Insulin Resistance and Insulin Resistance Syndrome (Frontiers in Animal Diabetes Research, 5)

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INSULIN RESISTANCE AND INSULIN RESISTANCE SYNDROME

Frontiers in Animal Diabetes Research Each volume of this series will be topic oriented with timely and liberally referenced reviews and provide in depth coverage of basic experimental diabetes research. Edited by Professor Anders A.F. Sima, Wayne State University, Detroit, USA and Professor Eleazar Shafrir, Hadassah University Hospital, Jerusalem, Israel.

Volume 1 Chronic Complications in Diabetes: Animal Models and Chronic Complications edited by Anders A.F. Sima Volume 2 Animal Models of Diabetes: A Primer edited by Anders A.F. Sima and Eleazar Shafrir Volume 3 Insulin Signaling: From Cultured Cells to Animal Models edited by George Grunberger and Yehiel Zick Volume 4 Muscle Metabolism edited by Juleen R. Zierath and Harriet Wallberg-Henriksson Volume 5 Insulin Resistance and Insulin Resistance Syndrome edited by Barbara Hansen and Eleazar Shafrir

This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.

INSULIN RESISTANCE AND INSULIN RESISTANCE SYNDROME

Edited by

Barbara Hansen School of Medicine University of Maryland Baltimore, USA and

Eleazar Shafrir Department of Biochemistry Hadassah University Hospital Jerusalem, Israel

London and New York

First published 2002 by Taylor & Francis 11 New Fetter Lane, London EC4P 4EE Simultaneously published in the USA and Canada by Taylor & Francis Inc 29 West 35th Street, New York, NY 10001 Taylor & Francis is an imprint of the Taylor & Francis Group This edition published in the Taylor & Francis e-Library, 2004. © 2002 Taylor & Francis All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. Every effort has been made to ensure that the advice and information in this book is true and accurate at the time of going to press. However, neither the publisher nor the authors can accept any legal responsibility or liability for any errors or omissions that may be made. In the case of drug administration, any medical procedure or the use of technical equipment mentioned within this book, you are strongly advised to consult the manufacturer’s guidelines. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book has been requested ISBN 0-203-21741-1 Master e-book ISBN

ISBN 0-203-27318-4 (Adobe eReader Format) ISBN 0-415-29197-6 (Print Edition)

CONTENTS Preface to the Series

vii

Preface

ix

Contributors

xi

1 Metabolism and Hypertension 1

Insulin Resistance and Glycogen Synthesis: Roles in Liver, Muscle and Adipose Tissue Hagit Eldar-Finkelman and Merav Yarkoni

2

Gestational Diabetes and Maternal Insulin Resistance in the C57BLKS/JLeprdb/ Mouse – A Unique Model for Understanding its Impact on the Fetus Jianhua Shao and Jacob E. Friedman

21

Role of Protein-Tyrosine Phosphatases in Insulin Action and Insulin Resistance: Recent Insights from Cellular and Animal Studies Barry J. Goldstein

35

3

3

4

Hypertension and Insulin Resistance in the Wistar Fatty Rat Masami Suzuki, Hiroyuki Odaka and Yasuo Sugiyama

51

5

Cardiovascular Disease in the Insulin-Resistant, Atherosclerosis-Prone JCR :LA-cp Rat J.C. Russell and S.E. Kelly

59

6

The C57BL/6J Mouse as a Model of Insulin Resistance and Hypertension Tonya Martin, Sheila Collins and Richard S. Surwit

73

2 Molecular and Genetic 7

Molecular Features of Insulin Resistance, Obesity and Type 2 Diabetes in Non-Human Primates Stephen V. Angeloni and Barbara Caleen Hansen

8

Effects of Genetic Alterations of Glut4 on Insulin Sensitivity Naira Gorovits, J. Skye Laidlaw, Mollie Ranalletta, Gloria Tannenbaum, Ellen B. Katz and Maureen J. Charron

9

Insulin Signaling Pathway and GLUT4-mediated Glucose Transport in the Insulin Resistant Muscle Daniel Konrad, Varinder K. Randhawa, Carol T.-L. Huang and Amira Klip

89

125

147

10

Which Genes are Important in the Development of Type 2 Diabetes? G.R. Collier, K. Walder, A. de Silva, S. Morgan, D. Segal, L. Kantham and G. Augert

175

11

Leptin and Insulin Resistance in Rodent Models Kevin L. Stark

187

v

vi

CONTENTS

12

Fat Feeding and Muscle Fat Deposition Eliciting Insulin Resistance E.W. Kraegen, G.J. Cooney, J.M. Ye and S.M. Furler

195

13

D-chiro-inositol and Insulin Resistance: An Allosteric Point of View Joseph Larner

211

14

Glucagon-like Peptide-1, Exendin and Insulin Sensitivity Andrew A. Young

235

15

Insulin Resistance and the Autonomic Nervous System L. Penicaud, C. Leloup, A. Lorsignol and T. Alquier

263

16

Hypothalamic Role in the Insulin Resistance Syndrome Anthony H. Cincotta

271

17

Insulin Resistance – Emerging Therapies for Affected Sites Julie S. Moyers and José F. Caro

313

3 Aging 18

Effect of Age on the Emergence of Insulin Resistance Nir Barzilai and Ilan Gabriely

337

19

Postnatal and Adult Insulin Sensitivity and Metabolism in Progeny of Nutritionally Compromised Mothers Clive J. Petry, Susan E. Ozanne and C. Nicholas Hales

349

Index

363

PREFACE TO THE SERIES Diabetes has been declared a major global health hazard by the WHO. Over the last few decades there has been an alarming increase in the incidence of diabetes particularly in densely populated areas such as India, China, southeast Asian countries and Arab nations. Even in North America and Europe the incidence of diabetes increases by 5% a year. The direct and indirect costs associated with diabetes are enormous. In the US they amounted to $137 billion in 1997 or a seventh of the total health care costs in this country. To avert this rapidly evolving global epidemic, it behoves the international biomedical community and responsible federal agencies and interest groups to intensify research into the causes of this disease and its complications, and to rapidly increase public awareness of the disease through education. Major advances have been made in diabetes research in animal models, contributing enormously to the understanding of etiopathology of this disease and its dreaded chronic complications. In particular factors in the areas of immunology, insulin signal transduction and insulin action as well as pathogenetic mechanisms involved in the development of the chronic complication have become clearer. The new knowledge gained is only slowly being translated to the beneﬁt of the patients and to serve as a basis for the development of new therapeutic modalities. The accumulation of this scattered information and ongoing publication of data from the interdisciplinary and critical reviews on diabetes in various animals is our fundamental motive. It is our hope that this book series on Frontiers in Animal Diabetes Research will be an efﬁcient vehicle for communicating extensive up-to-date review articles by the leading world experts in the ﬁeld. Each volume will be topic oriented with timely and liberally referenced reviews. It will ﬁll a gap in the spectrum of diabetes related journals and publications in as far as it will focus on all aspects of basic experimental diabetes research. As such we hope it will provide a valuable reference source for graduate students, research fellows, basic academic and pharmacological researchers as well as clinic investigators. Anders A.F. Sima Eleazar Shafrir

vii

PREFACE In 1936, Himsworth (Himsworth, 1936) observed that some diabetic patients require increasing amounts of insulin, and appear to become gradually insensitive to insulin, or “resistant” to the actions of insulin. Today, the term “insulin resistance” is applied to a broad range of biological observations, with an equally wide variety of potential underlying mechanisms. Animal models are being extensively used to examine both the causes of loss of insulin sensitivity, and the potential for reversal of this loss. Consensus has not yet developed around a speciﬁc and quantiﬁable deﬁnition of insulin resistance. Most commonly, the presence of insulin resistance has been inferred when a given amount of endogenous or exogenous insulin has shown less than its expected biological action. In truth, insulin resistance is substantially related to the methods used to seek it, and the methods today differ widely from in vitro cellular methods to whole body euglycemic or hyperglycemic clamps. Over the past twenty years the so-called “gold standard” for measuring in vivo (whole body) insulin resistance has been this “clamp”, as developed initially by DeFronzo, Tobin, and Andres (DeFronzo et al., 1979). During the earliest phases in the development of type 2 diabetes, insulin resistance gradually develops, accompanied by increasing fasting hyperinsulinemia and increasing beta cell responsiveness to a glucose stimulus. Animal models of obesity and diabetes have contributed to understanding the natural history of the development of insulin resistance and to identifying some of the factors underlying this insulin resistance. In the present volume, we have drawn together some of the leaders in the ﬁeld who provide their perspectives on the mechanisms of insulin resistance, as discerned from various animal models. While the exact mechanism underlying the appearance of resistance to insulin remains unknown, the authors here have examined many of the important factors. The contribution of age, from early perinatal to aged adulthood, has been considered. Insulin resistance is a major feature of the middle-aged onset, obesity-associated Metabolic Syndrome, sometimes referred to as Diabesity (Shafrir, 1993), diabetogenic obesity (Vague, 1956), or syndrome X (Reaven, 1988). This syndrome, described by many clinicians and investigators beginning nearly ﬁfty years ago (Vague, 1956), derives from observation of the frequent interactions of insulin resistance with obesity, dyslipidemia, glucose intolerance, cardiovascular disease, hypertension, and abnormalities in whole body metabolism, as previously reviewed (Hansen, 1999). These clinical features and their possible insulin resistanceassociated pathophysiology, as elucidated by a variety of animal models, are discussed in a series of chapters included here. Finally, many pieces of the complex signaling pathways involved in causing insulin resistance, or emerging as a consequence of insulin resistance have been identiﬁed at the molecular level. The human genome revolution has been paralleled by extensive study of the genetics of animal models. New targets for pharmaceutical and other therapeutic interventions, based in part on key observations made in animal models, are being identiﬁed, and these genetic and molecular studies are leading to growth in our understanding of insulin sensitivity and insulin resistance.

REFERENCES DeFronzo, R.A., Tobin, J.D. and Andres, R. (1979) Glucose clamp technique: A method for quantifying insulin secretion and resistance. Am. J. Physiol., 237, E214–E223. ix

x

PREFACE

Hansen, B.C. (1999) The metabolic syndrome X. In Hansen, B.C., Saye, J., Wennogle, L.P. (eds) The Metabolic Syndrome X. Convergence of Insulin Resistance, Glucose Intolerance Hypertension, Obesity and Dyslipidemias – Searching for the Underlying Defects. New York: Annals of New York Academy of Sciences, pp. 1–24. Himsworth, H.P. (1936) Diabetes mellitus: Its differentiation into insulin sensitive and insulin insensitive types. Lancet, i, 127–130. Reavean, G. (1988) Banting lecture 1988: Role of insulin resistance in human disease. Diabetologia, 30, 1595–1607. Shafrir, E. (1993) Animal models of syndrome X. Curr. Topics in Diab. Res., 12, 165–181. Shafrir, E. (1996) Development and consequences of insulin resistance: lessons from animals with hyperinsulinemia. Diabetes & Metabolism (Paris), 22, 122–151. Vague, J. (1956) The degree of masculine differentiation of obesities: A factor determining predisposition to diabetes, atherosclerosis, gout, and uric calculous disease. Am. J. Clin. Nutr., 4, 20–34.

CONTRIBUTORS T. Alquier UMR 5018 UPS-CNRS IFR 31 CHU Rangueil 1 Avenue Jean Poulhés 31403 Toulouse France

G.R. Collier Metabolic Research Unit School of Health Sciences Deakin University Geelong, Victoria 3217 Australia

Stephen V. Angeloni Obesity and Diabetes Research Center University of Maryland School of Medicine Baltimore, MD 21201 USA

Sheila Collins Duke University Medical Center Durham NC 27710 USA

G. Augert Merck-Lipha Lyon France

G.J. Cooney Garvan Institute of Medical Research St Vincent’s Hospital Sydney, NSW 2010 Australia

Nir Barzilai Department of Medicine Divisions of Geriatrics, Endocrinology, and the Diabetes Research and the Training Center Albert Einstein College of Medicine Bronx, NY 10461 USA

Hagit Eldar-Finkelman Department of Human Genetics and Molecular Medicine Sackler School of Medicine Tel Aviv University Israel

José F. Caro Endocrine Research Eli Lilly and Company Lilly Corporate Center Indianapolis, IN 46285 USA

Jacob E. Friedman Departments of Pediatrics, Biochemistry and Molecular Genetics University of Colorado Health Sciences Center 4200 East Ninth Avenue Denver, CO 80262 USA

Maureen J. Charron Department of Biochemistry Albert Einstein College of Medicine Bronx, NY 10461 USA

S.M. Furler Garvan Institute of Medical Research St Vincent’s Hospital Sydney, NSW 2010 Australia

Anthony H. Cincotta 158 Lake Road Tiverton, RI 02878 USA xi

xii

CONTRIBUTORS

Ilan Gabriely Divisions of Geriatrics, Endocrinology, and the Diabetes Research and the Training Center Department of Medicine Albert Einstein College of Medicine Bronx, NY 10461 USA Barry J. Goldstein Division of Endocrinology, Diabetes and Metabolic Diseases Jefferson Medical College Room 349 Alumni Hall Philadelphia, PA 19107 USA Naira Gorovits Department of Chemistry Albert Einstein College of Medicine Bronx, NY 10461 USA C. Nicholas Hales Clinical Biochemistry Department University of Cambridge Addenbrooke’s Hospital Hills Road, Cambridge UK Barbara Caleen Hansen Obesity and Diabetes Research Center University of Maryland School of Medicine Baltimore, MD 21201 USA

Ellen B. Katz Department of Biochemistry Albert Einstein College of Medicine Bronx, NY 10461 USA

S.E. Kelly Department of Surgery University of Alberta Edmonton, Alberta Canada

Amira Klip Programme in Cell Biology The Hospital for Sick Children Toronto, Ontario, M5G 1X8 Canada

E.W. Kraegen Garvan Institute of Medical Research St Vincent’s Hospital Sydney, NSW 2010 Australia

Daniel Konrad Programme in Cell Biology The Hospital for Sick Children Toronto, Ontario, M5G 1X8 Canada

Carol T.-L. Huang Programme in Cell Biology The Hospital for Sick Children Toronto, Ontario, M5G 1X8 Canada

J. Skye Laidlaw Department of Biochemistry Albert Einstein College of Medicine Bronx, NY 10461 USA

L. Kantham Metabolic Research Unit School of Health Sciences Deakin University Geelong, Victoria 3217 Australia

Joseph Larner Insmed Pharmaceuticals, Inc. 800 E. Leigh St Richmond, VA 23219 USA

CONTRIBUTORS

C. Leloup UMR 5018 UPS-CNRS IFR 31 CHU Rangueil 1 Avenue Jean Poulhés 31403 Toulouse France

L. Penicaud UMR 5018 UPS-CNRS IFR 31 CHU Rangueil 1 Avenue Jean Poulhes 31403 Toulouse France

A. Lorsignol UMR 5018 UPS-CNRS IFR 31 CHU Rangueil 1 Avenue Jean Poulhés 31403 Toulouse France

Clive J. Petry Clinical Biochemistry Department University of Cambridge Addenbrooke’s Hospital Hills Road, Cambridge UK

Tonya Martin Duke University Medical Center Durham NC 27710 USA

Mollie Ranalletta Department of Biochemistry Albert Einstein College of Medicine Bronx, NY 10461 USA

S. Morgan Metabolic Research Unit School of Health Sciences Deakin University Geelong, Victoria 3217 Australia

Varinder K. Randhawa Programme in Cell Biology The Hospital for Sick Children Toronto, Ontario, M5G 1X8 Canada

Julie S. Moyers Endocrine Research Eli Lilly and Company Lilly Corporate Center Indianapolis, IN 46285 USA Hiroyuki Odaka Pharmacology Research Laboratories II Takeda Chemical Industries Ltd 2-17-85 Juso honmachi Yodogawa-ku Osaka 532-8686 Japan Susan E. Ozanne Clinical Biochemistry Department University of Cambridge Addenbrooke’s Hospital Hills Road, Cambridge UK

J.C. Russell Department of Surgery University of Alberta Edmonton, Alberta Canada D. Segal Metabolic Research Unit School of Health Sciences Deakin University Geelong, Victoria 3217 Australia Jianhua Shao Departments of Pediatrics, Biochemistry and Molecular Genetics University of Colorado Health Sciences Center 4200 East Ninth Avenue Denver, CO 80262 USA

xiii

xiv

CONTRIBUTORS

A. de Silva Metabolic Research Unit School of Health Sciences Deakin University Geelong, Victoria 3217 Australia

Gloria Tannenbaum Department of Biochemistry Albert Einstein College of Medicine Bronx, NY 10461 USA

Kevin L. Stark Department of Metabolic Disorders Amgen One Amgen Center Drive Thousand Oaks, CA 91362 USA

K. Walder Metabolic Research Unit School of Health Sciences Deakin University Geelong, Victoria 3217 Australia

Yasuo Sugiyama Pharmacology Research Laboratories II Takeda Chemical Industries Ltd 2-17-85 Juso honmachi Yodogawa-ku Osaka 532-8686 Japan

Merav Yarkoni Department of Human Genetics and Molecular Medicine Sackler School of Medicine Tel Aviv University Israel

Richard S. Surwit Duke University Medical Center Durham NC 27710 USA Masami Suzuki Pharmacology Research Laboratories II Takeda Chemical Industries Ltd 2-17-85 Juso honmachi Yodogawa-ku Osaka 532-8686 Japan

J.M. Ye Garvan Institute of Medical Research St Vincent’s Hospital Sydney, NSW 2010 Australia

Andrew A. Young Amylin Pharmaceuticals Inc. 9373 Towne Center Drive San Diego, CA 92121 USA

Part 1

Metabolism and Hypertension

1. INSULIN RESISTANCE AND GLYCOGEN SYNTHESIS: ROLES IN LIVER, MUSCLE AND ADIPOSE TISSUE HAGIT ELDAR-FINKELMAN AND MERAV YARKONI Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel

INTRODUCTION Insulin resistance is a key factor in the pathogenesis of obesity and type 2 diabetes, the world’s most prevalent metabolic disorder. Insulin resistance is also the cause of insulin resistance syndrome, syndrome X, in which physiological abnormalities such as renal failure, hypertension and neuropathy cluster together. Insulin resistance is characterized by the failure of tissues to respond to insulin, resulting in reduced glucose intake to peripheral tissue and increased hepatic glucose output. Since the major portion of ‘whole body’ glucose is metabolized into glycogen (Bogardus et al., 1984; Roden and Shulman, 1999), defects in its metabolism may represent an important factor underlying the development of insulin resistance and type 2 diabetes. Glycogen synthase is the rate-limiting enzyme for glycogen synthesis, and its regulation is one of the few examples of complex, hierarchical, multisite phosphorylation. Studies to date have not fully uncovered the in vivo mechanisms regulating glycogen synthase; they have, however, established important concepts in cellular regulation such as enzyme regulation by reversible phosphorylation, the role of targeting molecules and function of signaling cascades. In this chapter we describe the regulation of glycogen synthase in type 2 diabetes, focusing on glycogen synthase in muscle, liver and fat. We discuss the concept that defects in glycogen synthase are early manifestations of insulin resistance. REGULATION OF GLYCOGEN SYNTHASE Glycogen synthase catalyzes the incorporation of the glycosyl residue from UDP-glucose into glycogen. Glycogen synthase is phosphorylated on multiple sites and is highly regulated by phosphorylation–dephosphorylation mechanisms, also termed covalent modiﬁcation. Different protein kinases can phosphorylate the enzyme in vitro, including c-AMP-dependent protein kinases (PKA), calmodulin dependent protein kinases, glycogen synthase kinase-3 (GSK-3), and casein kinase-2 (CK-2) (reviewed in Cohen, 1986; Roach, 1991). It is probable that only a few kinases are physiological kinases of glycogen synthase. Phosphorylation of glycogen synthase determines its activity. In general, increased phosphorylation of the enzyme inhibits its activity, while the extent of inactivation apparently depends on the speciﬁc site phosphorylated. Early in vitro studies implicated GSK-3 and PKA as the kinases that inhibit glycogen synthase (Cohen, 1986). Studies of rabbit muscle glycogen synthase identiﬁed its in vivo phosphorylation sites and correlated them with the corresponding kinase (Figure 1): at the N-terminus sites 2a and 2b phosphorylated by PKA and CK-1 3

4

HAGIT ELDAR-FINKELMAN AND MERAV YARKONI PKA CK-1

GSK-3

CK-2

PKA

5

1a 1b

a b c

N

2a

2b

3

C

Figure 1 Schematic presentation of the phosphorylation sites of muscle glycogen synthase. Site 2a is phosphorylated by PKA and promotes the phosphorylation of site 2b by CK-1, sites 1a and 1b are phosphorylated by PKA, site 5 is phosphorylated by CK-2 and promotes the phosphorylation of sites 3a, 3b and 3c by GSK-3.

respectively (Flotow and Roach, 1989); at the C-terminus two sites 1a and 1b phosphorylated by PKA (Parker et al., 1983); GSK-3 phosphorylates a cluster of 3 serine sites collectively termed ‘site 3’ (Parker et al., 1983; Woodgett and Cohen, 1984); and ‘site 5’ located downstream to ‘site 3’ phosphorylated by CK-2 (Fiol et al., 1987; Woodgett and Cohen, 1984). Administration of insulin to rabbit muscle led to a speciﬁc decrease in the phosphorylation of ‘site 3’ (Parker et al., 1983), indicating that dephosphorylation of this site is important for activation of the enzyme by insulin. Recent work using tissue cultured cells and molecular biology manipulations supported the notion that GSK-3 is a major physiological regulator of glycogen synthase (Cross et al., 1994; Eldar-Finkelman et al., 1996; Skurat et al., 1994). Insulin promotes dephosphorylation of glycogen synthase via inhibition of GSK-3 (Cross et al., 1994) and activation of a serine/threonine protein phosphatase (Dent et al., 1990). The phosphatase that is mainly responsible for dephosphorylation of glycogen synthase is the glycogen-bound form of type 1 protein phosphatase, PP1G, that comprise catalytic subunit and a targeting subunit (Dent et al., 1990). Activation of the phosphatase is controlled mainly by the targeting subunit that directs the enzyme to glycogen particles (Dent et al., 1990; Hubbard and Cohen, 1993). Tissue speciﬁc targeting subunits were identiﬁed in muscle and liver (Hubbard and Cohen, 1993). Recently, a protein targeting to glycogen (PTG) molecule was isolated from 3T3L1 adipocytes and was shown to assemble the enzymes involved in glycogen metabolism such as PP1, phosphorylase and phosphorylase kinase (Printen et al., 1997), and to promote glycogen synthesis when expressed in cells (Berman et al., 1998). Glycogen synthase is controlled by several allosteric effectors, of which the intracellular metabolite glucose 6 phosphate (G6P) is the most important (reviewed in Villar-Palasi and Guinovart, 1997). G6P can activate glycogen synthase even at highly phosphorylated states, and is therefore often used as an index of the enzyme activity. The enzyme activity assayed in the presence of high concentrations of G6P (6–10 mM) represents the activeinactive enzyme and is the total activity. The activity ratio is the ratio of activity assayed in the absence or presence of G6P and represents the fraction of active enzyme. Some studies measure the activity ratio in the presence of low or high concentrations of G6P in order to enhance the sensitivity of the enzyme (Guinovart et al., 1979); this ratio is usually termed as fraction velocity. The phosphorylation content of glycogen synthase determines its sensitivity to G6P; the more phosphorylated the less sensitive. Nevertheless, certain sites are more effective in changing the sensitivity of the enzyme to G6P (Embi and Cohen, 1980). As shown in Figure 2, phosphorylation of glycogen synthase by GSK-3 led to a shift to the right of the G6P dose response curve leading to a 10-fold increase in its Ka (i.e. the concentration of G6P required for half maximal activation of the enzyme). These results indicate that GSK-3 reduces glycogen synthase sensitivity to G6P.

GS activity (nmol/min/mg)

INSULIN RESISTANCE AND GLYCOGEN SYNTHESIS

5

20 15 10

+GSK-3

5

0

2

8

4 6 G6P (mM)

10

Figure 2 Effect of GSK-3 on allosteric activation of glycogen synthase by G6P. Glycogen synthase was phosphorylated in vitro by GSK-3 and then assayed for glycogen synthase activity in the presence of varied concentrations of G6P as indicated. Glycogen synthase activity is presented as UDPG incorporated into glycogen (nmol/min/mg protein) and results are an average of duplicated sample of one representative experiment.

Insulin ?

Glut-4

Insulin receptor

Glucose

IRS

HKII G6P

Pl3K

G1P

PKB –

?

Glycogen synthase

UDPG

GSK-3 PPIG –

+

Glycogen synthase

Glycogen synthesis

Figure 3 Activation of glycogen synthase by insulin. Insulin stimulates the activation of IRS/PI 3-kinase downstream pathway resulting in inhibition of GSK-3, and activation of the phosphatase PP1G. Insulin initiates the translocation of glucose transporters Glut4 from intracellular vesicles to the membrane, which promotes glucose entering into cells. Glucose is phosphorylated by hexokinaseII to glucose6-phopsphate (G6P) that binds and activates glycogen synthase. G6P is also an intermediate metabolite for utilization of UDP glucose (UDPG) required for glycogen synthesis.

6

HAGIT ELDAR-FINKELMAN AND MERAV YARKONI

In summary, activation of glycogen synthase by insulin is shared by two pathways (Figure 3). In one, the hormone induces glucose entry into cells, leading to increased concentrations of intracellular G6P. In the other, insulin causes dephosphorylation of the enzyme via activation of protein phosphatase. Concerted activation of both pathways is essential for efﬁcient activation of glycogen synthase and glycogen synthesis in the cell.

Glycogen Synthase in Diabetic Muscle The skeletal muscle is the major site of insulin-mediated glucose uptake, and glucose disposal into glycogen (Bogardus et al., 1984; Roden and Shulman, 1999). Even under hyperglycemic and hyperinsulinemic conditions, muscle glycogen synthesis represents a major pathway for glucose metabolism (Roden and Shulman, 1999). The good correlation between glycogen synthase activity and the in vivo action of insulin (Bogardus et al., 1984; Roden and Shulman, 1999), suggests that defects in glycogen synthesis may be related to insulin resistance and type 2 diabetes. The regulation of glycogen synthase by insulin in skeletal muscle has been debated for many years. Two theories, the push and pull hypotheses, were proposed (reviewed in Lawrence and Roach, 1997; Lawrence et al., 1997). In the push hypothesis, insulin induces glucose transport, thereby pushing glucose into glycogen. The pull theory argues that activation of glycogen synthase by insulin leads to the pull of glucose metabolites into glycogen. The push theory does not consider a dominant role for covalent activation of glycogen synthase in glycogen accumulation; instead, it suggests that the role of covalent modiﬁcation of the enzyme is to adapt the activity of the enzyme to the metabolic ﬂux (Shulman et al., 1995). The pull theory considers an active role for glycogen synthase in promoting glycogen synthesis that is not strictly related to glucose transport. Apparently, both theories are partially correct and their validity is dependent upon experimental conditions. Studies with transgenic mice clariﬁed this point by showing that overexpression of glucose transporters or glycogen synthase in skeletal muscle promoted glycogen synthesis in both cases indicating that each pathway can independently activate glycogen synthesis (Lawrence et al., 1997). Since insulin activates both glucose transport and glycogen synthase, the pathways are probably independent as well as complementary in activating glycogen synthesis by insulin in muscle. Defects in muscle glycogen synthesis may therefore result either from impaired glucose transport or from defects in signaling pathways upstream of glycogen synthase or, from both mechanisms. Most studies performed in muscle of type 2 diabetes subjects implicated loss of glycogen synthesis. Early studies employed tissue biopsy methodology and demonstrated that under euglycemic hyperinsulinemic clamp conditions, the sensitivity of glycogen synthase to insulin was extremely low in the muscle of type 2 diabetes subjects (Damsbo et al., 1991; Henry et al., 1996; Thorburn et al., 1990). In the presence of physiological insulin concentration, the enzyme was not stimulated at all. G6P dose-response curves of glycogen synthase from the diabetic muscle indicated that the enzyme is less sensitive to G6P, therefore is more phosphorylated (Damsbo et al., 1991). Loss of glycogen synthesis in diabetic muscle has also been demonstrated by nuclear magnetic resonance spectroscopy (NMR), a non-invasive methodology for monitoring in vivo concentrations of metabolite and metabolic ﬂuxes in the human muscle. 13C NMR studies demonstrated that under hyperglycemic hyperinsulinemic clamp conditions, rates of glycogen synthesis in muscle of type 2 diabetes subjects were reduced by 60% compared with non-diabetic matched controls (Roden and Shulman, 1999; Shulman, 1999). Interesting results came from NMR studies of normoglycemic offspring of type 2 diabetes patients that are known to have 40% increase in risk factor to develop diabetes: rates of

INSULIN RESISTANCE AND GLYCOGEN SYNTHESIS

7

glycogen synthesis in the offspring of type 2 diabetes were far lower than controls (Roden and Shulman, 1999). Reduced glycogen synthase activity was also shown in muscle of non-obese ﬁrst degree relatives of type 2 diabetes patients (Vaag et al., 1992b). These studies suggested that defects in glycogen synthesis may be an early stage in the pathogenesis of type 2 diabetes, and even precede hyperglycemia. What are the dominant factors that contribute to alterations in glycogen synthase in the diabetic muscle? This question was addressed in studies conducted with human muscle cells that separated the genetic factors from the in vivo external environment (Thorburn et al., 1991). Potential defects in the activation of glycogen synthase were examined in human myoblasts obtained from type 2 diabetic patients, which were differentiated into myotubes in culture dishes (Thorburn et al., 1991). Basal glycogen synthase activity and its responsiveness to insulin were signiﬁcantly reduced in cells obtained from type 2 diabetes patients (Thorburn et al., 1991). The enzyme was less sensitive to G6P and hence more phosphorylated in these cells (Thorburn et al., 1991). This ﬁnding that isolating the muscle cells from the in vivo milieu did not restore the defects in glycogen synthase activity suggested that genetic alterations are responsible at least in part for the insulin resistance in the diabetic muscle (Thorburn et al., 1991). Hyperglycemia and hyperinsulinemia are direct results of insulin resistance, but can also inﬂuence glucose metabolism in the muscle (Bak et al., 1991; Vaag et al., 1992a). The effects of hyperinsulinemia and hyperglycemia were studied in the human muscle cultured cells described in the previous paragraph (Thorburn et al., 1991). Under conditions of hyperglycemia, the activation of glycogen synthase by insulin was impaired in both control cells and cells from type 2 diabetic subjects. Hyperglycemia did, however, reduce glycogen synthase content levels in the muscle cells from type 2 diabetic patients (Nikoulina et al., 1997). Hyperinsulinemia enhanced basal glycogen synthase activity only in the muscle cells from type 2 diabetes patients, but abolished its acute responsiveness to insulin (Nikoulina et al., 1997). These studies indicated that both hyperglycemia and hyperinsulinemia contribute to impaired activation of glycogen synthase by insulin, but hyperinsulinemia can compensate for impaired basal glycogen synthase activity in the diabetic muscle (Nikoulina et al., 1997). The differences observed between the control and ‘diabetic cells’ also demonstrated the dominant role of genetic factors in causing insulin resistance in the muscle. The impaired activity of glycogen synthase in the diabetic muscle suggested that the enzyme is more phosphorylated, presumably by abnormal activity of a protein kinase. GSK-3 was implicated in regulating glycogen synthase in vitro (Woodgett and Cohen, 1984; Zhang et al., 1993) Figure 2), and in intact cells (Eldar-Finkelman et al., 1996). Recent studies also linked GSK-3 with insulin resistance via phosphorylation of IRS-1 (Eldar-Finkelman and Krebs, 1997). In the skeletal muscle of type 2 diabetic patients GSK-3 activity and its expression levels were signiﬁcantly higher than in healthy controls (Nikoulina et al., 2000). This activity could be directly responsible for impaired basal and insulin-induced glycogen synthase activity, as well as its impaired activation by G6P in the diabetic muscle. On the other hand, impaired activation of phosphatase could present another mechanism for impaired activation of glycogen synthase in the muscle (Kida et al., 1990). In vivo NMR spectroscopy examined the rate-limiting control step that contributes to reduction in glycogen synthesis of human muscle of type 2 diabetes subjects. In these studies the glucose transport step or the phosphorylation of glucose (i.e. hexokinase step) were shown to be the primary contributors for reduction of glycogen content in the diabetic muscle. Thus, the importance of defects in glycogen synthase itself was questionable (Roden and Shulman, 1999). In an attempt to determine which of the two steps is responsible for the defect in

8

HAGIT ELDAR-FINKELMAN AND MERAV YARKONI

glycogen accumulation, further studies employing 13C and 31P NMR were performed. Under hyperglycemic hyperinsulinemic clamp conditions, the intracellular concentration of glucose was far lower in muscle of type 2 diabetic subjects than non-diabetic controls; subsequently, G6P intracellular levels increased in response to insulin in controls, but did not change in type 2 diabetes patients. These results imply that a defect in glucose transport is mainly responsible for reduced glycogen synthesis in the diabetic muscle (Cline et al., 1999). In vivo studies in non-obese type 2 diabetes patients using radioactive tracers concluded that both glucose transport and glucose phosphorylation are severely impaired in type 2 diabetes (Bonadonna et al., 1996). Interestingly, the decrease in glucose phosphorylation was more prominent than the decrease in glucose uptake of muscle of type 2 diabetes (85% vs. 40% respectively), but it was not possible to determine which step is quantitatively more important (Bonadonna et al., 1996). It was suggested that the defect of both steps adds to the magnitude of insulin resistance in these patients (Bonadonna et al., 1996). It is noteworthy to mention that in the skeletal muscle of moderately overweight type 2 diabetic subjects, glycogen synthase activity was unchanged, but glucose uptake was severely impaired (Krook et al., 2000). These studies supported the view that covalent modiﬁcation is not a primary effector of defects in glycogen synthesis of the diabetic muscle, and speculated that reduction in glycogen synthase activity may be a consequence of obesity rather than diabetes (Krook et al., 2000). However, other studies refuted a correlation between obesity and glycogen synthase (Johnson et al., 1991). It is possible that the severity of insulin resistance and obesity is responsible for impaired activity of glycogen synthase. There is a wide range of animal model systems for diabetes, and some of them will be discussed here. The model system of rhesus monkeys that spontaneously develop obesity, insulin resistance and type 2 diabetes is closely related to human type 2 diabetes (Hansen and Bodkin, 1986). Studies in diabetic rhesus monkeys examined whether glycogen synthase is a potential early marker for the onset of diabetes. In the skeletal muscle of hyperinsulinemic (but nondiabetic) monkeys, glycogen synthase activation by insulin was 50% lower than that of controls (Ortmeyer et al., 1993b). In type 2 diabetic monkeys, insulin did not activate glycogen synthase and total enzyme activity was signiﬁcantly lower. These studies suggested a severe defect in glycogen synthase (presumably increased phosphorylation) in the type 2 diabetic muscle, with the defect appearing early and preceding the onset of overt diabetes (Ortmeyer et al., 1993b). Studies in genetically obese animals are widely used in diabetes research. The ob/ob mice are defective in leptin production, a key hormone regulating food intake, while the db/db mice are defective in leptin receptor (Chagnon and Bouchard, 1996). These animals have almost identical phenotypes and exhibit excess food intake, hyperphagia, insulin resistance and diabetes. The obese Zucker fa/fa rats carry the Fa mutation preventing a normal function of the leptin receptor (Chagnon and Bouchard, 1996). Despite the fact that genetic mutations found in animals are largely not present in man (reviewed in Chagnon and Bouchard, 1996), they provided many important insights into the molecular mechanism of eating behavior, obesity and diabetes. As in humans, insulin resistance in the animal muscle is primarily controlled by glucose uptake. In the ob/ob mice, impaired glucose uptake was the early defect detected in skeletal muscle and preceded the reduction in insulin receptor number (Grundleger et al., 1980; Le Marchand-Brustel et al., 1978; Ohshima et al., 1984). Reduction in glucose uptake was associated with reduced basal glycogen synthase activity ratios and its activation by insulin (Le Marchand-Brustel and Freychet, 1980; Liu et al., 1997; Ohshima et al., 1984). In the ob/ob diaphragm, the effective dose of insulin required to induce incorporation of 14C glucose into glycogen was greater than that required in lean animals (Cascieri et al., 1989). Similar

INSULIN RESISTANCE AND GLYCOGEN SYNTHESIS

9

observations were reported for the db/db mice (Benzo and Stearns, 1982). Studies in hind limb of Zucker fa/fa rats showed that glucose transport was depressed 50% in the absence of insulin, but muscle glycogen content did not change (Sherman et al., 1988). Nutritionally-induced diabetic model systems are favorable since changes in nutrition are dominant risk factors in the development of obesity and insulin resistance in afﬂuent societies. Certain animal strains are susceptible to high-fat diet induced obesity and type 2 diabetes and are widely used in diabetes research. In high-fat diet fed (HF) obese diabetic rats, a reduction in insulin-induced glucose uptake in skeletal muscle and soleus muscle was observed (Grundleger and Thenen, 1982; Zierath et al., 1997), and was associated with decreased insulin receptors and glycogen synthesis (Grundleger and Thenen, 1982; Zierath et al., 1997). In the perfused hindquarter of rats, glycogen synthase activity was similar in lean, HF rats, and in resistant rats that did not develop diabetes upon high-fat feeding (Pagliassotti et al., 1993). However, net glycogen synthesis was reduced only in the HF animals (Pagliassotti et al., 1993). Because the activity of insulin receptor tyrosine kinase was intact and glucose uptake was defective in the HF rats, it was suggested that this defect in glycogen synthesis is distal of the insulin receptor (Boyd et al., 1990). In summary, impaired glucose transport is the predominant determinant of insulin resistance in the muscle of humans and animals. The fact that expression levels of glucose transporter GLUT4 are unchanged in the diabetic muscle (Zierath et al., 1996) suggests that a post receptor defect impairs insulin-induced recruitment of GLUT4 to the membrane surface. The precise recruitment–translocation mechanism of GLUT4 is still not fully deﬁned. The decrease in glucose intake is the major cause for reduction in glucose deposition into glycogen and loss of glycogen is a general phenomenon in the diabetic muscle. Covalent activation of glycogen synthase is impaired in muscle of type 2 diabetes in many cases, but its importance in diabetic conditions is questionable. It appears that glycogen synthase activity (as assayed in vitro) does not necessarily reﬂect the capability to synthesize glycogen. Nevertheless, the presence of such defects in muscle represent an early marker for the development of insulin resistance.

Glycogen Synthase in Diabetic Liver The major roles of the liver are to sense blood glucose levels, and to store glycogen or produce glucose according to peripheral needs. In insulin resistance, glucose metabolism is abnormally regulated, leading to increased hepatic glucose output and impaired glucose tolerance. Hepatic glycogen synthase differs from the muscle isoform: it shares only 46% homology with the muscle enzyme and lacks the equivalent sites 1a and 1b phosphorylated by PKA (Bai et al., 1990). Studies to date indicate that insulin and glucose are major physiological effectors of glycogen synthesis in the liver (reviewed in Bollen et al., 1998; Guinovart et al., 1997). Glucose activates hepatic glycogen synthesis in various ways. Glucose binds and promotes dephosphorylation and inactivation of phosphorylase, preventing glycogenolysis, and leading to indirect activation of the phosphatase PP1G (Bollen et al., 1998). Glucose metabolite, glucose-6-phosphate (G6P) binds to glycogen synthase and promotes its dephosphorylation by PP1G (Guinovart et al., 1997; Villar-Palasi and Guinovart, 1997). Despite the fact that some studies have questioned the role of inhibition of phosphorylase in activation of glycogen synthase (Carabaza et al., 1992; Van de Werve and Jeanrenaud, 1984), it is currently appreciated that both pathways are required for activation of glycogen synthase by glucose (Bollen et al., 1998).

10

HAGIT ELDAR-FINKELMAN AND MERAV YARKONI

Compartmentalization of glycogen synthase represents another mechanism in its activation by glucose in which glucose initiates the aggregation and translocation of glycogen synthase from the cytoplasm to the cell periphery (Guinovart et al., 1997). The activation of glycogen synthase in the liver by insulin involves multiple pathways (reviewed in Bollen et al., 1998; Lavoie et al., 1999; Pugazhenthi and Khandelwal, 1995). Insulin initiates the phosphorylation of insulin receptor substrates (IRS) proteins which mediate the subsequent activation of phosphatidylinositol 3-kinase (PI 3-kinase), protein kinase B and inhibition of GSK-3 (Bollen et al., 1998; Lavoie et al., 1999; Pugazhenthi and Khandelwal, 1995). Insulin activates glycogen synthase via inhibition of phosphorylase, which in turn activates PP1G (Bollen et al., 1998). Glycogen synthase is also activated by insulin-induced cell swelling which mediates the activation of PI 3-kinase (Bollen et al., 1998). Additional effectors activating glycogen synthase by insulin such as atypical protein kinase C were recently identiﬁed (Lavoie et al., 1999). The insulin-resistant liver is resistant to insulin-induced suppression of hepatic glucose production. Insulin resistance may thus impair hepatic activation of glycogen synthase leading to reduction of glycogen. On the other hand, hyperinuslinemia could maintain a sustained activation of phosphatase and or inactivation of phosphorylase, resulting in increased glycogen synthase activation. Apparently, both scenarios play a role in the diabetic liver. Studies in hepatocytes isolated from fasted obese Zucker fa/fa rats showed that activation of glycogen synthase by effective glucose concentrations was much higher (at least 2-fold) than that of lean animals (Lavoie et al., 1991). The addition of the potent phosphatase inhibitor microcystine LR completely abolished glucose-induced activation of glycogen synthase in the cells (Lavoie et al., 1991). These results indicate that increased capacity of hepatic glycogen synthesis in the obese rats was a result of selective activation of PP1. Indeed, additional studies indicated that PP1 activity in livers of fasted obese Zucker fa/fa rats was 3-fold higher than that of their lean counterparts (Lavoie et al., 1991). It was proposed that hyperinsulinemia in the starved animals (which are also normoglycemic) may be the driving force for enhanced and sustained PP1 activity (Lavoie et al., 1991). Apparently, glycogen levels in the liver of obese rats were always higher even at different nutritional conditions or age (Koubi and Freminet, 1985; Margolis, 1987; Van de Werve and Jeanrenaud, 1987). Studies in the genetically obese ob/ob mice reported inconsistent results. Several studies indicated that glycogen synthase activity is impaired in the ob/ob mouse-liver. In hepatocytes of obese mice, phosphorylase was inhibited by glucose but this was not followed by activation of glycogen synthase as observed in controls (Van de Werve et al., 1983). These studies also indicated that glycogen levels in the liver of fed animals were similar among ob/ob and controls (Van de Werve et al., 1983). Another study showed that oral glucose administration to starved mice failed to produce signiﬁcant or sustained activation of hepatic glycogen synthase in ob/ob mice while such activation was observed in controls (Smith et al., 1983). The net glycogen synthesis after glucose administration was diminished whereas glycogen content was 14 times greater in the liver of the ob/ob mice (Smith et al., 1983). These results are somewhat puzzling; on the one hand glycogen synthase activity was impaired by glucose and on the other, glycogen content was high or unchanged in the ob/ob mice-liver (Smith et al., 1983; van de Werve et al., 1983). It was speculated that accumulation of hepatic glycogen in ob/ob mice inhibits PP1G activity, which in turn inhibit the activation of glycogen synthase (de Wulf and Hers, 1968; Smith et al., 1983). In contrast, other studies indicated that glycogen synthesis is enhanced in the liver of ob/ob mice. Studies of perfused liver with glucose showed that glycogen synthesis rates were much faster in the liver of ob/ob mice as compared with normal livers (Elliott et al., 1971). This

INSULIN RESISTANCE AND GLYCOGEN SYNTHESIS

11

could be well explained by studies showing that glucose utilization in hepatocytes of ob/ob mice was signiﬁcantly greater (Lahtela et al., 1990). Taken together, most studies indicated that the glycogen content in the liver of ob/ob mice is higher (Elliott et al., 1971; Lahtela et al., 1990; Lombardo et al., 1984; Smith et al., 1983) and thus implied potential, yet undeﬁned, abnormal regulation of glycogen in the ob/ob mice liver. Some reports indicated elevated levels of glycogen in the liver of db/db mice (Chan et al., 1975) while others, however, indicated no change in glycogen levels in db/db liver compared with controls (Roesler and Khandelwal, 1985; Stearns and Benzo, 1977). Different results were obtained for glycogen synthase activity in the db/db liver. In one study hepatic glycogen synthase and glycogen synthesis were markedly increased (Chan et al., 1975); another study, however, found only minor changes in the kinetic properties of glycogen synthase in the liver of db/db mice (Roesler and Khandelwal, 1986). Longitudinal studies examined whether these discrepancies were due to differences in the developmental stage of the db/db mice. As a whole, these studies could not ﬁnd considerable alterations in glycogen synthase activity or in glycogen content in the liver of the db/db mice (Roesler et al., 1990). It was found however that at early age (i.e. 5 weeks) there was a signiﬁcant increase in glycogen synthase activity in db/db mouse-liver (Roesler et al., 1990). The major difference was detected in glycogen structure. Apparently, liver glycogen particles from db/db mice were smaller and more homogenous compared with those from normal livers (Roesler et al., 1990). Glycogen from db/db mouseliver also exhibited different branching patterns suggesting different in vivo regulation of glycogen synthase by glycogen in the liver of the db/db mouse (Roesler et al., 1990). In gold thioglucose obese mice (GTG), which develop obesity, hyperglycemia and hyperinuslinemia following injection of GTG, an increase in glycogen synthase activity was reported (Chen et al., 1993): during the transition from the starved to the fed state, activation of hepatic glycogen synthase from obese mice was 2-fold that of controls. Glycogen content was signiﬁcantly higher in the liver of fasted or fed obese animals compared to lean animals (Chen et al., 1993). Similar observations were reported in humans. After a 3-day-fasting period, hepatic glycogen content of type 2 diabetes patients was 2-fold higher than that of controls (Clore et al., 1992). Studies in liver biopsies from obese insulin-resistant rhesus monkeys, indicated that glycogen synthase was activated in vivo by insulin (Ortmeyer and Bodkin, 1998) in a similar fashion to its activation in the liver of healthy monkeys (Ortmeyer et al., 1997). These studies concluded that glycogen synthase activity and or its activation by insulin is not defective, at least not at the level of covalent modiﬁcation (i.e. increased phosphorylation) in the diabetic monkey. In high-fat fed (HF) insulin-resistant and obese rats, hepatic glucose output suppression by insulin was impaired, but incorporation of glucose into glycogen and its content levels did not change signiﬁcantly (Oakes et al., 1997). In these rats, hepatic glycogen breakdown was smaller than in controls during starvation. In contrast, glyceride levels fell in the liver of diabetic rats but did not change in the lean controls (Schindler and Felber, 1986). The low breakdown of glycogen in HF animals demonstrated that energy consumption originated from triglycerides rather than glycogen (Schindler and Felber, 1986). In summary, hepatic glycogen synthesis is extremely sensitive to multiple factors that can alter activity of the enzyme. For example, activity of glycogen synthase is dependent on age, nutrition, light and dark cycle and diurnal rhythm of hepatic glycogen (Goriya et al., 1981; Roesler et al., 1990; Shulman and Rossetti, 1989). Thus, the inconsistent ﬁndings in studies of glycogen synthase or glycogen content in the liver could be attributed to small differences in experiment conditions. As a whole, it appears that hepatic glycogen storage is increased in the liver of diabetic animals. The excessive hepatic glycogen storage may be the driving force

12

HAGIT ELDAR-FINKELMAN AND MERAV YARKONI

behind increased glycogenolysis and subsequently increased hepatic glucose production in insulin resistance. Glycogen Synthase in Diabetic Fat Tissue The association of obesity and insulin resistance is well established although mechanisms responsible for this link are unclear. For many years, it looked as if free fatty acids produced by fat tissue are responsible in inducing insulin resistance in the peripheral tissue (Bergman et al., 2001; Johnson et al., 1991; Shulman, 1999). Recent discoveries detected other functions of fat tissue in mediating insulin resistance via production of hormones such as leptin (Muller et al., 1997) TNF-␣ (Hotamisligil et al., 1994) and resisitin (Steppan et al., 2001). Glycogen content in fat tissue is relatively small, and this is probably due to the fact that fat is the major energy storage in fat tissue. Thus, the role of fat glycogen in whole body glucose homeostasis is minor. Insulin activates glycogen synthase in adipocytes (Lawrence et al., 1977), and is controlled mainly by activation of PP1 (Brady et al., 1998). The use of lithium, a speciﬁc inhibitor of GSK-3, suggested that prolonged inhibition of GSK-3 represents another mechanism in activating glycogen synthesis in 3T3L1 adipocytes (Orena et al., 2000). Studies of insulin action in adipocytes from type 2 diabetic patients indicated that activation of downstream targets of the insulin cascade such as IRS-1, PKB and MAP kinase was impaired (Begum and Ragolia, 1998; Smith et al., 1999), as well as activation of glucose transport by insulin (Rondinone et al., 1999). There was no change in the basal or insulin-induced activation of glycogen synthase activity ratios in type 2 diabetes (Mandarino et al., 1986). Total enzyme activity, however, was much smaller in adipocytes from subjects with type 2 diabetes (Mandarino et al., 1986). In adipose tissue of type 2 diabetes rhesus monkeys, basal and total glycogen synthase activities were lower than controls (Ortmeyer et al., 1993a). Glycogen synthase was not activated by insulin in the tissue of diabetic animals indicating lack of covalent activation of the enzyme in the diabetic fat tissue (Ortmeyer et al., 1993a). Despite the fact that insulin-induced glucose uptake is lower in diabetic fat tissue (Rondinone et al., 1999; Smith et al., 1999), basal glucose uptake appears to be greater in the diabetic tissue (Czech et al., 1978). In adipocytes isolated from Zucker fa/fa rats glucose uptake was 5–10-fold greater than control (Czech et al., 1978). Studies performed in normal rats indicated that hyperinuslinemia might be the driving force for increased glucose uptake in fat tissue. In these studies, prior insulinization of normal rats resulted in decreased insulin-induced glucose uptake in muscle, however, glucose uptake in fat was signiﬁcantly greater (Cusin et al., 1992). Very few studies have evaluated glycogen synthase in the diabetic fat. It is therefore difﬁcult to establish a complete picture at this point. It appears that the capacity of fat tissue to expand or contract enables it to quickly adapt to drastic metabolic changes. In this regard, fat tissue enhances glucose uptake in response to hyperglycemia and or hyperinsulinemia. The excess of glucose entering into the tissue enhances lypogenesis and glycogen synthesis and eventually produces obesity. Glycogen Synthase in Muscle Liver and Fat: Studies in C57BL/6J Mice Model The data presented here show that despite the fact that general pathways control glycogen synthase, its regulation is uniquely adapted to its speciﬁc role in different cell types. It is also clear that glycogen metabolism is dependent upon metabolic conditions and hormonal milieu. In these studies we compared the regulation of glycogen synthase in liver, muscle, and fat of

INSULIN RESISTANCE AND GLYCOGEN SYNTHESIS

GS Activity (nmol/min/mg)

Liver

13

Muscle

Fat

16

3

8 12

6

2 8

4 2

4

0

0 0

5

10 15 G6P (mM)

20

1 0 0

5 10 15 G6P (mM)

20

0

5

10 15 G6P (mM)

20

Figure 4 Glycogen synthase activity in fed and starved conditions. Glycogen synthase activity was assayed in tissue extracts of fed or 30-h starved C57BL/6J mice in the presence of various concentrations of G6P as described. G6P dose response curves are presented for starved (ﬁlled circles) or fed (open circles) conditions. Results are a mean of 3 independent experiments SE.

Table 1 Glycogen synthase activity (nmol/min/mg) in diabetic mice. Glycogen synthase activity was assayed in tissues of obese/insulin resistant C57BL/6J mice (diabetic) and lean controls (control) as described (Eldar-Finkelman et al., 1996). The enzyme activity was assayed in the presence of low or high G6P concentrations. Results are a mean of 5 independent experiments SE, *p 0.05.

G6P (mM) Tissue Liver Muscle Fat

Control

Diabetic

Control

Diabetic

0.1

0.1

10

10

0.13 0.04 1.92 0.17 0.13 0.02

0.11 0.04 2.21 0.10 0.19* 0.04

8.35 0.92 12.15 0.23 2.0 0.02

10.1 1.5 12.3 1.0 4.3* 0.63

C57BL/6J mice at two metabolic situations – starvation and diabetes. Glycogen synthase activity and its allosteric activation by G6P in starved and fed animals, are presented in Figure 4. In agreement to previous studies, hepatic glycogen synthase activity was drastically reduced in the starved liver. Since total enzyme activity was reduced, we concluded that the enzyme levels were depleted in the starved liver. In muscle, starvation did not change the total activity of glycogen synthase, but reduced its sensitivity to G6P indicating that the enzyme is more phosphorylated (and less active). The changes in fat glycogen synthase in response to starvation were small but reproducible. In fat, total glycogen synthase activity reduced in starvation, but its sensitivity to G6P reduced as well. These results demonstrate that liver uses the most drastic pathway to ‘shut down’ glycogen synthesis in response to starvation in order to enhance glucose supply. In muscle on the other hand, covalent inhibition of the enzyme is sufﬁcient to reduce glycogen synthesis. Increased GSK-3 activity in starved muscle is probably the major cause for inhibition of enzyme (M. Yarkoni, unpublished results). Fat tissue is adapted to starvation by a small decrease in synthase activity and its protein levels. C57BL/6J mice develop obesity and type 2 diabetes as a result of high fat diet feeding (Surwit et al., 1988). We measured glycogen synthase activity in muscle, liver and fat tissues in HF and control animals (Table 1). No signiﬁcant changes were detected in glycogen synthase activity, total enzyme

14

HAGIT ELDAR-FINKELMAN AND MERAV YARKONI

activity or its sensitivity to G6P in muscle and liver among type 2 diabetic mice and controls. In contrast, total glycogen synthase activity increased 2-fold in fat tissue of diabetic mice. We assume that the total increase in glycogen synthase activity is a direct result of increased basal glucose uptake of adipocytes of type 2 diabetes (results from our laboratory). The elevation in glycogen synthase levels may be partially due to the fact that high activity of GSK-3 found in fat tissue of diabetic animals (Eldar-Finkelman et al., 1999) prevents covalent activation of the enzyme. Altogether, our results argue against a role for covalent modiﬁcation of glycogen synthase in diabetic tissues of C57BL/6J mice and suggest that changes in enzyme activity are mainly regulated by intracellular factors.

CONCLUDING REMARKS Defects in glycogen metabolism are primary factors in insulin resistance and type 2 diabetes, they are however, tissue speciﬁc. In skeletal muscle there are multiple defects in glycogen synthase leading to a signiﬁcant reduction in glycogen levels. Changes in glycogen synthase occur at very early stages of insulin resistance, and thus may serve as an early indicator for development of insulin resistance in the muscle. In the diabetic liver and fat, glycogen synthase activity is not necessarily defected, but glycogen metabolism is deﬁnitely abnormal in these tissues. One problem to consider is that glycogen synthase activity (as assayed in vitro) does not necessarily reﬂect the capability to synthesize glycogen. This is due to the fact that alterations in glycogen synthase activity caused by cellular factors (such as G6P or glycogen) are not evaluated in the conventional assays done on broken cells. Assessment of glycogen synthesis and glycogen content is required to have a better indication for in vivo glycogen synthase activity. Finally, drugs that increase insulin sensitivity usually improve glycogen synthase activity. It would be interesting to look if therapies that increase glycogen synthase activity improve insulin sensitivity in the diabetic tissue.

REFERENCES Bai, G., Zhang, Z.J., Werner, R., Nuttall, F.Q., Tan, A.W. and Lee, E.Y. (1990) The primary structure of rat liver glycogen synthase deduced by cDNA cloning. Absence of phosphorylation sites 1a and 1b. J. Biol. Chem., 265, 7843–7848. Bak, J.F., Moller, N., Schmitz, O., Richter, E.A. and Pedersen, O. (1991) Effects of hyperinsulinemia and hyperglycemia on insulin receptor function and glycogen synthase activation in skeletal muscle of normal man. Metabolism, 40, 830–835. Begum, N. and Ragolia, L. (1998) Altered regulation of insulin signaling components in adipocytes of insulin-resistant type II diabetic Goto-Kakizaki rats. Metabolism, 47, 54–62. Benzo, C.A. and Stearns, S.B. (1982) Glycogen synthase and phosphorylase activities in skeletal muscle from genetically diabetic (db/db) mice. Horm. Metab. Res., 14, 130–133. Bergman, R.N., Van Citters, G.W., Mittelman, S.D. et al. (2001) Central role of the adipocyte in the metabolic syndrome. J. Investig. Med., 49, 119–126. Berman, H.K., O’Doherty, R.M., Anderson, P. and Newgard, C.B. (1998) Overexpression of protein targeting to glycogen (PTG) in rat hepatocytes causes profound activation of glycogen synthesis independent of normal hormone- and substrate-mediated regulatory mechanisms. J. Biol. Chem., 273, 26421–26425. Bogardus, C., Lillioja, S., Stone, K. and Mott, D. (1984) Correlation between muscle glycogen synthase activity and in vivo insulin action in man. J. Clin. Invest., 73, 1185–1190.

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Bollen, M., Keppens, S. and Stalmans, W. (1998) Speciﬁc features of glycogen metabolism in the liver. Biochem. J., 336, 19–31. Bonadonna, R.C., Del Prato, S., Bonora, E. et al. (1996) Roles of glucose transport and glucose phosphorylation in muscle insulin resistance of NIDDM. Diabetes, 45, 915–925. Boyd, J.J., Contreras, I., Kern, M. et al. (1990) Effect of a high-fat-sucrose diet on in vivo insulin receptor kinase activation. Am. J. Physiol., 259, E111–E116. Brady, M.J., Bourbonais, F.J. and Saltiel, A.R. (1998) The activation of glycogen synthase by insulin switches from kinase inhibition to phosphatase activation during adipogenesis in 3T3-L1 cells. J. Biol. Chem., 273, 14063–14066. Carabaza, A., Ciudad, C.J., Baque, S. and Guinovart, J.J. (1992) Glucose has to be phosphorylated to activate glycogen synthase, but not to inactivate glycogen phosphorylase in hepatocytes. FEBS Lett., 296, 211–214. Cascieri, M.A., Slater, E.E., Vicario, P.P., Green, B.G., Bayne, M.L. and Saperstein, R. (1989) Impaired insulin-like growth factor I-mediated stimulation of glucose incorporation into glycogen in vivo in the ob/ob mouse. Diabetologia, 32, 342–347. Chagnon, Y.C. and Bouchard, C. (1996) Genetics of obesity: advances from rodent studies. Trends Genet., 12, 441–444. Chan, T.M., Young, K.M., Hutson, N.J., Brumley, F.T. and Exton, J.H. (1975) Hepatic metabolism of genetically diabetic (db/db) mice. I. Carbohydrate metabolism. Am. J. Physiol., 229, 1702–1712. Chen, C., Williams, P.F. and Caterson, I.D. (1993) Liver and peripheral tissue glycogen metabolism in obese mice: effect of a mixed meal. Am. J. Physiol., 265, E743–E751. Cline, G.W., Petersen, K.F., Krssak, M. et al. (1999) Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N. Engl. J. Med., 341, 240–246. Clore, J.N., Post, E.P., Bailey, D.J., Nestler, J.E. and Blackard, W.G. (1992) Evidence for increased liver glycogen in patients with noninsulin-dependent diabetes mellitus after a 3-day fast. J. Clin. Endocrinol. Metab., 74, 660–666. Cohen, P. (1986) Muscle glycogen synthase, The enzymes, Vol. 17A. Orlando, FL: Academic Press. Cross, D.A., Alessi, D.R., Vandenheede, J.R., McDowell, H.E., Hundal, H.S. and Cohen, P. (1994) The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem. J., 303, 21–26. Cusin, I., Rohner-Jeanrenaud, F., Terrettaz, J. and Jeanrenaud, B. (1992) Hyperinsulinemia and its impact on obesity and insulin resistance. Int. J. Obes. Relat. Metab. Disord., 16 (Suppl 4), S1–S11. Czech, M.P., Richardson, D.K., Becker, S.G., Walters, C.G., Gitomer, W. and Heinrich, J. (1978) Insulin response in skeletal muscle and fat cells of the genetically obese Zucker rat. Metabolism, 27, 1967–1981. Damsbo, P., Vaag, A., Hother-Nielsen, O. and Beck-Nielsen, H. (1991) Reduced glycogen synthase activity in skeletal muscle from obese patients with and without type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia, 34, 239–245. Dent, P., Lavoinne, A., Nakielny, S., Caudwell, F.B., Watt, P. and Cohen, P. (1990) The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature, 348, 302–308. De Wulf, H. and Hers, H.G. (1968) The interconversion of liver glycogen synthetase a and b in vitro. Eur. J. Biochem., 6, 552–557. Eldar-Finkelman, H., Agrast, G.M., Foord, O., Fischer, E.H. and Krebs, E.G. (1996) Expression and characterization of GSK-3 mutants and their effect on glycogen synthase activity. Proc. Natl. Acad. Sci. USA, 93, 10228–10233. Eldar-Finkelman, H. and Krebs, E.G. (1997) Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action. Proc. Natl. Acad. Sci. USA, 94, 9660–9664. Eldar-Finkelman, H., Schreyer, S.A., Shinohara, M.M., LeBoeuf, R.C. and Krebs, E.G. (1999) Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice. Diabetes, 48, 1662–1666.

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Elliott, J., Hems, D.A. and Beloff-Chain, A. (1971) Carbohydrate metabolism of the isolated perfused liver of normal and genetically obese–hyperglycaemic (ob-ob) mice. Biochem. J., 125, 773–780. Embi, N. and Cohen, P. (1980) Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem., 107, 519–527. Fiol, C.J., Mahrenholz, A.M., Wang, Y., Roeske, R.W. and Roach, P.J. (1987) Formation of protein kinase recognition sites by covalent modiﬁcation of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase-3. J. Biol. Chem., 262, 14042–14048. Flotow, H. and Roach, P.J. (1989) Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase. J. Biol. Chem., 264, 9126–9128. Goriya, Y., Bahoric, A., Marliss, E.B., Zinman, B. and Albisser, A.M. (1981) Diurnal metabolic and hormonal responses to mixed meals in healthy dogs. Am. J. Physiol., 240, E54–E55. Grundleger, M.L., Godbole, V.Y. and Thenen, S.W. (1980) Age-dependent development of insulin resistance of soleus muscle in genetically obese (ob/ob) mice. Am. J. Physiol., 239, E363–E371. Grundleger, M.L. and Thenen, S.W. (1982) Decreased insulin binding, glucose transport, and glucose metabolism in soleus muscle of rats fed a high fat diet. Diabetes, 31, 232–237. Guinovart, J.J., Gomez-Foix, A.M., Seoane, J., Fernandez-Novell, J.M., Bellido, D. and Vilaro, S. (1997) Bridging the gap between glucose phosphorylation and glycogen synthesis in the liver. Biochem. Soc. Trans., 25, 157–160. Guinovart, J.J., Salavert, A., Massague, J., Ciudad, C.J., Salsas, E. and Itarte, E. (1979) Glycogen synthase: a new activity ratio assay expressing a high sensitivity to the phosphorylation state. FEBS Lett., 106, 284–288. Hansen, B.C. and Bodkin, N.L. (1986) Heterogeneity of insulin responses: phases leading to type 2 (non-insulin-dependent) diabetes mellitus in the rhesus monkey. Diabetologia, 29, 713–719. Henry, R.R., Ciaraldi, T.P., Abrams-Carter, L., Mudaliar, S., Park, K.S. and Nikoulina, S.E. (1996) Glycogen synthase activity is reduced in cultured skeletal muscle cells of non-insulin-dependent diabetes mellitus subjects. Biochemical and molecular mechanisms. J. Clin. Invest., 98, 1231–1236. Hotamisligil, G.S., Budavari, A., Murray, D. and Spiegelman, B.M. (1994) Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha. J. Clin. Invest., 94, 1543–1549. Hubbard, M.J. and Cohen, P. (1993) On target with a new mechanism for the regulation of protein phosphorylation. Trends. Biochem. Sci., 18, 172–177. Johnson, A.B., Argyraki, M., Thow, J.C. et al. (1991) Impaired activation of skeletal muscle glycogen synthase in non-insulin-dependent diabetes mellitus is unrelated to the degree of obesity. Metabolism, 40, 252–260. Kida, Y., Esposito-Del Puente, A., Bogardus, C. and Mott, D.M. (1990) Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle. J. Clin. Invest., 85, 476–481. Koubi, H. and Freminet, A. (1985) Comparison of glycogen stores in 3- and 7-month-old lean and obese Zucker rats under fed and fasted conditions. Comp. Biochem. Physiol. B, 81, 103–110. Krook, A., Bjornholm, M., Galuska, D. et al. (2000) Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes, 49, 284–292. Lahtela, J.T., Wals, P.A. and Katz, J. (1990) Glucose metabolism and recycling by hepatocytes of OB/OB and ob/ob mice. Am. J. Physiol., 259, E389–E396. Lavoie, L., Band, C.J., Kong, M., Bergeron, J.J. and Posner, B.I. (1999) Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways. J. Biol. Chem., 274, 28279–28285. Lavoie, L., Bollen, M., Stalmans, W. and van de Werve, G. (1991) Increased synthase phosphatase activity is responsible for the super-activation of glycogen synthase in hepatocytes from fasted obese Zucker rats. Endocrinology, 129, 2674–2678. Lawrence, J.C., Jr., Guinovart, J.J. and Larner, J. (1977) Activation of rat adipocyte glycogen synthase by insulins. J. Biol. Chem., 252, 444–450. Lawrence, J.C., Jr. and Roach, P.J. (1997) New insights into the role and mechanism of glycogen synthase activation by insulin. Diabetes, 46, 541–547.

