppl. 3:U95–U100. Review. Evans, H. M., and Long, J. A. (1921). The effects of the anterior lobe administrated intraperitoneally upon growth, maturation, and oestrous cycle in the rat. Anat. Rec. 21, 62–63. Ezzat, S., Ezrin, C., Yamashita, S., and Melmed, S. (1993). Recurrent acromegaly resulting from ectopic growth hormone gene expression by a metastatic pancreatic tumor. Cancer 71, 66–70. Guistina, A., Barkan, A., Casanueva, F., Cavagnini, F., Frohman, F., Ho, K., Veldhuis, J., Wass, J., von Werder, K. and Melmed, S. (2000). Criteria for cure of acromegaly: A consensus statement. J. Clin. Endocrinol. Metab. 85, 526–529. Li, C. H., Dixon, J. S., and Liu, W. K. (1969). Human pituitary growth hormone. XIX. The primary structure of the hormone. Arch. Biochem. Biophys. 133, 70–91. Li, C. H., and Evans, H. M. (1944). The isolation of pituitary growth hormone. Science 99, 183–184. Ozata, M. M., Ozkardes, M. A., Beyhan, M. Z., Corakci, M. A., and Gundogan, M. M. (1997). Central and peripheral neural responses in acromegaly. Endocr. Pract. 3, 118–122. Pantanetti, P., Sonino, N., Arnaldi, G., and Boscaro, M. (2002). Self image and quality of life in acromegaly. Pitutitary 5, 17–19. Raben, M. S. (1957). Preparation of growth hormone from pituitaries of man and monkey. Science 125, 883–884. Roos, P., Fevold, H. R., and Gemzell, C. A. (1963). Preparation of human growth hormone by gel filtration. Biochim. Biophys. Acta 74, 525–531. Richert, S., Strauss, A., Fahlbusch, R., Oeckler, R., and von Werder, K. (1987). Psychopathologic symptoms and personalty traits in patients with florid acromegaly. Schweizer Arch. Neurol. Psychiatr. 138, 61–86. Rollo, C. D., Ko, C. V., Tyerman, J. G. A., and Kajiura, L. (2002). The growth hormone axis and cognition. Neuropeptides 36, 201–208. Ross, J. L., Feuillan, P., Kushner, H., Roeltgen, D., and Cutler, G. B., Jr. (1997). Absence of growth hormone effects on cognitive function in girls with Turner syndrome. J. Clin. Endocrinol. Metab. 82, 1814–1817. Sheppard, M. C. (2003). Primary medical therapy for acromegaly. Clin. Endocrinol. 58, 387–399. Trainer, P. J., Drake, W. M., Katznelson, L., Feda, P. U., Herman-Bonert, V., van der Lely, A. J., et al. (2000). Treatment of acromegaly with the growth hormone-receptor antagonist pegvisomant. N. Engl J. Med 342, 1171–1177. Tsolakis, A. V., Portela-Gomes, G. M., Stridsberg, M., Grimelius, L., Sundin, A., Eriksson, B. K., Öberg, K. E., and Janson, E. T. (2004). Malignant gastric ghrelinoma with hyperghrelinemia. Hygiea 112, 2. [Abstract] Van der Lely, A., Hutson, R. K., Trainer, P. J., Besser, G. M., Barkan, A. L., Katznelson, L., Klibanski, A., Herman-Bonert, V. et al. (2001). Longterm treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist. Arch. Intern. Med. 385, 1754–1759. Webb, S. M., Prieto, L., Bandia, X., et al. (2002). Acromegaly quality of life questionnaire (ACROQOL) a new health-related quality of life questionnaire for patients with acromegaly: Development and psychometric properties. Clin. Endocrinol. 57, 251–258.

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21 Human Aging and the Growth Hormone/Insulin-like Growth Factor-I Axis: The Impact of Growth Factors on Dementia E. ARVAT, R. GIORDANO, MICAELA PELLEGRINO, FABIO LANFRANCO, MATTEO BALDI, ANDREEA PICU, LORENZA BONELLI, and E. GHIGO Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, Italy

I. Introduction II. Age-Related Changes in Activity of the Growth Hormone (GH)/Insulin-like Growth Factor-I (IGF-I) Axis: Focus on Normal Aging III. GH/IGF-I Axis in Dementia: Focus on Alzheimer’s Disease IV. GH/IGF-I Axis in Down Syndrome: A Clinical Model of Anticipated Aging of the GH/IGF-I Axis V. Conclusions References

still a matter of debate. Although somatopause is likely to contribute to age-related changes in body composition, structure functions, and metabolism, we are now in front of the paradox of lifelong GH/IGF-I deficiency or resistance, resulting in prolonged life expectancy and GH replacement at advanced age, probably exerting antiaging effects. This evidence questions whether GH deficiency is a beneficial adaptation to aging. However, neuroendocrine studies provided evidence that brain aging is associated with peculiar age-related alterations in the control of the GH/IGF-I axis, mostly including growth hormone-releasing hormone (GHRH) deficiency and somatostatin hyperactivity, reflecting the age-related cholinergic impairment. This hormonal pattern is present in normal and demented elderly subjects and also in adults with Down syndrome; however, neuroendocrine distinction among these conditions is, at present, impossible.

This chapter focuses on the age-related changes in the activity of the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis as function of either normal or pathological brain aging. Particularly, the influence of the GH/IGF-I axis on cognitive functions and related disorders is considered. Given the well-known positive influence of GH/IGF-I on body composition, structure functions, and metabolism, potential clinical implications are discussed, taking into account evidence showing that, at least in animals, a deficiency in GH/IGF-I is associated with prolonged life. Studies regarding growth hormone deficiency (GHD) in adulthood definitely provided evidence that GH is more than simply a “growth hormone” and that it should more appropriately be renamed the somatotropic hormone. Its strong influence on body composition, metabolism, and structure functions, including central functions, is definitely demonstrated by how adult GHD patients benefit from rhGH replacement. Whether somatopause is simply physiology is

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION There is clear evidence that changes in endocrine functions in aging often reflect age-related impairment in the neuroendocrine control of pituitary function both in animals and in humans. The hypofunction of the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in elderly subjects is probably

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the most impressive example of decreased activity as a function of age-related changes in the neural control of somatotroph cells (Corpas et al., 1993; Muller et al. 1995; Ghigo et al. 1996; Arvat et al., 1999). GH secretion deeply varies; in fact, across the lifespan, its secretion is maximal at birth, clearly increases during puberty, and progressively decreases thereafter, showing a very low secretion in aging (Giustina and Veldhuis, 1998). Although mechanisms underlying the age-related variations of GH release include peripheral influences (i.e., gonadal steroids, adiposity), age-related changes in hypothalamic neuropeptides and neurotransmitters, mainly the neurohormones growth hormone-releasing hormone (GHRH) and somatostatin, seem to play a major role; they, in turn, are likely to reflect age-dependent changes in suprahypothalamic functions (Corpas et al., 1993; Muller et al., 1995; Ghigo et al., 1996; Arvat et al., 1999). The reduction in spontaneous and stimulated GH secretion in aging mostly depends on age-related changes in the neurotransmitter control leading to GHRH hypoactivity and absolute or relative somatostatin (SS) hyperactivity in the aged hypothalamus (Corpas et al., 1993; Muller et al., 1995; Ghigo et al., 1996; Arvat et al., 1999). In this context, the well-known cholinergic impairment in the aging brain involves hypothalamic pathways and contributes to the disrupted GHRH/somatostatin interplay, underlying the decreased function of the GH/IGF-I axis in normal as well as in demented elderly subjects or adults with Down syndrome (DS) (White et al. 1977; Wisniewski et al., 1978; Barthus et al., 1982; Rossor, 1982; Ghigo et al., 1993; Arvat et al., 1996). Also, age-related variations in the ghrelin system — a gastric hormone discovered as a natural GH secretagogue (GHS) and acting within the central nervous system (CNS) and the hypothalamus — could play a role in the decreased GH secretion that connotes aging (Kojima et al., 1999; Ghigo et al., 2001; van der Lely et al., 2004). It is well known that aging is associated with changes in body composition, metabolism, and structure functions, involving reduced lean mass, increased adiposity, decreased bone mass, and protein synthesis (Rudman et al., 1990; Corpas et al., 1993). It is impressive how these alterations are similar to those in young adults with GH deficiency (GHD) (de Boer et al., 1995; Toogod and Shalet, 1998). This evidence, together with demonstration of the agerelated declining activity of the GH/IGF-I axis, led to coin the neologism “somatopause”; it indicates the potential link between the age-related decline in GH and IGF-I levels and frailty in aging. The following hypothesis was that by restoring GH and IGF-I levels to young levels, it would be possible to counteract the age-related changes in body composition and metabolism as rhGH replacement is able to do in adult GHD (Rudman et al., 1990; Corpas et al., 1993; de Boer et al., 1995; Toogod and Shalet, 1998). However, GH and IGF-I also play remarkable central actions, e.g., on

brain plasticity and functions; in fact, changes in GH/IGF-I activity have been shown to be associated with cognitive and sleep disorders (Sartorio et al. 1996; van Cauter et al., 2000; Aleman et al., 2000; Compton et al., 2000; van Dam et al., 2000; Schneider et al., 2003). Following this line of reasoning, it was hypothesized that GH and/or IGF-I would also positively affect central functions associated with aging. As the age-related decline in the function of the GH/IGF-I axis reflects central alterations, it has been demonstrated that somatotroph function in aging could be restored more appropriately by GHRH and/or natural or synthetic GHS; these molecules, in turn, possess pleiotropic actions, including central activities, and this evidence added further complexity and opportunities of research. This chapter focuses on age-related changes in the activity of the GH/IGF-I axis as a function of either normal or pathological brain aging. The influence of the GH/IGF-I axis on cognitive functions and related disorders is also considered. Given the well-known positive influence of GH/IGF-I on body composition, structure functions, and metabolism, potential clinical implications are discussed, taking into account evidence showing that a deficiency in GH/IGF-I has been demonstrated to be associated with prolonged life.

II. AGE-RELATED CHANGES IN ACTIVITY OF THE GROWTH HORMONE (GH)/INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AXIS: FOCUS ON NORMAL AGING GH is secreted in pulses occurring approximately every 3 h with clear amplification during sleep, which, in turn, is affected by the GH/IGF-I axis. Sleep, physical exercise, and nutrition are age-related physiological parameters that importantly influence GH release (Ghigo et al., 1999; Giustina and Veldhuis, 1998). Both in animals and in humans, GH secretion undergoes clear age-related variations that are generally mirrored by IGF-I levels, the best marker of GH status, with the notable exception being at birth (Corpas et al., 1993; Chatelain et al., 1994; Ghigo et al., 1996; Arvat et al., 1999). Spontaneous pulsatile GH secretion is high in newborns, decreases in childhood, and maintains constant levels up to the onset of puberty, when it is clearly enhanced, particularly in term of pulse amplitude (Ghigo et al., 1999; Giustina and Veldhuis, 1998). Mean 24-h GH secretion then declines from puberty to adulthood, when it is more marked in women than in men (Ho et al., 1987). A further progressive fall in 24-h GH secretory rates occurs with aging (approximately 14% per decade); this is due to a decline in both daytime and nighttime GH secretory burst number and amplitude, particularly in women, so that no gender-related

21. Human Aging and the GH/IGF-I Axis

difference is present in aging (Zadik et al., 1985; Ho et al., 1987). The reduced GH levels observed in aging mostly reflect an age-related decrease in the GH production rate; in fact, while the daily GH production rate varies between 1.0 and 1.5 mg/day during puberty, elderly subjects can produce as little as 50 ␮g/day of GH, showing a hormonal secretion similar to that in hypopituitaric GH-deficient patients (de Boer et al., 1995; Veldhuis et al., 1995). IGF-I levels are low at birth despite GH hypersecretion, likely reflecting peripheral GH insensititivity and immaturity of the GH/IGF-I axis (Chatelain et al., 1994). Thereafter, from childhood to aging, at least in well-nourished subjects, IGF-I levels generally reflect the GH status, showing an increase at puberty and a progressive decline from puberty to ageing; in elderly subjects, IGF-I levels are often overlapping with those recorded in adult patients with severe GHD (Corpas et al., 1993; de Boer et al., 1995; Ghigo et al., 1996; Arvat et al., 1999). Interestingly, total IGF-I levels continue to decline progressively with advancing age, despite no further worsening of somatotroph insufficiency (Corpas et al., 1993; Arvat et al., 2000a). Nutritional alterations, which occur frequently in aged people, are associated with peripheral GH resistance and low IGF-I synthesis and could account for the progressive impairment of IGF-I levels (Thissen et al., 1994; Underwood et al., 1994; Ross and Chew, 1995; Arvat et al., 2000a). In fact, some peripheral GH resistance was suggested by a report in which the IGF-I response to rhGH in elderly subjects was found impaired (Lieberman et al., 1994). However, preserved IGF-I and IGFBP-3 responses to very low rhGH doses in the elderly were clearly demonstrated later on, ruling out the presence of GH resistance in aging, at least in well-nourished subjects (Arvat et al., 1998). In agreement with this assumption, free IGF-I levels have been reported even increased in the elderly (Janssen et al., 1998); the possibility that the reduced GH release in aging is due to exaggerated negative IGF-I feedback has, however, been ruled out (Chapman et al., 1997). Among peripheral factors influencing age-related changes in the GH/IGF-I axis are gonadal steroids and adiposity (Ho et al., 1987; Iranmanesh et al., 1991; Giustina and Veldhuis, 1998; Ghigo et al., 1999; Veldhuis et al., 1995). GH concentrations during 24 h are negatively correlated with body mass index and percentage of body fat, particularly abdominal visceral fat, which is known to increase in aging (Iranmanesh et al., 1991); in this context, an increased negative-free fatty acid (FFA) feedback action on somatotroph function has been hypothesized (Jorgensen et al., 1997). It is noteworthy, however, that age and body mass independently affect somatotroph function (Iranmanesh et al., 1991). Gonadal steroids have an important positive influence on GH secretion, playing a major role in the amplification of GH secretion at puberty. Either testosterone or estradiol has been shown able to modulate the GHRH/somatostatin interplay, exerting a positive influence on somatotroph

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function (Kerrigan and Rogol, 1992; Giustina and Veldhuis, 1998); however, testosterone positively affects whereas estradiol negatively affects IGF-I synthesis and secretion (Hernandez, 1995). Oral but not transdermal estrogen replacement augments GH pulsatility in postmenopausal women, likely reflecting the concomitant reduction of IGF-I levels (Bellantoni et al., 1996; Dawson-Hughens et al., 1986). However, testosterone augments 24-h pulsatile GH secretion and circulating IGF-I levels in elderly eugonadal men only at pharmacological doses (Gentili et al., 2002); some increase in IGF-I levels during testosterone treatment replacement has also been observed in hypopituitaric patients with severe GHD (Span et al., 2000). In all, despite gonadal steroids affecting the GH/IGF-I axis, there is no evidence supporting the hypothesis that the age-related decline in the function of the GH/IGF-I axis is dependent on the age-related change in the gonadal steroid milieu. As anticipated, the reduced function of the GH/IGF-I axis in aging mostly reflects age-related variations in the neural control of somatotroph secretion (Fig. 1). Most of the common stimuli of GH release, such as hypolgycemia, ␣2-adrenergic and cholinergic agonists, opioids, and galanin, showed a reduced stimulatory effect on GH in aged people (Corpas et al., 1993; Muller et al., 1995; Ghigo et al., 1996; Arvat et al., 1999). Even the GH responsiveness to GHRH has generally been reported reduced in the elderly, although about 30% of them still have a preserved GH response to the peptide (Corpas et al., 1993; Muller et al., 1995; Ghigo et al., 1996; Arvat et al., 1999). Nevertheless, it has clearly been demonstrated in both animals and humans that the pituitary GH releasable pool is not reduced with advancing age (Muller et al., 1995). However, hypothalamic GHRH synthesis and release are, at least relatively, reduced in the aged hypothalamus (Muller et al., 1995), and impairment of pituitary GHRH receptor and/or postreceptor mechanisms has also been reported (Ceda et al., 1986; Coiro et al., 1991). In agreement with an age-related impairment of GHRH release and/or action, prolonged treatment with GHRH has been shown able to restore GH pulsatility and to increase IGF-I levels to young levels, while theophylline, a phosphodiesterase inhibitor, restored the GH response to GHRH in aged subjects (Coiro et al., 1991; Corpas et al., 1992). Absolute or relative hyperactivity of hypothalamic somatostatinergic neurons in aging has also been shown and this, in turn, could lead to further intrahypothalamic suppression of GHRH-secreting neurons (Muller et al., 1995; Ghigo et al., 1996; Arvat et al., 1999). In agreement with the occurrence of a SS hypertone in aging, in elderly subjects, the GH response to GHRH is clearly enhanced by neuroactive substances acting via inhibition of hypothalamic somatostatin release and is even restored by arginine, an amino acid endowed with a strong GH-releasing effect mediated by SS inhibition (Ghigo et al., 1990a,

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YOUNG ELDERLY Acetylcholine Arginine Histamine Serotonin Opioids Dopamine Galanin α-adrenergic β-adrenergic

Acetylcholine Arginine Histamine Serotonin Opioids Dopamine Galanin α-adrenergic β-adrenergic

Galanin Dopamine α-adrenergic

SRIF

Galanin Dopamine α-adrenergic

GHRH SRIF

↓ GHRH

↓ GHRELIN

↓ (?) GHRELIN

GH

↓GH IGF-I gonadal steroids

↓ IGF-I

FFA

↓ gonadal steroids

Insulin

↑ FFA ↑ Insulin

FIGURE 1 Control of GH secretion as function of age.

1996; Giustina and Veldhuis, 1998). Thus, as also indicated by careful analysis of GH pulsatility in elderly subjects, concomitant GHRH hypoactivity and SS hyperactivity are likely to explain GH hyposecretion in aging. As anticipated, the age-related alterations in the activity of SS- and GHRHsecreting neurons, in turn, reflect age-related changes in the activity of neurotransmitter pathways (Fig. 1). Cholinergic pathways play a major stimulatory role in the control of somatotroph function via the inhibitory modulation of hypothalamic SS (Muller and Nisticò, 1989; Giustina and Veldhuis, 1998; Ghigo et al., 1999). Cholinesterase inhibitors stimulate GH release and potentiate the GH response to GHRH across the life span, whereas the opposite effect is exerted by muscarinic receptor blockers (Ghigo et al., 1999). However, in agreement with the well-known “cholinergic hypothesis of brain aging,” cholinergic agonists, such as pyridostigmine, increase but do not restore the GH response to GHRH in elderly subjects (Ghigo et al., 1990b, 1992; Giustina et al., 1992). Thus, hypothalamic cholinergic hypoactivity could, in turn, explain the SS hyperactivity leading to dampened GH secretion in aging. The catecholaminergic system regulates somatotropic function with dual action mediated by different receptors. The stimulatory effect of cathecolamines is mediated by ␣2-adrenergic receptor activation via a concomitant activation of GHRH- and inhibition of SS-secreting neurons (Muller and Nisticò, 1989; Giustina and Veldhuis, 1998; Ghigo et al., 1999). However, activation of the ␤-adrenergic receptor mediates the inhibitory effect of catecholamines via the

stimulation of hypothalamic SS release (Muller and Nisticò, 1989; Giustina and Veldhuis, 1998; Ghigo et al., 1999). There is evidence for reduced central catecholaminergic activity in brain aging; however, studies with adrenergic molecules in elderly subjects show controversial results. Clonidine has been reported to restore spontaneous and stimulated GH secretion in aged dogs (Cella et al., 1993), whereas somatotroph function in elderly humans is enhanced but not restored by clonidine treatment (Gil-Ad et al., 1984). Similarly, galanin, a neuropeptide that potentiates both basal and GHRH-stimulated GH secretion via concomitant activation of GHRH- and inhibition of SS-secreting neurons (Vrontakis et al., 1991) enhances but does not restore the GH response to GHRH in the elderly (Giustina et al., 1992); the same effect is shared by metenkephalin, a neuropeptide (Giusti et al., 1992), although the role of opioids in the control of somatotroph function is not definitively clarified. Interestingly, arginine, an amino acid, shares with cholinergic agonists the same effects on GH secretion, with the notable exception being aging (Ghigo et al., 1990a). Arginine is the nitric oxide (NO) precursor, and hypothalamic NO-secreting pathways where arginine serves to generate NO, a gaseous neurotransmitter, have been demonstrated; this would likely allow the consideration of the existence of central argininergic neurons (Grossman et al., 1997). Nevertheless, there is no definite evidence showing that arginine modulates GH secretion via NO-mediated mechanisms. Indirect evidence indicates that arginine acts via inhibition of hypothalamic SS. Like cholinergic agonists, arginine clearly increases both spontaneous and stimulated GH release, with a stimulatory effect

21. Human Aging and the GH/IGF-I Axis

higher in women than in men (Muller and Nisticò, 1989; Ghigo et al., 1999). Arginine strongly potentiates the GH response to GHRH across the life span (Ghigo et al., 1990a). Interestingly, at variance with cholinergic agonists, it fully restores the GH responsiveness to GHRH in elderly subjects, making it overlapping to that in young subjects and even in normally growing children (Ghigo et al., 1990a); this evidence indicates that the GH releasable pool is basically preserved in the aged pituitary and that SS hyperactivity is likely to have a major role in the age-related decline of somatotroph function. Age-related variations in the ghrelin system could also be involved in the pathogenesis of aging GH insufficiency. Ghrelin is a gastric hormone discovered as a natural ligand of the orphan GHS-R1a that, in turn, had been shown specific for synthetic GHS (Howard et al., 1996; Kojima et al., 1999; Ghigo et al., 2001; van der Lely et al., 2004). Ghrelin and synthetic GHS possess strong and dose-related GHreleasing effects both in humans and in animals by acting at the pituitary and mainly at the hypothalamic level via GHRH-secreting neurons (Kojima et al., 1999; Arvat et al., 2000b; Ghigo et al., 2001; van der Lely et al., 2004). The GH-releasing effect of ghrelin and GHS is gender independent but undergoes marked age-related variations increasing at puberty, persisting similar in adulthood, and decreasing with aging (Arvat et al., 2000c; Ghigo et al., 2001; Broglio et al., 2003). The reduced GH-releasing activity of ghrelin in aging probably reflects variations in GHRH and SS activity (Arvat et al., 2000c; Ghigo et al., 2001; van der Lely et al., 2004). However, the reduced GHS receptor number in the aged human hypothalamus suggested that an age-related decline in the ghrelin system could play a direct role in the age-related decline of GH (Ghigo et al., 2001; van der Lely et al., 2004). No clear evidence for an age-related decrease in ghrelin secretion has been demonstrated. Although it is at present questioned whether ghrelin really plays a major physiological role in the control of somatotroph function, treatment with ghrelin mimetics had been proposed as anabolic antiaging intervention in frail elderly subjects. Indeed, prolonged treatment with orally active, nonpeptidyl GHS such as MK-0677 has been shown able to restore spontaneous GH pulsatility and IGF-I levels in aged humans (Chapman et al., 1996); however, no definite evidence that elderly subjects really benefit by this treatment in term of anabolim has been provided yet (van der Lely et al., 2004). The strong similarity between signs and symptoms of adult GHD and age-related changes in body composition, structure functions, and metabolism, evidence that hypopituitaric patients with severe GHD benefit from GH replacement, and that elderly subjects are connoted by decreased activity of the GH/IGF-I axis suggested that they could benefit by restoring GH and IGF-I to young levels. This hypothesis led to clinical trials focusing on the effects of GH replacement and/or GH-releasing molecules aiming at

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“rejuvenating” the GH/IGF-I axis and meantime at becoming anabolic antiaging agents. The results of these trials were not as exciting as those in adult GHD. Basically, GH treatment was able to rejuvenate body composition and probably to protect muscle mass from the effects of acute diseases, allowing an earlier return to independent living after hip fractures (Rudman et al., 1990; Weissberger et al., 2003). However, no positive effect on muscle strength and bone mass has ever been clearly demonstrated (Rudman et al., 1990; Corpas et al., 1992; Toogod and Shalet, 1998; Blackman et al., 2002). Moreover, side effects have often been reported, probably reflecting overdosage and/or wide interindividual variations in GH sensitivity (Corpas et al., 1992; Toogod and Shalet, 1998; Blackman et al., 2002; Cummings and Merriam, 2003). In addition to the positive effects on body composition and metabolism, GH and IGF-I have been shown to exert relevant actions in the central nervous system, i.e., affecting sleep, cognitive functions, and neuronal cell survival (Sartorio et al., 1996; Aleman et al., 2000; Compton et al., 2000; van Cauter et al., 2000; van Dam et al., 2000; Schneider et al., 2003). This evidence implied the obvious hypothesis that GH replacement would also be effective in this context in pathological conditions of brain aging. As discussed in detail elsewhere in this book, GH and IGF-I act within the CNS at various levels in agreement with evidence that GH and IGF-I receptors are expressed in many brain areas, including the cerebral cortex, hippocampus, hypothalamus, and pituitary (Nyberg and Burman, 1996; Schneider et al., 2003). IGF-I and probably GH cross the blood–brain barrier (BBB), but it has to be recalled that these hormones are produced also within the CNS, suggesting that they could act via autocrine/paracrine mechanisms (Nyberg and Burman, 1996; Anlar et al., 1999; Coculescu, 1999). Both GH and IGF-I play a crucial role in the development and differentiation on the CNS and probably have distinct central actions reflecting distinct receptor distribution (Schneider et al., 2003). Particular emphasis has been given to the actions of the IGF system within the CNS where IGF-I exerts neurotrophic, neuroprotective, and metabolic effects, contributing to the negative feedback regulation of somatotroph function (Jones and Clemmons, 1995; Schneider et al., 2003). In addition to IGF-I and IGF receptors, IGF-binding proteins (IGFBPs), which are expressed in the CNS, play a relevant role by either modulating IGF-I activity or exerting direct actions (Jones and Clemmons, 1995; Lackey et al., 2002). Some central IGF I actions represent the rational basis to consider its potential usefulness in aging brain and neurodegenerative disorders; in fact, IGF-I (i) promotes neuronal myelinization; (ii) inhibits neuronal apoptosis; (iii) increases the activity of several neurotransmitters and their receptors, namely cholinergic pathways; and (iv) improves cerebral glucose metabolism (Jones and Clemmons, 1995;

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Lackey et al., 2002; Gasparini and Xu, 2003; Schneider et al., 2003). Experimental studies demonstrated that IGF-I and GH itself have a strong neuroprotective effect against degenerative, traumatic, and ischemic damages (Tsitouras et al., 1995; Nyberg, 2000; Liu et al., 2001a; Scheepens et al., 2001). In fact, GH-induced improvement of the CNS microvasculature contributing to the neuroprotective effect of GH would be IGF mediated (Liu et al., 2001a). Another important aspect to be considered is that within the CNS, IGF-I production, as well as either IGF-I or GH receptor density, declines with advancing age (Lai et al., 1993; Nyberg, 1997; Schneider et al., 2003). Several studies focused on the relationship between somatotroph function and parameters of CNS function in aging. GH secretion is associated with sleep rhythm and most GH is released during the slow wave sleep (SWS) phase (van Cauter et al., 1998). It is well recognized that both SWS and GH secretion decline with advancing age (van Cauter et al., 2000); moreover, GH and GH secretagogues positively affect sleep in aging, while neurotransmitters that rejuvenate sleep restore GH secretion in the elderly (Copinschi et al., 1997; Frieboes et al., 1997; Guldner et al., 1997; van Cauter et al., 2000). However, many neurotransmitters and neurohomones known to strongly influence GH secretion also affect the sleep pattern, such as GHRH, SS, ghrelin, cholinergic, and adrenergic pathways (Frieboes et al., 1997; Guldner et al., 1997; Steiger et al., 1998; van der lely et al., 2004; Steriade, 2004); all these factors clearly change during the life span, suggesting a common impairment in the neural control of both sleep and somatotroph function in aging. Whether there is a link between the decline in somatotroph activity and cognitive function during normal aging is still matter of debate. Both GH and IGF-I receptors have been reported decreased as a function of age in brain areas playing a critical role in cognitive processes, such as the hippocampus (Squire, 1992; Lai et al., 1993; Nyberg and Burman, 1996; Schneider et al., 2003). Some studies reported a positive correlation between total IGF-I levels or IGF/IGFBP3 ratio and cognitive function in elderly individuals (Morley et al., 1997; Aleman et al., 2000; Kalmijn et al., 2000), although these results have not been confirmed by others (Papadakis et al., 1995; Paolisso et al., 1997; Rollero et al., 1998). Interestingly, IGF but not GH levels correlate with cognitive paramenters in elderly subjects (Aleman et al., 2000). At present there is no evidence that treatments with rhGH or rhIGF-I improve cognitive parameters, memory, or mood in normal elderly subjects (Papadakis et al., 1996; Friedlander et al., 2001). These results are in contrast to those in young adult GHD patients in whom a positive effect of GH replacement therapy on cognitive function and well-being has been observed (de Boer et al., 1995; Sartorio et al., 1996; Cummings and Merriam, 2003). The mechanisms underlying the hyposomatotropinism in GHD and normal aging are different, which could explain the different impact of GH replacement on

cognitive function in elderly and adult GHD. In fact, aging is connoted not only by a decline in GH and IGF-I levels, but also in GH-binding sites that are reduced in many central areas, particularly in those devoted to the control of cognitive functions; the decline in central GH receptors is, therefore, likely to influence the effects of central rhGH actions. Moreover, many other hormonal changes occur during aging, e.g., reduction in (dehydroepiandrosterone (DHEA)/ dehydroepiandrosterone sulphate (DHEAS)) and gonadal steroids coupled with an increase in HPA activity (Orentreich et al., 1984; Seeman and Robbins, 1994; Baulieu, 2002); these hormones possess well-recognized neurotropic actions and contribute, in addition to the GH decline, to age-related cognitive dysfunctions. Although somatopause contributes to age-related changes in body composition, structure functions, and metabolism that connote the “frailty” in elderly subjects, information about the impact of GH and IGF-I axis on life expectancy is contradictory. In fact, we are now in front of the paradox of lifelong GH/IGF-I deficiency or resistance resulting in prolonged life expectancy and GH replacement at advanced age probably exerting antiaging effects; this questions whether GH deficiency is a beneficial adaptation to aging. That reduced GH and IGF-I levels or actions are associated with significant increases in both average and maximal life span have been clearly demonstrated in several animal models (Bartke, 2003). Mice homozygous for targeted disruption of the gene encoding the GH receptor/GH-binding protein are (Ghr-KO) GH resistant, as demonstrated by a profound suppression of IGF-I expression, low IGF-I levels, and decreased somatic growth and adult body size; these animals live 40–50% longer and show no decline of cognitive functions in comparison to normal siblings (Zhou et al., 1997; Coshigano et al., 2000; Kinney et al., 2001a). Hypopituitaric dwarf mice with primary GHD exhibit a major extension of their life span associated with reduced immune, collagen, and cognitive aging (Brown-Borg et al., 1996; Flurkey et al., 2001; Kinney et al., 2001b). Similarly, mice with GHRH receptor gene deletion and severe GHD live longer (Flurkey et al., 2001). Again, a significant increase in longevity is present in female mice with heterozygous KO of the gene encoding the IGF-I receptor (Holzenberger et al., 2003). This evidence fit well with studies of the genetics of aging in Drosophila showing that many mutations suppressing IGF–insulin signaling prolong life (Tissenbaum and Ruvkun, 1998; Guarente and Kenyon, 2000). However, in GH antisense transgenic rats, longevity is extended in heterozygotes that have a moderate reduction in IGF-I levels but is shortened in homozygotes showing marked suppression of IGF-I synthesis and secretion (Shimokawa et al., 2002). A negative correlation between adult body size and life span has been demonstrated in animals and often in humans (Li et al., 1996; Patronek et al., 1997; Weindruch

21. Human Aging and the GH/IGF-I Axis

and Sohal, 1997; Sonntag et al., 1999; Roth et al., 2002; Samaras and Elrick, 2002; Heilbronn and Ravussin, 2003); as, at least in rodents, caloric restriction is associated with a clear reduction in GH and IGF-I secretion, it has been hypothesized that caloric restriction-induced hypoactivity of the GH/IGF-I axis has a critical role in increasing longevity. Putative mechanisms linking reduced IGF-I with delayed aging and prolonged longevity in animal models probably include reduced insulin release and/or enhanced insulin sensitivity. In fact, decreased insulin sensitivity facilitates neuronal damage and accelerates the aging process. Moreover, reduced glucose utilization impairs repair processes in the brain, which are normalized by the improvement in glucose metabolism (Blum-Degen et al., 1995; Meier-Ruge and Bertoni-Freddari, 1996; Parr, 1997; Munch et al., 1998). The association of reduction of GH/IGF-I activity, improvement of insulin signaling, and prolonged longevity in animal models seems, however, in contrast with human findings showing that the reduced activity of the GH/IGF-I axis in normal aging and in adult GHD is associated with hyperinsulinism and insulin resistance (Rudman et al., 1990; de Boer et al., 1995; Toogod and Shalet, 1998). Moreover, both severe GH deficiency and GH excess have been found associated with reduced life expectancy in humans, although these alterations in life span could simply be the result of an increased risk of cardiovascular disease, diabetes, or cancer, instead of acceleration of the aging process (Colao et al., 2004; de Boer et al., 1995). All in all, there is a discrepancy between the potential role of GH and/or IGF-I on life expectancy and the quality of life. As suggested by Bartke (2003), it should be realized that even if GH in aging has no effects or a negative influence on life expectancy, producing objective improvements in body composition, metabolism, peripheral and central functions, well-being, and quality of life is a very reasonable clinical objective that is almost certain to be requested by most aged individuals.