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Lawrence, J.C., Jr., Skurat, A.V., Roach, P.J., Azpiazu, I. and Manchester, J. (1997) Glycogen synthase: activation by insulin and effect of transgenic overexpression in skeletal muscle. Biochem. Soc. Trans., 25, 14–19. Le Marchand-Brustel, Y. and Freychet, P. (1980) Alteration of glycogen synthase activation by insulin in soleus muscles of obese mice. FEBS Lett., 120, 205–208. Le Marchand-Brustel, Y., Jeanrenaud, B. and Freychet, P. (1978) Insulin binding and effects in isolated soleus muscle of lean and obese mice. Am. J. Physiol., 234, E348–E358. Liu, Y.L., Emilsson, V. and Cawthorne, M.A. (1997) Leptin inhibits glycogen synthesis in the isolated soleus muscle of obese (ob/ob) mice. FEBS Lett., 411, 351–355. Lombardo, Y.B., Hron, W.T., Sobocinski, K.A. and Menahan, L.A. (1984) A metabolic proﬁle of fed and fasting genetically obese mice at 4–5 months of age. Horm. Metab. Res., 16 (Suppl 1), 37–42. Mandarino, L.J., Madar, Z., Kolterman, O.G., Bell, J.M. and Olefsky, J.M. (1986) Adipocyte glycogen synthase and pyruvate dehydrogenase in obese and type II diabetic subjects. Am. J. Physiol., 251, E489–E496. Margolis, R.N. (1987) Hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities are increased in obese (fa/fa) hyperinsulinemic Zucker rats: effects of glyburide administration. Life Sci., 41, 2615–2622. Muller, G., Ertl, J., Gerl, M. and Preibisch, G. (1997) Leptin impairs metabolic actions of insulin in isolated rat adipocytes. J. Biol. Chem., 272, 10585–10593. Nikoulina, S.E., Ciaraldi, T.P., Abrams-Carter, L., Mudaliar, S., Park, K.S. and Henry, R.R. (1997) Regulation of glycogen synthase activity in cultured skeletal muscle cells from subjects with type II diabetes: role of chronic hyperinsulinemia and hyperglycemia. Diabetes, 46, 1017–1024. Nikoulina, S.E., Ciaraldi, T.P., Mudaliar, S., Mohideen, P., Carter, L. and Henry, R.R. (2000) Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes. Diabetes, 49, 263–271. Oakes, N.D., Cooney, G.J., Camilleri, S., Chisholm, D.J. and Kraegen, E.W. (1997) Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes, 46, 1768–1774. Ohshima, K., Shargill, N.S., Chan, T.M. and Bray, G.A. (1984) Adrenalectomy reverses insulin resistance in muscle from obese (ob/ob) mice. Am. J. Physiol., 246, E193–E197. Orena, S.J., Torchia, A.J. and Garofalo, R.S. (2000) Inhibition of glycogen-synthase kinase-3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes. J. Biol. Chem., 275, 15765–15772. Ortmeyer, H.K. and Bodkin, N.L. (1998) Lack of defect in insulin action on hepatic glycogen synthase and phosphorylase in insulin-resistant monkeys. Am. J. Physiol., 274, G1005–G1010. Ortmeyer, H.K., Bodkin, N.L. and Hansen, B.C. (1993a) Adipose tissue glycogen synthase activation by in vivo insulin in spontaneously insulin-resistant and type 2 (non-insulin-dependent) diabetic rhesus monkeys. Diabetologia, 36, 200–206. Ortmeyer, H.K., Bodkin, N.L. and Hansen, B.C. (1993b) Insulin-mediated glycogen synthase activity in muscle of spontaneously insulin-resistant and diabetic rhesus monkeys. Am. J. Physiol., 265, R552–R558. Ortmeyer, H.K., Bodkin, N.L. and Hansen, B.C. (1997) Insulin regulates liver glycogen synthase and glycogen phosphorylase activity reciprocally in rhesus monkeys. Am. J. Physiol., 272, E133–E138. Pagliassotti, M.J., Shahrokhi, K.A. and Hill, J.O. (1993) Skeletal muscle glucose metabolism in obesityprone and obesity-resistant rats. Am. J. Physiol., 264, R1224–R1228. Parker, P.J., Caudwell, F.B. and Cohen, P. (1983) Glycogen synthase from rabbit skeletal muscle; effect of insulin on the state of phosphorylation of the seven phosphoserine residues in vivo. Eur. J. Biochem., 130, 227–234. Printen, J.A., Brady, M.J. and Saltiel, A.R. (1997) PTG, a protein phosphatase 1-binding protein with a role in glycogen metabolism. Science, 275, 1475–1478. Pugazhenthi, S. and Khandelwal, R.L. (1995) Regulation of glycogen synthase activation in isolated hepatocytes. Mol. Cell. Biochem., 149–150, 95–101. Roach, P.J. (1991) Multisite and hierarchal protein phosphorylation. J. Biol. Chem., 266, 14139–14142.

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2. GESTATIONAL DIABETES AND MATERNAL INSULIN RESISTANCE IN THE C57BLKS/JLEPRdb/⫹ MOUSE – A UNIQUE MODEL FOR UNDERSTANDING ITS IMPACT ON THE FETUS JIANHUA SHAO AND JACOB E. FRIEDMAN Departments of Pediatrics, Biochemistry and Molecular Genetics University of Colorado Health Sciences Center, Denver, CO 80262

SUMMARY The heterozygous C57BLKS-Leprdb/ mouse is a genetic model of spontaneous GDM with features closely resembling those found in the human GDM. Beginning in late pregnancy, db/ mice develop impaired glucose tolerance (IGT) associated with hyperinsulinemia and extreme insulin resistance compared to wild-type / mothers. After delivery, IGT recovers and plasma glucose and insulin levels return to normal. The newborn fetuses from db/ mothers have higher insulin, normal to low glucose, increased birth and placental weight, and greater susceptibility to dietary-induced obesity compared to offspring from wild-type / mothers. This occurs in both / and db/ offsprings, suggesting that fetal imprinting of metabolism occurs in utero as a consequence of the maternal diabetic environment. Maternal insulin resistance in GDM is due to a novel mechanism for resistance including: (1) defects in tyrosine phosphorylation of insulin receptor without change of insulin receptor protein level, (2) low level of IRS-1 protein and increased serine phosphorylation, (3) increased expression and abnormal distribution of PI 3-kinase, Akt, and p70S6 kinase in skeletal muscle. These changes are unique to pregnancy, and are implicated as a mechanism for increased serine phosphorylation of IRS-1 and its degradation. Studies also show that improving maternal insulin resistance by overexpressing maternal GLUT4 in skeletal muscle reduces nutrient ﬂux to fetus and improves fetal development. Reducing excess energy intake in db/ mice during pregnancy also prevents spontaneous GDM and fetal macrosomia. However, leptin administration during gestation suggests that there are other factors beside maternal glucose concentration that determine the growth of the fetus. Thus, the db/ mouse model of spontaneous GDM is a useful model for understanding factors that trigger excess insulin resistance in pregnancy, promote fetal overgrowth, and lead to imprinting of fetal and adult metabolism.

INTRODUCTION: GESTATIONAL DIABETES MELLITUS Gestational diabetes mellitus (GDM) is one of the most common metabolic complications of pregnancy and is frequently predictive of later maternal type 2 diabetes mellitus. GDM is Address correspondence to: Jacob E. Friedman, Ph.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. Tel.: (303)-315–5130; Fax: (303)-315-3851; Email: [email protected] 21

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deﬁned as “carbohydrate intolerance of varying severity” with onset or ﬁrst recognition during pregnancy. GDM also results in fetal hyperinsulinemia, macrosomia, and an increased risk of future obesity. However, neither the cause nor the mechanisms have been adequately elucidated (Catalano et al., 1999; Gabbe, 1986). Uncomplicated pregnancy is characterized by insulin resistance and increased insulin secretion as a compensatory mechanism to maintain normal glucose tolerance. Although the insulin resistance of pregnancy appears to beneﬁt the fetus by assuring its adequate inﬂow of maternal nutrients, these adaptations result in a pathological state in the woman who develops GDM. An additional defect in insulin secretion and a more pronounced insulin resistance is characteristic of GDM, which together contribute to hyperglycemia. Women who continue to have impaired glucose tolerance (IGT) after delivery have an exceedingly high risk of developing type 2 diabetes documented as 80% in 5–10 years (Kjos et al., 1995). Thus, pregnancy can be considered a “stress test” for the subsequent development of type 2 diabetes. Moreover, fetal imprinting appears to occur in this intrauterine environment of nutrient excess and their offspring represent a high-risk group for the development of childhood obesity and IGT. GDM Effects on the Fetus GDM is associated with high rate of fetal macrosomia and other adverse outcomes seen in infants of mothers with preexisting diabetes. Infants born to women with GDM have increased amniotic ﬂuid insulin levels that predict a 10-fold increase in childhood obesity at 10–14 years, and 33% of these subjects had developed IGT by age 17 (Lindsay et al., 2000). In Pima Indians, the incidence of childhood DM at 10–14 yrs in offspring of GDM mothers was 20 times higher vs. offspring of non-diabetic mothers and 5-fold higher than that of prediabetic mothers who developed type 2 DM after pregnancy (Pettitt et al., 1993). Elevated glycemia is also associated with increased perinatal mortality. O’Sullivan et al. (1973) had reported that the perinatal mortality was 4-fold higher in untreated women with GDM when compared with women with a normal glucose tolerance test in the same time period. Pedersen (1967) hypothesized that maternal hyperglycemia was quickly transmitted to the fetus and that fetal hyperglycemia led to an exaggerated fetal insulin response. The high fetal insulin levels during gestation increase neonatal growth, leading to macrosomia, and also decrease fetal surfactant production, as well as neonatal hypoglycemia. This hypothesis has been conﬁrmed by several studies (Sacks et al., 1995; Pettitt et al., 1980; Sermer et al., 1995). Thus, understanding the mechanisms leading to excessive insulin resistance in GDM is critical to preventing maternal hyperglycemia, fetal hyperinsulinemia, and future obesity and diabetes. Animal Models of GDM: Advantages and Limitations Because of the inherent risks involved in studying pregnant women, research on the underlying mechanisms for human GDM has been limited. Thus, animal models are very important and have allowed this ﬁeld of investigation to go forward. There have been four different GDM models commonly used in the study of GDM: (1) streptozotocin (STZ) induced animal models (Harder et al., 2001; Cederberg et al., 2001; Sybulski and Maughan, 1971); (2) a partially pancreatectomized rat model (Foglia, 1970); (3) Chinese hamster model (Butler, 1967; Funaki and Mikamo, 1983); (4) spontaneous C57BLKS-Leprdb/ mouse model (Kaufmann et al., 1981; Ishizuka et al., 1999). The ﬁrst model relies on maternal STZ injection to produce diabetes during pregnancy. However, STZ-induced diabetes usually does not result

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23

in fetal overgrowth, and is in fact a model of type 1 diabetes. The partial pancreatectomy model also shares these concerns and entails a difﬁcult surgical procedure. The Chinese hamster model develops GDM spontaneously, however it is highly variable and the genes responsible are unknown. Thus, these animal models are not used commonly. In 1981, Kaufmann et al., reported a GDM model, the heterozygous leptin receptor deﬁcient (leprdb/) mouse. These animals develop spontaneous IGT during pregnancy, and the pups from these pregnancies are macrosomic compared to offspring of wild-type mothers, regardless of fetal genotype, suggesting that fetal imprinting occurs in utero as a consequence of the maternal diabetic environment. THE C57BLKS-LEPRdb/ MOUSE – A SPONTANEOUS GDM MODEL Historical Perspectives C57BLKS-Leprdb/ mice are heterozygous mice bearing a partial deletion of the long form of the leptin receptor. In 1966, at Jackson Laboratories, Bar Harbor, ME, a gene for diabetes (db) was discovered in mice of the C57BL/6J strain (Hummel et al., 1966). This db gene was proven to be the leptin receptor, which plays a key role in leptin’s ability to regulate appetite and energy expenditure. db is an autosomal recessive mutation with complete penetrance. The homozygous mutated mouse db/db is a commonly used obesity/diabetes mouse model and has been extensively characterized. The earliest abnormalities are obesity, an abnormally high level of circulating insulin and hyperglycemia (Hummel et al., 1966). The onset of diabetic symptoms in db/db on the C57BL/6J genetic background are severe and the mice have a shortened life span (3–6 months). The heterozygous C57BLKS-Leprdb/ mice do not develop diabetic symptoms in the nonpregnant state and have normal body weight with slightly elevated leptin levels compared with their wild-type C57BLK/6J / littermates (Ishizuka et al., 1999). Before pregnancy their fasting plasma glucose concentration and fasting insulin levels are similar to wild-type mice (Table 1). During the early stages of pregnancy (day 1–15) in C57BLKS-Leprdb/ mice there is no signiﬁcant IGT. However, generally beginning from 16 days of pregnancy, IGT becomes detectable. In some studies with this animal model a 3-h GTT protocol was used according to O’Sullivan criteria (Chick et al., 1970; Kaufmann et al., 1981). These studies show that at 18 days of pregnancy, the 1-, 2- and 3-h glucose values of C57BLKS-Leprdb/ mice were statistically higher than those of the normal mice and at one month after delivery, there was no signiﬁcant difference in the postpartum GTT of C57BLKS-Leprdb/ mice and control mice. We modiﬁed

Table 1

Characteristics of the mice (mean SE, n 6).

/ Non-preg / Pregnant db/ Non-preg db/ Pregnant

Body weight (g)

Fasting glucose (mg/dl)

Fasting insulin (␮U/ml)

19.08 6.58 29.50 2.09 20.87 6.79 32.38 9.37

119.12 10.56 118.55 7.56 116.37 0.04 122.36 6.29

0.22 0.03 0.97 0.19* 0.22 0.06 1.17 0.33**

/: C57BL/6J, db/: C57BLKS-Leprdb/. The mice were 12–14 weeks old, at 18 days of pregnancy. Fasting blood samples were collected through tails and after 6 h fasting. *p 0.05 between / pregnant and / non-preg mice. **p 0.05 vs. db/ non-preg mice.

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JIANHUA SHAO AND JACOB E. FRIEDMAN db/+ Non-preg db/+ Preg +/+ Non-preg +/+ Preg

Plasma glucose (mg/dl)

360 310 260 210 160 110 60 0

30 Time (min)

60

Figure 1 Plasma glucose concentrations during glucose tolerance test in pregnant C57BLKS-Leprdb/ and / control mice. Mice were fasted 12 h on day 18 of gestation and injected intraperitoneally with 2 g glucose per kg body weight. Blood samples were collected from tails at time zero, 30 and 60 min after injection. Values are means SE for 6–10 mice per group. *p 0.05, between db/ preg and / preg mice.

this 3-h GTT protocol after i.p. glucose injection and the plasma glucose concentration reached a maximal level around 30 min. Our studies showed that 98% of C57BLKSLeprdb/ pregnant mice had signiﬁcantly higher glucose levels at 30 min and 1 h and these elevated glucose levels met the diagnostic criteria for IGT (see Figure 1). At the end of 19 days of pregnancy, fasting plasma glucose levels in C57BLKS-Leprdb/ mice are still in the normal range despite impaired glucose tolerance, similar to most GDM human patients. The db/ pregnant mice are extremely insulin-resistant compared to wildtype pregnant controls based on the inability of injected insulin to reduce plasma glucose (Figure 2). The C57BLKS-Leprdb/ mice consumed ~13% more food and gained more weight during pregnancy compared to wild-type / controls (34), suggesting that the leptin receptor is not fully recessive with regard to fat mass and that heterozygosity at the leptin receptor may play a role in susceptibility to environmental conditions such as pregnancy favoring obesity and insulin resistance. Insulin Secretion It is well known that normal pregnancy reduces insulin sensitivity in peripheral tissues. To compensate for the reduced insulin sensitivity, insulin synthesis and secretion are upregulated. The ﬁnding of inadequate insulin secretion for a given degree of insulin sensitivity in the db/ model of GDM implies that the mice have a major defect in insulin secretion (Figure 3). It is well documented that insulin synthesis and secretion in response to glucose is elevated in the late stages of pregnancy both in human and animal models. During normal pregnancy, the volume of islets is enlarged by several mechanisms. The preproinsulin gene expression is upregulated and insulin secretion is increased in normal or C57BLKS-Leprdb/ pregnant mice (Table 1). Although islets of pregnant C57BLKS-Leprdb/ mice are larger than non-pregnant mice, circulating insulin levels were not high enough for compensating the severe insulin resistance. Women with GDM appear to have abnormalities in ﬁrst phase insulin secretion

GESTATIONAL DIABETES AND INSULIN RESISTANCE IN THE C57BLKS LEPRdb/ MOUSE db/+ Non-preg db/+ Control +/+ Non-preg +/+ Control

100 % of fasting glucose

25

80

60

40 0

15

30 Time (min)

60

Figure 2 Severe insulin resistance in GDM mice during an during insulin challenge test. At day 18 of gestation, the mice were fasted 6 h then injected i.p. with insulin (0.75 u/kg body wt). The blood was sampled from the tails at 15, 30 and 60 min time points after injection. Glucose values are means SE. *p 0.05 vs. non-pregnant control mice at the corresponding time point.

3

db/+ Non-preg

Insulin (U/ml)

2.5

db/+ Preg +/+ Non-preg

2

+/+ Preg

1.5 1 0.5 0 0

30 Time (min)

60

Figure 3 Plasma insulin concentrations during glucose tolerance test in pregnant C57BLKS-Leprdb/ and / control mice. Mice were fasted 12 h on day 18 of gestation and injected intraperitoneally with 2 g glucose per kg body weight. Blood samples were collected from tails at time zero, 30 and 60 min after injection. The insulin was measured by ELISA kit. Values are means SE for 6–10 mice per group. *p 0.05, between db/ Preg and / Preg mice.

that may contribute to the development of GDM (Kautzky-Willer et al., 1997; Kuhl, 1991; Buchanan et al., 1990). We suggest that leptin receptor may play an important role in the normal regulation of glucose metabolism in the islet to insure proper insulin secretion, as suggested by studies in islets from heterozygous fa/ mouse (Zhou et al., 1997). Accumulated data indicate that prolactin and placental lactogen upregulate islet function by: (1) increased insulin synthesis, (2) increased glucose utilization, oxidation and metabolism through elevated glucokinase, and (3) enhanced glucose-induced insulin release with reduced glucose stimulation threshold (Sorenson et al., 1987a,b; Weinhaus et al., 1998, 2000). Many studies demonstrated that glucocorticoids inhibit insulin synthesis and reduce glucose-stimulated insulin secretion by inhibiting insulin exocytic process (Philippe and Missotten, 1990;

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Lambillotte et al., 1997) suggesting that prolactin and glucocorticoids may counterregulate islet function at different sites. Studies are needed to clarify the mechanisms of the relative low insulin synthesis and secretion in GDM mice in addition to insulin resistance. Fetal Macrosomia Infant macrosomia and selective organomegaly are the hallmarks of the gestational diabetic pregnancy. Although the pathogenesis remains controversial, the maternal insulin resistance and hence, the degree of maternal hyperglycemia appear to be a major stimulus. Maternal hyperglycemia is believed to stimulate fetal pancreatic insulin secretion during development, which can stimulate fetal overgrowth. Although maternal weight in C57BLKS-Leprdb/ mice is slightly higher than normal control pregnant mice, the mean pups’ weight at birth is significantly higher. We also recently found that the wild-type pups from C57BLKS-Leprdb/ GDM mothers returned to normal body weight as adults, but particularly the female offspring were more likely to become obese on a high-fat diet compared to the wild-type offspring of normal mothers (Shao et al. in publication). Basal HGP was 25% higher ( p 0.05) in F1 female offspring on the high-fat diet compared to controls despite signiﬁcant fasting hyperinsulinemia, suggestive of hepatic insulin resistance in the F1 generation. The Pathology of Insulin Resistance in C57BLKS-Leprdb/⫹ Mice During normal pregnancy, insulin-mediated glucose disposal decreases by 50% from early to late pregnancy in humans and in rodents (Rossi et al., 1993; Catalano et al., 1991). Investigators have shown more pronounced insulin resistance in GDM patients compared to women with normal glucose tolerance during pregnancy (Kautzky-Willer et al., 1997; Ryan et al., 1985; Garvey et al., 1993), that may contribute to IGT in addition to defects in insulin secretion. Recent studies from our laboratory showed that the resistance to insulin-mediated glucose transport is more severe in skeletal muscle ﬁbers from GDM patients than in women who are pregnant with normal insulin tolerance. The total GLUT4 protein levels are normal in skeletal muscle of GDM (Garvey et al., 1992; Ishizuka et al., 1999). Thus, a defect in insulin signaling and/or translocation is responsible for insulin resistance to glucose transport. Whether the same defects combined with previous factors are present in GDM or whether there are additional mechanism(s) beyond normal pregnancy has yet to be resolved for GDM. Impaired insulin-mediated glucose uptake in skeletal of GDM most likely relates to impaired translocation or malfunction of GLUT4. Many studies have shown that insulin receptor binding afﬁnity and insulin receptor protein level are normal in pregnancy and in GDM patients (Shao et al., 2000; Puavilai et al., 1982; Moore et al., 1981; Camps et al., 1990). Therefore, altered post-receptor events of insulin-signaling system may play an important role in pregnancy-induced insulin resistance and GDM. Most studies of human skeletal muscle have found that insulin receptor tyrosine kinase (IRTK) activity is impaired in type 2 diabetes and in sub-populations of insulin resistant subjects (Kahn and Flier, 2000; Cusi et al., 2000; Shulman, 2000). One or more mechanisms might be involved in inhibition of insulinstimulated IRTK and IRS-1trate-1 phosphorylation, including phosphatase-mediated dephosphorylation (Goldstein et al., 1998), and phosphorylation of serine/threonine residues on IR and IRS-1 (Hotamisligil et al., 1996; Li et al., 1999; Ravichandran et al., 2001; Delahaye et al., 1998). However, neither mechanism has been demonstrated directly in humans with type 2 diabetes. Our recent data on human skeletal muscle suggest that insulin

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receptors from obese pregnant and GDM subjects are serine/theronine phosphorylated to a greater degree than IR from obese non-pregnant control subjects, and this is reversed by alkaline phosphatase treatment (Shao et al., 2000). These results suggest that impaired IRTK activity is not caused by obesity, but may play an important role in the natural insulin resistance of pregnancy as well as the basis for the higher intrinsic insulin resistance of GDM, by mechanisms that are currently not well understood. Using the db/ GDM mouse model, we have recently found that IRS-1 serine phosphorylation in pregnancy is associated with a redistribution of a pool of PI 3-kinase to the IR. This pool of PI 3-kinase increases skeletal muscle serine kinase activity. We hypothesize that the IR ␤ subunit may be a substrate for PI 3-kinase, and could inhibit IRTK activation during late pregnancy. We further hypothesize that redistribution of PI 3-kinase could trigger downstream serine kinases that inhibit IRS-1 associated PI 3-kinase activity, and increase IRS-1 degradation in pregnancy as shown in Figure 4.

Insulin

r-p

Se

Tp

Ser-p

p110

p85α

T-p

T-p

IRS-1

+

Ser-p

T-p

+

+

Degradation p

p

PKCλ/ζ

p

?

Akt

GLUT4

Serine kinase (S)

+ p

mTOR

+

+

p70S6k p

Figure 4 GDM is associated with re-distribution and up regulation of p85␣-PI 3-kinase to the insulin receptor and decreased IRS-1 binding to p85␣-PI 3-kinase in skeletal muscle. Upon stimulation by insulin, p85␣ binds to the insulin receptor and highly activates a PI 3-kinase/Akt/p70S6 kinase pathway. The solid arrow in GDM indicates increased Akt leading to p70S6 kinase activation pathway, rather than for GLUT4 translocation (broken arrow). Increased p70S6 kinase activity may promote phosphorylation of serine sites in IRS-1. Serine phosphorylation of IRS-1 inhibits the ability of IRS-1 to be further phosphorylated at tyrosine sites by the insulin receptor and inhibits binding with p85␣ to mediate insulin signaling. PKC activation had very little effect on GLUT4 translocation in GDM mice. Rather, the compartment for p70S6 kinase activation (solid arrow) is favored in GDM mice, which might be responsible for the greater insulin resistance to GLUT4 translocation in GDM.

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Several studies using cultured cells have shown that increased Ser/Thr phosphorylation of IRS-1 impairs insulin-induced tyrosine phosphorylation of IRS-1, PI 3-kinase activation, and glucose uptake (Tanti et al., 1994; De Fea and Roth, 1997). Moreover, persistent serine phosphorylation induces IRS-1 degradation (Egawa et al., 2000; Haruta et al., 2000; Pederson et al., 2001). The potential serine kinase(s) involved in IRS-1 serine phosphorylation include GSK3 (Orena et al., 2000), JNK1 (Aguirre et al., 2000), PKC (De Fea and Roth, 1997), and aPKC (Ravichandran et al., 2001; Liu et al., 2001) and Akt (Li et al., 1999; Paz et al., 1999). However, recent studies indicate a critical role of Ser/Thr phosphorylation and degradation of IRS-1 may be mediated by a rapamycin sensitive pathway (PI 3-kinase/Akt/m-TOR/p70S6 kinase), independent of ras/MAP kinase (Egawa et al., 2000; Haruta et al., 2000, Pederson et al., 2001). In addition, the cytokine TNF-␣ increases IRS-1 serine phosphorylation, which may inhibit IRTK activity (Hotamisligil et al., 1996). Our ﬁndings in human muscle also indicate that IRS-1 serine phosphorylation is increased in the basal state during late pregnancy (unpublished observations). Thus, increased serine kinase activity could account for the inhibition of IR and IRS-1 tyrosine phosphorylation, as well as IRS-1 degradation, and may contribute to insulin resistance of glucose transport found in skeletal muscle during pregnancy and GDM. The levels of Akt and p70S6 kinase, which can activate serine phosphorylation, are increased in pregnancy (Shao et al., 2002). In addition to these mechanisms, recent evidence points to an accumulation of muscle triglyceride as a major factor associated with insulin resistance to insulin-mediated glucose disposal in humans (Virkamaki et al., 2001). Increased muscle triglyceride content have been suggested to cause chronic activation of protein kinase C (PKC) isoforms. For example, in the diabetesprone sand rats (Psammonmys obesus), increased expression of protein kinase C in particular, correlates with increased diacylglycerol (DAG) in skeletal muscle (Ikeda, 2001), which appears to be responsible for activation of PKC. Since PKC can interfere with insulin signaling through Ser/Thr phosphorylation of the IR or IRS-1, an increase in muscle triglyceride, and therefore PKC over-expression could also play a role in increased serine phosphorylation in skeletal muscle during GDM.

Role of Placental Hormones in Insulin Resistance It is widely accepted that placental hormones reprogramme maternal physiology to an insulinresistant state in order to ensure adequate growth of the fetus, which requires 80% of its energy source as glucose. These hormones, primarily placental lactogen (hPL), placental growth hormone (hPGH), modify maternal physiology to ensure the successful growth of the fetus, often at the expense of causing pathological conditions in the mother. Historically, hPL was postulated to be the key hormone that mediates the insulin resistance of pregnancy. However, hPL has both insulin-like and anti-insulin effects. In vitro, it has been shown to increase lipolysis in adipocytes (Handwerger and Freemark, 2000), to stimulate growth of pancreatic islets during pregnancy in rats and stimulate insulin secretion in human islets (Brelje et al., 1993), suggesting that it has direct insulin-like actions in pancreatic beta cells. Furthermore, in isolated rat adipocytes, it appeared to have an insulin-like effect increasing glucose uptake and incorporation into glycogen, which may favor adipocyte accumulation in the mother (Handwerger and Freemark, 2000). Thus, it is still not clear whether hPL affects insulin signal transduction in skeletal muscle, the main target tissue for insulin-mediated glucose disposal. hPGH is another major hormone of pregnancy likely involved in insulin resistance. hPGH increases 6–8-fold with advancing gestation, and almost completely replaces

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pituitary growth hormone in the maternal circulation by ~20 weeks of pregnancy (Alsat et al., 1998). hPGH is a product of the human growth hormone variant gene (hGH-V), and a member of the growth hormone gene family that differs from pituitary growth hormone (GH) by 13 amino acids (Alsat et al., 1998). hPGH is not regulated by GH releasing hormone, and is secreted tonically rather than in a pulsatile fashion, unlike pituitary growth hormone. Although insulin resistance is a major consequence of excess growth hormone, GH also mimics the action of insulin in the short term (Brelje et al., 1993). Surprisingly, there have been no studies comparing the relative contributions of hPL or hPGH to the insulin signaling pathway in skeletal muscle. Our recent study shows that transgenic mice overexpressing hPGH exhibit a profound whole-body insulin resistance (Barbour et al., 2001). The animals have impaired IR tyrosine phosphorylation, normal IRS-1 levels, yet increased IRS-1 serine phosphorylation in skeletal muscle. The animals also have a noticeable increase in p85␣ expression and association with the IR. These data suggest that the insulin resistance of pregnancy might be caused, in part, by the anti-insulin effects of chronic hPGH elevation. TREATING INSULIN RESISTANCE IN C57BLKS-LEPRdb/ MICE, IMPACT ON THE FETUS GLUT4 Overexpression One of the utilities of the mouse model for spontaneous GDM is that treatment therapies aimed at reducing maternal insulin resistance can be evaluated for their impact on fetal growth. We investigated whether overexpression of the human GLUT4 gene in C57BLKSLeprdb/ mice had effects on maternal insulin resistance and their offspring development (Ishizuka et al., 1999). A 2–3-fold overexpression of GLUT4 in C57BLKS-Leprdb/ mice improved glucose-stimulated insulin secretion by 250%, and increased IR␤, IRS-1, and p85␣ phosphorylation 2-fold, despite no change in concentration of these proteins. Maternal fat mass and energy intake was reduced by GLUT4 overexpression, independent of food intake. Fetal body weight was still increased in offspring from db/ GLUT4 mothers despite GLUT4 overexpression by 2–3-fold ( p 0.05). However, fetuses from db/ mothers with 4–5-fold overexpression of GLUT4 weighed signiﬁcantly less compared with pups from / or C57BLKS-Leprdb/ mothers ( p 0.05). The results suggest that macrosomia may occur despite normoglycemia. One explanation for the increased fetal mass may be that adding excess substrates to the maternal compartment may increase placenta growth and transfer of substrates to the fetus. These results reveal that 4–6-fold GLUT4 overexpression in maternal skeletal muscle can prevent maternal hyperglycemia. However, fetal growth was clearly affected by low maternal glucose, suggesting that if maternal insulin resistance does not develop sufﬁciently the nutrient ﬂux to the fetus can be reduced. Leptin Administration and Insulin Resistance in Gestational Diabetes In humans and animals, plasma leptin increases early during gestation, derived primarily from the placenta (Masuzaki et al., 1997; Highman et al., 1998; Hoggard et al., 2000). The role of increased leptin during pregnancy on maternal–fetal metabolism and intrauterine growth remains unclear. There is no correlation between maternal leptin levels and fetal weight. However, it was reported that umbilical cord blood leptin levels are positively correlated with

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fetal insulin, birth weight, ponderal index (kg/cm3), and length and head circumference (Tamura et al., 1998; Ong et al., 1999; Lepercq et al., 1999), suggesting a potential relationship between placental leptin and fetal growth. Leptin normally reduces appetite and increases energy expenditure acting through the hypothalamus (Campﬁeld et al., 1995; Vaisse et al., 1996). Leptin also has direct metabolic effects on several tissues, resulting in increased glucose utilization and lipolysis (Sivitz et al., 1997; Barzilai et al., 1997; Bryson et al., 1999). The marked increase in maternal leptin, an appetite suppressant, suggests there is some form of maternal leptin resistance, or perhaps there is an alternative role for maternal leptin. Leptin also serves as a mitogen for a growing number of cell types, including endothelial cells, hematopoetic cells, lung epithelial cells, and pancreatic beta cells in vitro (Bouloumie et al., 1998; Gainsford et al., 1996; Tsuchiya et al., 1999; Morton et al., 1999). Leptin could therefore be acting as a placental mitogen in addition to stimulating growth of tissues in the developing fetus. To investigate whether leptin administration can reduce adiposity and thereby prevent GDM and neonatal overgrowth, we have injected recombinant human leptin-IgG during late pregnancy of C57BLKS-Leprdb/ and control mice, then examined energy balance, glucose and insulin tolerance, and fetal growth (Yamashita et al., 2001). Despite leptin resistance, exogenous human leptin administration lessened maternal weight gain and improved glucose tolerance in the db/ mouse. Leptin administration had only marginal effects on appetite, but signiﬁcantly reduced insulin resistance in pregnant db/ mice, in part through an improvement in skeletal muscle insulin signal transduction at the level of IRS-1. In spite of reduced energy intake and improved glucose tolerance, leptin administration did not reduce fetal overgrowth in db/ mothers. However, pair-feeding decreased placental leptin concentration and reduced fetal birth weight. Our results provide evidence that leptin administration during late gestation can reduce adiposity and improve glucose tolerance in the db/ mouse model of spontaneous GDM. However, fetal and placenta leptin levels are 1.3–1.5-fold higher in db/ mothers, and are subject to reduced negative feedback in response to leptin treatment. These data suggest that reducing total caloric intake directly appears to be the most effective means of reducing the fetal overgrowth. In summary, the heterozygous C57BLKS-Leprdb/ mouse is an ideal model for the study of mechanisms underlying insulin resistance in GDM and fetal macrosomia because: (1) the mice have a pathophysiology of insulin resistance similar to human GDM and are readily available; (2) the gene responsible for triggering GDM is known and its effects can be controlled for, (3) using a mouse model allows the introduction of other gene knock-outs/knockins into the model to test speciﬁc gene(s) and pathways in insulin resistance; (4) the model can be used to study modiﬁcations of maternal nutrient metabolism, potential drug therapy, and its impact on fetal growth and future diabetes. REFERENCES Aguirre, V., Uchida, T., Yenush, L., Davis, R. and White, M.F. (2000) The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J. Biol. Chem., 275, 9047–9054. Alsat, E., Guibourdenche, J., Couturier, A. and Evain-Brion, D. (1998) Physiological role of human placental growth hormone. Mol. Cell. Endocrinol., 140, 121–127. Barbour, L.A., Shao, J., Qiao, L., Pulawa, L., Jensen, D., Bartke, A., Draznin, B. and Friedman, J.E. (2002) Overexpression of human placental growth hormone causes severe insulin resistance in transgenic mice. Am. J. Obstet. Gynecol., 186, 512–517.

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Garvey, W.T., Maianu, L., Zhu, J.H., Hancock, J.A. and Golichowski, A.M. (1993) Multiple defects in the adipocyte glucose transport system cause cellular insulin resistance in gestational diabetes. Heterogeneity in the number and a novel abnormality in subcellular localization of GLUT4 glucose transporters. Diabetes, 42, 1773–1785. Goldstein, B.J., Ahmad, F., Ding, W., Li, P.M. and Zhang, W.R. (1998) Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases. Mol. Cell. Biochem., 182, 91–99. Handwerger, S. and Freemark, M. (2000) The roles of placental growth hormone and placental lactogen in the regulation of human fetal growth and development. J. Pediatr. Endocrinol. Metab., 13, 343–356. Harder, T., Aerts, L., Franke, K., Van Bree, R., Van Assche, F.A. and Plagemann, A. (2001) Pancreatic islet transplantation in diabetic pregnant rats prevents acquired malformation of the ventromedial hypothalamic nucleus in their offspring. Neurosci. Lett., 299, 85–88. Haruta, T., Uno, T., Kawahara, J., Takano, A., Egawa, K., Sharma, P.M., Olefsky, J.M. and Kobayashi, M. (2000) A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1. Mol. Endocrinol., 14, 783–794. Highman, T.J., Friedman, J.E., Huston, L.P., Wong, W.W. and Catalano, P.M. (1998) Longitudinal changes in maternal serum leptin concentrations, body composition, and resting metabolic rate in pregnancy. Am. J. Obstet. Gynecol., 178, 1010–1015. Hoggard, N., Hunter, L., Lea, R.G., Trayhurn, P. and Mercer, J.G. (2000) Ontogeny of the expression of leptin and its receptor in the murine fetus and placenta. Br. J. Nutr., 83, 317–326. Hotamisligil, G.S., Peraldi, P., Budavari, A., Ellis, R., White, M.F. and Spiegelman, B.M. (1996) IRS-1mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance. Science, 271, 665–668. Hummel, K.P., Dickie, M.M. and Coleman, D.L. (1966) Diabetes, a new mutation in the mouse. Science, 153, 1127–1128. Ikeda, Y., Olsen, G.S., Ziv, E., Hansen, L.L., Busch, A.K., Hansen, B.F., Shafrir, E. and MosthafSeedorf, L. (2001) Cellular mechanism of nutritionally induced insulin resistance in Psammomys obesus: overexpression of protein kinase Cepsilon in skeletal muscle precedes the onset of hyperinsulinemia and hyperglycemia. Diabetes, 50, 584–592. Ishizuka, T., Klepcyk, P., Liu, S., Panko, L., Gibbs, E.M. and Friedman, J.E. (1999) Effects of overexpression of human GLUT4 gene on maternal diabetes and fetal growth in spontaneous gestational diabetic C57BLKS/J Lepr(db/) mice. Diabetes, 48, 1061–1069. Kahn, B.B. and Flier, J.S. (2000) Obesity and insulin resistance. J. Clin. Invest., 106, 473–481. Kaufmann, R.C., Amankwah, K.S., Dunaway, G., Maroun, L., Arbuthnot, J. and Roddick, J.W., Jr. (1981) An animal model of gestational diabetes. Am. J. Obstet. Gynecol., 141, 479–482. Kautzky-Willer, A., Prager, R., Waldhausl, W., Pacini, G., Thomaseth, K., Wagner, O.F., Ulm, M., Streli, C. and Ludvik, B. (1997) Pronounced insulin resistance and inadequate beta-cell secretion characterize lean gestational diabetes during and after pregnancy. Diabetes Care, 20, 1717–1723. Kjos, S.L., Peters, R.K., Xiang, A., Henry, O.A., Montoro, M. and Buchanan, T.A. (1995) Predicting future diabetes in Latino women with gestational diabetes. Utility of early postpartum glucose tolerance testing. Diabetes, 44, 586–591. Kuhl, C. (1991) Insulin secretion and insulin resistance in pregnancy and GDM. Implications for diagnosis and management. Diabetes, 40 (Suppl 2), 18–24. Lambillotte, C., Gilon, P. and Henquin, J.C. (1997) Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. J. Clin. Invest., 99, 414–423. Lepercq, J., Lahlou, N., Timsit, J., Girard, J. and Mouzon, S.H. (1999) Macrosomia revisited: ponderal index and leptin delineate subtypes of fetal overgrowth. Am. J. Obstet. Gynecol., 181, 621–625. Li, J., DeFea, K. and Roth, R.A. (1999) Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway. J. Biol. Chem., 274, 9351–9356. Lindsay, R.S., Hanson, R.L., Bennett, P.H. and Knowler, W.C. (2000) Secular trends in birth weight, BMI, and diabetes in the offspring of diabetic mothers. Diabetes Care, 23, 1249–1254. Liu, Y.-F., Paz, K., Hershkovitz, A., Alt, A., Tennenbaum, T., Sampson, S.R., Ohba, M., Kuroki, T., LeRoith, D. and Zick, Y. (2001) Insulin stimulates PKCzeta-mediated phosphorylation of insulin

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receptor substrate-1 (IRS-1). A self-attenuated mechanism to negatively regulate the function of IRS proteins. J. Biol. Chem., 276(17), 14459–14465. Masuzaki, H., Ogawa, Y., Sagawa, N., Hosoda, K., Matsumoto, T., Mise, H., Nishimura, H., Yoshimasa, Y., Tanaka, I., Mori, T. and Nakao, K. (1997) Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat. Med., 3, 1029–1033. Moore, P., Kolterman, O., Weyant, J. and Olefsky, J.M. (1981) Insulin binding in human pregnancy: comparisons to the postpartum, luteal, and follicular states. J. Clin. Endocrinol. Metab., 52, 937–941. Morton, N.M., Emilsson, V., de Groot, P., Pallett, A.L. and Cawthorne, M.A. (1999) Leptin signalling in pancreatic islets and clonal insulin-secreting cells. J. Mol. Endocrinol., 22, 173–184. Ong, K.K., Ahmed, M.L., Sherriff, A., Woods, K.A., Watts, A., Golding, J. and Dunger, D.B. (1999) Cord blood leptin is associated with size at birth and predicts infancy weight gain in humans. ALSPAC study team – Avon longitudinal study of pregnancy and childhood. J. Clin. Endocrinol. Metab., 84, 1145–1148. Orena, S.J., Torchia, A.J. and Garofalo, R.S. (2000) Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes. J. Biol. Chem., 275, 15765–15772. O’Sullivan, J.B., Charles, D., Mahan, C.M. and Dandrow, R.V. (1973) Gestational diabetes and perinatal mortality rate. Am. J. Obstet. Gynecol., 116, 901–904. O’Sullivan, J.B. and Mahan, C.M. (1966) Glucose tolerance test. Variability in pregnant and nonpregnant women. Am. J. Clin. Nutr., 19, 345–351. Paz, K., Liu, Y.F., Shorer, H., Hemi, R., LeRoith, D., Quan, M., Kanety, H., Seger, R. and Zick, Y. (1999) Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function. J. Biol. Chem., 274, 28816–28822. Pedersen, J. (1967) Pathogenesis of the Characteristic Features of Newborn Infants of Diabetic Women, pp. 128–137. Baltimore: Williams and Wilkins. Pederson, T.M., Kramer, D.L. and Rondinone, C.M. (2001) Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation. Diabetes, 50, 24–31. Pettitt, D.J., Knowler, W.C., Baird, H.R. and Bennett, P.H. (1980) Gestational diabetes: infant and maternal complications of pregnancy in relation to third-trimester glucose tolerance in the Pima Indians. Diabetes Care, 3, 458–464. Pettitt, D.J., Nelson, R.G., Saad, M.F., Bennett, P.H. and Knowler, W.C. (1993) Diabetes and obesity in the offspring of Pima Indian women with diabetes during pregnancy. Diabetes Care, 16, 310–314. Philippe, J. and Missotten, M. (1990) Dexamethasone inhibits insulin biosynthesis by destabilizing insulin messenger ribonucleic acid in hamster insulinoma cells. Endocrinology, 127, 1640–1645. Puavilai, G., Drobny, E.C., Domont, L.A. and Baumann, G. (1982) Insulin receptors and insulin resistance in human pregnancy: evidence for a postreceptor defect in insulin action. J. Clin. Endocrinol. Metab., 54, 247–253. Ravichandran, L.V., Esposito, D.L., Chen, J. and Quon, M.J. (2001) Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin. J. Biol. Chem., 276, 3543–3549. Rossi, G., Sherwin, R.S., Penzias, A.S., Lapaczewski, P., Jacob, R.J., Shulman, G.I. and Diamond, M.P. (1993) Temporal changes in insulin resistance and secretion in 24-h-fasted conscious pregnant rats. Am. J. Physiol., 265, E845–E851. Ryan, E.A., O’Sullivan, M.J. and Skyler, J.S. (1985) Insulin action during pregnancy. Studies with the euglycemic clamp technique. Diabetes, 34, 380–389. Sacks, D.A., Greenspoon, J.S., Abu-Fadil, S., Henry, H.M., Wolde-Tsadik, G. and Yao, J.F. (1995) Toward universal criteria for gestational diabetes: the 75-gram glucose tolerance test in pregnancy. Am. J. Obstet. Gynecol., 172, 607–614. Sermer, M., Naylor, C.D., Gare, D.J., Kenshole, A.B., Ritchie, J.W., Farine, D., Cohen, H.R., McArthur, K., Holzapfel, S., Biringer, A. and et al. (1995) Impact of increasing carbohydrate intolerance on maternal-fetal outcomes in 3637 women without gestational diabetes. The Toronto tri-hospital gestational diabetes project. Am. J. Obstet. Gynecol., 173, 146–156.

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Seufert, J., Kieffer, T.J., Leech, C.A., Holz, G.G., Moritz, W., Ricordi, C. and Habener, J.F. (1999) Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus. J. Clin. Endocrinol. Metab., 84, 670–676. Shao, J., Catalano, P.M., Yamashita, H., Ruyter, I., Smith, S., Youngren, J. and Friedman, J.E. (2000) Decreased insulin receptor tyrosine kinase activity and plasma cell membrane glycoprotein-1 overexpression in skeletal muscle from obese women with gestational diabetes mellitus (GDM): evidence for increased serine/threonine phosphorylation in pregnancy and GDM. Diabetes, 49, 603–610. Shao, J., Qiao, L., Yamashita, H., Draznin, B. and Friedman, J.E. (2002) PI 3-kinase re-distribution is associated with insulin resistance in skeletal muscle during gestational diabetes mellitus. Diabetes, 51: 19–29. Shulman, G.I. (2000) Cellular mechanisms of insulin resistance. J. Clin. Invest., 106, 171–176. Sivitz, W.I., Walsh, S.A., Morgan, D.A., Thomas, M.J. and Haynes, W.G. (1997) Effects of leptin on insulin sensitivity in normal rats. Endocrinology, 138, 3395–3401. Sorenson, R.L., Brelje, T.C., Hegre, O.D., Marshall, S., Anaya, P. and Sheridan, J.D. (1987a) Prolactin (in vitro) decreases the glucose stimulation threshold, enhances insulin secretion, and increases dye coupling among islet B cells. Endocrinology, 121, 1447–1453. Sorenson, R.L., Johnson, M.G., Parsons, J.A. and Sheridan, J.D. (1987b) Decreased glucose stimulation threshold, enhanced insulin secretion, and increased beta cell coupling in islets of prolactin-treated rats. Pancreas, 2, 283–288. Sybulski, S. and Maughan, G.B. (1971) Use of streptozotocin as diabetic agent in pregnant rats. Endocrinology, 89, 1537–1540. Tamura, T., Goldenberg, R.L., Johnston, K.E. and Cliver, S.P. (1998) Serum leptin concentrations during pregnancy and their relationship to fetal growth. Obstet. Gynecol., 91, 389–395. Tanti, J.F., Gremeaux, T., van Obberghen, E. and Le Marchand-Brustel, Y. (1994) Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. J. Biol. Chem., 269, 6051–6057. Tsuchiya, T., Shimizu, H., Horie, T. and Mori, M. (1999) Expression of leptin receptor in lung: leptin as a growth factor. Eur. J. Pharmacol., 365, 273–279. Vaisse, C., Halaas, J.L., Horvath, C.M., Darnell, J.E., Jr., Stoffel, M. and Friedman, J.M. (1996) Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet, 14, 95–97. Virkamaki, A., Korsheninnikova, E., Seppala-Lindroos, A., Vehkavaara, S., Goto, T., Halavaara, J., Hakkinen, A.M. and Yki-Jarvinen, H. (2001) Intramyocellular lipid is associated with resistance to in vivo insulin actions on glucose uptake, antilipolysis, and early insulin signaling pathways in human skeletal muscle. Diabetes, 50, 2337–2343. Weinhaus, A.J., Bhagroo, N.V., Brelje, T.C. and Sorenson, R.L. (1998) Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy. Diabetes, 47, 1426–1435. Weinhaus, A.J., Bhagroo, N.V., Brelje, T.C. and Sorenson, R.L. (2000) Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology, 141, 1384–1393. Yamashita, H., Shao, J., Ishizuka, T., Klepcyk, P.J., Muhlenkamp, P., Qiao, L., Hoggard, N. and Friedman, J.E. (2001) Leptin administration prevents spontaneous gestational diabetes in heterozygous Lepr(db/) mice: effects on placental leptin and fetal growth. Endocrinology, 142, 2888–2897. Zhou, Y.T., Shimabukuro, M., Lee, Y., Koyama, K., Trieu, F. and Unger, R.H. (1997) Leptin normalizes the impaired response of proinsulin mRNA to long chain fatty acids in heterozygous Zucker diabetic fatty rats. J. Biol. Chem., 272, 25648–25651.

3. ROLE OF PROTEIN-TYROSINE PHOSPHATASES IN INSULIN ACTION AND INSULIN RESISTANCE: RECENT INSIGHTS FROM CELLULAR AND ANIMAL STUDIES BARRY J. GOLDSTEIN The Dorrance H. Hamilton Research Laboratories, Division of Endocrinology, Diabetes and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107, USA

INTRODUCTION Protein-tyrosine phosphatases (PTPases) play an integral role in the regulation of insulin action, by modulating the tyrosine phosphorylation state of the insulin receptor and the cellular substrate proteins that transmit the insulin signal. Extending earlier observations from cellular studies, recent work on the manipulation of speciﬁc PTPases in knock-out and transgenic animals has generated additional insight into the potential involvement of these regulatory enzymes in insulin action and glucose metabolism. To date, cellular and mouse models with altered expression of LAR (Leukocyte Antigen-Related), PTP1B, LRP (Leukocyte Antigen-Related Phosphatase) and SHP-2, have been carefully evaluated with respect to insulin signaling. In addition, an inactive form of SHP-2 and native LAR have also been overexpressed in transgenic mouse models. PTP1B has been most consistently shown to act as a negative regulator of insulin action, and animal studies have conﬁrmed that PTP1B plays a key role in glucose homeostasis and energy expenditure. Evidence has also shown that LAR can have a negative impact on cellular insulin signaling, although its exact physiological role has been more difﬁcult to discern from the knock-out and transgenic models. In contrast, SHP-2 positively inﬂuences post-insulin receptor signaling on mitogenic pathways in cellular studies, and animal models have conﬁrmed that SHP-2 has important effects on embryonic development as well as a contribution to glucose metabolism. Studies on reduced LRP expression in cellular systems show that this homolog is less likely to play an important role in insulin signaling. These recent studies have provided insight into the physiological role of speciﬁc PTPases in insulin action and have provided further evidence for PTP1B, in particular, as a key target for pharmaceutical intervention to enhance insulin action in type 2 diabetes and other insulin-resistant disease states. PTPASES AND THE REGULATION OF CELLULAR INSULIN ACTION Reversible tyrosine phosphorylation is well established as a central mechanism in the initiation of the insulin signal from the receptor to its proximate targets (Nystrom and Quon, 1999; Address correspondence to: Director, Division of Endocrinology, Diabetes and Metabolic Diseases, Jefferson Medical College, Room 349 Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107. Tel.: (215) 503-1272; Fax: (215) 9237932; Email: [email protected] 35

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Virkamaki et al., 1999). The regulation of insulin signaling is determined by the tyrosine phosphorylation state of the receptor, which augments its kinase activity, as well as the extent of tyrosine phosphorylation of its multiple cellular substrate proteins, which serve as docking sites for SH-2 domain containing adaptor molecules and transmit the downstream insulin signal. As a counter-regulatory inﬂuence to the activation of the receptor by insulin binding, speciﬁc cellular PTPases enzymes have been postulated to regulate insulin signaling in normal physiology and also to be involved in the pathogenesis of insulin-resistant disease states (reviewed in detail in Goldstein, 2000). In addition, the steady-state tyrosine phosphorylation of speciﬁc functional domains of the insulin receptor and IRS proteins is a potentially important mechanism for sorting of some of the pleiotropic responses of insulin in its target cells. The current view of reversible tyrosine phosphorylation in the regulation of the insulin receptor has led to a working hypothesis that the steady-state balance of these events is determined by the opposing actions of: (1) receptor autophosphorylation which activates the kinase activity and enhances the tyrosine phosphorylation of receptor substrate proteins; and (2) cellular PTPases, which deactivate the receptor kinase and dephosphorylate the receptor substrates. The development of this hypothesis has been supported by the ﬁndings that puriﬁed, tyrosine phosphorylated insulin receptor and IRS proteins studied in vitro retain their phosphorylation state (Goldstein et al., 2000; Haring et al., 1984; Kowalski et al., 1983). In intact or permeabilized cells in situ, however, dissociation of insulin from the insulin receptor is followed by rapid dephosphorylation of the receptor ␤-subunit and IRS proteins (Bernier et al., 1994; Haring et al., 1984; Mooney et al., 1997; Mooney and Anderson, 1989). By these concerted mechanisms, the pool size of fully activated insulin receptors appears to be tightly regulated in cells (White and Kahn, 1989). Studies with PTPase inhibitors have also provided additional support for the concept that PTPases are integrally involved in the regulation of insulin signaling. Insulin signaling can be enhanced by potent PTPase inhibitors such as vanadate and related compounds (Band et al., 1997; Fantus and Tsiani, 1998; Sekar et al., 1996). Furthermore, recent human clinical trials have also shown that vanadate or related compounds may potentially be effective as antidiabetic agents (see, e.g. Goldﬁne et al. (1995) and Cohen et al. (1995)). Chromium supplementation, which can enhance insulin action in some patients with type 2 diabetes, may act as a PTPase inhibitor (Cefalu et al., 1999).

THE PTPASE SUPERFAMILY OF ENZYMES The PTPases comprise an extensive family of proteins that exert both positive and negative inﬂuences on several pathways of cellular signal transduction and metabolism (Goldstein, 1998; Zhang, 1998). PTPases have in common a conserved ~230 amino acid domain that contains the PTPases signature sequence motif – (I/V)HCXAGXGR(S/T)G – which includes the cysteine residue that catalyzes the hydrolysis of protein-phosphotyrosine residues by the formation of a cysteinyl-phosphate intermediate. PTPases have been divided into two broad categories (Figure 1). The receptor-type enzymes have a general structure like a membrane receptor with an extracellular domain, a single transmembrane segment and one or two tandemly conserved PTPase catalytic domains. The nonreceptor-type enzymes have a single PTPase domain and additional functional protein segments that appear to be involved in their subcellular localization and protein–protein associations.

ROLE OF PTPASES IN INSULIN ACTION AND RESISTANCE Non-receptor PTPases PTP1B

37

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SS

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Figure 1 Schematic structure of PTPases implicated in the regulation of insulin signaling. The nonreceptor-type enzymes have a single PTPase domain and additional functional protein segments that affect subcellular localization and protein–protein associations. The receptor-type enzymes have a transmembrane segment like a membrane receptor and one or two tandemly conserved PTPase catalytic domains. The extracellular domain of LAR is composed of multiple cell adhesion molecule and immunoglobulin structural motifs. LRP has a short extracellular domain. See Goldstein (2000) for details.

Identiﬁcation of PTPases Relevant to the Insulin Action Pathway In order to implicate speciﬁc candidate PTPases for the insulin action pathway, recent studies have characterized the tissue expression of various PTPases, their subcellular localization, catalytic speciﬁcity for the insulin receptor, and effects of modulating PTPase abundance on insulin signaling in intact cells. Tissue distribution Since the restricted tissue distribution of some PTPases can be an important factor in determining their specialized cellular roles, identiﬁcation of PTPases expressed in insulin-sensitive tissues, including liver, skeletal muscle and adipose tissue, is crucial towards identifying candidates for the physiological regulation of insulin signaling. Liver contains a variety of PTPases including PTP1B, SHP-2, LAR and LRP (reviewed in Goldstein, 1998). Identiﬁcation of PTPases expressed in skeletal muscle has been of particular importance, since the abnormal glucose disposal associated with type 2 diabetes mellitus is largely dependent on defects on insulin action in this tissue (DeFronzo, 1998). RT-PCR and RNA blotting has shown that among others, LAR, LRP, PTP1B and SHP-2 are expressed in muscle (Hashimoto et al., 1992; Zhang and Goldstein, 1991; Freeman et al., 1992). Puriﬁcation of the major peaks of PTPase activity from rat skeletal muscle particulate and cytosol fractions by serial chromatographic techniques followed by immunoblotting revealed LAR in the particulate fraction and PTP1B and SHP-2 which were distributed between the cytosol and particulate fractions (Ahmad and Goldstein, 1995c). By immunodepletion techniques, these three enzymes were found to account for a signiﬁcant proportion of the total PTPase activity in the muscle homogenates (Ahmad and Goldstein, 1995c). In adipocytes, cDNA library screening revealed inserts for LRP, PTP1B, SHP-2 and LAR in decreasing order of abundance (Ding et al., 1994). Using RT/PCR techniques, Norris et al. (1997) identiﬁed twenty-four different PTPases in human liver, adipose tissue, skeletal muscle and endothelial cells. The most abundant in all four tissues were the receptor-type PTPases RPTP-␴ and LRP (RPTP-␣). Notably, the mRNA levels for LAR were low in skeletal muscle by

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both PCR and Northern blot analysis, although protein levels for this PTPase have been shown in other published studies to be relatively abundant by immunoblot analysis in rat skeletal muscle and in human muscle and adipose tissue (Ahmad et al., 1995, 1997; Ahmad and Goldstein, 1995c). Subcellular localization Since the highest speciﬁc PTPase activity towards the insulin receptor from a variety of tissue sources is present in the particulate fraction, a transmembrane or membrane-associated PTPase has been implicated in the regulation of insulin action (Ahmad and Goldstein, 1995c; Goldstein et al., 1991). In addition, the ﬁnding that the insulin receptor is rapidly dephosphorylated in situ in permeabilized cells supports the notion that subcellular localization is important for speciﬁc PTPases to have access to the insulin receptor and the IRS proteins (Mooney et al., 1997; Zhang et al., 1996). Among the candidate PTPases identiﬁed in insulin sensitive tissues, LAR and LRP are transmembrane proteins. PTP1B is expressed on the cytoplasmic face of the endoplasmic reticulum, anchored by its hydrophobic C-terminal segment and it may be membraneassociated by binding to a membrane bound adapter or subunit protein (Frangioni et al., 1992). PTP1B is also distributed to the cytosol of insulin-sensitive cell types (Ahmad et al., 1997; Goldstein, 1996; Ide et al., 1994). The other non-receptor type PTPase candidate, SHP-2, also distributed between the cytosol and particulate fractions, perhaps associated as SH2 domain/protein-phosphotyrosyl complexes with membrane-bound proteins or receptors (Ahmad and Goldstein, 1995a–c). LAR and PTP1B are also found in liver endosomes, where they may have a role in the dynamic dephosphorylation of the insulin receptor that occurs in this subcellular fraction (Ahmad and Goldstein, 1997). Substrate speciﬁcity PTPases will generally dephosphorylate a variety of substrates in vitro, albeit with different kinetic parameters. Numerous candidate PTPases have been shown to be active against the autophosphorylated insulin receptor, including each of the candidate enzymes indicated above (Goldstein, 1996; Hashimoto et al., 1992; Walchli et al., 2000). Some enzymes were shown to have regional speciﬁcity for certain phosphorylated domains of the receptor (Ramachandran et al., 1992). LAR, for example, was found to deactivate the receptor kinase 2–3 times more rapidly than either PTP1B or LRP, due to its activity in vitro to dephosphorylate the trisphosphorylated insulin receptor kinase domain 3–4 times more rapidly than either PTP1B or LRP (Hashimoto et al., 1992). Overall, however, most PTPases exhibit a relative lack of selectivity for tyrosine-phosphorylated protein and peptide substrates in vitro and important effects related to subcellular compartmentalization and access to relevant substrates in the in vivo environment. Manipulation of PTPase activity or abundance in intact cells and in the animal studies summarized below has provided the keenest insight into the potential physiological roles of these speciﬁc candidate PTPases.

LAR (LEUKOCYTE ANTIGEN-RELATED) Kulas and colleagues initially showed that LAR has a role in the physiological regulation of insulin receptor phosphorylation in intact cells, since overexpression of LAR antisense

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mRNA in hepatoma cells reduced LAR mass by 63% and signiﬁcantly ampliﬁed insulinstimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity (Kulas et al., 1995, 1996). Decreased LAR expression also resulted in an augmentation of additional post-receptor events including IRS-1 tyrosine phosphorylation, IRS-1 binding to the p85 subunit of PI 3-kinase, IRS-1 associated PI 3-kinase activity and the activation of both MAP kinase kinase (MEK) and MAP kinase. Since the reduced LAR mass led to a 3-fold increase in insulin-stimulated IRS-1 phosphorylation, and an increase in insulin receptor autophosphorylation by 150%, it appeared that LAR was acting directly on the insulin receptor in situ. Studies have also shown that LAR must be expressed eutopically, at the plasma membrane, to have its effects on insulin receptor signaling (Li et al., 1996; Zhang et al., 1996). A physical association between LAR and the insulin receptor was recently demonstrated by co-immunoprecipitation studies, where up to 8.6% of the LAR protein in the lysates of CHO-hIR cells transfected with LAR was found to be associated with the insulin receptor. In addition, insulin receptors and LAR may be co-localized in the same subcellular compartments since insulin stimulation of liver cells leads to internalization of LAR as well as the insulin receptor into the same endosomal fraction (Ahmad and Goldstein, 1997). Studies with transgenic mice lacking LAR expression have provided compelling data to support a role for this PTPase in the regulation of glucose metabolism and insulin action in intact animals. A line of transgenic mice was developed by random insertional mutagenesis with a gene trap vector targeted to transmembrane proteins (Skarnes et al., 1995). The homozygous knock-out mice have structurally abnormal mammary gland development and are signiﬁcantly smaller with less adipose tissue than control mice but otherwise appear to develop normally. The adult homozygous knock-out mice had signiﬁcantly lower fasting levels of insulin, glucose and triglycerides than controls, suggesting a heightened level of insulin sensitivity as might be expected from the loss of a PTPase that exerts a negative inﬂuence on the balance of insulin signaling (Ren et al., 1998). In glucose clamp studies, major abnormalities in insulin action were detected. The knock-out mice had 33% lower basal rates of hepatic glucose production and whole-body glucose disposal. At the high insulin infusion rate (20 mU/kg/min) hepatic glucose production was completely suppressed in control mice while it remained close to basal levels in the knock-out animals. Similarly, at the high insulin infusion rate, glucose disposal in control mice was increased by 3.4-fold over basal, while it increased by only 2-fold over basal in the knock-out mice. At a cellular level, insulin-stimulated receptor autophosphorylation and IRS-1 tyrosine phosphorylation was normal in the knock-out mice; however, insulinstimulated PI 3-kinase activity was reduced by 47% in the knock-out mice, indicating a post-receptor defect in insulin signaling. These ﬁndings in the LAR (/) mice suggests that secondary compensatory changes in insulin action have occurred at a post-receptor site in the adult mice, which may account for the observed insulin resistance in the clamp studies. A transgenic mouse strain overexpressing LAR ~6-fold in skeletal muscle driven by the creatine kinase promoter/enhancer was recently presented by Zabolotny et al. (1999). During insulin tolerance testing, these mice showed a signiﬁcantly decreased rate of fall in blood glucose accompanied by a 40–73% decrease in insulin-stimulated PI 3-kinase activity in muscle tissue ( p 0.05). These studies show that overexpression of LAR, selectively, in muscle can result in an insulin-resistant phenotype, providing further evidence for a role of this PTPase in insulin signaling and a potential role in the pathogenesis of insulin-resistant disease states (Ahmad et al., 1995, 1997).

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PTP1B In early studies, microinjection of PTP1B into Xenopus oocytes blocked insulin-stimulated S6 kinase activation and retarded insulin-induced oocyte maturation (Cicirelli et al., 1990; Tonks et al., 1990). Lammers et al. (1993) showed that overexpression of PTP1B almost completely dephosphorylated insulin proreceptors and ␤-subunits in the basal state, and reduced the phosphotyrosine content of the ligand-activated receptor ␤-subunits to less than 50% of the control level. Using an osmotic shock technique to load rat KRC-7 hepatoma cells with afﬁnity-puriﬁed antibodies that immunoprecipitate and neutralize the enzymatic activity of recombinant rat PTP1B, we provided further evidence that PTP1B served as a negative regulator of insulin signaling in intact cells (Ahmad et al., 1995). PTP1B antibody loading signiﬁcantly increased insulin-stimulated DNA synthesis and PI 3-kinase activity by 38–42% compared to control cells loaded with pre-immune IgG. We also determined that insulin-stimulated receptor kinase activity towards an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells and that insulin-stimulated receptor autophosphorylation and IRS-1 tyrosine phosphorylation were increased 2.0- to 2.2-fold. These data supported a role for PTP1B as a negative regulator with effects that promoted the dephosphorylation and deactivation of the insulin receptor itself. Further evidence, in support of PTP1B acting as a negative regulator of insulin action in cellular models, has been obtained in studies using transfection or transduction of various PTP1B constructs. Overexpression of active PTP1B up to 20-fold in 3T3-L1 adipocytes using adenovirus-mediated gene transduction resulted in a 50–60% decrease in insulin-stimulated receptor autophosphorylation, IRS-1 tyrosine phosphorylation and PI 3-kinase activation (Venable et al., 2000). However, in this study PTP1B overexpression had no effect on insulin-stimulated akt phosphorylation and glucose transport. In contrast, using isolated rat adipocytes, Chen et al. (1997) showed that transfection of active PTP1B reduced the insulinstimulated translocation of the GLUT4 glucose transporter. These divergent results suggest that cell-type speciﬁc differences may exist in responses to PTP1B also at various points in the insulin action pathway. Catalytically-inactive site-directed mutants of PTP1B have been demonstrated to physically associate with the activated insulin receptor in intact cells and to have dominant negative effects on insulin signaling in situ (Kenner et al., 1996; Seely et al., 1996). Using an inactive PTP1B mutant, Bandyopadhyay and colleagues (1997) also demonstrated that PTP1B is phosphorylated on multiple tyrosine residues in insulin-stimulated cells and that phosphorylation of these sites, as well as an active insulin receptor kinase was essential for the co-immunoprecipitation of PTP1B with the insulin receptor. We and other co-workers have also recently obtained evidence that PTP1B has a relatively high speciﬁc activity towards the tyrosine dephosphorylation of IRS-1 (Goldstein et al., 2000; Calera et al., 2000). The catalytic domains of bacterially-expressed candidate PTPases (PTP1B, SHP-2, LAR, LRP) were studied for their relative activity toward recombinant IRS-1 that was phosphorylated on tyrosine by puriﬁed insulin receptors in vitro. The speciﬁc activity of the various PTPases towards IRS-1 varied over three orders of magnitude; PTP1B had the highest speciﬁc activity, measuring 12 to 5000 times greater than the other candidate enzymes. When expressed as a ratio of IRS-1 dephosphorylating activity to that vs. a general phosphatase substrate (p-NPP) or a representative phosphotyrosine peptide, PTP1B exhibited a 2- to 190-fold higher activity than the other enzymes. Overall, these studies provide insight into the potential role of PTP1B in the regulation of insulin receptor activation as well as

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post-receptor pathways by the dephosphorylation of IRS-1, and possibly also other insulin receptor substrates. An important advance in our understanding of the physiological role of PTP1B not only in the regulation of insulin action, but also in the regulation of adiposity was the PTP1B knockout mouse ﬁrst reported by Kennedy and colleagues (Elchebly et al., 1999). The homozygous PTP1B knock-out mice had no obvious disease or tissue phenotype. Studies of insulin and glucose dynamics in the mice revealed a 50% decrease in fasting insulin levels with a 13% decrease in fasting glucose levels, suggesting a heightened degree of insulin sensitivity (Figure 2). Further studies were consistent with PTP1B acting as a negative regulator in insulin signaling, including less hyperglycemia during glucose tolerance testing and an accentuated drop in blood glucose during insulin tolerance testing in the knock-out mice (Figure 3). In further studies, the knock-out mice also exhibited an interesting resistance to weight gain with high-fat feeding and reduced levels of circulating triglycerides on both diets. Studies of insulin signaling components also revealed enhanced insulin receptor autophosphorylation and insulin-stimulated IRS-1 phosphorylation in skeletal muscle and liver. These signaling changes were not evident in fat cells, however, suggesting a potential mechanism for the tissue differences in insulin sensitivity and the apparent resistance to storage of triglycerides in adipose tissue. Additional insight into these physiological alterations was provided by Klaman et al. (2000) who independently developed a line of PTP1B null mice and conﬁrmed the phenotype described above by Elchebly and colleagues. In these mice, insulin-stimulated whole-body glucose disposal was also signiﬁcantly enhanced in a tissue speciﬁc fashion: insulin-stimulated

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Figure 2 Serum glucose and insulin concentrations in control, heterozygous and homozygous PTP1B knock-out mice. Animals were fed ad libitum or fasted overnight. Glucose (panel A) and insulin (panel B) concentrations were determined. The dark bars indicate the fed animals and the light bars indicate the fasted animals. The genotype of the homozygous (/) and heterozygous (/) knock-out mice and the normal controls (/) are as shown. Values are given as means – SEM. Compared to the wild type, *p 0.06 and **p 0.01. (Reproduced with permission from Elchebly et al. (1999).)

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Figure 3 Glucose and insulin tolerance tests in control (diamonds), heterozygous (squares) and homozygous PTP1B knock-out male mice (triangles) at age 10–14 weeks. Panel A, glucose tolerance; panel B, insulin tolerance. Data are presented as means – SEM. Compared to the wild type, *p 0.05 and **p 0.02. (Reproduced with permission from Elchebly et al. (1999).)

glucose uptake was elevated in skeletal muscle, while adipose tissue was unaffected. They also went on to show that leanness and resistance to weight gain during high-fat feeding in the PTPIB-deﬁcient mice was due to increased basal metabolic rate and total energy expenditure (Figure 4). Altogether, these results agree strongly in their identiﬁcation of PTP1B as a major regulator of not only insulin sensitivity, but also energy balance and body fat stores in vivo. The apparent speciﬁcity of PTP1B for the insulin signaling pathway was also demonstrated in the knock-out mouse studies. Previous work in cellular systems showed that PTP1B may have a role in the regulation of EGF receptor signaling (Flint et al., 1997; Liu and Chernoff, 1997; Milarski et al., 1993). However, EGF signaling was unaffected in the PTP1B knock-out mouse strain studied by Elchebly et al. (1999).

SHP-2 This widely expressed PTPase with two SH2 domains and a single catalytic PTPase domain (Freeman et al., 1992) associates with tyrosine-phosphorylated IRS-1 and IRS-2 by its SH2 domains in a process that activates its catalytic domain (Case et al., 1994; Kuhn et al., 1993, 1994; Sugimoto et al., 1994). In recombinant in vitro systems, SH-PTP2 can dephosphorylate the insulin receptor and IRS-1 (Kuhn et al., 1994), although studies in intact cells have failed

ROLE OF PTPASES IN INSULIN ACTION AND RESISTANCE Energy expenditure

Basal metabolic rate B ∗

40

+/+

38

–/–

36 34 ∗

0.54 0.52

kcal h–1 g–1

ml O2 min–1 kg–1

A

43

0.5

+/+ +/–

∗

–/–

0.48 0.46 0.44

32

0.42

30

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Figure 4 Basal metabolic rate and energy expenditure in PTP1B null mice. Data is shown for control (black bars), heterozygous (open bar) and homozygous (cross-hatched bars) mice (* p 0.05). (Reproduced with permission from Klaman et al. (2000).)

to demonstrate a direct interaction between SH-PTP2 and insulin receptors, or any effect of overexpression of catalytically active SH-PTP2 on insulin signaling (Milarski and Saltiel, 1994; Vogel et al., 1993; Yamauchi et al., 1995). Studies have also demonstrated a positive role for SH-PTP2 in insulin-induced mitogenesis (Milarski and Saltiel, 1994; Xiao et al., 1994). Thus, SH-PTP2 appears to play a positive role in downstream post-receptor signaling by insulin, but not involving the insulin receptor itself. An SHP-2 knock-out mouse was generated by Rabin and colleagues speciﬁcally to assess the role of SHP-2 in insulin action (Arrandale et al., 1996). The global importance of this PTPase in cellular function was apparent since the homozygous knock-out fetuses expired prior to day 10.5 of embryonic development. The studies of insulin signaling and glucose metabolism used hemizygous mice expressing half of the levels of SHP-2 protein found in the wild-type littermates. The hemizygous animals did not display any gross morphological abnormalities. Following careful and detailed evaluation, a variety of parameters and insulin responses were found to be unchanged in the hemizygous knock-out mice relative to control, homozygous mice expressing normal levels of SHP-2 protein. These included overall body weight, plasma insulin and glucose levels in fasting and glucose-challenged states, insulin-stimulated glucose uptake in skeletal muscle and isolated adipocytes, insulin inhibition of adipocyte lipolysis, insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1, and binding of the p85 adapter subunit of phosphatidylinositol 3-kinase to IRS-1. As summarized above, these data are consistent with the majority of studies using transfected cell systems, and help to conﬁrm that SHP-2 does not have a major role in the regulation of the metabolic actions of insulin. Using another approach, Maegawa et al. (1999) generated transgenic mice expressing a dominant negative mutant lacking the PTPase catalytic domain of SHP-2 in skeletal muscle, liver, and adipose tissue. By producing a protein segment containing the SH2 domains of SHP-2, this transgene blocks the insulin-induced association of IRS-1 with endogenous SHP-2 and prevents the activation and localization of the SHP-2 PTPase activity. In the transgenic mice, an impairment of glucose tolerance on glucose loading was evident, as well as insulin resistance with a 3-fold increase in fasting plasma insulin levels and impaired glucose disposal. Analysis of signal transduction pathways showed that tyrosine phosphorylation of IRS-l and

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stimulation of phosphatidylinositol 3-kinase and akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that blocking endogenous SHP-2 impaired insulin action, providing support for the physiological role of SHP-2 as an enhancer of insulin signaling in its target tissues that also involves glucose metabolism.

LRP (RPTP- ) LRP is a widely-expressed receptor-type PTPase that can dephosphorylate the insulin receptor in vitro (Hashimoto et al., 1992) and has been shown to act as a negative regulator of insulin receptor activation and insulin-stimulated translocation of GLUT4 in transfected cells (Lammers et al., 1997) (Cong et al., 1999; Jacob et al., 1998; Moller et al., 1995). Since overexpression of PTPases can often affect signaling pathways non-speciﬁcally, Arnott et al. (1999) used an oligonucleotide-based antisense strategy to speciﬁcally deplete endogenous LRP from 3T3-L1 adipocytes by 85–100%. Although LRP depletion inhibited c-Src activity by 80%, tyrosine dephosphorylation of the insulin receptor or IRS proteins was not altered (Arnott et al., 1999). These results are consistent with several studies by other authors showing a coupling of LRP to c-Src activation (den Hertog et al., 1993, 1994; Zheng et al., 1992) and suggested that endogenous LRP is a key regulator of c-Src activity in 3T3-L1 adipocytes and other cell types but not regulation of the insulin receptor or IRS phosphorylation.

CONCLUSIONS From the variety of PTPases found in insulin-sensitive tissues, the available data have thus far provided considerable support for a physiological role for PTP1B, and perhaps to a lesser degree LAR, in the negative regulation of the insulin action pathway. While signiﬁcant progress has been made in this area, much remains to be learned. PTPases have a complex cellular itinerary, with processing of pro-proteins, internalization events and protein interactions that are likely to have a signiﬁcant effect on their accessibility and interactions with substrates (Ahmad and Goldstein, 1997; Calera et al., 2000; Debant et al., 1996; Serra-Pages et al., 1994, 1998). Also, since both PTP1B and LAR have been shown to act at a site proximate to the insulin receptor itself and its immediate post-receptor substrates, it remains to be shown how they may work to regulate the initiation of the insulin signaling pathway in a coordinated fashion. Perhaps the greatest progress in this area has been the development of PTPases as key targets for novel therapeutics to treat insulin-resistant disease states and type 2 diabetes. The ﬁnding that insulin signaling can be enhanced by the speciﬁc inhibition of LAR or PTP1B in cellular systems and in the PTP1B knock-out mouse models, showed the potential clinical relevance to the management of these disorders with drugs directed at speciﬁc PTPases (Kennedy and Ramachandran, 2000; Klann et al., 2000). Recently, the possible success of this approach has been demonstrated with a series of novel, relatively speciﬁc inhibitors of PTP1B and other PTPases (Iversen et al., 2000; Malamas et al., 2000; Sarmiento et al., 2000). Work with some of these inhibitors has been successful in “proof of concept” studies that demonstrated they can effectively reduce fasting glucose and insulin levels in insulin-resistant ob/ob mice (Wrobel et al., 1999). At a cellular level, these inhibitors will provide novel tools for further characterization of the role of speciﬁc PTPases in signal transduction. Progress of these studies into

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human subjects to test the safety and efﬁcacy of these agents in diabetes and related disease states is anticipated over the next several years.

ACKNOWLEDGMENTS The work in the author’s laboratory is supported by NIH grants R01-43396 and R01 53388.

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Sarmiento, M., Wu, L., Keng, Y.F., Song, L., Luo, Z.W., Huang, Z.W., Wu, G.Z., Yuan, A.K. and Zhang, Z.Y. (2000) Structure-based discovery of small molecule inhibitors targeted to protein tyrosine phosphatase 1B. Journal of Medicinal Chemistry, 43(2), 146–155. Seely, B.L., Staubs, P.A., Reichart, D.R., Berhanu, P., Milarski, K.L., Saltiel, A.R., Kusari, J. and Olefsky, J.M. (1996) Protein tyrosine phosphatase 1B interacts with the activated insulin receptor. Diabetes, 45(10), 1379–1385. Sekar, N., Li, J.P. and Shechter, Y. (1996) Vanadium salts as insulin substitutes – mechanisms of action, a scientiﬁc and therapeutic tool in diabetes mellitus research. Critical Reviews in Biochemistry and Molecular Biology, 31(5–6), 339–359. Serra-Pages, C., Saito, H. and Streuli, M. (1994) Mutational analysis of proprotein processing, subunit association, and shedding of the LAR transmembrane protein tyrosine phosphatase. Journal of Biological Chemistry, 269, 23632–23641. Serra-pages, C., Medley, Q.G., Tang, M., Hart, A. and Streuli, M. (1998) Liprins, a family of lar transmembrane protein-tyrosine phosphatase-interacting proteins. Journal of Biological Chemistry, 273(25), 15611–15620. Skarnes, W.C., Moss, J., Hurtley, S.M. and Beddington, R.S. (1995) Capturing genes encoding membrane and secreted proteins important for mouse development. Proceedings of the National Academy of Sciences of the United States of America, 92(14), 6592–6596. Sugimoto, S., Wandless, T.J., Shoelson, S.E., Neel, B.G. and Walsh, C.T. (1994) Activation of the SH2containing protein tyrosine phosphatase, SH-PTP2, by phosphotyrosine-containing peptides derived from insulin receptor substrate-1. Journal of Biological Chemistry, 269, 13614–13622. Tonks, N.K., Cicirelli, M.F., Diltz, C.D., Krebs, E.G. and Fischer, E.H. (1990) Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes. Molecular and Cellular Biology, 10, 458–463. Ugi, S., Maegawa, H., Olefsky, J.M., Shigeta, Y. and Kashiwagi, A. (1994) Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs. FEBS Letters, 340, 216–220. Venable, C.L., Frevert, E.U., Kim, Y.B., Fischer, B.M., Kamatkar, S., Neel, B.G. and Kahn, B.B. (2000) Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/protein kinase B activation. Journal of Biological Chemistry, 275(24), 18318–18326. Virkamaki, A., Ueki, K. and Kahn, C.R. (1999) Protein–protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. Journal of Clinical Investigation, 103(7), 931–943. Vogel, W., Lammers, R., Huang, J.T. and Ullrich, A. (1993) Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. Science, 259, 1611–1614. Walchli, S., Curchod, M.L., Gobert, R.P., Arkinstall, S. and van Huijsduijnen, R.H. (2000) Identiﬁcation of tyrosine phosphatases that dephosphorylate the insulin receptor – A brute force approach based on substrate-trapping mutants. Journal of Biological Chemistry, 275(13), 9792–9796. White, M.F. and Kahn, C.R. (1989) The casacde of autophosphorylation in the ␤-subunit of the insulin receptor. Journal of Cellular Biochemistry, 39, 429–441. Wrobel, J., Sredy, J., Moxham, C., Dietrich, A., Li, Z.N., Sawicki, D.R., Seestaller, L., Wu, L., Katz, A., Sullivan, D., Tio, C. and Zhang, Z.Y. (1999) PTP1B inhibition and antihyperglycemic activity in the ob/ob mouse model of novel 11-arylbenzo[b]naphtho[2,3-d]furans and 11-arylbenzo[b]naphtho[2,3-d]thiophenes. Journal of Medicinal Chemistry, 42(17), 3199–3202. Xiao, S., Rose, D.W., Sasaoka, T., Maegawa, H., Burke, T.R., Roller, P.P., Shoelson, S.E. and Olefsky, J.M. (1994) Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction. Journal of Biological Chemistry, 269, 21244–21248. Yamauchi, K., Milarski, K.L., Saltiel, A.R. and Pessin, J.E. (1995) Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling. Proceedings of the National Academy of Sciences of the United States of America, 92, 664–668. Zabolotny, J.M., Kim, Y.B., Peroni, O.D., Pani, M.A., Neel, B.G. and Kahn, B.B. (1999) Subtle insulin resistance in transgenic mice overexpressing protein tyrosine phosphatase (PTP) LAR in muscle. Diabetes, 48(Suppl 1), A336.

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4.

HYPERTENSION AND INSULIN RESISTANCE IN THE WISTAR FATTY RAT

MASAMI SUZUKI, HIROYUKI ODAKA AND YASUO SUGIYAMA Pharmacology Research Laboratories II, Takeda Chemical Industries, Ltd., 2-17-85 Juso honmachi, Yodogawa-ku, Osaka 532-8686, Japan

INTRODUCTION The most common features of insulin resistance syndrome are obesity, insulin resistance/ hyperinsulinemia, dyslipidemia, impaired glucose intolerance and/or type 2 diabetes mellitus, and hypertension (Reaven, 1995; Hansen, 1999). Among these features, resistance to the metabolic effects of insulin and the compensatory hyperinsulinemia has been postulated to mediate human essential hypertension, especially when associated with obesity. Essential hypertensive subjects show reduced insulin sensitivity only through the non-oxidative pathway for glucose disposal, whereas obese and type 2 diabetes patients also have reduced glucose uptake through the oxidative pathway and reduced effects of insulin to suppress hepatic glucose output (Ferrannini et al., 1987; DeFronzo and Ferrannini, 1991). A causal link between hypertension and insulin resistance has not been clearly established, but several lines of evidence suggest that insulin resistance and the resultant hyperinsulinemia are causally related to hypertension in animals (Mark and Anderson, 1995; Sechi, 1999) and humans (Reaven, 1991; Landsberg, 1996).

HISTORICAL BACKGROUND The Wistar fatty rat was the ﬁrst rat model of obese type 2 diabetes established on the basis of the hypothesis that both an environmental factor and a genetic background of diabetes are needed to develop diabetes (Ikeda et al., 1981). Among the environmental factors, obesity is assumed to be the most powerful risk factor related to metabolic and hormonal abnormalities. In respect of metabolism, the diabetogenic effect of obesity is of special interest, because obesity increases the insulin requirement and decreases the insulin sensitivity in muscles, adipose tissue, and the liver. In fact, insulin resistance, frequently associated with obesity in humans and animals, characterizes type 2 diabetes. Zucker fatty rats develop obesity with hyperinsulinemia, hyperlipidemia, and glucose intolerance (Zucker and Antoniades, 1972), however, their blood glucose level is close to normal throughout their life, which suggests that additional factors are required to provoke diabetes in obesity. Therefore, the crossbreeding of the fa-gene carrier of the Zucker rat with the Wistar Kyoto rat was started because the Wistar Kyoto rat is less sensitive to insulin and has less tolerance to glucose than the Zucker rat. At the ﬁfth generation of backcrossing, male obese hybrids, Wistar fatty rats were found to be hyperglycemic in addition to hyperlipidemic and hyperinsulinemic, in association with severe obesity. These characteristics have been maintained through the later generations of backcrossing. As the symptom of diabetes in the fatty rat is similar to that in type 2 diabetes patients, Wistar 51

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fatty rat is used to clarify the mechanism of cause and development of diabetes (Hayakawa et al., 1996; Murase et al., 1998), diabetic complications (Suzuki et al., 1997) and to evaluate antidiabetic agents (Sugiyama et al., 1990).

INSULIN RESISTANCE IN WISTAR FATTY RAT Wistar fatty rats have abnormalities of glucose and lipid metabolism in the obese and insulin resistant conditions. Wistar fatty rats showed glucose intolerance, hypersecretion of insulin in response to oral glucose load and impaired response to exogenous insulin (Sugiyama et al., 1990). Insulin-stimulated glycogen synthesis and glycolysis in isolated soleus muscles, and insulin-stimulated glucose oxidation and lipogenesis in adipocytes were impaired in Wistar fatty rats as compared with those in lean littermates. Moreover, Wistar fatty rats decreased peripheral glucose utilization and increased hepatic glucose production compared to their littermates, indicating the presence of peripheral and hepatic insulin resistance in Wistar fatty rats. Impaired glucokinase mRNA expression was reported (Noguchi et al., 1993), but glucose transporter-2 gene expression was increased in Wistar fatty rats (Yamamoto et al., 1991). From these studies it was shown that insulin resistance existed not only in peripheral tissues but also in the liver of Wistar fatty rats. Recent studies imply a correlation between tumor necrosis factor-␣ (TNF-␣) and insulin resistance. TNF-␣, a cytokine produced predominantly in adipocytes, likely contributes to insulin resistance via down-regulation of the glucose transporter GLUT-4 mRNA, reduction in the insulin receptor tyrosine kinase autophosphorylation, and activation of a phosphotyrosine phosphatase that decreases insulin receptor substrate-1 (IRS-1) phosphorylation. Murase et al. (1998) previously reported that the mechanism of the insulin sensitizing activity of pioglitazone indicated that the PPAR␥ agonist activity was the principle cause of reduced muscle TNF-␣ production, where insulin resistance improved in Wistar fatty rats.

INSULIN RESISTANCE/HYPERINSULINEMIA-INDUCED FACTORS ASSOCIATED WITH HYPERTENSION IN WISTAR FATTY RAT The Wistar fatty rat develops hyperglycemia, hyperlipidemia, and hyperinsulinemia as features of insulin resistance followed by slight hypertension (Yoshimoto et al., 1997). In our study, administration of an insulin sensitizer, pioglitazone, ameliorated hyperglycemia, hyperlipidemia, and hyperinsulinemia, decreased systolic blood pressure to the level of normal rats and increased insulin sensitivity as assessed by glycemia response to exogenous insulin (Table 1, Figure 1). Hence, systolic blood pressure has a good relation to plasma insulin level and plasma glucose in insulin tolerance test (Figures 2 and 3). These results indicate that insulin resistance is one of the pivotal factors for hypertension in the fatty rats. The accumulated evidence on the association between insulin resistance/hyperinsulinemia and hypertension have suggested two hypotheses. First, insulin resistance leads to increased blood pressure via impaired vascular tone due to a decrease in insulin’s action. Second, hyperinsulinemia leads to increased blood pressure due to activation of the sympathetic nervous system and renin angiotensin system (with disruption of the sodium balance) and also due to change in the vascular structure, such as enhanced growth of vascular smooth muscle cells.

HYPERTENSION AND INSULIN RESISTANCE Table 1

53

Effect of pioglitazone on plasma components and blood pressure in Wistar fatty rats. Wistar fatty

Body weight (g) Plasma Glucose (mg/dl) Triglyceride (mg/dl) Insulin ( U/ml) Blood pressure Systolic (mmHg) Diastolic (mmHg) Heart rate (min1)

Wistar fatty pioglitazone

Wistar lean

737 44**

965 39##

528 35

364 28** 268 64** 1883 571**

124 14## 115 22## 437 150##

115 3 60 7 200 54

132 6** 95 14 270 20**

113 9## 87 9 298 23##

118 3 92 5 297 6

Thirty-week-old male Wistar fatty rats were administered with pioglitazone (50 ppm) as dietary mixture for 9 weeks. Mean SD (n 4 for lean group or n 10 for each fatty group). ## p 0.01 vs. fatty rat control, **p 0.01 vs. lean rat by Student’s t-test.

Insulin (0.2 U/kg)

Insulin (0.5 U/kg)

Insulin (1.0 U/kg)

Plasma glucose (mg/dl)

120 100 Wistar fatty

80 #

60 **

**

40

## **

**

0

60

##

Wistar lean

**

20 0

Wistar fatty + pioglitazone

##

120 180 Time (min)

**

240

Figure 1 Effect of pioglitazone on insulin tolerance in Wistar fatty rats. Wistar fatty and lean rats were given intraperitoneal injections of insulin at a dose of 0.2, 0.5, 1.0 U/kg. Mean SD (n 4 for lean group or n 10 for each fatty group). #p 0.05, ##p 0.01 vs. fatty rat control, **p 0.01 vs. lean rat by Student’s t-test.

Sympathetic Nervous System, Renin Angiotensin System When pressure natriuresis is reset, blood pressure increases to maintain sodium balance. However, the renal-pressure natriuresis mechanism is abnormal in the hypertensive state (Takenaka et al., 1991). An excess of dietary sodium intake increased plasma insulin and blood pressure, and the pressure-natriuresis response was shifted to the right and its slope was ﬂattened for Wistar fatty rats compared to normal rats (Suzuki et al., 1996). It suggests that salt sensitivity may change the abnormal pressure-natriuresis responses and contribute to an elevation of blood pressure in Wistar fatty rats.

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MASAMI SUZUKI ET AL.

Plasma insulin (µU/ml)

3500 3000 2500 2000 r = 0.756 p < 0.01

1500 1000 500 0 100 120 140 160 Systolic blood pressure (mmHg)

Hypoglycemic activity by exogeneous insulin (% of initial)

Figure 2 Relation between systolic blood pressure and plasma insulin level in Wistar fatty rats.

90 80 70 r = 0.706 p < 0.01

60 50 40 100

120

140

160

Systolic blood pressure (mmHg)

Figure 3 Relation between systolic blood pressure and hypoglycemic activity in insulin tolerance test in Wistar fatty rats. Plasma glucose level at 270 min after insulin injection in insulin tolerance test is used as the hypoglycemic activity in insulin tolerance test.

Wistar fatty rats are associated with mild hypertension, aortic medial wall thickening, and increased urinary norepinephrine excretion (Yoshimoto et al., 1997), or enhanced sympathetic nerve activity due to increase in sodium retention (Yamakawa et al., 1995). Suzuki et al. (1999) also reported that baroreﬂex is not impaired, in spite of elevation of blood pressure, and suggested that the raised sympathetic nerve activity may contribute to the development of hypertension in Wistar fatty rats. Administration of antihypertensive agents such as angiotensin II antagonist and candesartan cilexetil (TCV-116), suppressed the development of hypertension as well as glomerular ﬁltration rate change in Wistar fatty rats (Shibouta et al., 1996). The ﬁnding suggests that angiotensin II may be associated with the development of hypertension and progressive glomerulosclerosis in Wistar fatty rats. In fact, angiotensinogen is the unique substrate of renin in vivo the renin angiotensin system, and plasma angiotensinogen concentration as well as aortic angiotensinogen mRNA increased in Wistar fatty rats (Tamura et al., 1997).

HYPERTENSION AND INSULIN RESISTANCE

55

Vascular Structure The atherogenic effects of insulin through mitogenic action on vascular cells (Sowers et al., 1993) may be important in the pathogenesis of hypertension. Insulin stimulates the proliferation of vascular smooth muscle cells and increases DNA synthesis in the aorta. Balloon catheterization-induced carotid arterial intimal thickening and growth of vascular smooth muscle cells increased in Wistar fatty rats compared to normal rats (Igarashi et al., 1997), suggesting that the Wistar fatty rat has a disposition to deteriorate atherogenesis. The early atherosclerotic changes in vascular structure may be crucial to the development of hypertension via reduction in vessel compliance and vasodilative mechanisms in Wistar fatty rats. Vascular Tone Insulin-induced vasodilation is dependent on nitric oxide (NO) release (Steinberg et al., 1994). Resistance to insulin’s action on the arterial wall, with impaired insulin-dependent arterial vasodilation, has been observed in type 2 diabetes (Williams et al., 1996) and in insulin resistant patients with essential hypertension (Higashi et al., 1997). Since insulin treatment increases NO synthetase activity and reduces blood pressure in Zucker diabetic fatty rats, which are animal models for obese type 2 diabetes (Kawaguchi et al., 1999), it is possible that insulin resistance impairs vascular tone via decrease in NO production in Wistar fatty rats. Yoshimoto et al. (1995, 1996) reported that plasma arterial natriuretic peptide (ANP), natriuretic peptide receptors (NP-A and NP-B) mRNA in the aorta were all higher in Wistar fatty rats compared to normal rats. ANP is a cardiac hormone with potent natriuretic and vasorelaxant activity. It has been reported that obese animals and hypertensive subjects have an increased basal ANP concentration, probably due to body ﬂuid volume expansion and cardiac volume enlargement, but an impaired ANP response to an acute saline load in obese Zucker rat (McMurray and Vesely, 1993; Zeigler and Patel, 1991). So it is possible that insulin resistance leads to vasodilative dysfunction via an impaired natriuretic peptide system as well as via nitric oxide in the blood vessels of Wistar fatty rats.

CONCLUSION Several reports indicate that insulin resistance is one of the pivotal factors for hypertension in Wistar fatty rats. Further studies on Wistar fatty rat are expected to elucidate the mechanism of hypertension induced by insulin resistance and suggest an approach to treat hypertension in obese or type 2 diabetes.

REFERENCES DeFronzo, R.A. and Ferrannini, E. (1991) Insulin resistance: A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care, 14, 173–194. Ferrannini, E., Buzzigoli, G., Bonadonna, R., Giorico, M.A., Oleggini, M., Graziadei, L., Pedrinelli, R., Brandi, L. and Bevilacqua, S. (1987) Insulin resistance in essential hypertension. N. Engl. J. Med., 317, 350–357. Hansen, B.C. (1999) The metabolic syndrome X. Ann. N. Y. Acad. Sci., 892, 1–24.

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Hayakawa, T., Shiraki, T., Morimoto, T., Shii, K. and Ikeda, H. (1996) Pioglitazone improves insulin signaling defects in skeletal muscle from Wistar fatty (fa/fa) rats. Biochem. Biophys. Res. Commun., 223, 439–444. Higashi, Y., Oshima, T., Sasaki, N., Ishioka, N., Nakano, Y., Ozono, R., Yoshimura, M., Ishibashi, K., Matsuura, H. and Kajiyama, G. (1997) Relationship between insulin resistance and endotheliumdependent vascular relaxation in patients with essential hypertension. Hypertension, 29, 280–285. Igarashi, M., Takeda, Y., Ishibashi, N., Takahashi, K., Mori, S., Tominaga, M. and Saito, Y. (1997) Pioglitazone reduces smooth muscle cell density of rat carotid arterial intima induced by balloon catheterization. Horm. Metab. Res., 29, 444–449. Ikeda, H., Shino, A., Matsuo, T., Iwatsuka, H. and Suzuoki, Z. (1981) A new genetically obese-hyperglycemic rat (Wistar fatty). Diabetologia, 30, 1045–1050. Kawaguchi, M., Koshimura, K., Murakami, Y., Tsumori, M., Gonda, T. and Kato, Y. (1999) Antihypertensive effect of insulin via nitric oxide production in the Zucker fatty rat, an animal model for non-insulin-dependent diabetes mellitus. Eur. J. Endocrinol., 140, 341–349. Landsberg, L. (1996) Insulin sensitivity in the pathogenesis of hypertension and hypertensive complications. Clin. Exp. Hypertens., 18, 337–346. McMurray, R.W. and Vesely, D.L. (1993) Calorie-restricted weight reduction, blood pressure, and atrial natriuretic peptides. Nutrition, 9, 178–182. Mark, A.L. and Anderson, E.A. (1995) Genetic factors determine the blood pressure response to insulin resistance and hyperinsulinemia: a call to refocus the insulin hypothesis of hypertension. Proc. Soc. Exp. Biol. Med., 208, 330–336. Murase, K., Odaka, H., Suzuki, M., Tayuki, N. and Ikeda, H. (1998) Pioglitazone time-dependently reduces tumour necrosis factor- level in muscle and improves metabolic abnormalities in Wistar fatty rats. Diabetologia, 41, 257–264. Noguchi, T., Matsuda, T., Tomari, Y., Yamada, K., Imai, E., Wang, Z., Ikeda, H. and Tanaka, T. (1993) The regulation of gene expression by insulin is differentially impaired in the liver of the genetically obese-hyperglycemic Wistar fatty rat. FEBS Lett., 328, 145–148. Reaven, G.M. (1991) Insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hypertension. Parallels between human disease and rodent models. Diabetes Care, 14, 195–202. Reaven, G.M. (1995) Pathophysiology of insulin resistance in human disease. Physiol. Rev., 75, 473–486. Sechi, L.A. (1999) Mechanisms of insulin resistance in rat models of hypertension and their relationships with salt sensitivity. J. Hypertension, 17, 1229–1237. Shibouta, Y., Chatani, F., Ishimura, Y., Sanada, T., Ohta, M., Inada, Y. and Nishikawa, K. (1996) TCV116 inhibits renal interstitial and glomerular injury in glomerulosclerotic rats. Kidney International, 49, S115–S118. Sowers, J.R., Standley, P.R., Ram, J.L., Jacober, S., Simpson, L. and Rose, K. (1993) Hyperinsulinemia, insulin resistance, and hyperglycemia: contributing factors in the pathogenesis of hypertension and atherosclerosis. Am. J. Hypertens., 6, 260S–270S. Steinberg, H.O., Brechtel, G., Johnson, A., Fineberg, N. and Baron, A.D. (1994) Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J. Clin. Invest., 94, 1172–1179. Sugiyama, Y., Taketomi, S., Shimura, Y., Ikeda, H. and Fujita, T. (1990) Effects of pioglitazone on glucose and lipid metabolism in Wistar fatty rats. Arzneim-Forsch/Drug Res., 40, 263–267. Sugiyama, Y., Shimura, Y. and Ikeda, H. (1990) Effects of pioglitazone on hepatic and peripheral insulin resistance in Wistar fatty rats. Arzneim-Forsch/Drug Res., 40, 436–440. Suzuki, H., Ikenaga, H., Hayashida, T., Otsuka, K., Kanno, Y., Ohno, Y., Ikeda, H. and Saruta, T. (1996) Sodium balance and hypertension in obese and fatty rats. Kidney International, 49, S150–S153. Suzuki, H., Nishizawa, M., Ichikawa, M., Kumagai, K., Ryuzaki, M., Kumagai, H., Saruta, T. and Ikeda, H. (1999) Basal sympathetic nerve activity is enhanced with augmentation of baroreceptor reﬂex in Wistar fatty rats: a model of obesity-induced NIDDM. J. Hypertension, 17, 959–964.

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Suzuki, M., Yamada, Y., Yamasaki, H., Anayama, H., Sasaki, S., Odaka, H. and Ikeda, H. (1997) Nephropathy in genetically obese-diabetic Wistar fatty rats. Characterization and prevention. Jpn. Pharmacol. Ther., 25, 363–371. Takenaka, T., Suzuki, H., Ishii, N., Itoh, H. and Saruta, T. (1993) Role of NO on pressure-natriuresis in Wistar Kyoto and spontaneously hypertensive rats. Kidney International, 43, 205–211. Tamura, K., Umemura, S., Yamakawa, T., Nyui, N., Hibi, K., Watanabe, Y., Ishigami, T., Yabana, M., Tanaka, S., Sekihara, H., Murakami, K. and Ishii, M. (1997) Modulation of tissue angiotensinogen gene expression in genetically obese hypertensive rats. Am. J. Physiol., 272, R1704–R1711. Williams, S.B., Cusco, J.A., Roddy, M.A., Johnstone, M.T. and Creager, M.A. (1996) Impaired nitric oxide-mediated vasodilation in patients with non-insulin dependent diabetes mellitus. J. Am. Coll. Cardiol., 27, 567–574. Yamakawa, T., Tanaka, S., Tamura, K., Isoda, F., Ukawa, K., Yamakura, Y., Takanashi, Y., Kiuchi, Y., Umemura, S., Ishii, M. and Sekihara, H. (1995) Wistar fatty rat is obese and spontaneously hypertensive. Hypertension, 25, 146–150. Yamamoto, T., Fukumoto, H., Koh, G., Yano, H., Yasuda, K., Masuda, K., Ikeda, H., Imura, H. and Seino, Y. (1991) Liver and muscle-fat type glucose transporter gene expression in obese and diabetic rats. Biochem. Biophys. Res. Commun., 175, 995–1002. Yoshimoto, T., Naruse, M., Naruse, K., Fujimaki, Y., Tanabe, A., Muraki, T., Itakura, M., Hagiwara, H., Hirose, S. and Demura, H. (1995) Modulation of vascular natriuretic peptide receptor gene expression in hypertensive and hyperglycemia rats. Endocrinology, 136, 2427–2434. Yoshimoto, T., Naruse, M., Naruse, K., Arai, K., Imaki, T., Tanabe, A., Seki, T., Hirose, S., Muraki, T. and Demura, H. (1996) Vascular action of circulating and local natriuretic peptide systems is potentiated in obese/hyperglycemic and hypertensive rats. Endocrinology, 137, 5552–5557. Yoshimoto, T., Naruse, M., Nishikawa, M., Naruse, K., Tanabe, A., Seki, T., Imaki, T., Demura, R., Aikawa, E. and Demura, H. (1997) Antihypertensive and vasculo- and renoprotective effects of pioglitazone in genetically obese diabetic rats. Am. J. Physiol., 272, E989–E996. Zeigler, D.W. and Patel, K.P. (1991) Reduced renal responses to an acute saline in obese Zucker rats. Am. J. Physiol., 261, R712–R718. Zucker, L.M. and Antoniades, H.N. (1972) Insulin and obesity in the Zucker genetically obese rat “fatty”. Endocrinology, 90, 1320–1330.

5. CARDIOVASCULAR DISEASE IN THE INSULIN-RESISTANT, ATHEROSCLEROSIS-PRONE JCR:LA-cp RAT J.C. RUSSELL AND S.E. KELLY Department of Surgery, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION Driven by the increasing incidence of obesity in western societies, the insulin resistance syndrome is rapidly becoming a major cause of morbidity and mortality. The primary factor in this is its prominent association with cardiovascular disease. However, the nature of the underlying mechanisms leading to end-stage myocardial and other ischemic disease is still to date unclear. The clariﬁcation of these mechanisms remains a necessary step in the effective prevention and treatment of one of the primary determinants of cardiovascular disease. Atherosclerosis develops in humans at an early age and is present in most individuals if they live long enough (Stary, 1984, 1994). The lesions and vascular dysfunction associated with atherosclerosis are the root cause of most of the cardiovascular disease seen in humans. The most widely accepted theory of the origin of atherosclerosis has been one based on abnormalities of cholesterol metabolism and hypercholesterolemia. This has proven to be a highly successful paradigm that has provided a rational mechanism for atherogenesis, especially in individuals with genetic defects in the LDL receptor and resultant high LDL cholesterol levels (Goldstein and Brown, 1987; Hobbs et al., 1987). However, the cholesterol hypothesis does not account for all cardiovascular diseases, and recent evidence suggests that even the most effective cholesterol-lowering regimens reduce end-stage cardiovascular disease in humans only by 35% (Windler et al., 1998). This makes it clear that the processes leading to atherosclerosis, vascular dysfunction, and the infarction of organs such as the heart and brain are complex and multifactorial. The critical challenge is to identify those factors and mechanisms that lead to the end-stage disease. The metabolic syndrome, that is characterized by abdominal (android or male pattern) obesity, insulin resistance, and hypertriglyceridemia, is an independent major contributor to cardiovascular disease (Stout, 1985; Uusitupa et al., 1990; Stern, 1995). It is a distinct and different disorder from the hypercholesterolemic states. In common with type 1 diabetes, it is characterized in many cases by a transient hyperglycemia and in all cases by hyperinsulinemia, both basal and episodic. There is good evidence that hyperinsulinemia is directly damaging to the vascular endothelium, both in vivo and in vitro (Stout, 1985; Steiner, 1994; Richardson et al., 1998). In the JCR:LA-cp rat, treatments that improve insulin sensitivity and reduce plasma insulin levels also reduce the incidence of myocardial lesions (Russell et al., 1993, 1995, 1997b, 1998b). These ﬁndings suggest the existence of a major role for hyperinsulinemia in Address correspondence to: J.C. Russell, Department of Surgery, 275 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-6359; Fax: 780-407-7394; Email: Jim.Russell@ ualberta.ca 59

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endothelial damage and atherogenesis, and this concept is supported by recent observations that insulin inhibits the endothelium-dependent relaxation factor response of rat aortic rings (Karasu et al., 1995) and plays a role in human vascular function (Grundy, 1998). Very little work has been reported on the interaction between abnormal lipid metabolism and hyperinsulinemia in the induction of atherosclerosis and cardiovascular disease. VLDL hyperlipidemia, and the resulting hypertriglyceridemia, is well recognized as a component of the metabolic abnormalities of both type 1 and type 2 diabetes and is also recognized as another of the independent risk factors for cardiovascular disease (Uusitupa et al., 1990).

THE RAT AS A MODEL OF DISEASE A large number of rat strains with different characteristics and varying value for particular kinds of research have been developed. Due to its small size and the relatively low costs associated with it, the rat has become one of the most widely used mammalian animal models for research. This animal is also large enough to permit many of the physiological and metabolic studies that are not practical in smaller species such as the mouse. Although the rat is not phylogenetically close to humans, both species share many of the same characteristics, including an omnivorous diet. The glucose–insulin metabolism of the rat is also very similar to that of humans. There are signiﬁcant differences in lipid/lipoprotein status, with serum total cholesterol being much lower in the rat (50 mg/100 ml). The majority of the cholesterol is carried in the HDL fraction, and the VLDL and LDL contents are correspondingly low. Normal rats are highly resistant to dietary cholesterol and only develop hypercholesterolemia under such extreme and non-physiological conditions as a diet containing 10% cholesterol and treatment with thyrotoxic agents. In normal rats, this pattern is unfavorable to atherogenesis and has strengthened the view that this species is not a good model for the study of atherosclerosis. However, the recent development and characterization of genetic rat models that are atherosclerosis prone proves that this is not the case (Russell, 1995). In fact, the resistance of “normal” rats to atherosclerosis provides an essential negative (unaffected) control for experimental work. In addition, Wexler (1964) (Wexler et al., 1976) showed many years ago that intensively bred rats, of both sexes, develop myocardial infarcts. High doses of the synthetic catecholamine, isoproterenol, cause ischemic myocardial lesions similar to those seen in the JCR:LA-cp rat, and repeatedly bred or stressed animals are more sensitive to the drug (PericGolia and Peric-Golia, 1983; Tanaka et al., 1980; Tanaka, 1981). Thus, rats with the appropriate genetic background and treatment can be susceptible to a range of cardiovascular disease symptoms that are analogous to those seen in humans.

THE JCR:LA-cp RAT Background The JCR:LA-cp strain of rats is one of a number of strains incorporating the autosomal recessive cp gene ﬁrst isolated by Koletsky (1973, 1975). The gene was bred into two inbred strains at the National Institutes of Health by Hansen, and then two congenic strains, the LA/N-cp and SHR/N-cp, were created through repeated backcrossing (Greenhouse et al., 1988). At an early stage (the ﬁfth backcross), nucleus breeding stock of the LA/N-derived line was donated to the author and became the basis for a closed outbred colony. This colony retains some

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3% genetic contribution from the original Koletsky strain. It is maintained through a formal breeding protocol (Poiley, 1960) so as to retain the gene pool and not inadvertently select out certain (unknown) genes. This is essential, as the development of cardiovascular disease in rats is unique to this colony and the trait is clearly polygenetic. Genetic Defect In common with the other strains incorporating the cp gene, the JCR:LA-cp rats, if homozygous for the cp gene (cp/cp), are obese, insulin resistant, and hypertriglyceridemic (Dolphin et al., 1987; Russell et al., 1987, 1989, 1994, 1999; Vance and Russell, 1990). Rats that are heterozygous (/cp) or homozygous normal (/) are lean and not distinguishable from the parent LA/N strain in any respect. The cp gene has been shown to be a stop codon in the extracellular domain of the leptin receptor (ObR) (Wu-Peng et al., 1997). This leads to a complete absence of the ObR in the plasma membrane of the cp/cp animals. In contrast, the other obese rat gene, the fa or fatty gene, has been shown to result in an amino acid substitution, also in the extracellular domain of the ObR, that leads to a 10-fold reduction in binding afﬁnity for leptin. Thus, the fa/fa rat is leptin resistant as opposed to having no functional receptor. There are major metabolic and pathophysiological differences between cp/cp and fa/fa animals (Amy et al., 1988; Pederson et al., 1991; Chan et al., 1995). Most critically, the JCR:LA-cp strain is unique in the spontaneous development of atherosclerosis and myocardial ischemia. Neither the fa/fa Zucker (Amy et al., 1988) nor the cp/cp rats of the other cp strains develop cardiovascular disease (Russell et al., 1990). In contrast, the strain derived from the SHR/N-cp, now designated as “SHHF/Mcc-facp,” shows a mild perivascular ﬁbrosis as the only manifestation of vascular disease, but has a very high incidence of cardiomyopathy that culminates in congestive heart failure (Haas et al., 1995). The Zucker Diabetic Fatty (ZDF/Gmi-fa) rat exhibits the early transition from an obese, insulin-resistant state to overt type 2 diabetes with marked hyperglycemia (Etgen and Oldham, 2000). These complex differences in disease expression in closely related strains conﬁrm the complex polygenetic character of cardiovascular disease. The ob gene, coding for leptin itself, of the cp/cp rat is structurally normal and the DNA sequence is identical to that of the ob gene in the normal Sprague–Dawley rat (Vydelingum et al., 1995). The mRNA for the ob gene is overexpressed 10-fold in the white fat of the cp/cp rat, and this rat shows massive production of the gene product, leptin. Figure 1 shows the rapid rise in plasma leptin concentration in the cp/cp rat with age. As expected in the absence of the ObR, the leptin signal to the central nervous system is not recognized and the levels of neuropeptide Y (the strongest known mediator of eating) are thus signiﬁcantly elevated in the arcuate nucleus of the hypothalamus (Williams et al., 1990, 1992). Leptin also downregulates insulin secretion from the pancreas in normal animals (Emilsson et al., 1997). This leads to the cp/cp rat exhibiting an extreme hypersecretion of insulin that is much greater than that of the fa/fa rat (Pederson et al., 1991). This is consistent with the leptin-resistant status of the fa/fa rats and the absent-receptor status of the cp/cp rats. Metabolism Animals of the JCR:LA-cp strain that are cp/cp are detectably obese at 3 weeks of age. A modest hyperinsulinemia is present at three weeks and develops rapidly after ﬁve weeks of age (Russell et al., 1998a). The insulin resistance develops, such that, by eight weeks of age the male cp/cp rats no longer show any insulin-mediated glucose uptake or turnover. Increased

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J.C. RUSSELL AND S.E. KELLY 100

Leptin (ng/ml)

80 60 40 20 0

0

10

20 30 Age (weeks)

40

50

Figure 1 Plasma leptin concentrations in the JCR:LA-cp male rat as a function of age.

❍, /? rats; •, cp/cp rats. Values are mean SEM. (The authors thank Ms. Laura Atkinson for permission to reproduce the data.)

muscle triglyceride concentrations are already present in the cp/cp rats at four weeks of age, before the development of the insulin resistance. The muscle triglyceride is present in an intracellular form, in association with the mitochondria. Triglycerides are known to reduce insulin sensitivity, and this intracellular triglyceride may be the origin of the insulin resistance. We have also recently shown that the insulin-resistant state is accompanied by a reduction in the transendothelial transport of insulin that signiﬁcantly lowers insulin concentrations in peripheral tissue cells, especially during the postprandial phase (Wascher et al., 2000). Treatment of the rats from the time of weaning (3 weeks of age) with the potent hypotriglyceridemic drug, MEDICA 16, prevents the early accumulation of triglyceride in the muscle tissue and markedly suppresses the development of the hyperinsulinemia (Russell et al., 1998a). Plasma leptin levels have been shown to be reduced by treatments that also reduce plasma insulin levels, including the new insulin-sensitizing drug, S15261 (unpublished observations). While the mutant ObR is the core defect of the cp/cp rat, all evidence to date indicates that the hyperleptinemia is not the direct cause of the vascular dysfunction, but that it leads to, or exacerbates, the insulin-resistant status (Keiffer and Habener, 2000) which, in turn, is the determinant of the vasculopathy. Thus, the leptin defect leads to the metabolic syndrome which, in both animal models and humans, can have a number of ultimate causes. The metabolic syndrome, however induced, appears to give rise to complications of which cardiovascular disease is the most important. The female cp/cp rats show only a mild metabolic dysfunction, having both a much more modest hyperinsulinemia and a lower insulin-mediated glucose turnover and peripheral uptake (Russell et al., 1987, 1994). The reasons for this sexual dimorphism, which extends to vascular function and severity of end-stage disease, are not at all clear. The female cp/cp rats have lower plasma estrogen and higher testosterone levels than do the normal /? animals, consistent with similar observations in obese humans (Ivandic et al., 1998). Possibly, as a consequence of the hormonal imbalance, the cp/cp females are not fertile: while embryos introduced into the uterus appear to implant, they are not viable and leave only a visible implantation site. On the other hand, this failure to retain implanted concepti could be due to the extreme hyperleptinemia or the absence of the ObR signal. The female cp/cp rat may thus provide us with an understanding of as-yet-unrecognized mechanisms of infertility. The various aspects of the expression of dysfunction and disease in the female cp/cp rats are relatively unexplored and there are clearly major issues for future research.

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The profound peripheral insulin resistance in the male cp/cp rats leads to the diversion of glucose, derived from a carbohydrate diet, to triglyceride synthesis and VLDL secretion into the plasma. Thus, the marked VLDL hyperlipidemia is due to an elevated secretion of VLDL and not to reduced clearance (Russell et al., 1989; Vance and Russell, 1990). Lipoprotein lipase activity is 2.5- to 4-fold elevated in the fat of the cp/cp rats and is decreased 50% in the heart and skeletal muscle (Mantha et al., 2002). The modestly increased cholesterol levels are due to the obligatory cholesterol content of the VLDL particles and ﬂowthrough to the HDL fraction (Vance and Russell, 1990). In the cp/cp rat, cholesterol supplementation of the diet at even 0.25% leads to only a slight increase in total cholesterol concentrations in the plasma, but to a major increase in cholesterol ester levels in the VLDL fraction (Table 1 and Figure 2). Lean JCR:LA-cp rats and Wistar and Sprague–Dawley rats all show varying shifts in cholesterol esters from the HDL to the VLDL fractions on cholesterol feeding and an accompanying elevation in VLDL triglyceride. The signiﬁcant change in the male cp/cp rat towards a more Table 1 Effect on serum whole lipid concentrations of male JCR : LA-cp rats fed a diet containing 1% cholesterol from 3 to 12 weeks of age.

cp/cp control cp/cp cholesterol-treated /? control /? cholesterol-treated

Free cholesterol

Cholesteryl esters

Cholesterol

Phospholipids

Triglycerides

0.412 0.023 0.443 0.027 0.159 0.017 0.231 0.013*

1.61 0.05 1.89 0.09 1.15 0.09 1.17 0.06

2.02 0.06 2.33 0.12 1.31 0.08 1.40 0.06

1.86 0.10 1.75 0.13 0.503 0.038 0.715 0.045*

2.50 0.13 2.20 0.19 0.152 0.027 0.308 0.041*

Values are mean SD; 6 rats in each group; *p 0.01 vs. control.

Cholesterol ester levels mmol/l

0.4

0.3

0.2

0.1

0

2

4

6

8

10 12 14 16 Fraction number

18

20

22

24

Figure 2 Distribution of cholesterol esters by density fraction in the plasma of 12-week-old male cp/cp rats. Open bars, control animals; solid bars, rats fed a diet containing 1% cholesterol from 3 weeks of age. Values are mean SEM. The density of the fractions ranges from 1.009 g/ml (fraction #1) to 1.237 g/ml (fraction #25).

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atherogenic lipid/lipoprotein status occurs at a very modest level of cholesterol intake, suggesting that the cp/cp rat, like an element of the human population, is highly sensitive to dietary cholesterol. As discussed below, this sensitivity has very signiﬁcant effects on lesion formation in the arterial wall. Vascular Function Male cp/cp rats exhibit an enhanced noradrenergic contractile response and decreased nitric oxide (NO)-mediated relaxation of the arterial vessels (Figure 3) (McKendrick et al., 1998; O’Brien et al., 1998). The impairment of vascular relaxation in the cp/cp rat has both endothelium-dependent and -independent components (O’Brien et al., 1999), indicating the involvement of an important non-NO-mediated mechanism. A functional defect in NO metabolism has been conﬁrmed by our recent ﬁnding of a speciﬁc defect in NO-mediated relaxation in the coronary arteries of male cp/cp rats that is reversible with exogenous tetrahydrobiopterin (an obligatory co-factor of NO synthesis) (Brunner et al., 2000). This defect in NO metabolism and function may be an important contributor to vasospasm. It is also a marker for endothelial dysfunction, which develops as the hyperinsulinemic state becomes established. The new a-glucosidase inhibitor, miglitol (Bay m1099), essentially completely abolishes the postprandial glycemic and insulin responses and induces normal NO-mediated vascular relaxation, supporting the proposed critical role for hyperinsulinemia in endothelial damage and vascular dysfunction (Russell et al., 1999). Similarly, a reduction in plasma insulin levels through treatment with the insulin-sensitizing agents, D-fenﬂuramine and the novel S15261, was accompanied by improved vascular relaxation and reduced contraction (Russell et al., 1998b, 2000). We have recently conﬁrmed that the vascular dysfunction of the cp/cp rat is associated with increased phosphodiesterase 3A activity in the aorta (Nagaoka et al., 1998). In addition to endothelium-derived hyper-relaxation factor, cGMP-regulated mechanisms appear to be important in vascular relaxation (Kauffman et al., 1987). The role of phosphodiesterases in vascular function and NO metabolism is just beginning to be unravelled, but appears to be important in the cp/cp rat. Increased levels of plasminogen activator inhibitor-1 (PAI-1) are associated with atherosclerosis and thrombotic phenomena (Schneider et al., 1993; McGill et al., 1994). PAI-1 is (a)

(b) 100 Relaxation (%)

Force gram

1.5

1.0

0.5

0.0

75 50 25 0

–10 –9

–8

–7 –6 log [PE]

–5

–4

–9

–8

–7 –6 log [ACH]

–5

Figure 3 Contractile response of aortic rings from 12-week-old JCR:LA-cp male rats in response to phenylephrine (PE) (left panel) and relaxation in response to the NO-releasing agent, acetylcholine (ACh) (right panel). ❍, /? rats; •, cp/cp rats. Values are mean SEM. The contractility of cp/cp rings is greater, and the relaxation less, than in those of /? rats (p 0.01).

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Figure 4 Transmission electron micrograph of the aortic arch of a 9-month-old cp/cp rat, showing an intimal lesion and abnormal vascular smooth muscle cells in both the intima and the media (5000).

elevated in the plasma of patients with the metabolic syndrome and in atherosclerotic human aortas. We have also shown increased plasma concentrations of PAI-1 and mRNA expression in the hearts of male cp/cp rats (Schneider et al., 1998). Moreover, cultured aortic rings from male cp/cp rats secrete signiﬁcantly more PAI-1 than do those from either male / or female cp/cp animals. These ﬁndings are consistent with observations of thrombus accumulation and occlusion in the arterial system of the cp/cp rat (Russell and Amy, 1986a,b; Russell, 1995; Russell et al., 1998c,d; Richardson et al., 1998). Male cp/cp rats develop frank atherosclerotic lesions from an early age, with raised intimal lesions being present in the aortic arch of 100% of the rats by nine months of age (Russell, 1995; Russell et al., 1998c; Richardson et al., 1998). Accompanying the vascular lesions is a cumulative progression of ischemic lesions in the myocardium (Russell and Amy, 1986b). The intimal lesions contain frequent migrating and highly activated vascular smooth muscle cells (Figure 4). In the cp/cp rat, these cells, and many in the medial layers, have a very abnormal “spiky” morphology and appear to become lipid laden, in essence changing into foam cells (Russell et al., 1998d). The smooth muscle cells of the cp/cp rat have also been found to exhibit enhanced migration from aortic explants. Further, they are hyperproliferative in cell culture to at least nine passages (Figure 5) and hyper-respond to a number of cytokines, including insulin-like growth factor-1 (IGF-1) (Figure 6) (Absher et al., 1997). Consistent with these observations, increased activity of matrix metalloproteinases (MMPs), that are required for cellular migration, is seen in the aortic wall in association with vascular disease. The activation

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Cell number × 106

4

3

2

1 +/? cp/cp JCR:LA-cp

+/? cp/cp JCR:LA-cp

FA/FA fa/fa Zucker

Figure 5 Rate of migration of vascular smooth muscle cells from aortic explants from JCR:LA-cp and Zucker rats. The rate of migration of the cells from explants from male cp/cp rats was greater than that seen in explants from male /? rats (p 0.05). (The data are presented with the kind permission of Dr P.M. Absher.)

Relative growth rate (%)

160 140 120 100 80 Insulin

TGF-β

bFGF

IGF-1

Figure 6 Rate of proliferation in culture of vascular smooth muscle cells from the aorta of male JCR:LA-cp rats exposed to selected mitogens in vitro. Data shown are the rate of proliferation in medium containing the mitogen relative to that in control medium (100%); mean SEM. The rate was signiﬁcantly greater in the presence of insulin (p 0.001), TGF-␤ (p 0.005), bFGF (p 0.01) and IGF-1 (p 0.02). (The data are presented with the kind permission of Dr P.M. Absher.)

of MMP-2 has been reported to be promoted by thrombin, and this mechanism could be the origin of the enhanced MMP activity seen in the cp/cp rat with its thrombogenic character. The abnormal physical and growth characteristics of the vascular smooth muscle cells are prevented, or even reversed, by chronic exercise or severe food restriction, both of which strongly reduce plasma insulin levels (Absher et al., 1999), and also by a number of pharmaceutical treatments (Russell et al., 1995, 1997a,b, 1998d). Independent of the treatment of the animals, the rate of growth of aortic smooth muscle cells is a linear function of the plasma insulin concentration of the rat in vivo, as shown in Figure 7. Treatment of the rats with pharmaceutical agents that improve insulin sensitivity and/or reduce insulin levels also prevents the hypercontractility and improves relaxation of the vessels. Treatment with estradiol normalizes aortic contractility, and the selective estrogen receptor modulator, LY117018, reduces contractility

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Doublings (week)

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

50

100 150 200 250 Plasma insulin (mU/l)

300

Figure 7 Rate of proliferation of vascular smooth muscle cells from the aorta of JCR:LA-cp rats in vitro as a function of the plasma insulin concentration of the rat from which they were derived on sacriﬁce. The regression line indicates a signiﬁcant relationship (r 0.820, p 0.001). (The data are presented with the kind permission of Dr P.M. Absher.)

Figure 8 Transmission electron micrograph of the aortic arch of a 6-month-old cp/cp rat fed a diet containing 0.25% cholesterol from the age of 3 weeks. The intimal space contains abnormal vascular smooth muscle cells, lipid-containing foam cells, collagen ﬁbers, and amorphous material. The medial smooth muscle cells also show a highly abnormal morphology (5000).

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Figure 9 Section of the left ventricle of the heart of a 3-month-old cp/cp rat, showing an infarcted area secondary to a stressful episode 3 days previously. Hematoxin-and-eosin staining (5000).

below that of vessels from lean rats without any reduction in plasma insulin levels (Russell et al., 2001). Treatment of the cp/cp rats with cholesterol in the diet, even at the low dose of 0.2% in the feed, results in a signiﬁcant exacerbation of the vascular lesions at 26 weeks of age. Animals fed a 1% cholesterol diet show enhanced intimal lesions, with foam cell formation and apoptosis even at the very early age of 12 weeks, as in Figure 8. After cholesterol feeding, lean /? rats have also exhibited minor vascular damage, beyond the minimal lesions normally seen in these animals. Pilot data have shown increased activity of the matrix metalloproteinase MMP-2 in the aorta of the cp/cp rat subjected to a dietary cholesterol challenge. Interestingly, preliminary results from the study of a modiﬁed marine lipid compound showed a dramatic improvement in vascular function in the cp/cp rat, without alteration of either the plasma triglyceride or insulin levels. These ﬁndings emphasize the complex nature of the vascular pathophysiology and the utility of the animal model in unravelling these important mechanisms. An important series of observations has established that the male cp/cp rat is highly stress sensitive, showing a response that includes a marked increase in unesteriﬁed fatty acid levels (McArthur et al., 1998) and delayed changes in glutamate metabolism in the brain. Most interestingly, stressed animals develop large myocardial infarcts (Figure 9) that result in overt clinical signs of distress and arrhythmias and can rapidly be fatal.

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THE CARDIOVASCULAR DISEASE PROCESS IN THE JCR:LA-cp RAT Cardiovascular disease is best regarded as a symptomatic end-point rather than as a discrete disease with a deﬁnable cause. As such, it has a range of possible antecedent factors or dysfunctions, many of which may well interact to exacerbate the propensity to develop vascular dysfunction, atherosclerosis, and infarcts of distal tissues. From this perspective, cardiovascular disease has several recognized, major contributing factors; including dyslipidemias (both hypercholesterolemic and hypertriglyceridemic), hyperinsulinemic states (associated with both type 1 and type 2 diabetes), and hypertension. These are all strongly inﬂuenced by other factors such as the genetic background, environment (including stress and early developmental events), and diet of an individual. Unravelling the complex interactions between these factors is only possible in simpliﬁed animal model systems, and no single animal model is suitable for the study of all issues. The JCR:LA-cp rat offers a model in which to explore the mechanisms relating to the early, and vascular damaging, stages of the insulin resistance/hyperinsulinemic state and associated atherosclerosis. The strain shows the interactions with dietary factors, especially cholesterol, that are important in the human population and responds to a variety of interventions in ways that are very similar to those seen in humans. In terms of end-stage disease in the JCR:LA-cp rat, the vessel wall abnormalities are all consistent with a common origin in damaged endothelium and smooth muscle cells that must reﬂect altered intracellular metabolism. It seems quite clear that hyperinsulinemia, in itself, plays a signiﬁcant role in creating the vasculopathy; however, it is also clear that the underlying mechanisms must involve other factors, of which other cytokines and the dysfunction of the leptin system are probable elements. Our recent results also indicate a role for abnormal lipoproteins in exacerbating the vascular disease in the presence of these other factors. The vascular dysfunction, taken as a whole, leads to a propensity to vasospasm, clot formation, and impaired integrity of the vessel wall. The ultimate disease process of interest – the ischemic damage to the heart, brain, and other organs – follows. Effective prevention of the infarcts will come from understanding the metabolic dysfunctions and interactions underlying the vascular disease. Based on the whole-animal models currently available and rapidly evolving molecular techniques, identifying the mechanisms involved is no longer an unrealistic goal.

REFERENCES Absher, P.M., Schneider, D.J., Baldor, L.C., Russell, J.C. and Sobel, B.E. (1999) The retardation of vasculopathy induced by attenuation of insulin resistance in the corpulent JCR:LA-cp rat is reﬂected by decreased vascular smooth muscle cell proliferation in vivo. Atherosclerosis, 143, 245–251. Absher, P.M., Schneider, D.J., Russell, J.C. and Sobel, B.E. (1997) Increased proliferation of explanted vascular smooth muscle cells: a marker presaging atherogenesis. Atherosclerosis, 131, 187–194. Amy, R.M., Dolphin, P.J, Pederson, R.A. and Russell, J.C. (1988) Atherogenesis in two strains of obese rats. The fatty Zucker and LA/N-corpulent. Atherosclerosis, 69, 199–209. Brunner, F., Wölkert, G., Russell, J.C. and Wascher, T. (2000) Vascular dysfunction and myocardial contractility in the JCR:LA-corpulent rat. Cardiovascular Research, 36, 150–158. Chan, C.B., McPhail, R.M., Kibenge, M.T. and Russell, J.C. (1995) Increased glucose phosphorylation activity correlates with insulin secretory capacity of male JCR:LA-corpulent rat islets. Canadian Journal of Physiology and Pharmacology, 73, 501–508. Dolphin, P.J., Stewart, B., Amy, R.M. and Russell, J.C. (1987) Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat. Biochimica et Biophysica Acta, 919, 140–148.

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Emilsson, V., Lim, Y.L., Cawthorne, M.A., Morton, N.M. and Davenport, M. (1997) Expression of the functional leptin receptor in RNA in pancreatic islets and directly inhibitory action of leptin on insulin secretion. Diabetes, 46, 313–316. Etgen, G.J. and Oldham, B.A. (2000) Proﬁling of Zucker diabetic fatty rats in their progression to the overt diabetic state. Metabolism, 49, 684–688. Goldstein, J.L. and Brown, M.S. (1987) Regulation of low-density lipoprotein receptors: implications for pathogenesis and therapy of hypercholesterolemia and atherosclerosis. Circulation, 76, 504–507. Greenhouse, D.D, Michaelis, O.E. IV and Peterson, R.G. (1988) The development of fatty and corpulent rat strains. In New Models of Genetically Obese Rats for Studies in Diabetes, Heart Disease and Complications of Obesity (Hansen C.T. and Michaelis O.E., eds) Bethesda: National Institutes of Health, pp. 3–6. Grundy, S.M. (1998) Hypertriglyceridemia, atherogenic dyslipidemia, and the metabolic syndrome. (Review) American Journal of Cardiology, 81, 18B–25B. Haas, G.J., McCune, S.A., Brown, D.M. and Cody, R.J. (1995) Echocardiographic characterization of left ventricular failure adaptation in a genetically determined heart failure model. American Heart Journal, 130, 806–811. Hobbs, H.H., Brown, M.S., Russell, D.W., Davignon, J. and Goldstein, J.L. (1987) Deletion in the gene for the low-density-lipoprotein receptor in a majority of French Canadians with familial hypercholesterolemia. New England Journal of Medicine, 317, 734–737. Ivandic, A., Prpic-Krizevac, I., Sucic, M. and Juric, M. (1998) Hyperinsulinemia and sex hormones in healthy premenopausal women: relative contribution of obesity, obesity type, and duration of obesity. Metabolism, 47, 13–19. Karasu, C., Soncul, H. and Altan, V.M. (1995) Effects of non-insulin dependent diabetes mellitus on the reactivity of human internal mammary artery and human saphenous vein. Life Sciences, 57, 103–112. Kauffman, R.F., Schenck, K.W., Utterback, B.G., Crowe, V.G. and Cohen, M.L. (1987) In vitro vascular relaxation by new inotropic agents: relationship to phosphodiesterase inhibition and cyclic nucleotides. Journal of Pharmacology and Experimental Therapeutics, 242, 864–872. Keiffer, J. and Habener, J.F. (2000) The adipoinsular axis: effects of leptin on pancreatic ␤-cells. American Journal of Physiology, 278, E1–E14. Koletsky, S. (1973) Obese spontaneously hypertensive rats – a model for study of atherosclerosis. Experimental & Molecular Pathology, 19, 53–60. Koletsky, S. (1975) Pathological ﬁndings and laboratory data in a new strain of obese hypertensive rats. American Journal of Pathology, 80, 129–142. Mantha, L., Brindley, D.N., Russel, J.C. and Deshaies, Y. (2002) Developmental changes in adipose tissue and muscle lipoprotein lipase in the obese, atherosclerosis-prone JCR:LA cp/cp rat. International Journal of Obesity and Related Metabolic Disorders, 26, 308–317. McArthur, M.D., Graham, S.E., Russell, J.C. and Brindley, D.N. (1998) Exaggerated stress-induced release of non-esteriﬁed fatty acids in JCR:LA-corpulent rats. Metabolism, 47, 1383–1390. McGill, J.B., Schneider, D.J., Arfken, C.L., Lucore, C.L. and Sobel, B.E. (1994) Factors responsible for impaired ﬁbrinolysis in obese subjects and NIDDM patients. Diabetes, 43, 104–109. McKendrick, J., Salas, E., Dubé, G.P., Murat, J., Russell, J.C. and Radomski, M. (1998) Inhibition of nitric oxide generation unmasks vascular dysfunction in JCR:LA-cp rats. British Journal of Pharmacology, 124, 361–369. Nagaoka, T., Shirakawa, T., Balon, T.W., Russell, J.C. and Fujita-Yamaguchi, Y. (1998) Cyclic nucleotide phosphodiesterase 3 expression in vivo. Evidence for tissue-speciﬁc regulation of phosphodiesterase 3A or 3B mRNA expression in the aorta and adipose tissue of atherosclerosis-prone insulin-resistant rats. Diabetes, 47, 1135–1144. O’Brien, S.F., McKendrick, J.D., Radomski, M.W., Davidge, S.T. and Russell, J.C. (1998) Vascular wall reactivity in conductance and resistance arteries: differential effects of insulin resistance. Canadian Journal of Physiology and Pharmacology, 76, 72–76. O’Brien, S.F., Russell, J.C. and Davidge, S.T. (1999) Vascular wall dysfunction in JCR:LA-cp rats: effects of age and insulin resistance. American Journal of Physiology, 277, C987–C993.

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Pederson, R.A., Campos, R.V., Buchan, A.M.J., Chisholm, C.B., Russell, J.C. and Brown, J.C. (1991) Comparison of the enteroinsular axis in two strains of obese rats: the fatty Zucker and the JCR:LAcorpulent. International Journal of Obesity, 15, 461–470. Peric-Golia, L. and Peric-Golia, M. (1983) Aortic and renal lesions in hypercholesterolemic adult, male, virgin Sprague-Dawley rats. Atherosclerosis, 46, 57–65. Poiley, S.M. (1960) A systematic method of breeder rotation for non-inbred laboratory animal colonies. Animal Care Panel, 10, 159–161. Richardson, M., Schmidt, A.M., Graham, S.E., Achen, B., DeReske, M. and Russell, J.C. (1998) Vasculopathy and insulin resistance in the JCR:LA-cp rat. Atherosclerosis, 138, 135–146. Russell, J.C. (1995) The atherosclerosis-prone JCR:LA-corpulent rat. In Atherosclerosis X: Proceedings of the 10th International Symposium on Atherosclerosis (Woodford, F.P., Davignon, J. and Sniderman, A., eds) (Elsevier Amsterdam), pp. 121–125. Russell, J.C. and Amy, R.M. (1986a) Early atherosclerotic lesions in a susceptible rat model: the LA/N-corpulent rat. Atherosclerosis, 60, 119–129. Russell, J.C. and Amy, R.M. (1986b) Myocardial and vascular lesions in the LA/N-corpulent rat. Canadian Journal of Physiology and Pharmacology, 64, 1272–1280. Russell, J.C., Amy, R.M., Graham, S.E., Dolphin, P.J., Wood, G.O. and Bar-Tana, J. (1995) Inhibition of atherosclerosis and myocardial lesions in the JCR:LA-cp rat by ␤, ␤ -tetramethylhexadecanedioic acid (MEDICA 16). Arteriosclerosis, Thrombosis and Vascular Biology, 15, 918–923. Russell, J.C., Amy, R.M., Manickavel, V., Ahuja, S.K. and Rajotte, R.V. (1987) Insulin resistance and impaired glucose tolerance in the atherosclerosis prone LA/N-corpulent rat. Arteriosclerosis, 7, 620–626. Russell, J.C., Amy, R.M., Michaelis, O.E., McCune, S.M. and Abraham, A.A. (1990) Myocardial disease in the corpulent strains of rats. In Frontiers in Diabetes Research: Lessons from Animal Diabetes III, (Shafrir, E., ed.) (Smith-Gordon, London), pp. 402–407. Russell, J.C., Bar-Tana, J., Shillabeer, G., Lau, D.C.W., Richardson, M., Wenzel, L.M. et al. (1998a) Development of insulin resistance in the JCR:LA-cp rat: role of triacylglycerols and effects of MEDICA 16. Diabetes, 47, 770–778. Russell, J.C., Dolphin, P.J., Graham, S.E. and Amy, R.M. (1997a) Cardioprotective and hypolipidemic effects of nisoldipine in the JCR:LA-cp rat. Journal of Cardiovascular Pharmacology, 29, 586–592. Russell, J.C., Dolphin, P.J., Graham, S.E., Amy, R.M. and Brindley, D.N. (1998b) Improvement of insulin sensitivity and cardiovascular outcomes in the JCR:LA-cp rat by D-fenﬂuramine. Diabetologia, 41, 380–389. Russell, J.C., Graham, S.E., Amy, R.M. and Dolphin, P.J. (1998c) Cardioprotective effect of probucol in the atherosclerosis-prone JCR:LA-cp rat. European Journal of Pharmacology, 350, 203–210. Russell, J.C., Graham, S.E., Amy, R.M. and Dolphin, P. (1998d) Inhibition of myocardial lesions in the JCR:LA-corpulent rat by captopril. Journal of Cardiovascular Pharmacology, 31, 971–977. Russell, J.C., Graham, S.E. and Dolphin, P.J. (1999) Glucose tolerance and insulin resistance in the JCR:LA-cp rat: effect of miglitol (Bay m1099). Metabolism, 48, 701–706. Russell, J.C., Graham, S.E., Dolphin, P.J., Amy, R.M., Wood, G.O. and Brindley, D.N. (1997b) Antiatherogenic effects of long-term benﬂuorex treatment in male insulin resistant JCR:LA-cp rats. Atherosclerosis, 132, 187–197. Russell, J.C., Graham, S. and Hameed, M. (1994) Abnormal insulin and glucose metabolism in the JCR:LA-corpulent rat. Metabolism, 43, 538–543. Russell, J.C., Koeslag, D.G., Amy, R.M. and Dolphin, P.J. (1989) Plasma lipid secretion and clearance in hyperlipidemic JCR:LA-corpulent rats. Arteriosclerosis, 9, 869–876. Russell, J.C., Koeslag, D.G., Dolphin, P.J. and Amy, R.M. (1993) Beneﬁcial effects of acarbose in the atherosclerosis-prone JCR:LA-corpulent rat. Metabolism, 42, 218–223. Russell, J.C., McKendrick, J.D., Dubé, G.P., Dolphin, P.J. and Radomski, M.W. (2001) Effects of LY117018 and the estrogen analogue, 17a-ethinylestradiol, on vascular reactivity, platelet aggregation, and lipid metabolism in the insulin-resistant JCR:LA-cp rat: role of nitric oxide. Journal of Cardiovascular Pharmacology, 37, 119–128.