III. GH/IGF-I AXIS IN DEMENTIA: FOCUS ON ALZHEIMER’S DISEASE Neurotrophic factors regulate neuronal growth and survival in both central and peripheral nervous system (see other chapters in this book and Section II). As anticipated, the GH/IGF-I system exerts neurotrophic and neuroprotective actions against degenerative and ischemic damages in the CNS, which are the most important alterations responsible for cognitive, mood, and health impairment in aging. Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a progressive neurodegenerative disorder characterized by the insidious onset of cognitive

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impairment and a decline in memory, reason, and language, leading to inexorable progression of impaired self-sufficiency (Cummings, 2004). Brains of AD patients show extensive atrophy due to neuronal loss and accumulation of neurofibrillary tangles and neuritic plaques surrounded by neuroinflammation areas. Neuritic plaques consist of deposits of ␤-amyloid, the increase of which in the CNS is believed to have a crucial role in the pathogenesis of AD (Lackey et al., 2002; Gasparini and Xu, 2003). By altering glucose metabolism and mitochondrial functions, oxidative stress and free radicals have been shown to contribute to ␤-amyloid production and to the accumulation of senile plaques (Blum-Degen et al., 1995; Williams, 1995; Meier-Ruge and Bertoni-Freddari, 1996; Clutton, 1997; Sayre et al., 1997; Green and Reed, 1998; Munch et al., 1998). All of these morphological alterations are responsible for the impairment of neural pathways in several cerebral areas, including the cerebral cortex and limbic area, which play a crucial role in the modulation of cognitive function and mood. Indeed, a dramatic impairment of cholinergic activity, particularly in the cortical and hippocampal areas, occurs in AD, being much more pronounced that in normal brain aging (Perry et al., 1978; Barthus et al., 1982; Rossor, 1982). Increasing evidence points to a crucial role of the IGF system in the CNS as a protective factor against neurodegenerative processes, including those observed in AD (D’Ercole et al., 1996; Torres-Aleman, 2000; Lackey et al., 2002; Gasparini and Xu, 2003; Schneider et al., 2003). Apoptotic mechanisms seem to contribute to the neuronal alterations that connote AD, and ␤-amyloid induces apoptotic-related changes in cortical and hippocampal neurons (Barinaga, 1998; Mattson et al., 1998; Lackey et al., 2002; Gasparini and Xu, 2003). Because IGF-I is a potent antiapoptotic factor, this is probably one of the mechanisms underlying its positive effects on the neurodegenerative alterations of AD (Lackey et al., 2000; Gasparini and Xu, 2003). IGF-I has also been reported to specifically protect hippocampal neurons from ␤-amyloid-induced neurotoxicity, which is probably due to its ability to influence ␤-amyloid metabolism and clearance (Dorè et al., 1997; Carro et al., 2002; Lackey et al., 2002; Gasparini and Xu, 2003). Moreover, interactions between the IGF system and neurotransmitter pathways affected in AD have been demonstrated: the IGF-Iinduced increase in enzyme activities increases cholinergic, catecholaminergic, and dopaminergic functions (D’Ercole et al., 1996; Hwang and Choi, 1996; Dorè et al., 1997; Lackey et al., 2002). IGF-I has a positive effect on neuronal survival and acethylcholine release in hippocampal and cortical cholinergic neurons, areas deeply affected in AD (D’Ercole et al., 1996; Dorè et al., 1997). More recently, interaction of the IGF system with insulin and glucose metabolism within the CNS received much

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attention considering the hypothesis that central metabolic alterations may accelerate neurodegenerative processes, facilitating the processes leading to AD (Blum-Degen et al., 1995; Meier-Ruge and Bertoni-Freddari, 1996; Finch and Cohen, 1997; Munch et al., 1998; Ott et al., 1999; Lackey et al., 2002; Schneider et al., 2003). Finally, a central anti-inflammatory effect of IGF-I, mediated by the antagonism of the deleterious effects of TNF-␣, would be another mechanism explaining the IGF-I protecting action against AD cerebral alterations (Cacabelos, 1994; Pratico and Trojanowski, 2000; Gasparini and Xu, 2003). These data point toward a strict link between cerebral degenerative alterations of AD and cognitive and neurohormonal impairment, including the GH/IGF-I axis. Increasing evidence indicates that other neurohormones and growth factors, in addition to IGF-I, act successfully as neuroprotective factors in many neuropathological conditions (Lackey et al., 2002; Gasparini and Xu, 2003; Schneider et al., 2003). Based on this evidence and taking into account the agerelated derangement in the neuroregulation of somatotrope function leading to a decline in GH and IGF-I levels, several authors focused on the relationships among neurodegenerative alterations, cognitive impairment, and hormonal functions in AD, with particular attention to the GH/IGF-I axis. Like normal aging, AD is connoted by a GH hyposecretory state. However, studies comparing GH and IGF-I secretion in AD with that in normal elderly subjects provided conflicting results. Basal GH levels have been shown increased or unchanged in AD patients compared to age-matched controls (Steardo et al., 1984; Franceschi et al., 1988; Heuser et al., 1992; Ghigo et al., 1993; Tham et al., 1993). Regarding the IGF system, some studies reported IGF-I levels in AD lower than in young subjects but higher than in aged controls (Sara et al., 1982; Christie et al., 1987; Ghigo et al., 1993; Tham et al., 1993), whereas others showed IGF-I and IGFBP3 levels

in AD even lower than in age-matched controls (Mustafa et al., 1999; Murialdo et al., 2001). Slight age and/or nutritional differences between AD and normal aged subjects have been hypothesized to account for the controversial data so far reported. Murialdo et al., (2001) also showed higher IGFBP1 levels in AD than in normal aged subjects, associated with very low DHEAS levels; as both hormones modulate insulin signaling, a more pronounced impairment of insulin sensitivity and glucose metabolism in AD has been hypothesized as a factor contributing to worsening central functions. The GH responsiveness to provocative stimuli has also been evaluated in order to disclose potential neuroendocrine markers of AD possibly correlated to clinical parameters (Fig. 2). The GH response to GHRH has been reported similar, increased, or reduced in AD with respect to normal elderly subjects (Cacabelos et al., 1988; Nemeroff et al., 1989; Lesch et al., 1990; Murialdo et al., 1990; Heuser et al., 1992; Lamperti et al., 1992; Ghigo et al., 1993; Murialdo et al., 1993; Gomez et al., 1994). The GHRH-induced GH response has been found more marked in patients with early onset AD by some but not by others (Cacabelos et al., 1988; Heuser et al., 1992; Gomez et al., 1994). Again, a delayed GH response was observed by some but not by others (Nemeroff et al., 1989; Murialdo et al., 1990; Ghigo et al., 1993). By administering other neuroactive drugs able to stimulate GH secretion, such as clonidine, pyridostigmine, and apomorphine, other studies generally confirmed that AD shows a GH response as reduced as that in normal elderly (Lal et al., 1989; Murialdo et al., 1990; Heuser et al., 1992; Lamperti et al., 1992; Ghigo et al., 1993; Murialdo et al., 1993; Obermayr et al., 2003). In agreement with the well-known central cholinergic impairment in AD brain (Davies and Maloney, 1976; Perry et al., 1978; Barthus et al., 1982; Rossor, 1982;

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FIGURE 2 GH responses (mean ⫾ SEM) to various stimuli [GHRH ( ), PD ⫹ GHRH (▲), or ARG ⫹ GHRH (●)] in young, elderly, and Alzheimer’s disease (AD) subjects.

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Whitehouse et al., 1982), it was considered of major importance to study the effects of cholinergic agonists, such as pyridostigmine or rivastigmine, on the GH response to GHRH. Indeed, cholinergic agonists increase both spontaneous and GHRH-induced GH release in AD patients (Ghigo et al., 1993; Murialdo et al., 1990, 1993; Obermayr et al., 2003); however, the potentiating action of cholinergic agonists on the somatotroph response to GHRH was once again similar to that in normal elderly, although lower than in normal young subjects (Ghigo et al., 1990, 1993). These findings, therefore, confirmed the presence of neuronedocrine cholinergic derangement in the aging brain but demonstrated that, at least at the hypothalamic level, cholinergic pathways are not more impaired in AD than in normal elderly. This, in turn, implies that the age-dependent impairment of hypothalamic cholinergic activity is likely to lead to absolute or relative SS hyperactivity in brain aging independently of the presence of AD. In fact, AD patients, like normal elderly, show normal somatotroph responsiveness to GHRH combined with arginine (Ghigo et al., 1993). As arginine is likely to stimulate GH by inhibiting hypothalamic SS release, these findings further support the existence of hyperactivity of hypothalamic somatostatinergic neurons, probably reflecting peculiar impairment of the cholinergic control of hypothalamic somatostatin release in AD. Actually, this hypothesis disagrees with evidence that both SS immunoreactivity and receptors show peculiar reduction in many brain areas of AD (Davies et al., 1980; Rossor et al., 1980; Beal et al., 1985; Candy et al., 1985; Nemeroff et al., 1989). However, that the activity of tuberoinfundibular somatostatinergic neurons within the hypothalamus is indistinguishable by that in normal elderly subjects is also shown by evidence that the GH-inhibitory effect of atropine and pirenzepine, two cholinergic antagonists acting via the stimulation of hypothalamic SS release, is preserved in AD as well as in normal elderly (Murialdo et al., 1990; Lamperti et al., 1992). In all, the main message coming from neuroendocrine studies in AD is that it is impossible to differentiate patients with AD from normal elderly subjects. It might be that the experimental approaches used to test the neuroendocrine functions, namely neuroendocrine control of the GH/IGF-I axis, were not sensitive enough. However, at present, there is no evidence supporting the hypothesis that some peculiar hystopathological central alterations in AD are associated with peculiar neuroendocrine impairment. This assumption implies that treatments with GH or GH secretagogues in AD would be considered simply on the basis that these patients, likely normal elderly subjects, would benefit by restoring activity of the GH/IGF-I axis at a younger level; that is, at present, not demonstrated yet. However, given the relationship between brain alterations in AD and IGF-I/insulin signaling, the possibility exists that both peptides and/or their analogues could become

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candidates for drug therapy in neurodegenerative disorders. Some experimental studies suggest that small molecules insulin mimetics with better penetration into CNS as well as IGF-I analogues with very high affinity for IGFBPs, able to increase “free” IGF-I levels, might represent therapeutic tools for the treatment of neurodegenerative disorders, including AD (Loddick et al., 1998; Air et al., 2002)

IV. GH/IGF-I AXIS IN DOWN SYNDROME: A CLINICAL MODEL OF ANTICIPATED AGING OF THE GH/IGF/I AXIS Down syndrome (DS) is the most common genetic cause of mental and linear growth retardation. Short stature is, in fact, a classical somatic feature of DS patients, which has been reported, at least partially, responsive to treatment with exogenous rhGH (Anneren et al., 1993; Castels et al., 1993; Torrado and Castells, 1993). However, DS is a classical example of precocious brain aging. In fact, brains of adult DS patients share many neural abnormalities with normal aging and even with dementia (Wisniewski et al., 1978; Williams and Matthysse, 1986; Seidl et al., 2001). Cholinergic dysfunction and impairment of cognitive functions, such as learning or memory, as well as sleep disturbances, are common in the three conditions (Wisniewski et al., 1978; Yates et al., 1980; Barthus et al., 1982; Rossor, 1982; Williams and Matthysse, 1986). Moreover, hypothalamic alterations in DS have been reported, including a neuronal loss in arcuate and ventromedial nuclei, brain areas involved in the neural control of GH secretion (Wisniewski and Bobinski, 1991). Attention has been paid to the role of GH and IGF-I in DS; in fact, the GH/IGF-I axis is essential not only for body growth, but also for development and maintenance of the nervous system (Sara et al., 1981; van Cauter et al., 1998; Aleman et al., 2000; Compton et al., 2000; van Dam et al., 2000; Schneider et al., 2003). Fetal IGFs affect brain development, and soon after birth there is switch from fetal to adult forms of IGFs (Sara et al., 1981). Some authors reported a reduction of IGF-I levels in DS patients to an extent similar to that in GHD patients (Sara et al., 1983; Castells et al., 1992; Torrado and Castells, 1993; Barreca et al., 1994). Lack of an IGF-I increase in late childhood and puberty with persistently low IGF-I levels across the life span has been observed (Sara et al., 1983). It has been hypothesized that an impairment in GH secretion and/or activity could explain IGF-I insufficiency in DS (Castells et al., 1992; Barreca et al., 1994). Both hepatic and cerebral IGF-I receptors seem preserved in DS that, in fact, show normal IGF-I sensivity to exogenous GH administration as well as normal GH-binding

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proteins (Sara et al., 1984; Anneren et al., 1990; Barreca et al., 1994). Barreca et al., (1994) showed that IGF-I levels are, however, within the normal range in a consistent percentage of DS patients. In our hands, normal and DS pubertal children had similar circulating total IGF-I levels; IGF-I levels showed a progressive decrease thereafter in adulthood when DS patients showed IGF-I levels always within the age-related normal range (Arvat et al., 1996). Nutritional, lifestyle, and/or social differences may explain these controversial results. Data concerning spontaneous and stimulated GH secretion in DS are controversial, as both reduced and normal somatotroph responsiveness to several stimuli have been reported (Fig. 3). A low GH response to L-DOPA and clonidine, two indirect GH-releasing agents, has been reported in prepubertal DS patients, although a normal arginine- and GHRH-induced GH rise was reported in DS children (Castells et al., 1992; Pueschel, 1993; Barreca et al., 1994; Arvat et al., 1996; Castells et al., 1996; Beccaria et al., 1998). A normal GH response in DS children was also observed after maximal provocative stimuli, such as GHRH combined with pyridostigmine or arginine, as well as after hexarelin, a synthetic GH secretagogue mimicking ghrelin action (Arvat et al., 1996; Ragusa et al., 1996; Beccaria et al., 1998). Treatment with rhGH is generally not allowed in patients with DS; a notable exception is DS patients in whom concomitant severe GHD is demonstrated. However, adult DS patients show a reduction of the GH response to GHRH to an extent similar to that in normal elderly subjects and in AD patients (Ghigo et al., 1993; Arvat et al., 1996). As anticipated, precocious brain aging has been shown in DS, likely including anticipated impairment in the neural

pathways controlling GH release, namely cholinergic neurons (Wisiniewski et al., 1978; Yates et al., 1980; Williams et al., 1986). In agreement with this hypothesis, in adult DS patients the reduced GH response to GHRH is enhanced but not restored by pyridostigmine, an indirect cholinergic agonist inhibiting cholinesterase (Arvat et al., 1996); this evidence is reminiscent of that observed in both normal and demented elderly subjects (Ghigo et al., 1993). Once again, differently from pyridostigmine, arginine, that like pyridostigmine probably inhibits hypothalamic SS release, is able to completely restore the reduced GH response to GHRH in adult DS patients, making this similar to that in young adults as well as in normal children (Beccaria et al., 1998). These data demonstrate that, like normal and demented elderly subjects, adult patients with DS have a preserved pituitary-releasable GH pool across their life span, as well as precocious derangement of neural pathways, i.e., cholinergic and likely somatostatinergic neurons. The early brain aging in DS is therefore likely to involve alterations in hypothalamic cholinergic pathways that, in turn, probably induce SS hyperactivity; this could explain the overlap among the neuroendocrine behavior in DS, in AD, and in normal elderly subjects (Ghigo et al., 1993; Arvat et al., 1996; Beccaria et al., 1998). The early neuroendocrine aging in DS disclosed by neuropharmacological tests is fascinating from the pathophysiological point of view indicating that the GH/IGF-I axis in adult DS is already superimposable to that in normal and demented elderly subjects. Whether there is any way to generate antibrain-aging drug interventions and/or if it is useful treating adult DS patients with GH, GHRH, and/or GH secretagogues remains to be demonstrated.

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FIGURE 3 GH responses (mean ⫾ SEM) to various stimuli [GHRH ( ), PD ⫹ GHRH (▲), or ARG ⫹ GHRH (●)] in young, elderly, and Down syndrome (DS) subjects.

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21. Human Aging and the GH/IGF-I Axis

V. CONCLUSIONS Differently from the activity of the GH/IGF-I axis, basic and clinical research in the GH field does not seem aging. Studies about severe GHD in adulthood definitely provided evidence that GH is more than simply a “growth hormone” and that it should be renamed more appropriately as somatotropic hormone. Its strong influence on body composition, metabolism, and structure functions, including central functions such as sleep and cognition, is definitely demonstrated by how adult GHD patients benefit from rhGH replacement. Whether somatopause is simply physiology is still a matter of debate. Although somatopause is likely to contribute to the age-related changes connoting the “frailty” in elderly subjects, we are now in front of the paradox of lifelong GH/IGF-I deficiency or resistance resulting in prolonged life expectancy and GH replacement at advanced age probably exerting antiaging effects. This evidence questions whether GH deficiency is a beneficial adaptation to aging. By answering this question, one is not simply finding a new philosophical paradigm, but also the rational basis for antiaging drug interventions. Neuroendocrine studies provided evidence that brain aging is associated with peculiar age-related alterations in control of the GH/IGF-I axis, mostly including GHRH deficiency and absolute or relative somatostatin hyperactivity, which, at least partially, reflect the age-related cholinergic impairment. This picture is clear in both normal and demented elderly subjects and also in adults with Down syndrome; neuroendocrine distinction among these conditions is, however, impossible. Whether the present neuroendocrine knowledge is simply descriptive or antineuroendocrine-aging interventions could provide benefit to human beings is, at present, unknown.

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22 Cognitive Status of Adult Growth Hormone (GH)-Deficient Patients and GH-Induced Neuropsychological Changes JAN BEREND DEIJEN* and LUCIA. I. ARWERT † *

Department of Clinical Neuropsychology, Vrije Universiteit, van der Boechorststraat 1, 1081 BT Amsterdam, The Netherlands † Department of Endocrinology, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

I. Introduction II. Determination of the Role of Growth Hormone (GH) in Cognitive Functions III. Cognitive Effects of GH-Induced Hormonal Changes IV. Cognitive Status in Relation to GH Deficiency (GHD) and GH Treatment V. Meta-Analysis GH and Neuropsychological Functions VI. Cognitive Effects of Discontinued GH Treatment VII. GH Dosages and Cognitive Status in GH-Deficient Adults VIII. GH and Insulin-like Growth Factor-I Replacement in Syndromes Other than GHD IX. GH-Induced Cognitive Changes in Healthy Elderly Subjects X. Conclusion References

behind the relation between GH and cognitive functioning are not fully understood. There are indications that GH can cross the blood–brain barrier, whereas the choroid plexus, hypothalamus, putamen, and thalamus have been shown to possess binding sites for GH and IGF-I. Moreover, GH treatment has been found to reduce the concentration of homovanillic acid, a dopamine metabolite, in cerebrospinal fluid. As the hippocampus is known to contain high levels of dopamine, this structure may be particularly affected by GH treatment. Involvement of the aforementioned brain areas in cognitive functions might explain the connection between GH treatment and neuropsychological functions. There is conflicting evidence concerning the extent to which the somatotropic axis is involved in cognitive processes. However, evidence favors the notion that GH-deficient patients exhibit subnormal cognitive functioning but that this can be improved by at least 1 year of GH-therapy.

Adult patients with growth hormone deficiency (GHD) show a reduced sense of psychological well-being and are frequently found to have impaired cognitive functioning. It is also known that mood and cognitive functioning are positively related to the level of insulin-like growth factor-I (IGF-I), a serum marker for GH status. More specifically, IGF-I levels appear to be related to cognitive status, shortterm memory performance, and cognitive flexibility. GH substitution in GH-deficient patients appears to improve cognitive functioning, as assessed by tasks measuring attention, memory, and perceptual motor skill. The mechanisms

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION Hormones may alter brain function by affecting early brain development in utero, which is described as an “organizational process.” These are distinct from “activation processes,” which are characterized by temporarily changed peripheral and neural processes. Organizational effects are considered to permanently alter brain development, whereas activation processes temporarily affect specific behavior.

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Hormones’ activation effects may be partly dependent on organizational effects. Thus, aggressive behavior in boys may result from the temporal action of testosterone, which is superimposed on that hormone’s permanent effect on early brain development in boys (Buchanan et al., 1992). The distinction between a hormone’s permanent and temporal effects on brain function is important when considering the cognitive effects of growth hormone (GH) replacement in growth hormone-deficient (GHD) patients. In this context, it is important to be aware that GHD patients form a heterogeneous group. Further diagnosis can place them into one of a number of subgroups. In the first group, GH deficiency may be present from birth, whereas in others it may commence in early childhood (childhood-onset GHD, CO-GHD). Alternatively, pituitary function may only decline later in life (adult-onset GHD, AO-GHD). GH deficiency in AO-GHD patients is frequently caused by a pituitary tumor. Mental performance in these patients may be impaired either by the tumor itself or by the associated treatment (surgery and /or radiotherapy). In the second group, a distinction is made regarding the extent of pituitary failure. One diagnosis is isolated GHD (IGHD), in which only GH secretion is insufficient. Another is multiple pituitary hormone deficiency (MPHD), which involves GHD in addition to the impaired secretion of other pituitary hormones (i.e., Adrenocorticotropic Hormone (ACTH), Thyroid Stimulating Hormone (TSH), gonadotropins). IGHD is usually present from childhood and may thus be characterized as a chronic disease. Each of these subgroups may exhibit a different psychological profile, as the cognitive functioning of these patients can be related to a variety of factors. One such factor is subnormal brain development as a consequence of GHD in childhood. Other possibilities include a GH-specific disturbance in neural cell metabolism related to the low activity of the somatotropic axis or brain damage caused either by a tumor or by the associated surgical treatment and/or radiotherapy. Finally, inadequate replacement with thyroxin, adrenal steroids, or sex steroids may adversely affect psychological functions. Cognitive impairment may therefore be caused by various combinations of factors, each with its own implications for the efficacy of treatment. For instance, if cognitive impairment in CO-GHD patients appeared to be the result of disrupted brain development, there would seem to be little point in administering GH to normalize cognitive function. Indeed, abnormal cognitive functions have been observed in school-age children (ranging from 6 to 16 years of age) with congenital pituitary hormone deficiency. These children’s IQ scores, as determined by the Wechsler Intelligence Scale for Children (WISC-III) (mean 75), were all below the average value for the general population. As these individuals had been stabilized on a hormone replacement regimen, which included a daily injection of GH, the authors concluded that their subnormal IQs may have

resulted from abnormal brain development or from the impact of hypoglycemia or low thyroxin concentrations in early life (Brown et al., 2004). In such cases, fortunately, there are indications that subnormal IQ may be reversible. In the past few years, various studies in CO-GHD patients have shown that subnormal memory performance in these individuals can be normalized by GH treatment (Deijen et al., 1998; Stouthart et al., 2003). Accordingly, these observations suggest that cognitive impairments in GHD subjects are not related to subnormal brain development, but rather to reversible GH-specific disruption of neural cell metabolism. While much has been learned concerning the way in which GH and insulin-like growth factor-I (IGF-I) are related to cognitive functioning, the exact mechanisms underlying the relation between the GH/IGF-I axis and cognitive parameters are not yet fully understood. Memory impairments in GHD patients may be caused by a reversible disruption of neural cell metabolism (Deijen et al., 1998). This view is based on the observation that GH treatment changes the concentration of homovanillic acid (HVA) in the cerebrospinal fluid (CSF) of adult males (Burman et al., 1993, 1996; Johansson et al., 1995). High levels of dopamine are found in the hippocampus, a structure that plays an important part in learning and memory. A change in the availability of GH in the hippocampus may alter the dopamine turnover in this region, thereby influencing memory processes (Deijen et al., 1996). However, there are numerous mechanisms by which the effects of GH on cognitive functioning might be mediated. In addition to evidence that GH can cross the blood–brain barrier (Burman et al., 1996), it is known that binding sites for GH and IGF-I exist in the choroid plexus, hypothalamus, putamen, thalamus, and hippocampus (Adem et al., 1989; Nyberg, 2000). The number of GH receptors in these brain areas, which are involved in memory processes, declines with age (Lai et al., 1993). IGF-I and GH are also synthesized locally, in the brain (Han, 1995). Furthermore, IGF stimulates DNA and RNA synthesis, neurite formation, rates of protein synthesis, synaptogenesis, and neuronal repair. In addition, IGF potentiates acetylcholine release from the hippocampus, which also experiences a dramatic decline in IGF-I protein levels and receptor density as a result of aging (Cherrier et al., 2004). Finally, GH substitution in GH-deficient patients has been found to increase CSF levels of aspartate (Burman et al., 1996), an excitatory amino acid that is a ligand for the N-methyl-D-aspartate (NMDA) receptor. Activation of the NMDA receptor contributes to long-term potentiation of synaptic efficacy in the hippocampus, which is considered to be an inherent mechanism of memory consolidation in the mammalian brain (Gardner, 1995). Moreover, it has been shown that activation of NMDA receptors in the basal forebrain modifies attentional functions (Turchi and Sarter, 2001). Post-GH treatment attentional improvements may therefore be mediated by GH-induced

22. Cognitive Functioning and GH

activation of NMDA receptors in the basal forebrain. The aforementioned observations provide a biological context, which may explain how the GH–IGF-I axis is involved in cognitive functioning and how GH replacement may normalize intellectual functions in GHD patients.

II. DETERMINATION OF THE ROLE OF GROWTH HORMONE (GH) IN COGNITIVE FUNCTIONS Attempts to assess cognitive functions in GHD adults encounter serious methodological problems, as does the determination of the role of GH treatment in reducing cognitive impairments. First, in studies of the neuropsychological effects of GH, tests used to assess cognitive functions display a lack of uniformity. Cognitive function can be seen as the intellectual or mental processes that are active when an organism acquires knowledge. It can be subdivided into attention, cognition, and memory (Deijen et al., 1993). Memory, in turn, can be subdivided into sensory, short term, and long term. Many different cognitive tests can be used to assess these aspects of cognitive functioning. Accordingly, the cognitive functions assessed in one study may not always be comparable to those assessed in another study. A second problem concerns the most appropriate patient group to study. The group should ideally consist of patients with IGHD, as the situation in these patients is not complicated by additional hormone deficiencies (Johnston, 1997). However, because IGHD usually commences in childhood, we are dealing here with a chronic disease. In contrast, GH deficiency in patients with adult-onset disease is frequently caused by a pituitary tumor. The tumor itself, with the associated surgical treatment and/or radiotherapy, may cause brain damage, anxiety, and stress, all of which affect cognitive functions. This is illustrated by the results of a study in which attention, memory, and executive functions were assessed in patients receiving treatment for pituitary tumors (Peace et al., 1998). The group consisted of patients who had undergone transfrontal surgery (n ⫽ 23), patients who had undergone transsphenoidal surgery (n ⫽ 23), and patients with pituitary tumors who were treated with medication alone (n ⫽ 23). The results were compared with those obtained from healthy controls (n ⫽ 23). It was concluded that all patients treated for a pituitary tumor suffered significant cognitive impairment. The nature and severity of the impairment differed from group to group. Furman and Ezzat (1998) described the personal experience of one of the authors, who suffered from a pituitary macroadenoma with accompanying acromegaly. This account included observations and descriptions of memory impairment, word loss difficulties, and diminished hand–eye coordination. While there was some postoperative improvement in short-term memory, a degree of impairment remained.