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Russell, J.C., Ravel, D., Pégorier, J.-P., Delrat, P., Jochemsen, R., O’Brien, S.F. et al. (2000) Beneﬁcial insulin-sensitizing and vascular effects of S15261 in the insulin-resistant JCR:LA-cp rat. Journal of Pharmacology and Experimental Therapeutics, 295, 753–760. Schneider, D.J., Absher, P.M., Neimane, D., Russell, J.C. and Sobel, B.E. (1998) Fibrinolysis and atherogenesis in the JCR:LA-cp rat in relation to insulin and triglyceride concentrations in blood. Diabetologia, 41, 141–147. Schneider, D.J., Nordt, T.K. and Sobel, B.E. (1993) Attenuated ﬁbrinolysis and atherogenesis in type II diabetic subjects. Diabetes, 42, 1–7. Stary, H.C. (1984) Comparison of the morphology of atherosclerotic lesions in the coronary arteries of man with the morphology of lesions produced and regressed in experimental primates. In Regression of Atherosclerotic Lesions: Experimental Studies and Observations in Humans (Malinow, M.R. and Blaton, V.H., eds.) (Plenum Press, New York), pp. 235–254. Stary, H.C. (1994) Changes in components and structure of atherosclerotic lesions developing from childhood to middle age in coronary arteries. (Review) Basic Research in Cardiology, 89 (Suppl 1), 17–32. Steiner, G. (1994) Hyperinsulinemia and hypertriglyceridemia. Journal of Internal Medicine, 736 (Suppl), 23–26. Stern, M.P. (1995) Diabetes and cardiovascular disease: the “common soil” hypothesis. Diabetes, 44, 369–374. Stout, R.W. (1985) Overview of the association between insulin and atherosclerosis. Metabolism, 34, 7–12. Tanaka, M. (1981) Electron microscopic study of cardiac lesions induced in rats by isoproterenol and by repeated stress. Japanese Circulation Journal, 45, 1342–1354. Tanaka, M., Tsuchihashi, Y., Katsume, H., Ijichi, H. and Ibata, Y. (1980) Comparison of cardiac lesions induced in rats by isoproterenol and by repeated stress of restraint and water immersion with special reference to etiology of cardiomyopathy. Japanese Circulation Journal, 44, 971–980. Uusitupa, M.I.J., Niskanen, L.K., Siitonen, O., Voutilainen, E. and Pyorala, K. (1990) 5-year incidence of atherosclerotic vascular disease in relation to general risk factors, insulin level, and abnormalities in lipoprotein composition in non-insulin-dependent diabetic and nondiabetic subjects. Circulation, 82, 27–36. Vance, J.E. and Russell, J.C. (1990) Hypersecretion of VLDL, but not HDL, by hepatocytes from the JCR:LA-corpulent rat. Journal of Lipid Research, 31, 1491–1501. Vydelingum, S., Shillabeer, G., Hatch, G., Russell, J.C. and Lau, D.C.W. (1995) Overexpression of the obese gene in the genetically obese JCR:LA-cp rat. Biochemical & Biophysical Research Communications, 216, 148–153. Wascher, T.C., Wolkart, G., Russell, J.C. and Brunner, F. (2000) Delayed insulin transport across endothelium in insulin resistant JCR:LA-cp rats. Diabetes, 49, 803–809. Wexler, B.C. (1964) Spontaneous arteriosclerosis in repeatedly bred male and female rats. Journal of Atherosclerosis Research, 4, 57–80. Wexler, B.C., Iams, S.G. and Judd, J.T. (1976) Arterial lesions in repeatedly bred spontaneously hypertensive rats. Circulation Research, 38, 494–501. Williams, G., Cardoso, H., Domin, J., Ghatei, M.A., Russell, J.C. and Bloom, S.R. (1990) Disturbances of regulatory peptides in the hypothalamus of the JCR:LA-corpulent rat. Diabetes Research, 15, 1–7. Williams, G., Shellard, L., Lewis, D.A., McKibbin, P.E., McCarthy, H.D., Koeslag, D.G. et al. (1992) Hypothalamic neuropeptide Y disturbance in the obese (cp/cp) JCR:LA-corpulent rat. Peptides, 13, 537–540. Windler, E., Zyriax, B.-C. and Greten, H. (1998) Rationale and effectiveness of lipid therapy. In Atherosclerosis XI: Proceedings of the 11th International Symposium on Atherosclerosis (Jacotot, B. Mathé, D. and Fruchart, J.-C., eds) (Elsevier Science, Singapore), pp. 911–917. Wu-Peng, X.S., Chua, S.C. Jr., Okada, N., Liu, S.-M., Nicolson, M. and Leibel, R.L. (1997) Phenotype of the obese Koletsky (f ) rat due to Tyr763Stop mutation in the extracellular domain of the leptin receptor (Lepr). Diabetes, 46, 513–518.

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THE C57BL/6J MOUSE AS A MODEL OF INSULIN RESISTANCE AND HYPERTENSION TONYA MARTIN, SHEILA COLLINS AND RICHARD S. SURWIT Duke University Medical Center, Durham, NC 27710

INTRODUCTION While diabetes has long been known to be a risk factor for cardiovascular disease, we are only beginning to recognize the risks associated with elevated glucose and insulin levels in nondiabetic patients. Reaven (1988) described the existence of a constellation of metabolic abnormalities that seemed to occur together and to be related to the etiology of cardiovascular disease. He termed this clustering of anomalies “syndrome x” and deﬁned it as the coexistence of hyperinsulinemia, hyperlipidemia, and hypertension in non-diabetic individuals. Together, these features were seen as risk factors for both cardiovascular disease and type 2 diabetes (Reaven, 1988). When intra-abdominal obesity was later included as a risk factor, Bjorntorp (1993) suggested the term “metabolic syndrome”. More recent research has shown that increased fasting insulin, increased fasting glucose, and elevated hemoglobin A1c (HbA1c) all predict both the development of type 2 diabetes and cardiovascular disease. It has been suggested that the “metabolic syndrome” be called the “civilization syndrome” because the various metabolic components of the syndrome, as well as clinical diabetes, are coincident with lifestyle changes associated with Western urbanization (Bjorntorp, 1993). In addition to decreased exercise and increased caloric intake, the epidemiological observations provide evidence that the development of insulin resistance and type 2 diabetes is related to fat consumption and negatively related to carbohydrate consumption (Feskens et al., 1990; King et al., 1984; Marshall et al., 1991). During the past ten years it has become increasingly clear that elevated fasting glucose and insulin levels, even within what is typically considered the “normal” range, are nevertheless signiﬁcant risk factors for cardiovascular disease. Fasting blood glucose levels in the high end of the normal range independently predicted cardiovascular death among non-diabetic, healthy middle-aged men (Bjornholt et al., 1999), while glucose intolerance above the 95th percentile was associated with coronary-related death in a sample of civil servants (Fuller et al., 1983). Elevated levels of HbA1c in nondiabetics were an independent risk factor for increased mortality following myocardial infarction (Chowdhury et al., 1998). Finally, HbA1c was shown to be a continuously related factor for death from cardiovascular disease in a population of nearly 5000 men from the European Prospective Investigation into Cancer and Nutrition (Khaw et al., 2001). A large epidemiological study examining the link between insulin levels and coronary artery disease found that increased insulin sensitivity is associated with decreased atherosclerosis in Hispanic and non-Hispanic whites as well as in African-Americans (Haffner et al., 1997). Hyperinsulinemia, independent of hyperglycemia, has also been recognized as a risk factor for disease (Weyer et al., 2000). Elevations of fasting insulin, even within the normal range, have 73

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been linked to decreased vagal function in otherwise healthy individuals (Watkins et al., 2000). Hyperinsulinemia also has been shown to be a risk factor for the development of type 2 diabetes as well as cardiovascular disease (Haffner et al., 1997). In fact, it is now widely accepted that even in the absence of diabetes, insulin resistance is a risk factor for hyperlipidemia, hypertension and cardiovascular disease (Reaven, 1988; Bjorntorp, 1993).

DIET-INDUCED OBESITY IN C57BL/6J MICE An increase in dietary fat content has been shown to produce hyperglycemia, hyperinsulinemia and obesity in various strains of mice (West et al., 1992; Surwit et al., 1988) and in rats (Schemmel et al., 1970). In particular, the C57BL/6J (B6) mouse is lean and physically normal when restricted to low-fat chow. However, it develops a syndrome of obesity, hyperinsulinemia, hyperglycemia and hypertension when allowed ad libitum access to a high-fat diet (Surwit et al., 1988; Mills et al., 1993). Importantly, the B6 mouse develops this syndrome only in response to a high-fat diet (Surwit et al., 1995). Even when sucrose is used as the major carbohydrate source (Surwit et al., 1995), this animal remains normal throughout its early adult years if fat intake is restricted, although some spontaneous obesity can be seen in very old mice (Coleman, 1978). In marked comparison to B6, other strains such as the A/J mouse or the C57BL/KsJ (KsJ) are relatively resistant to these effects of a high-fat diet (Surwit et al., 1988, 1994, 1995, 1998; Rebuffe-Scrive et al., 1993). The development of insulin resistance, hyperglycemia and obesity in the B6 mouse closely parallels the progression of common forms of the human disease. For example, the onset of diabetes and obesity in humans occurs gradually and often in the presence of a high-fat diet (West and Kalbﬂeisch, 1971). Others have also noted that high-fat feeding can produce compromised immune function in B6 mice (Crevel et al., 1992) and the development of atherosclerosis (Paigen et al., 1987; Munday et al., 1998). For these reasons, the B6 mouse is an excellent model for the human metabolic syndrome. The development of obesity in B6 mice, that were fed a high-fat diet, is not a simple result of hyperphagia or low levels of physical activity. As shown in Figure 1, when fed a high-fat 17.5

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Figure 1 Feed efﬁciency (weight gained/kcal consumed) of B6 mice on either a high-fat or a low-fat diet. (Reprinted from Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W. and Collins, S. (2000) Diazoxide restores ␤3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 41, 3630–3637 with permission from Endocrinology Society Journals.)

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diet, B6 mice show increased feed efﬁciency (weight gained/kcal consumed) (Surwit et al., 1995; Brownlow et al., 1996; Parekh et al., 1998). An important point to recall is that these data contrast with models of leptin deﬁciency or leptin insensitivity. For example, the B6 mouse clearly develops obesity despite increased physical activity levels. Brownlow et al. (1996) reported that obese B6 mice are as equally active as their lean counterparts and nearly three times as active as A/J mice. By contrast, Yen et al. (1972) observed that the Lepob/Lepob and LepRdb/LepRdb models are hypoactive. Thus, even though the Lepob mutation is on the same B6 mouse strain, the complete absence of leptin has major effects that are separate from the obesity and diabetes that develops in the non-mutant B6 mouse challenged with high-fat feeding. From all these criteria, we conclude that the B6 mouse is an example of the obesity that develops as a result of the interaction of dietary content and genetic variables. For this reason, the B6 model is more consistent with human obesity than that which is found in single gene mutations consuming a very low-fat (4–10% calories from fat) diet. Although data on the relationship between hyperphagia, physical activity, and obesity in humans are conﬂicting, reports from several research groups show that, contrary to popular belief, obese subjects do not necessarily consume more calories than lean subjects (Braitman et al., 1985; Dreon et al., 1988; Romieu et al., 1988; Miller et al., 1990). Recent studies (Hunter et al., 1996, 1997) found a relationship between percent body fat, intra-abdominal fat, and physical activity in men and women. However, other studies (Maffeis et al., 1998; Waxman and Stunkard, 1980; Gazzaniga and Burns, 1993) have failed to ﬁnd a relationship between physical activity and body weight. Maffeis et al. (1998), Waxman and Stunkard (1980), and Gazzaniga and Burns (1993) noted that diet composition was one main risk factor for the development of obesity and Maffeis et al. (1998) determined that genetic background was another such factor. Thus, the development of the B6 mouse as a research tool to understand the mechanisms involved in the interactions between diet and genetic background will be invaluable for future research. Additionally, this animal model has recently been used in the discovery of multiple genes that govern the development of insulin resistance and obesity (Steppan et al., 2001; Fruebis et al., 2001).

GLUCOSE, INSULIN AND BODY COMPOSITION Rebuffe-Scrive et al. (1993) and Surwit et al. (1995) reported that diet-induced diabetes and obesity in the B6 mouse is characterized by selective deposition of fat in the mesentery, an observation consistent with the ﬁnding that abdominal obesity is an independent risk factor for diabetes in humans (Marshal et al., 1991; Feskens et al., 1995). The diabetes/obesity syndrome worsens with time and with increasing obesity. B6 mice, fed a high-fat diet for 16 weeks, developed adipocyte hyperplasia and hypertrophy, resulting in animals with a fat mass increased by 93% (Black et al., 1998). Even though fully manifested after 16 weeks of a high-fat diet, the diabetes/obesity syndrome is completely reversible even at this stage in these mice. Parekh et al. (1998) showed that reversal of the obesity/diabetes syndrome occurs when animals raised on a high-fat diet are later switched to a low-fat diet.

HYPERTENSION IN B6 MICE Diet-induced obesity and diabetes in B6 mice is similar to the human syndrome in that hypertension is part of the metabolic syndrome. Mills et al. (1993) showed that when fed a high-fat,

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high-sucrose diet, B6, but not A/J, mice showed a signiﬁcant elevation of systolic blood pressure as measured either by a tail cuff or intracarotid sensor. This increase in blood pressure was independent of dietary sodium content. In a second experiment using ganglionic blockade with Chlorisondamine, they demonstrated that the diet-induced increase in arterial blood pressure was due to an increase in SNS outﬂow to the heart and vasculature. While it remains to be seen whether or not this diet-induced hypertension is mediated by hyperinsulinemia, the syndrome of obesity, hyperinsulinemia and hypertension found in the B6 mouse closely parallels the (syndrome x/metabolic syndrome) described in humans (Bjorntorp, 1993).

ABNORMAL PANCREATIC FUNCTION IN B6 MICE Like humans with type 2 diabetes, B6 mice show a blunted insulin response to both glucose and non-glucose stimuli. Lee et al. (1995) demonstrated that when compared with A/J mice, isolated islets from B6 mice show an impaired insulin response to both glucose and the free fatty acid laurate. Diet-induced obesity further attenuated the response to glucose, but not to laurate. Thus, elevated free fatty acids from high-fat feeding might stimulate hyperinsulinemia in islets that show a blunted response to glucose. In a follow-up study, Wencel et al. (1995) showed that high-fat feeding signiﬁcantly attenuated the late phase insulin response from isolated islets in both A/J and B/6 mice, but that the effect was greater in B6 mice. In support of these studies, Ahren et al. (1997) also showed that glucose stimulated insulin release was blunted in these in B6 mice.

LEPTIN The discoveries of leptin and its receptor from the genetically leptin-deﬁcient B6 Lepob/Lepob (Zhang et al., 1994) and leptin-insensitive LepRdb/LepRdb mice (Tartaglia et al., 1995 and Lee et al., 1996) provided a major advance in our understanding of the molecular aspects of intercellular communication between adipose tissue and the central nervous system for the regulation of food intake and metabolic rate (reviewed in Schwartz and Kahn, 1999). However, the relationship between leptin and obesity and fuel metabolism in these genetic models of leptin-deﬁciency or leptin-insensitivity does not parallel the situation in the overwhelming majority of obese humans. The evidence suggests that most obese humans actually possess greatly elevated plasma leptin levels and appear to be leptin-resistant (Considine et al., 1996). As is the case in human obesity, leptin levels are elevated in obese/diabetic B6 mice (Surwit et al., 1997). However, the progression to this obese, hyperleptinemic state is preceded by a period, prior to the onset of obesity, wherein B6 mice on a high-fat diet actually have lower leptin levels than A/J mice on the same diet. In other words, an interesting difference between the obesity-prone B6 and the obesity-resistant A/J mouse is that it is only after B6 mice develop massive obesity that leptin levels exceed those of the A/J. Nevertheless, our efforts to augment this transiently depressed leptin pharmacologically were unable to prevent the development of insulin resistance or obesity in B6 mice on a high-fat diet (Surwit et al., 2000b). A similar phenomenon of low plasma leptin levels causing a predisposition to the development of obesity has been reported in a subgroup of Pima Indians (Ravussin et al., 1997). Similar to humans, B6 mice appear to be resistant to leptin despite these elevated leptin levels. Efforts now are best focused on trying to understand the basis of this leptin resistance.

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Lin (2000) recently demonstrated that the development of leptin insensitivity in B6 mice on a high-fat diet is progressive, with peripheral leptin resistance preceding central leptin resistance and changes in hypothalamic neuropeptides during the development of diet-induced obesity have begun to be examined (Ziotopoulou et al., 2000). Thus these studies with the B6 mouse and other diet-induced models of obesity and diabetes illustrate that these non-mutant models are more likely to provide a more relevant understanding of the progression of human obesity and leptin-resistance than genetic mutants. ADIPOSE TISSUE ␤-ARs The sympathetic nervous system plays an integral role in regulating both energy intake and energy expenditure in most mammals. Catecholamines increase lipolysis and decrease triglyceride-rich lipoprotein accumulation in WAT while, at the same time, increasing thermogenesis in BAT, thereby resulting in an overall decrease in total body fat stores (Wurtman, 1965; Rothwell, 1994). The actions of these catecholamines occur via the ␤-adrenergic receptors (␤-ARs), members of the G protein coupled receptor family. Three ␤AR subtypes ( ␤1AR, ␤2AR and ␤3AR) have been identiﬁed pharmacologically and by molecular cloning (Nahmias et al., 1991; Granneman et al., 1991; Emorine et al., 1989). Each of the three ␤ARs is coupled to Gs and the stimulation of intracellular cAMP levels, however more complex regulation involving interaction with members of the Gi family has also been reported (Soeder et al., 1999; Gerhardt et al., 1999; Cao et al., 2000). While all three ␤ARs are expressed in adipocytes, only the expression of the ␤3AR is adipocyte-speciﬁc, and relative levels of expression vary among species (Hollenga et al., 1990; Galitzky et al., 1997). In virtually all animal models of obesity, the ability of the ␤ARs to stimulate lipolysis is impaired. Not long after the discovery of the ␤3AR, the expression and function of the adipocyte ␤ARs from lean vs. leptin-deﬁcient C57BL/6J Lepob/Lepob mice were examined. A dramatic decrease in both ␤1- and ␤3-ARs mRNA levels were observed, whereas the expression of the ␤2AR remained unaltered, and these changes in ␤AR subtype expression were shown to be responsible for the inability to mobilize stored fat in response to ␤-agonists (Collins et al., 1994). Other models of congenital obesity such as LepRdb/LepRdb, tubby, fat, and the Zucker fatty rat show similar decreases in ␤3AR and ␤1AR expression (Muzzin et al., 1991; Collins et al., 1999). Like these monogenic models of obesity, the B6 mouse raised on a high-fat diet shows similar defects in ␤AR function and expression in adipocytes (Collins et al., 1997). As shown in Figures 2 and 3, Collins et al. (1997) reported that adenylyl cyclase activity, in response to ␤-adrenergic stimulation, is decreased in both white and brown adipose tissue of B6 mice fed a high-fat diet. In addition, the expression of the ␤1- (not shown) and ␤3-ARs (Figure 4) are severely reduced. Despite these defects in ␤AR expression in adipose tissue, under certain circumstances selective ␤3AR agonists can prevent or reverse obesity and diabetes in a variety of species (Arch et al., 1984; Nagase et al., 1996; Himms-Hagen et al., 1994; Collins et al., 1997; Casteilla et al., 1994; Sasaki et al., 1998a,b; Fisher et al., 1998). In addition, the blunted expression of ␤3AR appears to be modestly improved, as in the case of the Lepob/Lepob mice (Arbeeny et al., 1995) or nearly normalized in the case of diet-induced obese A/J mice (Collins et al., 1997). An important point concerning these studies is that the ability of selective ␤3AR agonists to stimulate lipolysis and thermogenesis, and normalize hyperglycemia and hyperinsulinemia, is dependent on genetic background (Collins and Surwit, 1996; Collins et al., 1997; Guerra et al., 1998). In responsive strains such as A/J, the effects of ␤3AR-agonist treatment can persist

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Figure 2 ␤-agonist-stimulated adenylyl cyclase activity in epididymal white adipose tissue (WAT) membranes from B6 mice. After the 16-week period on one of the three diets, gonadal WAT was removed and plasma membranes were prepared. ␤-agonist-stimulated adenylyl cyclase activity was determined in response to increasing doses of epinephrine (A) or CL316,243 (B). (Reprinted from Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W. and Collins, S. (2000) Diazoxide restores ␤3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 41, 3630–3637 with permission from Endocrinology Society Journals.)

even after four months of treatment, while efﬁcacy diminishes rapidly in the high-fat-fed B6 mouse (Collins et al., 1997). Although genetic and dietary models of obesity display various endocrine abnormalities, the presence of hyperinsulinemia and insulin resistance is the one most common variable (discussed in Collins et al., 1999 and 2001). In addition to being causally related to obesity, the condition of hyperinsulinemia and insulin resistance may itself lead to obesity. For these reasons, our current hypothesis is that hyperinsulinemia contributes to the inhibition in ␤AR expression and function in adipocytes. In support of this idea, treatment of 3T3-F442A adipocytes with insulin resulted in a rapid decrease in ␤3AR expression (Fève et al., 1994). In addition, a role for insulin in affecting ␤AR function in adipocytes is suggested by two sets of studies. The ﬁrst showed that suppressing hyperinsulinemia with the K-ATP channel agonist, diazoxide (Dz), results in an improved ability to stimulate lipolysis and a signiﬁcant loss of adipose tissue mass (Alemzadeh et al., 1996, 1998). More recently, we again directly examined

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Figure 3 ␤-agonist-stimulated adenylyl cyclase activity in brown adipose tissue (BAT) membranes from B6 mice. After the 16-week period on one of the three diets, interscapular BAT was removed and plasma membranes were prepared. ␤-agonist-stimulated adenylyl cyclase activity was determined in response to increasing doses of epinephrine (A) or CL316,243 (B). (Reprinted from Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W. and Collins, S. (2000) Diazoxide restores ␤3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 41, 3630–3637 with permission from Endocrinology Society Journals.)

the situation in the high-fat fed B6 mouse and showed that treatment with Dz resulted in an increase in the expression and function of the ␤3AR, as evidenced by increased cAMP production in response to selective ␤3AR agonist stimulation (Figure 5) (Surwit et al., 2000a). In addition, as shown in Figure 6, this increase in ␤3AR was accompanied by a signiﬁcant loss of WAT mass and percent body fat (Surwit et al., 2000a). Importantly, glucose transport into WAT, but not muscle, was increased in response to CLDz, suggesting that changes in insulin sensitivity in fat rather than muscle are critical to the development of diabetes, at least in the B6 model (Surwit et al., 2000a). Whereas ␤3AR has been estimated to account for as much as 90% of the ␤ARs at the cell surface in rodent adipose tissue (Fève et al., 1995), such is not the case with humans, and its expression and abundance in human adipose tissue has been controversial. However, a missense mutation in the human ␤3AR has been reported: the tryptophan at position 64 is replaced by an arginine (Clement et al., 1995; Widen et al., 1995; Walston et al., 1995), and

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Figure 4 Comparison of ␤3AR mRNA levels in EWAT depots of B6 mice. Total cellular RNA was prepared from pooled tissues of ﬁve mice in each diet group. Northern blot analyses measured the relative levels of ␤3AR mRNA, corrected for expression of cyclophilin. (Reprinted from Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W. and Collins, S. (2000) Diazoxide restores ␤3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 41, 3630–3637 with permission from Endocrinology Society Journals.)

1500 Dz 1200 EWAT cAMP (pmoles/mg/min)

LF 900 HF 600

300

0

0

–9

–8

–7

–6

–5

–4

–3

Log [CL316,243] (M)

Figure 5 The stimulation of adenylyl cyclase activity by the ␤3AR-selective agonist CL316,243 in membranes from animals fed the ◆, LF; ■, HF; or 嘷, Dz diets. The assays were incubated for 10 min. The cAMP produced was measured by radioimmunoassay. The data are expressed as picomoles of cAMP produced per mg of membrane protein/min of incubation. (Reprinted from Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W. and Collins, S. (2000) Diazoxide restores ␤3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 41, 3630–3637 with permission from Endocrinology Society Journals.)

this mutation has been associated with the development of obesity. In fact, greater than 50% of Pima Indians who have hereditary obesity carry this mutation. This mutation is found in approximately 25% of African-Americans and appears in 8–10% of the general population in both the US and Europe (Clement et al., 1995; Walston et al., 1995). The mutation is

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0.07

EWAT/Body weight (g/g)

0.06 0.05 0.04 0.03 0.02 0.01 0.00

LF

HF

CL

Dz

CLDz

Treatment

Figure 6 An estimate of the percent body fat in B6 mice fed low fat (LF), high fat (HF), high fat 0.001% CL316,243 (CL), high fat 0.32% diazoxide (Dz), or high fat both compounds (CLDz) for 1 month. Percent body fat was estimated by the weight of epididymal fat as a proportion of the total body weight. (Reprinted from Surwit, R.S., Dixon, T.M., Petro, A.E., Daniel, K.W. and Collins, S. (2000) Diazoxide restores ␤3-adrenergic receptor function in diet-induced obesity and diabetes. Endocrinology, 41, 3630–3637 with permission from Endocrinology Society Journals.)

associated with an increased capacity for weight gain and an increased maximum weight (Clement et al., 1995) as well as an increased risk for developing type 2 diabetes (NIDDM) (Widen et al., 1995). Importantly, Candelore et al. (1999) recently reported the discovery of potent and selective human ␤3AR antagonists. These compounds were shown to inhibit agonist-induced lipolysis in cells expressing human cloned ␤3AR or in isolated nonhuman primate adipocytes.

CONCLUSION In 1988, Reaven hypothesized that hyperinsulinemia was itself at the basis of a constellation of metabolic abnormalities which he termed “syndrome x” (Reaven, 1988). Later, others noted that “syndrome x” appeared to be linked to the dietary and other lifestyle habits of Westernized countries and might be particularly dependent upon the availability of a high-fat diet. Indeed, the research summarized above suggests that the B6 mouse is, in many ways, an excellent model of this syndrome. When raised on a high-fat diet, the B6 mouse develops signiﬁcant hyperinsulinemia, obesity, hyperglycemia, and hyperlipidemia and hypertension. Over the last ten years, a large body of research has developed supporting the role of both hyperinsulinemia in the pathophysiology of diabetes, as well as in other disease processes that are often seen as complications of diabetes. Research with the B6 mouse, as well as with other rodent models of obesity and diabetes, has provided persuasive evidence that hyperinsulinemia may play a key role in obesity, diabetes and the other abnormalities that occur in “syndrome x”. In particular, research in which hyperinsulinemia is attenuated with the K-ATP channel agonist diazoxide (Dz), has shown that the reduction of hyperinsulinemia in animals and humans facilitates weight loss in humans and improves glucose metabolism (Alemzadeh et al., 1996,

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1998). We have shown that one common feature of many obese murine models is the decreased expression and function of the ␤3AR (Collins et al., 1994, 1997, 1999; Muzzin et al., 1991). In the B6 mouse, the reduction of hyperinsulinemia with a K-ATP channel agonist, Dz, appears to restore ␤1- and ␤3-AR expression and function (Surwit et al., 2000a). This is accompanied by a reduction in obesity, hyperglycemia and hyperleptinemia even in animals maintained on a high-fat diet. Dz-treated animals also show enhanced UCP1 expression and improved glucose transport in adipocytes. Unfortunately, we did not assess blood pressure in Dz-treated animals. However, our data suggest that Dz-treated animals should show decreased sympathetic nervous system (SNS) activity as well as decreased blood pressure. Previous research in our laboratory has shown that exogenous leptin administration in the leptin deﬁcient Lepob/Lepob mouse produces increased SNS outﬂow to the peripheral organs (Collins et al., 1996). This, in turn, increases UCP1 expression and activity and leads to a decrease in adipose tissue mass and lower circulating leptin levels. Hyperinsulinemia, by interfering with the action of catecholamines on ␤1- and ␤3-ARs, could inhibit this feedback loop, thereby leading to decreased glucose uptake in adipocytes, and increased SNS outﬂow. Since we have previously shown that the hypertension that accompanies diet-induced obesity in B6 mice is mediated by increased SNS outﬂow to the vasculature (Surwit et al., 1995), this mechanism is entirely plausible. However, this theory may be of limited relevance to clinical conditions because of the uncertain role of UCP1 in human adipocytes. Nevertheless, the B6 mouse serves as an interesting model to study the multiple endocrine abnormalities that have been attributed to hyperinsulinemia.

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Part 2

Molecular and Genetic

7. MOLECULAR FEATURES OF INSULIN RESISTANCE, OBESITY AND TYPE 2 DIABETES IN NON-HUMAN PRIMATES STEPHEN V. ANGELONI AND BARBARA CALEEN HANSEN Obesity and Diabetes Research Center, University of Maryland School of Medicine, Baltimore MD 21201, USA

THE NATURAL HISTORY AND BACKGROUND OF INSULIN RESISTANCE, OBESITY AND TYPE 2 DIABETES IN NON-HUMAN PRIMATES Insulin resistance develops commonly during middle age in the rhesus monkey, in association with the gradual progressive development of obesity. Most monkeys who are demonstrably insulin resistant and obese (body fat 25%) subsequently go on to develop overt type 2 diabetes. These conditions are naturally occurring and become evident even as the monkeys are maintained on a “healthy heart diet” consisting of laboratory chow high in ﬁber and low in fat with near zero cholesterol. Insulin resistance and obesity develop in both free ranging monkeys, and in individually housed monkeys. In the free ranging monkey, the environmental conditions must provide protection from predators, minimization of exposure to diseases, and most importantly, the diet must be provided so as to permit ad libitum feeding. Some factors contribute to disease development in aging free ranging or laboratory animals, and other factors may prevent the development of obesity and diabetes. Our work with laboratory maintained monkeys has shown that calorie restriction (CR) at levels which maintain body fat between 17% and 22% is sufﬁcient to prevent the development of these diseases (Hansen and Bodkin, 1993). The monkeys in rhesus colonies in the United States are of random origin, coming from various breeding colonies. No monkeys originating in India have been imported to the United States since l977, and no systematic or selective breeding has taken place. Thus, they have not been genetically bred to express insulin resistance, obesity or diabetes. Some monkeys, maintained under identical environmental and dietary conditions, do not develop any of these conditions. The largest colony of longitudinally-studied normal or insulin resistant, obese and/or diabetic monkeys is currently held at the University of Maryland School of Medicine, and much of the current review will derive from studies using this colony. Many of these monkeys have been the subject of cross-sectional studies aimed at examining the potential role of candidate genes in the expression of these disorders under various conditions and at different time points in the development of each disease. The present review brings together, for the ﬁrst time, a wide range of molecular ﬁndings in rhesus monkeys deriving from speciﬁc studies of the insulin resistance, obesity or type 2 diabetes of this species. As noted below, many of the genes examined to date in the rhesus have been found to be remarkably similar in their cDNA’s and amino acid sequences to those that have been examined in humans. For this reason, monkeys have increasingly served as models for examining 89

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STEPHEN V. ANGELONI AND BARBARA CALEEN HANSEN

questions of gene expression and the mechanisms underlying insulin resistance, and its sequelae, obesity and type 2 diabetes.

OVERVIEW OF GENES ASSOCIATED WITH INSULIN RESISTANCE AND THE DEVELOPMENT OF OBESITY IN NON-HUMAN PRIMATES Alterations in the sequence or expression of a number of genes have been associated with the development of obesity and diabetes, and some of these genes have been speciﬁcally examined in non-human primates. The volume of sequence information available for humans and the mouse model has signiﬁcantly assisted in the study of the molecular features of insulin resistance in the non-human primate, but there is still limited sequence information available for the monkey genes. This review begins with an examination of the proinsulin molecule, including the structure of both insulin and its C-peptide. This is followed by consideration of the insulin receptor, its sequence, and its splice variants. The differential expression of these splice variants within different tissues and at various stages of the progression from normal to insulin resistant to overt diabetes in both monkeys and humans is reviewed. In non-human primates, only selected aspects of the insulin signaling system have been studied to date, and those are summarized. We have previously shown the critical role of adipose tissue in the development of insulin resistance. Speciﬁcally, prevention of excess adipose tissue prevents insulin resistance in primates (Hansen and Bodkin, 1993). Thus, genes expressed with a high degree of speciﬁcity in adipose tissue may hold clues to the understanding of mechanisms inducing insulin resistance at the whole body level. Finally, the newest pharmaceutical agents developed, or under development, for the treatment of insulin resistance have as their targets the family of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). In this review, we also examine reports on the sequence and/or expression of each of the PPARs ␣, ␥ and ␦, all of which appear to be involved in insulin sensitivity, and review studies that determine the effects of their ligands. Also discussed in this review is some of the work done on testing PPAR agonists in rhesus monkeys to develop evidence on receptor mechanisms of action and possible roles of these compounds in the prevention or treatment of insulin resistance.

THE INSULIN, PROINSULIN AND C-PEPTIDE MOLECULES While glucose levels in monkeys tend to be slightly lower than in humans, monkeys have plasma insulin levels that are four to ten times higher than humans (Table 1). This difference is most likely due to mechanisms regulating gene expression, protein stability/processing or other pancreatic/islet characteristics and is not due to the insulin sequence itself. Comparison

Table 1

Comparison of rhesus and human fasting plasma insulin levels.

Normal (lean) Obese Diabetic

Human ␮U/ml (pmol/l, pM)

Rhesus monkey ␮U/ml (pmol/l, pM)

1–9 (6–60) 10–100 (60–600) 0–40 (0–240)

20–70 (120–550) 50 500 (300–3000) 2 400 (12–2400)

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of human and monkey insulin sequences showed them to have the same amino acid sequence (Naithani et al., 1984). Further comparison of the amino acid sequences of insulin, proinsulin and preproinsulin from human, Cercopithecus aethiops (African green monkey), Macaca fascicularis (crab-eating macaque) and Macaca mulatta (rhesus monkey) have shown that although the insulin molecule is identical across all of these species, there are differences in the signal peptide just preceding the B chain, and there is a single amino acid substitution in the C-peptide portion of the preproinsulin and proinsulin molecules (Figure 1). The leucine (L) to proline (P) conversion at residue 61 in the monkey C-peptide sequence does not appear to interfere with using the human C-peptide assay for detecting the monkey C-peptide, and we have not seen disproportionate proinsulin plasma concentration in monkeys (Koerker et al., 1978). Whether or not the sequence differences effect the rate of processing of non-human primate preproinsulin to insulin, compared to other species is not known, but is a possibility. In the processing of human and rat preproinsulins to insulin, differences in speciﬁc amino acids can alter protease speciﬁcity and affect the rate of processing, sometimes in a species-speciﬁc manner (Sizonenko and Halban, 1991; Smeekens et al., 1992; Zhou and Lindberg, 1993). These changes are usually 4 residues from the arginine at 32 (R(32)) or the lysine at 64 (K(64)), or both sites of proinsulin and have been shown to alter the rate of proinsulin processing. The effect is believed to be mediated by altering the protein’s structure and affecting the binding of the endopeptidases PC2 and PC3 to proinsulin, which alters the rate of processing (Rhodes and Alarcon, 1994; Rhodes et al., 1992). The amino acids at these locations are important for maintaining the proper three-dimensional structure of the proinsulin molecule thus directing the sequential cleavage of proinsulin by PC2, then PC3 (Bailyes et al., 1991; Rhodes et al., 1992; Sizonenko et al., 1993; Weiss et al., 1990). It is therefore possible that some species-speciﬁc sequence differences may contribute to altering plasma insulin levels. However, this does not appear to be the case for monkeys. Human C. aethiops M. fascicularis M. mulatta

1 10 20 (1) (10) MALWMRLLPL LALLALWGPD PaaAFVNQHL CGSHLVEALY MALWMRLLPL LALLALWGPD PvpAFVNQHL CGSHLVEALY MALWMRLLPL LALLALWGPD PApAFVNQHL CGSHLVEALY FVNQHL CGSHLVEALY

Human C. aethiops M. fascicularis M. mulatta

51(30) TPKTRREAED TPKTRREAED TPKTRREAED TPKTRREAED

Human C. aethiops M. fascicularis M. mulatta

101(80) SLYQLENYCN SLYQLENYCN SLYQLENYCN SLYQLENYCN

(40) lQVGQVELGG pQVGQVELGG pQVGQVELGG pQVGQVELGG

(50) GPGAGSLQPL GPGAGSLQPL GPGAGSLQPL GPGAGSLQPL

(60) ALEGSLQKRG ALEGSLQKRG ALEGSLQKRG ALEGSLQKRG

(20) 50 LVCGERGFFY LVCGERGFFY LVCGERGFFY LVCGERGFFY (70) 100 IVEQCCTSIC IVEQCCTSIC IVEQCCTSIC IVEQCCTSIC

Figure 1 Amino acid sequence comparison of monkey and human preproinsulin. The sequences for human, C. aethiops and M. fascicularis preproinsulin were aligned with Align-X (Informax Inc. Bethesda MD). Amino acid residues that differ from human are in italics lower case. The signal peptide sequence is indicated as bold and the C-peptide sequence is underlined. The numbering system for proinsulin is in parentheses (#). Locus numbers for the human, C. aethiops and M. fascicularis sequences are AAH05255, CAA43405 and AAA36894, respectively. The M. mulatta sequence was reproduced from Naithani et al., 1984.

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THE INSULIN RECEPTOR AND ITS SPLICE VARIANTS Analysis of numerous mutations in the human insulin receptor have been linked to the development of insulin resistance and other complications that accompany some cases of type 2 diabetes (DeFronzo et al., 1992; Granner and O’Brien, 1992; Häring, 1993; Kahn and Goldﬁne, 1993). In addition to mutations, alteration in the proportion of alternative splicing of exon 11 from the proreceptor mRNA occurs producing two ␣ subunit isoforms. Different levels of expression of the two ␣ isoforms have been linked to and may contribute to the development of insulin resistance (Kellerer et al., 1993; Mosthaf et al., 1991, 1993; Norgren et al., 1993; Sesti et al., 1991). We therefore review reports on the structure of the insulin receptor and its splice variants in rhesus monkeys. The insulin receptor complex is composed of two ␣ and two ␤ subunits that form an ␣2␤2 heterotetramer. The mRNA transcript for both subunits is transcribed from the same gene and produces a large ␣–␤ proreceptor protein that is cleaved into the ␣ and ␤ subunits and undergoes several post-translational modiﬁcations (Hedo et al., 1981, 1983, 1987; Olson and Lane, 1987; Salzman et al., 1984). Alternative splicing of exon 11 from the transcript also occurs and produces two isoforms of the ␣ subunit with one isoform lacking the 12 carboxyl terminal amino acids coded for by the 36 base pairs (6p) of exon 11 (Moller et al., 1989; Mosthaf et al., 1990; Seino and Bell, 1989). The two ␣ subunit isoforms have different binding afﬁnities for insulin with the exon 11 A form binding insulin with an afﬁnity about two times stronger than the exon 11 B form (McClain, 1991; Mosthaf et al., 1990). The complete heterotetramer is positioned in the cell membrane with the ␤ subunits spanning the membrane and the ␣ subunits residing completely outside of the cell with class II disulﬁde bridges joining the ␣ subunits to the ␤ subunits (Boni-Schnetzler et al., 1987; Finn et al., 1990; Jacobs et al., 1979). As a result of these characteristics and organization, the primary function of the ␣ subunits is to bind insulin and the ␤ subunits possess protein tyrosine–kinase activity that mediates insulin’s action through a variety of signal transduction pathways. Monkeys, like humans, express both ␣ and ␤ isoforms of the insulin receptor (Huang et al., 1994, 1996). Comparison of the monkey amino acid sequence, for exons 9 through 12, to that of human, rat and mouse shows their homology to monkey being 99.5% for human, 90% for rat and 89.9% for mouse. In a longitudinal study of 19 monkeys with or without diabetes, while no insulin receptor mutations or allelic variations may be present, different levels of the ␣ subunit isoform expression correlated with the development of muscle and liver speciﬁc insulin resistance. As in humans and rats, there is tissue speciﬁc expression of the two ␣ isoforms (Figure 2A). As measured by RNA template speciﬁc reverse transcriptase PCR (RTPCR), monkeys have the lowest proportion of exon 11 mRNA in the liver (36.6 4.0%) and the highest proportion in the brain (97.0 1.0%) (Figure 2B). Monkey tissues with intermediate levels of exon 11 mRNA include muscle (67.0 3.9%), fat (77.2 2.8%), stomach (75.1 4.5%), heart (65.7 6.7%) and kidney (77.1 3.3%). This pattern of expression is similar to that of humans and rats except that monkeys tend to have higher levels of exon 11 expression (Goldstein and Dudley, 1990; Moller et al., 1989; Seino and Bell, 1989). This is, of course, counterintuitive, since with the relatively higher plasma insulin levels observed in non-human primates, one might expect greater expression of the insulin receptor isoform with the lower binding afﬁnity. While tissue differences are not surprising, given the need for tissue speciﬁc responses to insulin, in some tissues there are regional differences in the expression levels of the two splice variants. For example, in different muscles (vastus lateralis and rectus abdominis) and different fat regions (subcutaneous abdominal and intra-abdominal) from ﬁve monkeys (one normal,

MOLECULAR FEATURES OF INSULIN RESISTANCE

93

1

2

3

4

5

6

7

Tissue

M

F

L

S

H

K

B

% exon 11–

52.7

71.9

35.0

68.9

60.7

74.2

100

A Exon 11+ (382 bp)

Exon 11– (346 bp)

100

B

% exon 11–

75

50 M-2 (Normal) M-9 (Pre-DM) M-7 (Pre-DM) M-8 (Pre-DM) M-16 (NIDDM)

25

0 Muscle

Fat

Liver Stomach Heart Kidney Brain

Figure 2 RT-PCR analysis of insulin receptor exon 11 splice variant tissue distribution in rhesus monkeys. (A) Analysis of splice variants from M, normal rectus abdominis muscle; F, intra-abdominal fat; L, liver; S, stomach; H, heart; K, kidney and B, brain tissues. (B) Analysis of tissue speciﬁc exon 11 splice variant distribution from monkeys ranging from normal to type 2 diabetic (NIDDM). (Reproduced from Huang et al., 1994 with permission from the publisher.)

three prediabetic, and one late type 2 diabetic), there are regional differences for muscle but not fat. In muscle, there is a slight but signiﬁcant increase in the amount of exon 11 mRNA being expressed in the vastus lateralis (72.9 4.1%) vs. the rectus abdominis (67.0 3.9%). In contrast to the muscle differences, there is little difference in the percentage of exon 11 txranscript seen between subcutaneous fat with 80.3 1.7% and intra-abdominal fat having 77.2 2.8%. In muscle, differences in the amount of exon 11 transcript may be due to the type of muscle ﬁber present (fast vs. slow twitch) and/or the amount of intramuscular fat in the tissue (Johnson et al., 1973). In a study of 25 monkeys with varying degrees of diabetes from normal to overt, the percentage of exon 11 mRNA in the liver ranged from 23.0% to 53.9% with one very acutely ill diabetic monkey having 85% (Huang et al., 1996). While some studies indicate that an increase in the amount of muscle or liver expression of exon 11 transcript is associated with type 2 diabetes in humans (Kellerer et al., 1993; Mosthaf et al., 1991, 1993; Norgren et al., 1993; Sesti et al., 1991), other studies do not support this ﬁnding (Benecke et al., 1992; Hansen et al., 1993). These discrepancies in the literature may be due to a variety of factors one of which is differences in the methods of normalization used by different studies (Benecke et al., 1992; Hansen et al., 1993; Kellerer et al., 1993; Mosthaf et al., 1991). For example, in monkey muscle, there are no differences in

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STEPHEN V. ANGELONI AND BARBARA CALEEN HANSEN

the percentage of exon 11 transcripts when normoglycemic and hyperglycemic monkeys are compared (Figure 3A). However, when the monkeys are stratiﬁed into normal, prediabetic, early diabetic and late diabetic, the percentage of exon 11 was highest in hyperinsulinemic prediabetic (74.8 1.7%) and in late hyperinsulinemic early diabetic animals (64.2 3.9%) when compared to normal monkeys with 59.0 2.3% (Figure 3B and C). This indicates that in muscle an increase in the percentage of exon 11 transcripts is associated with insulin levels and not plasma glucose levels (Figure 3D). Similar discrepancies exist in the literature for the association of exon 11 variant expression in the liver. The percentage of exon 11 mRNA expression in the liver increases in monkeys with low glucose disappearance rates (KG). When monkeys were grouped according to fasting glucose levels, an increase in plasma glucose concentration was associated with an increase in liver exon 11 mRNA expression. Over all, monkeys with low fasting glucose levels have about 29.8 1.6% of exon 11 mRNA and this increases to about 39.2 2.9% in monkeys with elevated fasting glucose. While this increase in the high afﬁnity isoform of the insulin receptor in the liver is not as dramatic as seen in muscle, it is clearly linked to glucose concentration and not insulin as previously shown in muscle for some of the same monkeys. While these results indicate that increases in muscle exon 11 transcript expression are associated with increasing insulin levels and glucose, these results are not always supported by other reports in the literature. The differences arise from a number of factors including study group deﬁnitions, and different molecular lesions not involving insulin or insulin receptor functions contributing to disease development.

A

B

90 % exon 11–

% exon 11–

90 80 70 60 50

p = 0.07

70 60

Hyperglycemia (DM) (n =7)

Normal Pre-DM Early-DM (n = 6) (n = 6) (n = 2) NormoHyperinsulinemia insulinemia D

90 80 70 60 50 Normal Pre- and Early-DM Late DM (Normo(Hyper(Hypoinsulinemia) insulinemia) insulinemia) (n = 6) (n = 8) (n = 5)

Late DM (n = 5) Hypoinsulinemia

90

p < 0.05 % exon 11–

p < 0.005 % exon 11–

p < 0.05

50 Normoglycemia (Non-DM) (n =12)

C

80

80

p < 0.0001

70 60 50 Normo- and hypoHyperinsulinemia insulinemia (Normal and Late DM) (Pre- and Early-DM) (n =11) (n = 8)

Figure 3 Analysis of the proportion of insulin receptor exon 11 splice variants based on disease staging. (A) Monkeys grouped based on fasting glucose levels. (B) Monkeys separated into four groups based on plasma insulin levels. (C) Monkeys grouped based on insulin levels and combining pre- and earlydiabetic animals. (D) Monkeys grouped based on fasting plasma insulin. (Reproduced from Huang et al., 1994 with permission from the publisher.)

MOLECULAR FEATURES OF INSULIN RESISTANCE

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INSULIN SIGNALING AND DOWN STREAM EFFECTS While the interaction of insulin with the insulin receptor is an essential signal mechanism that assists the body with monitoring and regulating glucose levels, the effects of insulin binding its receptor would not be possible without a large number of additional proteins that participate in mediating the actions of this interaction. As noted, the ␣ subunit of the insulin receptor functions as the receiver of the signal and the ␤ subunit acts as the transducer by way of its kinase activity on a number of substrates residing in the cytoplasm. By acting on these cytoplasmic components, a number of signal transduction pathways are utilized which can alter the activity of other enzymes and change the pattern of genes being expressed (O’Brien and Granner, 1996). While some components of these signaling mechanisms are the same in all tissues, there are some that are tissue speciﬁc depending on how the organ needs to respond to the stimulus and how the tissue needs to communicate with other organs in the body. The organs playing a predominant role in regulating glucose and lipid metabolism are the liver, muscle, pancreas, adipose tissue and central nervous system. The initial steps in the signaling mechanism involve insulin receptor phosphorylation of proteins like the insulin receptor substrate protein family (IRS-1, 2, 3 and 4), Shc, Gab-1, p62doc and others (Lavan et al., 1997a,b; Sun et al., 1991, 1995; White et al., 1985; Yamanashi and Baltimore, 1997). One of the important down stream targets involved in regulating glucose clearance from the blood in response to increased insulin levels is glucose transporter 4 (GLUT4). Upon insulin stimulation via a PI3 kinase pathway, GLUT4 moves from the cytoplasm to the cell surface to facilitate glucose uptake by muscle and adipose tissue (Carpenter and Bodansky, 1990; Cushman and Wardzala, 1980; Suzuki and Kono, 1980). With muscle being the major source of glucose uptake in the body, mechanisms that disrupt or enhance this response can greatly alter an individual’s insulin sensitivity as measured by glucose clearance. Such alterations are exempliﬁed by CR, and thus the effects of CR on GLUT4 expression has been studied in monkeys. CR in monkeys, rodents and humans results in an increase in insulin sensitivity usually accompanied by a decrease in plasma glucose concentrations and plasma insulin levels (Bodkin et al., 1995; Cefalu et al., 1997; Hansen and Bodkin, 1993; Kemnitz et al., 1994; Lane et al., 1995; Masoro et al., 1992). This increase in insulin sensitivity may or may not be associated with an improvement in basal muscle glucose transport and often appears to be independent of IRS-1, PI3K and GLUT4 activities or expression (Cartee et al., 1994; Dean et al., 1998; Dean and Cartee, 1996; Gazdag et al., 1999; Gazdag et al., 2000). These studies indicate that as important as insulin signaling is for regulating glucose uptake and the assessment of insulin sensitivity, insulin insensitivity is not directly associated with alterations of normal GLUT4 activity, and that other insulin signaling pathways mediating changes in the activity of various enzymes are involved. Pathways involved with the use of glucose by muscle as either an immediate energy source or to replenish muscle glycogen stores have been identiﬁed as possible sources of perturbation contributing to the development of type 2 diabetes (Bogardus et al., 1984; Jucker et al., 1999; Ortmeyer et al., 1993; Rossetti et al., 1993; VillarPalasi and Larner, 1960). The role of speciﬁc glucose utilizing pathways in contributing to diabetes and obesity development or predisposition have been investigated as exempliﬁed by studies on the activity and enzymatic characteristics of skeletal muscle glycogen synthase (GS), a key enzyme in determining the fate of glucose utilization in muscle (Damsbo et al., 1991; Kida et al., 1992; Ortmeyer, 2001; Ortmeyer et al., 1993; Schalin-Jäntti et al., 1992). In a study on non-human primates (Ortmeyer, 2001), alterations in GS maximal catalytic activity (Vmax) were measured

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STEPHEN V. ANGELONI AND BARBARA CALEEN HANSEN

in monkeys on different dietary regimens and at different stages of diabetes development. When the monkeys in this study were stratiﬁed into ﬁve groups: very lean, calorie-restricted, normal, hyperinsulinemic and type 2 groups, no signiﬁcant difference in the basal and insulinstimulated skeletal muscle GS Vmax was observed (Figure 4B). However, when the ability of GS to bind its allosteric activator glucose-6-phosphate (G6P) was measured, there were marked differences in G6P binding afﬁnity (G6P Ka). Very lean and CR monkeys had the lowest basal and insulin-stimulated G6P K a values (highest afﬁnity for G6P) compared to hyperinsulinemic and type 2 diabetic monkeys (Figure 4A). When comparing normal monkeys to very lean, CR, hyperinsulinemic and type 2 monkeys, the G6P Ka of normal monkey GS decreased by about half (a 2-fold increase in afﬁnity) upon insulin stimulation, but did not change signiﬁcantly in the other groups (Figure 4A). The similar Vmax of GS from these 41 monkeys suggests that most of them share a GS that has the same enzymatic characteristics, indicating they are likely to have the same amino acid sequence or at least no signiﬁcant sequence variations that dramatically alter the proteins activity (sequence analysis of GS from these monkeys was not actually done). These data also indicate that other factors are altering the enzyme’s basal activity by affecting the enzymes ability to bind G6P. Because the activity of GS is dependant on its phosphorylation state, with active GS being more dephosphorylated, signaling pathways and other factors affecting the ratio of active/inactive GS will effect the magnitude of insulin activation of GS. Factors that can contribute to individual variations in this metabolic setting are hormonal levels (Craig and Larner, A

3 a G6P K a of skeletal muscle GS (mmol/l)

c 2

1

b

b

0 B Skeletal muscle GS Vmax (nmol/min/mg protein)

25 20 15 10 5 0 Very lean

Normal Hyperinsulinemic

DM

Calorierestricted

Figure 4 Analysis of G6P binding afﬁnity and GS activity. Monkeys ranged from very lean to obese hyperinsulinemic to type 2 diabetic. Normal and calorie-restricted were also studied. (A) G6P Ka of GS in skeletal muscle. (B) Skeletal muscle GS Vmax. The statistical signiﬁcance compared to insulinstimulated is p 0.004 (a), p 0.05 for normal, hyperinsulinemic and diabetic (b), and p 0.05 for VL, normal and CR animals (C). (Reproduced from Ortmeyer, 2001 with permission from the publisher.)

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1964; Lawrence and Zhang, 1994; Villar-Palasi and Larner, 1960) and nutritional status. As a result, in the study of Ortmeyer 2001, the magnitude of insulin stimulated vs. basal changes in G6P Ka and skeletal muscle G6P concentration varies for each individual monkey regardless of health status as shown in Figure 5. Despite these individual variations, the patterns that emerge are as follows. In normal monkeys, while insulin stimulates GS activity, it is not accompanied by an increase in muscle G6P concentration, indicating that an insulin signal mechanism other than that driving glucose transport is being used. In addition, monkeys with an increase in insulin-stimulated G6P K a were characterized by having a low basal G6P K a (high afﬁnity), high basal GS activity (highly dephosphorylated), along with the lowest percent body fat and lowest plasma glucose levels. These monkeys were usually very lean or CR. The high afﬁnity of GS for G6P in these animals may be involved in the beneﬁts of CR. CR appears to result in a setting of the metabolic sensitivity of an individual by making GS more active (dephosphorylated) resulting in it having a high afﬁnity for its allosteric activator G6P. This may constitute another advantage of CR and the beneﬁt it bestows on the body in preventing the onset of obesity and diabetes by priming the body to work more efﬁciently. In addition, the unexpected phosphorylation of GS that was observed in response to insulin in some of the CR monkeys revealed that there are still individual factors (probably genetic) that may act to resist the beneﬁts of CR. Determining which of these factors may be inherent aspects of genetically determined insulin resistance can be accomplished using advanced molecular genetic approaches, together with more detailed sequence information. In addition to regulating the activity of enzymes as discussed above, the action of insulin also leads to alterations in the expression of many genes, the analysis of which may reveal more about the molecular nature of insulin resistance. A number of signal transduction pathways lead from the insulin receptor’s tyrosine–kinase activity to transcription factors in the cytosol or nucleus. While a number of genes are known to be regulated by insulin, some of the transcription factors involved have only recently been identiﬁed. One of the key candidates for

Insulin-stimulated minus basal skeletal muscle G6P (nmol/mg dry weight)

1.0

0.5

Very lean

0.0

Normal Hyperinsulinemic –0.5

Diabetic Calorie-restricted r = 0.80, p < 0.0001

–1.0 –4

–3 –2 –1 0 1 2 Insulin-stimulated minus basal G6P Ka of skeletal muscle GS (mmol/l)

3

Figure 5 Analysis of individual variations in the changes of basal skeletal muscle GS G6P Ka and G6P concentrations. The changes in each of 41 monkeys are plotted for insulin stimulated minus basal changes in skeletal muscle G6P concentration vs. G6P binding afﬁnity. (Reproduced from Ortmeyer, 2001 with permission from the publisher.)

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mediating the transcriptional regulatory properties of insulin is FKHR, a member of the forkhead family of transcription factors. In the presence of insulin, FKHR activation of the insulin-like growth factor-binding protein-1 (IGFBP-1) gene is inhibited (Tomizawa et al., 2000; Unterman et al., 1994). Insulin regulation of gene expression can also be mediated through other pathways whose transcription factors have not been identiﬁed. This is exempliﬁed by the study of Lochhead et al., 2001 who showed that the inhibition of glucose6-phosphatase and phosphoenolpyruvate carboxykinase gene expression are regulated through a GS kinase-3 pathway whose ﬁnal target transcription factors have not yet been identiﬁed (Lochhead et al., 2001). Given the high degree of homology between human and non-human primate gene sequences and metabolic pathways, it will be interesting to see how the nonhuman primate model plays a role in discovering new genes and pathways contributing to disease development or predisposition.

LEPTIN AND THE LEPTIN RECEPTOR Adipose tissue is both a site of insulin resistance involved in obesity and diabetes as well as a major endocrine organ that releases cytokines that may increase or decrease insulin sensitivity. Leptin and adiponectin are two such hormones released by adipose tissue into the circulation. Leptin itself is a 16 kDa protein secreted by adipose tissue. It is the product of the obesity (ob) gene which is about 20 kb long consisting of 3 exons and two large introns on human chromosome 7q31.3 (Campﬁeld et al., 1995; Isse et al., 1995; Zhang et al., 1994). In homozygous ob/ob mice, administration of leptin resulted in a number of behavioral and physiological changes which included a decrease in food consumption, an increase in physical activity, increased energy expenditure, a reduction in body weight and the lowering of plasma glucose and insulin concentrations to normal levels (Campﬁeld et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995; Weigle et al., 1995 and K. Stark, this volume). These and a variety of other responses in different tissues, are mediated by leptin binding to its receptor in a number of different tissues as will be discussed below. Isolation and sequence analysis of a monkey leptin transcript showed the messenger RNA to be about 770 bases long, much shorter than the human transcript of about 4.5 kb (Hotta et al., 1996). Northern blot analysis of several monkey tissues conﬁrmed the transcript size and demonstrated that it is only expressed in adipose tissue, as occurs in humans and rodents (Considine et al., 1995; Funahashi et al., 1995; Murakami and Shima 1995; Ogawa et al., 1995). The predicted amino acid sequence of the monkey transcript codes for a protein of 167 amino acids with a sequence homology of 91% to human leptin and 84% to mouse leptin. To determine if plasma leptin levels or gene expression levels are associated with the development of diabetes, normal, hyperinsulinemic and insulin-resistant type 2 diabetic monkeys were studied (Hotta et al., 1996). The results show that leptin expression levels are highest in subcutaneous adipose tissue of hyperinsulinemic monkeys, slightly lower in normal monkeys and lowest in type 2 diabetic monkeys while there were no differences in levels of the control transcript HSP83 (Figure 6A and B). While these differences were not signiﬁcant, expression levels of leptin were generally correlated to body weight and fasting plasma insulin levels (Figure 6C and D). In monkeys, evidence supporting the hypothesis that leptin expression is regulated by insulin is not strong. For example, euglycemic hyperinsulinemic clamps producing maximal insulin stimulation of young normal monkeys showed no changes in ob mRNA expression levels from basal levels in either omental or subcutaneous adipose tissue (Figure 7A and B). While this is in conﬂict with some reports in the literature (Becker et al., 1995; Cusin

MOLECULAR FEATURES OF INSULIN RESISTANCE A

B

6 HSP83 mRNA (AU, ×103)

ob mRNA (AU, ×104)

2.5 2.0 1.5 1.0 0.5

4

2

0

0 Normal

C

HyperType 2 insulinemic diabetes

Normal

D

3

r = 0.514, p < 0.01 ob mRNA (AU, ×104)

2

1

0 0

10

HyperType 2 insulinemic diabetes

3

r = 0.528, p < 0.01 ob mRNA (AU, ×104)

99

20

Body weight (kg)

30

2

1

0

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Figure 6 Messenger RNA expression levels of leptin in monkey subcutaneous adipose tissue. Expression levels for leptin mRNA (A) and the control transcript HSP83 mRNA (B) were compared in adipose tissue from normal, hyperinsulinemic and type 2 (NIDDM) monkeys. Correlation of leptin mRNA expression to body weight (C) and fasting plasma insulin concentration (D) in the three groups. Normal monkeys □, hyperinsulinemic monkeys 嘷, type 2 monkeys ■. (Reproduced from Hotta et al., 1996 with permission from the publisher.)

et al., 1995; Moinat et al., 1995; Saladin et al., 1995; Trayhurn et al., 1995), it coincides with another study showing that insulin did not regulate leptin in cultured adipose cells (Murakami et al., 1995). In monkeys, the strongest correlation between a physiological parameter and leptin is between plasma leptin levels and increasing body weight, although this relationship includes wide variation for any given level of body fat content. The action of leptin is mediated by binding of the hormone to the leptin receptor which is expressed in a number of different tissues. The gene for the leptin receptor is on human chromosome 1p31 from which a number of isoforms can be expressed. The receptor is a member of the gp130 family of cytokine receptors and mediates the action of leptin through the JAK/STAT signaling pathway (Darnell, 1996; Ghilardi et al., 1996; Vaisse et al., 1996). Because the receptor is expressed in a number of different tissues, leptin has multiple physiological effects on tissues such as brain, liver, kidney, ovary, adipose tissue, pancreas and skeletal muscle (Ciofﬁ et al., 1996; Cohen et al., 1996; Fei et al., 1997; Ghilardi et al., 1996; Lee et al., 1996; Tartaglia et al., 1995). In these various tissues, leptin has been reported to affect insulin signaling in hepatic cells (Cohen et al., 1996) as well as to affect lipid and glucose metabolism

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Figure 7 Basal and insulin-stimulated leptin mRNA expression in different adipose tissues. Tissues obtained before and at the end of a euglycemic/hyperinsulinemic clamp in monkeys. (A) Subcutaneous and (B) omental adipose tissue. (Reproduced from Hotta et al., 1996 with permission from the publisher.)

(Berti et al., 1997) and to alter gene expression in muscle, adipose tissue and pancreatic ␤-cells (Zhou et al., 1997). In pancreatic ␤-cells, it is also capable of inhibiting insulin secretion (Emilsson et al., 1997; Kieffer et al., 1997; Koyama et al., 1997). Isolation and characterization of monkey leptin receptor cDNAs indicated that at least two alternatively spliced isoforms are produced (Hotta et al., 1998). The long form of the monkey leptin receptor is 96% homologous to the human long form. The short form of the monkey was homologous to the mouse ob-ra. In the monkey sequence of the long isoform, the two R-S-X-R-S motifs and the C/R motif characteristic of class I cytokine receptors were conserved as well as the motifs required for JAK and STAT interactions (Chen et al., 1996). In the monkey, two additional variations of the long and short forms were identiﬁed and designated as long form 2 and short form 2. Both of these variants had the same additional 31 amino acid insert starting at residue 889. These additional splice variants were not homologous to other known mouse and human isoforms (Ciofﬁ et al., 1996; Lee et al., 1996), so it is not clear if they represent a monkey speciﬁc splicing event or improperly spliced messages. Northern blot analysis for leptin receptor mRNA in several monkey tissues identiﬁed a transcript of about 6 kb that was most abundant in the liver and expressed at slightly lower levels in adipose tissue. Expression of the receptor in other tissues such as muscle, kidney, stomach, duodenum and colon was much lower or not detectable. RT-PCR analysis also showed the long form being expressed at higher levels in subcutaneous adipose tissue, the hypothalamus and choroid plexus compared to muscle, kidney, stomach, duodenum, colon, heart, lung, cortex, cerebellum, thalamus and corpus callosum. These patterns of expression are like those in humans but differ slightly from mice. In monkeys and humans, total leptin receptor expression is higher in liver than in kidney, but in the mouse it is higher in the kidney (Ciofﬁ et al., 1996; Tartaglia et al., 1995). Expression of the long form in monkeys and humans is higher in adipose tissue, hypothalamus and choroid plexus while in the mouse very little is expressed in adipose tissue and choroid plexus (Fei et al., 1997; Ghilardi et al., 1996; Mercer et al., 1996). Such species-speciﬁc differences may not interfere with the use of mouse models for examining the mechanisms of leptin action; however, they indicate that some facets of the mouse model are not likely to be as predictive of the human metabolic responses as a non-human primate model where tissue speciﬁc expression patterns are the same as in

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humans. These kinds of differences are important when considering using the model for accurately assessing the efﬁcacy and potential side effects of candidate therapeutic compounds. To determine if there was a relationship between leptin receptor expression and various obesity and diabetes characteristics, Hotta et al. studied 26 rhesus monkeys (M. mulatta) for linkage of receptor expression levels to a number of obesity and diabetes characteristics (Hotta et al., 1996). The results of this study showed that the levels of total and long leptin receptor mRNA expression varied from monkey to monkey. In addition, there was no correlation of receptor mRNA expression to body weight, fasting plasma insulin levels, plasma glucose levels, or plasma leptin concentrations in these monkeys. There was also no difference in expression levels of insulin treated diabetic monkeys vs. untreated monkeys. These data indicate that in these 26 monkeys, changes in leptin receptor expression levels are not a contributing factor to their development of insulin resistance, obesity or diabetes. The role of leptin and the leptin receptor in the development of obesity and diabetes does not always involve changes in the expression level of these proteins as occurs in ob and db mice. This is indicated in the monkey model by the lack of relationship between mRNA levels of total and the long form of the leptin receptor with body weight, fasting plasma insulin or glucose levels, severe diabetes or body mass index. However, there are clinical case reports where, like in the ob/ob mouse model, low plasma leptin levels are associated with obesity in humans (Forooqi et al., 1999; Hager et al., 1998; Montague et al., 1997; Strobel et al., 1998). Most studies in humans, as in monkeys, have reported general elevations in leptin levels with obesity, but with wide variability (Considine et al., 1996b; Lonnqvist et al., 1995; Weigle et al., 1997). In addition, while there have been case reports in humans where mutations in leptin or the receptor have been linked to disease development (Considine et al., 1996a; Ghilardi et al., 1996; Hager et al., 1998; Hixson et al., 1999; Montague et al., 1997; Strobel et al., 1998), no such mutations have yet been identiﬁed in monkeys.

ADIPONECTIN Adiponectin is another plasma protein that is released by adipose tissue. In addition to adiponectin, other adipocytokines like tumor necrosis factor-␣ (TNF-␣), leptin, resistin and plasminogen activator inhibitor-1 are released by adipose tissue and alterations in their expression or activity may contribute to the development of insulin resistance, diabetes and obesity (Aizawa-Abe et al., 2000; Auwerx et al., 1988; Caro et al., 1996; Dunbar et al., 1997; Hotamisligil et al., 1993; Juhan-Vague et al., 1989; Masuzaki et al., 1999; Matsuzawa et al., 1999; Shek et al., 1998; Shimomura et al., 1996a, 1999; Steppan et al., 2001). Studies in humans have shown that decreased plasma adiponectin levels are associated with the development of cardiovascular disease concomitant with obesity (Arita et al., 1999; Hotta et al., 2000; Ouchi et al., 1999). Adiponectin decreases the expression of adhesion molecules in endothelial cells by modulating NF␬B signaling and disrupts the attachment of the monocyte cell line THP-1 cells to aortic endothelial cells (Ouchi et al., 1999, 2000). Adiponectin also suppresses the secretion of TNF-␣ in human monocytes and macrophages (Yokota et al., 2000). These ﬁndings suggest that adiponectin inhibits a variety of processes involved with the development of arteriosclerosis and has anti-atherogenic and anti-inﬂammatory properties. As in humans where decreases in adiponectin are associated with obesity and diabetes, the same associations are seen in rhesus monkeys (Hotta et al., 2001a). In this study, lean monkeys had signiﬁcantly higher plasma concentrations of adiponectin than obese and diabetic monkeys. Comparison of adiponectin levels in the three groups studied differed from the pattern

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seen for leptin. Lean monkeys had the lowest leptin levels, diabetic monkeys had slightly higher levels and the highest leptin levels were seen in the obese group. In addition, these patterns of adiponectin and leptin levels showed tight but opposite correlations with fat weight. At the gene and protein level, the sequence of monkey adiponectin is about 96%

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Figure 8 Longitudinal changes of several physiological markers through phases 1–9, ranging from normal to severe diabetes (phase 9). Analysis of plasma adiponectin in arbitrary units (AU), plasma leptin in ng/ml, body weight in kg, fat weight in kg, fasting plasma glucose in mmol/l, fasting plasma insulin in pmol/l, insulin-stimulated glucose uptake rate (M), and glucose disappearance (KG) in monkeys. (Reproduced from Hotta et al., 2001a with permission from the publisher.)

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homologous to human adiponectin and its expression is conﬁned to adipose tissue as determined by Northern blot analysis (Maeda et al., 1996; Nakano et al., 1996). The development of diabetes occurs over a period of years and can be divided into eight or nine phases (Hansen and Bodkin, 1986). In a longitudinal study by Hotta et al., (2001), the change in a number of factors, including adiponectin and leptin, was monitored through these phases. In this study, monkeys in phase 1 were young (10 years old), lean (22% body fat) and had normal fasting insulin and glucose concentrations (Figure 8). Their glucose disappearance rates (K G ) and insulin-stimulated glucose uptake (M ) were in the normal range indicating that all the monkeys were insulin responsive. During phase 2, plasma adiponectin declined slightly and leptin increased slightly. During phases 3–7, adiponectin concentrations declined to their lowest values by phase 6 and leptin concentrations increased to their highest values by phase 7. This decrease in adiponectin and increase in leptin occurred at the same time as the monkeys aged and progressed to being obese (increased body weight and fat weight), insulin resistant, and diabetic (increased fasting plasma glucose and insulin concentrations) as shown in Figure 8. This progression also accompanies a decrease in responsiveness to insulin as measured by a decrease in both the glucose uptake rate, during a euglycemic hyperinsulinemic clamp, and K G rates during an intravenous glucose tolerance test (see graphs of Figure 8). When the nondiabetic monkeys were divided into obese hyperadiponectin or obese hypoadiponectin groups and compared to lean monkeys, there was no signiﬁcant difference in the body weight, body fat or plasma glucose levels in the obese monkeys (Figure 9). However, when the obese monkeys were compared based on insulin-stimulated M rates, monkeys with lower adiponectin had

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lower M values indicating greater insulin resistance, and were hyperinsulinemic (Figure 9A, D and E). Since adiponectin is secreted by adipose tissue and the amount of fat tissue can vary between individuals, expression levels of the adiponectin gene could be partly a function of fat content and thus reﬂected in plasma concentrations. Analysis of adiponectin mRNA levels vs. fat weight showed no relationship between them (Figure 10A) and no correlation of plasma adiponectin concentrations to adiponectin mRNA levels (Figure 10B). These data suggest that the regulation of adiponectin plasma concentration is controlled by a post-transcriptional mechanism. Taken together, these data indicate that hypoadiponectinemia is linked to insulin resistance. In addition, the role of adiponectin in inhibiting biological responses that lead to cardiovascular disease provides additional information about the mechanism contributing to the occurrence of this complicating condition accompanying the development of obesity and diabetes. In non-human primate models, the progression of dietary induced coronary artery disease progresses in the same way as it does in humans, suggesting that many of the underlying causes may be the same between monkeys and humans (Rudel et al., 1990; Strong et al., 1994; Weingand, 1989). The exact molecular mechanisms that link insulin signaling to the changing patterns of adiponectin plasma concentrations are gradually being unraveled. NUCLEAR RECEPTORS The past ten years have seen an explosion in research aimed at understanding the roles of nuclear receptors in insulin resistance and dyslipidemia. Among the proteins that are involved

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with the regulation of lipid metabolism and adipose tissue differentiation, the PPARs have emerged to be among some of the most important as a result of their being targets for therapeutic compounds in the treatment of type 2 diabetes and dyslipidemia. The PPARs are members of the nuclear hormone receptor family of ligand dependent transcription factors. Some of the other members of this family include the estrogen receptors, progesterone receptor, thyroid hormone receptors, glucocorticoid receptors, vitamin D receptor, and the retinoid receptors RAR and RXR. All members of this family are involved with regulating the transcription of target genes by forming homodimeric or heterodimeric complexes whose activities are mediated by binding their speciﬁc ligands. As members of this family, the PPARs are represented by three separate genes on human chromosomes 22q12–q13.1 (PPAR␣), 6p21.2–p21.1 (PPAR␦) and 3p25 (PPAR␥). In non-human primates, PPAR␣ and PPAR␥ have been the most studied and recently attention has turned to PPAR␦ with the development of a PPAR␦ speciﬁc ligand.

PPAR␣ The PPAR␣ receptor is the target of ﬁbrate compounds. In rodent models, these compounds increase peroxisomal fatty acid oxidation and microsomal ␻-hydroxylation in the liver (Lock et al., 1989; Reddy and Lalwai, 1983). Other target tissues expressing PPAR␣ in rodents are the kidney and intestine (Braissant et al., 1996; Kliewer et al., 1994; Mukherjee et al., 1994). In the rodent model, ﬁbrates act by altering the expression patterns of a number of genes involved in lipid metabolism (Berthou et al., 1995; Catapano, 1992; Hertz et al., 1995; Lefebvre et al., 1997; Schoonjans et al., 1996; Staels et al., 1995). These changes in gene expression patterns result in lowering plasma triglyceride (TG) and cholesterol levels associated with increased hepatic uptake and catabolism of fatty acids (Catapano, 1992; Hertz et al., 1995; Lefebvre et al., 1997; Schoonjans et al., 1996; Staels et al., 1995; Willson et al., 2000). There are, however, species-speciﬁc differences in rodent and human responses to the ﬁbrates. These differences may be due to different tissue expression patterns of PPAR␣. In rodents, expression was highest in the liver, kidney and intestine, while in humans it was highest in skeletal muscle followed by liver, kidney and adrenal (Braissant et al., 1996; Kliewer et al., 1994; Mukherjee et al., 1994; Palmer et al., 1998; Su et al., 1998). In addition, ﬁbrates do not stimulate a peroxisome proliferation response in the primate liver or cultured primate hepatocytes as occurs in rodents (Blaauboer et al., 1990; Cornu-Chagnon et al., 1995; Graham et al., 1994; Makowska et al., 1992). Another difference is that in humans, ﬁbrates increase high density lipoprotein-cholesterol (HDL-C) plasma concentrations by increasing apolipoproteins AI and AII (apoAI and apoAII), while in rodents plasma HDL-C concentrations drop concomitant with a decrease in apoAI and apoAII (Berthou et al., 1996; Berthou et al., 1995; Staels and Auwerx, 1998). As a result of these differences, rodents are not the best model system for predicting the human metabolic response to these compounds. To test a better model system, Winegar et al. studied the effects of fenoﬁbrate on obese monkeys (Winegar et al., 2001). To ensure that any differences encountered were not due to differences in PPAR␣ peptide sequence, the sequence for PPAR␣ was derived from M. mulatta cDNA. Comparison of the human and monkey nucleotide sequences showed a 97% identity and at the amino acid sequence level the proteins were 99% homologous. Consistent with the high degree of sequence similarity between human and monkey PPAR␣, the monkey receptor showed similar transcriptional activation responses to fenoﬁbrate, bezaﬁbrate and gemﬁbrozil as was seen with the human

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receptor. When the levels of PPAR␣ protein in different monkey tissues were examined, PPAR␣ was found to be highest in skeletal muscle followed by liver, heart and brown adipose tissue. These similarities between human and monkey are also reﬂected in the way the two species respond to fenoﬁbrate. As occurs in humans, obese monkeys treated with up to 30 mg/kg b.i.d. had serum TG levels 50% lower than baseline. With this treatment, serum apolipoprotein CIII (apoCIII) concentrations also dropped 29% lower than basal levels. In these obese monkeys, total serum cholesterol did not change, but low density lipoprotein cholesterol (LDL-C) concentrations decreased 22%, while HDL-C increased as much as 35%. During the decrease in LDL-C, apolipoprotein B-100 (apoB) decreased by as much as 70%. In contrast, while HDL-C increased, apoAI concentrations did not increase. Treatment of these obese monkeys also reduced insulin levels by as much as 40%. Characteristics that did not change in these obese normal glycemic monkeys were body weight, food consumption and plasma glucose concentrations. These characteristics demonstrate the beneﬁts of using the insulin-resistant non-human primate in studies of PPAR functions and activation.

PPAR␥ PPAR␥ is another member of this nuclear hormone family of transcription factors that has been studied in monkeys. Like PPAR␣ it is one of a number of proteins that plays a important role in the regulation of fat and lipid metabolism. Two isoforms of PPAR␥ are expressed by alternative splicing of the gene on human chromosome 4p15.1. The splicing event produces transcripts coding for PPAR␥1 and PPAR␥2 which differ at their amino termini (Chen et al., 1993; Tontonoz et al., 1994b; Zhu et al., 1993). The two isoforms also differ in their tissue distribution with PPAR␥2 being highly expressed in adipocytes and PPAR␥1 being expressed in many tissues (Chawla et al., 1994; Tontonoz et al., 1994b,c; Vidal-Puig et al., 1996). The potency of the PPAR␥s in regulating adipose tissue development is made evident by studies showing that completely differentiated ﬁbroblasts or cultured myoblasts transform into adipocytes when PPAR␥ expression is up-regulated or cells are transfected with PPAR␥ expressing constructs (Hu et al., 1995; Tontonoz et al., 1994c). In addition, PPAR␥ is a key factor for regulating the expression of genes required to maintain the adipocyte phenotype (Ailhaud et al., 1992; MacDougald and Lane, 1995; Spiegelman and Flier, 1996; Zheng et al., 1996). The receptor’s ability to have such a profound effect on altering a cell’s characteristics is directly related to the function of the protein as a transcription factor that can dramatically alter the pattern of gene expression. PPAR␥ forms a heterodimer with the retinoid X receptor ␣ (RXR␣) (Cha et al., 2001; Kliewer et al., 1992). This PPAR␥–RXR␣ heterodimer has been shown to regulate the expression of genes like adipocyte P2 (aP2), phosphoenolpyruvate carboxylase (PEPCK), and apolipoprotein E, all of which are important for adipogenesis and lipid metabolism (Galetto et al., 2001; Tontonoz et al., 1994a, 1995). During natural physiological responses, these activities are regulated by ligands for both partners of the heterodimers. While the natural ligands for PPAR␥ are not clear, fatty acids and their derivatives are capable of binding PPAR␥ with afﬁnities that appear to be too low for credible ligands. While these natural compounds may be the actual ligands, there are a number of synthetic compounds with higher binding afﬁnities, some of which function as therapeutic agents. Of these synthetic compounds, the thiazolidinedione (TZD) drugs are among the most useful because of their ability to increase insulin sensitivity in insulin-resistant type 2 diabetics (Saltiel and

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Olefsky, 1996). Other synthetic compounds include cloﬁbrate, 5,8,11,14-eicisatetraynoic acid and Wy14,643 (Chawla et al., 1994; Green and Wahli, 1994). While the true identity of the native ligands for PPAR␥ may still be debated, it is clear that the receptor is involved in regulating the expression of a number of genes for speciﬁc metabolic processes, and alterations in receptor expression or function are recognized as contributing not only to obesity and diabetes, but to other diseases such as cancer (Fajas et al., 2001). How PPAR␥1 and PPAR␥2 are involved together in regulating normal gene expression is not clearly deﬁned, but could be related to altering signal transduction pathways or the relative levels of isoform expression. As noted above, while PPAR␥2 is most abundant in adipose tissue, PPAR␥1 is expressed in all tissues at considerably lower levels. Studies of mice with ablated brown fat, showed slightly increased expression levels of PPAR␥1 to PPAR␥2 while in two other mouse models (gold thioglucose and ob/ob) there were no changes in expression levels (Vidal-Puig et al., 1996). In humans, it has been reported that in obese patients, the expression of PPAR␥2, but not PPAR␥1 was increased in adipose tissue (Vidal-Puig et al., 1997). In tissues expressing both isoforms, changing ratios of isoform expression may play a role in receptor activity. A similar shift in the expression of isoforms for another nuclear hormone receptor, the progesterone receptor, has been identiﬁed as a mechanism that alters receptor activity and contributes to the development of some cancers (Graham et al., 1996; McGowan and Clarke, 1999). Whether or not a similar mechanism is being used by the PPAR␥–RXR complex is not known. In rhesus monkeys, as in humans, the PPAR␥ gene expresses two different transcripts, PPAR␥1 and PPAR␥2 as a result of alternate promoter utilization and splicing. The splicing site is at 8 bp from the translation start site. In addition, the PPAR␥1 isoform in monkeys is further spliced into PPAR␥1-a and PPAR␥1-b transcripts (Hotta et al., 1998). As also occurs in humans, the monkey PPAR␥2 is homologous to PPAR␥1 with PPAR␥2 having an additional 30 amino acids on the amino terminus. The splicing of the mRNAs for the two transcripts occurs in the 5 untranslated region of PPAR␥1 with the PPAR␥2 speciﬁc exon providing the additional 30 N-terminal amino acids. The splicing event that produces the PPAR␥1-a and PPAR␥1-b transcripts also occurs at the 8 bp site but different exons are placed upstream. The 5 untranslated sequences for the ␥1-a and ␥1-b messages are the same from 9 to 88 bp after which they are completely different. Whether the divergence beyond 88 bp represents another splice or indicates another exon that is spliced in at 8 bp has not been determined. No matter what is taking place with mRNA splicing, the proteins being expressed from the two possible PPAR␥1 transcripts are the same. While M. mulatta has two PPAR␥1 isoforms, RT-PCR analysis of their tissue distribution showed that both splice forms were expressed at roughly equivalent low levels in muscle, subcutaneous adipose tissue, liver, heart, kidney, stomach, duodenum and colon (Hotta et al., 1998). In contrast, PPAR␥2 was expressed predominantly in subcutaneous adipose tissue, followed by stomach, colon, duodenum and kidney. In obese monkeys, adipose expression of PPAR␥2 was higher than PPAR␥1 compared to normal monkeys. In these same animals, adipose expression of C/EBP␣, LPL and GLUT4 were not increased in obese monkeys, which is in contrast to their increased expression noted in obese rodent models (Dugail et al., 1992; Hainault et al., 1991; Rolland et al., 1995; Shimomura et al., 1992, 1996b). In addition, there was no correlation in normal, type 2 diabetic or obese monkeys with the levels of expression of C/EBP␣, LPL, GLUT4, aP2 or PEPCK to fasting plasma insulin, body weight or adiposity. However, there was a direct correlation of total PPAR␥ expression with the levels of C/EBP␣, LPL and GLUT4 as well as a direct correlation of the

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PPAR␥2/total PPAR␥ ratio to fasting plasma insulin levels and body weight (adiposity). Surprisingly there was no correlation of total PPAR␥, PPAR␥1 or PPAR␥2 expression to adiposity or fasting plasma insulin. It should be noted that in these studies only differences in mRNA levels were measured. For some of these data, analysis of expression at the protein level and identiﬁcation of changes in signal transduction pathways may show more striking differences and possibly clearer associations with the physiological characteristics of insulin resistance, obesity and diabetes. In addition, age and individual metabolic differences in the absence of overt and deﬁned genetic or metabolic perturbations may have contributed to the variability, thus reducing associations. The age factor is especially important because as an animal ages there are signiﬁcant changes in fat mass. In humans, monkeys and rodent models, fat mass, fat cell size and fat cell number are known to increase through middle age and into early old age (Bertrand et al., 1978, 1980; Hirsch and Knittle, 1970; Jen et al., 1985; Kirkland and Dobson, 1997; Silver et al., 1993). This is followed by a decline in fat mass in old age as a result of a decrease in cell size without a change in cell number (Bertrand et al., 1980; Kirkland and Dobson, 1997; Silver et al., 1993). During the development and maintenance of adipose tissues, several genes are involved in addition to PPAR␥. Some of these are adipocyte determination- and differentiation dependent factor 1/sterol regulatory element binding protein 1 (ADD1/SREBP1), CCAAT/ enhancer binding protein ␣ (C/EBP␣), LPL, GLUT4, aP2 and adipsin (MacDougald and Lane, 1995; Spiegelman and Flier, 1996). In rodent models, expression of LPL, aP2 and adipsin decreased with age (Kirkland and Dobson, 1997; Kirkland et al., 1993a,b). In monkeys, the expression of PPAR␥ as well as ADD1/SREBP1, LPL, GLUT4 and C/EBP␣ decreased in the adipose tissue of older individuals (Hotta et al., 1999). However, the expression of adipsin did not decrease with age. In addition, there was no correlation of PPAR␥2/ total PPAR␥ ratio to age but this ratio was signiﬁcantly correlated with body weight and fasting insulin levels as noted above. In summary, while PPAR␥ is an important player in adipose tissue development, it is not the only factor. The lack of a large number of signiﬁcant correlations is in part due to the number of other factors that can impact on adipose tissue characteristics as an individual ages. Among these factors are the hormonal changes that accompany the aging process. This is exempliﬁed by studies of estrogen receptor ␣ knockout mice where both male and female mice are subject to an increase in adipose tissue development accompanied by insulin resistance and glucose intolerance (Heine et al., 2000).

PPAR␦ Of the three PPARs, the function of PPAR␦ is the least understood. The functions of ␣ and ␥ as sensors and effectors of lipid and carbohydrate metabolism were recognized through their roles as mediators of ﬁbrate and glitizone drug activities (Willson et al., 2000; Xu et al., 1999). While the tissue distributions of PPAR␣ and PPAR␥ are primarily in the liver and adipose tissue respectively, PPAR␦ is expressed in a number of different tissues (Braissant et al., 1996). Recently, work using cellular techniques and non-human primate models has shed light on some of the roles of PPAR␦ (Oliver et al., 2001). In this study, the authors showed that the compound GW501516 had a binding afﬁnity for human PPAR␦ of 1.1 0.1 nM. They also demonstrated that in some human cell lines, like THP1 macrophages, treatment with GW501516 resulted in increased expression of the ATP-binding cassette A1 protein

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(ABCA1). At doses of up to 1 M, GW501516 did not induce adipocyte differentiation, bind RXR, or have activity on PPAR␣ or PPAR␥. The regulation of ABCA1 is key to understanding the role of PPAR␦ since ABCA1 is involved in cholesterol metabolism by functioning as a cholesterol transporter (Henke et al., 1998). In addition, THP1 cells treated with GW501516 showed not only an increase in expression of ABCA1 but also an increase in cholesterol efﬂux to apoA1. Because of the distribution of PPAR␦ expression in a number of tissues, it may be functioning in all of these tissues to promote cholesterol efﬂux. In addition, such a widespread distribution of PPAR␦ expression may also support its possible role in the association between arteriosclerosis and obesity/diabetes. Further, it may explain the effectiveness of some ﬁbrates in treating atherosclerosis and coronary disease in some patients (Rubins et al., 1999; Staels and Auwerx, 1998; Staels et al., 1998). The regulation of ABCA1 expression and its involvement in cholesterol metabolism points to an impact of PPAR␦ receptor activity on metabolic processes that contribute to the development of cardiovascular disease and other disorders that are part of the metabolic syndrome X. To study this involvement, Oliver et al., 2001 used obese monkeys expressing the features of the metabolic syndrome X. Some facets of this syndrome were powerfully reversed by GW501516 treatment. Obese monkeys, like humans, frequently have low serum HDL-C, high insulin, and high LDL-C concentrations (Hansen, 2000). In this particular study, the animals were also hyperinsulinemic but had normal glucose concentrations. Treatment of these monkeys with GW501516 produced a signiﬁcant increase in HDL-C as well as decreases in LDL-C and VLDL-TG. Compared to the vehicle, treatment with GW501516 produced an 80% increase in HDL-C, as well as a 29% and 50% decrease in LDL-C and VLDL-TGs, respectively. Analysis of apolipoprotein concentrations showed increases of 43% for apoAI, 21% for apoAII and 46% for apoCIII with GW501516 treatment. Analysis of the lipoprotein particle size distribution, using two different methods, indicated that the GW501516 induced increases in HDL-C and accompanying decreases in LDL-C and VLDL-TG were due to an increase in the number of HDL particles while fewer but larger LDL-C and VLDL-TG particles were produced. The concomitant increase in apoAI and apoAII is expected considering they make up a major component of the HDL-C particle. However, the increase in apoCIII was unexpected even though it is a component of VLDL and HDL particles. Usually, ﬁbrates targeted at PPAR␣ (in the liver) result in a lowering of TGs with decreases in apoCIII as well as decreases in LDL and VLDL cholesterol concentrations (Brown et al., 1999; Rubins et al., 1999; Staels et al., 1995). The fact that the PPAR␦ agonist GW501516 can lower LDL, VLDL and increase HDL and apoCIII may suggest that PPAR␦ can regulate a step (or steps) in the lipid metabolic pathways that can shift or direct apoCIII between HDL-C and VLDL synthesis. In addition, insulin levels were lowered suggesting improvement in insulin sensitivity, although this was not measured directly. This study suggests that PPAR␦ agonists could be useful for minimizing lipid and cholesterol related risk factors associated with the development of cardiovascular disease as shown for other PPAR agonists in humans (Clee et al., 2000; Grundy, 1998). While the clinical use of the ﬁbrates and glitizone drugs is well established for a number of features of the metabolic syndrome X, further study of PPAR␦ agonists are needed to sort out the mechanisms of action in view of its wide tissue distribution. This will be achieved by understanding the molecular details of PPAR␦ regulation of these pathways in a number of tissues, including liver and adipose tissue, particularly in humans and in non-human primates as there are a number of differences in the lipid metabolic pathways and PPAR drug responses between rodents and humans (Dietschy et al., 1993; Suckling and Jackson, 1993).

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GALECTIN-12 There are a variety of genetic and lifestyle factors that contribute to the development of insulin resistance, obesity and diabetes. In the case of obesity, regulating the growth of adipose tissue is a key component of managing or preventing the disease and with mitigating the severity of the type 2 diabetes associated with it. The identiﬁcation of adipose speciﬁc genes has been and remains an important target for determining the role of adipose tissue in insulin resistance and for targeted interventions to improve insulin sensitivity and the metabolic syndrome X. Recently another new gene, expressed solely in adipose tissue has been identiﬁed, and its product determined. This gene is galectin-12, a member of the galectin family of lectins that binds the ␤-galactoside residues on some glycoproteins (Barondes et al., 1994a, b). This family of lectins contains 12 members which all share one or two copies of a highly conserved carbohydraterecognition domain (CRD) (Cherayil et al., 1990; Dunphy et al., 2000; Gitt and Barondes, 1986; Gitt et al., 1992; Wada and Kanwar, 1997; Yang et al., 2001). The galectins appear to participate in regulating a variety of biological functions, such as cell adhesion, cell migration, cell growth and apoptosis (Barondes et al., 1994b; Bernerd et al., 1999; Hadari et al., 2000; Perillo et al., 1995; Wada and Kanwar, 1997; Wada et al., 1997). While some of the galectins are expressed in a number of tissues, others are expressed in a tissue speciﬁc manner where they perform tissue speciﬁc functions (Dyer et al., 1997; Gitt et al., 1992, 1995, 1998; Huﬂejt et al., 1997; Madsen et al., 1995). Recently, a cDNA coding for the expression of galectin-12 was characterized by Hotta et al., (2001b) from a human adipose tissue cDNA library. The cDNA contained a 1008 bp open reading frame coding for a protein of 336 amino acids with a predicted molecular weight of 37,450 Da. Comparison of the human sequence to the mouse sequence showed an 81% identity at the amino acid sequence level and galectin-12 contains two tandem arranged CRDs at amino acids P48 to V177 and P211 to L331. The two CRDs were connected by the linker sequence Q178 to V210. In addition, the sequence of the ﬁrst CRD was highly conserved while the second site differed from the consensus sequence. This suggested that galectin-12 has a different substrate binding afﬁnity compared to other isoforms. Comparison of galectin-12 binding with that of galectin-8 indicated that it did bind more weakly to lactosyl-agrose. Analysis of the genomic sequence in Genbank indicated that the gene contained 9 exons. In addition, the 5 ﬂanking region of the gene lacked a TATA consensus sequence but contains two SP1 binding sites, four AP-2 sites, a CCAAT/enhancer-binding site and a C/EBP binding site (Briggs et al., 1986; Mitchell et al., 1987; Thiesen and Bach, 1990). Analysis of several clones from monkey identiﬁed two clones that lacked 27 nucleotides from 598 to 624 which are encoded by exon 6. Whether or not these represent new alternative splice forms has not been determined. Analysis of the tissue distribution by northern blotting in mice and humans, showed that galectin-12 could not be detected in any tissues except adipose tissue (Yang et al., 2001). This adipose speciﬁc expression may be driven by the C/EBP promoter element which is also found in other adipose speciﬁc genes like PPAR␥, leptin and aP2 (Akira et al., 1990; Christy et al., 1989; Clarke et al., 1997; He et al., 1995; Herrera et al., 1989; Johnson, 1993). In 3T3-L1 cells, galectin-12 mRNA was not detectable in cells before differentiation but was detected 3 days after induction of differentiation. Members of the galectin family function either after being secreted from the cell and binding to extra cellular proteins or they function within the cell. Most of the galectins that function by binding proteins on the cell surface contain two functional CRDs, yet galectin-12 may have only one functional CRD. To determine how galectin-12 may function Hotta et al., studied the subcellular localization of the protein

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using several methods (Hotta et al., 2001b). In mature 3T3-L1 cells, immunocytochemical localization with a rabbit polyclonal antibody showed that galectin-12 was located primarily in the nucleus with a dotted pattern in the cytosolic region. Further analysis of mature 3T3-L1 cells by subcellular fractionation into nuclear, mitochondrial and cytosolic fractions indicated that the protein is localized in the nuclear and mitochondria fractions, but not the cytosol. Since the galectins mediate a number of biological functions, among which is the regulation of cell growth and apoptosis, analysis of galectin-12 expression in monkey and rat models was performed to monitor any changes associated with changes in fat content. In lean, obese and diabetic monkeys, there were no signiﬁcant differences in mRNA levels of galectin-12 in either subcutaneous or omental adipose tissue. However, in long-term CR monkeys, the levels of galectin-12 mRNA in adipose tissue were considerably higher than was seen in monkeys fed ad libitum. As in monkeys, Zucker rats have no differences between lean and the obese rats, but there was a considerable increase in galectin-12 mRNA when the rats were treated with troglitazone, an insulin sensitizer. Accompanying this increased expression induced by troglitazone was an increase in the number of apoptotic cells in adipose tissue. In attempting to control or even reduce the growth of adipose tissue, the regulation of galectin-12 expression by troglitazone appears to be one mechanism for achieving this goal. Since troglitazone and a number of galectins are known to induce apoptosis (Bernerd et al., 1999; Hadari et al., 2000; Okuno et al., 1998; Perillo et al., 1995; Wada and Kanwar, 1997; Wada et al., 1997), analysis of galectin-12’s effect by over expression in COS-1 cells was performed (Hotta et al., 2001b). TUNEL analysis of cells transfected with a galectin-12 expressing construct showed more TUNEL positive nuclei than in control cells. These data indicate that galectin-12 is capable of inducing apoptosis and its strict tissue speciﬁc expression pattern suggests it is involved with regulating adipose and possibly adipose tissue growth. Whether or not alterations in galectin-12 expression or function occurs in speciﬁc cases of obesity or diabetes remains to be determined. It is likely that a number of other functional and regulatory adipose tissue speciﬁc genes will be identiﬁed in the near future, and a better understanding of the interactions of these genes in adipose tissue development, insulin sensitivity and physiology will almost certainly lead to a more mechanistic understanding of insulin resistance.

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EFFECTS OF GENETIC ALTERATIONS OF GLUT4 ON INSULIN SENSITIVITY NAIRA GOROVITS, J. SKYE LAIDLAW, MOLLIE RANALLETTA, GLORIA TANNENBAUM, ELLEN B. KATZ AND MAUREEN J. CHARRON*

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA

INTRODUCTION Glucose is the main monosaccharide that supplies carbon and energy for almost all cells in humans, making the entry of the glucose into cells a crucial step in life supporting processes. Tight regulation of blood glucose levels, insuring adequate glucose ﬂux into tissues, is required in mammals where glucose is the primary energy source of brain tissue (Lund-Andersen, 1979; Pardridge, 1983). Glucose uptake depends on different factors including expression of appropriate glucose transporter proteins and hormonal regulation of their function. Alteration in insulin sensitivity is a primary feature of the pathogenesis of type 2 diabetes mellitus (Kahn et al., 2000; Mauvais-Jarvis and Kahn, 2000; Nakae et al., 2001). Therefore, studying insulin action at the molecular level provides a means for understanding the mechanisms of insulin resistance, and the potential for revealing new targets for the treatment of insulin resistance and type 2 diabetes. The molecular manipulation of genes to investigate the mechanisms of insulin action and resistance is a powerful tool of contemporary science (Katz et al., 1996; Mauvais-Jarvis and Kahn, 2000). This “gene knockout” technology allows scientists to generate homozygous mutant mice (nulls) in which the effects of the total lack of a particular gene product can be studied. Heterozygous null mutants provide answers to the questions of gene dosage and the compensatory abilities of single alleles. In addition, reconstitution of multiple gene alterations can be achieved through the mating of homo/heterozygous knockout animals with various mutations. Finally, to avoid lethal homozygous mutations or abolish a gene’s expression in a speciﬁc tissue, tissue speciﬁc knockouts and conditional/inducible mutants can be generated utilizing the Cre/LoxP system (Rajewsky et al., 1996). Mutations have been introduced at key steps of the insulin-signaling cascade to elucidate the mechanisms of insulin resistance and the development of type 2 diabetes mellitus (for reviews, see Kahn et al., 2000; Mauvais-Jarvis and Kahn, 2000; Nakae et al., 2001). The ﬁrst step of insulin signaling is binding of the hormone to its receptor (IR) on target tissues. The inactivation of the murine IR resulted in pups exhibiting a severe form of diabetes and a very short life span (Accili et al., 1996; Joshi et al., 1996). A single allele of IR appeared to compensate for the lack of the other since, depending on the genetic background, only about 10%

* Address correspondence to: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. Tel.: 718-430-2852; Fax: 718-430-8676. 125

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of IR/ mice developed type 2 diabetes (Accili et al., 1996; Bruning et al., 1997). To avoid the extreme phenotype of a general knockout, the Cre/LoxP system was used to create tissue speciﬁc IR knockout mice including muscle (MIRKO) (Bruning et al., 1998), ␤-cell (␤IRKO) (Kulkarni et al., 1999), liver (LIRKO) (Michael et al., 2000), and neuron (NIRKO) knockout mice (Bruning et al., 2000). MIRKO mice showed impaired insulin stimulated glucose uptake into skeletal muscle accompanied by altered fat metabolism. Exercise stimulation of glucose uptake into MIRKO soleus muscle appeared to be unaltered (Wojtaszewski et al., 1999). ␤IRKO mice exhibited loss of insulin secretion and deterioration of glucose tolerance revealing the necessity of appropriate insulin and IR association for normal pancreatic ␤-cell function (Kulkarni et al., 1999). IR deﬁciency in the liver of LIRKO mice led to severe glucose intolerance in 2-month-old mice with fasting glucose levels returning to control levels at the age of 4 months (Michael et al., 2000). Finally, neuron-speciﬁc disruption of IR in NIRKO mice resulted in impaired energy homeostasis with diet induced obesity, hyperinsulinemia and hypertriglyceridemia (Bruning et al., 2000). These studies conﬁrm that normal expression of IR in these key tissues is essential for maintenance of glucose homeostasis and that there is no alternative pathway that can compensate for the lack of IR. The next step in the insulin signaling cascade involves the insulin receptor substrate proteins (IRS1–4). IRS-1 null mice exhibited retarded embryonic and postnatal development and decreased insulin/IGF stimulated glucose uptake in vivo and in vitro. However, because of compensatory insulin secretion by hyperplastic ␤-cells, these mice displayed nearly normal glucose tolerance (Araki et al., 1994; Tamemoto et al., 1994; Terauchi et al., 1997). This residual insulin action led to the discovery of the IRS-2 molecule (Kadowaki et al., 1996). Consequently, targeted disruption of IRS-2 yielded mice which developed type 2 diabetes due to an 80% reduction in ␤-cell mass and peripheral insulin resistance (Kubota et al., 2000; Withers et al., 1998). IRS-3 and IRS-4 knockout mice demonstrated normal or slightly impaired growth and glucose homeostasis, respectively, pointing to a secondary role for these proteins in maintenance of glycemia (Fantin et al., 2000; Liu et al., 1999). PI3 kinase, the next step in the insulin-signaling pathway, controls many cellular functions including proliferation, apoptosis, and cell motility and adhesion (Toker and Cantley, 1997). Knockout mice containing a homozygous mutation in the p110 catalytic subunit of PI3 kinase appeared normal (Sasaki et al., 2000). However, further investigation revealed that p110 was necessary for normal immune system function, speciﬁcally thymocyte development and T cell activation. Deleting only p85␣ PI3 kinase regulatory subunit, resulted in viable mice with increased insulin sensitivity (Terauchi et al., 1999). Whereas, when the Pik3r1 gene that encodes for all three regulatory subunits, p85␣ and its two smaller variants, p55␣ and p50␣, was deleted, the mice died perinatally from hypoglycemia and liver necrosis demonstrating the importance of expressing all three variants (Fruman et al., 2000). Protein kinase B/Akt has been identiﬁed as a downstream target of insulin and various growth factors that signal through PI3 kinase (Burgering and Coffer, 1995). Recently, a knockout mouse with a disruption of Akt2, the isoform found mainly in insulin-sensitive tissues (Calera et al., 1998), has been generated (Cho et al., 2001a). The Akt2 null animals have mild fasting hyperglycemia. The in vivo glucose uptake into the glycolytic EDL muscle is blunted at low insulin (0.33 nM), but is normal at a maximal concentration of insulin (13.3 nM). The oxidative soleus muscle shows no decrease in glucose uptake under insulin stimulation. In addition, the ablation of Akt2 had no effect on the level of GLUT4 expressed in skeletal muscle. Isolated adipocytes showed a mild decrease in insulin stimulated glucose uptake. The effects of Akt2 ablation on the liver were more severe. Insulin was not able to normally suppress hepatic glucose production. Taken together, these results suggest that Akt2 is necessary for

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normal glucose homeostasis (Cho et al., 2001a). Modiﬁcations of Akt1 gene have been performed in vitro and in vivo, including the construction of conditionally and constitutively active forms of the molecule, the creation of a dominant negative mutant of Akt and generation of Akt1 null mouse (Cho et al., 2001b; Kohn et al., 1996, 1998; van Weeren et al., 1998). The animal model appeared to be metabolically normal, but displayed a striking impairment in both fetal and postnatal growth suggesting a non-redundant functions of Akt2 and Akt1 with respect to metabolic actions of insulin and organismal growth. These and other studies identiﬁed Akt as a key molecule involved in many signaling pathways such as those leading to glycogen synthesis, glucose transport and growth (Brozinick and Birnbaum, 1998; Cho et al., 2001b; Kohn et al., 1996; Summers and Birnbaum, 1997; van Weeren et al., 1998; Wang et al., 1999). Every step in the insulin-signaling cascade contributes to maintaining normal glucose homeostasis, including the effector molecules, the glucose transporters GLUT1–5, which have been studied extensively (for reviews see Czech and Corvera, 1999; Mueckler, 1994; Olson and Pessin, 1996). Recently, new members of the glucose transporter family have been cloned including GLUTx1/GLUT8 (Carayannopoulos et al., 2000; Doege et al., 2000b; Ibberson et al., 2000), two distinct proteins named GLUT9 (Doege et al., 2000a; Phay et al., 2000) and GLUT10 (McVie-Wylie et al., 2001). GLUT8 has been shown to transport glucose. This new transporter is expressed in mouse blastocyst and a wide variety of adult tissues (Carayannopoulos et al., 2000; Doege et al., 2000b; Ibberson et al., 2000; Reagan et al., 2001). These include testis, liver, spleen, brain stem, cerebellum, hippocampus, hypothalamic principal and non-principal neurons, adrenal gland, brown adipose tissue, lung, muscle, kidney, tongue and white adipose tissue. Two independent groups have identiﬁed two distinct new glucose transporters originally referred to as GLUT9 (Doege et al., 2000a; Phay et al., 2000). The expression of one (currently GLUT6) is restricted to brain and lymphoid tissue (spleen and peripheral blood leukocytes) and it has also been shown to transport glucose (Doege et al., 2000a). The expression of GLUT9 was shown in kidney and liver with very low expression in lung, placenta, leukocytes, skeletal muscle and heart (Phay et al., 2000). Finally, the most recent addition to this family of transporters, GLUT10, is proposed to be an NIDDM susceptibility gene (McVie-Wylie et al., 2001). The expression of its 4.3 kb mRNA is highest in the liver and pancreas. GLUT10 was mapped to an area of chromosome 20 that contains several markers of type 2 diabetes. The research in our laboratory focuses on GLUT4, the insulin responsive glucose transporter expressed in muscle, heart and adipose tissue (Kahn, 1992; Mueckler, 1994). GLUT4 translocates from an intracellular compartment to the plasma membrane upon insulin stimulation where it facilitates glucose transport (Pessin et al., 1999; Simpson et al., 2001). Reduced insulin stimulated glucose transport resulting from defects in different signaling molecules and/or impaired insulin stimulated translocation of GLUT4 to the plasma membrane has been implicated in the development of type 2 diabetes (Shepherd and Kahn, 1999). The translocation of GLUT4 to the plasma membrane can also be activated by muscle contraction or ischemia/hypoxia independent of insulin (Klip and Marette, 1992). Using homologous recombination, our laboratory generated mice lacking GLUT4 (Katz et al., 1995; Stenbit et al., 2000). These homozygous GLUT4 null and heterozygous GLUT4 knockout (GLUT4/) mice will be presented in detail in this review. The non-diabetic phenotype of GLUT4 null mouse stimulated the search for alternative pathways for glucose transport Charron et al., 1999). Additional studies with targeted overexpression of GLUT4 in the skeletal, adipose and heart tissues of normal, high fat fed or genetic models of diabetes demonstrated the improvement of glucose homeostasis despite insulin signaling defects (for review Charron et al., 1999; Galuska et al., 1998; Katz et al., 1996). Transgenic complementation of GLUT4 in fast twitch muscle restored insulin stimulated glucose transport and

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prevented the development of hyperinsulinemia and hyperglycemia in GLUT4/ mice that are genetically predisposed to diabetes (Tsao et al., 1999). Subsequently, Cre/LoxP gene targeting was used to selectively disrupt the GLUT4 gene in heart (Abel et al., 1999), adipose (Abel et al., 2001) and muscle tissue (Zisman et al., 2000). Heart GLUT4 knockout mice did not exhibit any perturbations in glucose homeostasis (Abel et al., 1999). Lack of GLUT4 only in adipose tissue resulted in mice with glucose intolerance and hyperinsulinemia (Abel et al., 2001). Muscle speciﬁc GLUT4 knockout mice developed mild insulin resistance and glucose intolerance whereas mice heterozygous for the muscle speciﬁc GLUT4 deletion exhibited a somewhat intermediate phenotype (Zisman et al., 2000). Each of these models will be discussed in detail later in the chapter. Understanding the roles of the existing and potential players involved in the maintenance of normal glucose homeostasis and insulin sensitivity requires careful analysis of each molecule. Below, we focus on one piece of this enormous puzzle; speciﬁcally, what is known and what is yet to be discovered about the function of GLUT4 through the use of the GLUT4 knockout model. GENERAL PHENOTYPE OF GLUT4 NULL AND GLUT4/ KNOCKOUT MICE Since the development of the GLUT4 null mouse in 1995, our laboratory has been striving to elucidate the mechanisms responsible for its anti-diabetic phenotype (Katz et al., 1995; Stenbit et al., 1997). Although complete ablation of GLUT4 does not cause overt type 2 diabetes, the lack of the insulin sensitive glucose transporter does affect whole body glucose homeostasis on multiple levels. As expected, GLUT4 null mice have impaired insulin tolerance. Surprisingly, the mice exhibit normal glucose and insulin levels in both the fed and fasted state. Morphologically, the GLUT4 null mice exhibit signiﬁcant cardiac hypertrophy and dramatically reduced body weights attributable to the almost complete lack of white adipose tissue. GLUT4 null sera contains depressed levels of lactate and free fatty acids in both the fed and fasted state as compared to controls reﬂecting the altered pattern of substrate utilization by GLUT4 null (Katz et al., 1995). The morphological alterations and the changes in metabolism were originally thought to be responsible for the somewhat shortened life span seen in the GLUT4 null mice born soon after the derivation of the line (Katz et al., 1995). Subsequently, after years of breeding the GLUT4 null into a more homogenous genetic background (i.e. CD1 and C57/Bl6), the GLUT4 null mice now live a normal life span. Ablation of only one allele of GLUT4 results in a majority of the male GLUT4/ mice developing a diabetic phenotype with hyperglycemia and hyperinsulinemia (Stenbit et al., 1997). Until 2–4 months of age, male GLUT4/ mice express normal levels of GLUT4 in both muscle and adipose tissue. Subsequently, GLUT4 content in the GLUT4/ mice decreases approximately 50%, 26% and 46% in adipose tissue, EDL, and soleus, respectively, as compared to normal age matched controls. Concurrently, the male GLUT4/ mice develop pre-diabetic and diabetic phenotypes while the females remain normal. The GLUT4/ mice can be divided into three phenotypic groups according to fed serum glucose and insulin concentrations: N/N-normal fed glycemia (200–220 mg/dl) and normal insulin (10 ng/ml), prediabetic N/H-normal fed glycemia and hyperinsulinemia (25 ng/ml), or diabetic H/H-hyperglycemia (350 mg/dl) and hyperinsulinemia (25 ng/ml). After 5 months of age, the majority of GLUT4/ mice develop a diabetic phenotype. Under a euglycemic/hyperinsulinemic clamp diabetic GLUT4/ mice exhibit a 50% decrease in glucose disappearance as compared to controls, indicating insulin resistance (Stenbit et al., 1997). In addition, glucose uptake of EDL and soleus of diabetic GLUT4/ is decreased

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34% and 38% as compared to controls (Stenbit et al., 1997). Morphological consequences of the diabetic phenotype include hearts with hypertrophied myocytes and focal necrosis and livers that contain extensive micro- and macro-steatosis. WHOLE BODY EFFECTS OF GLUT4 ABLATION The morphological effects of partial and complete disruption of GLUT4 on the major GLUT4 expressing tissues (muscle, fat and heart) and those involved in whole body glucose metabolism (liver) were assessed (Table 1). A longitudinal growth study was conducted on both sexes of the GLUT4 null and GLUT4/ mice at 9, 18 and 24 weeks of age. Since insulin-like growth factor 1(IGF-1) has been shown to affect whole body and organ growth (Harel and Tannenbaum, 1995; Le Roith et al., 2001), serum IGF-1 levels were also measured. The following details of this study show that altering whole body GLUT4 content manifests itself morphologically in multiple organs. GLUT4 null mice of both sexes exhibit a signiﬁcant 5–7% decrease in body length as compared to controls (Table 1). Also at all ages, GLUT4 null mice show signiﬁcantly reduced Table 1 Morphological characteristics of male and female GLUT4 null, Glut4/ and control mice aged 9, 18 and 24 weeks. Genotype/ Length Age (wks) (cm) Male W. T 9 9.2 0.26 18 10.0 0.41 24 10.0 0.21 GLUT4/ 9 8.9 0.18* 18 9.2 0.35* 24 9.3 0.41* GLUT4/ 9 9.6 0.28* 18 10.6 0.28* 24 10.4 0.24* Female W. T 9 8.8 0.26 18 9.3 0.31 24 9.7 0.25 GLUT4/ 9 8.7 0.40 18 8.9 0.41* 24 9.6 0.39* GLUT4 / 9 9.3 0.29 18 9.6 0.18 24 10.2 0.34*

Body weight (g)

Heart weight (g)

Liver weight (g)

Gonadal Perirenal Serum fat weight (g) fat weight (g) IGF-1

29.4 2.30 42.3 1.70 46.2 2.60

0.14 0.01 0.16 0.01 0.21 0.03

1.3 0.13 2.0 0.37 2.5 0.50

0.8 0.20 1.7 0.30 1.7 0.20

0.2 0.06 0.4 0.09 0.6 0.10

169.1 11.21 191.3 15.72 152.5 12.1

26.9 2.80 0.21 0.01* 1.4 0.20 0.4 0.10* 28.1 3.30* 0.29 0.17* 1.3 0.20* 0.4 0.30* 33.6 3.90 0.24 0.04* 1.7 0.40* 0.7 0.20*

0.1 0.04* 167.69 14.49 0.2 0.10* 143.92 14.88 0.3 0.10* 135.88 9.66

35.3 3.30* 0.17 0.02* 1.7 0.20* 1.1 0.30* 46.6 3.80* 0.18 0.02 2.9 0.70* 1.0 0.70* 45.9 3.90 0.19 0.02 2.8 0.80 1.1 0.80*

0.3 0.10* 160.37 8.43 0.4 0.10 167.00 10.55 0.4 0.10* 180.61 17.88

21.6 1.70 30.2 3.10 33.3 4.30

0.4 0.20 1.5 0.40 1.9 0.70

0.1 0.06 0.3 0.02 0.5 0.23

0.1 0.10* 0.2 0.07* 0.3 0.30*

0.05 0.02* 137.16 12.9 0.11 0.10* 129.44 8.17 0.13 0.10* 133.69 21.11

0.12 0.01 0.12 0.01 0.13 0.01

1.0 0.11 1.3 0.14 1.4 0.20

20.0 2.00* 0.19 0.02* 1.0 0.14 23.9 2.60* 0.22 0.03* 1.2 0.29 25.7 3.30* 0.2 0.03* 1.3 0.28

23.1 2.40 0.13 0.02 1.13 0.21 0.3 0.14 35.9 5.10* 0.15 0.02* 1.6 0.28* 1.7 0.57 41.5 7.30* 0.15 0.02 1.8 0.68* 2.7 1.40

* Signiﬁcance is attributed to p 0.05 as compared to control values at the same age.

140.97 11.19 137.82 6.18 160.61 11.33

0.1 0.07 159.8 7.05 0.5 0.10* 146.76 11.19 0.8 0.50 164.91 10.68

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perirenal and gonadal fat mass while the hearts exhibit a 1.5–2-fold increase in weight as compared to controls when corrected for body weight. Finally, livers of GLUT4 null males weigh 35% less than controls at age 18 and 24 weeks. However, livers from female GLUT4 null mice when corrected for body weight weigh more than controls. In contrast to GLUT4 null mice, both sexes of GLUT4/ mice exhibit a 5% increase in body length at all ages as compared to control mice (Table 1). These studies conﬁrm that expression of GLUT4 is essential for normal skeletal development (Maor and Karnieli, 1999). In addition, the GLUT4/ mice demonstrate a sexually dimorphic proﬁle when considering gonadal fat pad weight. Female GLUT4/ mice, which do not become diabetic, exhibited no differences in gonadal fat pad mass compared to controls. Male GLUT4/ mice had larger gonadal fat pads than controls at 9 weeks, which became comparatively smaller with age. The differences seen in male GLUT4/ gonadal fat pad mass may be attributable to the development of diabetes with age. Male GLUT4/ mice exhibit an 18% increase in cardiac mass compared to controls at the age of 9 weeks, while the females show a similar increase at 18 weeks. Both sexes of GLUT4/ mice demonstrate a 25–30% increase in liver weight as compared to control. Histological examination showed that this increase in weight correlates with extensive steatosis present in the GLUT4/ liver. Because of the differences in length, body weight and organ weights seen in GLUT4 null and GLUT4/ mice as compared to controls, it was expected that serum IGF-1 levels would be altered. Surprisingly, there was no difference in fed or fasted levels of IGF-1 among the genotypes studied (Table 1). These data suggest that IGF-1 does not play a role in the altered phenotypes of GLUT4 null and GLUT4/ mice. However, the lack or alterations of GLUT4 expression in muscle and adipose tissue and the consequent changes in energy substrate metabolism have profound effects on the morphological development of the homozygous and heterozygous GLUT4 knockout mice.

EFFECT OF GLUT4 MANIPULATION ON MUSCLE GLUCOSE UPTAKE AND PERFORMANCE GLUT4 is expressed in insulin sensitive tissues with muscle accounting for about 80% of insulin stimulated glucose uptake (DeFronzo et al., 1981). Insulin stimulation, muscle contraction, ischemia and hypoxia result in translocation of GLUT4 from an intracellular pool to the plasma membrane where it mediates glucose transport (Klip and Marette, 1992; Pessin et al., 1999). Reduction in insulin stimulated translocation of GLUT4 to the plasma membrane has been implicated in the development of type 2 diabetes (Shepherd and Kahn, 1999). Therefore, it was expected that the ablation of one or both alleles of GLUT4 would have a severe effect on muscle development and metabolism resulting in insulin resistance and diabetes. The majority of male GLUT4/ mice exhibit a progressive decrease in whole body GLUT4 content accompanied by the development of hyperinsulinemia and hyperglycemia. Muscle insulin resistance was demonstrated by an in vitro assessment of glucose transport in isolated oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles from GLUT4/ mice (Stenbit et al., 1997). In these studies a 45% reduction in GLUT4 protein levels in skeletal muscle resulted in a 34–38% decrease in insulin stimulated glucose transport in both EDL and soleus in vitro (Stenbit et al., 1997). In vivo studies utilizing hyperinsulinemic/ euglycemic clamp showed a 65% decrease in muscle glucose uptake rate in prediabetic GLUT4/ mice as compared to controls. In addition, GLUT4/ mice had a 2–2.4-fold

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reduction in the rate of glycolysis and glycogen synthesis without altered muscle glycogen synthase activity (Rossetti et al., 1997). These studies in the GLUT4/ mice demonstrated for the ﬁrst time the inability of one allele of GLUT4 to express adequate GLUT4 levels for maintaining whole body glucose homeostasis and correct glucose partitioning. Because several studies have established the importance of proper GLUT4 function in maintaining whole body glucose homeostasis, the ablation of GLUT4 was expected to lead to fetal mortality or at least a diabetic phenotype. The unexpected survival of GLUT4 null mice that do not develop diabetes prompted studies on the skeletal muscle, the tissue containing the most GLUT4. Intriguingly, GLUT4 null soleus and EDL exhibited different capacities for insulin stimulated glucose transport (Stenbit et al., 1996). The in vitro analysis of EDL from both sexes of GLUT4 null mice demonstrated an expected proﬁle of slightly reduced basal glucose uptake with no insulin stimulated glucose uptake. The contrasting result was seen in the soleus. In the male GLUT4 null soleus a 2.3-fold compensatory increase in basal glucose uptake was measured; however, there was no stimulatory effect of insulin on glucose transport. The female soleus exhibited normal basal levels of glucose transport with a small, yet signiﬁcant, increase upon insulin stimulation (Stenbit et al., 1996). This unexpected compensatory phenomenon in GLUT4 null muscle was proposed to be due to a novel facilitated glucose transport activity (Stenbit et al., 1996). Thus, the complete elimination of GLUT4 unveiled this compensatory activity in skeletal muscle. Experiments similar to those described above were conducted in muscle speciﬁc GLUT4 knockout mice (Zisman et al., 2000). These mice exhibited mild insulin resistance and glucose intolerance. Surprisingly, there was an 80% reduction in basal glucose uptake in EDL and soleus with no decrease in GLUT1 protein content. The inability of insulin to stimulate muscle glucose transport in vitro suggests that the compensatory mechanisms unmasked by complete ablation of the functionally dominant GLUT4 in our model were not initiated in the muscle speciﬁc knockout. The muscles in the muscle speciﬁc GLUT4 knockout still express about 5% of the normal amount of GLUT4 protein in their muscles which could inhibit or mask any expression or function of a compensatory insulin independent glucose transport system (Zisman et al., 2000). In addition, the Lox/Lox genotype exhibited decreased GLUT4 protein content in adipose tissue suggesting that 3 regulatory elements in the GLUT4 gene may have been affected (Zisman et al., 2000). This reduction in adipocyte GLUT4 could affect adipose tissue function and cause some of the effects on whole body glucose homeostasis seen in this model (Zisman et al., 2000). Indeed, in our GLUT4/ model of diabetes, the ﬁrst lesion to be seen is a signiﬁcant reduction of GLUT4 content in adipose tissue followed by a reduction in muscle GLUT4 (Stenbit et al., 1997). Further evidence for the existence of a novel GLUT4 independent transporter system responsible for the compensatory glucose transport in GLUT4 null muscle was found in the ability of cytochalasin B to cause a dose dependent inhibition of glucose transport activity in GLUT4 null soleus (Ryder et al., 1999a). The potential existed that overexpression of GLUT1 and/or ectopic expression of other known GLUTs was responsible for the increase in basal glucose transport in male soleus and the partially preserved insulin stimulated glucose transport in female soleus of GLUT4 null mice. However, no increase in either total or plasma membrane GLUT1 protein content in GLUT4 null soleus or EDL muscles or expression of any other known GLUT was found (Ryder et al., 1999a; Stenbit et al., 1996). Consequently, expression of a novel GLUT4 independent glucose transport activity was proposed (Charron et al., 1999; Ryder et al., 1999a; Stenbit et al., 1996). There are several documented pathways for stimulation of glucose transport in muscle that are insulin independent (Czech and Corvera, 1999). One such pathway is activated by

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hypoxia or exercise/contraction (Goodyear and Kahn, 1998; Klip and Marette, 1992; Rodnick et al., 1992). An in vitro study demonstrated that the compensatory glucose uptake system in the GLUT4 null muscle could not be activated by exposing the muscle to 45 min of hypoxia followed by 20 min of normoxia (Zierath et al., 1998). This study demonstrated that GLUT4 is necessary for hypoxic stimulation of glucose transport in both oxidative and glycolytic muscles. High-energy phosphate stores appeared to be unaltered in the EDL and soleus of female GLUT4 null mice under normoxic and hypoxic conditions (Zierath et al., 1998). Male soleus muscles, however, exhibited a signiﬁcant (30–45%) decrease in ATP levels under both hypoxic and normoxic conditions (Zierath et al., 1998). On the other hand, an exhaustive bout of swimming (e.g. 6 rounds, each lasting 30 min) led to a 2-fold increase in glucose transport in GLUT4 null EDL, highlighting the different effects of hypoxia and intense exercise on muscle glucose uptake (Ryder et al., 1999b). Since glucose uptake into GLUT4 null EDL was unable to be triggered by insulin, a different mechanism/pathway of stimulation of the compensatory glucose transport must be active in the swim exercised GLUT4 null muscle. Indeed, GLUT4 null EDL muscle demonstrated a small, yet signiﬁcant increase (1.6-fold) of glucose transport activity following electrical stimulation that mimics the effects of exercise (Ryder et al., 1999b). In contrast, electrically stimulated contraction did not alter the in vitro glucose transport rate in EDL and soleus from muscle speciﬁc GLUT4 knockout mice (Zisman et al., 2000). The reason for this discrepancy may lie in the balance between glucose partitioning and energy needs in the muscle under hypoxia or exercise/contraction conditions. It has been shown that the amount of glycogen stored in the rat fast-twitch gastrocnemius muscles correlated inversely with the stimulatory effect of insulin on glucose transport (Derave et al., 2000). GLUT4 is necessary for the acute repletion of glycogen after exercise (Ryder et al., 1999b). Thus, the low-level expression of GLUT4 protein in the muscle speciﬁc GLUT4 knockout may provide adequate ﬂow of glucose to glycogen under acute exercise conditions avoiding depletion of its stores. In addition, 10 min of electrical stimulation may be insufﬁcient to exhaust the energy depots in the muscle speciﬁc GLUT4 knockout mouse (Zisman et al., 2000). Perhaps the depletion of glycogen stores in GLUT4 null EDL after an exhaustive bout of swim exercise represents a more potent stress than that induced by hypoxia or brief electrical stimulation and, thus, provokes increased glucose uptake (Ryder et al., 1999b). Contractile function following in vitro electrical stimulation was also studied in GLUT4 null EDL (Ryder et al., 1999b). Isometric tension developed by muscle during 0.2 ms contraction was measured every 2 min for a total of 10 min and appeared to be similar between wild-type and GLUT4 null muscle (Ryder et al., 1999b). Similarly, when the tetanic torque was normalized per unit muscle mass, the isometric contractile properties studied in the intact dorsal ﬂexor complex appeared to be unaltered in GLUT4 null compared to controls (Gorselink et al., 2002). However, the maximal peak power was signiﬁcantly lower in GLUT4 null muscle complex even after normalization per unit muscle mass. One of the principal determinants of peak power output is the ﬁber type composition of muscle (Bottinelli et al., 1991). The peak power of rat skinned ﬁbers was demonstrated to be 1.5 times lower in fasttwitch oxidative (IIA) than in fast-twitch glycolytic (IIB) type ﬁbers. In fact, there is a shift in ﬁber type composition in GLUT4 null EDL and tibialis anterior muscles from glycolytic ﬁbers to fast-twitch oxidative ﬁbers (Gorselink et al., 2002). In addition, subjecting dorsal ﬂexors to a series of 150 shortening contractions demonstrated increased muscle fatigability in GLUT4 null compared to wild type control mice (Gorselink et al., 2002). The comparison of work output between control and GLUT4 null dorsal ﬂexors showed a substantial decline in the parameters of GLUT4 null muscle. The glycogen

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levels were signiﬁcantly lower in dorsal ﬂexors of GLUT4 null mice, suggesting that appropriate glucose targeting to glycogen through GLUT4 is essential for sustained muscle performance. To establish the metabolic fate of glucose in GLUT4 null muscle, studies were performed using a pentaacetate ester of D-glucose that is taken up independent of the glucose transporter system (Ladriere et al., 1999; Malaisse et al., 1997). Incorporation of D-[U-14]C glucose into glycogen was measured in diaphragm, soleus, and EDL muscles of GLUT4 null mice. Glucose incorporation into glycogen was only 34% ( p 0.005) of wild type values. However, there was no signiﬁcant difference ( p 0.3) in the incorporation of D-[U-14]C glucose pentaacetate into glycogen between GLUT4 null and control tissues. Catabolic parameters of D-glucose and its metabolism, including the oxidation of D-[U-14]C glucose and generation of 14C labeled lactic acid and amino acids, were not different between muscles derived from GLUT4 null and control mice. Thus, the defect in D-glucose metabolism in GLUT4 null muscle primarily affects synthesis of glycogen and not catabolism of glucose.

CARDIAC PROFILE OF GLUT4 NULL AND GLUT4/ MICE GLUT4 is responsible for the majority of glucose uptake in the heart with the rest being taken up through GLUT1 and GLUT3 (Egert et al., 1999; Grover-McKay et al., 1999). Normally the heart relies upon fatty acids to meet its energy needs (Opie, 1992a; Opie, 1992b). As availability of free fatty acids (FFA) decreases, or when faced with hypoxic/ischemic conditions, the heart switches to glucose as its primary substrate for energy production. Since GLUT4 is the major glucose transporter in the heart, ablation of GLUT4 was expected to affect the performance of the heart. As mentioned above, the GLUT4 null heart is severely hypertrophied (Katz et al., 1995). When compared to age and weight matched controls, the GLUT4 null hearts show a 1.4–1.5fold increase in heart to body weight ratio (Katz et al., 1995). Histological examination of the GLUT4 null heart showed hypertrophied myocytes and marked necrosis, including interstitial and replacement ﬁbrosis, predominantly in the left ventricle (LV) (Stenbit et al., 2000). Magnetic resonance imaging of 16-week-old mice revealed an increase in LV thickness, without a decrease in LV chamber diameter as compared to age matched controls. The LV free wall is increased 1.5-fold in GLUT4 null hearts as compared to controls, while the septum, anterior and posterior free walls show a 1.3-fold increase ( p 0.05). Both the anterior–posterior and left free wall-septum internal diameters were not signiﬁcantly different in GLUT4 null hearts from controls. The degree of hypertrophy in the GLUT4 null heart described above differs from the milder hypertrophy reported in the cardiac speciﬁc GLUT4 knockout, G4H/ mice, suggesting a role for GLUT4 in normal cardiac morphological development (Abel et al., 1999). Longitudinal image analysis demonstrated GLUT4 null hearts to have comparable ejection fraction to control hearts (Stenbit et al., 2000). Upon stimulation with isoproterenol, the GLUT4 null hearts failed to mount the appropriate increase in left ventricular pressure response suggesting a downregulation of ␤-adrenergic receptors (Stenbit et al., 2000). However, the hemodynamic proﬁle, including LV and arterial pressure tracings, of GLUT4 null mice were normal. These studies indicated that the cardiac hypertrophy present in the GLUT4 null mice was not due to pressure overload (Stenbit et al., 2000). One of the hallmarks of pathological cardiac hypertrophy is the reversion of contractile proteins troponin and myosin from the adult isoforms to their fetal counterparts (Buttrick et al.,

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1994; Chien et al., 1991; Dorn et al., 1994; Mercadier et al., 1981; Resnick, 1992; Scheuer and Buttrick, 1987; Swynghedauw, 1986). The purpose of this switch in isoform that results in diminished contractile performance is thought to be conservation of ATP (Buttrick et al., 1994; Dorn et al., 1994; Scheuer and Buttrick, 1987; Swynghedauw, 1986). However, GLUT4 null hearts do not exhibit altered levels of myosin, nor do they switch to the fetal isoform (V1 to V3) illustrating the uniqueness of GLUT4 null cardiomyocyte hypertrophy (Stenbit et al., 2000). Furthermore, although GLUT4 null hearts express the appropriate isoforms of troponin (TnI and TnT), the levels are signiﬁcantly increased (52–62% and 78%, respectively) in GLUT4 null hearts as compared to controls. These alterations in TnI and TnT in the GLUT4 null heart could have effects on the rate of phosphorylation, thereby reducing calcium sensitivity in myocytes (Malhotra, 1990; Malhotra et al., 1995; Redaelli et al., 1998). Most models of hypertrophy are coincident with a switch in myocardial substrate use from fatty acids to glucose (Neely et al., 1972; Vary et al., 1981). Medium chain acyldehydrogenase (MCAD) and long chain acyldehydrogenase (LCAD), enzymes involved in fatty acid oxidation, are decreased in the GLUT4 null heart (Stenbit et al., 2000). Male and female GLUT4 null mice exhibit similar decreases in MCAD (47% and 49%) and LCAD (31% and 34%), respectively, compared to controls. Even though the unique GLUT4 null heart exhibited compromised contractility at baseline conditions, it was able to maintain a high energetic proﬁle. NMR revealed normal glucose uptake in GLUT4 null hearts even though they lack the major glucose transporter (Stenbit et al., 2000). Other known GLUTs were measured in the GLUT4 null heart with only GLUT1 showing a modest upregulation compared to controls (Katz et al., 1995). The normal glucose uptake seen in the GLUT4 null heart is accompanied by a 3-fold increase in glycogen synthesis rates. This varies from the cardiac speciﬁc GLUT4 knockout mice which show a 3-fold increase in GLUT1 expression and consequently a 3-fold increase in basal glucose uptake with no further increase with insulin stimulation (Abel et al., 1999). The low levels of MCAD and LCAD in conjunction with the glucose uptake data suggest that the GLUT4 null heart uses glucose as its primary source of energy. These studies, combined with the muscle glucose uptake experiments, were the catalysts for the search for novel glucose transporters. The GLUT4/ mice exhibit cardiac pathology commonly associated with type 2 diabetes (Stenbit et al., 1997). The global disruption of one allele of GLUT4 results in hypertrophied cardiomyocytes, interstitial ﬁbrosis and necrosis (Stenbit et al., 1997). As is typical in most type 2 diabetics, the diabetic GLUT4/ mice also suffer from hypertension (Stenbit et al., 1997). Although the diabetic GLUT4/ mice exhibit increased arterial pressure, there is no difference in cardiac contractile performance when compared to controls (Stenbit et al., 1997). It is evident from the data presented above that the morphological and functional characteristics of the GLUT4 null, GLUT4/ and cardiac speciﬁc GLUT4 knockout hearts are very different. However, all of the models conﬁrm the need for adequate levels of GLUT4 expression and/or function for normal cardiac growth and function.

ADIPOSE TISSUE Adipose tissue is one of the main sites of insulin regulated glucose uptake (Kahn, 1992; Kahn and Flier, 2000). Type 2 diabetes is often associated with increased adipose tissue mass coincident with increased adipocyte cell size, and insulin resistance (DeFronzo et al., 1981; Shepherd and Kahn, 1999). The increased cell size of diabetic adipocytes is associated with an increased

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volume of stored triacylglycerides. Not only is adipose tissue important for insulin sensitivity, it is also involved in the production of hormones and factors important in energy homeostasis. These include adipsin, TNF-␣, leptin, and ACRP30 which play critical roles in energy balance and insulin sensitivity (Ailhaud et al., 1992; Hwang et al., 1997; Scherer et al., 1995; Spiegelman et al., 1993). INSULIN SIGNALING AND GLUCOSE UPTAKE IN THE GLUT4 NULL ADIPOCYTES Insulin signaling was studied in GLUT4 null adipocytes from 3–4-month-old females. Individual adipocyte cell size/lipid content is reduced 60%, and the protein content of GLUT4 null adipocytes is decreased 42% compared to controls (Tsao et al., 1997). Under basal conditions, adipose tissue uses GLUT1 as the major glucose transporter. During insulin stimulation, GLUT4 is responsible for the majority of glucose transport (Kahn, 1992). When comparing basal adipocyte glucose transport there was no difference between GLUT4 null and control mice (Li et al., unpublished observations). This is consistent with normal expression levels of GLUT1 mRNA and protein content in GLUT4 null fat. Maximal insulin stimulated glucose transport in control adipocytes was 5.6-fold higher than under basal conditions. As expected, adipocytes from GLUT4 null mice showed no increase in glucose transport upon insulin stimulation. Because the small size of GLUT4 null adipocytes could be due to development arrest at the pre-adipocyte stage, speciﬁc markers of adipocyte differentiation and genes involved in fatty acid synthesis were examined (Li et al., unpublished observations). When compared to controls, the differentiation marker PPAR␥2, fatty acid synthase and hexokinase II mRNA levels were actually increased 50%, 8.4-fold and 3.7-fold, respectively. There were no differences in expression levels of the differentiation markers C/EBP␣ and aP2 between GLUT4 null and control adipocytes. Thus, GLUT4 null adipocytes, although smaller, were shown to be mature differentiated adipocytes. Insulin signaling in vivo was assessed by measurement of phosphorylation of downstream signaling molecules after portal vein insulin injections in both GLUT4 null and control mice (Li et al., unpublished observations). Activity of upstream regulators of the insulin signaling pathway were also examined. Normal basal and insulin stimulated IRS-1 and IRS-2 associated PI3 kinase activity was seen in GLUT4 null adipose tissue. However, when equalizing the data on a per cell basis, there was a 65–71% decrease in phosphorylation of the downstream signaling molecules Akt, p44/42 and p38 in GLUT4 null adipocytes. ADIPOSE SPECIFIC REDUCTION OF GLUT4 Using Cre/LoxP DNA recombination, mice were generated with adipose speciﬁc reduction of GLUT4 (Abel et al., 2001). These mice have a 70–99% reduction of GLUT4 in white and brown adipose tissues with normal GLUT4 expression levels in skeletal muscle and heart. Adipose speciﬁc GLUT4 knockouts, unlike the GLUT4 null, have normal body weights, gonadal fat pad weights, adipocyte size and number, and heart weights when assessed at 16–38 weeks of age. Isolated adipocytes from adipose speciﬁc GLUT4 knockout mice have a 40% reduction in basal glucose uptake despite normal levels of GLUT1 mRNA. The insulin stimulated glucose uptake in isolated adipocytes was reduced by as much as 72%. This is consistent with adipose tissue glucose uptake data from GLUT4/ mice in which the level of glucose uptake was proportional to the level of GLUT4 protein content (Li et al., 2000). Based on

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NAIRA GOROVITS ET AL. Insulin stimulated glucose uptake

Leptin

Leptin

ACRP30

ACRP30 Wild type

GLUT4+/–

Adipose GLUT4–/–

GLUT4 null

Basal glucose uptake

Figure 1

Glucose uptake and cytokine secretion in adipose tissue from various GLUT4 knockout models.

euglycemic/hyperinsulinemic clamps and insulin tolerance tests the female adipose speciﬁc knockout mice, like the GLUT4 nulls, appear to be insulin resistant. However, unlike GLUT4 null mice, the circulating free fatty acids and triglycerides are normal in adipose speciﬁc knockout mice. In addition, muscle and liver triglyceride content are also normal in these mice. In conjunction with the normal morphology of the adipose tissue, it would appear that these animals have normal lipid metabolism with impaired glucose uptake. Comparison of adipose speciﬁc knockout mice, which show only a reduction in GLUT4 content, with the GLUT4 null, which has a total ablation of GLUT4, provides insight into the differences of no GLUT4 protein versus low level expression of GLUT4 and the metabolic interplay among speciﬁc tissues (Figure 1).