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In another study, 65 patients with pituitary tumors were subjected to a neuropsychological assessment. Three different groups were compared, one of which consisted of 38 patients with pituitary tumors who had undergone radiotherapy. A second group consisted of 27 patients with pituitary tumors who were not treated with radiotherapy. Twenty-one subjects with a chronic illness served as controls. Cognitive functioning was assessed using a variety of tests to measure executive functioning, verbal and visual memory, and premorbid intelligence (Grattan-Smith et al., 1992). Memory performance was shown to be impaired in both groups with pituitary tumors. There were no differences in performance between radiotherapy and nonradiotherapy groups in any of the neuropsychological tests. Both groups performed at a poorer level than controls with respect to executive functioning, verbal, and visual memory. However, no use was made of stimulation tests to assess pituitary functions, and there was only a brief reference to the fact that patients received thyroxin replacement or bromocriptine. As stated earlier, in addition to the adverse effects on cognition caused by a tumor, psychological functions in MPHD patients may be impaired by inadequate replacement with thyroxin, adrenal steroids, or sex steroids (Burman and Deijen, 1998). A final methodological hurdle is the choice of the reference group. The cognitive functions of a GHD group should ideally be compared with those of a reference group that differs from the patients group only in terms of GH deficiency. In an ideal world, the reference group should be comparable in terms of stature, previous medical treatment, hormone deficiencies other than GH, and medical problems. Because no such ideal group exists, a normal healthy reference group is used most frequently. This may be inappropriate, however, as impairments in GH deficiency may be the result of psychiatric disturbances related to the chronic nature of the disease rather than to the disease itself. Patients with another chronic disease, such as diabetes mellitus, may therefore be more useful as a control group (Johnston, 1997). Some previous studies in the field of GH research and psychological functions have indeed used control groups that consisted of patients with a chronic disease. However, these studies tended to focus on quality of life (QoL) rather than the assessment of cognitive functions. For instance, Lynch et al. (1994) compared 41 adults suffering from GHD with diabetes patients matched in terms of age and gender. These workers found a higher incidence of psychiatric morbidity and mood disorder in the GHD patients. Comparing GHD patients with a similar reference group, Wallymahmed et al. (1999) found a decreased QoL in GHD patients compared to diabetic patients. Page et al. (1997) compared QoL in GHD patients with patients who had undergone mastoid surgery. They found similar scores in both groups, based on QoL questionnaires (Page et al., 1997). Rikken et al. (1995) studied 210 CO-GHD patients (93 IGHD and 111 MPHD) and compared their test results in

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terms of social functioning with short, healthy control subjects. While these workers found no difference between the IGHD group and the MPHD group in terms of social functioning, the GHD patient group as a whole was characterized by a higher percentage of singles, less prestigious jobs, and a lower income than the control subject group. Clearly, very few studies on QoL included a reference group with a chronic disease. Furthermore, studies on neuropsychological functions made no use whatsoever of a chronic disease group as a reference. The methodological problems (uniformity of cognitive tests and selection of most appropriate patient group and control group) should be kept in mind when considering the impact of a study on the cognitive aspects of GH deficiency and GH treatment.

III. COGNITIVE EFFECTS OF GH-INDUCED HORMONAL CHANGES The GH–IGF-I axis exerts major effects on a wide variety of tissues, as well as influencing the action of other hormones through a series of complex interactions. GH replacement in GHD patients is associated with changes in the pituitary–thyroidal axis, the pituitary–adrenal axis, and the pituitary–gonadal axis (Jorgensen et al., 1999). GH and IGF-I are known to increase the peripheral conversion of thyroxin (T4) to triiodothyronine (T3). GH substitution in healthy subjects and GHD patients is accompanied by a decrease in free T4 (fT4) and reversed T3 levels, an increase of free T3 (fT3 ) levels, and an increased T3/T4 ratio (Grunfeld et al., 1988;Moller et al., 1992). IGF-I partly mediates the effect of GH on the peripheral conversion of T4 to T3 (Hussain et al., 1996). In a placebo-controlled crossover study of 4 months of GH therapy in GHD patients, the main effect on thyroid function was increased peripheral T4 to T3 conversion, without significantly affecting TSH levels or thyroid hormone secretion by the thyroid gland (Jorgensen et al., 1992). Another study revealed a transient effect of GH substitution in GHD patients, which caused T4 levels and f T4 levels to decrease and f T3 levels to increase in the first 3 months of GH substitution. Values returned to pretreatment values thereafter (Kalina-Faska et al., 2004). This effect is clinically relevant and supports the idea of measuring both T3 and T4 in patients with GH substitution. GH inhibits cortisol–cortisone interconversion by inhibiting the enzyme 11␤-hydroxysteroid dehydrogenase (11␤-HSD1). As a result, the activity of this enzyme is increased in GHD patients and the conversion of inactive cortisone to active cortisol is enhanced. GH substitution in GHD patients leads to an inhibition of 11␤-HSD1 and to a decrease of mean serum circulating cortisol levels (Rodriguez-Arnao et al., 1996; Swords et al., 2003).

Because GH substitution restores normal cortisol metabolism, dose adjustments could be necessary when initializing GH therapy in GHD patients (Gelding et al., 1998). GH and IGF-I have no major influence on estrogen action. However, estrogens do have an effect on GH action, as estrogens enhance GH secretion, increase the concentration of GH-binding proteins (Baumann, 2001), and inhibit GH action (Leung et al., 2003). Patients with GHD have subnormal androgen levels, which normalize with GH substitution (Isidori et al., 2003). GH stimulates androgen production directly, through the adrenal gland, or by influencing 11␤-HSD1 and the hypothalamus–pituitary–adrenal axis. Testosterone administration has been reported to increase serum IGF-I levels in young and older men (Ferrando et al., 2002; Veldhuis et al., 1997). These findings indicate that GH replacement may have additional psychological effects, as a result of the increased conversion of thyroxin to T3, the decreased conversion of cortisone to cortisol, and the enhanced production of androgens. For instance, an excess or deficiency of the thyroid hormone can cause mental disturbances, such as depression or mania. Anger scores have been found to be positively correlated with serum-free T3 levels in GH-deficient adults (Deijen et al., 1996). Excess cortisone is known to produce adverse emotional effects, such as anxiety and agitation, whereas cortisone-deficient patients suffer from worsened cognitive functioning, reduced energy, and a decreased arousal. In addition, a sex hormone such as testosterone is known to have profound effects on behavior, which may differ from those exerted by the GH/IGF axis (Erlanger et al., 1999). For instance, a state of anxiety was positively correlated to serum testosterone in GH-deficient adults (Deijen et al., 1996). One study examined the relationship between exogenous testosterone administration and cognitive abilities in a population of healthy older men. Twentyfive healthy, community-dwelling volunteers, aged from 50 to 80, completed a randomized, double-blind, placebocontrolled study. Participants received weekly intramuscular injections of either 100 mg testosterone-enanthate or placebo (saline) for 6 weeks. Cognitive evaluations were conducted at baseline, week three, and week six of treatment using a battery of neuropsychological tests. In the treatment group, circulating total testosterone had increased by an average of 130% from baseline at week three and 116% at week six. Also, in the treatment group, the aromatization of testosterone caused estradiol to increase by an average of 77% at week three and 73% at week six. Relative to the baseline and the placebo, significant improvements in cognition were observed for spatial memory (recall of a walking route), spatial ability (block construction), and verbal memory (recall of a short story) in these older men treated with testosterone (Cherrier et al., 2001). A second study was undertaken to determine whether these cognitive improvements (Cherrier et al.,

22. Cognitive Functioning and GH

2004) were linked to possible changes in IGF-I, IGF-II, and IGF-related binding proteins (IGFBPs) induced by testosterone administration. Following cognitive testing, serum levels of total IGF-I, IGF-II, and IGFBPs were determined for this purpose. Regarding the association between IGF and cognitive functions, after 6 weeks of testosterone administration, higher IGF-I and IGF-II levels were found to be associated with improved spatial memory, spatial reasoning, and verbal fluency. Increased levels of estradiol were associated with a decline in divided attention performance. Finally, testosterone treatment did not produce changes in serum IGF-I, IGF-II, and IGFBPs. The authors conclude that testosterone, estradiol, and IGF-I may have independent and selective effects on cognitive functioning.

IV. COGNITIVE STATUS IN RELATION TO GH DEFICIENCY (GHD) AND GH TREATMENT A. Cognitive Impairments in Adult CO-GHD Patients and Effects of GH Replacement Results of the studies reviewed later indicate that both CO-IGHD and CO-MPHD patients exhibit cognitive dysfunction. Patients with IGHD and MPHD both demonstrate poorer performance with respect to several memory components; they also have a lower IQ than healthy controls. In addition, their IQ score and educational level appear to be positively related to IGF-I concentration, suggesting that subnormal cognitive performance is specifically related to GHD (Deijen et al., 1996). Twenty years ago, there was very little published work evaluating the cognitive performance of GHD patients and/or the neuropsychological effects of treatment. One of the earliest reports concerns an open study in 11 CO-GHD patients aged 4–18 (four MPHD, seven IGHD) whose cognitive performance was assessed after 1 year of GH treatment (Abbott et al., 1982). No treatment effects were found for intellectual functioning and visual-motor integration skills. Intelligence and academic achievement were found to be positively correlated only with socioeconomic status. They were independent of the condition of hypopituitarism. A subsequent study in even fewer CO-GHD patients evaluated the level of cognitive functions in 5 patients aged between 22 and 36 (three males, two females) (Almqvist et al., 1986). All of these patients had previously completed GH treatment, 4 of them 3 years prior to their inclusion in the study, and the remaining patient 4 months prior to inclusion. All patients were MPHD and were receiving concomitant substitution therapy. Mental status was determined by means of five cognitive tests, covering memory and attention, as well as language and arithmetic skills. GH treatment (biosynthetic methionyl human GH or native

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human GH in random order intramuscularly 8 IU three times a week) was given for two 4-week periods, separated by a washout period of at least 4 weeks. Cognitive tests were administered at the beginning and at the end of each treatment period. Regrettably, the patients’ cognitive functional levels prior to treatment were not compared to normal reference values. Therefore, there is no way of knowing whether their baseline cognitive functions were subnormal. With respect to treatment, however, it was found that 4 weeks of GH replacement improved memory function, as assessed by a face recognition test. In terms of their effect on cognition, there was no difference between biosynthetic GH and human GH replacement. In only one of the older studies were IQ scores determined separately for IGHD and MPHD patients. This involved a sample of 42 young adults (23 males and 19 females; 14 IGHD; 28 MPHD) who had been treated previously with GH (Galatzer et al., 1987). This distinction led to the observation that the distribution of IQ scores for patients with IGHD was in the upper part of the curve, whereas that of patients with MPHD was skewed toward the lower part. It therefore seems that a combined deficiency of pituitary hormones during a critical period of brain development may be more harmful than GHD alone (Burman and Deijen, 1998). Some years after these uncontrolled studies, a report was published concerning a double-blind crossover study in which the cognitive effects of recombinant methionyl GH were compared with a placebo in three women and three men, aged between 20 and 38 (Degerblad et al., 1990). Five of these patients suffered from MPHD, the other was diagnosed as IGHD. The patients received GH or placebo for two periods of 12 weeks, in random order. Cognitive functions were assessed by verbal and nonverbal learning tests. Attention was assessed by a reaction–time test and a digit–symbol substitution test. GH was not found to have any beneficial effects on cognition. The authors ascribe the absence of an effect to the tests used, indicating that they may lack sufficient sensitivity. However, the small sample size and short treatment duration can only reveal large effects. Small effects may remain undetected. Several additional studies on GH treatment and cognitive functions have been carried out in the last few years. In one of them, Sartorio et al. (1995) evaluated the psychometric profile of eight adult patients with CO-GHD (three IGHD, five MPHD). This was done using nonverbal scales of the Wechsler Adult Intelligence Scale (WAIS) before and after a 6-month period of recombinant GH therapy. The patients exhibited a normal intellectual profile prior to this course of treatment. After 6 months of treatment, patients presented higher scores on the symbol–number association subtest of the WAIS, indicating that intellectual functioning had improved. More recently, a study on baseline and posttreatment cognitive functions was performed in a relatively large

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sample of patients. First, an evaluation of baseline cognitive functioning in GH-deficient men was presented by comparing cognitive function in 48 men with CO-GHD (31 with MPHD and 17 with IGHD) with an age-matched control group (Deijen et al., 1996). The patients were aged from 19 to 37, with a mean age of 27. In all patients, the diagnosis of GHD had been established during childhood on the basis of growth retardation, skeletal immaturity, and a subnormal GH response to at least two GH provocative tests. The control group consisted of 41 healthy men aged from 19 to 37, with a mean age of 27. The measurement of cognitive functions involved iconic, short-term, and long-term memory tests. Perceptual-motor skill (i.e., eye/ear–hand/foot coordination) and IQ score were also determined. Results of this study clearly indicated that cognitive performance was subnormal in GHD patients. They obtained poorer results than control subjects in the memory tests. In addition, GHD patients had a lower IQ score (mean IQ ⫽ 89) than controls (mean IQ ⫽ 97). There was no difference in perceptual-motor skill between patients and control subjects. The same results were obtained after the IGHD and MPHD groups were tested separately against controls, with both groups showing a subnormal IQ, iconic-, short-term-, and long-term memory. Finally, in the patient group as a whole, the IQ score was positively correlated with serum IGF-I concentration. Summarizing these results, GHD patients clearly exhibit a subnormal IQ score and memory performance, whereas their perceptual-motor capabilities are normal. As IQ score is related to the level of IGF-I and subnormal cognitive function is observed in both IGHD and MPHD patients, this subnormal cognitive functioning seems to be specifically related to GHD rather than to other pituitary hormone deficiencies, for example. The study included a second treatment phase in which the 48 CO-GHD men were assigned randomly to placebo treatment or GH replacement. Placebo treatment was given for a period of 6 months. All groups eventually received GH replacement for a period of 2 years. Iconic memory, short-term memory, long-term memory, and perceptualmotor skill were checked at 6-month intervals. GH treatment was considered to be physiological if the observed IGF-I levels were within the normal range. It was viewed as supraphysiological if serum IGF-I rose to a value exceeding the normal upper limit for the patient’s age and gender. During the placebo-controlled phase of the study, changes in short- and long-term memory were positively correlated to the GH-induced changes in serum IGF-I concentration. At 6 months, however, only the group receiving supraphysiological GH treatment showed improvements in short-term and long-term memory. This did not apply to the group of patients whose serum IGF-I had been normalized. After 1 year of treatment, memory functioning was found to have normalized in both groups of patients. This was maintained

throughout the second year of treatment. No changes were observed in perceptual-motor skill (Deijen et al., 1998). These results demonstrate that GH replacement improves memory function in adults with CO-GHD, but not their perceptual-motor skills. Supraphysiological treatment accelerates the recovery of memory performance, although the long-term effects are no different from those achieved using physiological GH replacement. A study on short-term and long-term memory in patients with CO-GHD evaluated the effects of 1 year of GH treatment following a 1-year period of GH discontinuation. Memory was assessed at baseline and at 6 and 12 months into a period of GH treatment. The subjects were 10 males and 10 females, aged between 17 and 27 (5 IGHD, 15 MPHD). No improvements in memory were found during the 1-year period of treatment. Nevertheless, positive correlations were found between IGF-I and short-term memory performance. On the basis of these findings, it was concluded that patients with a normal serum IGF-I concentration require at least 1 year of treatment before their memory improves (Stouthart et al., 2003). The general picture that emerges with respect to cognitive functioning in CO-GHdeficient patients is far from unequivocal. There are contradictory findings regarding the cognitive status of GHD patients. However, studies with a larger sample size indicate that cognitive status (including IQ) is subnormal in GH-deficient patients. The impairment seems even more pronounced in MPHD than in IGHD. Conflicting results have also been reported in connection with GH treatment. Additional evidence shows that GH treatment improves cognitive functioning in CO-GHD patients. Yet in the case of normal physiological GH replacement, it appears that a minimum treatment period of 1 year is required to achieve an improvement in cognitive status.

B. Event-Related Potentials during Selective Attention in CO-GHD Event-related potentials are electrical potentials generated in the brain, which can be measured on the scalp. External stimuli or events activate neurons. These potentials are generated by the synchronized activation of large numbers of neurons. The effect of low levels of GH and IGF-I on performance and event-related potentials during a selective attention task was studied in 10 childhood-onset GHD patients (Lijffijt et al., 2003). GH-deficient patients’ performance in the selective attention task, as well as their electrophysiologic recordings, was compared to those of healthy controls. CO-GHD patients had attentional deficits, as indicated by their poor detection of occasional visual target patterns. The patients did not have slower reaction times, so the speed of attentional processes appeared to be normal. The reduced quality of attentional performance was reflected by a reduction in an attention-related potential associated

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with the activity of the anterior cingulate cortex. Results of this study show that selective attention is disrupted in patients with CO-GHD. They also suggest that the accuracy of attention and the associated activity of the anterior cingulate cortex depend on the functional status of the somatotropic axis.

C. Cognitive Impairments in Adult AO-GHD and Effects of GH Replacement Patients with adult-onset GHD frequently report lapses of attention, difficulty in concentrating, and forgetfulness (Hunt et al., 1993;McGauley et al., 1996;Rosen et al., 1994). Similar results were found by Burman et al. (1995), who observed that patients with AO-GHD exhibited significantly more problems with cognitive function, particularly concentration ability as assessed by the Hopkins Symptom Check List (HSCL), than the healthy reference group. The effect of GH replacement in these 36 AO-GHD patients was examined in a 21-month crossover, placebocontrolled, double-blind trial. The active and placebo treatment periods each lasted for 9 months, and there was an intervening 3-month washout period. When separate items of the HSCL were analyzed, it was found that GH treatment produced a greater improvement in concentration problems (classified as cognitive impairment) than the placebo. Baum et al. (1998) conducted an 18-month, randomized, double-blind, placebo-controlled trial in 40 AO-GHD men. Numerous psychometric tests were administered to assess verbal and performance IQ, working memory, verbal learning, executive cognitive function and selective attention. At baseline, IQ scores were normal, but GHD patients showed a relative impairment on verbal learning and visual memory (below the mean but within normal limits). No significant changes in cognitive function were observed after 18 months of GH therapy. As this chronic low-dose GH replacement therapy produced no beneficial cognitive effects, the authors concluded that acquired GHD in adult men is not associated with significant alterations in cognitive function. The incidence of mental disorders and the prevalence of mental distress and cognitive dysfunction were investigated in 33 hypopituitary women (aged from 39 to 77) with AO-GH deficiency (Bulow et al., 2002). All of these individuals were treated for pituitary disease, 29 had undergone surgery for a pituitary tumor, 25 had received radiotherapy, and 15 had visual dysfunction. These GH-deficient women were investigated cross-sectionally for mental functioning, and the results were compared with 33 controls matched for gender, age, smoking habits, educational level, and residence. Several tests were performed to measure cognitive functioning (vocabulary, perceptual speed, spatial ability, verbal memory, spatial learning, and reaction time). In addition, the incidence of mental disorders was calculated.

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The AO-GHD women had a higher incidence of mental disorders and lower scores on a majority of the neuropsychological tests than the control subjects. From this handful of studies in AO-GHD, it may be concluded that AO-GHD patients, like those suffering from CO-GHD, exhibit subnormal neuropsychological functions, although IQ seems to be normal. Treatment produces conflicting results, as one study showed that a 9-month period of GH treatment increased concentration ability while another indicated that 18 months of GH therapy had no cognitive effects. Given the small number of studies involved, it can only tentatively be concluded that mental status is reduced in AO-GHD and that GH treatment may possibly improve cognitive functions in these patients.

D. Cognitive Effects of GH Replacement in a Mixed Group of AO and CO-GHD In a double-blind, placebo-controlled trial, nine GHD adults were assessed using a battery of neuropsychological tests (including attention and memory measurements) before and after a 6-month period of GH substitution (Soares et al., 1999). There were improvements in attention, vocabulary, picture arrangement, and comprehension after 6 months of GH therapy. Given that this study incorporated controls, the positive results in terms of cognitive functions cannot be easily dismissed.

V. META-ANALYSIS GH AND NEUROPSYCHOLOGICAL FUNCTIONS We performed a meta-analysis of a selection of studies on GH treatment and cognitive functions, disregarding the type of patients involved (i.e., AO-GHD/CO-GHD; IGHD/MPHD). Only those studies that produced quantitative data on the effect of GH therapy on cognition in GHD adults were eligible for inclusion. Another criterion was that study designs could be either placebo controlled, crossover/parallel or open. One or more of the measured outcomes had to concern cognitive functioning. All case reports, review articles, and studies in which cognitive functioning was determined by a test or questionnaire of unknown psychometric quality were excluded. Nor were studies dealing with GH therapy for other diseases (Turner syndrome, Prader–Willi syndrome, fibromyalgia, etc.) included in the meta-analysis. Only four studies met the criteria for inclusion in this meta-analysis of GH treatment and cognition in patients. These are shown in Table I. Because most studies lacked a control group, the effect of GH substitution on cognitive performance could not be compared with placebo treatment. Using the selected studies, it was only possible to

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TABLE 1 Selected Studies on the Effects of Growth Hormone (GH) Treatment and Cognitive Functioninga First author, year

N (sex)

Mean age in years (range)

CO-AO GHD

IGHD/MPHD

Trial design

Duration GH therapy

Cognitive assessment

Deijen, 1998

48 (all males)

27 (19–37)

CO

Both

Placebo controlled, open

6, 12–24 months

Memory (short term) Memory (long term)

Soares, 1999

9 (6 males, 3 females)

39.4 (28–52)

AO/CO

Both

Randomized controlled, open

6–12 months

Picture arrangement, vocabulary, comprehension, verbal fluency

Sartorio, 1995

8 (all males)

29.6 (25–34)

CO

Both

Open

6 months

Nonverbal WAIS (seven items)

Stouthart, 2003

20 (10 males, 10 females)

21 (17–27)

CO

Both

Open

12 months

Memory (short term) Memory (long term)

a AO-GHD, adulthood-onset GH deficiency; CO-GHD, childhood-onset GH deficiency; IGHD, isolated GH deficiency; MPHD, multiple hormone deficiency; WAIS, Wechsler Adult Intelligence Scale.

analyze the effects of GH treatment for a 6-month period, and during a period that was averaged across all studies. We therefore derived d values (effect sizes) by calculating the difference before and after GH therapy, divided by the pooled standard deviation of the two groups. The calculated effect sizes (d) were pooled. Effect size d ⫽ 0.2 was defined as a small effect, d ⫽ 0.5 as a medium-sized effect, and d ⫽ 0.8 as a large effect (Cohen, 1977). Heterogeneity was calculated using the Q test. This test revealed that the d values were not distributed homogeneously, so we used a random effects model. The effect of GH on cognition was calculated using 19 d values obtained from four studies. After 6 months there was a significant increase (p ⫽ 0.003) in cognitive functioning in GHD patients (d ⫽ 0.28, 0.09 ⫺ 0.46 [CI]). The group of 19 d values was not homogeneous (Q ⫽ 26.3, p ⫽ 0.09). The total effect of GH on cognitive functioning was calculated from 34 “effect sizes” from the four studies. This effect was small, but significant (d ⫽ 0.24, 0.11 ⫺ 0.37 [CI], p ⬍ 0.001). The mean duration of GH therapy was 11.1 months. The d values were not distributed homogeneously (Q ⫽ 58.5, p ⬍ 0.01). It was not possible to analyze differences in outcomes in the IGHD and MPHD group and/or CO- and AO-GHD patients. As cognitive function was mostly studied in CO-GHD, separate data from these groups are not available. The observed effects of GH on cognition are small, but significant. The small effect sizes of a 6-month period of GH treatment and of the averaged treatment durations (11 months) may be a consequence of the wide variety of memory tasks used. While GH substitution may indeed improve some fields of cognition, the pooling of all these different tests means that any effect found is rather small. Another possibility is that IGF-I, rather than GH, is the main factor that influences

cognition directly. Finally, as the d values were not distributed homogeneously, results may have been distorted by moderator variables such as gender, medical history (with or without radiotherapy), or severity of GHD.

VI. COGNITIVE EFFECTS OF DISCONTINUED GH TREATMENT There is only one published study dealing with the possible decline of cognitive functioning after discontinuation of GH treatment in late adolescence. It also addressed the question of whether differences in neuropsychological parameters could be discerned between those patients with persisting GH deficiency and those exhibiting restored GH secretion capacity, within the normal range. To this end, a 2-year prospective longitudinal study was carried out in 40 CO-GHD patients (ranging from 16 to 21 years of age, with a mean age of 19), commencing with discontinuation of GH therapy at final height (Wiren et al., 2001). Discontinuation followed a stabilization period of 3 months, during which all subjects received GH treatment. After 1 year, 21 patients were retrospectively assigned to a GH-deficient group and 19 to a GH-sufficient group. Cognitive function tests revealed that the baseline scores on the digit–symbol test (which measures short-term memory, visual acuity, and motor speed) were higher in the GH-sufficient group than in the GHD group. In addition, after 2 years, the trail-making test B score was lower in the GHD group than in the GH-sufficient group. This indicates that long-term GH deficiency seems to impair abilities such as information processing, planning, and cognitive flexibility, as measured by this motor tracking task.

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VII. GH DOSAGES AND COGNITIVE STATUS IN GH-DEFICIENT ADULTS The optimal GH substitution dose for GHD adults is still open to debate. During childhood, the optimal GH dose can be monitored using measurements of a child’s linear growth. Serum IGF-I values are considered to be a good marker of GH status in GHD adults. Individual dose adjustments should be made to optimize GH substitution, as age, gender, and adiposity influence the responsiveness to GH substitution. The goal of GH substitution in adults is to minimize side effects (prevent overtreatment) and to optimize beneficial clinical effects (prevent undertreatment). It is recommended that GH substitution in adults should start at a low dose of GH and that this should be increased on the basis of measured serum IGF-I values and the clinical response. Regarding the inadequate replacement of GH, patients with an excess of this hormone (which gives rise to acromegaly or gigantism) are known to exhibit mood fluctuations (Bleuler, 1951; Richert et al., 1987). Thus, while administering the correct dose of GH to a given patient is important in terms of physical functions, it also has major implications for psychological functions. While a low dose of GH may result in insufficient GH replacement and may be cognitively ineffective, a high dose of GH may even be harmful to neuropsychological functions. Indeed, many years ago, animal experiments demonstrated that the behavioral effects of cognition-enhancing drugs exhibit a bell-shaped dose–effect curve (Pepeu et al., 1989). It has even been postulated that a bell-shaped dose–effect curve is a typical feature of all drugs acting on cognitive processes (Martinez and Kesner, 1991). With respect to human studies, it was found that the way in which different doses of glucose (10, 25, 50 g) affected memory performance in elderly subjects conformed to an inverted-U dose–response curve. The subjects performed best after ingesting 25 g of glucose, the intermediate dose (Parsons and Gold, 1992). Such a bell-shaped dose–effect curve may also account for the relationship between cognitive processes and GH treatment. This means that while intermediate doses enhance cognitive processes, low doses of GH have no effect and higher doses either have no effect or may even impair cognitive functions. It is not known why treatments that modulate memory storage are characterized by an inverted-U dose–response curve. One quite dated but nevertheless valuable explanation is that if there are a finite number of receptors, the drug effect should be proportional to the fraction of receptors occupied by the drug, and the maximum effect should occur when all receptors are occupied. However, if the receptor shows tachyphylaxis or fatigue (due to its constant occupation by agonist drugs), then greater activity should lead to greater fatigue. Hence, larger doses should produce smaller effects than a lower (i.e., optimal) dose (Day, 1979). While a larger than optimal dose may speed up cognitive recovery, this may

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involve an increased risk of side effects. Indeed, as is already mentioned in Section IVA, supraphysiological GH treatment was observed to accelerate the recovery of memory performance, but in the long term the effects were no different than those achieved with physiological GH doses (Deijen et al., 1998).

VIII. GH AND INSULIN-LIKE GROWTH FACTOR I REPLACEMENT IN SYNDROMES OTHER THAN GHD A. IGF Treatment in Postmenopausal Women The impact of 1 year of IGF-I replacement therapy on psychological parameters was studied in 16 healthy postmenopausal women (mean age 70.6) (Friedlander et al., 2001). In a double blind, placebo-controlled trial, subjects injected themselves with IGF-I (15 ug/kg, twice daily) or placebo. They were assessed at baseline and at 6 and 12 months into a period of treatment. Memory, which was tested by name–face recall and numerical digit recall, was not improved by IGF-I treatment. It was concluded that 1 year of IGF-I treatment is not an effective means of enhancing memory in older postmenopausal women.

B. GH Substitution and Cognition in Prader–Willi Syndrome The effect of GH administration on cognition was studied in children with Prader–Willi syndrome (Haqq et al., 2003). In a 12-month, double-blind, placebo-controlled, crossover design trial, 12 subjects (ranging from 4.5 to 14.5 years of age) were randomized to GH (0.043 mg/kg/day) or placebo for 6 months. Thereafter, subjects crossed over to the other intervention for a period of 6 months. Behavioral and cognitive assessments were performed at baseline and at 6 and 12 months using the Wechsler Intelligence Scale for children. At baseline, all subjects demonstrated moderate mental retardation. No significant changes were observed after GH intervention.

C. GH Substitution and Cognition in Turner Syndrome The effects of GH substitution on cognition in Turner syndrome were assessed in a long-term, double blind, placebo-controlled trial (Ross et al., 1997). Twenty girls (with a mean age of 9.9) received GH (0.1 mg/kg, three times weekly) and another 20 girls (with a mean age of 9.3) received placebo. Treatment durations ranged from 1 to 7 years. Neuropsychological functions were assessed using various tests to measure attention, affect recognition, visual-motor and visual-spatial skill, language, memory

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(verbal and nonverbal), academic achievement, and general cognitive skills. GH treatment was found to have no effect on any of the psychological (or neuropsychological) functions assessed in these Turner syndrome patients.

D. Intelligence in Subjects with GH Receptor Deficiency The intellectual ability of subjects with GH receptor deficiency was evaluated and compared to their relatives and community controls (Kranzler et al., 1998). The intelligence of 18 patients with GH receptor deficiency (12 males and 6 females) of school age (mean age 11.5 ⫾ 2.7 years), 42 relatives, and 28 community controls was measured using nonverbal psychometric and chronometric tests of intelligence. The intellectual ability of the patients with GH receptor deficiency was not significantly different from that of their relatives and was comparable to that of the control subjects. It was concluded from this study that the gene effect causing GH receptor deficiency is not related to intelligence. Further it was concluded that GH-induced IGF-I production is not required for normal brain growth in utero nor for the postnatal development of intelligence. Available data on GH and IGF replacement in patient groups other than GHD point to the conclusion that such interventions have no proven beneficial effects on cognitive functions. In addition, the disruption of GH and IGF-I activity associated with GH receptor deficiency is not harmful in terms of brain development.