ADIPOSE TISSUE CYTOKINES There is an evolving role for adipose tissue in regulating central and peripheral insulin sensitivity and energy homeostasis (Abel et al., 2001; Spiegelman and Hotamisligil, 1993). Not only does adipose tissue respond to hormones and cytokines, but it also secretes them to help regulate other metabolic functions (Kahn and Flier, 2000; Spiegelman and Flier, 2001). GLUT4 null adipocytes were shown to have normal levels of leptin mRNA expression and protein content (Li et al., unpublished observations). However, given the reduced level of adipose tissue in GLUT4 null mice, the circulating serum levels of leptin were reduced 54% as compared to controls. This is consistent with the adipose speciﬁc GLUT4 knockout mice, which have a linear relationship between body fat and serum leptin (Abel et al., 2001). In addition, GLUT4 null mice exhibited normal circulating serum levels of ACRP30, a peptide hormone expressed exclusively in adipose tissue (Hu et al., 1996; Scherer et al., 1995). This is the result of a 2-fold increase in mRNA expression and a 3.4-fold increase in protein content of ACRP30 in adipose tissue of GLUT4 nulls as compared to controls. ACRP30 mRNA has been reported to be downregulated in both human and murine models of obesity (Hu et al., 1996). Insulin stimulation of 3T3-L1 adipocytes has been shown to enhance the secretion of ACRP30 (Bogan and Lodish, 1999). The elevation of plasma free fatty acids normally seen in

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mice after a high fat test meal or intravenous administration of lipid was signiﬁcantly reduced in mice treated with the globular head domain of ACRP30 (Fruebis et al., 2001). These results suggest a peripheral role of ACRP30 in energy homeostasis. The ability of GLUT4 null mice to maintain normal levels of ACRP30 despite signiﬁcantly less adipose tissue suggests that ACRP30 plays a role in helping to maintain the non-diabetic phenotype of the GLUT4 null.

GLUT4 VESICLE TRAFFICKING IN GLUT4 NULL ADIPOSE TISSUE GLUT4 resides in specialized intracellular compartments, distinct from recycling endosomal compartments (Martin et al., 1996; Pessin et al., 1999; Rea and James, 1997; Sevilla et al., 1997). As mentioned previously, the majority of insulin stimulated glucose transport results from the translocation of intracellular GLUT4 vesicles to the plasma membrane (Charron et al., 1989; James et al., 1989). The formation and translocation of the vesicle which contains GLUT4 and insulin regulated aminopeptidase (IRAP) was examined in GLUT4 null and control adipose tissue and skeletal muscle ( Jiang et al., 2001). Total IRAP protein was signiﬁcantly increased 1.6-fold in GLUT4 null adipocytes and decreased 40% in skeletal muscle and 60% in heart of GLUT4 null mice as compared to controls. Furthermore, in the basal state, IRAP content in the plasma membrane of the GLUT4 null adipocyte was 1.9-fold higher than in controls. Surprisingly, no further increase in IRAP translocation under insulin stimulation was observed in GLUT4 null adipocytes or skeletal muscle. In contrast, sortilin localization was not altered in GLUT4 null adipocytes, suggesting normal trafﬁcking of the recycling endosomal pathway. The mistargeting of IRAP to the plasma membrane of GLUT4 null cells suggests malformation or altered sequestration of the insulin regulated intracellular compartment, resulting in constitutive recycling. These studies comparing the formation and trafﬁcking of insulin responsive vesicles in GLUT4 null and control tissues strongly suggest that the presence of GLUT4 in the insulin regulated compartment is important not only for the correct biogenesis of the compartment but also for its proper retention and cycling.

INSULIN SIGNALING AND GLUCOSE UPTAKE IN THE GLUT4/ ADIPOCYTES Studies were conducted to determine the role of insulin signaling and glucose uptake in the adipocytes to the progression of diabetes in the GLUT4/ (Li et al., 2000). Control and GLUT4/ mice had similar body weights and epididymal fat pad weights. However, isolated adipocytes were 35 0.02% larger in diabetic GLUT4/ mice as compared to controls. This is seen in human diabetics, who exhibit larger adipocytes compared to non-diabetic adipocytes (DeFronzo et al., 1981; Shepherd and Kahn, 1999). All adult male GLUT4/ mice showed a 50% reduction in GLUT4 protein content in adipose tissue irrespective of their phenotype. No difference in GLUT1 protein content was measured in GLUT4/ adipose tissue compared to controls. Therefore, no difference in basal glucose uptake was measured between GLUT4/ and control adipocytes. Upon insulin stimulation, both prediabetic and diabetic GLUT4/ adipocytes demonstrated reduced glucose uptake at 1 nM and 5 nM insulin when compared to controls suggesting reduced insulin sensitivity. Maximum insulin stimulated glucose uptake achieved by GLUT4/ adipocytes was approximately 50% of controls, which corresponds to the 50% reduction in GLUT4 content.

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Table 2

NAIRA GOROVITS ET AL.

Insulin signaling in GLUT4/ adipocytes. Values are expressed as percentage of control.

GLUT4⫹/⫺ adipocytes Normal Prediabetic Diabetic

Insulin stimulated tyrosine phosphorylation of

Protein content

IR␤

IRS-1

IRS-3

IR

IRS-1

p85

ND ↓64 5% ↓88 1%

ND ↓71 4% ↓barely detectable

ND ↓35 1% ↓77 3%

ND ND ↓42 3%

ND ND —

ND ND ↑80 3%

ND no differences.

Insulin signaling begins with tyrosine phosphorylation of the insulin receptor ␤ subunit (IR␤) upon insulin binding. N/N GLUT4/ demonstrate normal tyrosine phosphorylation of the IR␤ subunit, IRS-1 and IRS-3 (Li et al., 2000). Prediabetic and diabetic GLUT4/ have reduced tyrosine phosphorylation of IR␤, IRS-1 and IRS-3 as compared to controls (Table 2). Only in the diabetic GLUT4/ adipocyte was the IR content reduced, while the p85 content was increased when compared to controls (Table 2). In agreement with these ﬁndings, the insulin stimulated PI3 kinase activity in adipocytes was reduced in both prediabetic and diabetic GLUT4/ compared to controls (Table 2). However, coincident with the higher p85 phosphorylation, diabetic GLUT4/ adipocytes have an increase in p85-associated PI3 kinase activity. These studies suggest that GLUT4 is the limiting factor in glucose uptake into adipocytes during the progression of insulin resistance and diabetes. In addition, these studies suggest that the required level of expression of the components in the insulin signaling pathway is adaptable. Furthermore, these altered expression levels did not contribute to a further reduction in glucose transport after a decline in GLUT4 content in adipocytes.

SUMMARY Manipulation of the genes thought to be important in insulin signaling and glucose transport has produced many new and unexpected insights into the function and necessity of the molecules involved in the insulin response. Before genetically engineered null mice were generated, it was thought that almost normal life would not be possible without GLUT4, or IRS-1, or insulin receptor expression in skeletal muscle. Analysis of the whole body GLUT4 knockout models has revealed more details about the role of GLUT4 in individual tissues and the whole body in maintaining glucose homeostasis (Figure 2). These studies suggest that GLUT4 plays a central role in glucose partitioning to oxidative or glycolytic pathways in skeletal muscle and adipose tissues. A certain minimum expression of GLUT4 also appears necessary for normal cardiac and adipose tissue morphology and function. The results of experiments with the GLUT4 heterozygous mice show that a single allele of GLUT4 is able to compensate for the loss of the other only for a limited amount of time. As the male GLUT4/ mice age, decompensation results in loss of GLUT4 protein content in insulin responsive tissues leading to insulin resistance and diabetes. The unexpected phenotype of the GLUT4 null mice with the compensatory glucose transport demonstrated in GLUT4 null muscle stimulated the search for novel transporters related to the glucose transporter family (GLUT1–5). This quest was facilitated by the rapid pace of human and mouse genome sequencing, and resulted in the discovery of four new glucose transporter-like molecules, GLUT8–10. The challenge now is to understand the function and role of these new transporters in maintaining glucose homeostasis.

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139

Hypertrophy = Hemodynamic profile = Basal glucose uptake

GLUT4 null

Glycogen synthesis rate Troponin (TnI and TnT) MCAD LCAD GLUT1

Skeletal muscle =/ basal glucose transport insulin stimulated glucose transport

Adipose tissue

Exercise stimulated glucose transport No hypoxia stimulated glucose transport = GLUT1 Cell size/lipid content

=/

High energy phosphates

=Basal glucose transport

Fatigability

No insulin stimulated glucose transport

Glucose incorporation into glycogen

PPARγ 2 mRNA Fatty acid synthase mRNA Hexokinase II mRNA ACRP30 mRNA = Leptin mRNA

Figure 2 Major features of GLUT4 null mouse.

The question remains whether any of these new transporters is responsible for the maintenance of normal glycemia in the GLUT4 null mouse. Undoubtedly, information about the role of each of these new transporters in glucose homeostasis, metabolism and insulin sensitivity will come from future targeting studies on mice.

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9. INSULIN SIGNALING PATHWAY AND GLUT4-MEDIATED GLUCOSE TRANSPORT IN THE INSULIN RESISTANT MUSCLE DANIEL KONRAD1,2, VARINDER K. RANDHAWA1,3, CAROL T.-L. HUANG1,2 AND AMIRA KLIP1,3 1

Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada 2 Institute of Medical Science, University of Toronto, Ontario, Canada 3 Department of Biochemistry, University of Toronto, Ontario, Canada

INTRODUCTION Insulin is an extraordinary hormone that has been implicated in various cellular events including lipogenesis, cell growth and differentiation, glycogen and protein synthesis, and glucose metabolism. It is now evident that systemic glucose homeostasis and metabolism consists of complex interactions between multiple factors, hormones and tissues. Insulin increases glucose uptake into peripheral tissues, mainly muscle and fat, and decreases hepatic glucose output. The regulation of blood glucose by insulin is achieved by metabolic interactions among muscle, liver and adipose tissue. Insulin resistance occurs in both type 1 (insulin-dependent) and type 2 (non-insulindependent) diabetes. In type 1 diabetes, insulin resistance has been largely ascribed to the prevailing hyperglycemia, but the exogenously maintained hyperinsulinemia may also be a contributing factor. Therefore, insulin resistance in type 1 diabetes occurs typically in individuals whose blood glucose levels are not well-controlled, in times of stress, or in case of superimposed illness (e.g. infections). Primary (i.e. genetic) and secondary (i.e. environmental) factors contribute to insulin resistance in type 2 diabetes. In the “garden variety” type 2 diabetes, insulin resistance precedes overt diabetes and plays an important role in the pathogenesis of the disease. However, insulin resistance is not sufﬁcient to cause overt diabetes; abnormalities in ␤-cell function must also be present (Polonsky et al., 1996). Once hyperglycemia develops, there is a secondary insulin resistance in type 2 diabetes akin to the acquired insulin resistance of type 1 diabetes. In all cases, insulin resistance manifests as a poor response of glucose uptake to stimulation by insulin, notably in skeletal muscle. The cellular mechanisms underlying these changes are still poorly understood. Whether of primary (i.e. genetic) or secondary (i.e. acquired) origin, insulin resistance within muscle cells could result from defects in any aspect of the insulin signaling cascade. This includes the levels and activities of the known early insulin signaling molecules, the translocation, docking or fusion with the plasma membrane of the insulin-sensitive glucose transporter 4 (GLUT4), or Address correspondence to: Amira Klip, Programme in Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada. Tel.: 416-813-6392; Fax: 416-813-5028; Email: [email protected] 147

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the regulation of the intrinsic activity of GLUT4. This chapter analyzes current studies examining possible causes of insulin resistance of glucose uptake in skeletal muscle. INSULIN SIGNALING PROTEINS Insulin Receptor The insulin receptor is a heterotetrameric protein that consists of two ligand-binding ␣-subunits and two transmembrane ␤-subunits. Binding of insulin to the ␣-subunit of the receptor leads to conformational changes and activation of the tyrosine kinase activity of its ␤-subunits. This results in trans-autophosphorylation of the ␤-subunits uncovering kinase activity toward proteins termed insulin receptor substrates. There are two isoforms (A and B) of the human insulin receptor that result from differential splicing of a single gene product (Ullrich et al., 1985). Insulin receptor mutations are rare, but will lead to extreme insulin resistance, for example, type A syndrome of insulin resistance (leprechaunism) and acanthosis nigricans (Krook and O’Rahilly, 1996). Several animal models of insulin receptor (IR) gene disruptions and mutations exist. For example, disruption of the insulin receptor in mice was characterized by early postnatal death due to extreme hyperglycemia and developmental defects (Accili et al., 1996). The contribution of the insulin receptor in different tissues and its implications for whole-body glucose homeostasis were analyzed by tissue-speciﬁc knockout of the insulin receptor. In muscle-speciﬁc insulin receptor knockout (MIRKO) mice, whole body glucose homeostasis was near normal and the mice failed to develop diabetes despite the severe insulin resistance seen in isolated skeletal muscle (Bruning et al., 1998). A similar pattern was observed in transgenic mice overexpressing a dominant-negative insulin receptor in muscle (Moller et al., 1996). In MIRKO mice, insulin-stimulated glucose uptake into muscle was decreased by 80%. In contrast, contractionstimulated glucose uptake into muscle was normal (Wojtaszewski et al., 1999). Notably, insulinstimulated glucose transport into white adipocytes was increased 3-fold (Kim et al., 2000). Therefore, selective insulin resistance in muscle appears to promote the redistribution of substrates to adipose tissue, leading to increased body fat mass, elevated triglycerides and free fatty acids. This suggests that insulin resistance in skeletal muscle contributes to the altered fat metabolism present in type 2 diabetics. It is likely that, in vivo, muscle activity is used as a major inﬂux route for glucose into muscle of MIRKO mice, explaining the absence of overt diabetes. In skeletal muscle of type 2 diabetic patients, reduced autophosphorylation and tyrosine kinase activity of the insulin receptor was found to occur during euglycemic clamp studies (Nolan et al., 1994). Similarly, a decrease in insulin receptor phosphorylation was found in liver and skeletal muscle of ob/ob mice (Folli et al., 1993). Reduced tyrosine phosphorylation of the insulin receptor could be the result of either decreased afﬁnity of the insulin receptor towards insulin or modiﬁcations of the receptor by kinases and/or phosphatases (discussed in the next section). However, they may be the result, rather than the cause, of an altered metabolism in type 2 diabetes. Indeed, a more recent study showed no decrease of the insulin-induced IR autophosphorylation in isolated skeletal muscle from type 2 diabetic patients (Krook et al., 2000). Insulin Receptor Substrates-1/2 The insulin receptor substrate-1 (IRS-1) was the ﬁrst docking protein found to bind to the insulin receptor (White et al., 1985). Three other IRS-proteins have now been identiﬁed in mammals: including IRS-2, IRS-3 and IRS-4. Of these, IRS-1 and IRS-2 co-ordinate the

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essential effects of insulin and insulin-like growth factor (IGF). Binding to the insulin receptor occurs through the phosphotyrosine-binding (PTB) domain of the IRS protein and the phosphorylated NPXY motif in the juxtamembrane region of the insulin receptor. The pleckstrin homology (PH) domain helps targeting the IRS proteins to the membrane and the insulin receptor. Recent data suggest tissue-speciﬁc roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo: it was proposed that IRS-1 is required to mediate insulin action in muscle and adipose tissues while IRS-2 is needed in liver and muscle (Kido et al., 2000; Withers, 2000 #106). However, a contribution of IRS-2 to glucose uptake in brown fat was also documented (Fasshauer et al., 2000). Polymorphisms of IRS-1 were found to be more prevalent in type 2 diabetic subjects compared to controls. Among them, the G972R polymorphism is the most common one (Almind et al., 1996; Imai et al., 1997; Laakso et al., 1994). Overexpression of this polymorphism in L6 skeletal muscle cells compromised activation of PI 3-kinase by insulin (discussed in the next section) and was paralleled by reduced glucose uptake and GLUT4 translocation (Hribal et al., 2000). This ﬁnding could explain the in vivo insulin resistance observed in genetic carriers of this polymorphism. Targeted disruptions of the different IRS isoform-encoding genes were also performed in mice to examine their role in the pathophysiology of diabetes. In the IRS-1 knockout mice, there was mild insulin resistance and growth retardation, yet no frank diabetes was evident (Tamemoto et al., 1994). The insulin resistance was mainly ascribed to decreased insulin stimulation of peripheral glucose metabolism (Previs et al., 2000). Both IRS-2 and ␤-cell compensation likely prevented the development of diabetes in these mice despite the lack of IRS-1 (Araki et al., 1994; Withers et al., 1998). The IRS-2 knockout mice showed a quite different phenotype characterized by severe hyperglycemia, which was attributed to decreased peripheral glucose uptake, unsuppressed hepatic gluconeogenesis, and a lack of ␤-cell compensation (Withers et al., 1998). Interestingly, only hyperglycemic knockout mice had reduced basal, exercise-, and submaximally insulin-stimulated 2-deoxyglucose uptake (Higaki et al., 1999). The authors therefore suggested that the onset of diabetes in the IRS-2 knockout mice might be due to hyperglycemia-induced insulin resistance rather than to a primary defect in skeletal muscle glucose transport. In a recently published study, whole-body glucose utilization was markedly impaired in both IRS-1 and IRS-2 knockout mice (Previs et al., 2000). To further analyze the role of IR and IRS in insulin resistance, combined heterozygous null mutations of the insulin receptor and IRS-1 or IRS-2 were generated in mice (Bruning et al., 1997; Kido et al., 2000). Although both types of heterozygous mice ([IR/IRS-1]/ and [IR/IRS-2]/) were diabetic, the extent of insulin resistance in skeletal muscle differed. [IR/IRS-1]/ mice developed severe insulin resistance in skeletal muscle, whereas [IR/IRS2]/ mice showed only mild insulin resistance. Collectively, these studies suggest that IRS-1 plays a more important role in mediating insulin action in skeletal muscle compared to IRS-2. Serine phosphorylation of IRS-1 has been shown to interfere with the phosphorylation of its tyrosine residues (Paz et al., 1997). The proposed kinases responsible for this effect are protein kinase C, mitogen-activated protein kinase, glycogen synthase kinase 3, JNK1, and even phosphatidylinositol 3-kinase (PI 3-kinase) (Kellerer et al., 1999; Aguirre et al., 2002). This mechanism is thought to reﬂect a negative feedback loop of insulin signaling. Therefore, stimulation of these signalling pathways may play a role in the development of insulin resistance. Phosphatidylinositol 3-Kinase Phosphatidylinositol 3-kinase is a heterodimeric protein that consists of a catalytic subunit p110 with a phospholipid-binding domain and serine kinase activity, as well as a regulatory subunit with

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SH2 domains that bind to phosphotyrosine residues. Such binding activates the p110 subunit to phosphorylate inositol phospholipids on the D3 position of the inositol ring. Several isoforms of both the catalytic and regulatory subunits have been described (for review, see Taha and Klip, 1999). PI 3-kinase activation is one of the best understood functions of the IRS molecules. PI 3-kinase is a key player in insulin-stimulated glucose uptake into cells since PI 3-kinase inhibitors block insulin-induced glucose transport in muscle and fat cells (Cheatham et al., 1994; Tsakiridis et al., 1995). Dominant-negative mutants of the regulatory p85␣ subunit also abolish the effect of insulin on glucose transport and GLUT4 translocation (Hara et al., 1994), while massive overexpression of constitutively-active forms of PI 3-kinase elevate glucose uptake and GLUT4 translocation in adipocytes (Katagiri et al., 1996; Tanti et al., 1996). Although insulin-stimulated PI 3-kinase activity is necessary for GLUT4 translocation, it is not sufﬁcient to stimulate glucose uptake at physiological levels of activation (Isakoff et al., 1995; Wiese et al., 1995). Moreover, a permeable version of the major product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate-acetoxy-methyl ester (PIP3-AM), was found to be required but was, on its own, insufﬁcient to stimulate glucose uptake ( Jiang et al., 1998). Therefore, mechanisms in addition to PI 3-kinase activation are required to elicit glucose transport. Recently, insulin was found to induce the tyrosine phosphorylation of a protein named Cbl in 3T3 L1-adipocytes. Cbl in turns binds CAP (c-Cbl-associated protein) to form a complex that dissociates (Baumann et al., 2000). The complex is thought to generate a PI 3kinase-independent signal required for the regulation of GLUT4 translocation since expression of the N-terminus of CAP reduced the stimulation of GLUT4 translocation by insulin. A common polymorphism of the p85␣ subunit, M326I, was associated with decreased insulin sensitivity in homozygous individuals, but its frequency is not elevated in diabetes (Hansen et al., 1997). Recently, a novel heterozygous mutation, R409Q, was detected in a subject with severe insulin resistance. This mutation was associated with lower insulinstimulated PI 3-kinase activity in cell culture, implying that this mutation may have contributed to the observed insulin resistance (Baynes et al., 2000). In addition to these reports of primary defects in PI 3-kinase as likely contributors to diabetes, reduced IRS-1 phosphorylation and PI 3-kinase activity were found in skeletal muscle of lean and obese type 2 diabetic (Bjornholm et al., 1997) and obese non-diabetic persons (Cusi et al., 2000; Goodyear et al., 1995). In high-fat fed mice, reduced PI 3-kinase activity in muscle was accompanied by low glucose uptake and GLUT4 translocation (Zierath et al., 1997). Given the integral role of PI 3-kinase on insulin-induced glucose uptake, it is plausible that a defect that lowers PI 3-kinase activity could further exacerbate insulin resistance. Examples of animal models of insulin resistance and the status of PI 3-kinase in these are listed in Table 1. Akt/Protein Kinase B Akt (also protein kinase B, PKB) is a serine/threonine kinase downstream of PI 3-kinases. Akt1 (PKB␣) is the major isoform activated by insulin in muscle, hepatocytes and adipocytes (Walker et al., 1998). Akt2 (PKB␤) is the major isoform activated in adipocytes, although it is also activated in muscle, whereas Akt3 (PKB␥) is only activated by insulin in cell culture models (Walker et al., 1998). All of the Akt isoforms are activated through dual input: PIP products of PI 3-kinase bind to the PH domain of Akt, and the Ser/Thr kinase is then activated by phosphorylation on two residues (serine and threonine in Akt1) via 3 -phophatidylinositoldependent kinase 1 (PDK1) and a yet uncharacterized PDK2. PDK1 itself is activated by PI 3-kinase. Therefore, PI 3-kinase inhibitors, such as wortmannin, block the stimulation of Akt elicited by insulin.

↓↓ ↓↓ ↓↓ ↓ (glucose disposal) ↓ ↓ ↔ ↓

↓ ↓ ↑ ↓ ↓ ↓ ↔ ↓↓ ↔ ↓↓ n.d. ↓↓ ↓ ↓↓ ↓↓

↓ ↓↓ ↓↓ ↑↑ ↓ ↔ ↔ ↓↓ ↓

↓↓ ↓ ↓↓ ↓↓ ↔

↓↓ ↓

n.d. ↓↓ (adipocytes) n.d. n.d.

↓ (glucose disposal)

↓↓ ↔ ↓

↓ ↓↓ (glucose disposal) ↓↓

Glucose transport

n.d. ↓↓ n.d.

Akt activity

↓ means 50% decrease; ↓↓ 50% decrease; ↔ no change compared to control; n.d. not determined.

Human, type 2 diabetic Skeletal muscle Skeletal muscle Adipocytes Human, muscle damage Skeletal muscle Zucker rat Skeletal muscle White adipose tissue Liver White adipocytes Sprague–Dawley rat (model of insulin resistance) Skeletal muscle (induced hyperglycemia) Skeletal muscle (denervation) day 1 day 3 Skeletal muscle (glucosamine infusion) Goto–Kakizaki rat Skeletal muscle Skeletal muscle type I type II White adipocytes 3T3-L1-adipocytes Glucosamine Osmotic shock

PI 3-Kinase activity

Alterations in insulin-stimulated PI 3-kinase, Akt activity and glucose transport in different models of insulin resistance.

Subject/system

Table 1

Heart et al., 2000 Chen et al., 1999

Kanoh et al., 2000

Krook et al., 1997 Song et al., 1999

Kurowski et al., 1999 Turinsky and DamrauAbney, 1998 Kim et al., 1999

Nawano et al., 1999 Carvalho et al., 2000

Kim et al., 2000

Del Aguila et al., 2000

Krook et al., 1998 Kim et al., 1999 Rondinone et al., 1999

Reference

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Akt activity has been implicated in insulin-induced glucose transport since overexpression of wild-type or constitutively-active mutants of Akt1 in several cell lines elevate glucose transport and GLUT4 translocation to levels similar to, or greater than, those achieved with insulin (Kohn et al., 1996; Tanti et al., 1997; Ueki et al., 1998; Wang et al., 1999). It has been suggested that Akt participates in the mobilization of GLUT4 from the GLUT4 storage compartment (which selectively utilizes the SNARE complex consisting of SNAP-23, syntaxin-4 and VAMP-2), but not from the transferrin receptor- and GLUT1-containing vesicles derived from recycling endosomes (Foran et al., 1999). In support of this scenario, a dominantnegative Akt enzyme partially inhibited (by about 20%) the GLUT4 translocation of cultured primary rat adipocytes in response to insulin (Cong et al., 1997), and a less leaky Akt mutant (AAA-Akt) reduced GLUT4 translocation in muscle cells by 70% (Wang et al., 1999). Moreover, microinjected antibodies to Akt prevented GLUT4 translocation in 3T3-L1 adipocytes to a signiﬁcant degree (Hill et al., 1999). In general, the literature has been controversial with respect to the levels and activity of Akt in humans with type 2 diabetes, as well as in different models of insulin resistance or diabetes (see Table 1). For example, a decreased insulin stimulation of Akt activity was found in skeletal muscle and adipocytes from type 2 diabetic patients (Krook et al., 1998). Therefore, a role of reduced Akt activity in the development of insulin resistance was suggested. However, in a recent study, there was no change in the insulin-dependent activation of Akt1 or Akt2 from skeletal muscle of type 2 diabetics, even though insulin-stimulated PI 3-kinase activity was diminished (Kim et al., 1999). The latter study suggested that defects in Akt are unlikely to be the cause of the observed insulin resistance in type 2 diabetics. The controversy regarding alterations in Akt activity in insulin resistant and type 2 diabetic patients supports the notion that the defect in diabetes can occur at different levels. In skeletal muscle from the spontaneously diabetic Goto-Kakizaki (GK) rats, insulinstimulated Akt activity was signiﬁcantly lower compared to that in non-diabetic control animals (Krook et al., 1997). Phlorizin treatment for 4 weeks restored this activity by normalizing hyperglycemia in these rats. Muscle ﬁber type-speciﬁc defects in PI 3-kinase activity were observed: the IRS-1-associated PI 3-kinase activity was lowered in the oxidative soleus but not in the glycolytic extensor digitorum longus muscle whereas Akt activity was reduced in both (Song et al., 1999). The obese Zucker rat also showed a reduction in muscle (30%) and adipose tissue (21%) but an increase in liver (37%) of the insulin-induced Akt1 activity. Akt2 activity was decreased in muscle (56%) and liver (35%), and increased in adipose tissue (24%) (Kim et al., 2000). The PI 3-kinase activity was reduced in all of these tissues. The glucose transport response to insulin was diminished by 70% in adipose tissue, suggesting that Akt1 was primarily involved in glucose uptake. In another diabetic animal model, hyperglycemia lowered insulin-induced glucose uptake by 70% and Akt activity by 60%, but had no effect on PI 3-kinase activity in rat skeletal muscle (Kurowski et al., 1999). Finally, in the denervated soleus and plantaris muscle of mice, the drop in Akt1 activity was time-dependent: one day after denervation glucose uptake was reduced but Akt1 activity was unchanged. Two days later, Akt1 activity was reduced (Turinsky and Damrau-Abney, 1998). Contrary to these results, glucosamine infusion in rats, leading to insulin resistance, was associated with reduced insulinstimulated PI 3-kinase but not Akt activity in skeletal muscle (Kim et al., 1999). All of the models of insulin resistance discussed above showed a compromised response of glucose uptake to insulin (Figure 1B). However, the role of Akt is still unclear even though most recent studies have shown decreased Akt activity paralleling reduced glucose uptake. In addition, most of the studies showed a larger reduction in PI 3-kinase than in Akt activities. One study showed normal Akt activity even though PI 3-kinase activity was clearly decreased,

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Akt-activity (fold-stimulation over ND)

A p < 0.001 1

0 Co

1

2 3 4 5 6 7 8 9 10 11 12 Diabetic/insulin-resistant

Glucose transport (fold-stimulation over ND)

B p < 0.001 1

0 Co

1

2 3 4 5 6 7 8 9 10 11 12 Diabetic/insulin-resistant

Figure 1 Comparison of Akt activity or glucose transport in diabetic/resistant subjects vs. their controls (data from Table 1). (A) The average insulin-stimulated Akt activity in the analyzed studies was reduced by 43% compared to controls. This difference was statistically signiﬁcant (Student’s t-test, p 0.001, relative to control). (B) The average insulin-stimulated glucose transport was reduced by 47% (Student’s t-test, p 0.001). 1. Krook et al., 1998; 2. Kim et al., 1999; 3. Rondinone et al., 1999; 4. Del Aguila et al., 2000; 5. Kurowski et al., 1999; 6. Turinsky and Damrau-Abney, 1998; 7. Kim et al., 1999; 8. Krook et al., 1997; 9. Song et al., 1999; 10. Kanoh et al., 2000; 11. Heart et al., 2000; 12. Chen et al., 1999. ND non-diabetic.

suggesting that PI 3-kinase is in substantial excess and that its full activation is not necessary for the full activation of downstream signaling events (Kim et al., 1999). Other studies suggest that the relationship between Akt activity and glucose uptake is time-dependent (i.e. dependent on duration of the altered metabolism) (Turinsky and Damrau-Abney, 1998) or tissuespeciﬁc (Kim et al., 2000). An overall analysis of these studies shows a clear reduction in insulin-stimulated Akt activity in diabetic/resistant subjects (Figure 1A). We therefore conclude that reduced Akt activity probably contributes to insulin resistance, but defects at other levels are also important. Protein Kinase C-/ The protein kinase C (PKC) family consists of at least 12 serine/threonine kinases, including the conventional (␣, ␤, ␥), novel (␦, ␧, ␩, ␷) and atypical (␨, ␭) isoforms. There is extensive

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evidence that PI 3-kinase activates the atypical PKC isoforms ␨ and ␭. The insulin-stimulated activation of PKC-␨ and ␭ is blocked by the PI 3-kinase inhibitor wortmannin (Bandyopadhyay et al., 1997; Kotani et al., 1998). Moreover, it was proposed that insulin signals downstream of PI 3-kinase diverge into Akt- and atypical PKC-dependent pathways (Kotani et al., 1998). However, both Akt and the atypical PKC isoforms have been reported to induce GLUT4 translocation. In rat adipocytes transfected with kinase-inactive forms of either atypical PKCs, an 85% reduction of insulin-stimulated GLUT4 translocation was observed (Bandyopadhyay et al., 1999). Pharmacological inhibitors of atypical PKC isoforms reduced glucose uptake signiﬁcantly. Earlier studies had shown a 60% inhibition of insulin-induced translocation of GLUT4 by using kinase-dead, dominant-negative mutants of atypical PKC isoforms (Bandyopadhyay et al., 1999; Kotani et al., 1998; Standaert et al., 1997). Hence, both Akt and the atypical PKCs ␨ and ␭ seem to play a key role in the regulation GLUT4 transport to the plasma membrane because inhibition of each of these kinases led to partial inhibition of GLUT4 translocation. These results may be reconciled by a model postulating regulation of two different intracellular vesicle pools of GLUT4: the PKC-␨ and ␭-dependent recycling endosomes, where GLUT4 co-localizes with GLUT1 and the transferrin receptor, and an Akt-dependent specialized GLUT4 pool (Fletcher and Tavare, 1999). Alternatively, it is also conceivable that atypical PKC and Akt inhibit translocation out of the same pool but act at different steps of this translocation (e.g. GLUT4 budding, mobilization or fusion with the plasma membrane). Regulation of GLUT4 trafﬁc out of the recycling pool may depend on other, unidentiﬁed signals. The above observations should prompt examination of the status of atypical PKCs in diabetic animal and human tissues. To date, only the diabetic GK rat has been examined. The reduced glucose uptake displayed by this model was accompanied by a reduction in PI 3kinase activation and in PKC-␨ and ␭ activities (Kanoh et al., 2000). Rosiglitazone, a thiazolidinedione, reversed the defects in glucose uptake and PKC-␨ and ␭ activity, but had no effect on PI 3-kinase and Akt activities. Summarizing, it is conceivable that Akt and PKC have input at different steps leading to GLUT4 translocation and glucose uptake. Further studies should clarify the role of the PKC isoforms in glucose transport and especially in insulin resistance.

METABOLIC ENDPOINT: GLUT4 MEDIATED GLUCOSE UPTAKE GLUT4 Translocation To date, ﬁve functional glucose transporters have been described (termed GLUT1–4, 8) and exhibit tissue-speciﬁc distribution. We will focus on GLUT4, the predominant insulinresponsive glucose transporter and therefore a major target for insulin resistance. Insulin stimulates the translocation of GLUT4 from intracellular pools to the plasma membrane, a prerequisite for insulin-stimulated glucose-uptake (Cushman et al., 1998). The transporters cycle to and from the cell surface in the basal state. Insulin increases their rate of exocytosis and may decrease their rate of endocytosis (Czech and Buxton, 1993; Yang et al., 1996). The impact of the insulin signaling proteins Akt and atypical PKC’s in GLUT4 trafﬁc was outlined above. Abnormal tissue-speciﬁc GLUT4 expression and GLUT4 protein content are evident in diabetes, as has been previously reviewed (Klip et al., 1994; Tirosh et al., 2000). In summary, patients with type 2 diabetes showed normal GLUT4 protein content in skeletal muscle (Andersen et al., 1993; Garvey et al., 1992; Kahn et al., 1992) whereas both GLUT4 mRNA

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and protein levels were reduced in adipocytes (Garvey et al., 1991). However, because of the small contribution of adipose tissue in whole body glucose disposal, this observed reduction hardly contributes to the diabetes-associated reduction in glucose uptake except in the case of morbidly obese individuals. In animal (Goodyear et al., 1991; Marette et al., 1992) as well as in human skeletal muscle (Gaster et al., 2000), GLUT4 expression is ﬁber type-dependent and decreases with age. Therefore, it has been proposed that insulin resistance, especially age-related, could be caused by changes in muscle ﬁber composition (Gaster et al., 2000). This contention is supported by the ﬁnding that heterozygous GLUT4 knockout mice showed features of insulin resistance (Stenbit et al., 1997) suggesting that a reduction in GLUT4 expression could lead to the development of insulin resistance and diabetes. Consistent with this notion, the development of insulin resistance in GLUT4/ mice could be prevented by transgenic complementation of GLUT4 in skeletal muscle (Tsao et al., 1999). In addition, overexpressing GLUT4 selectively in skeletal muscle of insulin-resistant obese mice diminished their insulin resistance (Tsao et al., 2000). As expected, targeted disruption of GLUT4 selectively in skeletal muscle led to severe insulin resistance (Zisman et al., 2000). These ﬁndings underscore the importance of the skeletal muscle in whole body glucose metabolism. Yet, as stated above, in the cohorts of type 2 diabetic patients studied to date there is no obvious diminution in muscle GLUT4 protein content.

Regulation of the Intrinsic Activity of GLUT4 There has been an ongoing debate about whether or not increased GLUT4 translocation to the plasma membrane can fully account for the insulin-stimulated increase in glucose uptake (Zierler, 1998). A survey of the literature shows a discrepancy between the extent of GLUT4 translocation and the stimulation of glucose uptake in response to insulin in rat (Goodyear et al., 1991; Marette et al., 1992), mouse (Brozinick et al., 1996; Deems et al., 1994), and human muscle (Guma et al., 1995). This discrepancy occurred in both, control and insulin resistant subjects (Table 2 and Figure 2). All of these studies used subcellular fractionation to examine GLUT4 translocation. Although powerful, this method does not allow one to distinguish GLUT4 vesicles incorporated into the plasma membrane from docked but unfused GLUT4 vesicles. This discrepancy between GLUT4 translocation and glucose uptake was not found when the ATB-BMPA photolabelling method was used (Hansen et al., 1998; Ryder et al., 1999; Wilson and Cushman, 1994), probably because this latter method allows only the detection of active transporters (Ferrara and Cushman, 1999; Harrison et al., 1992). Hence, the changes in the GLUT4 transporter number always parallels glucose uptake. In addition, recent studies have suggested that insulin-stimulated GLUT4 translocation in rat skeletal muscle (Hansen et al., 1998), L6 muscle cells (Sweeney et al., 1999) and 3T3-L1 adipocytes (Hausdorff et al., 1999; Sweeney et al., 1999) is not sufﬁcient to achieve the maximal increase in glucose uptake. Therefore, we have supported the concept that the intrinsic activity of the translocated GLUT4 is subject to regulation. We have evidence to suggest that the p38 mitogen-activated protein (MAP) kinase may modulate the intrinsic activity of GLUT4. The p38 MAP kinase pathway plays an essential role in regulating many other cellular process including inﬂammation, cell differentiation, cell growth and death (Ono and Han, 2000). Insulin causes phosphorylation of p38 MAP kinase in L6 myotube (Tsakiridis et al., 1996) and increases its activity in 3T3-L1 adipocytes (Sweeney et al., 1999) and rat skeletal muscle (Somwar et al., 2000). Furthermore, we have

Klip et al., 1990 Goodyear et al., 1991

King et al., 1992 Lund et al., 1993 Rosholt et al., 1994 Brozinick et al., 1994 Wilson and Cushman, 1994 Hansen et al., 1998 Brozinick et al., 1999 Deems et al., 1994 Brozinick et al., 1996 Zierath et al., 1997 Ryder et al., 1999 Deems et al., 1994; Ramlal et al., 1996 Brozinick et al., 1996 Wang et al., 1996

1.8-fold (GLUT4 i.b. on PM) 2.5-fold (GLUT4 i.b. on white muscle PM) 1.6-fold (GLUT4 i.b. on red muscle PM) 1.5-fold (GLUT4 i.b. on white muscle PM) 1.7-fold (GLUT4 i.b. on red muscle PM) 1.8-fold (CB binding); 1.4-fold (GLUT4 i.b. on PM) 6-fold (ATB-BMPA photolabelling 2.0-fold (CB binding to PM) 1.5-fold (GLUT4 i.b. on PM) 8.0-fold (ATB-BMPA photolabelling) 3.2-fold (ATB-BMPA photolabelling) 4.8-fold (ATB-BMPA photolabelling) 1.4-fold (GLUT4 i.b. on PM) 4.1-fold (GLUT4 i.b. on PM) 7.7-fold (ATB-BMPA photolabeling and GLUT4 i.p.) 2.8-fold (ATB-BMPA photolabeling and GLUT4 i.p.) 3.1-fold (GLUT4 i.b. on PM) 2.6-fold (GLUT4 i.b. on PM) 3-fold (e.m. ultrathin slices)

Marette et al., 1992

Guma et al., 1995

1.7-fold (GLUT4 i.b. on PM)

Reference

perf. h.q., A/V glc: glucose uptake into perfused hindquarter, arterio-venus glucose difference; h.q., 3OMG: 3-O-methylglucose uptake into muscles of the perfused hindquarter; quad.: quadriceps.

Human muscle 3.4-fold (isolated muscle strips) Rat muscle 2.7-fold (perf. h.q., A/V glc) 3.6-fold (white muscle PM vesicles) 2.2-fold (red muscle PM vesicles) 2.2-fold (perf. h.q., 3OMG white quad.) 2.5-fold (perf. h.q., 3OMG red quad.soleus) 4.5-fold (in PM vesicles) 6.0-fold (isolated soleus) 3.0-fold (in PM vesicles) 17-fold (perf. h.q., 2dG uptake) 8.6-fold (isolated soleus) 3.1-fold (isolated epitrochlearis) 5.2-fold (isolated soleus) Mouse muscle 3-fold (isolated soleus) 8-fold (perf. h.q.) 4.8-fold (isolated soleus) 2.8-fold (isolated soleus) Mouse muscle overexpressing GLUT4 2.5-fold (hyperinsulinemic clamp) 10-fold (perf. h.q.) 3-fold (isolated epitrochlearis)

GLUT4 in plasma membranes (change by insulin)

Discrepancies between GLUT4 translocation and glucose uptake in response to insulin in skeletal muscle.

Glucose uptake (change by insulin)

Table 2

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157

Glucose uptake (fold)

20 15 10 5 0 0

5 10 15 20 GLUT4 translocation (fold)

Figure 2 Comparison of GLUT4 translocation vs. glucose uptake in skeletal muscle. Each point represents the insulin stimulated GLUT4 translocation and the corresponding insulin stimulated glucose uptake in skeletal muscle of insulin-sensitive humans, rats or mice (data from Table 2). Results are expressed relative to the corresponding basal values ascribed a value of 1.0. Notice that most points fall above the hypothetical dotted line traced for a 1 : 1 relationship between glucose uptake and GLUT4 translocation. This observation strongly suggests that glucose uptake is stimulated beyond GLUT4 translocation.

previously shown that inhibition of p38 MAP kinase reduced insulin-stimulated glucose transport but did not affect GLUT4 translocation in 3T3-L1 adipocytes and L6 GLUT4myc myotubes (Sweeney et al., 1999). These L6 myotubes express a myc-epitope in the ﬁrst exofacial loop of GLUT4, thus allowing the parallel measurements of GLUT4 translocation and glucose uptake induced by insulin in intact cells. In addition, we recently found a signiﬁcant reduction in insulin-stimulated glucose uptake in 3T3-L1 adipocytes expressing a dominantnegative p38 MAP kinase mutant (R. Somwar, A. Klip, unpublished results). In rat skeletal muscle, p38 MAP kinase ␣ and ␤ isoforms were both activated by insulin and contraction. An inhibitor of p38 MAP kinase, SB203580, caused a signiﬁcant reduction in this activation. In addition, insulin and contraction-stimulated glucose uptake was inhibited by this compound (Somwar et al., 2000). We therefore propose that insulin stimulates two independent signals contributing to glucose transport: the PI 3-kinase signals leading to GLUT4 translocation and the p38 MAP kinase signals leading to activation of the recruited glucose transporter at the membrane (Figure 3). This hypothesis is further supported by the ﬁnding that the cell permeant phosphatidylinositol 3,4,5-trisphosphate (PIP3), the natural product of PI 3-kinase, does not stimulate glucose uptake in 3T3-L1 adipocytes ( Jiang et al., 1998) although it leads to GLUT4 translocation to the plasma membrane (G. Sweeney and A. Klip, submitted). However, the permeant PIP3 was able to rescue the insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with wortmannin ( Jiang et al., 1998). The above discussion, therefore, identiﬁes two mechanisms that contribute to insulinstimulated glucose uptake: GLUT4 translocation and activation. It is therefore important to examine which of these are altered on diverse animal and human cases of insulin resistance. In several animal models (e.g. STZ rat, Zucker rat, GK rat), reduced GLUT4 translocation to the plasma membrane was shown along with a reduction in glucose uptake (Brozinick et al., 1994; Hansen et al., 1998; Klip et al., 1990; Zierath et al., 1997). Also, a lower GLUT4 translocation was found in insulin-resistant (Garvey et al., 1998) and type 2 diabetic human subjects (Garvey et al., 1998; Ryder et al., 2000; Zierath et al., 1996). Therefore, suboptimal translocation

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IR

PI3K Akt/aPKCs

p38 MAPK

GLUT4 GLUT4 translocation activation

Stimulation of glucose uptake

Figure 3 Model of insulin-stimulated glucose uptake. Stimulation of glucose transport by insulin consists of two events: PI 3-kinase-dependent translocation of GLUT4 to the plasma membrane and p38 MAPK-dependent stimulation of GLUT4 intrinsic activity.

of GLUT4 to the plasma membrane seems to contribute to insulin resistance. Table 3 summarizes these results. Interestingly, in some of these studies (Brozinick et al., 1994; Hansen et al., 1998a; Klip et al., 1990) the reduction in the amount of translocated GLUT4 was smaller than the decrease in the extent of glucose uptake (Table 3). This implies that beyond a diminution in GLUT4 translocation, decreased intrinsic activity of GLUT4 may further reduce glucose uptake in diabetes or insulin resistance. Indeed, in high-fat fed rats, a lessened insulin-stimulated glucose uptake was found but GLUT4 translocation was not altered compared to control rats (Rosholt et al., 1994). Further evidence that the intrinsic activity may be subject to regulation was found in mice overexpressing GLUT1 in skeletal muscle. In these mice, insulin-stimulation did not further increase glucose uptake even though insulin-stimulated GLUT4 translocation was normal (Hansen et al., 1998b). Methodological improvements leading to an accurate determination of GLUT4 translocation in animal and human tissues will help to further study the importance of impaired translocation and intrinsic activity of GLUT4 in insulin resistance and diabetes.

MODULATORS OF INSULIN SIGNALING The preceding sections have analyzed known protein–protein interactions required for the insulin-stimulated mobilization and incorporation of GLUT4 glucose transporter vesicles at the cell surface, as well as for the stimulation of glucose uptake. Several additional modulators of insulin signaling that regulate glucose uptake have been identiﬁed. These are discussed below in the context of insulin resistance. Phosphotyrosine Phosphatases The insulin-induced IR-tyrosine phosphorylation allows the receptor to engage the insulin signaling pathway and mediate glucose transport. To modulate such signals emanating from

3.4-fold (strip) 6.2-fold (w.b.) 2.7-fold (h.q., A/V) 4.5-fold (PMv) 17-fold (h.q.,2dG) 3.0-fold (PMv) 3.1-fold (m. epi.) 4.8-fold (f. sol.)

3.2-fold 7.4-fold 4.9-fold 2.5-fold 4.0-fold 2.0-fold 1.9-fold 3.7-fold

1.7-fold (PM/i.b.) 2.8-fold (PM/i.b.) 1.8-fold (PM/i.b.) 1.8-fold (PM/i.b.) 1.3-fold (PM/i.b.) 2.0-fold (PM/i.b.) 7.0-fold (photo/i.p.) 7.7-fold (photo/i.p.)

1.3-fold 1.5-fold 1.3-fold no gain 1.5-fold 2.0-fold 5.5-fold 3.2-fold

Experimental Zierath et al., 1996 Garvey et al., 1998 Klip et al., 1990 King et al., 1992 Brozinick et al., 1994 Rosholt et al., 1994 Zierath et al., 1997 Hansen et al., 1998a

Reference

h.q. A/V.: glucose uptake into perfused hindquarter, arterio-venus glucose difference; PMv: glucose uptake into isolated PM vesicles; h.q.,2dG: 2-deoxyglucose; uptake selective muscles of the perfused hindquarter; f.sol.: 2-deoxyglucose uptake into isolated female rat soleus; m. epi.: 3-O-methylglucose uptake in isolated male rat epitrochlearis; strip: glucose uptake into isolated, human muscle strips; w.b.: whole body glucose uptake in humans during euglycemic clamp; photo/i.p.: ATB-BMPA photolabelling followed by GLUT4 immunoprecipitation; PM/i.b.: isolated plasma membranes immunoblotted for GLUT4.

Human type 2 (d) Human type 2 (d) Streptozotocin rat (d) Zucker rat (r) Zucker rat (r) Fat-fed rat (r) Fat-fed rat (r) Fat-fed mouse (r)

Control

Control

Experimental

Insulin dependent GLUT4 on PM

Insulin dependent glucose uptake

Effect of insulin resistance (r)/diabetes (d) on glucose uptake and GLUT4 in skeletal muscle.

Diabetes model

Table 3

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the IR, phosphotyrosine phosphatases must exist to turn off the insulin signal. Although no IR-speciﬁc phosphatase has yet been identiﬁed, several candidate tyrosine phosphatases have been described. These include members of the protein-tyrosine phosphatase (PTPase) family (i.e. PTP-␣, 1B, 1C, and 1D) and a leukocyte antigen-related (LAR) phosphatase. In skeletal muscle of obese or type 2 diabetic patients, the total membrane-bound tyrosine phosphatase activity is increased. PTP-1B and LAR phosphatase, both known to dephosphorylate the IR and IRS-1, were mainly responsible for this increase (Virkamaki et al., 1999). IR tyrosine kinase activity promotes the interaction of PTP-1C through its C-terminus with the autophosphorylated IR, and thereby stimulates its activity (Uchida et al., 1994). A similar type of protein–protein interaction has been observed for PTP-1D (also SHP-2 or Syp) with the IR and IRS-1 (Vogel et al., 1993). These interactions are likely mediated by the SH2 domains on the PTPases. The need for proximity between PTPases and the IR or IRS-1 in vivo led to a recent study that examined the physiological localization of these signaling components in rat adipocytes (Calera et al., 2000). PTP-␣ and LAR were found largely in the plasma membrane and somewhat in the heavy microsomes. This pattern was similar to the IR distribution, all determined in subcellular fractions. The PTP-1B and IRS-1 molecules were fractionated into light microsomes and the cytosol, while PTP-1D was only present in the cytosolic fraction. In addition, insulin caused a redistribution of PTP-␣ and PTP-1B which mimicked that of the IR and IRS-1, respectively. Therefore, these latter PTPases were suggested to modulate insulin signaling at the level of the IR and IRS-1 in the internal membranes of rat adipocytes (Calera et al., 2000). To clarify the role of PTP-1B in insulin action in vivo, two different groups recently generated PTP-1B-deﬁcient mice by either disruption (Elchebly et al., 1999) or deletion (Klaman et al., 2000) of the gene encoding for the PTP-1B mouse homologue. Both [PTP-1B]/ mice displayed improved insulin sensitivity and insulin-stimulated glucose utilization, have low adiposity and failed to become obese or insulin-resistant upon high fatfeeding. Interestingly, the PTP-1B knockout mice showed tissue-speciﬁc effects: an increase in the insulin sensitivity of only skeletal muscle, and a decrease in fat cell mass but not adipocyte number (Klaman et al., 2000). Since PTP-1B has been implicated in the negative regulation of the IR, the state of phosphorylation of the receptor was examined in the [PTP-1B]/ mice. In comparison to [PTP-1B]/ mice, the PTP-1B-deﬁcient mice displayed an increased and prolonged phosphorylation state of the IR speciﬁcally in the liver and muscle tissues following an insulin injection (Elchebly et al., 1999). In contrast, PTP-1B overexpression in 3T3-L1 adipocytes resulted in a 50–60% decrease in the phosphorylation of the IR and IRS-1, and the phosphotyrosine or IRS-1-associated activity of PI 3-kinase (Venable et al., 2000). The activity of Akt and glucose transport however, were unaltered by PTP-1B overexpression (Venable et al., 2000). Clearly, more work will have to be done to ﬁnd a causal relationship between tyrosine phosphatase activity and the insulin signaling defects leading to reduced glucose uptake under insulin-resistant states. In this regard, the use of newly developed and highly selective inhibitors of members of the PTPase family (Iversen et al., 2000) should serve as valuable tools to examine the function of speciﬁc PTPases in liver, muscle and adipose tissues.

Other Protein Kinase C Isoforms We have alluded earlier to the possible role of the atypical PKCs in insulin-stimulated GLUT4 translocation. Other PKC family members also appear to participate in insulin signaling albeit as negative inputs, and have therefore been implicated in insulin resistance and diabetic

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complications. PKC-mediated signaling events generally require the translocation of the enzyme from the cytosol to subcellular membranes along with additional activation by intracellular calcium, diacylglyceride (DAG) or phosphatidylserine. Under insulin-resistant conditions, the distribution, activity and expression levels of certain PKC isoforms were altered. For example, higher PKC ␣, ␤, ␦ and ␧ but lower PKC ␪ levels were found to be membrane-associated in soleus muscle from lean diabetic GK rats (Avignon et al., 1996). The total membrane-associated PKC activity was unchanged in human skeletal muscle from obese, insulin-resistant subjects vs. lean, insulin-sensitive subjects. However, total PKC ␤ content (and PKC ␤ activity under basal conditions) was elevated whereas total PKC ␪, ␩ and ␮ contents were diminished. In this latter study, the IR tyrosine kinase activity was also reduced due to serine hyperphosphorylation (Itani et al., 2000). In support of an abnormal elevation in PKC levels in type 2 diabetes, higher DAG levels associated with heightened PKC serine kinase activity arose in hyperinsulinemic conditions in human skeletal muscle (Virkamaki et al., 1999). More speciﬁcally, PKC inhibitors or activators increased or decreased, respectively, the IR tyrosine phosphorylation, PI 3-kinase activity and insulin-stimulated 2deoxyglucose transport in muscle and adipocytes from obese, insulin-resistant subjects (Cortright et al., 2000). A parallel observation has been made in cells in culture, where high glucose/high insulin caused hyperphosphorylation of IRS-1 leading to its downregulation. PKC ␪ mimicked these effects and was therefore proposed to be a causal mediator of insulin resistance (Kellerer et al., 1998). Future studies will have to examine carefully the contribution of the various PKC isoforms to insulin resistance. Moreover, the mechanism whereby these PKC enzymes induce resistance at the level of the IR and IRS-1 (or other insulin signaling molecules leading to glucose transport) will have to be further explored.

Peroxisome Proliferator-Activated Receptor-␥ Peroxisome proliferator-activated receptor-␥ (PPAR-␥), an orphan nuclear receptor, is a key regulator of adipogenesis. Human tissues express 3 isoforms (␥1, ␥2 and ␥3) due to differential splicing at the 5 end of the PPAR-␥ mRNA. These isoforms are most abundantly expressed in adipose tissue. PPAR-␥ binds the insulin-sensitizing thiazolidinediones with high afﬁnity. Administration of PPAR-␥ agonists to insulin-resistant animals and humans causes transcription of several genes implicated in adipocyte differentiation, improves insulin sensitivity and lowers plasma glucose levels and blood pressure (Auwerx, 1999). The resultant gain in the number of small adipocytes probably decreases the production of other metabolites, such as TNF-␣ and free fatty acids which have also been associated with insulin resistance (Auwerx, 1999; Virkamaki et al., 1999). On the other hand, heterozygous [PPAR-␥]/ mice were protected from insulin resistance due to adipocyte hypertrophy, leptin (which regulates satiety) overexpression and hypersecretion, and reduced fat mass under a high-fat diet (Kubota et al., 1999). Insulin itself induces PPAR-␥ expression in adipocytes. In turn, the expression of GLUT4 and CAP, as well as the insulin-stimulated tyrosine phosphorylation of c-Cbl, are induced by PPAR-␥ (Auwerx, 1999). These proteins are implicated in insulin-stimulated glucose transport (see earlier section). To date, several mutations in the PPAR-␥ gene have been characterized. One of these mutations, P115Q, occurs near a regulatory serine phosphorylation site and results in a constitutively-active PPAR-␥. As a result, this mutation no longer enables MAP kinase to phosphorylate the adjacent serine residue. Human carriers of this mutation, though extremely obese, are not highly insulin-resistant (Virkamaki et al., 1999). In keeping with this observation,

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overexpression of this mutant in 3T3-L1 ﬁbroblasts resulted in enhanced adipogenesis. Moreover, the presence of this mutation in obese diabetic animal models led to whole body insulin-sensitization. A dominant negative mutation, P12A, also improved insulin sensitivity, but this time by decreasing insulin receptor activity and not adipogenesis. Thus, this P12A mutation is typically associated with a low body mass index. More recently, two heterozygous mutations (P467L and V290M) in the ligand-binding domain of PPAR-␥ were discovered. Carriers of these mutations exhibit impaired tissue insulin sensitivity and transcriptional activity regulated by PPAR-␥ (Barroso et al., 1999). In addition to the PPAR-␥-speciﬁc mutations, defects in other regulators of adipogenesis, such as the CCAATT enhancer binding proteins (C/EBP), adipocyte differentiation and determination factor 1/sterol regulatory element binding proteins (ADD-1/SREBP) and other transcriptional cofactors, also affect PPAR-␥ expression and function (Auwerx, 1999; Wu et al., 1999). Together, these ﬁndings underscore the importance of the adipogenic activity of PPAR-␥ for activation of the insulin signaling molecules regulating insulin-stimulated glucose transport. Elucidation of the mechanisms controlling adipose tissue development will therefore be crucial to understanding the etiology of insulin resistance. Plasma Cell Differentiation Factor-1 The plasma cell differentiation factor-1 (PC-1) is a membrane glycoprotein with ectonucleotide pyrophosphatase activity. Its expression is typically associated with insulin resistance (Pizzuti et al., 1999). In fact, higher PC-1 levels in human ﬁbroblasts, muscle and adipose tissue correlates with a high body mass index and a decrease in insulin-stimulated glucose transport. Its overexpression inhibited the IR tyrosine kinase activity by direct interaction of PC-1 with the IR ␣-subunit, and subsequently insulin signaling was reduced (Maddux and Goldﬁne, 2000). PC-1 was also found to be highly expressed in skeletal muscle of a group of obese subjects (Youngren et al., 1996) and of patients with gestational diabetes (Shao et al., 2000), potentially explaining the observed downregulation of IR tyrosine phosphorylation. Moreover, elevated expression of PC-1 showed negative correlation with insulin sensitivity as observed in an intravenous insulin tolerance test and in vitro assays of muscle IR tyrosine kinase phosphorylation and activity. Finally, a polymorphism in exon 4 of the PC-1-encoding gene, K121Q, was linked to insulin resistance in healthy non-obese, non-diabetic individuals by higher glucose and insulin levels during oral glucose tolerance testing and euglycemic clamp studies (Pizzuti et al., 1999). Human carriers of the K121Q mutation showed higher risk of becoming hyperinsulinemic and insulin resistant. Moreover, IR autophosphorylation was decreased in cultured skin ﬁbroblasts from the K121Q mutation carriers than noncarriers (Pizzuti et al., 1999). The link between dysregulated PC-1 function and resistance in the insulin signaling pathway of insulin-sensitive tissues will have to be examined in more detail.

IN VITRO MODELS OF INSULIN RESISTANCE In order to understand the pathogenesis of insulin resistance, it is crucial to dissect the individual variables. This goal is facilitated by the use of cellular models of insulin resistance, which have been instrumental to discern the defects in insulin signaling that arise in a controlled environment. We analyze next the possible interrelationship between several conditions

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used to generate in vitro models of insulin resistance and the resultant downregulation of the cellular processes governing insulin-stimulated glucose uptake. Collectively, these in vitro systems serve not only as valid models to study insulin resistance, but also to provide evidence of the cellular mechanisms affected by hyperglycemia, hyperinsulinemia and increased reactive oxygen species (ROS) production. These three conditions arise in type 2 diabetes and are thought to contribute to impaired insulin action.

High Glucose/High Insulin In vivo, hyperglycemia and hyperinsulinemia appear to have deleterious effects on both insulin secretion and insulin action. In vitro, incubation of muscle strips with insulin and glucose correlates with impaired glucose transport in obese subjects with or without type 2 diabetes and lean type 2 diabetic patients (Zierath et al., 2000). Insulin resistance can also be induced by incubating cells in culture with high levels of glucose and/or insulin. Pre-exposing primary cultured adipocytes to a combination of insulin and glucose for 24 h results in a dosedependent reduction of insulin-stimulated glucose transport rates by decreasing the glucose transporters at the cell surface (Garvey et al., 1987). Exposure of cultured adipocytes to high glucose along with high insulin levels has also led to impaired insulin signaling, glucose uptake and GLUT4 translocation upon insulin stimulation (McClain and Crook, 1996; Virkamaki et al., 1999; Zierath et al., 2000). Similarly, high insulin levels induce insulin resistance of glucose transport in cultured primary human skeletal muscle cells, which was further impaired by the addition of high glucose to the medium (Ciaraldi et al., 1995). Table 4 highlights changes to various insulin signaling components (IRS-1 phosphorylation, Akt) and GLUT4 trafﬁc under different insulin-resistant conditions. The in vitro results described above are also in line with the well-known inverse correlation between insulin-stimulated glucose clearance and high fasting blood glucose (hyperglycemia) in type 2 diabetes (Del Prato et al., 1993; Lillioja et al., 1993). Other evidence for the contribution of high glucose to insulin resistance includes the ability of experimentally-induced euglycemia to restore insulin-stimulated glucose transport in skeletal muscles from type 2 diabetic individuals (Kahn et al., 1991; Krook et al., 1997; Rossetti et al., 1987; Zierath et al., 1994). All of these ﬁndings beg the question of how high insulin or high glucose levels mechanistically bring about changes in the protein components that facilitate glucose transport in response to insulin. There are results to suggest that the insulin resistance evident under hyperglycemia or hyperinsulinemia may arise from the inappropriate metabolic consequences of glucose disposal. Presently, PKC activation and ﬂux into the hexosamine pathway are considered possible mediators of induced insulin resistance. Hyperglycemia has been shown to increase DAG levels, which in turn stimulates PKC-translocation to subcellular membranes (see Zierath et al., 2000). This is thought to lead to an increase in the PKCmediated serine phosphorylation of the IR and IRS-1, as discussed earlier in this chapter (see section on Other PKCs). Consequently, insulin action downstream of IR tyrosine phosphorylation is reduced (see Table 4). Indeed, incubation of rat adipocytes in high glucose has led to an impairment in IRS-1 tyrosine phosphorylation and attenuation of PI 3-kinase activity (Pillay et al., 1996). How the additional availability of glucose for passage through the hexosamine pathway could contribute to insulin resistance is discussed in some detail below. In any event, more work must still be done to explore these two possibilities, PKC activation and an increase in hexosamine-derived metabolites, as inducers of insulin resistance.

— —

Free fatty acids C2C12 myocytes 3T3-L1 adipocytes ↔ —

↓↓ —

↓ means 50% decrease; ↓↓ means 50% decrease; ↔ no change compared to control; — not determined.

↔ —

— ↓↓

↓ ↓↓ (LDM) ↔ (TCL)

↔ ↓↓

— —

— ↓ —

↔ ↓ ↔

— ↓ ↔

— ↓↓ ↔

Akt activity

— ↔ — — ↓↓

PI 3-Kinase activity — — ↓↓ ↔ ↓

— ↔ ↓ — ↓↓

— ↔ ↓ — ↓

IRS-1 phosphorylation

High glucose/high insulin 3T3-L1 adipocytes 3T3-L1 adipocytes 3T3-L1 adipocytes 3T3-L1 adipocytes L6-Myc myotubes Glucosamine 3T3-L1 adipocytes 3T3-L1 adipocytes 3T3-L1 adipocytes Oxidative stress 3T3-L1 adipocytes 3T3-L1 adipocytes

IR phosphorylation

In vitro cellular models of insulin resistance.

Mechanism to induce insulin resistance cell type

Table 4

— ↔

↓ —

↓ ↓↓ ↓↓

↓ — ↓ ↔ ↓

GLUT4 translocation

— ↓

↓ —

↓ ↓ —

↓↓ ↓ ↓ ↓ ↓

Glucose uptake

Schmitz-Peiffer et al., 1999 Van Epps-Fung et al., 1997

Rudich et al., 1998 Tirosh et al., 1999

Nelson et al., 2000 Hresko et al., 1998 Heart et al., 2000

Thomson et al., 1997 Ross et al., 2000 Ricort et al., 1995 Nelson et al., 1995 Huang et al., 2002

Reference

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Glucosamine Normally, a small proportion of intracellular glucose is shunted to the hexosamine pathway to produce the UDP-GlcNAc that is required for protein glycosylation. Several studies have shown a positive correlation between UDP-GlcNAc and the observed insulin resistance. It is thought that the increased availability of this metabolite may lead to abnormal glycosylation of proteins, purportedly those governing GLUT4 translocation, glucose transport activity and/or the ﬂux of glucose into glycogen (Marshall et al., 1991). As with glucose, glucosamine also enters cells via the GLUT family glucose transporters, and is readily phosphorylated by hexokinase, like glucose-6-phosphate, to produce glucosamine-6-phosphate (Robinson et al., 1993). This latter compound is used to synthesize UDP-GlcNAc. To test whether glucoseinduced insulin resistance is due to abnormally high UDP-GlcNAc intracellularly, numerous studies have examined the effects of glucosamine on insulin action in muscle and fat. Glucosamine is not normally present in the circulation in signiﬁcant amounts, but exogenously administered glucosamine is readily taken up by tissue resulting in elevated intracellular levels of UDP-GlcNAc. Glucosamine infusion into rats in vivo induces severe insulin resistance in skeletal muscle. Such insulin resistance correlates with increased activity of the rate-limiting enzyme glutamine: fructose-6-phosphate aminotransferase (GFAT) of the hexosamine pathway (Rossetti et al., 1995). Interestingly, transgenic overexpression of GFAT in mice markedly reduced whole-body glucose disposal (Hebert et al., 1996). As well, GFAT activity was more highly induced by glucose and insulin in human skeletal muscle cultures from type 2 diabetic subjects vs. non-diabetic controls (Daniels et al., 1996). The above observations have prompted the analysis of possible alterations in the insulin signaling pathway. Glucosamine infusion into rats decreased the activation of PI 3-kinase by insulin in skeletal muscle (Kim et al., 1999). More direct observations have been made in cells in culture. In rat skeletal L6 myotubes, glucose or glucosamine incubation caused a downregulation of glucose transport (Davidson et al., 1993). In 3T3-L1 adipocytes, glucosamine caused a dose- and time-dependent impairment in insulin-stimulated glucose uptake and GLUT4 translocation but did not alter the insulin-induced phosphorylation of the IR, IRS1/2 or PI 3-kinase activity. However, Akt and p70 S6 kinase stimulation by insulin were impaired (Heart et al., 2000). When glucosamine was the only source of energy, IR and IRS-1 phosphorylation, IRS-1-associated PI 3-kinase and Akt activity, and the translocation of GLUT4 and GLUT1 to the plasma membrane were all reduced. These changes could be prevented by supplementing the culture medium with an alternate energy source (Hresko et al., 1998). These authors had suggested that perhaps the insulin resistance caused by glucosamine in vivo was only the result of reductions in ATP. However, it is unlikely that the effect of glucosamine in vivo is due to exhaustion of ATP levels, given the availability of glucose in the circulation, as well as other energy sources. Moreover, most in vitro studies generating insulin resistance via glucosamine had included glucose in the medium. In conclusion, the glucosamine-infusion in vivo and in vitro alters PI 3-kinase and glucose uptake, but the mechanisms involved are not delineated, and action in the whole organism may differ from that involved in the local insulin resistance observed in cells in culture. In addition, ongoing studies comparing the effects of high glucose and glucosamine show differences in their affected pathways. Future emphasis should be given to examine the effect of glucosamine, in vivo and in vitro on Akt and PKC.

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Oxidative Stress Hyperglycemia, hyperinsulinemia and an altered antioxidant capacity, all components of insulin resistance, are suspected to generate oxidative stress in type 2 diabetes (Yaworsky et al., 2000). Normally, a natural intracellular balance exists between oxidants and antioxidant defenses. Under oxidative stress, increased free radical production or decreased antioxidant capacity alters this equilibrium, resulting in oxidative damage by ROS such as hydrogen peroxide, hydroxyl radicals or superoxide anions. In diabetes, the high levels of glucose and insulin contribute to oxidative damage via elevations in glucose oxidation, non-enzymatic glycation and polyol accumulation (Yaworsky et al., 2000). All these condition can be found to lead to oxidative stress through free radical production, protein oxidation, and reductant (NADPH) or antioxidant (glutathione) depletion. Recently, it was shown that, in addition to high glucose, high insulin or glucosamine, insulin resistance can also be generated in vitro by culturing cells in the presence of enzymatic systems that produce reactive oxygen species (such as glucose oxidase (GO) or xanthine oxidase). 3T3-L1 adipocytes pre-exposed to GO and glucose (a hydrogen peroxide-generating system) for 18 h displayed reduced cellular capacity to increase glucose transport in response to insulin. The underlying mechanism appeared to be via a decrease in GLUT4 protein and mRNA levels (Rudich et al., 1997). Signiﬁcant reductions in insulin-stimulated lipogenesis and glycogen synthase a activity were also found in these cells. Similar results were obtained in L6 myotubes. Shorter exposure of 3T3-L1 adipocytes to low levels of H2O2 generated by GO/glucose caused an impaired redistribution of IRS-1 and PI 3-kinase from the cytosol to the low density microsomal fractions upon insulin stimulation (Rudich et al., 1998; Tirosh et al., 1999). This correlated with a decrease in the IRS-1 and p85-associated PI 3-kinase activities recovered in the light microsomal fraction. However, the overall activity of PI 3-kinase in cell extracts was normally stimulated. Notably, Akt activation was also severely impaired in response to insulin (Tirosh et al., 1999). These results suggest that oxidative stress abnormally affected insulin signaling downstream of PI 3-kinase, likely through delocalizing the enzyme, and thereby diminished GLUT4 translocation. A more in-depth analysis of how oxidative stress contributes to insulin resistance by altering the insulin-induced cellular compartmentalization of certain insulin signaling components is reviewed elsewhere (Tirosh et al., 2000), and these ﬁndings are summarized in Table 4. The three in vitro models of insulin resistance discussed above are not the only cellular models being used to explore the defects in insulin signaling. Studies analyzing the impact of factors such as TNF-␣ and free fatty acids, have been described as well. Space limitations preclude us from reviewing them here. Table 4 provides a summary of the various models of insulin resistance used in isolated cell systems, and the consequent abnormalities observed in insulin signals and GLUT4 translocation. Systematic analyses of this type should lead to a better understanding of the cellular basis of insulin resistance. CONCLUSION There is ample evidence that the pathogenesis of insulin resistance is heterogeneous. Alterations of the insulin signaling pathway at different levels are associated with different models of insulin resistance and in patients with type 2 diabetes. Further insights in the pathophysiology of these mechanisms will contribute to a better understanding of insulin resistance and hopefully will lead to improvements in therapy offered to type 2 diabetic patients.