IX. GH-INDUCED COGNITIVE CHANGES IN HEALTHY ELDERLY SUBJECTS IGF-I levels and cognitive functioning decrease with aging. Features of aging resemble those of GHD, suggesting that a decreased level of circulating IGF-I may be involved in age-related cognitive decline. The expression of GH and IGF-I receptors in the hippocampus declines with increasing age (Lai et al., 1993). This has led to suggestions that GH or IGF-I substitution in such healthy elderly subjects may be beneficial to cognition. Several studies in healthy elderly subjects have demonstrated correlations between serum IGF-I levels and cognitive performance (Dik et al., 2003;Kalmijn et al., 2000; Papadakis et al., 1995;Rollero et al., 1998). Others revealed that IGF-I levels were linked to the age-related decline in cognitive function (Vitiello et al., 1999). A study in 22 healthy elderly men and women (ranging from 65 to 86 years of age) revealed a positive correlation between IGF-I levels and scores on the Mini Mental State Examination (MMSE) (Rollero et al., 1998). In another study in 49 healthy elderly subjects (above 75 years of age), a positive relation was found

between IGF-I and cognition, as measured with the MMSE score (Paolisso et al., 1997). Higher IGF-I levels were associated with better performance in tests of mental processing speed in healthy elderly men (n ⫽ 25, mean age ⫽ 69) (Aleman et al., 1999). IGF-I levels were also correlated with other tests sensitive to aging, such as the digit symbol substitution test and concept shifting task, measuring speed of information processing. Tests not sensitive to aging (information and vocabulary, Benton line and Brus reading) showed no correlation with IGF-I in 17 men aged from 66 to 76 (Aleman et al., 2000). A prospective study on circulating IGF-I, IGFBP-3, and cognitive function in 186 elderly subjects (ranging from 55 to 80 years of age) showed that higher total IGF-I and total IGFBP-3 levels were associated with a less pronounced cognitive decline over the following 2 years, as measured with the MMSE (Kalmijn et al., 2000). Dik et al. (2003) found that IGF-I levels in 1318 elderly men and women (ranging from 65 to 88 years of age) were directly related to information processing speed, memory, fluid intelligence, and MMSE scores. IGF-I levels below 9.4 nmol/liter were negatively associated with both the level of information processing speed and its decline over a 3-year period. A cross-sectional survey in 56 healthy males (ranging from 20 to 84 years of age) revealed a high degree of correlation between the IGF-I /GH ratio and cognitive functioning, as determined using four different tasks for measuring visual and verbal memory (Morley et al., 1997). In a study in 49 healthy centenarians (mean age 100.4), cognitive functioning was assessed by clinical dementia rating (Arai et al., 2001). Centenarians with lower IGF-I levels had a higher prevalence of definitive dementia. One of the few studies of GH treatment in healthy subjects tested a hypothesis that 6 months of GH treatment would reverse the muscle weakness and functional decline associated with aging. The subjects were 52 healthy ambulatory men (aged from 70 to 85, with a mean age of 75) whose baseline IGF-I factor levels were less that the 10th percentile found in younger healthy adults (Papadakis et al., 1996). Participants were randomly allocated to a GH treatment group or a placebo group. Treatment involved the administration of 0.03 mg rhGH per kilogram of body weight, three times a week. The subjects’ physical and cognitive parameters were evaluated at 2, 4, 8, 12, 18, and 24 weeks of treatment. Cognitive assessments consisted of the trails B test, the MMSE, and the digit-symbol substitution test. The active treatment group showed a significant speed improvement in the trails B test relative to the placebo group. However, the GH treatment group’s scores in the other cognitive tests showed no improvement. The trails B test is a motor-tracking task, which measures information processing, planning, and cognitive flexibility. As these are all functions thought to be controlled by the frontal cortex, this result can be interpreted as an improvement in executive functions. However, there

22. Cognitive Functioning and GH

was no indication of an improvement in the other cognitive tests. In addition, numerous statistical tests indicated that the improved trails B score might be due to chance. Accordingly, the authors refrained from concluding that GH treatment improves cognitive functions. This result supports the authors’ view that GH should not be used to preserve or improve functional ability in healthy, functionally intact elderly men. However, they do suggest that controlled studies in functionally impaired elderly subjects would enhance our understanding of the clinical use of GH. Growth hormone-releasing hormone (GHRH) in rats has been shown to increase memory performance (Thornton et al., 2000). A report on GHRH and cognitive function in humans also revealed positive effects. Cognitive function was investigated in 89 healthy elderly men and women (mean age 68) both before treatment and after 5 months of treatment either with a placebo or with GHRH (Vitiello et al., 2003). GHRH treatment resulted in improved psychomotor and perceptual processing speed relative to the placebo, as indicated by improvements on the Wechsler Adult Intelligence Scale (WAIS performance IQ and WAIS picture arrangement), findings A’s, verbal sets, and the single-dual task. The observed cognitive improvements were independent of baseline cognitive capacity. This result supports the notion that an agerelated decline in the somatotrophic axis may be involved in cognitive decline and that it may be possible to counteract this by GHRH supplementation. Another relatively recent study explored the stimulatory effect of a nutritional supplement on the GH–IGF-I axis and cognition (Arwert et al., 2003). Fortytwo healthy subjects (14 men and 28 women, aged from 40 to 76) received either 5 g of a nutritional supplement (containing glycine, glutamine, and niacin) or a placebo, twice daily for a period of 3 weeks. Serum GH, IGF-I, and cognitive function were assessed at baseline and again after 3-weeks. The nutritional supplement was found to increase serum GH levels by 70% relative to the placebo; however, there was no change in circulating IGF-I levels. The elevated GH levels were not associated with any cognitive improvement. Correlation analyses, however, revealed that individual increases in IGF-I (unlike GH) were associated with memory improvement. This study showed that a mixture of glycine, glutamine, and niacin can enhance GH secretion, even though this did not improve the subjects’ memory. These data further suggest that cognitive changes seem to be more strongly associated with changes in IGF-I than in GH.

X. CONCLUSION Data on neuropsychological functioning in GHD patients are limited and controversial. While by no means unequivocal, the general picture that emerges with respect to cognitive functioning in GHD patients does seem to indicate that their

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cognitive status is subnormal. Although both CO-GHD and AO-GHD patients exhibit an impaired neuropsychological status, IQ only appears to be subnormal in the former. In addition, evidence suggests that neuropsychological impairment is even more pronounced in MPHD than in IGHD. As CO-GHD mainly involves IGHD, a subnormal IQ may be specifically associated with IGHD, whereas neuropsychological impairment is more severe in MPHD. These interpretations seem to contradict one another. Accordingly, further studies need to be performed to shed more light on differences in the neuropsychological profile within the various subgroups of GHD patients. As yet, we can only tentatively conclude that GHD patients exhibit a subnormal neuropsychological status. The same problems arise with regard to GH treatment, for which similarly conflicting results have been reported. There is some evidence that GH treatment improves cognitive functioning in CO-GHD and AO-GHD patients. However, the achievement of a substantial cognitive improvement appears to require at least 1 year of treatment. Unfortunately, there are few studies on GH treatment and cognition and even fewer controlled studies in that field. As a result, our meta-analysis was based on a very limited number of studies indeed. Moreover, it was only possible to analyze data from uncontrolled studies. Results of this meta-analysis indicate that both 6-month periods of treatment and averaged treatment durations of 11 months improve cognitive function, albeit with a small effect size. It is possible that this small effect size is caused by the lack of uniform cognitive tests, as while GH substitution may improve specific cognitive domains, any such effect would be attenuated when the various tests are pooled. Also, the results may have been distorted by moderator variables such as gender, medical history (with or without surgery and/or radiotherapy), or the severity of GHD, resulting in a small treatment effect. Thus, heterogeneous patient groups complicate interpretation of the effects of GH substitution in these GHD patients. In addition, the cognitive deficits found in GHD patients could result from GH or IGF-I deficiency. A number of studies are cited, in Section IX, as reporting correlations between serum IGF-I levels and cognitive performance in healthy elderly subjects. Similarly, the report by Arwert et al. (2003) revealed that individual increases in IGF-I (unlike GH) were associated with memory improvement. This indicates that IGF-I levels appear to be specifically related to cognitive status. As yet, there are no data on the effect of IGF-I administration in GHD patients. Further studies are required to distinguish between the effects of GH and IGF-I on brain function. Recombinant IGF-I is available, although it is very expensive. Its use has only been studied in postmenopausal women (Friedlander et al., 2001). Relatively high doses were required to achieve normal IGF-levels. Furthermore, the administration of IGF-I reduces residual GH production by negative feedback. Not only may IGF-I treatment be too

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costly for it to be used in aging subjects, but its positive effect on cognitive functioning has still to be proven. Another way to increase IGF-I levels is by administering orally active growth hormone secretagogues. Treatment with MK-0677, for instance, increased serum IGF-I levels by 84% in hip-fracture patients compared to an increase of 17% for the placebo group (Bach et al., 2004). However, the effect of this GH secretagogue on cognitive performance has not yet been investigated. As most of the findings presented in this chapter apply to open studies, it may well be the case that GH treatment produces no improvement whatsoever in the cognitive functions of GHD patients. In order to confirm any beneficial effects of treatment, additional placebo-controlled studies are needed. Also, the cognitive tests used should be more uniform in nature. At this point, reports concerning the existence and magnitude of any beneficial effects of GH treatment on cognitive function in GHD adults should be treated with caution. Despite this reservation, it must be stressed that the putative relationship between the GH–IGF-I axis and cognitive functioning is based on firm biological foundations. As described earlier, GH treatment changes the concentration of the dopamine metabolite HVA in cerebrospinal fluid. The importance of this is that dopamine is known to play an important part in learning and memory. Also the presence of binding sites for GH and IGF-I in the choroid plexus, hypothalamus, putamen, thalamus, and hippocampus may be important for cognitive processes. Finally, GH substitution may activate the NMDA receptor by increasing aspartate levels in the CSF. This may contribute to memory consolidation in the brain. The existence of these biological mechanisms makes it likely that GH treatment can indeed improve cognitive functions in GHD patients. However, a great deal of research needs to be done before more definitive conclusions can be drawn. Finally, it can be concluded that GH replacement has no proven beneficial effects on cognitive functions in patients other than those suffering from GHD. It has been suggested that GH might have cognition-enhancing effects in healthy elderly subjects. However, while some data suggest that GHRH might be effective in this regard, there is none to support the notion that GH itself either preserves or improves functional ability in healthy elderly subjects. Nor have any studies been conducted to determine whether GH therapy might be of clinical relevance in cognitively impaired elderly subjects.

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Ross, J. L., Feuillan, P., Kushner, H., Roeltgen, D., and Cutler, G. B., Jr. (1997). Absence of growth hormone effects on cognitive function in girls with Turner syndrome. J. Clin. Endocrinol. Metab. 82, 1814–1817. Sartorio, A., Molinari, E., Riva, G., Conti, A., Morabito, F., and Faglia, G. (1995). Growth hormone treatment in adults with childhood onset growth hormone deficiency: Effects on psychological capabilities. Horm. Res. 44, 6–11. Soares, C. N., Musolino, N. R., Cunha, N. M., Caires, M. A., Rosenthal, M. C., Camargo, C. P., and Bronstein, M. D. (1999). Impact of recombinant human growth hormone (RH-GH) treatment on psychiatric, neuropsychological and clinical profiles of GH deficient adults: A placebo-controlled trial. Arq Neuropsiquiatr. 57, 182–189. Stouthart, P. J., Deijen, J. B., Roffel, M., and Delemarre-van de Waal HA (2003). Quality of life of growth hormone (GH) deficient young adults during discontinuation and restart of GH therapy. Psychoneuroendocrinology 28, 612–626. Swords, F. M., Carroll, P. V., Kisalu, J., Wood, P. J., Taylor, N. F., and Monson, J. P. (2003). The effects of growth hormone deficiency and replacement on glucocorticoid exposure in hypopituitary patients on cortisone acetate and hydrocortisone replacement. Clin. Endocrinol. (Oxf.) 59, 613–620. Thornton, P. L., Ingram, R. L., and Sonntag, W. E. (2000). Chronic [D-Ala2]-growth hormone-releasing hormone administration attenuates age-related deficits in spatial memory. J. Gerontol.A Biol.Sci.Med.Sci. 55, B106-B112. Turchi, J., and Sarter, M. (2001). Bidirectional modulation of basal forebrain N-methyl-D-aspartate receptor function differentially affects visual attention but not visual discrimination performance. Neuroscience 104, 407–417. Veldhuis, J. D., Metzger, D. L., Martha, P. M., Jr., Mauras, N., Kerrigan, J. R., Keenan, B., Rogol, A. D., and Pincus, S. M. (1997). Estrogen and testosterone, but not a nonaromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)-insulinlike growth factor-I axis in the human: Evidence from pubertal pathophysiology and sex-steroid hormone replacement. J. Clin. Endocrinol. Metab. 82, 3414–3420. Vitiello, M. V., Merriam, G. R., and Mazzoni, G. (2003). Growth hormone releasing hormone treatment improves cognitive function in healthy older men and women. Gerontologist 43, S1-S4. Vitiello, M. V., Merriam, G. R., Moe, K. E., Drolet, G., Barsness, S., Kletke, M., and Schwartz, R. S. (1999). IGF-I correlates with cognitive function in healthy older men and estrogenized women. Gerontologist 39, 6. Wallymahmed, M. E., Foy, P., and MacFarlane, I. A. (1999). The quality of life of adults with growth hormone deficiency: Comparison with diabetic patients and control subjects. Clin. Endocrinol. (Oxf.) 51, 333–338. Wiren, L., Johannsson, G., and Bengtsson, B. A. (2001). A prospective investigation of quality of life and psychological well-being after the 1deficiency during childhood. J. Clin. Endocrinol. Metab. 86, 3494–3498.

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23 Growth Hormone and Insulin-like Growth Factor-I in Alzheimer’s Disease JOSÉ MANUEL GÓMEZ Servicio de Endocrinología y Nutrición, Hospital Universitario de Bellvitge, Facultad de Medicina, Universidad de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain

I. Introduction II. Growth Hormone and Insulin-like Growth Facter-I (IGF-I) and Cognitive Decline in the Elderly III. Interactions between IGF-I System and Alzheimer’s Disease IV. Growth Hormone in Alzheimer’s Disease V. Growth Hormone Response in Alzheimer’s Disease VI. Somatostatin in Cerebrospinal Fluid in Alzheimer’s Disease VII. Relationship Between Alzheimer’s Disease Treatment and IGF-I System and Growth Hormone Response to Stimuli VIII. Possible Usefulness of Growth Hormone/IGF-I Axis in Alzheimer’s Disease Treatment References

neuronal survival and plays a role in neuronal rescue during degenerative diseases. The investigations of GH-releasing stimulation tests, especially of GHRH in AD, are equivocal and, in some cases, contradictory. When a cholinesterase inhibitor, such as rivastigmine, a drug for AD, is acutely administered, the area under the curve of the GH response to GHRH doubles, showing that rivastigmine is a powerful drug in the enhancement of GH release. Consequently, an emerging clinical target for improving the quality of life with aging, or for improving the clinical manifestations of AD, may be the activation of GH/IGF-I, which rejuvenates the axis, resulting in an overall physiological benefit, with a potential for preventing or reversing detrimental age-related or AD changes in the brain.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by insidious onset with cognitive impairment. It affects mental function and is of the amnesic type. Neuropathological analysis of AD-affected brains reveals extensive atrophy and an accumulation of neurofibrillary tangles. Taken together, neurochemical changes in the brain in patients with AD indicate multiple disturbances, and it seems likely that the changes are secondary to more fundamental changes in the brain. There is a physiological decline of the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis with aging, and the possibility that the GH/IGF-I axis is involved in cognitive deficits has been recognized for several years. The IGF-I is a potent neurotrophic as well as a neuroprotective factor found in the brain, with a wide range of actions in both the central and the peripheral nervous systems. IGF-I is a critical promoter of brain development and

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. INTRODUCTION Dementia disorders are syndromes in which there is evidence of decline in memory and thinking, deterioration of emotional functions, and an impairment of motor performance sufficient to restrict functioning in daily life. The classification of dementias must be made at different levels, and usually they are subgrouped as primary degenerative dementias, vascular dementias, and secondary dementias; this classification implies that the syndrome is heterogeneous from the etiological and pathogenetic points of view. Alzheimer’s disease (AD) is the most common dementing illness in the elderly and is a progressive neurodegenerative disorder characterized by insidious onset with cognitive impairment and affected mental function, amnesic type of memory

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impairment, visuospatial deficits, deterioration of language and self-sufficiency with aggressive behavior, and an inexorable progression; the disease is a leading cause of morbidity and mortality in the geriatric population (McKhann et al., 1984). Neuropathological analysis of AD-affected brains reveals extensive atrophy due to neuronal loss and an accumulation of neurofibrillary tangles and neuritic plaques, surrounded by a tract of neuroinflammation and a loss of neurons. These events are observed not only in the cerebral cortex, but also in other brain regions, such as the hypothalamus, thalamus, and brain stem. Neuritic plaques consist of deposits of variously sized peptides, collectively called ␤-amyloid, and it is now widely accepted that the key player in the pathological cascade leading to AD is initiated by the accumulation of amyloid plaques in the brain. The mechanisms of plaque formation are not entirely clear: it is not evident whether different factors, such as vascular or immune dysfunction, calcium dysregulation, free radical accumulation, or other disturbances, are the origin of plaque formation or a consequence of it (Butterfield et al., 2002). Within this context, reducing AD amyloidosis represents one of the main therapeutic strategies for AD. However, AD is associated with significant structural and functional changes in many peptides in the brain, and it is well known that these peptides are important for communication. Some peptides function as neurotransmitters or neuromodulators. The most consistent change within the brain is the degeneration of cholinergic projections from the basal forebrain to the hippocampus and neocortex (Davis et al., 1999). Cortical somatostatin concentrations have also been reported to be reduced in varying degrees in AD patients, with a loss of cortical somatostatin receptors and diminished cerebrospinal somatostatin concentrations (Atack et al., 1988; Gottfries, 1990). The infracortical white matter of the adult human cerebral cortex, named interstitial cells, in AD shows somatostatin expression and is susceptible to develop the AD-related cytoskeletal changes; in this case the progression in cytoskeleton changes is accompanied by a loss of somatostatin (van de Nes et al., 2002). Neuropeptides are synthesized as large inactive polypeptide precursors that undergo endoproteolytic cleavage at specific basic residue sites to produce biologically active peptides; this conversion is often mediated by protein convertases, but the content of the proprotein convertase 2 responsible for the processing of neuropeptide precursors is more similar in AD patients than in controls (Winsky-Sommerer et al., 2003). AD is also associated with significant structural and functional changes in the noradrenergic system (Mattews et al., 2002), in corticotropin-releasing factor immunoreactivity (Davis et al., 1999), and in opioid peptides (Heilig et al., 1995; Nyberg, 2004). Taken together, the neurochemical changes reported in the brain in patients with AD indicate multiple disturbances, and at the present level of knowledge it is not possible to single out any of these

changes as being of especial significance for the disease; it seems likely that the changes are secondary to more fundamental alterations in the brain.

II. GROWTH HORMONE AND INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AND COGNITIVE DECLINE IN THE ELDERLY A. Physiological Decline of Growth Hormone and IGF-I with Aging There is a physiological decline of the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis with aging (Corpas et al., 1993; Gómez et al., 2003, 2004), and the possibility that the GH/IGF-I axis is involved in cognitive deficits has been recognized for several years. The highaffinity IGF-binding proteins (IGFBP1 to 6) of the IGF family have also been involved in IGF-I regulation, and it is important to include the IGF-independent properties, particularly those of IGFBP3 (Le Roith et al., 2001). A small proportion of circulating IGF-I is detected in the free or readily dissociate state, which is thought to be the metabolically active form, and more than 99% of total IGF-I in serum is complexed with binding proteins and acid-labile subunit; thus, free IGF-I measures the true IGF-I that is available to the tissues (Frytsk et al., 1995). The amount of free IGF-I is dependent on a complex interplay between the production rate and the concentrations of IGF-I and IGFBP3, but it is still a matter of debate as to what extent IGF-I bioactivity is maintained by circulating and/or tissuederived IGF-I (Le Roith et al., 2001). The strongly significant decline in plasma IGF-I, free IGF-I, and IGFBP3 between the ages of 15 and 70 (Gómez et al., 2003) may be the result of a number of processes: (1) the age decline in GH secretion, which has been well documented (Corpas et al., 1993), and (2) the decline in the number of GH receptors in the liver. It remains uncertain whether plasma IGFBP3 concentrations are directly stimulated by GH alone, by IGF-I alone, or by both GH and IGF-I, but the reported decrease in IGFBP3 is probably a consequence of a decrease in the secretion of GH and IGF-I (Corpas et al., 1993; Le Roith et al., 2001).

B. Growth Hormone and IGF-I and Age-Related Decline In population-based studies of elderly subjects, serum total IGF-I and the total IGF-I to IGFBP3 ratio were inversely related to cognitive decline in the next years of follow-up (Rollero et al., 1998; Alemán et al., 1999; Kalmijn et al., 2000). As GH secretion is one of the main regulators of circulating IGF-I and IGFBP3, this finding

23. GH and IGF-I in Alzheimer’s Disease

suggests that GH secretion plays a important role in agerelated cognitive decline. Although free IGF-I concentrations are probably a better indicator of GH secretion than total IGF-I, and free IGF-I is the major IGF-I component responsible for GH suppression (Chapman et al., 1998), free IGF-I concentrations are not associated with cognitive decline (Kalmijn et al., 2000) In addition, some authors have observed that the Mini-Mental State Examination scores in elderly subjects are not related to basal GH or GH response after GHRH stimulation, whereas they are positively related to IGF-I concentrations (Rollero et al., 1998). These studies suggest that factors other than GH secretion are involved in the relationship of the IGF-I axis to cognitive decline as parameters that reflect protein caloric malnutrition and decreased physical activity that possibly take part in affecting IGF-I function in subjects with mild cognitive impairment; reciprocally, IGF-I decrement might affect the neuronal function. It has been demonstrated that healthy centenarians have a plasma IGF-I/IGFBP-3 molar ratio greater than those in aged subjects, and such a parameter is associated with a lesser amount of fat mass, a more favorable insulin action, a more favorable lipidic profile, and a positive association with the degree of cognitive function (Paolisso et al., 1997). In a prospective population-based study in older persons with a 3-year follow-up, low IGF-I status was associated with lower levels of quality of life and a greater decline in cognition (Rollero et al., 1998). In children, GH deficiency, a state characterized by low IGF-I concentrations, has been associated with significant cognitive deficits (Burman and Deijen, 1998), and also adult men with childhood GH deficiency had lower memory function compared to controls; this function improved to normal scores within 1 year of GH replacement therapy (Deijen et al., 1998).

C. ␤-Amyloid and IGF-I Axis in Alzheimer’s Disease Amyloid precursor protein, a precursor of ␤-amyloid, is one the molecules involved in the pathogenesis of AD, but the molecular mechanisms of cytotoxicity have not yet been completely clarified. Studying the citotoxic mechanism of London type 2, neuroprotective factors, IGF-I and humanin, protect the neurons of the amyloid precursor protein toxicity (Niikura et al., 2004). However, plasma IGF-I concentrations have been shown to be low in family members carrying the Swedish amyloid precursor protein (APP 670/671) mutation, with or without AD, and without any differences in GH and prolactin concentrations; these results indicate that the mechanisms regulating GH and prolactin are preserved and that those regulating IGF-I may be affected in AD patients (Mustafa et al., 1999). It has been demonstrated that IGF-I serum concentrations in AD were found to be in the lower part of the reference range for

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adult subjects or below; they were lowered in patients with more advanced cognitive deterioration, and these concentrations were in correlation with midarm circumference, the decrease in which is thought to reflect proteic caloric malnutrition (Rollero et al., 1998). The ability of IGF-I to cross the blood–brain barrier strongly suggests that IGF-I interacts directly with the brain (Dik et al., 2003). Alternatively, IGF-I may be a biomarker of another parameter that may influence cognitive function, such as cortisol. Glucocorticoids influence the production of IGF-I and modify its systemic and neurotrophic biological activity by inducing changes in IGF-binding proteins; in AD cases, total IGF-I and IGFBP-3 were lowered, whereas IGFBP-1 was higher than in controls and cortisol was directly — and IGF-I inversely — correlated with cognitive impairment (Murialdo et al., 2001).

III. INTERACTIONS BETWEEN IGF-I SYSTEM AND ALZHEIMER’S DISEASE A. IGF-I System as a Neurotrophic Factor IGF-I is a potent neurotrophic as well as neuroprotective factor found in the brain: virtually all brain regions possess IGF-I-binding sites, and IGF-I receptors are expressed ubiquitously. Consequently, this factor is a pleiotropic polypeptide with a wide range of actions in both the central and the peripheral nervous systems. IGF-I is a critical promoter of brain development and neuronal survival and acts as a trophic factor in both the developing and the adult central nervous systems; it also plays a role in neuronal rescue during degenerative diseases (Venters et al., 2001). Transport of peripheral IGF-I across the blood–brain barrier is accomplished by receptor-mediated transport across endothelial cells. IGF-I reverses the effects of ␤-amylin and amylin-induced neurotoxicity in hippocampal cells and is involved in facilitating neuronal repair and in promoting neuronal survival in the entorhinal cortex, which is affected in AD. Interactions between the IGF-I system and the neurotransmitters affected in AD include stimulation of the catecholaminergic enzyme activity of tyroxine hydroxylase, dopamine ␤-hydroxylase, and phenylethanolamine N-methyltransferase. IGF-I also increases choline acetyltransferase expression, supports the survival of cholinergic neurons, and modulates acetylcholine release in hippocampal and cortical neurons affected in AD. ␤-Amylin induces apoptosis-related changes in cortical and hippocampal cells, including caspase activation and mitochondrial membrane dysregulation. IGF-I induces the B-cell leukemia-2 gene product (Bcl-2) family that regulates apoptosis and is able to suppress caspase-1-mediated cell death through a mechanism

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independent of the expression of Bcl-2. IGF-I also activates antiapoptotic pathways involving Ras, phosphatidylinositol 3-kinase, protein kinase B, and Bad modulator and may be involved in maintaining mitochondrial membrane potential; those IGF-I decreased levels leave the cell more susceptible to noxious stimuli (Lackey et al., 2000). Furthermore, accumulated evidence indicates that IGF-I protects neurons against cell death induced by amyloidogenic derivates via the activation of intracellular pathways implicating phosphatidylinositide 3/Akt kinase, of the family which belongs to the transcription factor FKHRL1 phosphorylation or production of free radicals (Zheng et al., 2000) (Table I). IGF-I may carry out neurotrophic activity, as it protects and even rescues rat hippocampal primary culture neurons from ␤-amylin toxicity (Dore et al., 1997). Studies in primary cultures have shown the properties of IGF-I that are able to rescue neurons against ␤ toxicity (Dore et al., 1999). IGF-I is actively transported through the blood–brain barrier, but its specific mechanism for this process is poorly understood; because IGF-I is also produced in endothelial and smooth muscle cells, the possibility exists that both plasma IGF-I and vascular-derived IGF-I are the primary sources of IGF-I for the brain. Total IGF-I concentrations in the brain decrease by 30–40% with age, and probably the reduction in vessel density is one the factors that contribute to the decline in brain IGF-I concentrations (Sonntag et al., 2000). The high expression of IGF-I in vasculature has led some authors to propose that both GH and IGF-I have an important role in blood vessel growth and repair. In rats, the decrease in plasma IGF-I is accompanied by an increase

Table I Neuroprotective Actions of IGF-I 1. Stimulation in catecholaminergic enzyme activity of tyroxine hydroxylase 2. Stimulation of dopamine ␤-hydroxylase and phenylethanolamine N-methyltransferase 3. Increases choline acetyltransferase 4. Expression and support of the survival of cholinergic neurons 5. Influences the antiapoptotic pathways involving Ras, phosphatidylinositol 3-kinase, protein kinase B, and Bad modulator 6. Maintains the mitochondrial membrane potential 7. Protects neurons against cell death induced by amyloidogenic derivates via phosphatidylinositide 3/Akt kinase, family of transcription factor FKHRL1 phosphorylation, or production of free radicals 8. Reverses the effects of ␤-amylin and amylin-induced neurotoxicity in hippocampal cells 9. Alzheimer-associated neuronal thread protein increases neuronal death by apoptosis and may be mediated by impaired insulin/IGF-I signaling 10. Neuroprotective actions of IGF-I may be inhibited by IGFBP3

in perivascular IGF-I, and administration of IGF-I during transient ischemia reduces the resulting area of infarct (Guan et al., 1996).

B. Tumor Necrosis Factor ␣ and Its Relationship with IGF-I Effects However, tumor necrosis factor ␣ (TNF␣) is both a promoter and an inhibitor of neurodegeneration. TNF␣ is a proinflammatory cytokine that promotes neuronal death in vitro as well as in vivo, and TNF␣ and IGF-I are potently induced in the same areas of the central nervous system in response to neurodegenerative conditions. TNF␣ is also implicated in a variety of degenerative diseases, including AD (Collins et al., 2000). Emerging possibilities are that this cytokine and IGF-I interact with each other (Venters et al., 2001), that AD is associated with the dysregulation of proinflammatory cytokines in the brain and serum TNF␣, and that a haplotype for TNF␣ is associated with AD (Collins et al., 2000), but the role of TNF␣ in AD is not clear. In experimental studies it has been demonstrated that only one concentration of TNF␣ is protective over neurons, while the neuroprotective effects of IGF-I are linear, and that the interactions between IGF-I and TNF␣ receptors may exceed the relevance of their individual roles. TNF␣ production may promote neurodegeneration not through the direct killing of neurons, but rather through the inhibition of IGF-I survival signaling (Venters et al., 2001).