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Itani, S.I., Zhou, Q. et al. (2000) Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity. Diabetes, 49, 1353–1358. Iversen, L.F., Andersen, H.S. et al. (2000) Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B. J. Biol. Chem., 275, 10300–10307. Jiang, T., Sweeney, G. et al. (1998) Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem., 273, 11017–11024. Kahn, B.B., Rosen, A.S. et al. (1992) Expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle of humans with insulin-dependent diabetes mellitus: regulatory effects of metabolic factors. J. Clin. Endocrinol. Metab., 74, 1101–1109. Kahn, B.B., Shulman, G.I. et al. (1991) Normalization of blood glucose in diabetic rats with phlorizin treatment reverses insulin-resistant glucose transport in adipose cells without restoring glucose transporter gene expression. J. Clin. Invest., 87, 561–570. Kanoh, Y., Bandyopadhyay, G. et al. (2000) Thiazolidinedione treatment enhances insulin effects on protein kinase C-zeta /lambda activation and glucose transport in adipocytes of nondiabetic and Goto-Kakizaki type II diabetic rats. J. Biol. Chem., 275, 16690–16696. Katagiri, H., Asano, T. et al. (1996) Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes. J. Biol. Chem., 271, 16987–16990. Kellerer, M., Lammers, R. and Haring, H.U. (1999) Insulin signal transduction: possible mechanisms for insulin resistance. Exp. Clin. Endocrinol. Diabetes, 107, 97–106. Kellerer, M., Mushack, J. et al. (1998) Protein kinase C isoforms alpha, delta and theta require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells). Diabetologia, 41, 833–838. Kido, Y., Burks, D.J. et al. (2000) Tissue-speciﬁc insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2. J. Clin. Invest., 105, 199–205. Kim, J.K., Michael, M.D. et al. (2000) Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle. J. Clin. Invest., 105, 1791–1797. Kim, Y.B., Nikoulina, S.E. et al. (1999) Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes [see comments]. J. Clin. Invest., 104, 733–741. Kim, Y.B., Peroni, O.D. et al. (2000) Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats. Diabetes, 49, 847–856. Kim, Y.B., Zhu, J.S. et al. (1999) Glucosamine infusion in rats rapidly impairs insulin stimulation of phosphoinositide 3-kinase but does not alter activation of Akt/protein kinase B in skeletal muscle. Diabetes, 48, 310–320. King, P.A., Horton, E.D. et al. (1992) Insulin resistance in obese Zucker rat (fa/fa) skeletal muscle is associated with a failure of glucose transporter translocation. J. Clin. Invest., 90, 1568–1575. Klaman, L.D., Boss, O. et al. (2000) Increased energy expenditure, decreased adiposity, and tissue-speciﬁc insulin sensitivity in protein-tyrosine phosphatase 1B-deﬁcient mice. Mol. Cell. Biol., 20, 5479–5489. Klip, A., Ramlal, T. et al. (1990) Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle. Biochem. Biophys. Res. Commun., 172, 728–736. Klip, A., Tsakiridis, T. et al. (1994) Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J., 8, 43–53. Kohn, A.D., Summers, S.A. et al. (1996) Expression of a constitutively active Akt Ser/Thr kinase in 3T3L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J. Biol. Chem., 271, 31372–31378. Kotani, K., Ogawa, W. et al. (1998) Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes. Mol. Cell. Biol., 18, 6971–6982. Krook, A., Bjornholm, M. et al. (2000) Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes, 49, 284–292. Krook, A., Kawano, Y. et al. (1997) Improved glucose tolerance restores insulin-stimulated Akt kinase activity and glucose transport in skeletal muscle from diabetic Goto-Kakizaki rats. Diabetes, 46, 2110–2114.

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10.

WHICH GENES ARE IMPORTANT IN THE DEVELOPMENT OF TYPE 2 DIABETES?

G.R. COLLIER,1,2 K. WALDER,1 A. DE SILVA,1 S. MORGAN,1 D. SEGAL,1 L. KANTHAM,1 AND G. AUGERT3 1

Metabolic Research Unit, School of Health Sciences, Deakin University, Geelong, Victoria, 3217 Australia 2 Autogen Limited, Melbourne, Victoria, Australia 3 Merck-Lipha, Lyon, France

Diabetes mellitus is rapidly becoming one of the world’s major public health problems, and there is every reason to believe that over the next decade the epidemic of type 2 diabetes will continue to escalate. Diabetes affects large numbers of people from a wide range of ethnic groups and at all social and economic levels throughout the world. A minority of patients suffer from type 1 diabetes which is caused by pancreatic beta-cell failure, however type 2 diabetes is far more common and is characterized by tissue resistance to insulin that cannot be overcome by beta-cell hypersecretion. It has been predicted that the number of adults with diabetes in the world will rise from 135 million in 1995 to 300 million in the year 2025 (Seidell, 2000). The major part of this increase will occur in developing countries. Diabetes has a prevalence rate of approximately 7% in the Australian population with higher rates in many non-Caucasian populations such as the Australian Aborigines (Australian Diabetes, Obesity and Lifestyle Report, 2001). The true prevalence of diabetes is higher than the ﬁgures above suggest, because a large proportion of type 2 diabetes remains undiagnosed in the community. In 2000 the prevalence of impaired glucose metabolism (impaired glucose tolerance or impaired fasting glycemia) in the Australian population was 16% (Australian Diabetes, Obesity and Lifestyle Report, 2001). Diabetes is among the top ten causes of death in westernized societies (Murray and Lopez, 1996). In Australia, diabetes is the seventh leading cause of death and contributes to signiﬁcant illness, disability, poor quality of life and premature mortality (Australian Bureau of Statistics, 1999). Yet mortality statistics greatly underestimate the true rate of diabetes-related mortality because diabetes is frequently under-reported on death certiﬁcates. Diabetes is often mentioned as a contributory cause on death certiﬁcates but the underlying cause is more frequently coded as cardiovascular disease or renal disease (Colagiuri et al., 1998). If the burden of cardiovascular disease attributable to diabetes is included, diabetes becomes the third leading cause of disease burden in Australia (Australian Institute of Health and Welfare, 2000). The health care direct and indirect costs associated with diabetes in 1997 in the United States alone were an estimated $98 billion (American Diabetes Association, 1998). The primary features of type 2 diabetes are hyperglycemia, defects in insulin action and secretion and abnormalities in carbohydrate, fat and protein metabolism. Type 2 diabetes is characterized clinically as the presence of hyperglycemia in the fasting state, with defects in insulin secretion and action. Pathophysiologically there are two major defects, a decrease in the tissue response to insulin, leading to insulin resistance, and a failure of the pancreatic beta-cells to compensate for this insulin resistance by increasing insulin secretion. The metabolic disturbances 175

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created by chronic hyperglycemia along with the strong association between diabetes, obesity, hypertension and hyperlipidemia, lead to a list of long-term complications including cardiovascular disease, peripheral vascular disease, retinopathy, nephropathy and neuropathy (International Diabetes Foundation Taskforce, 1999). A positive association between obesity and the risk of developing type 2 diabetes has been repeatedly observed in both cross-sectional and prospective studies (World Health Organization, 1998). Adult weight gain, the degree of obesity and the duration of obesity all independently predict the risk of type 2 diabetes (Wannamethee and Shaper, 1999). The risk associated with obesity is particularly high when excess fat is distributed intra-abdominally (Samaras and Kelly, 1998). The increasing prevalence of obesity in westernized communities is thought to be due to an increasingly available high-fat, energy-dense food supply, combined with an increasingly sedentary lifestyle. Mechanization of many types of work, and changes in transport are causing increasing numbers of people to be sedentary most of the time (Bray, 2000; Boyko et al., 2000). The mechanisms linking obesity with diabetes are not well known, but may include elevated levels of circulating free fatty acids, tumor necrosis factor-␣ and/ or the recently discovered hormone ‘resistin’ (Bergman and Ader, 2000; Moller, 2000; Steppan et al., 2001). Clustering of type 2 diabetes in certain families and ethnic populations, and the high concordance rate (50–95%) for diabetes in monozygotic twins, points to a strong genetic basis for the disease (Groop, 1999). Clearly, interactions between genes and the environment are important in the pathogenesis of type 2 diabetes, but it remains unclear how these interactions occur. The comprehensive longitudinal studies of diabetes conducted in Pima Indians over the last 30 years, conﬁrm that both genetic and environmental factors play a critical role in the pathogenesis of the disease. A high fat diet and low levels of physical activity in adulthood markedly increase the risk of developing type 2 diabetes in this population. In addition, the high prevalence of diabetes in Pima Indians relative to other populations and the familiality of the disease and its precursors, support a substantial genetic basis (Knowler et al., 1990, 1991; Sakul et al., 1997). Due to the complex inheritance and interaction with the environment, identifying the genes involved in type 2 diabetes is difﬁcult. It is generally accepted that genetic factors play a signiﬁcant role in the development of type 2 diabetes, however the nature of the key genes involved is not known. A small percentage of cases can be explained by single gene defects, including those known to cause Maturity Onset Diabetes of the Young (MODY). As shown in Table 1, these single gene defects generally affect key components of insulin action, for example, insulin receptor, glucokinase, or a set of transcription factors associated with MODY. However, all of the known single gene defects associated with diabetes thus far account for only a small proportion (2–5%) of cases of type 2 diabetes, and are usually associated with Table 1

Examples of monogenic causes of diabetes.

MODY:

HNF4␣ (MODY 1) Glucokinase (MODY 2) HNF1␣ (MODY 3) Insulin promoter factor-1 (MODY 4) HNF1␤ (MODY 5) Insulin gene mutations (familial hyperproinsulinemia) Insulin receptor mutations (leprechaunism, Robinson–Mendenhall syndrome) Maternally inherited mitochondrial gene mutations (Adapted from Kahn et al., 1996.)

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atypical cases such as severe familial insulin resistance or diabetes with a very early age of onset. Signiﬁcant contributions of mutations in these genes to more general cases of type 2 diabetes has not been established, and it is highly likely that a number of other genes are involved in the pathogenesis of typical cases of type 2 diabetes. Searching for individual genes, candidate by candidate, in a complex disease with polygenic or oligogenic origins, such as type 2 diabetes, is a daunting and near-impossible task. Candidate genes are normally selected from physiological pathways known to be important in the regulation of carbohydrate or fat metabolism, and include a wide variety of genes (some examples are shown in Table 2). Difﬁculties in proving association between these genes and diabetes arise because each individual gene may play only a minor role in determining the ﬁnal (measurable) phenotype, thus making the detection of association very difﬁcult. However, such a gene may be vital for the development of type 2 diabetes when in combination with variants in a number of other genes. Hundreds of candidate gene association studies have been performed in recent decades identifying very few (if any) plausible, replicable relationships between individual genes and diabetes or its sub-phenotypes (e.g. fasting glucose and insulin concentrations, measures of insulin secretion and action). An alternative approach to searching for type 2 diabetes-causing genes is the use of genomewide scans in large family groups to identify chromosomal regions linked with the disease. It is then a major undertaking to identify the speciﬁc genetic variant within this region that is causally related to the linked phenotype (a process known as ﬁne mapping). Genome-wide linkage scans for diabetes and related sub-phenotypes have been performed in a number of populations, and at least ﬁfteen different chromosomal regions thought to harbor diabetescausing genes have been identiﬁed (Table 3). Given the great investment of time and money into genome-wide linkage-based approaches, the results in terms of identiﬁcation of disease-causing genes has thus far been

Table 2

Examples of potential candidate genes for diabetes.

GLUT1 GLUT4 Hexokinase II GSK-3 Glycogen synthase Glucagon receptor gene

Hormone-sensitive lipase Fatty acid binding proteins IRS1/2 Shc PI3 kinase PPAR␥

(Adapted from Moller et al., 1996, Pederson, 1999.)

Table 3

Genome-wide linkage scans for type 2 diabetes.

Population

Region(s) linked with diabetes

Reference

Mexican Americans

2q37 15 20q13 11q, 6q, 9q, 1q, 7q 20p 10q, 3p, 4q, 9p 1q21–1q23

Hanis et al., 1996 Cox et al., 1999 Zouali et al., 1997 Hanson et al., 1998 Ghosh et al., 1999 Duggirala et al., 1999 Elbein et al., 1999

3q27-qter, 1q21–q24, 2p21–16, 10q26, 20p, 20q

Vionnet et al., 2000

French Pima Indians Finnish Mexican Americans Utah (Northern European ancestry) French

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disappointing. The task of statistical analysis of linkage data and ﬁne-mapping to identify genes has proved to be very difﬁcult in the case of type 2 diabetes, as it has in other multifactorial, polygenic diseases. It is hoped that continued improvements in technologies useful for ﬁne-mapping and analysis of data will facilitate the deﬁnitive identiﬁcation of disease-causing genes from these scans in the near future. Until such time, the real impact of these studies is difﬁcult to quantitate. To date, only one genome-wide linkage study has progressed successfully through ﬁnemapping and positional cloning to the identiﬁcation of a potential candidate gene, Calpain 10 (Horikawa et al., 2000). This ﬁnding was the result of screening of the human genome for susceptibility genes for type 2 diabetes in a group of Mexican American affected sib pairs (Hanis et al., 1996). Calpain 10 is a ubiquitously expressed nonlysosomal cysteine protease, part of a family implicated in the regulation of intracellular signaling, cell proliferation and differentiation. In Pima Indians, individuals with the UCSNP-43 G/G genotype had reduced expression of calpain 10 in skeletal muscle that was associated with reduced glucose oxidation rates, indicative of insulin resistance (Baier et al., 2000). The calpain family of proteases therefore represent an interesting new candidate pathway in the pathogenesis of type 2 diabetes. However, the studies that resulted in the identiﬁcation of calpain 10 took approximately 10 years and enormous resources, and although new technologies may shorten the time taken from population collection to identiﬁcation of candidate genes, the task should not be underestimated. The time and ﬁnancial investment required for such a study should be a sobering thought for any research group embarking on this long and difﬁcult road to gene discovery. Overall, DNA-based approaches for the discovery of genes that contribute to the development of type 2 diabetes have not been very successful despite substantial investment of time and money. It is possible that the etiology of a complex disease such as type 2 diabetes is not easily solved using current DNA-based approaches, due to the multiple gene–gene and gene–environment interactions. Large numbers and/or variable combinations of small gene defects leading to the ﬁnal disease state also complicate these analyses and make identiﬁcation of important genes problematic. However, there are alternatives in the ﬁeld of gene discovery in type 2 diabetes, such as RNA (gene expression) or proteomics-based strategies. Gene expression-based technologies may prove to be a more rewarding approach to identify diabetes candidate genes. Moreover, the use of this technology, when combined with appropriate animal models will be a very powerful tool to understand the underlying mechanisms of human polygenic diseases. There are a number of RNA-based technologies available to identify differentially expressed genes in different samples. These include differential display polymerase chain reaction (ddPCR), representational difference analysis (RDA) and cDNA microarrays. Both ddPCR and RDA have been successfully used to identify novel genes involved in energy metabolism. For example, in our laboratory, ddPCR was used to identify beacon, a novel polypeptide involved in the regulation of energy balance, which is differentially expressed in the hypothalamus of obese, diabetic and lean, non-diabetic Israeli sand rats (Collier et al., 2000). The RDA technique was used to identify genes differentially expressed in adipocytes exposed to rosiglitazone, a PPAR␥ agonist used to treat type 2 diabetes. One of the identiﬁed genes, resistin, is a soluble molecule that may provide a link between obesity and type 2 diabetes (Steppan et al., 2001). To date there have been very little data published using cDNA microarrays to examine gene expression in type 2 diabetes. Each of these methods has distinct advantages and disadvantages in identifying differentially expressed genes. Both ddPCR and RDA are used to identify genes that are expressed more highly in one sample relative to another. RDA uses a subtractive hybridization method to enrich a cDNA library for differentially expressed genes

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while ddPCR relies on identiﬁcation of differentially ampliﬁed cDNA transcripts derived from two RNA samples. In contrast, cDNA microarrays use competitive hybridization of labeled cDNAs to cDNA probes (each representing a distinct gene) attached to a solid support. The advantages of RDA and ddPCR are that they potentially generate target genes quickly and cheaply and use equipment found in most modern laboratories. However, both these techniques are labor-intensive and typically identify small numbers of differentially expressed genes. Moreover, the binary nature of RDA and ddPCR limit their application to simple univariate comparison experiments. In contrast, cDNA microarrays can provide quantitative and comparative gene expression measurements for many thousands of genes and from large numbers of experimental samples. It is notable that the production and use of cDNA microarrays requires the purchase of expensive hardware and cDNA clone sets, and are labor intensive to establish. However, the technique is ideal for multivariate analyses aimed at understanding the underlying mechanisms associated with a disease. Currently, clustering algorithms are commonly used to identify genes that exhibit similar expression patterns (Eisen et al., 1998), however, efforts are underway to construct models of gene transcription from microarray data that identify sets of genes that participate in particular biological processes (Kim et al., 2000). It is probable that cDNA microarray technology will prove to be of great utility in identifying key molecules and mechanisms in type 2 diabetes. The power of new technologies to detect differential gene expression is ideally suited to studies utilizing appropriate animal models of human disease. Very few polygenic, outbred rodent models of obesity and type 2 diabetes exist. One such model, Psammomys obesus (Israeli Sand Rat), has been studied extensively in our laboratory (Barnett et al., 1994, 1995; Habito et al., 1995; Collier et al., 1997a,b, Lewandowski et al., 1997; Walder et al., 1997, 1998, 1999; Collier et al., 2000). Psammomys obesus are gerbil-like rodents native to the desert regions of the Middle East and Northern Africa. In their native habitat, Psammomys obesus remain lean and free from diabetes on a diet consisting mainly of saltbush (Atriplex Halimus) (Shafrir and Glutman, 1993). However, when taken into a laboratory setting and allowed ad libitum access to standard rodent chow, a proportion of Psammomys obesus develop varying degrees of obesity, insulin resistance and type 2 diabetes (Barnett et al., 1994; Kalderon et al., 1986). It is the heterogeneity of the metabolic response to a relatively energy dense diet that makes Psammomys obesus an excellent animal model of human obesity and type 2 diabetes. Previous studies have identiﬁed a number of metabolic disturbances in obese, diabetic Psammomys obesus relative to their lean littermates. These include hyperglycemia, hyperinsulinemia, insulin resistance, hyperphagia, obesity and dyslipidemia (Barnett et al., 1994; Habito et al., 1995; Collier et al., 1997a; Walder et al., 2000). Diabetic Psammomys obesus have marked hepatic insulin resistance (Ziv et al., 1996) and a defective insulin receptor-signaling pathway (Kanety et al., 1994; Shafrir and Ziv, 1998). Hyperproinsulinemia, reduced pancreatic insulin storage capacity and beta-cell apoptosis have also been observed in these animals (Gadot et al., 1994; Bar-On et al., 1999). A recent study demonstrated increased expression of protein kinase C epsilon in skeletal muscle of diabetic Psammomys obesus that may be causally related to the development of insulin resistance (Ikeda et al., 2001). Investigation of leptin in Psammomys obesus showed elevated plasma leptin concentrations in obese, diabetic animals (Walder et al., 1997), and resistance to the effects of peripheral (intraperitoneal) leptin administration in obese but not lean animals (Walder et al., 1999). It is possible that the leptin resistance exhibited in this study may be contributing to the development of obesity in Psammomys obesus. The most important aspect of the development of obesity and type 2 diabetes in Psammomys obesus, is that cross-sectional analysis of the animal population reveals heterogeneous distributions

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of blood glucose, plasma insulin and body weight (Barnett et al., 1994; Walder et al., 2000). These distributions are almost identical to the patterns observed in cross-sectional studies of human populations, including the inverted U-shaped relationship between circulating glucose and insulin concentrations known as “Starlings curve of the pancreas” (Zimmet et al., 1979; DeFronzo, 1988). In addition, we have shown that genetic factors account for over 50% of the variation in body weight and approximately 25% of the variation in blood glucose and insulin concentration in Psammomys obesus (Walder et al., 2000). Furthermore, obesity in Psammomys obesus increases the risk of developing type 2 diabetes, and obese, diabetic animals are more prone to dyslipidemia and cataracts than their lean, non-diabetic littermates (Walder et al., 2001, unpublished data). Therefore, Psammomys obesus represents a unique, polygenic animal model of obesity and type 2 diabetes that closely resembles the human metabolic syndrome (Zimmet et al., 1989; Reaven et al., 1988; Stern, 1995). In our laboratory, the use of modern technologies to detect differential gene expression, in combination with an excellent animal model such as Psammomys obesus, has resulted in the identiﬁcation of novel genes important in the development of type 2 diabetes. Using ddPCR, we compared gene expression in the hypothalamus of obese, diabetic and lean, non-diabetic Psammomys obesus and identiﬁed a novel gene we called beacon. The beacon gene was identiﬁed as a gene product overexpressed in the hypothalamus of obese, diabetic animals. Further studies in a larger group of animals demonstrated that the beacon gene was expressed in the hypothalamus in direct proportion to the body fat content of the animals (Collier et al., 2000). The beacon gene is expressed ubiquitously throughout the body and encodes a small protein of seventy-three amino acids. The human beacon gene consists of 2194 nucleotides arranged into ﬁve exons and four introns and has been mapped to chromosome 19. The Psammomys obesus beacon gene is composed of four exons, has a shorter 5 untranslated region, and consequently lacks the ﬁrst exon present in the human gene (Genebank Accession: AF318186; Collier et al., 2000). The identiﬁcation of a candidate gene is only the ﬁrst step in determining its importance in the development of diabetes. It is necessary to determine the physiological function of the protein produced by the novel gene and to validate the potential of this protein as a therapeutic target. Knowledge of the complete sequence of the human genome has not yet added extensively to our understanding of the function of the expected gene products. The outlook of one gene/one function is slowly being replaced by gene function in the context of interconnecting signal transduction and metabolic pathways, and there is increased realization that it is crucial to understand the function of gene products in a physiological context. Recent years have seen the addition of various new tools to the existing classical biochemical techniques for understanding the role of proteins of unknown function. The research tools and strategies for functional studies that have been applied in our own and various other laboratories are outlined in Figure 1, and will be discussed here in the context of functional analysis of the beacon protein. We produced 25 mg of pure beacon protein by expression in Escherechia coli as a fusion protein with GST and afﬁnity puriﬁcation. The beacon protein produced has been used for production and screening of monoclonal and polyclonal antibodies, which in turn were used for immunohistochemical studies that localized the beacon protein to the retrochiasmatic area of the hypothalamus. This region has previously been implicated in the regulation of energy balance, thus providing support for a role for beacon in obesity and type 2 diabetes (Collier et al., 2000). Gain or loss of function studies provide evidence of a novel protein’s effects in both cell systems and intact animals. We are currently conducting experiments to investigate the effects

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Target gene

Protein production in bacteria, yeast, insect or mammalian cells

Affinity purification

Antibody production

Immunolocalization by immunohistochemistry and western blot

Gain or loss of function studies

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Expression transient and /or stable in relevant eukaryotic cells (e.g. C2C12, L6, 3T3-L1, Min6)

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Antisense oligonucleotides treatments of relevant eukaryotic cells (e.g. C2C12, L6, 3T3-L1, Min6)

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Yeast two hybrid

Central and/or peripheral administration of Biophysical methods plasmon surface resonance - Protein - Specific antibody - Gene using high expression Studies with intact cells adenoviral and retroviral vectors FRET and BRET technologies Co-expression and co-immunoprecipitation of two potential interacting partners

Study changes in metabolic functions Changes in expression levels normal vs. disease states

Physiological and biochemical changes

Figure 1 Tools for validation of functional role of target gene.

of increased beacon gene expression using recombinant adenoviral transfection both in cell systems and in Psammomys obesus, and inhibition of beacon gene expression using antisense oligonucleotides. Endpoints to be measured in the cell systems, such as 3T3-L1 adipocytes and H4IIE hepatocytes, include basal and insulin-stimulated glucose uptake, fat uptake and glycogen synthesis. Studies in Psammomys obesus will reveal effects of altered beacon gene expression on body weight, blood glucose and plasma insulin, and indirect calorimetry will be used to investigate effects on substrate utilization and energy expenditure. Taken together, these studies will provide data to help determine the importance of beacon in the development of obesity and type 2 diabetes. Almost all proteins fulﬁll their functional role via interaction with other proteins. Identiﬁcation of interacting proteins and any knowledge of their functional properties can provide clues for positioning the candidate protein in a deﬁned signal transduction pathway. Protein–protein interactions can be identiﬁed using co-expression technologies such as the yeast 2-hybrid system (Fields and Song, 1989; Gyuris et al.,1993; Durfee et al., 1993; Vidal et al., 1996), or biophysical techniques such as Fluorescence and Bioluminescence Resonance Energy Transfer (Damelin and Silver, 2000; Angers et al., 2000) or Biacore. All of these techniques aim to identify interactions between two proteins. Using the yeast 2-hybrid method, we have identiﬁed a novel kinase that interacts with beacon, thus providing a novel candidate pathway in the pathogenesis of obesity and type 2 diabetes. Importantly this pathway now provides a target for the development of chemical interventions to decrease beacon action, which can then be developed further as a potential therapeutic agent in the treatment of obesity. Finally, it is important to conﬁrm the predicted function of a candidate protein by direct application in vivo in a relevant animal model. We demonstrated physiological effects of beacon in Psammomys obesus by intracerebroventricular (ICV) administration of beacon for 7 days, which increased food intake and body weight gain in a dose-dependent manner (Figure 2) and resulted in a 2-fold increase in hypothalamic expression of NPY (Collier et al., 2000). Coadministration of beacon and NPY for 7 days enhanced this orexigenic effect and resulted in a profound increase in food intake and body weight that was signiﬁcantly greater than the response seen for each peptide when administered separately (Figure 3) (Collier et al., 2000).

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Figure 2 Effect of ICV beacon administration on food intake (A) and body weight (B) in Psammomys obesus. Animal were treated with 3, 15 or 30 g beacon per day for 7 days, and cumulative food intake and change in body weight are shown; Saline; 3 g; 15 g; 30 g. *p 0.05 vs. saline group. Mean change in body weight

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Figure 3 Effect of ICV administration of beacon and/or NPY on food intake (A) and body weight (B) in Psammomys obesus. Animal were treated with 15 g beacon, 15 g NPY, or 15 g of both beacon and NPY per day for 7 days, and cumulative food intake and change in body weight are shown. Saline; beacon; NPY; NPY beacon. *p 0.05 vs. saline group; #p 0.05 vs. NPY group; **p 0.05 vs. NPY, beacon, and saline groups.

The increase in body weight observed after beacon treatment was predominately due to an increase in fat mass (Walder et al., 2001). Further studies using indirect calorimetry have demonstrated no effect of beacon administration on substrate oxidation, physical activity or total energy expenditure (Walder et al., 2001). These results suggest, that beacon acts in the hypothalamus to increase food intake, which subsequently results in increased body weight, and more speciﬁcally, increased fat mass. These observed in vivo effects of beacon support the concept that beacon has an important role in the maintenance of energy balance in Psammomys obesus, thereby making this gene a potential target for the design of therapeutics for the control of obesity and type 2 diabetes. There are clearly many different strategies that can be utilized to identify and characterize genes that are important in the development of type 2 diabetes. DNA-based approaches are demanding in terms of time and money, but may lead to the identiﬁcation of causative genes. RNA-based (gene expression) approaches, such as microarrays, can provide extensive data about the physiological processes that contribute to the disease phenotype, and provide multiple targets for therapeutic intervention. In either case, the samples used in the investigation are

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of paramount importance. We have shown that the gene expression approach, in combination with an excellent animal model such as Psammomys obesus, can provide novel genes and pathways such as beacon that may be important in the disease process and provide novel therapeutic approaches. As the functional characterization of beacon, and other novel genes discovered in our laboratory using this approach, continues, it is anticipated that we will soon be able to compile a deﬁnitive list of genes that are important in the development of type 2 diabetes. More importantly, it is hoped that such knowledge will facilitate the development of therapeutic strategies to effectively treat and prevent the spread of this disease.

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Wannamethee, S.G. and Shaper, A.G. (1999). Weight change and duration of overweight and obesity in the incidence of type 2 diabetes. Diabetes Care, 22, 1266–1272. World Health Organization (1998) Obesity. Preventing and managing the global epidemic. Report of a WHO consultation on Obesity. Geneva: World Health Organization. Zimmet, P. (1989) Non-insulin-dependent (type 2) diabetes mellitus: Does it really exist? Diabetes Medicine, 6, 728–735. Zimmet, P., Whitehouse, S. and Kiss, J. (1979) Ethnic variability in the plasma insulin response to oral glucose in Polynesian and Micronesian subjects. Diabetes, 28, 624–628. Ziv, E., Kalman, R., Hershkop, K., Barash, V., Shafrir, E. and Bar-On, H. (1996) Insulin resistance in the NIDDM model Psammomys obesus in the normoglycemic, normoinsulinemic state. Diabetologia, 39, 1269–1275. Zouali, H., Hani, E.H., Philippi, A., Vionnet, N., Beckmann, J.S., Demenais, F. et al. (1997) A susceptibility locus for early-onset non-insulin dependent (type 2) diabetes mellitus maps to chromosome 20q, proximal to the phosphoenolpyruvate carboxykinase gene. Human Molecular Genetics, 6(9), 1401–1408.

11.

LEPTIN AND INSULIN RESISTANCE IN RODENT MODELS KEVIN L. STARK

Department of Metabolic Disorders, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91362, USA

INTRODUCTION In recent years, the knowledge of the molecular mechanisms controlling body weight has exploded (for a recent review, see Spiegelman and Flier, 2001), due in large measure to the techniques of molecular biology. However, the concept of an endocrine feedback system controlling body fat was presciently suggested in 1953 based upon data from classical experiments (Kennedy, 1953). Since body fat tends to be stable over long periods of time, the proposed regulatory system maintained a balance between energy consumption and expenditure. The presence of a blood-borne hormone released from adipose tissue which sends a negative feedback signal to the central nervous system (CNS) was then conﬁrmed with parabiosis experiments in which animals made hyperphagic and obese by hypothalamic lesions induced dramatic weight loss in normal animals (Hervey, 1958). The application of this parabiosis technique to mouse models of obesity resulted in the identiﬁcation of strains of mice which genetically lacked either the postulated hormone (ob/ob) or the response to the hormone (db/db) (Coleman and Hummel, 1969; Coleman, 1973). Both strains of mice exhibited hyperphagia, decreased energy expenditure, and early onset obesity. More than 20 years passed until molecular cloning techniques resulted in the identiﬁcation and characterization of the genes mutated in these mice. The product of the ob gene, named leptin (Zhang et al., 1994) is a protein secreted from adipose tissue. When injected into normal or ob/ob mice, a reduction in food intake, body weight, and adiposity was produced (Halaas et al., 1995; Pellymounter et al., 1995). The db gene was found to encode a protein with homology to the cytokine receptor gene family, and was found to be highly expressed in the CNS. This receptor was predicted to encode an extracellular ligand binding domain and an intracellular domain responsible for signaling through the JAK (janus kinase)-STAT (signal transducers and activators of transcription) pathway (Chua et al., 1996; Vaisse et al., 1996; Ghilardi et al., 1996; Bjorbeak et al., 1997). Thus, both the signal and the receptor were identiﬁed. Not surprisingly, however, the notion of a blood-borne hormone secreted from adipose tissue that exclusively and bi-directionally regulates obesity via the CNS has proved overly simplistic. Leptin is now appreciated to play a role in many physiological settings, including energy balance, reproduction, immune function and development (for review, see Ahima and Flier, 2000a). In addition, it is now appreciated that while the absence of leptin produces a robust signal and resulting phenotype, the presence of abundant leptin may not. Thus, an emerging vision of leptin’s role may be to signal the absence of energy stores and effect a switch in metabolism from the fed to the fasted state. This does not minimize the potential physiologic 187

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signaling by high leptin levels, rather it emphasizes the need to understand more completely the precise molecular sequelae of “leptin resistance”. It is perhaps ﬁtting that leptin, one of the newest hormones, is being investigated for its interplay with insulin, one of the oldest. It is also interesting that while the actions of insulin in the periphery have historically been emphasized, the role of this hormone in the CNS is being increasingly appreciated. This is conversely symmetrical with leptin, in that the peripheral effects of this hormone are receiving increased scrutiny. This review will provide an overview of the emerging role of leptin and insulin resistance.

ADIPOSE AS AN ENDOCRINE ORGAN The role of adipose tissue as a storage depot for energy is well appreciated. In times of caloric excess, energy is stored within adipocytes as glycogen, protein and triglycerides. However, in addition to leptin, adipocytes express and secrete a number of cytokines and peptide hormones, including TNF-␣, interleukin-6, angiotensinogen, plasminogen-activator-inhibitor-1, sex steroids and glucocorticoids (for review, see Ahima and Flier, 2000b). A recent addition to this list is resistin, a secreted protein which may play a role in mediating insulin resistance (Steppan et al., 2001). Therefore, the adipocyte is capable of exerting inﬂuence on both local (paracrine) and distant (endocrine) tissues based upon the nutritional and endocrine status of the whole animal. Adipose tissue is the main tissue from which leptin is secreted (although low levels have been found in other tissues, such as placenta, skeletal muscle, and the brain). The expression of leptin is regulated by a number of factors, including thyroid-stimulating hormone (Pinkey et al., 1998), infection (Bornstein et al., 1998), cytokines (Sarraf et al., 1997), and glucocorticoids (De Vos et al., 1995). However, the most investigated regulator of leptin levels is the quantity of energy stored in fat. Both increased number and increased size of adipocytes are correlated with an increased level of leptin mRNA (Maffei et al., 1995; Hamilton et al., 1995; Lonqvist et al., 1995). However, the changes in leptin expression upon fasting or feeding are exaggerated with respect to the corresponding changes in adiposity (Saladin et al., 1995; Frederich et al., 1995; Boden et al., 1996), suggesting that leptin levels may also reﬂect shorter-term effects on energy balance. Therefore, in rodents at least, fasting levels of leptin may signal the degree of adiposity, while the incremental response to leptin during a fast/ refeed cycle may signal insulin mediated energy ﬂux.

LEPTIN AND INSULIN The link between leptin expression and nutritional status is likely via insulin stimulated glucose utilization (Mueller et al., 1998), possibly via glucose ﬂux in the hexosamine pathway (Wang et al., 1998). Expression of leptin temporally and qualitatively follows the rise in insulin following feeding (Saladin et al., 1995; Sinha et al., 1996). Insulin can also stimulate leptin secretion in isolated adipocytes (Rentsch and Chiesi, 1996), suggesting that this is a direct effect on the fat cells. In addition to increased body weight and food intake, mice deﬁcient in either leptin (ob/ob) or the leptin receptor (db/db) have severe insulin resistance. This insulin resistance develops early in the life of the animal, and out of proportion to the degree of obesity observed. Injection of leptin into these mice results in a dramatic decrease in both glucose and insulin.

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This effect occurs within hours, and prior to changes in food intake or adiposity, and at doses which produced no effects on body weight (Halaas et al., 1995; Weigle et al., 1995). Although the ob/ob mice are exquisitely sensitive to the effects of leptin, injection of leptin into normal mice also produces increased sensitivity to insulin (Campﬁeld et al., 1995; Pelleymounter et al., 1995). Injection of low doses into the cerebral ventricles increased glucose utilization and decreased the amount of hepatic glycogen in normal mice (Kamohara et al., 1997). The precise mechanism by which leptin produces this insulin sensitivity is not completely clear, and there is evidence for both central and peripheral effects.

LEPTIN AND CNS EFFECTS Since a major site of leptin receptor expression is within the hypothalamus, much of leptin’s effects have been ascribed to a central mechanism. This is supported by the fact that lesions of the hypothalamus can produce hyperinsulinemia and insulin resistance. The form of the leptin receptor that can transduce a signal (long-form or OBRb) is expressed on a number of hypothalamic neurons implicated in the control of energy balance. This includes neurons expressing CART (cocaine and amphetamine regulated transcript) and POMC (proopiomelancortin) as well as a distinct set of neurons expressing AGRP (agouti-related protein) and NPY (neuropeptide Y; for a recent review, see Flier, 2001). While the precise hypothalamic pathways are not well understood (and particularly the interplay between pathways), the melanocortin pathway is clearly implicated. This system serves as a negative regulator for food intake and obesity. Therefore, decreased signaling through the melanocortin 4 receptor (MC4R) by genetic (Huszar et al., 1997) or biochemical (Graham et al., 1997) approaches increased obesity. Stimulation of the pathway by administration of MC4R agonists decreased obesity. Appropriately, leptin has been found to regulate the expression of genes which encode an agonist (Thornton et al., 1997) and antagonist for the MC4R (Shutter et al., 1997). In any case, these central effects are relayed to the periphery by a variety of means including endocrine (dopamine, serotonin, glucocorticoids, thyroid), behavioral (activity, food intake), and autonomic activity (sympathetic outﬂow). The precise effect that each of these pathways may have on each speciﬁc target organ is an important area of investigation (Speigelman and Flier, 2001). It is important also to consider the role of insulin in the context of the CNS. In some respects, insulin and leptin have interesting parallels. Like leptin, insulin circulates (Schwartz et al., 1997) and enters the brain (Baura et al., 1996) at levels proportional to adiposity. Insulin receptors are present on neurons involved in mediating energy balance (Baskin et al., 1988) and CNS administration of insulin decreases food intake (Woods et al., 1979). The role of insulin signaling in the CNS has been recently highlighted by the creation of mice which lack expression of the insulin receptor in speciﬁc neurons (Bruning et al., 2000). The resulting mice developed increased adipose mass, hyperleptinemia, mild insulin resistance and dyslipidemia, as well as reproductive deﬁciencies. Since obesity developed in the face of elevated leptin levels, this suggests a link between centrally mediated insulin resistance and leptin resistance.

LEPTIN AND THE PERIPHERY In addition to effects on neurons within the CNS, there is also evidence that leptin may exert effects directly on peripheral tissues. Although many peripheral tissues do not express an

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abundance of the leptin receptor, appropriate signal transduction mechanisms are activated in peripheral tissues of animals injected with leptin (Kim et al., 2000). Although it is difﬁcult to separate the actions of leptin in the CNS from those mediated only through peripheral sites, recent reports suggest a potential peripheral mechanism. Numerous studies have shown that mice develop insulin resistance when fed a high-fat diet (Furler et al., 1997; Kim et al., 1996; Zierath et al., 1997), and this insulin resistance is correlated with increased visceral fat mass (Kim et al., 2000) and triglyceride accumulation within the skeletal muscle (Ebeling et al., 1998; Phillips et al., 1996). Reductions in the visceral fat mass by surgery, ␤3 agonists, caloric restriction (Cases and Barzilai, 2000) leptin injection and substituting ﬁsh oil for corn oil in the high fat diet (Kim et al., 2000) all partially ameliorate insulin resistance. Experiments using isolated soleus muscle preparations have demonstrated that a high-fat diet abolishes the stimulatory effect of leptin on lipid oxidation (Steinberg and Dyck, 2000). Thus, during the pathogenesis of obesity, leptin resistance in peripheral tissues (skeletal muscle and liver) may lead to excess triglyceride accumulation by relieving the inhibitory effect of leptin on free fatty acid synthesis. Excess triglyceride levels have been proposed to lead to a variety of “lipotoxic” complications, including increased ceramide formation and nitric-oxide mediated apoptosis (Shimabukuro et al., 1998). The ability of ﬁsh oil substitution in a high-fat diet to partially reverse leptin resistance may be explained by increased signaling of the leptin receptor due to changes in biophysical properties (e.g. ﬂuidity) of the cell membrane. Changes in membrane composition have been demonstrated to inﬂuence insulin receptor signaling (Borkman et al., 1993). It is presently impossible to precisely ascribe the relative contribution of central vs. peripheral effects of leptin on insulin resistance. However, while peripheral effects have been demonstrated and are no doubt important, the actions of leptin in the CNS seem to play a preponderant role. For example, mice with hypothalamic lesions fail to respond to leptin with changes in insulin sensitivity, and direct injections of leptin into the brain elicit profound metabolic changes.

LEPTIN AND LIPODYSTROPHY Since triglyceride accumulation and increased adiposity (particularly visceral adiposity) are associated with insulin resistance, a remarkable ﬁnding is that a lack of adipose tissue can produce the same outcomes. This condition, known as lipoatrophy, results from a deﬁciency in the amount of mature, functioning adipocytes. This should be contrasted from conditions in which otherwise normal adipocytes are depleted of lipid stores, such as starvation and extreme exercise, since these conditions are not associated with insulin resistance. While much of the information about the consequences of lipoatrophy and lipodystrophy is gleaned from mouse models, it is important to note that a diverse collection of lipodystrophic syndromes exist in human (for review, see Reitman et al., 2000). The human syndromes can originate from genetic mutations, infection by HIV, and autoimmune destruction of fat cells. Patients with this syndrome have insulin resistance, low leptin levels, severe hypertriglyceridemia, hepatomegaly and muscle hypertrophy (Pardini et al., 1998). Lipoatrophic mice have been generated by expressing modiﬁed transcription factors under the control of an adipocyte-speciﬁc promotor. These transcription factors were designed to block development of adipocytes (A-ZIP/F-1 model, Reitman, 2000) or increase the expression of genes regulating fatty acid synthesis (SREBP-1c model, Shimomura et al., 1998). Both animal models show a correlation between the degree of white adipose tissue present and the

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degree of insulin resistance observed as well as leptin levels. In addition, the insulin resistance is attenuated in the A-ZIP/F-1 model by the surgical transplantation of white adipose tissue (Gavrilova et al., 2000a; Kim et al., 2000a). Interestingly, the amount of fat transplanted also correlates with the improvement in insulin sensitivity. The models differ, however, in their response to leptin administration. Leptin treatment of the SREBP-1c model resulted in a dramatic improvement in glucose and insulin levels (Shimomura et al., 1999). Since there is very little adipose tissue in which to store lipids in these mice, there is lipid accumulation in other tissues such as liver and skeletal muscle. In this regard, leptin administration was very effective in reversing the hepatic steatosis. Thus, even when adipose depots are dystrophic, leptin can suppress the high level of lipid accumulation in these animals. Whether this occurs predominantly by a direct action of leptin on lipid synthesis or turnover, or via restoration of more “normal” adipose tissue, and secretion of other factors, remains to be determined. In the A-ZIP/F-1 mice, which have a near complete loss of adipose tissue, leptin injections had a much smaller effect (Gavrilova et al., 2000). Human patients with lipodystrophy also have a wide variation in the degree of adipose tissue loss, and it will be interesting to see the effects of leptin in these patients. CONCLUSIONS Although much progress has been made in elucidating individual control systems for adiposity, feeding behavior, energy balance, lipid metabolism and glucose utilization, the current challenge is two-fold. One, we must achieve a deeper understanding of the precise molecular events that mediate the effects of these control systems (e.g. leptin and insulin resistance), and become more sophisticated in our understanding of how these events may be tailored to the needs of individual tissues and organs. Second, the challenge of integrating all these systems into a cohesive, understandable, uniﬁed whole is essential to understanding the role of these systems in pathophysiology. REFERENCES Ahima, R.S. and Flier, J.S. (2000a) Leptin. Ann. Rev. Physiol., 62, 413–437. Ahima, R.S. and Flier, J.S. (2000b) Adipose tissue as an endocrine organ. Trends in Endrocrin. Metabol., 11(8), 327–332. Baskin, D.G., Wilcox, B.J., Figlewicz, D.P. and Dorsa, D.M. (1988) Insulin and insulin like growth factors in the CNS. Trends Neurosci., 11, 107–111. Baura, G.D., Foster, D.M., Kaiyala, K., Porte, D., Kahn, S.E. and Schwartz, M.W. (1996) Insulin transport from plasma into the central nervous system is inhibited by dexamethasone in dogs. Diabetes, 45(1), 86–90. Bjorbaek, C., Uotani, S., da Silva, B. and Flier, J.S. (1997) Divergent signaling capacities of the long and short isoforms of the leptin receptor. J. Biol. Chem., 272, 32686–32695. Borkman, M., Storlein, L.H., Pan, D.A., Jenkins, A.B., Chisholm, D.J. and Campbell, L.V. (1993) The relation between insulin sensitivity and the fatty acid composition of skeletal-muscle phospholipids. New England J. of Med., 328, 238–244. Boden, G., Chen, X., Mozzoli, M. and Ryan, I. (1996) Effect of fasting on serum leptin in normal human subjects. J. Clin. Endocrinol. Metab., 81, 3419–3423. Bornstein, S.R., Licinio, J., Tauchnitz, R., Engelmann, L., Negrao, A.B. and Chrousos, G.P. (1998) Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm in cortisol and leptin secretion. J. Clin. Endocrinol. Metab., 83, 280–283.

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Steppan, C.M., Bailey, S.T., Bhat, S., Brown, E.J., Banerjee, R.R., Wright, C.M., Patel, H.R., Ahima, R.S. and Lazar, M.A. (2001) The hormone resistin links obesity to diabetes. Nature, 409(6818), 307–312. Thornton, J., Cheung, C., Clifton, C. and Steiner, R. (1997). Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice. Endocrinology, 138, 5063–5066. Vaisse, C., Halaas, J.L., Horvarth, C.M., Darnell, J.E. Jr., Stoffel, M. and Friedman, J.M. (1996) Leptin activation of Stat3 in the hypothalamus of wild type and ob/ob mice but not db/db mice. Nat. Genet., 14, 95–97. Wang, J.L., Liu, R., Hawkins, M., Barzilai, N. and Rossetti, L. (1998) A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature, 393(6686), 684–688. Weigle, D.S., Bukowski, T.R., Foster, D.C., Holderman, S., Kramer, J.M., Lasser, G., Loften Day, C.E., Prunkard, D.E., Raymond, C. and Kujiper, J.L. (1995) Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J. Clin. Invest., 96, 2065–2070. Woods, S., Lotter, E., McKay, L. and Porte, D.J. (1979) Chromic intracerebroventricular infusion of insulin reduces food intake and body weight of baboons. Nature, 282, 503–505. Zierath, J.R., Houseknecht, K.L., Gnudi, L. and Kahn, B.B. (1997) High-fat feeding impairs insulin stimulated GLUT4 recruitment via an early insulin-signaling defect. Diabetes, 46, 215–223. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425–432.

12.

FAT FEEDING AND MUSCLE FAT DEPOSITION ELICITING INSULIN RESISTANCE

E.W. KRAEGEN, G.J. COONEY, J.M. YE AND S.M. FURLER Garvan Institute of Medical Research, St Vincent’s Hospital, Sydney, NSW 2010, Australia

INTRODUCTION Skeletal muscle insulin resistance, generally deﬁned as a reduced ability of insulin to stimulate tissue utilization and storage of glucose, is an early and major perturbation in the conditions now characterized as “syndrome X” or “insulin resistance syndrome”. These states which include obesity, type 2 diabetes mellitus, dyslipidemia and hypertension, are known to be inﬂuenced by dietary factors, and are reaching epidemic proportions in many societies with a high caloriﬁc intake. In this review we consider the growing body of evidence which links insulin resistance with an increase in fatty acid availability in muscle and liver, the major target tissues for insulin action where insulin resistance occurs. We also consider possible causal links whereby increased muscle lipid accumulation can result in impaired insulin signaling and insulin resistance, and therapeutic options for ameliorating insulin resistance based on a “lipid-lowering” approach. It is now ten years or more since it was found in animal studies that triglyceride accumulates in muscle coincident with diet-induced insulin resistance (Storlien et al., 1991; Kraegen et al., 1991). Studies in 2000 and 2001, reviewed here, have added a lot to the story, both clarifying the relevance of lipid accumulation to insulin resistance in humans, and at the other end of the spectrum, indicating very plausible mechanisms whereby lipid accumulation can generate insulin resistance in basic animal and cell-based studies. Evidence is considered for a “lipid supply hypothesis of insulin resistance”. That is “an oversupply and/or accumulation of lipid in muscle and liver leads to changes in metabolism at the level of substrate competition, enzyme regulation, intracellular signaling and/or gene transcription that account for the insulin resistance seen in states such as syndrome X and type 2 diabetes”.

OVERVIEW OF LIPID METABOLISM AND INSULIN RESISTANCE Muscle lipid accumulation will occur over a period when the total supply of lipid to muscle from all sources (systemic fatty acids, VLDL-triglycerides, gut-derived chylomicrons) exceeds the rate of muscle fatty acid oxidation over that period. The relative contribution of the sources of excess lipid to muscle in insulin resistant states is not known in quantitative terms, and a combination of factors may be a possibility, combined with the possibility of impaired fatty acid oxidation. Rates of muscle lipid supply and utilization also vary considerably according to Address correspondence to: Edward W. Kraegen, Garvan Institute of Medical Research, St Vincent’s Hospital, Sydney, NSW 2010, Australia. Tel.: +61 2 9295 8206; Fax: +61 2 9295 8201; Email: [email protected] 195

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Figure 1 Some of the interactions, discussed in the text, whereby an increased muscle uptake of fatty acids, derived from circulating fatty acids or triglycerides, may lead to increased muscle lipid accumulation and insulin resistance. LCACoAs, long chain acyl CoAs; DAG, diacylglycerol (diglyceride); PKCs, protein kinase C isoforms.

skeletal muscle type, but in the context of this review it is perhaps relevant that red oxidative muscle, with high levels of intramyocellular lipid (Hwang et al., 2001), is also most prone to dietary fat-induced insulin resistance (Kraegen et al., 1986). Figure 1 sets the scene for many of the metabolic interactions to be discussed in this chapter. Muscle lipid accumulation is evident as increased levels of either stored muscle triglyceride or of the metabolically active form of lipid, the long chain acyl CoAs (LCACoAs). This lipid excess may inﬂuence metabolism acutely by changing substrate availability or by altering key enzyme activities by allosteric regulation. Excessive mitochondrial FFA oxidation may impair glucose oxidation via the classic Randle cycle (Randle et al., 1963). On the other hand a reduction in the mitochondrial transfer of LCACoAs, perhaps by negative regulation of carnitine palmitoyl transferase (CPT-1) by malonyl CoA (Ruderman et al., 1999), may lead to accumulation of cytosolic LCACoAs and subsequent insulin resistance (Oakes et al., 1997a; Prentki and Corkey, 1996; Ruderman et al., 1999). Changes in speciﬁc fatty acid levels as a result of cytosolic FFA accumulation (e.g. speciﬁc diacylglycerols etc.) may alter insulin signaling pathways via protein kinase C activation (Schmitz-Peiffer et al., 1997a) or ceramide production (Schmitz-Peiffer et al., 1999) and/or may change gene expression. Lastly, humoral signals derived from adipose tissue, such as TNF-␣ (Hotamisligil and Spiegelman, 1994; Shimabukuro et al., 1997), interleukin-6 (Fernandez-Real et al., 2000) or the recently described hormone resistin (Steppan et al., 2001) may also act in muscle to alter lipid metabolism and/or inhibit insulin action.

ACUTE SYSTEMIC FATTY ACID ELEVATION CAN CAUSE MUSCLE INSULIN RESISTANCE One convincing demonstration of the importance of lipid availability in the onset of muscle insulin resistance is that it is now clear that acute elevation of fatty acid levels, such as can be

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generated by triglyceride/heparin infusion, can cause whole body, muscle and liver insulin resistance (Boden, 1997; Chalkley et al., 1998; Grifﬁn et al., 1999). The effect in muscle takes several hours to occur (e.g. see Boden, 1997; Chalkley et al., 1998; Grifﬁn et al., 1999) and is particularly associated with a reduction in insulin-mediated muscle glycogen synthesis (Boden et al., 1991, 1994; Kelley et al., 1993; Kim and Youn, 1997; Jucker et al., 1997). Thus the effects of increased FFA availability on insulin signaling and/or on glycogen synthesis appears to take a number of hours to develop. This lack of a short-term effect confounded studies in the late 1980s, which tried to link fatty acid availability to muscle insulin resistance (Bevilacqua et al., 1990; Wolfe et al., 1988; Jenkins et al., 1988). With the beneﬁt of hindsight these older studies were too short to show insulin resistance. Newer evidence now suggests that there is a signiﬁcant elevation of muscle triglyceride and total LCACoAs after 3–5 h of triglyceride/heparin infusion when insulin resistance occurs (Chalkley et al., 1998), and that this is in fact very similar to what happens when muscle insulin resistance is induced chronically in rats over several weeks by a high fat diet (Oakes et al., 1997a; Chen et al., 1992). To summarize the current position, we know of no evidence to suggest that the lipid-related processes leading to insulin resistance in rat muscle (whether by several hours direct systemic fatty acid elevation, or by several weeks high fat feeding) are intrinsically different, other than the obvious difference in duration of effects. Defects in insulin signaling have been found in both cases (Zierath et al., 1997; Grifﬁn et al., 1999). Given the strong probability that excess fatty acid availability causes insulin resistance during lipid infusion, we consider it highly likely that excess fatty acid accumulation causes high fat diet-induced insulin resistance (rather than the theoretically possible converse of insulin resistance causing muscle lipid accumulation). This view is strengthened by recent reports that transgenic mouse models which have musclespeciﬁc enhanced ability to take up fatty acids, either by fatty acid transporter (CD36) (Ibrahimi et al., 1999) or human lipoprotein lipase (Ferreira et al., 2001) overexpression, develop in vivo insulin resistance. Possible mechanisms (Figure 1) include but go beyond the classic Randle cycle, and are considered in more detail later.

MUSCLE TRIGLYCERIDE CONTENT AND INSULIN RESISTANCE Animal feeding studies in the 1980s and early 1990s established an association between muscle lipid accumulation and insulin resistance. Rats on high fat diets develop insulin resistance ﬁrst in the liver (Kraegen et al., 1991), and insulin resistance then develops as triglyceride accumulates in muscle giving rise to a signiﬁcant association between muscle insulin resistance and triglyceride content (Kraegen et al., 1991; Storlien et al., 1991). Supporting this association, changes in dietary fat subtype, for example, by increasing omega-3 fats, lessened both insulin resistance and muscle triglyceride accumulation (Storlien et al., 1991). Now the muscle lipid–insulin resistance association has also been demonstrated in a number of animal models (e.g. Shimabukuro et al., 1997; Russell et al., 1998; Pan et al., 2001; Dobbins et al., 2001). Also strengthening the association was the discovery that various metabolic manipulations of chronically high fat fed rats which lessen muscle lipid accumulation, such as prior exercise, overnight fasting, acutely reducing food fat content (Oakes et al., 1997a), or PPAR agonist drug treatment (Oakes et al., 1997b; Ye et al., 2001b), also signiﬁcantly improve insulin sensitivity. In our various animal high fat diet studies, we almost universally use high fat diets which are isocaloric with high carbohydrate control diets; this suggests that the association of muscle lipids and insulin resistance is related to a relative macronutrient availability rather than to body obesity per se, although evidence suggests that overnutrition and development of obesity,

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as might be the case in many humans, will exacerbate the problem. In summary, based on animal studies, there is a compelling case that a high fat dietary intake extended for more than a few days will cause muscle lipid accumulation and insulin resistance, with both being readily reversible when the source of lipid oversupply is removed or when there is an accelerated rate of muscle lipid utilization. Much work is going on to resolve whether muscle lipid accumulation is also likely to be a cause of insulin resistance in humans. A problem recognized by workers in the area has been the likelihood of contamination of muscle biopsies with small amounts of adipose tissue (often seen as fat “marbling” of muscle), and we have estimated that a 2% fat contamination could account for all of the triglyceride measured in a muscle biopsy (Ellis et al., 2000). This could possibly be the case in early studies showing very high muscle triglyceride in type 2 diabetes subjects (Falholt et al., 1988), but later studies with careful muscle biopsy on a non-diabetic male Pima Indian group (Pan et al., 1997) reported a signiﬁcant association of muscle triglyceride content with insulin resistance. In this study insulin resistance was more associated with muscle triglyceride content than with more general measures of adiposity such as body mass index. Another muscle biopsy study on non-diabetic women with subsequent neutral lipid staining and histologic assessment of intramuscular triglyceride content, has shown a negative correlation with insulin-mediated activation of glycogen synthase (Phillips et al., 1996). Since 1998, there has been increasing use of proton magnetic resonance spectroscopy (MRS), methodology described by Boesch et al. (1997) and Szczepaniak et al. (1999), to quantify and distinguish intra-myocellular (IMCL) and extra-myocellular (EMCL) lipid content in human and rat studies. Using these techniques, several recent clinical studies show an association between intramyocellular lipid accumulation and peripheral insulin resistance (Krssak et al., 1999; Jacob et al., 1999; Perseghin et al., 1999). It is interesting that this association includes non-diabetic “at risk” subjects who have an increased chance of developing diabetes. Very recently Dobbins et al. (2001) used a rat model to speciﬁcally show that peripheral insulin-mediated glucose disposal negatively correlates with IMCL when lipid accumulates either as a result of a high fat diet or by pharmacologically impairing rates of fat oxidation. This interesting study not only highlights the two contributory ways in which intracellular lipids may accumulate in muscle and putatively lead to insulin resistance, but also indicates that the Randle cycle cannot be the principal mechanism of fatty acid-induced muscle insulin resistance. Therefore, we believe there has been strong support over the last two years for the muscle lipid oversupply hypothesis of insulin resistance. However, there are some confounding aspects. The ﬁrst is the oft-quoted comment that with exercise training there is both an enhanced storage of muscle triglyceride and enhanced insulin sensitivity (Kelley and Mandarino, 2000). However, a close scrutiny of one quoted paper indicates that while there is evidence of greater muscle triglyceride mobilization during exercise, basal levels between trained and untrained subjects are very similar (Hurley et al., 1986). The issue needs further investigation, perhaps using the MRS technique. Second, some have suggested that there may be differing compartmentation of triglyceride in the myocyte in trained athletes (Boesch et al., 1997; Vock et al., 1996) or that cyclical muscle lipid depletion/repletion in athletes somehow protects against insulin resistance (Kelley and Mandarino, 2000). At this stage none of these issues have been resolved. This problem is less of an issue if muscle triglyceride itself is not the culprit causing insulin resistance. If we take the view that the steps causing insulin resistance emanate from cytosolic accumulation of metabolically active forms of lipid such as the LCACoAs (Figure 1), then it is possible with the enhancement in oxidative capacity of muscle of exercise-trained subjects, or in type 1 vs. type 2 muscle ﬁber types, that accumulation of cytosolic LCACoAs may be negatively related to the ability of the muscle to utilize fatty acids, rather than simply being positively correlated to muscle IMCL.

FAT FEEDING, INSULIN RESISTANCE AND MUSCLE FAT DEPOSITION r 2 = 0.342; p = 0.0137

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0.2 0.15 0.1 0.05 0 1

2 3 4 5 6 7 LCACoA (nmol/g wet weight)

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Figure 2 Negative correlation between leg muscle long chain acyl CoA (LCACoA) and whole body insulin sensitivity in human subjects assessed by euglycemic hyperinsulinemic clamp. GIR: clamp glucose infusion rate. FFM: body fat free mass. (From Ellis et al., 2000.)

To better understand the metabolic steps which might link lipid metabolism to impairment of insulin action in muscle, over the last several years, we have been directly measuring total muscle LCACoAs and examining their relationship to insulin resistance (Oakes et al., 1997a; Ellis et al., 2000). Although the HPLC methodology (based on Corkey, 1988) is considerably more complex than measurement of triglyceride, it is not confounded by adipose cell contamination to any appreciable degree, as the LCACoAs are present in a much lower amount in adipose cells than in muscle cells (Ellis et al., 2000). The ﬁrst step in the metabolism of fatty acids in any tissue is the activation of the fatty acid to its LCACoA derivative by the enzyme acyl CoA synthase. The LCACoAs can then be transported into the mitochondria via the action of acyl carnitine transferase and oxidized in the beta-oxidation pathway. Alternatively the LCACoAs may be esteriﬁed to mono- and diglyceride and stored as triglyceride or incorporated into phospholipid in membranes (for a review see Faergeman and Knudsen, 1997). The concentration of LCACoAs is increased by fasting (Carroll et al., 1983; Ellis et al., 2000) and decreased by insulin (Oakes et al., 1997a), and is therefore an indication of the extent of fat metabolism in a tissue. LCACoA levels are increased in muscle from fat-fed insulin resistant rats and there is a close link between insulin resistance and increased LCACoAs in the muscle of these animals (Oakes et al., 1997c). This link is strengthened by results from another study where amelioration of fat diet-induced insulin resistance by one of several acute diet or exercise manipulations, was closely related to the ability of insulin to suppress muscle LCACoAs (Oakes et al., 1997a). In human studies initial results suggest an association between total muscle LCACoAs in human muscle obtained during knee surgery, and previously determined insulin sensitivity (Figure 2) (Ellis et al., 2000). Thus there appears to be increasing evidence for an accumulation of muscle lipid in insulin resistant states in humans. Newer clinical data becoming available should clarify the role of lipid accumulation in the pathophysiology of muscle insulin resistance.

ADIPOSE BODY DEPOTS AND FFA FLUXES TO MUSCLE While dietary changes, either increasing or reducing lipid, can readily alter muscle lipid accumulation in rodents, the situation in humans seems more complex. A number of studies have

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suggested that peripheral insulin sensitivity is not easily altered in humans by short term dietary changes (Storlien et al., 1996). A possible explanation, consistent with the muscle lipid supply hypothesis of insulin resistance, is that chronic levels of intramyocellular lipid in humans are much harder to vary by dietary means than in rodents. Even when there is significant weight loss in obese subjects it is not clear whether elevated muscle lipids signiﬁcantly decline, with reports suggesting (Goodpaster et al., 2000) and refuting this (Malenfant et al., 2001). This apparent species difference is sobering for those performing rodent studies, given the ease of reversing high fat diet-induced insulin resistance in the rat (Oakes et al., 1997a). Nevertheless, while factors determining muscle lipid accumulation in humans and rodents may differ, their accumulation may have very similar implications for potency of insulin action. It may be that where genetic factors are involved, as in many human (and animal) insulin resistant states, muscle lipid accumulation and/or insulin resistance may be harder to manipulate simply by dietary changes. Resolving this will require a better knowledge of mechanistic links between muscle lipids and insulin action in the two species. Lipid accumulation differs between humans and rodents in two respects. First there is greater dependence on adipose stores in humans for overall lipid supply; second the modulation of fatty acid utilization may have a greater inﬂuence in humans. There may be a link between increased fatty acid supply from fat depots in humans, particularly associated with increased central adiposity, and lipid accumulation in muscle. A number of studies, making use of DEXA or CT scanning, have shown strong correlations between central abdominal fat and insulin resistance (Carey et al., 1996a; Park et al., 1991; Ross et al., 1996; Simoneau et al., 1995). The greater insulin sensitivity of women compared to men is also in accord with a gender difference in intra-abdominal fat stores (Carey et al., 1996b), which again suggests that intra-abdominal fat may be a speciﬁc determinant of insulin resistance. When both subcutaneous abdominal fat and thigh muscle attenuation, an (indirect measures of lipid content) were estimated in the same obese subjects by computed tomography, both predicted insulin sensitivity (Goodpaster et al., 1997). However it has been difﬁcult to demonstrate an acute increase in FFA ﬂux to muscle in subjects with increased visceral obesity – in fact the reverse has been reported in regional catheterization studies of premenopausal women (Colberg et al., 1995). Currently it is not clear in quantitative terms how much increased FFA uptake into muscle, and reduced utilization of fatty acids, each contribute to muscle triglyceride accumulation. A glucose oversupply to muscle may contribute to reduced utilization of fatty acids. Increased glucose availability and oxidation can inhibit lipid oxidation (Sidossis et al., 1996), producing a “glucose–fatty acid cycle in reverse” (Wolfe, 1998). Some possible mechanisms for this have been proposed and recently reviewed (Ruderman et al., 1999). In principle, these mechanisms propose that various factors may lead to a high level of muscle malonyl CoA which in turn inhibits CPT-1 and mitochondrial transfer of LCACoAs. In support of this concept, we have found that in a rat model of chronic glucose infusion (to produce 3-fold normal basal glucose turnover), muscle becomes insulin resistant and malonyl CoA increases, presumably inhibiting fatty acid oxidation. Muscle triglyceride and LCACoAs are signiﬁcantly increased in these animals (Laybutt et al., 1997, 1999). More work is needed to examine the magnitude, source, turnover and regulatory factors of lipid storage in muscle in relation to insulin resistance. To begin with, we collaborated in a study (Oakes et al., 1999), to develop a non-metabolizable fatty acid tracer to quantify tissue uptake of fatty acids in animal models. Recent application of this technique has indicated increased efﬁciency of fatty acid uptake into muscle of rats adapted to a high fat diet (Furler et al., 2001). This adaptation makes sense in that it may enhance utilization of the dominant available energy fuel, but it may also contribute to accumulation of muscle lipids and insulin resistance.

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It would also be useful to use appropriate tracers to examine muscle uptake of fatty acids derived from VLDL-triglycerides or chylomicrons, but this is difﬁcult and remains to be done. There is increasing interest in the postprandial state, and it is recognized that with Western society dietary habits there is a considerable period of each day spent in a postprandial state. Normal regulation of fatty acid metabolism in this state may be crucial in controlling the daily average lipid supply to liver, muscle and other tissues (Evans et al., 1999). It has been suggested that a disruption of the normal role of insulin in the postprandial period may underlie many of the lipid abnormalities associated with insulin resistance (Frayn, 1998). In addition, there is reported evidence that a state characterized by excessive plasma triglyceride elevation following standardized oral fat intake (Schrezenmeir et al., 1997) may be associated with syndrome X. Whether these subjects also have excess muscle triglyceride accumulation is not clear, but it seems quite possible.

BEYOND THE RANDLE CYCLE Randle and co-workers (Randle et al., 1963) proposed that the availability and oxidation of free fatty acids reduces glucose oxidation and therefore glucose uptake. The proposed mechanism involves the metabolites of increased fatty oxidation inactivating the key enzyme of glucose oxidation, pyruvate deydrogenase (PDH). Increased fatty acid oxidation is also thought to produce an increased cytosolic content of citrate which can allosterically inhibit the activity of 6-phosphofructokinase (PFK-1), another key enzyme in the regulation of glucose metabolism (Randle et al., 1988; Randle, 1998). However, the uptake and oxidation of circulating fatty acids by tissues is far more rapid than the 2–3 h needed to see whole body insulin resistance after acute increases in fatty acid availability. This is not to say that the Randle cycle does not have an effect in some circumstances. For example, mouse transgenic models with overexpression of muscle CD36 (Ibrahimi et al., 1999) or human lipoprotein lipase (Ferreira et al., 2001) have shown increased fat oxidation and development of insulin resistance. However, we would argue that further assessment of muscle lipid-related parameters (e.g. LCACoAs, DAGs etc., see Figure 1) may be necessary to clarify involvement of other lipid-related factors. Another example is acute pharmacological inhibition of fatty acid oxidation by etomoxir in fat-fed, insulin resistant rats, leading to improved insulin action by restoring oxidative glucose disposal (predicted by the Randle cycle); but this treatment had little ability to overcome the impaired insulin stimulation of glycogen synthesis or the decreased glycogen synthase activity in muscle of fat-fed rats (Oakes et al., 1997c). Furthermore, when etomoxir is administered chronically (Dobbins et al., 2001), there is accumulation of muscle triglyceride and development of insulin resistance in rats. This latter ﬁnding is contrary to that which would be predicted by the Randle cycle, where reducing fat oxidation should improve insulin-mediated glucose utilization. This and human data (Colberg et al., 1995) suggest that mechanisms other than Randle cycle effects are involved in the induction of insulin resistance by increased availability of fatty acids.