C. Insulin and IGF-I in Alzheimer’s Disease AD patients were also reported to have increased fasting glucose and insulin concentrations, thus indicating insulin resistance in AD etiology; glucose utilization is decreased in AD, as are the glucose transporters GLUT-1 and GLUT-3 levels in the brains of AD patients. An emerging body of evidence suggests that an increased prevalence of insulin abnormalities and insulin resistance in AD may contribute to the disease (Watson and Craft, 2003); in AD patients, hyperinsulinism induced without hyperglycemia enhances memory significantly (Craft et al., 1999). Both insulin and IGF-I belong to the same protein family and are important modulators of brain function, and the ability of insulin/IGF-I to modulate neuronal excitability and synaptic plasticity underlies the modulatory effects of these hormones on cognitive processes (Craft et al., 2000). Also, insulin as IGF-I concentrations are altered in many other types of human degenerative diseases (Busiguina et al., 2000). Impaired insulin sensitivity and resultant increased glucose concentrations may potentiate neuronal death and induce other complications. Studies associating AD and diabetes mellitus are conflicting, but there are others that document an increased incidence of AD and vascular dementia in

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diabetes mellitus (Skoog, 1998). Thus, changes in IGF-I and insulin, among other pathological alterations, may be an additional factor involved in the progression of neurodegenerative processes in AD. Resistance to insulin is closely interrelated with IGF-I signaling in the sense that IGF-I resistance originates insulin resistance and vice versa (Carro et al., 2004). Regulation of brain ␤-amyloid by insulin has a double-sided effect on brain. It stimulates neuronal release of ␤-amyloid and, at the same time, contributes to the extraneuronal accumulation of ␤-amyloid by competing for the insulin-degrading enzyme that degrades insulin and ␤-amyloid. The ultimate activity of insulin is to increase brain ␤-amyloid. IGF-I appears to stimulate brain ␤-amyloid elimination through a process that includes stimulation of neuronal ␤-amyloid release and clearance from the brain parenchyma (Gasparini and Xu, 2003). The effect of IGF-I in ␤-amyloid clearance is mediated by enhancing transport of the ␤-amyloid carrier proteins, albumin and transthyretin into the brain through the choroid plexus, with increased levels of ␤-amyloid in the cerebrospinal fluid and this process is blocked by TNF␣ (Venters et al., 2001).

D. Mechanisms of Action of IGF-I in Alzheimer’s Disease On the basis of decreased IGF-I concentrations in AD, some authors have suggested that disrupted IGF-I input into the brain may be involved in the pathogenesis of amyloidosis and that changed IGF-I signaling may potentially lead to amyloidosis (Carro et al., 2002). The disruption of IGF-I signaling in the coroid plexus is sufficient to trigger pathological changes such as those observed in AD and increases in brain ␤-amyloid as a consequence of its lower clearance. IGF-I resistance at the blood–brain barrier is also a pathogenic event in AD, and local alterations in IGFBPs, together with altered cytokine signaling, especially TNF␣ production, may therefore lead in AD to a loss of IGF-I input at the blood–brain barriers. Furthermore, Alzheimerassociated neuronal thread protein is one protein that accumulates in cortical neurons and colocalizes with phospho-tau-containing cytoskeletal lesions in brains with AD; overexpression of this protein results in increased neuronal death mediated by apoptosis and mitochondrial dysfunction and may be mediated by impaired insulin/IGF-I signaling. Neurons with abundant insulin or IGF-I receptors may be particularly vulnerable to the adverse effects of Alzheimer-associated neuronal thread protein (de la Monte and Wands, 2004). A novel factor, termed humanin, antagonizes neurotoxicity by various types of familial AD genes, which are mutants of amyloid precursor protein with a clear action specificity, inactivating neurotoxicity by polyglutamine repeat or superoxide dismutase 1 mutants, and which can

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also inhibit neurotoxicity by other AD-relevant insults; humanin makes other familial AD genes ineffective, such as amyloid precursor protein stimulation, antiamyloid precursor protein antibody, and other ␤-amyloid peptides (Hashimoto et al., 2001).

E. Insulin-like Growth Factor-Binding Protein-3 in Alzheimer’s Disease IGFBP3 has been shown to have a function in the regulation of cell survival, not only through the regulation of free IGF-I, but also via several IGF-independent effects. In particular, it has been shown to induce apoptosis; however, IGFBP3 interacts with humanin, and this connection may be important in AD as a target for future drug development. ␤-Amyloid deposition in cerebral blood vessel walls is one of the key features of AD. Studying the mechanisms of ␤-amyloid toxicity, a comparative cDNA expression array was performed to detect the differential gene expression of untreated human pericytes. The increase in cellular mRNA of IGFBP3 expression was confirmed (Rensink et al., 2002), and an increased expression of IGFBP3 has been related to the inhibition of cell growth and to the induction of apoptosis (Buckbinder et al., 1995). The neuroprotective actions of IGF-I may be inhibited by IGFBP3 by sequestering IGF-I from its receptor, thereby inhibiting cell proliferation and inducing apoptosis (Rensink et al., 2002).

IV. GROWTH HORMONE IN ALZHEIMER’S DISEASE Chronic GH deficiency, coupled with the decline in skeletal muscle mass and protein synthesis, contributes to frailty in the elderly and suggests that hormonal deficiency may be a factor in age-related disability. IGF-I has an important trophic role in neuronal function and limits neuronal loss after ischemic damage (Guan et al., 1996). The majority of evidence suggests that GH does not cross the blood–brain barrier, although it is known that hypophysectomy decreases both GH and IGF-I mRNA in brains restored by GH administration (Sonntag et al., 2000). Some studies suggest that GH treatment (HernbergStahl et al., 2001; Mesa et al., 2003) improves alertness, vitality, and mood and induces a sense of well-being, but some data suggest that GH may have an impact on brain function by increasing plasma IGF-I concentrations or by regulating brain IGF-I levels. The effect of long-term GH-releasing hormone in animals has a specific influence on spatial memory, but probably this effect is mediated by an increase of IGF-I in the brain (Sonntag et al., 2000).

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V. GROWTH HORMONE RESPONSE IN ALZHEIMER’S DISEASE A. Neuroendocrine Regulation of Growth Hormone in Alzheimer’s Disease Some authors have indicated that neuroendocrine regulation of GH is damaged more selectively and severely in AD patients than in patients suffering from other dementias at the hypothalamus (Higashi et al., 1994). We know the difficulty involved in separating AD from vascular dementia by clinical methods and the possibility that this classification may be wrong in a number of patients. Neurochemical investigation of postmortem human brain material of AD patients has shown multiple changes in the neurotransmitter metabolism: the most consistent changes are the degeneration of cholinergic projections, but cortical somatostatin concentrations have also been reported with a loss of cortical somatostatin receptors, and multiple region-specific neurotransmitter system abnormalities in the AD hypothalamus have been documented (Gottfries, 1990). Consequently, the hypothesis that the abnormal function of some of these neuropeptide systems may contribute to the symptoms of AD is supported by some studies (Raskind et al., 1986). Somatostatin concentrations are widely, but consistently, reported to be reduced to 21–62% of control values in the temporal cortex, and the clinical relevance of altered somatostatin in AD is also suggested by reports of correlation between the degree of cognitive impairment and the reduction of somatostatin concentrations in the brain. The cerebrospinal fluid levels of somatostatin are also lower in AD patients (Raskind et al., 1986; Atack et al., 1988); this decrease is more severe in patients with an earlier age of AD onset (Patel, 1992).

B.Growth Hormone Response to Growth Hormone-Releasing Hormone An initial study reported that patients with early onset AD showed an exaggerated GH response to GHRH, whereas those with late onset showed a normal response (Cacabelos et al., 1988), and that as a consequence, the GHRH test might constitute a useful antemortem marker for AD, especially in early stages of the disease. Later studies demonstrated a normal but delayed GH response to GHRH, suggesting that somatostatin deficiency is primary in those regions of the brain unrelated to neuroendocrine regulation of the GH response to GHRH (Nemeroff et al., 1989). Other studies of GH response to the GHRH provocative test and of TSH response to TRH in primary dementias, AD, parkinsonism with dementia, progressive supranuclear palsy, or vascular dementia, showed no differences among the different

groups. Nevertheless, the response was higher in all patient groups than in normal subjects; in AD patients there was a group of patients with an exaggerated GH response (52%), a group with a normal GH response (39%), and a group with a blunted GH response (9%). Among the four patients with early onset AD, two showed an exaggerated response of GH to GHRH and two showed a normal response. The pattern of TSH response to TRH was similar to that of GH to GHRH, but without any correlation between the responses of the two hormonal axes (Gómez Sáez et al., 1991; Gómez et al., 1994). It would appear that the GH response to GHRH cannot be useful as a diagnostic aid in AD patients because the response is similar to that in other dementias, especially those of vascular origin. When AD patients were examined concurrently, the anatomical findings assessed by computed tomography, the results of neuropsychological tests and GH response to GHRH or TSH response to TRH, we can demonstrate the lack of correlation between the severity of the AD and the hormonal responses to their stimuli, thus suggesting that psychometric and morphologic assessment discrimination of AD may be more accurate in the diagnosis and assessment of dementia than hormonal responses (Gómez et al., 2000). Results of several studies addressing this point show varied results: superimposable responses of GH to GHRH than responses of GH to GHRH in controls; a blunted GH to GHRH response in AD patients; higher GH concentrations in the morning; and a greater increase of GH to GHRH in AD patients than in controls (Higahsi et al., 1994) (Table II). More recent studies demonstrate the reduced release of GH to GHRH in AD patients (Obermayr et al., 2003). In general, investigations of GH-releasing stimulation tests, especially of GHRH in AD, are equivocal and, in some cases, contradictory. Factors that may contribute to the inconsistent findings include (1) the variability of GHRH stimulated among control groups, (2) the age and sex of patients, (3) the lack of uniformity in test procedures, and (4) the variability and the lack of reproducibility of the GHRH test either in controls or in AD patients (Ghigo et al., 1993; Skare et al., 1994).

Table II Different Patterns of Response of GH to Its Stimuli in Alzheimer’s Disease Patients 1. Higher GH concentrations in the morning 2. Superimposable GH response to GHRH than GH response to GHRH in controls 3. Blunted GH response to GHRH in Alzheimer’s disease patients 4. Higher increase of GH after GHRH in Alzheimer’s disease patients 5. Blunted GH response to clonidine in Alzheimer’s disease patients, with higher levels of aggression

23. GH and IGF-I in Alzheimer’s Disease

C. Growth Hormone Response to Clonidine AD is also associated with significant structural and functional changes in the noradrenergic system, and the GH response to a clonidine challenge reflects the sensitivity of the postsynaptic ␣2-noradrenergic receptors because GH secretion is stimulated through the activation of postsynaptic ␣2-noradrenergic receptors in the hypothalamus. Decreased activity of presynaptic noradrenergic neurons has been associated with an augmentation of the GH response to clonidine. A blunted GH response to clonidine reflects noradrenergic overactivity that has been demonstrated in normal aging. When AD patients were divided into those with a preserved GH response and those with a blunted GH response, patients with a blunted response had higher levels of aggression; they also showed greater reductions in aggression following a treatment with a ␤ blocker, pindolol, compared to those undergoing placebo therapy. These findings are consistent with the fact that patients with a blunted GH response have a reflection of postsynaptic ␣2-adrenergic receptor compensatory downregulation in response to noradrenergic overactiviy in aggressive AD patients (Herrmann et al., 2004a,b). In a postmortem study, noradrenergic neuron loss in the rostral locus ceruleus was correlated positively with aggressive behaviors, and those studies suggest that aggressivity in AD may result from greater noradrenergic neuron loss in the locus ceruleus associated with preserved postsynaptic noradrenergic receptors in a variety of brain regions (Matthews et al., 2002).

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VII. RELATIONSHIP BETWEEN ALZHEIMER’S DISEASE TREATMENT AND IGF-I SYSTEM AND GROWTH HORMONE RESPONSE TO STIMULI A. Treatment of Alzheimer’s Disease No antiamyloid therapies are currently available, and treatment is based on neuroprotective approaches, antioxidants, anti-inflamatory agents, cholinesterase inhibitors, the management of neuropsychiatric symptoms and behavioral disturbances, and general medical treatment (Ritchie et al., 2004; Cummings, 2004). A central feature of AD is impaired cholinergic function in the brain, with a loss of enzyme activity in the cholinergic system. Cholinesterase plays a major role in the metabolic degradation of acetylcholine and has been shown previously to play an important role in AD (Davidsson et al., 2001). Cholinesterase inhibitors are approved for the treatment of mild-to-moderate AD and should be considered as a standard of care for AD patients. Four cholinesterase inhibitors are available— tacrine, donezepil, rivastigmine, and galantamine—and pivotal clinical trials have shown changes on the AD assessment scale and may help affected patients maintain, with significant benefits, their ability to perform everyday activities (Edwards et al., 2004). The combination of cholinesterase inhibitors with memantine, a N-metil-D-aspartic receptor antagonist, is superior to cholinesterase inhibitors therapy (Farlow et al., 2003).

B. Treatment with Cholinesterase Inhibitors VI. SOMATOSTATIN IN CEREBROSPINAL FLUID IN ALZHEIMER’S DISEASE Although the hypothalamus contains the highest concentration of somatostatin in the human brain, the cerebral cortex and the spinal cord probably contribute to cerebrospinal somatostatin concentrations. Previous studies reported that somatostatin cerebrospinal concentrations were reduced to varying degrees in AD patients, thus suggesting that dysfunction of the cortical somatostatin system in AD is reflected in reduced cerebrospinal fluid concentrations (Raskind et al., 1986; Atack et al., 1988) or is decreased consistently in AD (Nilsson et al., 2001). Other studies have reported that somatostatin cerebrospinal concentrations have not been found to be reduced (Doraiswamy et al., 1991); in this regard there are no differences between AD patients and vascular dementia patients (Heilig et al., 1995; Gómez et al., 1996). In both groups, AD patients and vascular dementia patients, there was no correlation between GH response to GHRH and the sex, stage of the disease, and somatostatin cerebrospinal concentrations (Gómez et al., 1996).

Evidence shows that GH secretion and IGF-I system concentrations are reduced in normal elderly subjects as well as in patients with AD. Cholinesterase inhibitors such as pyridostigmine are able to elicit GH secretion when administered alone and to enhance the GH response to GHRH. Several previous studies have demonstrated the effects of pyridostigmine, a substance probably acting via the inhibition of hypothalamic somatostatin. After acute pyridostigmine administration the GH to GHRH response is potentiated both in elderly and in AD patients (Ghigo et al., 1993) and in those suffering from other neurological diseases (Gómez et al., 1993). When a cholinesterase inhibitor such as rivastigmine, a drug for AD, was administered acutely, the area under the curve of the GH response to GHRH nearly doubles, showing that rivastigmine is a powerful drug for enhancing GH release (Obermayr et al., 2003). In controls, subsequent to 8 weeks of treatment with the very well-tolerated selective cholinesterase inhibitor donezepil, the area under the curve of the GH response to GHRH increased by 52%, and, remarkably, IGF-I concentrations increased by 28%, which means a significant and probably clinically relevant shift of IGF-I concentrations to those seen in younger subjects (Huber et al., 2004).

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No long-term studies have examined whether such a new therapeutic intervention could delay or reverse some of the manifestations of the decrease of GH in the elderly or the manifestations as a consequence of lower IGF-I concentrations in AD. Therefore, an emerging clinical target for improving the quality of life with aging or for improving the clinical manifestations of AD may be the activation of GH/IGF-I, which rejuvenates the axis to result in an overall physiological benefit and a potential for preventing or reversing detrimental age-related or AD changes in the brain (Smith, 2000)

potential in the treatment of cognitive impairment (Doggrell, 2004). A group of AD patients has been treated with GM1 ganglioside by continuous injection into the frontal horns of the lateral ventricles for 12 months; the progression of deterioration was stopped and neuropsychological assessment improved with a increase in cerebrospinal concentrations of monoamine metabolites and somatostatin (Svennerholm et al., 2002).

References VIII. POSSIBLE USEFULNESS OF GROWTH HORMONE/IGF-I AXIS IN ALZHEIMER’S DISEASE TREATMENT Much research has been done over the past decades on the role of IGF-I in the maintenance of normal homeostasis, but limited attention has been given to the potential significance of IGFs in the central nervous system. Trophic as well as neuromodulatory roles of IGFs in the brain are well known. Their effects, particularly those of IGF-I on key markers in AD brains, namely cholinergic dysfunction, neuronal amyloid toxicity, tau phosphorylation, and glucose metabolism, suggest the potential usefulness of this factor in the treatment of neurodegernerative diseases. Avenues and bases for further investigation have been exposed in an effort to further understanding of AD and other neurodegenerative diseases. However, the poor availability of IGF-I, and its penetration of the brain, hampers progress in this regard (Dore et al., 2000). However, the GH secretagogue receptor is a target of new drugs that can reverse the age-related reduction in the amplitude of GH pulsatility and IGF-I concentrations, as has been demonstrated with MK-0677, whose effects over the GH/IGF-I axis were sustained and accompanied by a better lean/fat ratio and a modest increase in strength (Smith, 2000). IGFBP3 has been shown to have a function in the regulation of cell survival via several IGF-independent effects; in particular, it has been shown to induce apoptosis. It also interacts with humanin, and this connection may be important in AD as a target for drug development because its high potency, full efficacy, and the strict selectivity of its activity against a very wide spectrum of AD insults disclose a possibility that humanin and its additional derivatives may be quite useful as new therapeutic reagents for AD cases (Rensink et al., 2002; Ikonen et al., 2003). FK960 is a somatostatin-releasing agent, and after its administration to rhesus monkeys it is distributed to the entire brain; the activation of somatostatinergic neurotransmission with FK960 is associated with cognitive impairment, thus suggesting that it is a drug with considerable

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Raskind, M. A., Peskind, E. R., Lampe, T. H., Risse, S. C., Taborsky, G. J., Jr., and Dorsa, D. (1986). Cerebrospinal fluid vasopressin, oxytocin, somatostatin, and ␤-endorphin in Alzheimer’s disease. Arch. Gen. Psychiatr. 43, 382–388. Renksink, A. A. M., Gellekink, H., Otte-Höller, I., ten Donkelaar, H. J., de Waal, R. M. W., Verbeek, M. M., and Kremer, B. (2002). Expression of the cytokine leukemia inhibitory factor and pro-apoptotic insulin-like growth factor binding protein-3 in Alzheimer’s disease. Acta Neuropathol. (Berl.) 104, 525–533. Ritchie, C. W., Ames, D., Clayton, T., and Lai, R. (2004). Metaanalysis of randomized trials of the efficacy and safety of donazepil, galantamine, and rivastigmine for the treatment of Alzheimer’s disease. Am. J. Geriatr. Psychiatr. 12, 358–369. Rollero, A., Murialdo, G., Fonzi, S., Garrone, S., Gianelli, M. V., Gazzerro, E., Barreca, A., and Polleri, A. (1998). Relationship between cognitive function, growth hormone and insulin-like growth factor-I plasma levels in aged subjects. Neuropsychobiology 38, 73–79. Skare, S. S., Dysken, M. W., and Billington, C. J. (1994). A review of GHRH stimulation test in psychiatry. 36, 249–265. Skoog, I. (1998). Status of risk factors for vascular dementia. Neuroepidemiology 17, 2–9. Smith, R. G. (2000). The aging process: Where are the drug opportunities?. Curr. Opin. Chem. Biol. 4, 371–376. Sonntag, W. E., Lynch, C., Thornton, P., Khan, A., Bennet, S., and Ingram, R. (2000). The effects of growth hormone and IGF-1 deficiency on cerebrovascular and brain ageing. J. Anat. 197, 575–585. Svennerholm, L., Brane, G., Karlsson, I., Lekman, A., Ramstom, I., and Wikkelso, O. C. (2002). Alzheimer disease-effect of continuous intracerebroventricular treatment with GM1 ganglioside and a systematic activation programme. Dem. Geriatr. Cogn. Disor. 14, 128–136. van de Nes, J. A. P., Sandmann-Keil, D., and Braak, H. (2002). Interstitial cells subjacent to entorthinal region expressing somatostatin-28 immunoreactivity are susceptible to development of Alzheimer’s diseaserelated cytoskeletal changes. Acta Neuropathol. (Berl.) 104, 351–356. Venters, H. D., Broussard, S. R., Zhou, J.-H., Bluthé, R. M., Freund, G. G., Johnson, R. W., Dantzer, R., and Kelley, K. W. (2001). Tumor necrosis factora and insulin-like growth factor-I in the brain: Is the whole greater than the sum of its parts? J. Immunol. 119, 151–165. Watson, G. S., and Craft, S. (2003). The role of insulin resistance in the pathogenesis of Alzheimer’s disease: Implications for treatment. CNS Drugs 17, 27–45. Winsky-Sommerer, R., Grouselle, D., Rougeot, C., Laurent, V., David, J. P., Delacourte, A., Dournaud, P., Sidah, N. G., Lindberg, I., Trottier, S., and Epelbaum, J. (2003). The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer’s disease. Neuroscience 122, 437–447. Zheng, W. H., Kar, S., Dore, S., and Quirion, R (2000). Insulin-like growth factor-I (IGF-1): A neuroprotective trop factor acting via Akt kinase pathway. J. Neural. Transm. Suppl. 60, 261–272.

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24 Growth Hormone Antagonists: A Pharmacological Tool in Present and Future Therapies JOHN J. KOPCHICK, LINGUA QIU, ELAHU GOSNEY, CHAD KELLER, AMANDA PALMER, and SUDHA SANKARAN Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701

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role in growth promotion. GH regulates many genes, including insulin-like growth factor-I (IGF-I) in the liver and other tissues (Fig. 1). IGF-I itself is a potent growth factor that acts on many tissues (Humbel, 1990). GH also promotes metabolic changes, including both anabolic and catabolic events. GH deficiency results in dwarfism in children and metabolic changes in adults. However, hypersecretion of GH due to a GH-secreting pituitary adenoma is responsible for a syndrome called acromegaly (Melmed, 1990; Melmed et al., 1983; Orme et al., 1998). GH contains 191 amino acids with a molecular mass of approximately 22,000 Da. It possesses four ␣ helices and two disulfide bonds (Fig. 2) (Abdel-Meguid et al., 1987; de Vos et al., 1992). The growth-promoting activity of GH is a result of GH binding to its predimerized membrane receptor (R), which results in GH-induced intracellular signal transduction (Gent et al., 2002; Ross et al., 2001; Harding et al., 1996b). Although the mechanisms underlying the signal transduction are not fully known, it has been demonstrated that GH binds to two identical preformed GHRs using two distinct sites on the GH molecule, termed site 1 and site 2 (Fig. 3A). Following binding of one GH molecule to the preformed GHR dimer, tyrosine phosphorylation of GHR and subsequent phosphorylation and activation of signaling molecules such as Janus Kinase 2 (JAK2) and members of signal transducers and activators of transcription family, in particular STAT5, occur (Foster et al., 1988; Stred et al., 1990, 1992; Stubbart et al., 1991; Wang et al., 1992, 1993, 1994). Other reported intracellular signaling systems include

Introduction The Discovery of Growth Hormone Antagonists Development of Pegvisomant Preclinical Trials Clinical Trials Other Uses of Pegvisomant Conclusion References

Structure–function analysis of growth hormone (GH) resulted in the discovery of a growth hormone antagonist (A). The GHA competes with native GH and inhibits its proper or functional binding to and activation of GH receptors. Subsequent development of a GHA (Pegvisomant) resulted in a novel drug for the treatment of acromegalic individuals. The ability of Pegvisomant to normalize elevated circulating IGF-I levels in patients with acromegaly serves as a new therapeutic approach toward this disorder. Also, the potential role of GHAs in the treatment of certain types of cancer, as an insulin sensitizer, and in treatment of diabetes end organ damage is discussed.

I. INTRODUCTION Growth hormone (GH) is a protein that belongs to the somatotrophin/prolactin family. It is secreted by the pituitary gland (Evans et al., 1921; Li et al., 1944) and plays a major

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Stress, sleep, exercise + Somatostatin GHRH

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FIGURE 3 Schematic representation and comparison of the interactions GH

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FIGURE 1 Regulation of GH secretion and its effects on target tissues. GH secretion is regulated by somatostatin and growth hormone-releasing hormone (GHRH) produced in the hypothalamus. Stress, sleep, and exercise also enhance GH secretion indirectly. GH is released into the circulation in secretory spikes. GH then acts on target tissues such as liver, muscle, adipose, and bone by binding to its cell membrane receptors (GHRs). The secretion of insulin-like growth factor I (IGF-I) from these tissues is enhanced as a response to the activation of GH. IGF-I is a potent growth-promoting factor. High plasma concentrations of IGF-I can repress GH release by a negative feedback. Adapted from Kopchick et al. (2000). (See color plate 19)

pathways through protein kinase C (Tollet et al., 1991; Gorin et al., 1990) and mitogen-activated protein (MAP) kinase (Moller et al., 1992; Winston et al., 1992; Anderson 1992; Campbell et al., 1992; Harding et al., 1995).

FIGURE 2 The crystal structure of porcine (p) GH. The four ␣ helices appear as cylinders. Other loops appear as round tubes. The third ␣ helix is represented in green. Adapted from Abdel-Meguid et al. (1987). (See color plate 20)

of GH/GHR and GHA/GHR. (A) Proper binding of GH to cell surface GHR via sites, 1 and 2 in a preformed dimer results in intracellular signal transduction. (B) Nonfunctional interaction of GHA with preformed GHR dimer does not induce subsequent signal transduction. Notice that the site 2 interaction is structurally “improper.” Adapted from Kopchick (2003b). (See color plate 21)

The discovery of GHAs was derived from structure–function analysis of GH. Glycine at position 119 of third ␣ helix of bovine (b) GH (Fig. 2) or glycine-120 of human (h)GH was found to be especially critical for the biological activity of GH (Chen et al., 1991b; Chen et al., 1991a, 1994). A single amino acid substitution of this glycine with arginine, lysine, or a variety of amino acids resulted in loss of the growth-promoting activity of GH (Chen et al., 1991b). Genes encoding bGH-G119R or hGHG120R, when expressed in transgenic mice, compete with endogenous GH for the GHR and result in dwarf mice with decreased serum IGF-I (Chen et al., 1990, 1991a). These results were the first to reveal the activity and discovery of a GHA. Interestingly, the GHA bound to GHRs in the context of a dimerized GHR and was found to be internalized similarly to native GH (Harding et al., 1996b; Ross et al., 2001). However, GH-induced intracellular signaling is inhibited by the GHA (Fig. 3B). The discovery of antagonistic activity of these GH analogs provided the basis for development of the GHA as a new type of drug. It was recognized that potential clinical indications for a GHA include disorders with increased endogenous levels of GH or GH activity, such as acromegaly, or those with GH as a major pathophysiological factor. Development of the GHA resulted in a new drug termed Pegvisomant (Somavert), a GH antagonist that includes a single amino acid substitution of glycine to lysine at position 120. (Note: We will use name Pegvisomant throughout this chapter to describe the GHA that is in clinical use.) In addition to the G120R amino acid substitution, the molecule has several other additional amino acid substitutions and addition of several polyethylene glycol (PEG) moieties that improves its half-life and reduces its immunogenicity (Ross et al., 2001; Kopchick

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et al., 2002; Goffin et al., 1999). Pegvisomant has been used successfully to normalize elevated circulating IGF-I levels in acromegalic individuals who have undergone surgery or radiation therapy and been found to be resistant to somatostatin analogs (Trainer et al., 2000; van der Lely et al., 2001; Kopchick 2003a). Other studies suggest that GHAs may have a potential role in the treatment of microvascular disorders, including diabetic nephropathy and retinopathy (Bellush et al., 2000; Smith et al., 1997; Flyvbjerg et al., 1999, 2000). Due to the importance of the GH/IGF-I axis in growth-stimulating activity, GHA may play a role in the treatment of cancer. This has been supported by several experimental studies of GHA in breast, brain, and colon cancer (Pollak et al., 2001; McCutcheon et al., 2001; Friend, 2001; Friend et al., 1999). This chapter describes the experimental methodologies that resulted in the discovery of GHAs, the in vitro and in vivo data that led to the approval of clinical use of Pegvisomant, and further experimental exploitation of GHAs for other clinical indications, including the treatment and prevention of diabetic end organ damage and certain types of cancer.

II. THE DISCOVERY OF GROWTH HORMONE ANTAGONISTS The three-dimensional structure of porcine (p) GH was determined (Fig. 2) using single crystal X-ray diffraction techniques to a resolution of 2.8 Å (Abdel-Meguid et al., 1987). It was found to contain four antiparallel ␣-helical regions. Following determination of the three-dimensional structure of pGH, Chen et al. (1990) observed that bGH and pGH share ⬎90% amino acid sequence identity and, therefore, it was considered likely that the two molecules had a similar three-dimensional structure. When the ␣-helical structures of bGH were aligned into two-dimensional Edmundson wheel projections, it became apparent that an amphiphilic ␣-helical region existed within the third ␣ helix between amino acid residues 109 and 126 (Brems et al., 1988; Chen et al., 1990). This same amphiphilic ␣ helix was observed to be present in hGH between the amino acid residues 110 and 127. Within the amphiphilic segment of the third ␣ helix of bGH, the amino acids residues 117, 119, and 122 were positioned such that an imperfect amphiphilic ␣ helix was formed. As an important aside, a tryptic peptide containing the third ␣ helix of bGH had been found to exhibit low but significant growthpromoting activity (Hara et al., 1978), thus the third ␣ helix appeared to be important for biological activity. In order to determine the importance of this amphiphilic segment of the third ␣ helix of bGH relative to its growthpromoting biological activities, Chen et al. (1990) engineered a mutant bGH gene, pBGH10⌬6-M8, that encoded the following amino acid residue substitutions: E117L,

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G119R, and A122 D. These substitutions resulted in an idealized amphiphilic third ␣-helical segment. Binding studies were performed on both the wild-type bGH and the bGH-M8 and both bound GHRs in mouse liver preparations with similar affinities (Chen et al., 1990; Okada et al., 1992). To determine the in vivo activity of the bGH-M8 analog, transgenic mouse lines were generated using both the bGH and the bGH-M8 gene. Serum concentrations of bGH ranged from 0.5 to 6 ␮g/ml and concentrations of bGH-M8 ranged from 0.6 to 5 ␮g/ml in the transgenic mice. Giant bGH mice had a mean growth ratio of ⬇1.2–1.6 for females and 1.4–1.6 for males. Surprisingly, bGH-M8 transgenic mice exhibited a growth ratio of between 0.58 and 1.00 compared to their nontransgenic litter mates. This ratio was found to be directly proportional to the circulating concentration of bGH-M8 in serum. Based on the finding that there was no significant difference in the binding affinity of bGH-M8 compared to bGH to liver membrane preparations and that the bGH-M8 transgenic mice exhibited a dwarf phenotype comparative to their nontransgenic littermates, it was surmised that GH contained distinct and separate growth-promoting and receptor-binding domains (Chen et al., 1990). Considering that the amino acid sequence comparison of growth hormones shows that G-117 and A-122 are relatively conserved and that Gly-119 is conserved in all the members of the GH gene family (Watahiki et al., 1989), it was hypothesized that these amino acids may play a vital role in the biological activities of growth hormones (Chen et al., 1990). One possible mechanism for the resultant dwarf bGH-M8 transgenic mice involved insulin-like growth factors (IGF) signaling. IGF-I is produced in many tissues following binding of GH to the GHR (Froesch et al., 1985; Zapf et al., 1986) (Fig. 1). IGF-I is known to decrease GH production in the pituitary via a negative feedback mechanism (Tannenbaum et al., 1983). The dwarf mouse phenotype may have resulted from the bGH-M8 protein acting as an in vivo functional antagonist to mouse GH and, therefore, decreased serum IGF-I levels (Chen et al., 1991a). This idea was confirmed by the observation that bGH-M8 transgenic mice were found to have a serum IGF-I level approximately half that their nontransgenic litter mates (Chen et al., 1991a). The same study showed that giant bGH transgenic mice had serum IGF-I levels of nearly double their nontransgenic littermates. Also, pituitary levels of mGH were shown to be high in bGH-M8 transgenic mice and low in bGH transgenic mice compared to their respective nontransgenic littermates (Chen et al., 1991a). Further studies of bGH-M8 showed that it lacked the ability to stimulate preadipocyte differentiation in vitro of 3T3-F442A cells while retaining the ability to bind to the GHR with the same affinity as bGH. It also inhibited the ability of bGH to promote preadipocyte differentiation in this cell line (Okada et al., 1992). Together, these studies indicated that bGH-M8 was the first example of a GHA.

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To further investigate the biological growth-promoting activity of the third ␣ helix of bGH, single amino acid substitution analogs were generated at E117, G119, and A122 (Chen et al., 1991b). These substitutions were E117L, A122 D, and G119 to R, K, L, P, or W. GHR-binding assays revealed that there were no significant differences in affinities between analogs and bGH. Transgenic mouse lines were generated using each of the different single mutated genes and the resulting mice were monitored for growth rates (Fig. 4). bGHE117L mice grew at rates similar to bGH wild-type mice with a growth ratio of ⬇1.7 compared to their nontransgenic littermates. Surprisingly, transgenic mice expressing the bGHG119R and all of the other G119 substitutions exhibited a dwarf phenotype when the serum concentrations of these analogs were relatively high. Thus, another type of GHA was discovered, i.e., one in which only glycine in the third ␣ helix was replaced by other amino acids. As an aside, mice that expressed bGH-A122D were statistically smaller then their

FIGURE 4 Representative transgenic mice and Edmondson wheel projection of the third ␣ helix of bGH. Transgenic mice overexpressing bGH-E117L, bGH-G119R, and bGH-A122D are shown. Adapted from Chen et al. (1991b). (See color plate 22)

FIGURE 5 The third ␣ helix of GH is represented by a space-filling model. The “cleft” around glycine in native GH (right) is filled at the same site in GHA (left) due to substitution by an amino acid with a large side group, in this case arginine. Adapted from Chen et al. (1991b). (See color plate 23)

nontransgenic littermates; however, they did not achieve the same degree of growth suppression as bGH-G119R mice even when they exhibited high serum levels of the bGH-A122D. According to computer modeling of the structure of the third ␣ helix of GH, Gly 119 was positioned near the middle of the helix. Also, Gly possesses the smallest side chain of all of the amino acids, namely a hydrogen atom. The location of Gly in the third ␣ helix results in a cleft (Fig. 5). We hypothesized that this cleft may be critical for the growthpromoting activity of GH (Chen et al., 1991b). If this was correct, then it was entirely possible that Gly was the only acceptable amino acid residue at position 119 because of its small side chain. Continuing the pursuit of the GHA, we began work with hGH (Chen et al., 1994). Within the third ␣ helix of hGH, Gly 120 corresponded to the Gly 119 of bGH. By making single amino acid substitutions at the G120 of hGH, it should have been possible to create an hGHA (Chen et al., 1994). For this purpose the following hGH analogs were created: G120A, G120R, and I4A. The N terminus I4A substitution was created because it had been shown previously that this region and the third ␣ helix were portions of hGH-binding site 2 (Cunningham et al., 1991a; de Vos et al., 1992). Receptor binding assays showed that hGH, hGH-G120-A, hGH-G120R, and hGH-I4A all bound to GHR with similar affinities. To check the ability of hGH and hGH analogs to induce intracellular signaling events, namely tyrosine phosphorylation of Stat-5, IM-9 cells were used. hGH, hGH-G120A, and hGHI4A all induced the phosphorylation of Stat-5. hGH-G120R did not but did antagonize hGH-induced tyrosine phosphorylation. Transgenic mouse lines containing hGH, hGH-G120A, or hGH-I4A all possessed giant phenotypes; however,

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hGH-G120R transgenic mice were dwarf similar to what was found for bGH G119R. Also, IGF-I was increased in giant mice and decreased in dwarf mice. Thus, hGH-G120R was an effective hGHA. Overall, studies revealed that glycine in the third ␣ helix of GH was important for activity and that substituting it with a variety of amino acids, other than alanine, resulted in GHAs.

III. DEVELOPMENT OF PEGVISOMANT There were potential clinical indications for a GHA; however, a major obstacle existed. Because of the fast clearance of hGH by the kidneys, the GH antagonist was also expected to possess a short serum half-life of approximately 30 min. Clark et al. (1996) were able to significantly increase the half-life of the hGH molecule by reacting with polyethylene glycol-5000 (PEG5000). In this reaction, PEG5000 binds to 1 of 10 amine groups located within hGH. This modification resulted in an overall decreased clearance of hGH and an increase in the time for the molecule to reach peak levels in the blood. The clearance rates continued to decrease with more pegylation. The final half life of the modified hGH was increased to 2 days. The increase in the half-life of hGH led to another problem. When the molecule was modified with PEG5000, the binding affinity of site 1 was reduced greatly. This was apparently due to the steric effects of PEG on the ability of GH to bind GHRs (Clark et al., 1996). Prior to this work, Cunninham et al. (1991b) revealed that combinations of specific amino acid substitutions resulted in an hGH molecule with increased binding affinity. The effects of these amino acid substitutions turned out to be additive. A total of eight amino acid substitutions were found to increase the affinity of GH for its receptor. These mutations occurred at site 1 of GH and were H18D, H21N, R167N, K168 A, D171S, K172 R, E174S, and I179T (Cunningham et al., 1991b; Fuh et al., 1992). Thus, an hGH antagonist was developed that included eight amino acid substitutions in site 1 and the original G120K substitution in site 2 along with several PEG additions. This molecule was termed B2036-PEG and was also called Pegvisomant. It was found that pegylation of the molecule decreases binding to membrane receptors, but also reduces clearance, immunogenicity, and its interaction with the GH-binding protein (Ross et al., 2001). Thus, relatively high doses of Pegvisomant would be required to lower serum IGF-I (see later). Also, Pegvisomant binds to a receptor dimer and induces internalization but not subsequent GH-dependent intracellular signaling (Ross et al., 2001). Thus, importance of the eight amino acid substitutions in Pegvisomant is not one of increased receptor-binding affinity. By

removal of two potential Pegylation sites (lysines), the GH site 1 was now free from pegylation. Thus, the molecule was able to interact with the GHR (Ross et al., 2001). Debate has occurred over the mechanism of action for the GH antagonist. Fuh et al. (1992) suggested that the antagonist prevented formation of the receptor dimmer. However, subsequent studies refuted this (Harding et al., 1996a; Ross et al., 2001) by showing that both hGH and hGH-G120R associate with dimerized receptors and were internalized at similar rates after binding the receptor (Fig. 5). Thus, GHAs do not prevent GHR dimerization but prevent proper or functional GHR dimerization.

IV. PRECLINICAL TRIALS Trials were conducted with Pegvisomant to determine its effectiveness in primates. Studies were carried out in which 1.0 mg/kg was administered to ovariectomized female rhesus monkeys. In the first study, five monkeys were treated (Wilson, 1998). It was found that serum IGF-I levels in the Pegvisomant-treated group were decreased by 61% and reached the maximum suppression level by day 10. When Pegvisomant treatment was stopped, IGF-I levels returned to control levels after 14 days. The GH concentrations were also elevated while Pegvisomant was being administered. Thus, Pegvisomant seemed to “work” quite well in decreasing IGF-I levels in monkeys.

V. CLINICAL TRIALS Acromegaly is a chronic and debilitating disease marked by hypersecretion of GH from the pituitary. The increase in plasma GH levels results in elevated plasma insulin-like growth factor-I (IGF-I) concentrations. In greater than 98% of cases, this disease is caused by a GH-secreting pituitary adenoma (Melmed, 1990). Physical markers of this endocrine disorder include enlargement of the hands, feet, forehead, and jaw. Other common symptoms include fatigue, headache, excessive sweating, and numbness of the hands. This relatively rare disease is associated with high morbidity with an average twofold increase in mortality that is largely due to cardiovascular disease (Holdaway et al., 2004; Clayton, 1997, 2003; Bengtsson et al., 1988; Alexander et al., 1980). Prior to the development of Pegvisomant, all available treatment options directly targeted the pituitary to decrease the secretion of GH through surgery, radiotherapy, or drug therapy. Thanks to the development of Pegvisomant, there is now an alternative treatment strategy to combat the

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detrimental effects of acromegaly. Pegvisomant acts not by inhibiting GH secretion but by blocking GH signaling and action through a competitive, antagonistic interaction with the GHR. Surgical treatment of acromegaly is accomplished by transphenoidal surgery to remove as much of the adenoma as possible, thus eliminating the bulk of the GH-secreting tissue in the pituitary. The efficacy of surgery depends on multiple factors, the most important of which is the choice of surgeon (Lissett et al., 1998; Bourdelot et al., 2004). With a single consistent and highly skilled surgeon, remission rates can be 64% or higher; however, remission rates are 33% or lower in some study groups treated by multiple surgeons with less expertise (Gittoes et al., 1999; Yamada et al., 1996; Lissett et al., 1998). Radiotherapy is generally considered an adjunctive treatment option that is used when surgery and/or medical treatment is not successful or is refused (Melmed et al., 2002). Radiotherapy will halt further tumor growth in 99% of patients and does predictably reduce growth hormone levels in most patients (Eastman et al., 1992). A problem with radiotherapy is that the benefits are delayed; they are sometimes not evident for 10 to 15 years. Also, there is a high incidence of hypopituitarism that presents itself 2 to 25 years after treatment (Arafah et al., 2001). Ten years after irradiation, as many as 80% of patients will experience hormone insufficiency due to hypopituitarism (Barrande et al., 2000). Normalization of GH levels has been reported in 7% and 66% of patients after 2 and 15 years, respectively (Barrande et al., 2000). Other studies have reported even lower success rates (Thalassinos et al., 1998; Gutt et al., 2001; Cozzi et al., 2001). There is much controversy surrounding the efficacy of radiotherapy in the treatment of acromegaly and further debate will undoubtedly continue (Thorner, 2003). Medical treatment for acromegaly includes dopamine agonists and somatostatin analogs, both of which have been used for several years. Dopamine agonists decrease GH secretion in acromegalic patients. These drugs have relatively low success rates, with GH normalization from 20 to 46% depending on the specific drug used (Jaffe et al., 1992; Abs et al., 1998). Native somatostatin inhibits release of GH from the pituitary. Synthetic somatostatin analogs, octreotide and lanreotide, have been designed to mimic the actions of somatostatin and to reduce GH secretion to a greater degree. Lanreotide and octreotide can normalize GH levels in 56 and 49% of the treated patients, respectively, while they can normalize IGF-I in 66 and 48% (Freda, 2002). A microsphere-incorporated form of octreotide, sandostatin LAR, may be more effective in that GH and IGF-I normalization has been reported between 70 and 80%, respectively (Turner et al., 1999). Overall, this leaves 30–50% of acromegalic individuals in need of other therapeutic options.

A. Phase I Following two separate studies in which rhesus monkeys were subjected to sc injections of Pegvisomant (described earlier), a phase I clinical trial of Pegvisomant was performed in healthy young men (mean age 25 years) in a randomized, placebo-controlled, single rising-dose study. Subjects were placed randomly into either the placebo group (n ⫽ 12) or one of four treatment groups receiving a 0.03-, 0.1-, 0.3-, or 1.0-mg/kg (n ⫽ 6) dose of Pegvisomant via a single subcutaneous injection. There was a dosedependent rise in serum Pegvisomant concentrations with highest levels detected after approximately 36 h. Concentrations in excess of 5000 ␮g/liter were observed in the 1.0-mg/kg group 24–144 h after administration. A dosedependent drop in serum IGF-I levels reached statistical significance on day 3 for the 0.3- and 1.0-mg/kg dose groups. Serum IGF-I levels fell 28% for the 0.3-mg/kg group at 72 h and decreased by 49% for the 1.0-mg/kg group on day 5. Serum GH levels did not change substantially. However, there was an extremely small, yet statistically significant, rise in mean GH levels beyond 48 h in the 1.0-mg/kg group (Thorner et al., 1999).

B. Phase II A 6-week, double-blind, placebo-controlled phase II study of Pegvisomant showed that the drug significantly reduces serum IGF-I levels in acromegalic patients. Forty-six patients with active acromegaly received once weekly subcutaneous injections of either placebo or Pegvisomant at doses of 30 or 80 mg/week. Mean serum IGF-I levels decreased from baseline after 6 weeks in both the 30- and 80-mg/week groups by 16 and 31%, respectively, and did not change in the placebo group. After 6 weeks, free IGF-I levels in the 80-mg/week group showed an even more dramatic drop of 47%. Importantly, no subjects withdrew from the study due to adverse effects and the drug was well tolerated (van der Lely et al., 1998). It was suspected that the low rate of IGF-I normalization was due to the pharmacokinetics of the drug. Due to the half-life of the drug, pharmacokinetic modeling predicted that a higher average serum concentration would be achieved if the same weekly dose was divided and administered daily. It was thought that more constant serum levels of Pegvisomant would be more effective and would lower IGF-I levels more dramatically; thus, daily injections were administered.

C. Phase III In a phase III-randomized, double-blind, placebocontrolled study of 112 patients with acromegaly, Pegvisomant significantly lowered IGF-I levels and

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resulted in marked clinical improvement (Trainer et al., 2000). Patients with serum IGF-I levels of at least 1.3 times the upper limit of the age-adjusted normal range were eligible for this study. The patients were divided into two cohorts: those with IGF-I levels 1.3 to 2 times the upper age-adjusted normal range and those above 2 times the upper limit. The patients were then selected randomly to receive either placebo or a daily dose of 10, 15, or 20 mg of Pegvisomant. All patients receiving Pegvisomant were administered a loading dose of 80 mg at the baseline visit to achieve a steady-state serum drug concentration. At baseline, 2, 4, 8, and 12 weeks after initial treatment, both clinical and laboratory assessments were completed. The evaluation consisted of a physical examination and a questionnaire that assessed five symptoms of acromegaly (soft tissue swelling, arthralgia, headache, excessive perspiration, and fatigue) with a score of 0 (no symptoms) to 8 (incapacitating). Ring size using standardized European jewelers’ rings on the fourth digit of the right hand and serum levels of several proteins were also tested. Magnetic resonance images of the pituitary were taken at baseline and at the completion of the 12-week study. Dose-dependent decreases in serum concentrations of free IGF-I, IGFBP-3, and acid-labile subunit, as well as a dose-dependent increase in the number of patients with normal IGF-I levels, were observed in all three treatment groups while no change was seen in the placebo group. The percentage of patients achieving age-adjusted normal IGF-I levels at any visit after baseline for 10-, 15-, and 20-mg doses was 54, 81, and 89%, respectively. Significant decreases in soft tissue swelling, excessive perspiration, and fatigue accompanied the fall in IGF-I levels in all Pegvisomant groups, whereas they increased slightly in the untreated group. Ring size evaluation confirmed the decrease in soft tissue swelling. At the 12-week evaluation, mean ring size had decreased by 0.1 sizes in the placebo group and by 0.8, 1.9, and 2.5 sizes in groups receiving 10, 15, and 20 mg, respectively (Trainer et al., 2000). The decrease in serum IGF-I concentrations coincided with increases in serum growth hormone levels in a dose-dependent manner. Groups receiving 15 and 20 mg Pegvisomant showed statistically significant increases in growth hormone levels at 12 weeks. The mean rise in growth hormone levels for 10-, 15-, and 20-mg Pegvisomant groups were 2.7, 9.2, and 14.4 ng/ml, respectively. Magnetic resonance imaging showed that tumor volume did not change significantly for any patient, and no significant mean tumor volume change was observed in any of the Pegvisomant-treated group (Trainer et al., 2000). The frequency of adverse effects was low and similar in the placebo and all three Pegvisomant-treated groups. Injection site reactions were reported in 6 patients receiving Pegvisomant and 0 receiving placebo. All reactions were mild and did not require treatment. One patient withdrew

from the study due to persistent headaches. Another patient, who was receiving 15 mg, displayed a serious adverse effect on the liver and subsequently withdrew from the study due to abnormally high levels of serum aspartate aminotransferase (389 U per liter) and alanine aminotransferase (904 U per liter). Viral tests were negative, ultrasonography of the liver was normal, and serum bilirubin and alkaline phosphatase concentrations were normal. Discontinuation of Pegvisomant brought the abnormal enzyme levels to normal, but they rose again upon a 4-week rechallenge of 10 mg/day Pegvisomant. After discontinuation of the rechallenge, enzyme levels again returned to normal. No change in mean serum levels of alanine aminotransferase or aspartate aminotransferase was seen in the groups receiving Pegvisomant (Trainer et al., 2000).

D. Long Term On completion of phase III trials, questions concerning the safety and efficacy of long-term treatment with Pegvisomant remained. These questions were answered by a long-term treatment study. This study included 160 patients treated with Pegvisomant an average of 425 days and accumulated 186 patient years (van der Lely et al., 2001). Patients were eligible for this study if their serum IGF-I levels were 1.3 times the upper limit of the age-adjusted normal range. Patients were categorized into three cohorts based on the length of continuous daily treatment with Pegvisomant for at least 6, 12, or 18 months. These cohorts were designed in a cumulative fashion. For example, patients included in the 18-month cohort were also included in the 12- and 6-month cohorts. Serum IGF-I concentrations were normalized in 97% of those treated for at least 12 months (n ⫽ 90) (Fig. 6). Both fasting serum insulin and glucose concentrations fell in all three cohorts. Fasting insulin concentrations fell in the 6-, 12-, and 18-month cohorts by 23.0 to 15.8 mU/liter, 23.0 to 12.4 mU/liter, and 23.3 to 12.4 mU/liter, respectively. Similarly, fasting glucose concentrations fell in the 6-, 12-, and 18-month cohorts from 1053 to 862 mg/liter, 1053 to 906 mg/liter, and 984 to 904 mg/L, respectively (van der Lely et al., 2001). Mean serum GH levels rose in a dose-dependent fashion for all treatment cohorts, and time course analysis revealed that the increases paralleled the drop in IGF-I levels. In the 6-, 12-, and 18-month cohorts, GH levels increased from baseline from 10.9 to 23.1 ␮g/liter (115%), 13.2 to 25.6 ␮g/liter (95%), and 19.2 to 33.8 ␮g/liter (74%), respectively. This rise in GH did not appear to affect tumor growth or decrease the efficacy of Pegvisomant. Tumor size was analyzed by magnetic resonance imaging (MRI). Of the 131 patients who had paired baseline and the most recent MRI scans, 78 had been treated previously with radiation therapy whereas 53 were untreated. In those who received radiation therapy previously, mean tumor volume decreased by 0.126 cm3. In the

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FIGURE 6 Basal and lowest serum IGF-I concentrations in 90 acromegalic patients receiving Pegvisomant at a daily dosage of 5–40 mg/day for more than 12 months. Age-adjusted normal serum IGF-I levels are indicated by the shaded area. Normalization of IGF-I levels was achieved in 97% of patients. Figure kindly provide by A.J. van der Lely and adapted from van der Lely et al. (2001). (See color plate 24)

53 patients who had not received radiation therapy, mean tumor volume increased by 0.103 cm3. Similarly, all patients who had at least 12 months pass between baseline scan and final scan saw a mean tumor volume change of ⫺0.067 cm3. Two patients had large, globular tumors impinging the optic nerve at baseline and both showed a progressive increase in tumor size requiring treatment. The cause of this progression is unknown and did not correlate with duration of the Pegvisomant treatment (van der Lely et al., 2001). Reported adverse effects included injection site reactions and upper respiratory tract infections. These were generally mild and did not require treatment with the exception of seven cases of pneumonia, a gluteal abscess, and a case of urosepsis (van der Lely et al., 2001). Thus, Pegvisomant was found to be a safe and efficacious new drug for the treatment of acromegaly.

in inhibition of retinal neovascularization, despite a failure to reduce hypoxia-stimulated retinal vascular endothelial growth factor (VEGF) or VEGF receptor mRNA and protein expression (Smith et al., 1997). This indicates distinct functions for IGF-I and VEGF in angiogenesis (Smith et al., 1997). In a clinical trial including 13 type I diabetic and 12 type 2 diabetic patients with proliferative diabetic retinopathy, Pegvisomant was administered for 12 weeks. While a majority of patients had improved IGF-I levels, this trial did not provide any data to indicate a beneficial role of Pegvisomant in the treatment of proliferative diabetic retinopathy (Beck et al., 2001). This does not completely preclude the use of Pegvisomant in the treatment of retinopathy. Perhaps a larger and/or longer study with subjects who have less severe retinopathy could yield favorable results.

E. Retinopathy

VI. OTHER USES OF PEGVISOMANT

A role of GH in diabetic retinopathy has been speculated since 1953 when it was observed that pituitary ablation relieved the condition (Poulsen, 1953). GH has been implicated to promote angiogenesis along with other growth factors; a function that can be associated with neovascularization in the retina (Jaffe et al., 1992; Chanson et al., 2000). Several studies have since shown the ability of somatostatin analogs to prevent the progression of diabetic retinopathy (Grant et al., 2002). In a study of nondiabetic ischemia-induced retinal neovascularization, inhibition of GH (and consequently IGF-I) by a GH antagonist expressed in transgenic mice also resulted

GH and IGF-I excess has been implicated in a number of pathological states, metabolic disorders such as diabetes, deteriorative disorders such as nephropathy and retinopathy and neoplastic disorders in such organs as prostrate, breast, thyroid, and bone (Pollak et al., 2004). GH also possesses an anti-insulin-like activity that can result in insulin resistance (Roelfsema et al., 1985). Elevated levels of GH cause increased cholesterol levels and a consequent increase in nonesterified fatty acids due to lipolysis, all of which are atherogenic markers (Bolinder et al., 1986; Keller et al.,

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1991; Li et al., 2003). GH affects a variety of tissues and energy metabolism. In such cases, GH interacts directly with target cells and the effect is seemingly independent of IGF-I levels. Pegvisomant prevents functional dimerization of the GHR. This, in turn, downregulates the signaling cascade initiated by GH binding. The consequences of this are a decrease in serum IGF-I levels, as shown in the clinical trial mentioned earlier. Thus, Pegvisomant could have therapeutic uses in the treatment of pathologies induced by high levels of GH or IGF-I. It is not feasible to cite all of these studies here, but the results have been reviewed (Kopchick, 2003a; Kopchick et al., 2002) and the following references are enlightening (Muller et al., 2004; Parkinson et al., 2000, 2003).

A. In the Treatment of Diabetes As early as 1937, the link between GH and diabetes could be speculated. Young (1937) was able to demonstrate that injections of anterior pituitary preparations induced permanent diabetes, which was later confirmed when highly purified GH was administered (Campbell et al., 1950). This connection was further strengthened when end organ damage due to diabetes was arrested following pituitary ablation (Poulsen, 1953). Studies on either acromegalics or diabetics link GH excess to diabetes (Press, 1988; Sonksen et al., 1993). Using GH transgenic mice, the direct role of GH in the development of diabetic nephropathy has been established (Yang et al., 1993). Among the anti-insulin-like activities of GH, its effect on insulin sensitivity is the primary cause of GH–IGF-I axismediated diabetic end organ damage (Flyvbjerg, 1990). High levels of GH cause insulin resistance, which in turn decreases both glucose transport and glucose metabolism, resulting in hyperglycemia (Okada et al., 2001). These elevated glucose levels affect IGF-I output by the liver, which leads to feedback stimulation of increased GH production. Theoretically, a reduction of GH levels should ameliorate insulin insensitivity. In liver IGF-I-deficient (LID) mice, which have high levels of GH due to low lGF-1 levels, expression of a GH antagonist (via animal mating) resulted in an increase in insulin-stimulated glucose uptake or insulin sensitivity. This was seen in muscle and white adipose tissues. The uptake was mainly achieved by reduction in the elevated GH levels, despite low IGF-I levels (Yakar et al., 2004). Thus, the GH antagonist acts to relieve the insulin resistance found in these mice. The use of GHA improved insulin sensitivity significantly in mice. In a human clinical trial, insulin resistance was alleviated and insulin sensitivity and glucose tolerance were improved with Pegvisomant treatment (Muller et al., 2001a,b). In a study involving acromegalic individuals who were treated previously with somatostatin analogs,

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Pegvisomant administration improved plasma glucose levels, insulin sensitivity, and ␤-cell secretory function significantly (Drake et al., 2003). In other studies where patients were given Pegvisomant, the results were similar with improvements seen in the glycated hemoglobin levels as well (Rose et al., 2002). In studies involving adolescents with insulin-dependent diabetes mellitus (IDDM), suppression of the GH pulse with pirenzepine was found to decrease the dawn rise in insulin requirements (Dunger et al., 1991), suggesting that the use of a GHA might reduce the amount of insulin needed in IDDM patients. Cortisol and cortisone are interconverted by the two isoenzymes of 11 ␤-hydroxysteroid dehydrogenase. Conversion of the inactive cortisone to cortisol is inhibited in patients with acromegaly. Treatment of acromegalic patients with Pegvisomant relieves the inhibition and also returns the accelerated cortisol clearance to normal (Trainer et al., 2001). Together, these studies are indicative of the positive effects of reducing chronically elevated levels of GH action via a GH antagonist as it relates to diabetes indications.

B. In the Treatment of Nephropathy Nephropathy is one of the major complications associated with GH excess. This could result from pathological changes in the kidney due to direct effects of GH on renal tissue, particularly the glomerulus (Flyvbjerg et al., 1990; Flyvbjerg, 2000). Nephropathy is associated with glomerulosclerosis and mesangial expansion. About 30% of renal failures are attributed to diabetic nephropathy, which is characterized by mesangial proliferation, accumulation of glomerular extracellular matrix, increased urinary albumin excretion, and glomerulosclerosis (Ayala et al., 2004). The development of glomerulosclerosis in mice is independent of IGF-I levels. GH may induce glomerulusspecific ␣ I (or IV) collagen and lamilin B1 synthesis and accumulation. This leads to thickening of the glomerular matrix followed by renal hypertension and consequent sclerosis (Esposito et al., 1996). GH transgenic mice treated with streptozotocin have been used as animal models of type I diabetes. In both control and giant GH transgenic mice, streptozotocin treatment resulted in the elevation of glycated hemoglobin and severe glomerulosclerosis. In animals that express GH antagonists, although the glycated hemoglobin levels rise after streptozotocin treatment, glomerulosclerosis is not detected (Chen et al., 1996). In another study, placebo-treated diabetic animals had increased levels of kidney IGF-I. When they were given the GHR antagonist, kidney weights, IGF-I levels, and glomerular volume were normalized (Flyvbjerg et al., 1999). A similar suppression of streptozotocin-induced glomerulosclerosis was observed when GHAs were used to treat diabetic rats (Bellush et al., 2000; Segev et al., 1999).

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Because nephropathy was seen as a consequence of elevated GH, studies to improve renal deterioration were also carried out with somatostatin analogs. Renal problems such as glomerular filtration rate and kidney size were improved both in acromegalic and in type I diabetic patients following treatment. Thus, both somatostatin analogs and GHAs may provide possible new means for the treatment of renal complications due to diabetes and/or GH excess.

C. In the Treatment of Cardiovascular Disease Increased premature mortality due to cardiovascular disease is one of the major causes of acromegaly-related morbidity (Clayton, 2003; Matta et al., 2003). Chronically elevated levels of GH are associated with increased levels of triglycerides and lipoproteins (Takeda et al., 1982). Subsequently, hypertriglyceridemia and hyperlipidemia are risk factions for atherosclerosis. Total serum cholesterol levels along with low-density lipoprotein (LDL) levels are returned to normal with the use of Pegvisomant in patients with active acromegaly, along with alleviating insulin resistance (Parkinson et al., 2002).

D. In the Treatment of Cancer Elevated IGF-I levels are implicated with tumorogenesis (Pollak et al., 2004). GH, either directly or via IGF-I, has also been implicated as an important factor in the growth of malignant tumors. Numerous preclinical studies have identified IGF-I as a potent growth-inducing factor for different cancer types (Khandwala et al., 2000). For example, in the central nervous system, tumor cells that express IGF-I and GHRs are increasingly responsive to IGF-I stimulation (Glick et al., 1991, 1992). In gastrointestinal neoplasms, IGF-I m-RNA levels are elevated and the addition of IGF-I to serum-free media increased the growth of mouse colon carcinoma cells (Lahm et al., 1992; Tricoli et al., 1986). Experiments with gastric cancer, pancreatic cells, and hepatoma cell lines show growth stimulation by IGF-I (Tsai et al., 1988; Thompson et al., 1990; Ohmura et al., 1990). In pulmonary neoplasms, IGF-I stimulates growth and removal of IGF-IR by silencing shows marked antitumor activity (Lee et al., 1996; Rotsch et al., 1992; Jaques et al., 1988; Nakanishi et al., 1988; Minuto et al., 1988). In thyroid cancers, IGF-I has an additive effect on cell proliferation in combination with TSH (Onoda et al., 1992; Minuto et al., 1989; Tode et al., 1989). In ovarian cancers, IGF-I expression is enhanced significantly (Conover et al., 1998; Karasik et al., 1994; Yee et al., 1991). Similar growth stimulatory effects were seen in breast cancer (Furstenberger et al., 2003), cancer of reproductive organs and prostrate (Chan et al., 1998; Shaneyfelt et al., 2000; Shi et al., 2001, 2002), and bladder cancers.

Given the high association between IGF-I and cancer, a number of studies were conducted with GHAs to see if its capability to reduce IGF-I ameliorates the progression of the tumors. In experiments studying mammary cancers, GHA transgenic mice had significantly reduced IGF-I levels and the animals exhibited lower chemically induced tumor levels in comparison to controls (Pollak et al., 2001). In a study of hepatic malignancy, the GHA in association with a topoisomerase inhibitor reduced tumor size and hepatic metastasis. In colon cancers, the xenografted tumor cells were reduced in number when compared to untreated controls. When treated with GHAs, meningioma tumors xenografted onto nude mice showed a significant decrease in IGF-IBP3 and tumor regression was also seen (McCutcheon et al., 2001). It was demonstrated that activation of the GH/IGF-I axis increases the growth of meningiomas and the inhibition of the axis with a GHA-inhibited tumor growth (Friend et al., 1999). In mice, the use of GHA to determine its effect on meningiomas and on colon and breast cancer cell lines is being evaluated. These experiments, albeit preliminary, are yielding positive feedback on the possibility of the use of the GHA in the treatment of these types of cancers (Friend, 2001). It must be maintained that although IGF-I is implicated in a number of cancers, IGF-I expression is under the control of other hormones such as estradiol, gonadotropins, and TSH. IGF-I levels can also be altered by nutritional state and developmental stage. In addition, heterogeneity of tumors and tumor types makes their response to GHA difficult to predict. The possibility of a combinatory use of Pegvisomant with other agents might prove to be of higher efficacy than either agent alone.

VII. CONCLUSION Use of a protein structure/function approach toward the understanding of the topology of the GH molecule resulted in the unexpected discovery of several types of GHAs. During these studies, 1 amino acid out of 191 (namely glycine in the third ␣ helix of GH) was found to be crucial for biological activity. Substitution of this glycine residue for a variety of amino acids converted GH from a growth promoter to a growth suppressor. This fundamental finding established that GHAs could in fact be produced and used for suppression of GH activity. The development of Pegvisomant and its use in clinical situations where elevated levels of GH have been show to be detrimental progressed quickly. The GH antagonist Pegvisomant has been approved in the United States and Europe for use in acromegalic individuals. Future studies and results for cancer and diabetes indications are eagerly awaited.

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Acknowledgments JJK is supported in part by the State of Ohio’s Eminent Scholar Program, which includes a gift from Milton and Lawrence Goll; by DiAthegen LLC; and by NIH Grants R01 AG19899-02 and R01 CA099904-01. We thank Darlene Berryman, Ph.D., for her critical comments related to this review.

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Thorner, M. O., Strasburger, C. J., Wu, Z., Straume, M., Bidlingmaier, M., Pezzoli, S. S., Zib, K., Scarlett, J. C., and Bennett, W. F. (1999). Growth hormone (GH) receptor blockade with a PEG-modified GH (B2036- PEG) lowers serum insulin-like growth factor-I but does not acutely stimulate serum GH. J. Clin. Endocrinol. Metab. 84, 2098–2103. Thorner, M. O. (2003). Controversy: Radiotherapy for acromegaly. Clin. Endocrinol. (Oxf.) 58, 136–137. Tode, B., Serio, M., Rotella, C. M., Galli, G., Franceschelli, F., Tanini, A., and Toccafondi, R. (1989). Insulin-like growth factor-I: Autocrine secretion by human thyroid follicular cells in primary culture. J. Clin. Endocrinol. Metab. 69, 639–647. Tollet, P., Legraverend, C., Gustafsson, J. A., and Mode, A. (1991). A role for protein kinases in the growth hormone regulation of cytochrome P4502C12 and insulin-like growth factor-I messenger RNA expression in primary adult rat hepatocytes. Mol. Endocrinol. 5, 1351–1358. Trainer, P. J., Drake, W. M., Katznelson, L., Freda, P. U., Herman-Bonert, V., van der Lely, A. J., Dimaraki, E. V., Stewart, P. M., Friend, K. E., Vance, M. L., Besser, G. M., Scarlett, J. A., Thorner, M. O., Parkinson, C., Klibanski, A., Powell, J. S., Barkan, A. L., Sheppard, M. C., Malsonado, M., Rose, D. R., Clemmons, D. R., Johannsson, G., Bengtsson, B. A., Stavrou, S., Kleinberg, D. L., Cook, D. M., Phillips, L. S., Bidlingmaier, M., Strasburger, C. J., Hackett, S., Zib, K., Bennett, W. F., and Davis, R. J. (2000). Treatment of acromegaly with the growth hormone-receptor antagonist pegvisomant. N. Engl. J. Med. 342, 1171–1177. Trainer, P. J., Drake, W. M., Perry, L. A., Taylor, N. F., Besser, G. M., and Monson, J. P. (2001). Modulation of cortisol metabolism by the growth hormone receptor antagonist pegvisomant in patients with acromegaly. J. Clin. Endocrinol. Metab. 86, 2989–2992. Tricoli, J. V., Rall, L. B., Karakousis, C. P., Herrera, L., Petrelli, N. J., Bell, G. I., and Shows, T. B. (1986). Enhanced levels of insulin-like growth factor messenger RNA in human colon carcinomas and liposarcomas. Cancer Res. 46, 6169–6173. Tsai, T. F., Yauk, Y. K., Chou, C. K., Ting, L. P., Chang, C., Hu, C. P., Han, S. H., and Su, T. S. (1988). Evidence of autocrine regulation in human hepatoma cell lines. Biochem. Biophys. Res. Commun. 153, 39–45. Turner, H. E., Vadivale, A., Keenan, J., and Wass, J. A. (1999). A comparison of lanreotide and octreotide LAR for treatment of acromegaly. Clin. Endocrinol. (Oxf.) 51, 275–280. van der Lely, A. J., Hutson, R. K., Trainer, P. J., Besser, G. M., Barkan, A. L., Katznelson, L., Klibanski, A., Herman-Bonert, V., Melmed, S., Vance, M. L., Freda, P. U., Stewart, P. M., Friend, K. E., Clemmons, D. R., Johannsson, G., Stavrou, S., Cook, D. M., Phillips, L. S., Strasburger, C. J., Hackett, S., Zib, K. A., Davis, R. J., Scarlett, J. A., and Thorner, M. O. (2001). Long-term treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist. Lancet 358, 1754–1759.

van der Lely, A. J., Lamberts, S. W., Barkan, A., Panadya, N., Besser, G. M., Trainer, P. J., Bonnert, V., Melmed, S., Clemmons, D. R., Rose, R., Vance, M. L., Thorner, M. O., Zib, K., Davis, R. J., Bennett, W. F., and Scarlett, J. A. (1998). A six week, double blind, placebo controlled study of a growth hormone antagonist, B2036-PEG (Trovert) in acromegalic patients. In “The Endocrine Society 80th Annual Meeting,” p. 57, New Orleans, LA. Wang, X., Moller, C., Norstedt, G., and Carter-Su, C. (1993). Growth hormone-promoted tyrosyl phosphorylation of a 121-kDa growth hormone receptor-associated protein. J. Biol. Chem. 268, 3573–3579. Wang, X., Uhler, M. D., Billestrup, N., Norstedt, G., Talamantes, F., Nielsen, J. H., and Carter-Su, C. (1992). Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase. J. Biol. Chem. 267, 17390–17396. Wang, X., Xu, B., Souza, S. C., and Kopchick, J. J. (1994). Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors. Proc. Natl. Acad. Sci. USA 91, 1391–1395. Watahiki, M., Yamamoto, M., Yamakawa, M., Tanaka, M., and Nakashima, K. (1989). Conserved and unique amino acid residues in the domains of the growth hormones: Flounder growth hormone deduced from the cDNA sequence has the minimal size in the growth hormone prolactin gene family. J. Biol. Chem. 264, 312–316. Wilson, M. E. (1998). Effects of estradiol and exogenous insulinlike growth factor I (IGF-I) on the IGF-I axis during growth hormone inhibition and antagonism. J. Clin. Endocrinol. Metab. 83, 4013–4021. Winston, L. A., and Bertics, P. J. (1992). Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins. J. Biol. Chem. 267, 4747–4751. Yakar, S., Setser, J., Zhao, H., Stannard, B., Haluzik, M., Glatt, V., Bouxsein, M. L., Kopchick, J. J., and LeRoith, D. (2004). Inhibition of growth hormone action improves insulin sensitivity in liver IGF-I-deficient mice. J. Clin. Invest. 113, 96–105. Yamada, S., Aiba, T., Takada, K., Ozawa, Y., Shimizu, T., Sawano, S., Shishiba, Y., and Sano, T. (1996). Retrospective analysis of long-term surgical results in acromegaly: Preoperative and postoperative factors predicting outcome. Clin. Endocrinol. (Oxf.) 45, 291–298. Yang, C. W., Striker, L. J., Kopchick, J. J., Chen, W. Y., Pesce, C. M., Peten, E. P., and Striker, G. E. (1993). Glomerulosclerosis in mice transgenic for native or mutated bovine growth hormone gene. Kidney Int. Suppl. 39, S90–S94. Yee, D., Morales, F. R., Hamilton, T. C., and Von Hoff, D. D. (1991). Expression of insulin-like growth factor-I, its binding proteins, and its receptor in ovarian cancer. Cancer Res. 51, 5107–5112. Zapf, J., and Froesch, E. R. (1986). Insulin-like growth factors/somatomedins: Structure, secretion, biological actions and physiological role. Horm. Res. 24, 121–130.

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25 Growth Hormone Replacement Therapy and Life Quality: Future Perspectives PETER SÖNKSEN St. Thomas’ Hospital and King’s College London and Southampton University Hospital and Medical School

I. Case Study: Neuroprotective Effect of Growth Hormone in Spinal Cord Injury II. Growth Hormone Replacement and Life Quality in the Frail Elderly III. Growth Hormone Replacement and Quality of Life in Adults with Growth Hormone Deficiency References

to remain independent longer. Clinical trials over a period of at least 2 to 3 years are needed. The use of growth hormone in the treatment of adults with growth hormone deficiency has been challenged by the United Kingdom government advisory body [National Institute of Clinical Excellence (NICE)] and this advice is often adopted unthinkingly by other countries. NICE has issued guidelines advising rhGH replacement only in individuals who score 11 or above on a commercial quality of life questionnaire (QoL-AGHDA) that has not in the author’s opinion been validated properly. This advice comes from a government body made up of doctors, economists, and epidemiologists but containing no endocrinologists and carefully ignoring any advice it received. The author recommends ignoring the advice of NICE and using the advice developed by the Growth Hormone Research Society Consensus Workshop in 1997 and published in a top peer-reviewed international journal.

The neuroprotective actions of insulin-like growth factor-I (IGF-I) and growth hormone (GH) are considered in the context of a case study of the author’s spinal cord injury. Local generation of IGF-I in the spinal cord under the influence of growth hormone (or through IGF-I/BP3 delivered systemically) may well be useful in mitigating damage from injury to the spinal cord and the rest of the central nervous system. The author believes that it was of benefit to him personally but formal trials are obviously needed before this could be established as a new form of treatment. Growth hormone treatment of the elderly has been shown to be of benefit in building/retaining lean body mass and may well be of value in retaining cognitive function. Many studies have obscured beneficial effects through overdosage, as the elderly are very sensitive to growth hormone. Side effects can virtually be eliminated through individual dose titration using IGF-I and/or a collagen marker to monitor a safe and effective dose. It is anticipated that judicious use of GH will help prevent/delay frailty and possibly dementia in a significant proportion of elderly people, allowing them

THE SOMATOTROPHIC AXIS IN BRAIN FUNCTION

I. CASE STUDY: NEUROPROTECTIVE EFFECT OF GROWTH HORMONE IN SPINAL CORD INJURY As I lay immobile in the neurological intensive care unit within the University of Utah Hospital in Salt Lake City, it was not surprising that my mind dwelled rather a lot on

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neuroprotection. I had just been helicoptered off the ski slopes after an accident that left me face down in the snow fully conscious but unable to move any limb. I had made an immediate self-diagnosis of cervical cord injury but was soon somewhat encouraged by the (thorough) examination in the emergency room that showed that the lesion was at C4/5 but was incomplete. I was aware of several publications suggesting that insulin-like growth factor I (IGF-I) was neuroprotective (Chapters 6 and 18) but I knew that none was available for clinical use. I knew that growth hormone crossed the blood–brain barrier and most likely had a hand in regulating “autocrine” IGF-I production within the central nervous system. Should I try and persuade my doctors to give me recombinant human growth hormone (rhGH)? Would it do any good? From my knowledge of endocrinology I knew that at 69 I was well into the somatopause. Indeed, having volunteered for several student projects, I knew that my GH and IGF-I levels were pretty low and my GH response to maximum exercise was, at best, pathetic. Would rhGH replacement do me any harm? Again, I knew that this was extremely unlikely. So my decision was to go for it; the only problem was to convince my doctors. After “stabilization” I was transferred to the neurorehabilitation ward under the care of a young ex-New Yorker with whom I soon developed a good rapport. His policy with “elderly” (⬎50) men and spinal injuries was to put them on testosterone replacement routinely, as free testosterone levels were always low and pilot studies of testosterone replacement had convinced him that it was of tangible benefit. He wanted to start me on testosterone patches (manufactured locally by Watson Laboratories) and after I had given him a seminar on the importance of growth hormone (as well as testosterone) in maintaining body composition and its possible role in neuroprotection, he agreed to put me on both. In the course of the next 2 months of intensive rehabilitation in Utah I regained movement in all four limbs and was brushing my teeth, eating with implements with large rubber handles, and taking a few steps with the help of three physical therapists and a large walking frame. After transfer to the United Kingdom I spent another 2 months in a hospital being rehabilitated by an equally energetic physiotherapist and was discharged home just in time for Easter (2003), walking independently with two elbow crutches and with control over bowel and, a few days later, over bladder too. Not bad when more than one United Kingdom rehabilitation doctor told me to expect 12–15 months in the hospital. Did growth hormone and testosterone do anything for me? Of course I cannot be sure but I have done extremely well for a man of my age with such a severe injury. By Easter, 4 months after my injury, my weight had fallen

from 88 to 82 kg but my lean body mass had remained unchanged at 75.6 kg and my fat mass had fallen from 16 to 6 kg (Tanita). I had managed to retain my muscle bulk even if the neurological connections were not 100%. I do not have any doubt that growth hormone and testosterone were beneficial in this respect but like any good clinical anecdote, it needs to be tested formally in a randomized control trial. So this is my first future perspective: An investigation of the neuroprotective effect of growth hormone ⫹/⫺ testosterone/oestrogen (by patch) in “elderly” (⬎50) men/women with acute neurological lesions. Clearly there will be a need for stratification before randomization. Head injuries, spinal chord injuries and strokes should each be able to provide enough for a definitive multicenter study. Although not available at present, IGF-I is in the pipeline for clinical use. A combination of IGF-I with equimolar amounts of IGFBP3 is being developed as a therapeutic product by Insmed1 and clinical trials with this evaluating its neuroprotective functions in vivo (with and without growth hormone) would be of great importance and interest.

II. GROWTH HORMONE REPLACEMENT AND LIFE QUALITY IN THE FRAIL ELDERLY Many have commented on the similarity between “normal” aging and growth hormone deficiency in adults (GHDA). Aging is associated with a progressive decline in lean body mass (and neuronal mass), and in a substantial proportion of “healthy” elderly, this inevitably leads to frailty that eventually dominates their lives and threatens their independence. Each illness, be it simply influenza or hospitalization for treatment of any condition, results in a further fall in lean body mass (muscle and bone). These stepwise reductions in key skeletal structures are rarely reversible. Although exercise programs have been shown to be capable of increasing lean body mass in such people, its use in preventing or reversing frailty has been limited, as compliance is poor in everyone and suitable exercise programs impossible in many. Like patients with organic GHDA, these people are extremely sensitive to GH. The many trials of rhGH replacement carried out so far in elderly people have shown the potential benefit of GH in terms of reactivating bone remodeling and rebuilding lean body mass. Unfortunately, all the trials to date have been either of too short a duration to show significant

1 http://www.insmed.com /secondary.asp?title⫽Overview§ion⫽ productpipeline

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effects on enhancing the bony skeleton and increasing muscle strength and performance or have used too high a dose of GH, resulting in unwanted side effects masking the underlying beneficial changes. It has now been well documented that rhGH replacement needs to be individually tailored to the sensitivity to GH of each patient (just like insulin in people with diabetes but a little easier). What is more, it needs to be started at a very low dose and increased gradually over weeks or months based on the individual’s response. There are several GH-dependent markers of the GH response that can be used to titrate the dose of rhGH. The most familiar is IGF-I, which has proven reliable and safe in most people’s experience, but it does have limitations and may give erroneous guidance in people with diabetes, eating disorders, or pancreatic disease. Others include the markers of collagen and bone remodeling: the carboxyterminal propeptide of type I procollagen (PICP), the carboxy-terminal cross-linked telopeptide of type I collagen (ICTP), the procollagen type III telopeptide (P-III-P), and osteocalcin. All have been shown to increase in a dose-dependent manner after administration of rhGH. Each has a different half-life, varying from days in the case of the collagen markers to months in the case of osteocalcin. Their limitations and usefulness in monitoring GH replacement are not as well known as with IGF-I,

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but enough is known to say that they should be used in trials of rhGH administration in the elderly. Growth hormone acts synergistically with gonadal steroids (both estrogen and testosterone) and both normally play a key role in the development and maintenance of the normal skeleton and its associated muscles, ligaments, and cartilage. The benefits of estrogen HRT on the female skeleton are well known, although the whole question of giving HRT to protect the female skeleton has been dominated by fear of a slight increased risk of breast cancer and its lack of effect in preventing cardiac disease. Additionally, all data on possible risks of breast cancer and heart disease are from studies on orally administered HRT, which in itself interferes with the GH/IGF-I axis. In the prevention and treatment of frailty associated with aging it is logical to look at the effects of testosterone or estrogen/progestogen alone or in combination with GH (and GH alone). We have completed such a study in 80 healthy elderly men average age 70 (Giannoulis et al., 2005.) using a fixed dose of testosterone (5 mg/day) by patch and an individually tailored dose of rhGH over a total period of 6 months. By slowly adjusting the rhGH dose, side effects were virtually eliminated (but inevitably the duration of rhGH treatment reduced). Although the study was of only 6 months duration, several important findings are summarized in Fig. 1.

FIGURE 1 Growth hormone in the prevention and treatment of frailty.

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A dose of rhGH that increased the IGF-I level of an individual to the upper half of the age-specific reference range was more effective than an equivalent dose of testosterone that raised plasma testosterone to a similar degree. Although testosterone alone had no measurable effect on these end points, it augmented the effects of rhGH. It is of particular interest that the rhGH and testosterone groups had an increase in maximum aerobic capacity and an increase in one of the measures of muscle strength (not shown), while these end points tended to continue their fall in the other groups during the course of the study. Patient-reported outcomes were also monitored using a newly developed age-related hormone deficiency (A-RHDQoL) questionnaire.2 The testosterone-alone group reported a worsening of the negative impact of age-related hormone deficiency on quality of life at the end of the study, but the growth hormone and growth hormone plus testosterone groups showed an improvement. Cognitive function was not measured but may well also be improved by growth hormone and testosterone (see Chapter 22). It is interesting in this context that the selective cholinesterase inhibitors used in the treatment of Alzheimer’s seem to potentiate GH secretion and increase IGF-I levels (Chapter 23). So my second future perspective is an investigation into the preventative effects of growth hormone ⫹/⫺ testosterone/oestrogen on frailty, cognition, and quality of life in the elderly. In this case “elderly” needs to be much older than when one is looking at neuroprotective effects, as the trials need to be carried out over at the very least 2 to 3 years and preferably longer. There are two types of trial needed here, the first type using surrogate end points such as body composition, bone density, markers of bone remodeling, muscle strength, aerobic capacity, and quality of life. These need a minimal duration of 2 years to show an effect on bone remodeling. The second type needs to be open ended and should be randomized but probably not double blind. These trials should be to hard end points such as fractures, falls, independence, and dementia (see Chapters 21 and 23). Although this will involve daily injections of rhGH in the active treatment arm, this should not be a problem, as modern delivery systems make administration simpler than brushing one’s teeth and anyone capable of living alone is capable of taking their own injection (and this can be made an entry criterion). Both types of trials will of course need to look at safety issues. 2 For access and licence to use the A-RHDQoL questionnaire, contact the copyright holder, Clare Bradley Ph.D., Professor of Health Psychology, Health Psychology Research, Royal Holloway, University of London, Egham, Surrey, TW20 0EX. Email: [email protected]

Although these trials will be expensive and need to be done with injections of rhGH, if they show benefit (and in my opinion the likelihood is that they will), the potential market becomes enormous. It will also reopen research into GH secretagogues, as once there has been “proof of concept” with rhGH, the potential market for secretagogues then becomes even greater as they will be usable by more frail people and there will be less cost limitation.

III. GROWTH HORMONE REPLACEMENT AND QUALITY OF LIFE IN ADULTS WITH GROWTH HORMONE DEFICIENCY One patient, a thirty-five year old female was treated in addition with hGH, 3 mg three times a week. After two months of hGH she noted increased vigor, ambition and sense of well-being. Observations will be needed in more cases to indicate whether the favourable effect was more than coincidental. (Raben, 1962)

Another clinical anecdote but this one has now been confirmed in many well-designed and well-executed randomized control trials, although controversy still exists. My last “perspective” is to revisit something that is not new but is current and has got rather bogged down in political and economic issues to the point where it has become what one might well consider a medical scandal. Although it is “local” to the United Kingdom, its impact is far reaching, as many countries follow policies adopted by our National Health Service (NHS). The National Institute of Clinical Excellence (NICE) is, as you might guess from its name, a government agency within the United Kingdom. Its original concept was a sound one: to educate doctors and other medical personnel about ineffectual medicines that were being prescribed in large amounts and thus “wasting” taxpayers’ money going into the health service budget that could be better spent on effective treatments. After doing a good job in this area they moved onto more dodgy ground, reviewing expensive and new medications and producing guidelines on their use, independent of the “product licence” that had already been issued independently on the basis of efficacy and safety. Although every doctor still has the right to prescribe any drug that he or she thinks is appropriate for a given patient, a general practitioner (GP) who prescribes a drug (and nearly all prescriptions come from the GP in the United Kingdom, often on the advice of a hospital specialist) for use outside the NICE guidelines (which may differ considerably from the product licence) may find himself or herself vulnerable. There is considerable pressure to conform and, in

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the case in question, rhGH, this means that many patients with GHDA are not getting access to rhGH. Despite clear clinical documentation of the syndrome of growth hormone deficiency in adults (GHDA) with features effecting soma as well as psyche (Cuneo et al., 1992), NICE has chosen to recommend rhGH replacement only for those with poor quality of life and multiple hormone deficiency3: 1 Guidance 1.1 Recombinant human growth hormone (somatropin) treatment is recommended for the treatment of adults with growth hormone (GH) deficiency only if they fulfil all three of the following criteria.

• They have severe GH deficiency, defined as a peak GH response of less than 9 mU/litre (3 ng/ml) during an insulin tolerance test or a cross-validated GH threshold in an equivalent test. • They have a perceived impairment of quality of life (QoL), as demonstrated by a reported score of at least 11 in the disease-specific ‘Quality of life assessment of growth hormone deficiency in adults’ (QoL-AGHDA) questionnaire. • They are already receiving treatment for any other pituitary hormone deficiencies as required. Knowing quite a lot about the background to these recommendations and having chaired the Growth Hormone Research Society (GRS) Consensus Workshop producing their “guidelines,” I can see that the NICE guidelines are not about treating patients, but about rationing access to rhGH and are of no clinical merit. One of the key working papers considered by NICE was produced by pharmacists, economists, and epidemiologists who either never sought advice from endocrinologists or sought it and then ignored it. NICE itself had no endocrinologist on the panel considering GH; they also ignored recommendations from many individual and several official groups of British endocrinologists. There appears to be severe bias against recommending rhGH. The report also makes what I consider derogatory remarks about highest quality peer-reviewed papers from top experts published in first rate journals: “Many of the available studies were of poor quality” the (anonymous) authors of the report proclaim! Their bias is well illustrated by the comments they made on a paper from my group published in The Journal of Clinical Endocrinology and Metabolism (Gibney et al., 1999): Quality-of-life evidence from observational trials 4.1.11 A 10-year study provided the longest period of observational follow-up of replacement therapy in GH deficiency. This study included patients who had previously 3

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participated in an RCT. Of the 24 patients in the original study, ten patients who had received GH continuously for 10 years were compared with 11 who had not. For the group receiving GH, QoL — as measured by the NHP — was improved over baseline in the domains of energy level and emotional reactions. Overall score was also improved. There was no change in the untreated group. However, the two groups may not be comparable because there are several reasons why patients may not continue treatment. Two shorter observational studies (12 months) reported improvements in overall NHP scores after GH treatment.”

The authors of the Gibney et al. (1999) paper had examined in detail to see if there were any differences between the two groups but could find none and the editor and referees of what is probably the world’s number one endocrine journal did not challenge this. The failure of one group to obtain long-term rhGH replacement was largely determined by where they lived and local prescribing policies within the NHS (the so-called “post code lottery”). I can only hope that my medical colleagues everywhere will treat these guidelines with the contempt that they deserve and follow those produced by the Growth Hormone Research Society4 that were produced and unanimously agreed upon by 31 international experts in the field. Although people with GHDA often have an impaired quality of life, it is by no means universal. In most peoples’ experience it is around 50% from whom one can elicit appropriate symptoms. It was only through the use of generic health questionnaires that the syndrome was discovered in the first place. Patients who we had been seeing for years never spontaneously complained, but when faced with a questionnaire that asked direct questions, they answered truthfully. Of course, they also answered truthfully when we asked for the symptoms directly but we had never known what questions to ask. We had not been aware of the problem. The existence and extent of the problem came as a shock to us all. In our original study on rhGH replacement in GHDA (Salomon et al., 1989; McGauley, 1989), we used three well-established generic questionnaires to measure quality of life. They all showed concordant results, indicating a serious problem that we had been completely unaware of. The 60-item general health questionnaire (GHQ) has the added advantage that, as a screening instrument for psychiatric morbidity in primary care, it produces a numerical score that indicates the extent of psychiatric disturbance on a continuum. Thus in untreated GHD patients, 42% scored high enough to be identified as a psychiatric case (Table I). 4

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TABLE I General Health Questionnaire (GHQ) GHQ at entry ⬎ 11:

33%

GHQ at entry ⬎ 10:

42%

GHD

Controls

Entry (GHQ)

10.9 ⫾ 2.5

Entry (Likert)

52 ⫾ 4.7

1.9 ⫾ 1.1

p ⫽ 0.002

31.3 ⫾ 2.9

p ⫽ 0.001

In this study, 42% were ill enough to justify a psychiatric referral; this is almost identical to the 46% found by Lynch et al. quoted by Murray and Shalet in Chapter 16. It is not difficult to see that there is a very considerable burden of disease “out there” needing treatment. NICE recommends screening patients with GHDA using the questionnaire developed by Pharmacia, specifically for GHDA — the “QoL-AGHDA” — and only treating patients who score high with this instrument: “They have a perceived impairment of quality of life (QoL), as demonstrated by a reported score of at least 11 in the disease-specific ‘Quality of life assessment of growth hormone deficiency in adults’ (QoL-AGHDA) questionnaire.” It is of interest that they accept this “commercial” and poorly validated questionnaire over instruments such as the GHQ that have been extremely well validated in many countries. However, not all patients benefit from rhGH replacement and there are many who are perfectly happy with their quality of life (scoring zero on the NHP) without GH replacement. It has not been uncommon in our experience, however, for a patient in this category to have participated in a double-blind study and continue to score zero on the NHP during both the rhGH and the placebo phases only to see their NHP score rising (worsening) after stopping rhGH as they realize retrospectively how much GH had benefited them! Impaired quality of life is only one of the features of the syndrome GHDA (Cuneo et al., 1992) and it is obviously

clinically wrong just to treat some features and ignore others (how could a responsible body like NICE do this?). The benefits of rhGH replacement are not restricted to improving quality of life; there are many other issues. It is not appropriate to deal with all features here but the issue of “quantity of life” fits well with quality of life. Several studies have shown that with GHDA in the absence of GH replacement, the mortality rate from cardiovascular disease is about twice that in the normal population. GHDA has also been shown to be associated with excess risk factors for cardiovascular disease that most likely account for this and these usually normalize after rhGH replacement. There are many other compelling reasons why all patients with GHDA should be offered the opportunity to take rhGH replacement. As a clinician, I would like to end with another case study. Mrs. Sheila B, a housewife in her forties, presented with severe depression with active suicidal thoughts (sufficiently severe to lead to an emergency admission from the out-patient clinic). She had previously been treated by surgery and radiotherapy for a pituitary tumor and was on full hormone replacement, apart from GH. She had read about potential benefits of GH in patients like herself and asked her GP to refer her to St. Thomas’ Hospital, London. She was started on GH (after tests to confirm diagnosis) even though there was a considerable amount of residual pituitary tumor. She fully understood the potential risk that her tumor might be reactivated by the GH but was adamant that we should treat her with GH. Needless to say, she responded very well to treatment, both psychiatrically and physically. As an expert patient she generously gave her time to explain articulately to undergraduate medical students the physical and psychological sequelae of GHD and her response to treatment. On one occasion when she could not come to a teaching session she sent the apologia shown in Fig. 2. My third and final perspective is: When treating adults with GHDA, observe the GRS guidelines for treatment, not those of NICE.

FIGURE 2 Photograph and letter reproduced with the patient’s consent.

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References Cuneo, R., Salomon, F., McGauley, G., and Sonksen, P. H. (1992). The growth hormone deficiency syndrome in adults. Clin.Endocrinol. 37, 387–397. Giannoulis, M., Sonksen, P. H., Umpleby, M., Breen, L., Pentecost, C., Whyte, M., McMillan, C. V., Bradley, C., and Martin, F. C. (2005). HRT for men? The effects of growth hormone and/or testosterone in healthy elderly men: A randomized controlled trial.–Submitted for publication. Gibney, J., Wallace, J. D., Spinks, T., Schnorr, L., Ranicar, A., Cuneo, R. C., Lockhart, S., Burnand, K. G., Salomon, F., Sonksen, P. H., and

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Russell-Jones, D. L. (1999). The effects of 10 years of recombinant human growth hormone (GH) in adult GH-deficient patients. J. Clin. Endocrinol. Metab. 84(8), 2596–2602. McGauley, G. A. (1989). Quality of life assessment before and after growth hormone treatment in adults with growth hormone deficiency. Acta Paediatr. Scand. Suppl. 356, 70–72; discussion 73–74. Raben, M. S. (1962). Clinical use of human growth hormone. N. Engl. J. Med. 266, 82–86. Salomon, F., Cuneo, R., Hesp, R., and Sonksen, P. H. (1989). The effects of treatment with recombinant human growth hormone on body composition and metabolism in adults with growth hormone deficiency. N. Engl. J. Med. 321, 1797–1803.

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Index

A acromegaly and brain function, 263–266 brain effects during growth hormone hypersecretion, 264–265 overview, 263–264 treatment of growth hormone-producing tumors, 265–266 adult cell genesis by IGF-1 and GH, 130–133 physiological and functional correlates to, 133–141 functionality of newborn cells, 133–134 IGF-1 and adult human brain, 136–141 overview, 133 physiological endogenous regulation of neurogenesis, 133 proliferation or cell survival, 134 roles of IGF-1-mediated neurogenesis, 134–136 adult-derived hippocampal progenitors (AHPs), 122, 129 Adult Growth Hormone Deficiency Assessment (AGHDA), 217–219, 221–223, 234–241, 326 adult hippocampal progenitors, 129–130 affinity chromatography, 89 AGHDA (Adult Growth Hormone Deficiency Assessment), 217–219, 221–223, 234–241, 326 aging, 134, 136, 162–168, see also Alzheimer’s disease age-related changes in activity of GH/IGF-1 axis, 268–273 age-related decreases in plasma IGF-1, 196–204 overview, 196 in vitro studies, 196–197 in vivo studies, 197–204 GH/IGF-1 axis in dementia, 273–275 GH/IGF-1 axis in Down syndrome, 275–280 and growth hormone/insulin-like growth factor-1 axis, 267–280 loss of IGF-1 function contributing to brain aging and disease, 208–209

overview, 267–268 quality of life in deficiency of GH, 222 quality of life in GH replacement therapy, 322–324 AHPs (adult-derived hippocampal progenitors), 122, 129 Akt activation, 186–187, 192 Alzheimer’s disease (AD), 181, 196, 198, 273–276 Alzheimer-associated neuronal thread protein, 299 b-amyloid in, 297 growth hormone in, 295–304 cognitive decline in elderly, 296–297 growth hormone response, 300–301 overview, 295–296 possible usefulness of IGF-1 axis in treatment, 302–304 insulin-like growth factor-1 in, 295–304 cognitive decline in elderly, 296–297 IGF-1 axis in treatment, 302–304 IGF-1 system as neurotrophic factor, 297–298 insulin and IGF-1 in Alzheimer’s disease, 298–299 insulin-like growth factor-binding protein-3, 299 mechanisms of action of IGF-1 in Alzheimer’s disease, 299 overview, 295–296, 297 tumor necrosis factor ␣ and its relationship with IGF-1 effects, 298 AMP-activated protein kinase (AMPK), 26 angiogenesis, 134, 314 anxiety, effect of GH on, 255–256 appetite effect of GH on, 256 regulation by ghrelin, 25–27 arcuate nucleus (ARC), 25 astrocytes, 133–134, 147–151, 153–161 astroglial calcium wave, 147 astroglial cells, see astrocytes attention, effect of GH on, 248–252

335

B b-amyloid, 297 basic fibroblast growth factor (bFGF), 158–159 BBB, see blood–brain barrier BCB (brain–cerebrospinal fluid barrier), 66, 197 BDNF (brain-derived neurotrophic factor), 206, 210 behavior, effect of GH on, 254–255 bFGF (basic fibroblast growth factor), 158–159 bGH (bovine growth hormone), 143, 151–152, 156, 263, 309–311 blood-borne IGF-I, 205 blood–brain barrier (BBB), 66, 71–74, 107, 109, 181, 271, 297–299 absence of saturable transport system for GH at, 73 lack of direct interaction of GH and insulin, 73–75 transport systems for IGF-1 at, 72–73 whether growth hormone crosses, 71–72 blood–cerebrospinal fluid (CSF) interface, 207 bovine growth hormone (bGH), 143, 151–152, 156, 263, 309–311 brain. see also blood–brain barrier (BBB) brain, distribution of ghrelin, 24 brain–cerebrospinal fluid barrier (BCB), 66, 197 brain-derived neurotrophic factor (BDNF), 206, 210 bromodeoxyuridine (BrdU), 125–129, 185

C calorie restriction (CR), 169–178 cancer, Pegvisomant in treatment of, 316–320 cardiovascular disease, Pegvisomant in treatment of, 316 cellular signaling, 150–151 central nervous system (CNS), 3, 71–73, 77–83, 87–88, 95, 115, 121–136, 143–144, 160–161, 268, 271–273 astrocytes in, 147–149 connexin expression in, 144 GH and adult proliferation and differentiation in, 132–133

336

Index

central nervous system (CNS)—(Continued) GHR mRNA in, 97–100 somatotrophic and lactotrophic systems in, 108–111 brain production of GH and prolactin, 111 distribution of GH and prolactin receptors in brain, 109 overview, 108 signal transduction pathway, 109–111 transport of GH and prolactin into brain, 108–109 cerebrospinal fluid (CSF), 65–69, 156–158, 187, 207, 246, 282, 292 CGD (constitutional growth delay) children, 248 cholinesterase inhibitors, 301–302 class I cytokine receptor superfamily, 35–36 cloning of growth hormone receptor (GHR), 96–97 cognitive/mental function cognitive decline in elderly, 296–297 conditions where GH/IGF-1 axis affects, 161–168 aging and depression, 162–168 exercise, 162 overview, 161–162 and discontinued GH treatment, 288 effect of GH on cognitive deficits, 248–252 GH deficiency and GH treatment, cognitive status in relation to, 285–287 GH dosages and cognitive status in GH-deficient adults, 289 GH-induced cognitive changes in healthy elderly subjects, 290–294 GH-induced hormonal changes, cognitive effects of, 284–285 column electrophoresis, 89 connexin43, regulation of by GH and IGF-1, 156–159 IGF-1 system and connexin43/gap junctions, 159 overview, 156 systemic versus local pathways, 156–158 connexins, 143–147, 154–156, 159–161 constitutional growth delay (CGD) children, 248 corticotropin-releasing hormone (CRH), 229 CR (calorie restriction), 169–178 CRH (corticotropin-releasing hormone), 229 CSF (blood–cerebrospinal fluid) interface, 207 CSF (cerebrospinal fluid), 65–69, 156–158, 187, 207, 246, 282, 292 c-Src utilization to activate Ras-like small GTPases, 39–40 Cushing’s disease, 240 cx43 gene, 143–147, 154–157, 159–162

D DCT (digit cancellation test), 250–251 dementia, 295, 321 and GH/IGF-1 axis, 267–280 age-related changes in activity of, 268–273 and Alzheimer’s disease, 273–275 and Down syndrome, 275–280 overview, 267–268

depression, 134–135, 162–168, 255 diabetes, Pegvisomant in treatment of, 315 digit cancellation test (DCT), 250–251 DNA hypomethylation, 5 DNA methylation, 4–5 Down syndrome (DS), 266–267 GH axis in, 275–280 IGF-1 axis in, 275–280 “dual effect hypothesis,” 4

E EC (enterochromaffin), 23–24 elderly, see aging; Alzheimer’s disease electrophoresis, 89 embryogenesis, 79, 82 endocrinological treatment, 247 energy, effect of GH on, 255 enterochromaffin (EC), 23–24 ERK (extracellular signal-regulated kinase) cascades, 48 estrogen, 323 exercise, 135, 162, 185 extracellular signal-regulated kinase (ERK) cascades, 48

F FAK (focal adhesion kinase), 38–39 FK960 reagent, 302 focal adhesion kinase (FAK), 38–39 FoxO family transcription factors, 178

G gap junctional plasticity and cortical functions, 159–168 astrocytes and learning/enriched environment, 159–160 conditions where GH/IGF-1 axis affects cognitive/mental function, 161–168 aging and depression, 162–168 exercise, 162 overview, 161–162 overview, 159 significance of astroglial gap junctions for learning and memory, 160–161 gap junction coupling (GJC), 143 –147, 152–155, 158–161 gastrointestinal organs, distribution of ghrelin, 23–24 GCL (granule cell layer) neurons, 122–124, 130, 133, 185 GEF (guanine nucleotide exchange factor), 39–40 “genetic epistemology,” 246 genetic labeling, 129 GH, see growth hormone (GH) GHAs, see growth hormone antagonists (GHAs) GHBP (growth hormone-binding proteins), 36, 95–97, 99–101, 162 GHD, see growth hormone deficiency (GHD) GHIS (growth hormone insensitivity syndrome), 99 GHR, see growth hormone receptors (GHR)

ghrelin, 7–8, 113, 271–272 distribution of, 23–24 knockout mouse, 27 physiological functions of, 24–27 appetite regulation, 25–27 growth hormone-releasing activity, 24–25 purification and identification of, 23 regulation of secretion, 27 ghrelin-immunoreactive cells, 24 GHRH (growth hormone-releasing hormone), 5–9, 21–22, 24–25, 27–28, 111–112, 182, 194, 196–197, 227–229, 255, 267–276, 277, 291–292, 300–301, 308 GHS-R (growth hormone secretagogue receptors), 21–23, 27–28 GHSs (growth hormone secretagogues), 21–22, 27–28, 182, 227, 268, 271, 276, 292 GJC (gap junction coupling), 143 –147, 152–155, 158–161 glial plasticity, 206 gliogenesis, 133 glucocorticoids, 12, 297 glucose–insulin system, 173–177 in anti-GH (tg/-) rats in comparison with CR rats, 173–175 insulin signaling in longevity models, 175–176 in longevity models, 173 overview, 173 selected parameters for insulin resistance, 176–177 stress response, 177–180 glucose metabolism, 159 GMC-SF (granulocyte macrophage colonystimulating factor), 35–36 GPCR (G-protein-coupled receptor), 21–23 G-protein-coupled receptor (GPCR), 21–23 granule cell layer (GCL) neurons, 122–124, 130, 133, 185 granulocyte macrophage colony-stimulating factor (GMC-SF), 35–36 granulose cells, 146 growth hormone (GH), 4–9, 21, 65–69, 71–74, 77–83, 87–92, 95–101, 107–115, 121–136 absence of saturable transport system for at blood–brain barrier, 73 actions in mammalian brain, 183–193 cognitive role, 192–193 endocrine, paracrine, and autocrine sources of growth hormone and IGF-1 act on brain, 184 IGF-1 promotes neurogenesis from early stages of development, 184–185 overview, 183 adult cell genesis by, 130–133 in Alzheimer’s disease, 295–304 cognitive decline in elderly, 296–297 growth hormone response, 300–301 overview, 295–296 possible usefulness of IGF-1 axis in treatment, 302–304 autoregulation, 9 and cellular signaling, 150 in cerebrospinal fluid (CSF) compartment, 66–68

337

Index conditions where GH/IGF-1 axis affects cognitive/mental function, 161–168 aging and depression, 162–168 exercise, 162 overview, 161–162 discontinued GH treatment, cognitive effects of, 288 factors affecting biosynthesis and secretion in pituitary, 5–9 GH and IGF-1, 8–9 growth hormone-releasing hormone and somatostatin, 6–7 Leptin and Ghrelin, 7–8 overview, 5–6 thyroid hormones, 8 future perspectives, 12–19 GH axis in dementia, 273–275 GH axis in Down syndrome, 275–280 GH deficiency and GH treatment, cognitive status in relation to, 285–287 GH dosages and cognitive status in GH-deficient adults, 289 GH-induced cognitive changes in healthy elderly subjects, 290–294 GH-induced hormonal changes, cognitive effects of, 284–285 GH receptor deficiency, intelligence in subjects with, 290 hypersecretion, 263–266 and IGF-1 in nervous system, 78–79 impact on dementia, 267–280 increased concentrations, effects on human brain, 263–266 during growth hormone hypersecretion, 264–265 overview, 263–264 treatment of growth hormone-producing tumors, 265–266 lack of direct interaction of GH and insulin, 73–75 mechanisms regulating biosynthesis and secretion, 4–5 meta-analysis GH and neuropsychological functions, 287–288 overview, 4 quality of life in deficiency of, 215–226 effect of long-term GH replacement on, 223 effect of untreated GH deficiency on, 217–218 effects of GH replacement on in GHD hypopituitary adults, 219–222 following discontinuation of childhood GH therapy, 219 importance of, 215–216 measurement of, 216–217 natural history of in adults with untreated GHD, 219 overview, 215 potential biases in open studies of, 224–226 predictors of improvements in with GH replacement, 223–224 specific patient groups, 222 whether GH causative in impaired quality of life of hypopituitary adults, 218–219

regulation of connexin43/gap junctional coupling by, 151–152, 156–159 IGF-1 system and connexin43/gap junctions, 159 overview, 156 systemic versus local pathways, 156–158 replacement in childhood, psychological importance of, 245–262 effect of GH on cognitive and academic performance, 248–252 effect of GH on psychological well-being, 252–256 future directions, 258–262 GH secretion versus sensitivity, 246 location of action of GH, 246 methodological difficulties in measurement, 256–258 modes of action of GH, 246 overview, 245 typical cognitive development and critical windows of development, 246–247 replacement in syndromes other than GHD, 289–290 replacement therapy, and quality of life, 321–327 cost–benefit considerations, 240–244 effects of GH deficiency and GH therapy on QoL in specific conditions, 239–240 in GHD adults, 235–239 quality of life measurements, 234–235 therapeutic mechanisms of GH replacement, 240 role in cognitive functions, 283–284 secretion, 5, 193–194 signaling, 35–42 signal transduction pathways activated by, 37–45 c-Src utilization to activate Ras-like small GTPases, 39–40 FAK, 38–39 JAKs, 37–38 overview, 37 Ras-like small GTPases, 39 SOCS, 41–45 Src family, 38 STATs, 40–41 sites of action in developing mammalian brain, 77–78 transcription, 4–5, 8 growth hormone (GH)/ insulin-like growth factor-I (IGF-I) axis, 295–296, 302, 309, 323 growth hormone antagonists (GHAs), 307–320 clinical trials, 311–314 long term, 313–314 overview, 311–312 phase I, 312 phase II, 312 phase III, 312–313 retinopathy, 314 development of Pegvisomant, 311 discovery of, 309–311 other uses of Pegvisomant, 314–320 overview, 314–315 in treatment of cancer, 316–320

in treatment of cardiovascular disease, 316 in treatment of diabetes, 315 in treatment of nephropathy, 315–316 overview, 307–309 preclinical trials, 311 growth hormone-binding proteins (GHBP), 36, 95–97, 99–101, 162 growth hormone deficiency (GHD), 65–69, 87, 130, 136, 150, 162, 227, 229–230, 233–241, 245–258, 267–277, 281–292, 307–309, 322, 325 adult onset (AO), 193, 236, 240, 287–288, 291 cancer survivors, 222, 224–225 childhood onset (CO), 236, 240, 248–258, 282–288, 291 tests for, 234–235 growth hormone insensitivity syndrome (GHIS), 99 growth hormone modulation, embryonic and postnatal growth under, 12 growth hormone-producing tumors, 263–265 growth hormone promoter, 5 growth hormone receptors (GHR), 35–42, 66, 78–79, 87–92, 95–101, 107–114, 132, 150, 156–158, 171–175, 184, 246, 257, 307–311, 315–316 cloning of, 96–97 dimerization, 311 gene expression, regulation of, 100–103 GHR mRNA, in central nervous system (CNS), 97–100 purification of, 87–93 growth hormone-releasing hormone (GHRH), 5–9, 21–22, 24–25, 27–28, 111–112, 182, 194, 196–197, 227–229, 255, 267–276, 277, 291–292, 300–301, 308 Growth Hormone Research Society, 325 growth hormone secretagogue receptors (GHS-R), 21–23, 27–28 growth hormone secretagogues (GHSs), 21–22, 27–28, 182, 227, 268, 271, 276, 292 guanine nucleotide exchange factor (GEF), 39–40

H hGH (human growth hormone), 36, 78, 109. see also growth hormone (GH) HI (hypoxic-ischemic) injury, 183 hippocampal progenitors, adult, 129–130 hippocampus, 130–131 differentiation, 130–131 proliferation, 130 in vitro, 131 histone acetylation, 8 homovanillic acid (HVA), 246, 282, 292 Hopkins Symptom Check List (HSCL), 287 hormone iodination, 89 HSCL (Hopkins Symptom Check List), 287 human choroid plexus, 91, 101, 109 human growth hormone (hGH), 36, 78, 109. see also growth hormone (GH) HVA (homovanillic acid), 246, 282, 292 hyperglycemia, 173–175 hyperinsulinemia, 173

338

Index

hyperinsulinism, 273 hyperphagia, 114 hyperprolactinemia, 265 hypogonadism, 265 hypopituitaric growth hormone deficiency, 269 hypopituitarism, 312 hypopituitary adults, 215–225 hypothalamic cholinergic hypoactivity, 270 hypothalamic GH-releasing hormone, 21 hypothalamus–pituitary–adrenal axis, 284 hypothyroidism, 8 hypoxic-ischemic (HI) injury, 183

I IDDM (insulin-dependent diabetes mellitus), 315 IGF, see insulin-like growth factor (IGF) IGF-1, see insulin-like growth factor-1 (IGF-1) IGF-1R (insulin-like growth factor receptors), 47–49, 80–81, 150–157, 246 IGFBPs, see insulin-like growth factor binding proteins (IGFBPs) IGFs (insulin-like growth factors), 3, 47–55 immunohistochemistry, 127–128 impulsivity, effect of GH on, 251 insulin, 3, 72–73, 170–177, 298–299, 315–316 insulin-dependent diabetes mellitus (IDDM), 315 insulin-like growth factor (IGF), 77–83, 112–114 expression in nervous system, 80 IGF-binding proteins, 81–86 insulin-like growth factor-1 (IGF-1), 8–9, 27, 47–61, 65–69, 71–74, 77–86, 99–100, 112–114, 121–136, 143–144, 149–162, 169–178, 181–198 actions in mammalian brain, 183–193 cognitive role, 192–193 effects on synaptic structure, function, and strength, 189–192 endocrine, paracrine, and autocrine sources of growth hormone and IGF-1 act on brain, 184 estrogen and IGF-1, 192 exertion of prosurvival/antiapoptotic actions, 185–187 IGF-1 promotes neurogenesis from early stages of development, 184–185 neuroprotective effects pre- and postinjury, 187–189 overview, 183 adult cell genesis by, 130–133 age-related decreases in plasma IGF-1, and development of neurodegenerative diseases, 196–204 overview, 196 in vitro studies, 196–197 in vivo studies, 197–204 in Alzheimer’s disease, 295–304 cognitive decline in elderly, 296–297 IGF-1 system as neurotrophic factor, 297–298 insulin and IGF-1 in Alzheimer’s disease, 298–299 insulin-like growth factor-binding protein-3, 299

mechanisms of action of IGF-1, 299 overview, 295–296, 297 possible usefulness of IGF-1 axis in treatment, 302–304 tumor necrosis factor ␣ and Its Relationship with IGF-1 effects, 298 and cell genesis in adult brain, 124–125 and cellular signaling, 150–151 in cerebrospinal fluid (CSF) compartment, 66–69 conditions where GH/IGF-1 axis affects cognitive/mental function, 161–168 aging and depression, 162–168 exercise, 162 overview, 161–162 effect of decreased plasma levels on age-related cognitive deficits, 194–196 IGF-1 axis in dementia, 273–275 IGF-1 axis in Down syndrome, 275–280 impact on dementia, 267–280 age-related changes in activity of IGF-1 axis, 268–273 Alzheimer’s disease, 273–275 Down syndrome, 275–280 overview, 267–268 loss of IGF-1 function contributing to brain aging and disease, 208–209 and neuroprotection, 205–211 loss of IGF-1 function contributing to brain aging and disease, 208–209 maintenance of brain cell function, 207–208 mechanisms of, 208 neuroprotective signaling, 206 overview, 205–206 perspectives, 209–211 regulation of new brain cell populations, 206–207 neuroprotective effects of, 125–126 and ocular neovascularization, 54–55 overview, 10–12, 47–49 regulation of connexin43 by, 156–159 IGF-1 system and connexin43/gap junctions, 159 overview, 156 systemic versus local pathways, 156–158 regulation of connexin43/gap junctional coupling by, 151–152 signaling, 48–49, 209 tissue-specific expression of, 10 transport systems for at blood–brain barrier, 72–73 insulin-like growth factor 2 (IGF-2), 48 insulin-like growth factor binding proteins (IGFBPs), 11, 47–55, 69, 82–83, 151–159, 188, 194, 285, 290, 296, 299, 302, 313, 322 function, 52–53 IGFBP-related proteins (IGFBP-Rps), 50–51 overview, 49–50 proteolysis, 53–55 regulation of expression, 51–52 structure, 50–51 insulin-like growth factor receptors, 112 insulin-like growth factor receptors (IGF-1R), 47–49, 80–81, 150–157, 246

insulin-like growth factors (IGFs), 3, 47–55 insulin receptor (IR), 175–176 insulin tolerance, 235 insulin transport systems, at blood–brain barrier (BBB), 72–73 intelligence quotient, effect of GH on, 248 IQ scores, 282, 285–287 IR (insulin receptor), 175–176

J Janus kinase (JAK), 35, 37–42, 107–108, 110, 114 Janus kinase 2 (JAK2), 307

K KIMS database, 241

L lactotrophic systems in central nervous system, 108–111 brain production of GH and prolactin, 111 distribution of GH and prolactin receptors in brain, 109 overview, 108 signal transduction pathway, 109–111 transport of GH and prolactin into brain, 108–109 LDL (low-density lipoprotein) levels, 316 learning, 135, 136, 251–252 leptin, 7–8, 26 liver IGF-1-deficiency (LID), 315 longevity models for growth hormone–insulinlike growth factor 1/insulin axis, 170–172 longitudinal bone growth, 4 low-density lipoprotein (LDL) levels, 316

M magnetic resonance imaging (MRI), 313 mammalian ghrelin homologues, 23 MAPK (mitogen-activated protein kinase), 37–40, 131, 151, 308 MAP kinase pathway, 171, 183, 186–187, 191 maturity, psychological, effect of GH on, 254–255 memory, effect of GH on, 251–252 Mini Mental State Examination (MMSE), 290 mitogen-activated protein kinase (MAPK), 37–40, 131, 151, 308 MMSE (Mini Mental State Examination), 290 molecular sieve chromatography, 89 mood effect of GH on, 255 and somatotropic axis, 229–232 motivation, effect of GH on, 255 MPHD (multiple pituitary hormone deficiency), 282–288 MRI (magnetic resonance imaging), 313 mRNA transcript for GHR in rat, 97 multiple pituitary hormone deficiency (MPHD), 282–288 myoblast differentiation, 4

339

Index

N National Institute of Clinical Excellence (NICE), British, 216, 245, 321, 324–326 nephropathy, 314–316 “neural stem cell,” 122 neural stem cell, defined, 122–123 neurodegeneration, 298 neurodegenerative diseases, 181, 196–204 neuroendocrine regulation, 300 neurogenesis, 11, 114–115, 121–125, 132–136, 184 conditions affecting, 124 hippocampal, 122, 130, 133–135 history, 121–122 postnatal, 122 two regions with, 123–124 neurons, 133 neuroprotection, and insulin-like growth factor-1, 205–211 loss of IGF-1 function contributing to brain aging and disease, 208–209 maintenance of brain cell function, 207–208 mechanisms of, 208 neuroprotective signaling, 206 overview, 205–206 perspectives, 209–211 regulation of new brain cell populations, 206–207 neuroprotective effects, 11 NF-_B activation, 197 NHP (Nottingham Health Profile), 217–224, 234, 239 NICE (National Institute of Clinical Excellence), British, 216, 245, 321, 324–326 nitric oxide signaling pathways, 191 N-methyl-D-aspartate (NMDA) receptors, 195–196, 198, 282–283, 292 nonverbal learning disorder (NVLD), 251 Northern blot analysis, 10, 98 Nottingham Health Profile (NHP), 217–224, 234, 239 nuerotransmissional plasticity, 149 NVLD (nonverbal learning disorder), 251

O obesity, 7 ocular neovascularization, 54–55 oligodendrocytes, 133–134, 148, 188

P PACAP (pituitary adenylate cyclase-activating polypeptide), 5–6 paraventricular nucleus (PVN), 25 Pegvisomant, 311–320 development of, 311 overview, 314–315 in treatment of cancer, 316–320 in treatment of cardiovascular disease, 316 in treatment of diabetes, 315 in treatment of nephropathy, 315–316 perception, effect of GH on, 251

perinatal stage of CNS development, 11 PGWB (Psychological General Well-Being Schedule), 217–224 physiological neuroprotective network, 205 Piaget, 246–248 pindolol, 301 pituitary distribution of ghrelin, 24 signaling mechanisms of GH secretion in, 5 pituitary adenylate cyclase-activating polypeptide (PACAP), 5–6 PKC (protein kinase C) pathway, 5–7 pleiotropic effects, 233, 245 PLP (proteolipid protein), 188 postmenopausal women, IGF treatment in, 289 Prader–Willi syndrome, 288–289 preparative zone electrophoresis in novel multibuffer system, 89 proapoptotic pathways, 208 prolactin, 107–115, 170 differential actions of GH and prolactin in brain, 111–113 cognitive function, 113 feedback regulation of hormone secretion, 111–112 maternal behavior and neuroendocrine adaptation to pregnancy and lactation, 112–113 overview, 111 potential interaction with GH in brain function, 113–119 food intake, 113–114 overview, 113 tuberoinfundibular development, 113 prolactin (PRL) receptors, 88–92, 107–114 protein kinase C (PKC) pathway, 5–7 proteolipid protein (PLP), 188 psychiatric symptoms, and somatotropic axis, 229–232 Psychological General Well-Being Schedule (PGWB), 217–224 psychological maturity, effect of GH on, 254–255 Purkinje cells, 189, 191 PVN (paraventricular nucleus), 25 pyridostigmine, 301

predictors of improvements in with GH replacement, 223–224 specific patient groups, 222 whether GH causative in impaired quality of life of hypopituitary adults, 218–219 effect of GH on, 256 and growth hormone replacement therapy, 321–327 in adults with growth hormone deficiency, 235–239, 324–327 cost–benefit considerations, 240–244 effects of GH deficiency and GH therapy, 239–240 in frail elderly, 322–324 neuroprotective effect of GH in spinal cord injury, 321–322 overview, 321 QoL measurements, 234–235 therapeutic mechanisms of GH replacement, 240

R rapid eye movement (REM) sleep, 228–229 Ras-like small GTPases, 39 RAS-MAPK pathway, 171 rats effect of IGF-1 decreased plasma levels on age-related cognitive deficits, 194–196 glucose–insulin system in anti-GH (tg/-) rats in comparison with CR rats, 173–175 mRNA transcript for GHR in, 97 restraint stress in water (RSW), 101 reactive nitrogen species (RNS), 177 reactive oxygen species (ROS), 177, 208 recombinant human insulin-like growth factor 1 (rhIGF-1), 143, 152–153, 155, 158 REM (rapid eye movement) sleep, 228–229 retinal pigment epithelium (RPE), 54–55 reversed transcriptase polymerase chain reaction (RT-PCR), 99, 111 rhIGF-1 (recombinant human insulin-like growth factor 1), 143, 152–153, 155, 158 RNS (reactive nitrogen species), 177 ROS (reactive oxygen species), 177, 208 RPE (retinal pigment epithelium), 54–55 RT-PCR (reversed transcriptase polymerase chain reaction), 99, 111

Q quality of life (QoL), 283–284, 321–326 in deficiency of growth hormone, 215–226 effect of long-term GH replacement on, 223 effect of untreated GH deficiency on, 217–218 effects of GH replacement on in GHD hypopituitary adults, 219–222 following discontinuation of childhood GH therapy, 219 importance of, 215–216 measurement of, 216–217 natural history of in adults with untreated GHD, 219 overview, 215

S school achievement, effect of GH on, 248 SDS (sodium dodecyl sulfate)–polyacrylamide gel electrophoresis, 90 secretagogues, 8 self-esteem, effect of GH on, 253–254 SGZ (subgranular zone), 123–124 signal tranducers and activator of transcription (STATs), 40–41 signal transduction pathways activated by growth hormone, 37–45 c-Src utilization to activate Ras-like small GTPases, 39–40 FAK, 38–39

340 signal transduction pathways activated by growth hormone—(Continued) JAKs, 37–38 overview, 37 Ras-like small GTPases, 39 SOCS, 41–45 Src kinase family, 38 STATs, 40–41 sleep effect of GH on, 255 and somatotropic axis, 228–229 slow wave sleep (SWS), 228–229 SOCS (suppressors of cytokine signaling), 41–45, 110 sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, 90 somastatin hyperactivity, 267–270, 275 somatic growth, 3–4 “somatomedin hypothesis,” 4 somatostatin, 6–7, 9, 182, 301 somatotrophic systems in central nervous system, 108–111 brain production of GH and prolactin, 111 distribution of GH and prolactin receptors in brain, 109 overview, 108 signal transduction pathway, 109–111 transport of GH and prolactin into brain, 108–109 somatotrophin, see growth hormone (GH) somatotrophs and cell genesis in adult brain, 124–125 hormonal manipulation of somatotrophic axis, 127–130

Index neuroprotective actions of somatotrophic axis, 125–127 somatotropic axis in psychologic functioning, 227–232 mood and psychiatric symptoms, 229–232 overview, 227–228 sleep, 228–229 Src kinase family, 38 ß-amyloid, 298–299 STAT proteins, 40–41, 107–108, 110–111 STATs (signal tranducers and activator of transcription), 40–41 stomach, distribution of ghrelin, 23–24 stress, 134–135, 256 subgranular zone (SGZ), 123–124 suppressors of cytokine signaling (SOCS), 41–45, 110 SWS (slow wave sleep), 228–229 synaptogenesis, 124

T tachyphylaxis, 289 testosterone, 323–324 TGF-␤1, 158–159 thyroid hormones, 8 thyroid-stimulating hormone (TSH), 170–171 thyroxine, 158 transgenic animals, 132 transgenic brain tissue, 184 TSH (thyroid-stimulating hormone), 170–171 tumors, growth hormone-producing, treatment of, 265–266

Turner syndrome, GH substitution and cognition in, 289–290

U ultrasonography, 313 upstream stimulatory factory (USF), 4 USF (upstream stimulatory factory), 4

V vagus nerve, and appetite regulation by ghrelin, 26–27 vascular endothelial growth factor (VEGF), 54–55, 314 verbal and performance IQ discrepancy, effect of GH on, 250–251 vigor/vitality, effect of GH on, 255 visual-motor skills, effect of GH on, 251 visual-spatial deficits, effect of GH on, 248–252

W Wechsler Adult Intelligence Scale (WAIS), 285, 289 World Health Organization (WHO), 233–234

X X-ray crystallography, 36

Z zone electrophoresis, 87–89, 91–92