FATTY ACID METABOLITES AND INSULIN ACTION IN MUSCLE Because of the consistent link between insulin action and lipid accumulation in muscle it is worth considering which actions of lipid metabolites might regulate insulin action in muscle. LCACoAs have been shown to directly modify the activity of key enzymes of glucose metabolism

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Hexokinase activity (U/g wet wt)

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Figure 3 Palmitoyl CoA (5 mol/l, solid histogram) adds to the effect of glucose-6-phosphate (G6P) alone (open histogram) to inhibit hexokinase activity in rat soleus muscle homogenate. p 0.001 for effect of palmitoyl CoA. (From Thompson and Cooney, 2000.)

in liver such as glycogen synthase (Wititsuwannakul and Kim, 1977), glucokinase (Tippett and Neet, 1982), glucose-6-phosphatase (Fulceri et al., 1995) and acetyl CoA carboxylase (Nikawa et al., 1979). Recently we have also demonstrated that LCACoAs at concentrations in the physiological range, can reduce the activity of hexokinase in rat and human skeletal muscle (Figure 3) (Thompson and Cooney, 2000). As can be seen (Figure 3) this inhibition was additive to the well-described inhibition of hexokinase by glucose-6-phosphate. It is therefore quite possible that some of the inhibitory effects of lipid oversupply on glucose metabolism are direct interactions with the glucose metabolic pathway. Intracellular lipid species may also modulate the activity of the insulin signaling cascade. There is clear evidence that diacylglycerol-activated members of the protein kinase C family of serine/threonine kinases (PKC theta and PKC epsilon) are translocated and activated in muscle of fat-fed, insulin resistant rats (Schmitz-Peiffer et al., 1997a). PKC epsilon was also activated in the presence of increased LCACoA levels in the muscle of glucose-infused, insulin resistant rats (Laybutt et al., 1999). Individual fatty acid species may also have speciﬁc effects, with palmitate, oleate and linoleate having differential effects on IRS-1 and PKB phosphorylation in a cell line derived from murine muscle (Schmitz-Peiffer et al., 1999). Ceramide production may mediate some of the effects of palmitate on insulin signaling and direct addition of ceramides to muscle cells mimics effects on glycogen synthesis of adding palmitate (Schmitz-Peiffer et al., 1999). Another way in which the fatty acid composition and content of the diet may alter lipid and glucose metabolism is by regulation of gene expression ( Jump and Clarke, 1999). The recent discovery of nuclear hormone receptors which are regulated directly by fatty acids or fatty acid metabolites provides at least one mechanism for these effects although not all the actions of fatty acids on gene expression are direct (Duplus et al., 2000). The most widely studied of the fatty acid-activated nuclear hormone receptors are the peroxisome proliferator activated receptors (PPARs) and in particular PPAR␣ and PPAR␥ which are important for fat oxidation in the liver (Lemberger et al., 1996) and adipose tissue proliferation (Spiegelman, 1998), respectively. There are also reports of other nuclear receptors such as hepatic nuclear factor-4␣ being regulated by fatty acids or their derivatives such as speciﬁc long chain acyl CoAs (Hertz et al., 1998). The observation that several hours of triglyceride/heparin infusion is enough to alter speciﬁc gene expression (Fabris et al., 2001) clearly demonstrates that fatty acid mediated gene regulation may be a major mechanism by which increased lipid availability inﬂuences insulin action.

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A signiﬁcant correlation has also been demonstrated between the fatty acid composition of muscle membranes and insulin action at a tissue and whole body level in humans and animals (Pan et al., 1994, Borkman et al., 1993). Although the mechanisms by which membrane phospholipid composition inﬂuences insulin action are not clear, several possibilities have been proposed. These include changing membrane ﬂuidity which could affect insulin and other membrane receptor action (Storlien et al., 1997), release of different diacylglycerol molecules after phospholipase activity with altered modulatory effects on cellular processes such as PKC activity (Storlien et al., 1996), and altered cellular energy expenditure inﬂuencing accumulation of intracellular triglyceride (Storlien et al., 1997).

PHARMACOLOGICAL MANIPULATION OF MUSCLE LIPID ACCUMULATION PPAR␥ Agonists These speciﬁcally ameliorate insulin resistance (Spiegelman, 1998; Saltiel and Olefsky, 1996; Smith, 1997). The PPAR-␥ receptor is principally (but not only) expressed in adipose tissue (Lehmann et al., 1995). Its activation by compounds such as the thiazolidinediones (TZDs) has been shown to have a key role in adipogenesis by increasing the gene expression of a number of key enzymes and/or transport proteins involved in adipose tissue metabolism, recently reviewed (Spiegelman, 1998; Smith, 1997). Evidence suggests that the TZDs might principally affect muscle metabolism “from a distance” by acting in adipose tissue to favor retention of lipid (Oakes et al., 1994, 2001). Of direct relevance to mechanisms discussed in this review is the fact that recent data demonstrate that TZDs lower lipid accumulation in muscle of insulin resistant rats. Rosiglitazone, while enhancing insulin-mediated muscle glucose uptake and glycogen synthesis, also signiﬁcantly reduces muscle triglyceride and DAG levels (Oakes et al., 1997b), and corrects some biochemical effects (e.g. chronic PKC isozyme activation, Schmitz-Peiffer et al., 1997b) which are associated with muscle lipid accumulation and insulin resistance in the high fat-fed rat (Schmitz-Peiffer et al., 1997a). While the “modulation of lipid supply” hypothesis seems most plausible for TZD action in vivo, there are other possibilities. For example, there may be local direct actions in muscle (Bahr et al., 1996; Park et al., 1998), or TZDs may modulate a “signal” from adipose tissue, such as TNF-␣ (Spiegelman, 1998) or resistin (Steppan et al., 2001), which otherwise could inﬂuence insulin sensitivity. Further work is needed to clarify the various actions of TZDs. However, it is clear that these agents have a powerful effect on reducing systemic fatty acid supply and increasing insulin sensitivity. PPAR␣ Agonists PPAR␣ is strongly expressed in liver, and in line with this the liver seems to be the principal target of action of PPAR␣ agonists (e.g. ﬁbrates, WY-14643). PPAR␣ activation leads to, inter alia, expression of genes involved in beta oxidation of fatty acids, and it could be argued that reducing lipid by “burning it off ” is more desirable than sequestering it in adipose tissue, as is likely with the PPAR␥ agonists. PPAR␣ is also expressed in muscle, but its effects there are not clear at this stage. Nevertheless, while PPAR␣ agonists might act differently to PPAR␥ agonists one might predict that they should also lead to a reduction in muscle lipid accumulation and to amelioration of insulin resistance. This was recently demonstrated using the high

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fat-fed rat model of insulin resistance (Ye et al., 2001b). Similarly in a diet-induced insulin resistant rat, bezaﬁbrate signiﬁcantly altered muscle phospholipid composition and lowered muscle triglyceride (Matsui et al., 1997). Because several of the lipid-lowering agents act with mutually distinct mechanisms and/or on differing principal tissue targets, it is possible that combined therapy will prove to have desirable synergistic effects. More work needs to be done, particularly in relation to effects on insulin resistance, but it is interesting that compounds with combined actions are available or are being developed. Examples are etoﬁbrate, with combined PPAR␣ (ﬁbrate) and nicotinic acid-like action (Bocos and Herrera, 1996). Compounds with combined PPAR␣ and PPAR␥ action are also under development and show promise in rodent models (Murakami et al., 1998; Hegarty et al., 2001; Ye et al., 2001a). The expectation is that compounds with PPAR␣ activity will show less tendency to weight gain than PPAR␥ activity (Guerre-Millo et al., 2000; Ye et al., 2001b), but further experience with their application to humans is needed. CONCLUSION From the above discussion it is clear that there are a number of potential mechanisms by which fatty acids might inﬂuence glucose metabolism and insulin action. The Randle glucose–fatty acid cycle represents only one mechanism, and perhaps makes only a minor contribution. Total muscle lipid availability (from systemic and intracellular sources), as well as speciﬁc fatty acids, may act to regulate enzymes of fat and glucose metabolism and enzymes which alter insulin signaling pathways. The added action of fatty acids to bind speciﬁc nuclear receptors and transcription factors provides another direct mechanism whereby different dietary lipids could inﬂuence metabolism. Lastly, the principles described here are fundamental to understanding metabolic effects of various lipid-lowering agents, particularly in relation to improving insulin sensitivity in insulin resistant states. REFERENCES Bahr, M., Spelleken, M., Bock, M., Vonholtey, M., Kiehn, R. and Eckel, J. (1996) Acute and chronic effects of Troglitazone (CS-045) on isolated rat ventricular cardiomyocytes. Diabetologia, 39, 766–774. Bevilacqua, S., Buzzigoli, G., Bonadonna, R., Brandi, L.S., Oleggini, M., Boni, C. et al. (1990) Operation of Randle’s cycle in patients with NIDDM. Diabetes, 39, 383–389. Bocos, C. and Herrera, E. (1996) Comparative study on the in vivo and in vitro antilipolytic effects of etoﬁbrate, nicotinic Acid and cloﬁbrate in the rat. Environ. Toxicol. Pharm., 2, 351–357. Boden, G. (1997) Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes, 46, 3–10. Boden, G., Chen, X.H., Ruiz, J., White, J.V. and Rossetti, L. (1994) Mechanisms of fatty acid induced inhibition of glucose uptake. J. Clin. Invest., 93, 2438–2446. Boden, G., Jadali, F., White, J., Liang, Y., Mozzoli, M., Chen, X. et al. (1991) Effects of fat on insulin stimulated carbohydrate metabolism in normal men. J. Clin. Invest., 88, 960–966. Boesch, C., Slotboom, J., Hoppeler, H. and Kreis, R. (1997) In vivo determination of intra-myocellular lipids in human muscle by means of localized H-1-MR-Spectroscopy. Magn. Reson. Med., 37, 484–493. Borkman, M., Storlien, L.H., Pan, D.A., Jenkins, A.B., Chisholm, D.J. and Campbell, L.V. (1993) The relation between insulin sensitivity and the fatty acid composition of skeletal-muscle phospholipids. N. Engl. J. Med., 328, 238–244. Carey, D.G., Jenkins, A.B., Campbell, L.V., Freund, J. and Chisholm, D.J. (1996a) Abdominal fat and insulin resistance in normal and overweight women – direct measurements reveal a strong relationship in subjects at both low and high risk of NIDDM. Diabetes, 45, 633–638.

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13.

D-CHIRO-INOSITOL AND INSULIN RESISTANCE: AN ALLOSTERIC POINT OF VIEW JOSEPH LARNER Principal Scientist, Insmed Pharmaceuticals, Inc., 800 E. Leigh St., Richmond, Va. 23219, USA

INTRODUCTION In this chapter, we ﬁrst consider general characteristics of insulin resistance to develop our theme that insulin resistance is multilayered with reversible and non-reversible elements. Next, we review our work on the discovery of D-chiro-inositol (DCI) and its role as a component and precursor of an inositol phosphoglycan putative mediator in insulin action and lack in insulin resistance. Third, we summarize data using DCI in repletion experiments in animals, clinical trials in naive type 2 diabetic subjects, and women with polycystic ovary syndrome (PCOS) emphasizing its action to reverse signs of insulin resistance in the Metabolic Syndrome X. Fourth, we discuss a rationale for repletion therapy based on results using the G/K type 2 diabetic rat model. This model has a demonstrated severe defect in conversion of myo-inositol to chiro-inositol compared to Wistar controls together with a demonstrated “back door” incorporation of labeled DCI into tissue phospholipids in both G/K and Wistar control rats. In concluding remarks, we provide an explanation for our allosteric view of insulin resistance. This review is centered on our own research and is not meant in any way to minimize contributions from other laboratories. These laboratories have studied mechanisms of insulin resistance from different points of view resulting in the extensive literature on this subject. Clearly, insulin resistance in animal models and humans is multi-factorial in nature and beyond the scope of a single review. For example, contributions from many other authors have been collected in a volume devoted to this subject and detailed in reference [1].

CHARACTERISTICS OF INSULIN RESISTANCE Insulin Resistance in Carbohydrate Metabolism is Primarily but not Solely Post-Receptor Much research has been devoted to measuring glucose disposal in control subjects compared to type 2 diabetic subjects using tightly monitored insulin clamp conditions. A seminal study carried out in Pima Indian subjects [2], clearly demonstrates the post-receptor nature of insulin resistance. Glucose disposal and glycogen synthase (GS) activation state were compared, the latter in muscle biopsy samples from control and type 2 diabetic subjects administered insulin in a dose-responsive fashion (Figure 1). In diabetic subjects both glucose disposal measured as carbohydrate storage (Figure 1A), and GS activation (Figure 1B), insulin action curves were shifted to the right (i.e. an increase in apparent Km shown by the cross-hatched bars) with decreased Vmax; kinetic changes that clearly support a post-receptor mechanism. 211

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Figure 2 (A) Insulin-stimulated glucose metabolism in lean control subjects (LC), obese non-diabetic control subjects (OC) and obese type 2 diabetic subjects (OD). Glucose disposal was determined as nonoxidative and oxidative. Left set of bars – basal, middle set of bars – clamp 1 (see the text) and right set of bars – clamp 2 (see the text). Note the greatest decrease in obese non-diabetic subjects and obese diabetic subjects is non-oxidative glucose disposal. (B) Insulin dose-response of GS activity state (muscle biopsy) in 3 sets of subjects as in (A) Note the right-shifted curves with obese non-diabetic subjects and more pronounced right-shifted curve with obese diabetic subjects. Note similarity with Figure 1B (Adapted from Ref. 3).

Further studies in a European population (Figure 2) compared type 2 diabetic subjects to control subjects and further evaluated the inﬂuence of obesity per se [3]. Again, both glucose disposal and GS activation state in muscle were determined, in response to insulin. As seen in Figure 2[A], non-oxidized and oxidized glucose disposal was greatest in lean controls, and decreased progressively in obese controls, and further in obese diabetic subjects. Two euglycemic, hyperinsulinemic clamp protocols were used in these studies; clamp 1 at 20 and clamp 2 at 80 mU insulin (per m2)min1. These insulin concentrations were chosen to mimic

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those found in obese type 2 diabetic subjects. The largest decrement was in non-oxidative glucose disposal. Figure 2[B] illustrates the insulin dose-response determined by activation of GS in muscle biopsies. Note the progressive right shift in the desensitized curves progressing from lean control subjects to obese control subjects to obese diabetic subjects with an apparent progressive decrease in Vmax. A similar kinetic result was seen when adipocytes were assayed for both pyruvate dehydrogenase (PDH) and GS activation after insulin administration in control subjects, obese subjects and type 2 diabetic subjects (Figure 3) (4). As discussed below, experimental evidence indicates that GS is the rate-limiting enzyme for non-oxidative glucose disposal and PDH a rate-limiting enzyme for oxidative glucose disposal. Figure 3[A] illustrates activated PDH

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Figure 3 (A) Insulin dose-response of adipocyte PDH activity state in control subjects, obese subjects and type 2 diabetic subjects. Note the decreased activation with insulin in the obese subjects with decreased Vmax and the completely inactive enzyme activity of the type 2 diabetic subjects. (B) Insulin dose-response of adipocyte GS activity state in control subjects, obese subjects and type 2 diabetic subjects. Left panel, enzyme assayed with low glucose 6-P (1 mM), right panel, enzyme assayed with high glucose 6-P (10 mM). Again, note markedly decreased enzyme activity of the type 2 diabetic subjects at all insulin concentrations (markedly decreased Vmax). (Adapted from Ref. 4.)

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(PDHa) as a function of administered insulin in human adipocytes prepared from subcutaneous fat biopsies. The adipocyte enzyme activity from control subjects was activated about 2-fold with increased insulin, while activation was blunted in adipocytes from obese individuals, and completely unreactive in adipocytes from diabetic subjects (decreased Vmax). When GS was examined, a familiar pattern of right-shifted curves with depressed Vmax was observed. As shown in Figure 3[B] when assayed with either 1.0 mM glucose 6-phosphate as allosteric activator (left panel) or 10 mM glucose 6-phosphate (right panel), GS activation state in adipocytes was markedly reduced in diabetic individuals at all insulin concentrations examined. No signiﬁcant differences were seen in the obese subjects. Thus, PDH activities were right shifted with depressed Vmax in insulin dose-responses in obese subjects and diabetic subjects and GS activities similarly right shifted with depressed Vmax in diabetic subjects. These data provide strong kinetic evidence for a post-receptor defect in insulin action. In a scholarly analysis, Olefsky and Kolterman [5] pointed out that right-shifted insulin doseresponse curves without a decrease in Vmax (termed decreased “insulin sensitivity”) may be consistent with insulin receptor defects. However, when decreased Vmax is also observed, (termed decreased “insulin responsiveness”) this suggests a post-receptor defect in insulin action. These investigators found evidence in mild obesity and early stage diabetic subjects for receptor defects (right-shifted curves with no decreased Vmax). With more severe obesity or in diabetic subjects with more pronounced insulin resistance, clearly decreased Vmax with right-shifted curves was observed. Thus, “pure” receptor defects cannot be ruled out in early stages of obesity or early type 2 diabetes. As discussed subsequently, the reversibility of insulin resistance with weight reduction, particularly of pure receptor defects should be considered in this context. With regard to the insulin receptor itself, elegant studies in mice of dual organ speciﬁc muscle, and islet ␤ cell receptor deletions clearly lead to insulin resistance and type 2 diabetes (6). In a limited number of human diseases with severe insulin resistance (hyperandrogenism, acanthosis nigricans, leprechanism and Type A insulin resistance with lipotropic diabetes), mutations in the human insulin receptor have been documented (7). However, when examined, insulin receptor mutations have not been found in the vast majority of subjects at risk for type 2 diabetes, including Pima Indians (8) Caucasian subjects (9) and Rhesus monkeys (10). Thus, while a small percentage of coding sequence mutations in the insulin receptors of type 2 diabetic subjects have not been ruled out, there is no compelling evidence in the vast majority of type 2 diabetic subjects for coding sequence mutations. In summary, in the vast majority of human type 2 diabetes, direct evidence for genetic alterations of the insulin receptor itself has not been established; however, kinetic evidence for a post-receptor mechanism(s) has been demonstrated and conﬁrmed (2–4).

Insulin Resistance in Carbohydrate Metabolism is Primarily in Skeletal Muscle with the Bulk in Non-Oxidative rather than Oxidative Metabolism The skeletal muscle mass now is recognized as the major site for insulin resistance in glucose metabolism (11). Careful studies in normal human muscle have shown that oxidative glucose metabolism is more sensitive to low physiological insulin concentrations with PDH the initial and rate-limiting enzyme. At higher insulin concentrations, non-oxidative glucose metabolism is more responsive to the hormone with GS, the rate-limiting enzyme (12). Both enzymes are regulated covalently by insulin in a similar manner. Both are activated by covalent dephosphorylation (13,14). Although both non-oxidative and oxidative carbohydrate metabolism are impaired in insulin resistance, as shown in Figure 2A, non-oxidative glucose disposal is quantitatively the more important site for insulin resistance (3). Thus, measurement of

D-CHIRO-INOSITOL AND INSULIN RESISTANCE

215

GS activation state with insulin in biopsy specimens has become one clinical measure of the severity of insulin resistance. Many published studies have focused on measuring glucose transport, glycogen synthesis, as well as GS activation state as potential mechanisms of the non-oxidative component of insulin resistance at the mechanistic level (15). Insulin Resistance is Multilayered with Reversible and Non-Reversible Elements Both hyperglycemia and hyperlipidemia induce insulin resistance by mechanisms which may be considered at least in part metabolic in origin. Hyperglycemia acts not only by glycosylation of proteins forming reversible and irreversible cross-linked products (16), but also induces insulin resistance by generating glucosamine (17). Glucosamine, by an incompletely understood mechanism, clearly induces insulin resistance (17). Hyperlipidemia, either endogenous or via administered lipids, also induces insulin resistance (18). Obesity per se, as illustrated in Figure 2, is associated with insulin resistance in the absence of diabetes. As will be discussed subsequently, PCOS with or without accompanying obesity, is another example of insulin resistance without diabetes. Euglycemic hyperinsulinemic clamp studies in 65 non-diabetic obese and 58 type 2 obese diabetic subjects established insulin resistance as a factor both in obese non-diabetic subjects as well as in obese type 2 diabetic subjects (19). Estimates indicated that 60–75% of the observed insulin resistance was due to type 2 diabetes with an estimated 25–40% attributable to the obesity per se (19). Visceral fat deposits, chieﬂy omental and mesenteric are correlated more with insulin resistance in skeletal muscle than peripheral fat deposits (20). Recently, intramuscular lipid accumulation in skeletal muscle has been demonstrated by CT imaging, magnetic resonance spectroscopy and direct staining (21,22). There was a direct correlation between the amount of intramuscular lipid with severity of insulin resistance (22). Clearly, insulin resistance is observed with and without obesity. Some parameters of this metabolic insulin resistance (hyperglycemia, hyperlipidemia and hyperinsulinemia) are reversible with caloric restriction and weight loss (22). Long-term caloric restriction in Rhesus monkeys (and rats) dramatically prevents obesity, maintains health, delays development of type 2 diabetes, increases life span and maintains glucose tolerance (23). Insulin resistance characterized by impaired tyrosine auto-phosphorylation of the solubilized, lectin puriﬁed, insulin receptor in adipocytes from humans with type 2 diabetes was restored essentially to normal with weight reduction (24). When further quantitated with anti-receptor and antiphosphotyrosine antibodies, about 43% of the receptors which bound insulin were tyrosine auto-phosphorylated in lean subjects and obese non-diabetic subjects. In diabetic subjects, the ﬁgure was decreased to 14%. After weight reduction, the ﬁgure rose substantially in diabetic subjects, providing again strong evidence for the reversibility of the receptor defect (24). Insulin resistance observed with lipid accumulation particularly in muscle and its reversal with caloric restriction constitutes another example (22). The data cited on reversibility of insulin resistance with reduction of obesity is in no way meant to imply that obesity per se does not also have a genetic component (vide infra). The clear cut distinction between non-reversible and reversible signs of insulin resistance point to a multilayered etiology. Evidence for stable (non-reversible) insulin resistance is discussed in the next two sections. Insulin Resistance has a Non-Reversible and Presumably Genetic Component It is well established that type 2 diabetes has a genetic basis. The concordance rate of identical twins is high in the order of ~70% (25). There is a great variation in the incidence and prevalence

216

JOSEPH LARNER

within ethnic groups, Pima Indians, for example, have a prevalence of about 50%. Offspring of two diabetic parents have an 80% lifetime risk of developing diabetes (26). Although evidence exists to suggest type 2 diabetes is polygenic in nature, some data indicate insulin resistance per se may be determined by a single gene. Lean normal glycemic offspring of one type 2 diabetic parent presents a trimodal distribution of clamp “M” values compatible with a single major gene inﬂuence (27). Recent work has established calpain 10 on chromosome 2 as a genetic site of mutations in Mexican Americans with diabetes (28). Another study has identiﬁed mutations in the short arm of chromosome 3 in Mexican Americans and suggested that 2 enzymes, namely glycogen branching enzyme (GBE1) and peroxisomal branched-chain acyl-CoA oxidase (ACOX2) may be involved (29). Additional studies in Ashkenazie Jews and Finnish families have indicated that type 2 diabetes susceptibility locus is present on chromosome 20q (30). Signiﬁcant progress is being made to identify the multiple genes associated with an increased risk for disease development including obesity. Several studies have now shown that abdominal obesity has a genetic basis. For example, recent family studies in Quebec have shown a linkage to chromosome 12q 24.3 (31). The irreversible (and likely genetic) component of insulin resistance itself is best demonstrated in breeding studies with rats and complemented by detailed analysis of glucose tolerance tests in humans. “Normal” Wistar rats were selected based on slightly abnormal glucose tolerances (~10%) and inbred for ~30 generations, to establish a stable, lean, mild type 2 diabetes model. The Goto Kakizaki (G/K) rat model exhibits both insulin resistance and defective islet ␤ cell insulin secretion (32). Inbreeding of this genetic tendency present in “normal” animals, produces a stable, non-obese type 2 diabetic model, which mirrors human type 2 diabetes with both insulin resistance and islet ␤ cell secretory defects. Thorough glucose tolerance studies in humans (33) are similar to those in rats and reveal approximately 25% of “normal” subjects manifest a distinct degree of insulin resistance as determined by plasma glucose disappearance rates and/or by slightly elevated plasma insulin values during the tests (33). A strikingly high percentage of “normal” individuals, therefore, is presumably at risk for development of type 2 diabetes. This high incidence possibly is related to the hypothesis of a “thrifty gene” leading to enhanced survival via fat storage during periods of food deprivation (34). In summary, post-receptor insulin resistance of carbohydrate metabolism is multilayered with reversible metabolic component(s) as well as non-reversible presumably genetic components.

Further Evidence for a Genetic Basis for Insulin Resistance Considerable evidence has implicated impaired glucose uptake as well as impaired glycogen synthesis as mechanisms of insulin resistance. Many mechanistic studies have focused on glycogen synthesis and GS activity state, in part because the covalent phosphorylation– dephosphorylation mechanisms of GS activation and deactivation are more readily studied than the more complex regulation of glucose transport. While further work needs to be done, the basic principles of GS activation by dephosphorylation and inactivation by phosphorylation are understood and can be quantiﬁed by well-deﬁned assays. Using independent methods of NMR technology, there is clear evidence that glycogen synthesis is impaired in type 2 diabetic subjects (35). We will not discuss the many studies on the control of glucose transport by insulin. In insulin resistance, studies thus far point to an impaired recruitment of glucose transporters to the cell membrane as a possible deﬁcit (36). Needless to say, both impaired glycogen synthesis and impaired glucose transport activation could reﬂect a common signaling mechanism.

D-CHIRO-INOSITOL AND INSULIN RESISTANCE

217

50

*

Control 40

GS mean (%)

*

Diabetic

*

30

* 20

10

0 0

250

500

750

1000

1250

1500

1750

2000

Insulin (mU/ml)

Figure 4 Insulin dose-response of human skin ﬁbroblast GS activity of control subjects and type 1 diabetic subjects. Again, note the depressed enzyme activity in the diabetic subjects (decreased Vmax) compared to control subjects. (Adapted from Ref. 37 with modiﬁcation.)

In cultured skin ﬁbroblasts and muscle cells insulin-stimulated glycogen synthesis from glucose and GS activation are defective in cells from diabetic subjects compared to cells from control subjects. In an early study of human skin ﬁbroblasts, Craig et al. (37) demonstrated an impaired ability to activate GS in those from a small number of type 1 diabetic subjects. As seen in Figure 4, the insulin dose-response was clearly shifted to the right with decreased Vmax in the diabetic ﬁbroblasts compared to controls (37). A certain percentage of type 1 diabetic subjects is known now to also have insulin resistance, not reversed by tight control, likely with a genetic basis (38). This is reasonable since ~25% of “normal” humans have some slight degree of insulin resistance (see above). Wells et al. (39) next demonstrated the impaired ability of insulin to activate glycogen synthesis in skin ﬁbroblasts from type 2 diabetic subjects selected from a strong family history of diabetes compared to matched controls. Other insulin sensitive actions including [3H] uracil incorporation into DNA were not affected. More recently, Henry et al. (40) demonstrated an inability to activate GS by insulin in cultured human myotubes from type 2 diabetic subjects compared to controls. While there is still controversy (41), the bulk of evidence supports the concept of a stable form of insulin resistance in cultured cells determined by a decreased ability of insulin to activate GS. Thus, both animal inbreeding experiments as well as studies in cultured human cells strongly implicate a genetic component for insulin resistance. An Argument for an Insulin Speciﬁc Signaling Defect as the Basis for Insulin Resistance of Non-Oxidative Glucose Metabolism Glucose and insulin independently activate GS and glycogen synthesis via separate signaling pathways. In experiments with rat adipocytes and mouse skeletal muscle, Lawrence and

218

JOSEPH LARNER

Larner (42) and Oron and Larner (43) distinguished two separate mechanisms for GS activation using glucose alone or insulin alone. Both pathways, although separate, clearly augment each other and thus, physiologically operate cooperatively. With glucose alone, GS activation required the formation of a phosphorylated hexose. Thus, glucose, other hexoses, and 2 deoxy-glucose phosphorylated in the 6 position were effective, while 3-O-methyl-glucose, which is not phosphorylated in the 6 position, was ineffective. Glucose 6-P allosterically activates phosphoprotein phosphatase 1 (PP1) to dephosphorylate GS by binding to synthase and making synthase a more accessible substrate for dephosphorylation (44). Glucose 6-P accumulation, therefore, provides a plausible allosteric mechanism for signaling via glucose alone to activate GS dephosphorylation and activation. In the case of insulin alone, allosteric activation of phosphoprotein phosphatase 2C (PP2C) by a chiro-inositol containing inositol phosphoglycan putative mediator has been shown by Huang et al. (45) to allosterically activate PP1 via dephosphorylation and inactivation of two speciﬁc peptide inhibitors of PP1, INH-1-P and DARPP32-P. These peptides are active as inhibitors only when phosphorylated. Thus, insulin alone would act via a separate allosteric mechanism distinct from that of glucose, but physiologically would synergize when glucose is present. In vivo studies in human muscle have shown that hyperglycemia differs from insulin in directing glucose to oxidative and non-oxidative pathways (46). With this in mind, we may now consider the following clinical data: muscle biopsy studies show that GS activation by insulin is impaired in type 2 diabetic subjects as well as in their 1st degree relatives (47). In control experiments, Beck-Nielsen et al. (47) have shown that in contrast to insulin, elevated glucose concentrations alone are capable of activating GS under conditions where insulin is ineffective. These results argue that the GS activation defect associated with insulin resistance resides speciﬁcally in the insulin signaling rather than in the glucose signaling pathway, and probably not in the enzymatic components themselves. In conclusion, although both glucose and insulin are capable of activating GS by allosteric effectors, the defect in insulin resistance appears to reside speciﬁcally in the insulin signaling effector.

THE ROLE OF A CHIRO-INOSITOL DEFICIT IN INSULIN RESISTANCE Discovery of D-Chiro-Inositol in a Putative Insulin Mediator Preparation Employing two enzyme assays to search for putative insulin mediators, we puriﬁed two inositol phosphoglycan species from rat liver to single spot purity on thin layer chromatography plates (48). A slower migrating species (Figure 5, lanes 2 and 3) contained myo-inositol and glucosamine and inhibited cAMP kinase and adenylate cyclase (49a,b). It explained the wellknown actions of insulin to counteract the effects of epinephrine and glucagon. A second, faster migrating species (Figure 5, lanes 5 and 6) contained DCI and galactosamine (48a). It activated PDH phosphatase, PP2C, and, as mentioned above, also PP1 via removal of the inhibiting inﬂuence of INH-1-P or DARPP-32-P (45). PDH phosphatase dephosphorylates and thus activates PDH, a rate-limiting enzyme of oxidative carbohydrate metabolism (12) (see model Figure 10). Phosphatase 2C and PP1 dephosphorylate and activate GS (50,51), the rate-limiting enzyme of non-oxidative glucose metabolism. The faster moving species activated both PDH phosphatase and PP2C, both Mg dependent enzymes in contrast to PP1, by shifting their Mg dose response curves to the left, thus sensitizing the phosphatases to their required metal Mg (52a). Of interest, this in vitro action of the chiro-inositol

D-CHIRO-INOSITOL AND INSULIN RESISTANCE

1

2

3

4

5

219

6

Figure 5 Thin layer chromatogram of 2 puriﬁed inositol phosphoglycans from rat liver. Plates were developed in isopropanol : pyridine : acetic acid : water (8 : 8 : 1 : 4) and sprayed with ninhydrin. Lanes 1 and 4 galactosamine standards. Lanes 2 and 3 cAMP-kinase inhibitors from control and insulin pretreated rats. Lanes 5 and 6 PDH phosphatase stimulators from control and insulin pretreated rats (Adapted from 48b with modiﬁcation).

containing inositol phosphoglycan duplicates the in vivo action of insulin acting on fat tissue, an argument for seriously considering the faster migrating inositol phosphoglycan as a true insulin mediator (52b). Chiro-Inositol Deﬁcit and Insulin Resistance Initially, to deﬁne the roles of the two inositol phosphoglycan putative mediators, we evaluated their turnover by measuring the excretion of their respective inositols in 24 h urine collections. We found an increased excretion of myo-inositol, and a decreased excretion of chiro-inositol in type 2 diabetic subjects, a mixed population at the University of Virginia, Pima Indian subjects, ﬁrst degree relatives of type 2 diabetics and a certain percentage of type 1 diabetic subjects (53) (Table 1). Similar ﬁndings were observed in a primate model of type 2 diabetes, the Rhesus monkey (53), (Table 2) and the G/K rat (32). Expressed as a ratio of myo-inositol to chiro-inositol, the differences become more striking and are independent of units. In the monkey, we found a progressive increase in myo-inositol excreted, and a reciprocal progressive decrease in chiro-inositol excreted as animals progressed from normal to obese non-diabetic to obese diabetic (53) (Table 2). The altered urinary inositol excretion pattern found in the obese non-diabetic animals, compelled us to consider that the urine ﬁndings were more closely related to the underlying insulin resistance than the diabetes per se.

220 Table 1

JOSEPH LARNER Mean urinary excretion of inositols in 24-h specimens in six groups of subjectsa.

Non-diabetic control UVAb Non-diabetic control Pimac Type 2 diabetes UVA Type 2 diabetes Pima Type 1 diabetes UVA Non-diabetic relatives of type 2 diabetics UVA

Chiro-inositol (␮mol/d)

Myo-inositol (␮mol/d)

Ratio myo/chiro

Low (10 ␮mol/d) chiro-inositol, % of subjects

36.1 6.6 52.3 10.6 13.2 3.6d 11.1 7.2d 18.5 5.7 6.8 1.6e

91 11 112 14 270 55d 244 90 251 32e 90 23

2.5 2.1 20.4 22.0 13.6 13.2

31.8 20.0 72.0e 77.8d 62.8d 75.0d

Source: Adapted from Craig et al. (53b). a Plus-minus values are means SE. b UVA University of Virginia Medical Center. c Pima Pima Indian specimens from Clinical Diabetes and Nutrition Section, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona. d p 0.01 compared to corresponding control group. e p 0.001 compared to corresponding control group.

Table 2

Urinary excretion of chiro-inositol and myo-inositol in monkeys.

Monkey no.

Diagnosis

Chiro-inositol (␮mol/day)

Myo-inositol (␮mol/day)

1 2 3 4 Mean SE

Normal Normal Normal Normal —

18.5 3.1 1.8 3.7 6.7 2.3

21.9 5.9 6.7 3.5 9.5 2.4

5 6 7 8 9 10 11 Mean SE

Obese Obese Obese Obese Obese Obese Obese —

1.7 3.6 2.9 0.3 ND ND ND 1.2 0.2

7.0 6.5 11.3 3.1 72.2 63.2 4.9 24.0 4.7

12 13 14 15 Mean SE

Diabetic Diabetic Diabetic Diabetic —

1.0 ND ND ND 0.2 0.1

169.0 61.4 312.5 61.3 151.1 34.3

Source: Adapted from Kennington et al. (53a). ND, not detectable (limit of detection 0.5 mol/l).

To further examine this question, we directly measured insulin resistance in Rhesus monkeys by several tests including glucose disappearance rates, hyperinsulinemic euglycemic clamp “M” values, as well as muscle and fat biopsy measurements of GS and phosphorylase activity states. These tests were compared to chiro-inositol excretion in urine. We observed

D-CHIRO-INOSITOL AND INSULIN RESISTANCE 10 9 8 7 6 5 4 3 2 1

221

Si (×10–4· min–1µU–1ml–1)

Y = 2.373 + 0.042X R = 0.766

0

20 40 60 80 100 120 140 Urinary chiro-inositol (µmol/day)

PDH phosphatase (% stimulation)

Figure 6 Urinary chiro-inositol excretion in 24 h vs. insulin sensitivity determined by Bergman’s minimal model method in Japanese subjects. Note the relationship between insulin sensitivity and urinary chiro-inositol excretion. See the text for further discussion. S i insulin sensitivity index, ● type 2 diabetic subjects, □ impaired glucose tolerance subjects, ▲ normal subjects. (Adapted from Ref. 55.)

900 Control

600

Type 2 diabetic

300

0 Hemodialysate

Muscle

Urine

Figure 7 PDH phosphatase stimulatory inositol phosphoglycan bioactivity in control subjects and type 2 diabetic subjects. Bioactivity in hemodialysate, autopsy derived skeletal muscle, and urine was decreased about 50% in diabetic subjects compared to control subjects (Adapted from 56). See text for further discussion.

and reported linear inverse correlations with urinary chiro-inositol excretion with each of the insulin sensitivity tests (54). In independent tests in Japan, (55) urinary chiro-inositol excretion was measured in control subjects, non-diabetic subjects with impaired glucose tolerance and type 2 diabetic subjects. The type 2 diabetic subjects had the lowest chiro-inositol excretion, the normal subjects the highest and the subjects with impaired glucose tolerance, intermediate values (Figure 6). To determine whether the decrement of chiro-inositol was solely in urine or more widespread in the body, further studies were conducted examining human autopsy muscle, hemodialysate and again urine of type 2 diabetic and control subjects. An approximately 50% decrement in chiro-inositol content and putative mediator bioactivity (PDH phosphatase

222

JOSEPH LARNER

activation) was observed in muscle tissue extracts, urine and hemodialysate of type 2 diabetic subjects compared to controls (Figure 7) with no decrement in the myo-inositol phosphoglycan bioactivity (assayed by cAMP-kinase inhibition) (56) (not shown). All the studies thus demonstrated a widespread tissue decrement in chiro-inositol and its putative mediator bioactivity associated with insulin resistance and diabetes.

REPLETION THERAPY WITH D-CHIRO-INOSITOL Administration of D-Chiro-Inositol to Animals – Brief Summary With the demonstration that a chiro-inositol deﬁcit in urine was associated with insulin resistance in primates (54), humans (53) and G/K rat (32), and a tissue deﬁcit associated with diabetes in humans, DCI was administered initially to low dose STZ rats (a model of type 2 diabetes) (57), then to Rhesus monkeys (57), and lastly to three human populations, subjects with impaired glucose tolerance, subjects with early type 2 diabetes, and women with PCOS, both obese and lean (58–60). In both animal models, DCI was effective in reducing hyperglycemia and accelerating glucose disposal. All subjects received the compound orally with no adverse effects noted. In human subjects, signs of insulin resistance, including elevated lipid proﬁles, hyperglycemia, hyperinsulinemia, systolic and diastolic blood pressure, body mass index, body weight, signs of Syndrome X were reversed (61). Evidence for DCI acting as an insulin sensitizer has been obtained in Rhesus monkeys (62). DCI was administered to Rhesus monkeys during a euglycemic, hyperinsulinemic clamp and muscle biopsy samples taken to measure GS activation. Samples from animals administered DCI in addition to maximal amounts of insulin demonstrated a further activation of GS above that of maximal insulin alone (62). Presumably DCI acts by incorporation into putative mediator precursor phospholipids bypassing an endogenous block in biosynthesis from myoinositol (see model Figure 10). It is then released as active inositol phosphoglycan putative mediator by insulin (or IGF 1), thus sensitizing the action of the hormone. Further evidence for sensitization of insulin action has now also been obtained with H4IIE hepatoma cells. Preincubation of cells with DCI (10–200 M) clearly left-shifts insulin dose-response curves for glycogen synthesis and GS activation. Myo-inositol and L-chiro-inositol are inactive thus demonstrating speciﬁcity for the D isomer (63). Results of Two Clinical Trials with Administration of D-chiro-Inositol to Naive Type 2 Diabetic Subjects and to Women with PCOS DCI was administered orally, 1200 mg once a day for 28 days to 110 subjects with mild type 2 diabetes in a multicenter, randomized, double-blind, placebo-controlled trial (59). No subject had received prior anti-diabetic medication. A 3 h oral glucose tolerance test (OGTT) was performed at the initiation of the study and again after 28 days of therapy. Basal demographic characteristics were similar in the treatment and placebo groups (Table 3). DCI was well tolerated without signiﬁcant changes in safety parameters. DCI-treated subjects showed statistically signiﬁcant decreases in diastolic blood pressure, and lipid parameters including triglycerides, cholesterol, and LDL cholesterol (Table 3). Subjects with elevated free fatty acid levels (0.57 mEq/l) were separately evaluated since their insulin resistance was presumably somewhat more severe. They had a 27% decrease in FFA, a 20% decrease in

Table 3

Demographics and subject characteristics at baseline and at the end of the study (EOS) for all subjects. DCI (N 57) Baseline

Age (yr) Sex (% female) Duration of diabetes (years) Body mass index (kg/m2) Waist-to-hip ratio Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mg/dl) Glucose AUC 3 h (mg/dl/min) HbA1C (%) Fasting plasma insulin (␮U/ml) Triglycerides (mg/dl) Cholesterol (mg/dl) LDL Cholesterol (mg/dl) Free fatty acid (mEq/l)

54.68 10.34 54 2.2 4.07 33.4 6.57 0.91 0.07 131.4 14.0 80.6 7.7 143.4 22.8 42904 6631 6.82 0.84 15.63 9.2 153.2 100.1 191.1 36.9 126.8 31.4 0.45 0.16

Placebo (N 53) EOS

Baseline

33.3 6.68 0.91 0.07 128.8 13.0 77.7 8.1a 142.0 34.0 41707 7913 6.71 0.84 15.58 12.3 135.8 75.5b 182.8 33.7a 121.1 28.1c 0.42 0.18

57.51 10.22 62 3.2 5.19 33.1 7.04 0.92 0.08 133.5 19.9 80.4 9.3 138.4 25.9 42310 7208 6.79 0.99 15.42 8.9 173.7 85.3 187.2 34.5 120.4 34.9 0.49 0.18

EOS

33.1 7.11 0.91 0.08 131.1 15.9 79.1 8.3 142.9 33.6 42335 8953 6.73 1.11 17.40 13.0 191.9 114.9 188.4 38.4 117.1 40.7 0.48 0.20

Data are means SD. Use the following conversions for SI units: glucose – from mg/dl to mmol/l, multiply by 0.0556; insulin – from U/ml to pmol/l, multiply by 6.0; triglycerides – from mg/dl to mmol/l, multiply by 0.0113; cholesterol – from mg/dl to mmol/l, multiply by 0.0259; and LDL cholesterol – from mg/dl to mmol/l, multiply by 0.0259. a p 0.01 for comparison with baseline within treatment group. b p 0.01 for comparison of effect between treatment groups. c p 0.05 for comparison with baseline within treatment group.

Table 4 Demographics and subject characteristics at baseline and at the end of the study (EOS) for subjects with elevated free fatty acids (0.57 mEq/l). DCI (N 14) Baseline Age (yr) Sex (% female) Duration of diabetes (years) Body mass index (kg/m2) Waist-to-hip ratio Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mg/dl) Glucose AUC 3 h (mg/dl/min) HbA1C (%) Fasting plasma insulin ( U/ml) Triglycerides (mg/dl) Cholesterol (mg/dl) LDL Cholesterol (mg/dl) Free fatty acid (mEq/l)

52.5 11.35 71 3.9 7.52 33.0 7.31 0.89 0.06 130.9 13.7 82.1 7.50 145.1 27.0 45211 8021 6.87 0.86 15.93 7.67 236.1 148.5 200.9 39.9 122.1 31.1 0.67 0.07

Placebo (N13) EOS

Baseline

32.7 7.38a 0.88 0.07 123.0 14.4b 77.0 8.87a 139.1 44.2 41064 8561a 6.60 0.83a 15.86 8.77 175.5 76.5a 187.1 34.1a 118.9 25.9 0.49 0.20b

58.08 10.36 77 1.1 1.37 32.5 4.38 0.91 0.08 137.2 27.6 79.2 11.30 132.2 30.8 41457 9206 6.71 1.23 16.46 5.19 218.3 117.8 190.9 34.4 115.5 35.4 0.74 0.15

EOS

32.3 4.23 0.92 0.09 133.4 13.8 78.8 7.73 139.2 48.1 43186 11443 6.65 1.32 17.0 5.66 270.9 162.5a 188.3 24.9 103.0 32.1a 0.69 0.25

Data are means SD. Use the following conversions for SI units: glucose – from mg/dl to mmol/l, multiply by 0.0556; insulin – from U/ml to pmol/l, multiply by 6.0; triglycerides – from mg/dl to mmol/l, multiply by 0.0113; cholesterol – from mg/dl to mmol/l, multiply by 0.0259; and LDL cholesterol – from mg/dl to mmol/l, multiply by 0.0259. a p 0.05 for comparison with baseline within treatment group. b p 0.01 for comparison with baseline within treatment group.

224

JOSEPH LARNER

triglyceride levels, a 7% decrease in total cholesterol levels and surprisingly, a statistically signiﬁcant decrease in HbA1C concentrations (Table 4). There was also a 49 mg/dl decrease in the 2 h OGTT glucose concentration (not shown) and a 9.2% reduction in the OGTT AUC as well as a decrease in the body weight (not shown), body mass index, systolic and diastolic blood pressure. These data are also shown graphically in Figure 8. Thus, DCI administration to a population of naive type 2 diabetic subjects improved both lipid and glycemic parameters as well as other aspects of the metabolic Syndrome X. Decreases in lipid parameters appear a more sensitive index of response than the changes in glycemic parameters. Surprisingly, a signiﬁcant reduction in HbA1C was observed after 28 days of therapy. Studies conducted in women with PCOS are particularly instructive. As previously mentioned, the disorder is a paradigm for normoglycemic insulin resistance. Non-diabetic women with PCOS, regardless of whether they are obese or lean, have insulin resistance and compensatory hyperinsulinemia (64–68). This is due to both a form of insulin resistance intrinsic to PCOS (69–71) and, in many cases, acquired insulin resistance due to obesity (72). The importance of this observation is that hyperinsulinemia appears to play an important

p = 0.03

p < 0.05

300 275 250 225 200 175

210 200 190 180 170

150 125 Baseline EOS Placebo

160

Baseline EOS INS-1 p = 0.005

0.6 0.4 0.2

Baseline EOS Placebo

Baseline EOS INS-1 p = 0.04

48000 Glucose AUC (mg/dl/min)

0.8

FFA (mEq/L)

p = 0.037

220 Cholesterol (mg/dl)

Triglyceride (mg/dl)

325

46000 44000 42000 40000 38000 36000

0.0

Baseline EOS Placebo

Baseline EOS INS-1

Baseline EOS Placebo

Baseline EOS INS-1

Figure 8 DCI administration improved triglyceride, cholesterol, free fatty acid and glucose AUC in type 2 diabetic subjects with elevated free fatty acid (data are means SE). Placebo group (N 13) was treated for 30.1 0.82 days and the DCI group (N 14) was treated for 29.3 0.38 days. p values are for the change from baseline to the EOS within a treatment group. The change in triglycerides and glucose AUC in the DCI-treated subjects differed signiﬁcantly from the change in the placebo group (p 0.012 and p 0.02, respectively). To convert triglyceride from mg/dl to mmol/l, multiply by 0.0113, and to convert cholesterol from mg/dl to mmol/l, multiply by 0.0259.

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pathogenetic role in the hyperandrogenism and the chronic anovulation of both obese and lean women with PCOS (73–75). Moreover, the PCOS is associated with other metabolic abnormalities likely to be linked to insulin resistance: glucose intolerance (76–78), dyslipidemia (79–81), hypertension (79,82) and atherosclerosis (83). This constellation of ﬁndings in non-diabetic individuals is, as previously discussed, termed Syndrome X or dysmetabolic syndrome (61). The fundamental pathogenetic mechanism appears to be insulin resistance. It was hypothesized that insulin resistance in these women relates in part to a deﬁciency in a DCI-containing inositol phosphoglycan putative mediator, and that administration of DCI would replenish DCI-containing inositol phosphoglycan stores and improve insulin sensitivity. To test this hypothesis, Nestler et al. (60) measured fasting serum sex steroids and performed OGTTs before and after 6–8 weeks of oral administration of 1200 mg DCI, or placebo once daily in 44 obese women with PCOS. Serum progesterone levels were obtained weekly to monitor for ovulation. In 22 women given DCI, the mean (SE) area under the serum insulin curve after oral glucose administration decreased signiﬁcantly from 13.4 0.5 to 5.1 0.3 mU/ml/min. Notably, glucose tolerance normalized in the six women who had impaired glucose tolerance at baseline, consistent with an increase in insulin sensitivity. Simultaneously, serum free testosterone levels, diastolic and systolic blood pressure and plasma triglyceride concentrations decreased in the women treated with DCI, whereas none of these variables changed in the women administered placebo. Similarly, 19 of the 22 women (86%) who received DCI ovulated, compared with only 6 of the 22 women (27%) in the placebo group. A further study demonstrated that a dose of 600 mg DCI was similarly effective as 1200 mg, and that 100 mg was ineffective (unpublished). To further explore the nature of insulin resistance in PCOS, the effects of DCI administration to lean women with PCOS were subsequently assessed. This study tested the hypothesis that DCI deﬁciency in PCOS is unrelated to adiposity (84). Twenty lean (BMI: 20.0–22.4 kg/m2) women with PCOS received either 600 mg DCI or placebo once daily for 6–8 weeks. OGTTs were performed and serum sex steroids were measured before and after therapy. To monitor for ovulation, serum progesterone concentrations were determined weekly. In the 10 women given DCI, the mean (SE) area under the plasma insulin vs. time curve after oral administration of glucose decreased signiﬁcantly by 36% while the area under the plasma glucose vs. time curve decreased signiﬁcantly by 17%. These combined results were consistent with an overall increase in insulin sensitivity. Simultaneously, the serum free testosterone concentration, systolic and diastolic blood pressures, and plasma triglycerides decreased in the women treated with DCI. In contrast, these values did not change in the placebo group. Six of the 10 women (60%) in the DCI group ovulated compared to only 2 of 10 women (20%) in the placebo group. Collectively, these ﬁndings in both obese and lean women with PCOS are consistent with the concept that DCI deﬁciency (presumably leading to a deﬁciency of a DCI-containing inositol phosphoglycan putative mediator) contributes to insulin resistance in women with PCOS. They further suggest that DCI deﬁciency in women with PCOS is unrelated to adiposity, since it appears to be present in lean women with PCOS as well. Finally, the ﬁndings of both studies suggest that DCI stores can be repleted, and normal insulin sensitivity apparently restored, by exogenous oral DCI administration in both obese and lean women with PCOS, as well as in subjects with early type 2 diabetes.

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RATIONALE FOR REPLETION THERAPY A defect or several defects (presumably genetic in nature) in the signaling pathway involving the biosynthesis, release or degradation of the chiro-inositol phosphoglycan putative mediator could explain the results observed, that is, the chiro-inositol and putative mediator deﬁcit and its reversal by administered DCI. However, it is more attractive to consider a deﬁcit in chiroinositol synthesis itself since the deﬁcit is reversed by administration of the substance. Further, we have demonstrated a defect in conversion of myo-inositol to chiro-inositol in the G/K rat (see Figure 9A). Our working hypothesis is shown in the model of (Figure 10). As shown, myo-inositol conversion to chiro-inositol is blocked and thus its incorporation into the lipid phosphoinositol glycan precursor is abrogated. Normally with insulin action, the phospholipase is activated via a G-protein related mechanism and the precursor GPI-phospholipid is cleaved at the cell surface to release the phosphoinositol glycan mediator. Evidence for the occurrence of the normal G-protein linked insulin-stimulated signaling events is cited in a recent review (85). The released phosphoinositol glycan then reenters the cell through an ATP dependent uptake system (86a) and allosterically activates the key phosphoprotein phosphatases, 1,2C and PDH that in turn dephosphorylate and activate GS and PDH (45,48,52). As mentioned, the in vivo separate “back door” incorporation of labeled DCI into tissue phospholipids has been demonstrated in G/K type 2 diabetic rats and Wistar control rats thus bypassing the defective conversion of myo-inositol to DCI (89). A Na dependent co-transporter for myo-inositol and DCI has also been demonstrated (86b). Conversion of Myo-Inositol to Chiro-Inositol Sources of DCI include dietary (e.g. legumes, citrus fruit, etc.), generation from myo-inositol via epimerization of OH #3 or generation from an unknown source. Clearly, food sources are insufﬁcient to supply amounts required. Epimerization of more abundant myo-inositol is, therefore, an attractive possibility, particularly since it has already been experimentally demonstrated (87–89). We have demonstrated that [3H] myo-inositol is epimerized to [3H] chiro-inositol in rats in vivo and in tissue cultured ﬁbroblasts in vitro (87,88). Using [3H]myo-inositol as precursor, we clearly demonstrated its conversion to [3H]chiro-inositol after separating the two inositols and demonstrating the presence of [3H]chiro-inositol. Studied in vivo in Sprague–Dawley rats, the highest conversions occurred in the insulin sensitive tissues, liver and muscle (approximately 8%). When blood phospholipids were examined, a 60% conversion was observed (87). In vitro studies in ﬁbroblasts transfected with the human insulin receptor demonstrated that [3H]myo-inositol was converted to [3H]chiro-inositol chieﬂy at the phospholipid level and the conversion was stimulated by added insulin (88). These experiments demonstrated that a conversion of myo-inositol to chiro-inositol in vivo as well as in vitro does indeed occur. A Defect in Conversion of Myo-Inositol to Chiro-Inositol In a recent study we compared the conversion of [3H] myo-inositol to [3H] chiro-inositol in control Wistar and G/K type 2 diabetic rats (89). After a 3-day labeling period designed to achieve isotopic equilibrium, we observed again excellent conversions (20–30%) in liver, fat and muscle, insulin-sensitive tissues of the Wistar rat. However, in the G/K rat, there was

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Figure 9 (A) Conversion of [3H]myo-inositol to [3H]chiro-inositol in control Wistar and type 2 diabetic G/K rats. Rats were pre-injected intraperitoneally with [3H]myo-inositol (1 mC) over a 31 e day period in 10 divided doses until isotopic equilibrium was obtained (urine excretion). Tissues were then harvested, digested in 6 N HCl for 48 h, lyophilized, inositols puriﬁed through mixed-bed ion exchange resins and C18 Sep-Pak cartridges, inositols separated and counted. As seen, liver, fat and muscle had conversions of [3H]myo-inositol to [3H]chiro-inositol of 25–30%. This was reduced in the G/K rat to 3–5%. (B) Back conversion of [3H]chiro-inositol to [3H]myo-inositol in control Wistar and type 2 diabetic G/K rats. Note the extremely low conversion in cell tissues examined (1% or less). (Adapted from Ref. 89).

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D-chiro-Inositol and Insulin signal transduction O

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Figure 10 Model of the proposed mechanism of action of insulin to release DCI phosphoglycan and a block in the conversion of myo-inositol to DCI. Also shown is the “back door” mechanism of reincorporation of DCI into chiro-inositol containing GPIs. A Na dependent DCI co-transporter has been demonstrated (86b) as well as the “back door” reincorporation into GPIs (89). Note the release of the inositol phosphoglycan to the extracellular milieu, and its reentry into cells via an ATP-dependent transporter (83a) to allosterically activate PP2C and PDH phosphatases activating GS, the rate-limiting enzyme of non-oxidative glucose metabolism and PDH, a rate-limiting enzyme of oxidative glucose metabolism. See the text for further discussion.

a marked reduction in the conversion to very low levels 3–5% (Figure 9A). When [3H] chiroinositol conversion to [3H] myo-inositol was determined (Figure 9B), it was extremely low ~1%. Thus, in vivo, the conversion is essentially unidirectional from myo-inositol- to chiro-inositol. In summary, a defect in epimerization of myo-inositol to chiro-inositol is present in the insulin-sensitive tissues of the G/K rat, providing an attractive hypothesis to explain the deﬁciency in chiro-inositol and its effectiveness of repletion therapy.

CONCLUDING REMARKS As discussed in this review, post-receptor mechanisms in the control of glucose disposal via non-oxidative and oxidative mechanisms appear defective in the insulin resistant state. Insulin resistance and obesity are clearly multifaceted with both reversible (metabolic) and irreversible (genetic) aspects. An argument has been put forward based upon the fact that the activities of two critical and rate-limiting enzymes of non-oxidative and oxidative glucose metabolism are under covalent control by insulin in a similar manner (activated by dephosphorylation). One

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can therefore begin to approach the mechanism of their defective activation by insulin in insulin resistance. The argument developed with GS hinges on the demonstration of two separate activation mechanisms with glucose alone and with insulin alone. The activation of GS by hyperglycemia is not defective in insulin resistance but it is clearly defective with insulin. The tentative conclusion is that the insulin signaling system per se is defective since the enzymatic apparatus responds to, and is activated by, hyperglycemia presumably through a separate allosteric mechanism. The argument then turns to the allosteric activation mechanism induced by insulin. Here we rely upon data that a speciﬁc chiro-inositol phosphoglycan putative mediator may be defective in tissues and decreased in urine of diabetic subjects with insulin resistance and that this may be reversed (at least in part) by administration of the precursor inositol, DCI, which acts as a pro-drug and restores the metabolic deﬁcit toward normal. Further studies with type 2 diabetes in the G/K rat, a model developed by inbreeding Wistar rats speciﬁcally for insulin resistance demonstrate a failure to convert myo-inositol to chiro-inositol in insulin-sensitive tissues. Future studies will delineate the possible genetic signiﬁcance of the defect in converting myo-inositol to chiro-inositol as well as other components in this novel insulin signaling system to insulin resistance in the G/K rat model and whether these ﬁndings are applicable to insulin resistance in human diabetes and PCOS. ACKNOWLEDGMENTS We wish to thank M. Sleevi, G. Allan, A. Rogol, S. Taylor, C. Newgard, G. Barrett, E. Grollman for their comments after reviewing this paper; M. Sleevi for providing the ﬁgures and S. Lamm for her help in typing the manuscript. We also thank J. Nestler for providing the discussion of therapy of PCOS. REFERENCES 1. Larner, J. and Taylor, S.I. (eds) (1998) Mechanisms of insulin resistance. In Larner, J. and Taylor, S.I. (eds), J. Basic and Clin. Physiol. Pharm. Mech. Insulin Resistance, 9, 2–4. Freund Publ., London. 2. Young, A.A., Bogardus, C., Wolfe-Lopez, D. and Mott, D.M. (1988) Muscle glycogen synthesis and disposition of infused glucose in humans with reduced rates of insulin-mediated carbohydrate storage. Diabetes, 37, 303–308. 3. Damsbo, P., Vaag, A., Hother-Nielsen, O. and Beck-Nielsen, H. (1991) Reduced glycogen synthase activity in skeletal muscle from obese patients with and without type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia, 34, 239–245. 4. Mandarino, L.J., Madar, Z., Kolterman, O.G., Bell, J.M. and Olefsky, J.M. (1986) Adipocyte glycogen synthase and pyruvate dehydrogenase in obese and type 2 diabetic subjects. Am. J. Physiol., 251, E489–496. 5. Olefsky, J.M. and Kolterman, O.G. (1981) Mechanisms of insulin resistance in obesity and non-insulin-dependent (type 2) diabetes. Am. J. Med., 70, 151–168. 6. Mauvais-Jarvis, F., Virkamaki, A., Michael, M.D., Winnay, J.N., Zisman, A., Kulkarni, R.N. and Kahn, C.R. (2000) A model to explore the interaction between muscle insulin resistance and ␤-cell dysfunction in the development of type 2 diabetes. Diabetes, 49, 2126–2134. 7. Taylor, S.I. and Arioglu, E. (1998) Syndromes associated with insulin resistance and acanthosis nigricans. In Larner, J. and Taylor, S.I. (eds), J. Basic and Clin. Physiol. Pharm. Mech. Insulin Resistance, 9, 419–439. Freund Publ., London.

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55. Suzuki, S., Kawasaki, H., Satoh, Y., Ohtomo, M., Hirai, M., Hirai, A., Hirai, S., Onoda, M., Matsumoto, M., Hinokio, Y., Akai, H., Craig, J., Larner, J. and Toyota, T. (1994) Urinary chiro-inositol excretion is an index marker of insulin sensitivity in Japanese type 2 diabetes. Diabetes Care, 17, 1465–1468. 56. Asplin, I., Galasko, G. and Larner, J. (1993) Chiro-inositol deﬁciency and insulin resistance: A comparison of the chiro-inositol and the myo-inositol containing insulin mediators isolated from urine, hemodialysate and muscle of control and type 2 diabetic subjects. Proc. Natl. Acad. Sci. USA, 90, 5924–5928. 57. Ortmeyer, H.K., Huang, L.C., Zhang, L., Hansen, B.C. and Larner, J. (1993) Chiro-inositol deﬁciency and insulin resistance II. Acute effects of D-chiro-inositol administration in streptozotocin-diabetic rats, normal rats given a glucose load and spontaneously insulin-resistant Rhesus monkeys. Endocrinology, 132, 646–651. 58. Larner, J., Allan, G., Kessler, C., Reamer, P., Gunn, R. and Huang, L.C. (1999) Phosphoinositol glycan-derived mediators and insulin resistance. Prospects for diagnosis and therapy. J. Basic and Clin. Physiol. and Pharm., 9, 127–137. 59. Kessler, C., Allan, G. and Larner, J. (1999) Effects of D-chiro-inositol (INS-1) on glycemic control and lipid proﬁles in a naive type 2 diabetic population. Late breaking Abs. Presidential Session Am. Diabetes Assn. Mtg., San Diego, California (1999). 60. Nestler, J.E., Jakubowicz, D.J., Reamer, P., Gunn, R. and Allan, G. (1999) Ovulatory and metabolic effects of d-chiro-inositol in the polycystic ovary syndrome. N. Eng. J. Med., 340, 1314–1320. 61. Reaven, G.M. (1988) Banting Lecture 1988. Role of insulin resistance in human disease. Diabetes, 37, 1595–1607. 62. Ortmeyer, H.K., Bodkin, N.L., Hansen, B.C. and Larner, J. (1995) In vivo d-chiro-inositol activates skeletal muscle glycogen synthase and inactivates glycogen phosphorylase in rhesus monkeys. J. Nutr. Biochem., 6, 499–503. 63. Bond, A., Smith, L., Hawkins, E., Green, D., Grollman, E., Sleevi, M. and Larner, J. (2001) D-chiroinositol speciﬁcally sensitizes H4IIE hepatoma cells (unpublished personal communication). 64. Apter, D., Butzow, T., Laughlin, G.A. and Yen, S.S. (1995) Metabolic features of polycystic ovary syndrome are found in adolescent girls with hyperandrogenism. J. Clin. Endocrinol. Metab., 80, 2966–2973. 65. Dunaif, A. and Finegood, D.T. (1996) Beta-cell dysfunction independent of obesity and glucose intolerance in the polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 81, 942–947. 66. Dunaif, A., Segal, K.R., Futterweit, W. and Dobrjansky, A. (1989) Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes, 38, 1165–1174. 67. Ehrmann, D.A., Sturis, J., Byrne, M.M., Karrison, T., Rosenﬁeld, R.L. and Polonsky, K.S. (1995) Insulin secretory defects in polycystic ovary syndrome. Relationship to insulin sensitivity and family history of non-insulin-dependent diabetes mellitus. J. Clin. Invest., 96, 520–527. 68. Legro, R.S., Kunselman, A.R., Dodson, W.C. and Dunaif, A. (1999) Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: A prospective, controlled study in 254 affected women. J. Clin. Endocrinol. Metab., 84, 165–169. 69. Ciaraldi, T.P., El-Roeiy, A., Madar, Z., Reichart, D., Olefsky, J.M. and Yen, S.S.C. (1992) Cellular mechanisms of insulin resistance in polycystic ovarian syndrome. J. Clin. Endocrinol. Metab., 75, 577–583. 70. Dunaif, A., Xia, J., Book, C.B., Schenker, E. and Tang, Z. (1995) Excessive insulin receptor serine phosphorylation in cultured ﬁbroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome. J. Clin. Invest., 96, 801–810. 71. Jahanfar, S., Eden, J.A., Warren, P., Seppala., M. and Nguyen, T.V. (1995) A twin study of polycystic ovary syndrome. Fertil. Steril., 63, 478–486. 72. DeFronzo, R.A. and Ferrannini, E. (1991) Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia and atherosclerotic cardiovascular disease. Diabetes Care, 14, 173–194. 73. Nestler, J.E. (1997) Role of hyperinsulinemia in the pathogenesis of the polycystic ovary syndrome, and its clinical implications. Sem. Reprod. Endocrinol., 15, 111–122.

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74. Nestler, J.E. and Jakubowicz, D.J. (1996) Decreases in ovarian cytochrome P450c17a activity and serum free testosterone after reduction in insulin secretion in women with polycystic ovary syndrome. N. Eng. J. Med., 335, 617–623. 75. Nestler, J.E. and Jakubowicz, D.J. (1997) Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17a activity and serum androgens. J. Clin. Endocrinol. Metab., 82, 4075–4079. 76. Dahlgren, E., Janson, P.O., Johansson, S., Mattson, L.-A., Lindstedt, G., Crona, N., Knutsson, F., Lundberg, P.-A. and Oden, A. (1992) Women with polycystic ovary syndrome wedge resected in 1956 to 1965: A long-term follow-up focusing on natural history and circulating hormones. Fertil. Steril., 57, 505–513. 77. Ehrmann, D.A., Barnes, R.B., Rosenﬁeld, R.L., Cavaghan, M.K. and Imperial, J. (1999) Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome. Diabetes Care, 22, 141–146. 78. Legro, R.S., Finegood, D. and Dunaif, A. (1998) A fasting glucose to insulin ratio is a useful measure of insulin sensitivity in women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 83, 2694–2698. 79. Rebuffe-Scrive, M., Cullberg, G., Lundberg, P.A., Lindstedt, G. and Bjorntorp, P. (1989) Anthropometric variables and metabolism in polycystic ovarian disease. Horm. Metab. Res., 21, 391–397. 80. Wild, R.A. (1995) Obesity, lipids, cardiovascular risk, and androgen excess. Am. J. Med., 98, 27S–32S. 81. Wild, R.A., Alaupovic, P. and Parker, I.J. (1992) Lipid and apolipoprotein abnormalities in hirsute women. I. The association with insulin resistance. Amer. J. Obstet. Gynecol., 166, 1191–1196. 82. Bjorntorp, P. (1996) The android woman – a risky condition. J. Intern Med., 239, 105–110. 83. Talbott, E.O., Guzick, D.S., Sutton-Tyrrell, K., McHugh-Pemu, K.P., Zborowski, J.V., Remsberg, K.E. and Kuller, L.H. (2000) Evidence for association between polycystic ovary syndrome and premature carotid atherosclerosis in middle-aged women. Arterioscler. Thromb. Vasc. Biol., 20, 2414–2421. 84. Iuorno, M.J., Jakubowicz, D.J., Dillon, P., Eisner, J.R., Gunn, R.D., Allan, G. and Nestler, J.E. (2000) Lean women with the polycystic ovary syndrome: d-chiro-inositol reduces serum insulin and androgens, improves ovulation, and beneﬁcially affects syndrome X by decreasing blood pressure and serum triglycerides. Program & Abstracts of the 82nd Annual Meeting of the Endocrine Society, 400. 85. Larner, J. and Huang, L.C. (1999) Identiﬁcation of a novel inositol glycan signaling pathway with signiﬁcant therapeutic relevance to insulin resistance: An insulin signaling model using both tyrosine kinase and G-proteins. Diabetes Rev., 7, 217–231. 86. (a) Alvarez, J.F., Sanchez-Arias, J.A., Guadano, A., Estevez, F., Varela, I., Feliu, J.E. and Mato, M. (1999) Transport in isolated rat hepatocytes of the oligosaccharide that mimics insulin action. Biochem. J., 274, 369–374. (b) Ostlund, R.E., Jr., Seemayer, R., Gupta, S., Kimmel, R., Ostlund, E.L. and Sherman, W.R. (1996) A stereospeciﬁc myo-inositol/D-chiro-inositol transporter in Hep G2 liver cells. J. Biol. Chem., 271, 10073–10078. 87. Pak, Y., Huang, L.C., Lilley, K.J. and Larner, J. (1992) In vivo conversion of [3H]myo-inositol to [3H]chiro-inositol in rat tissues. J. Biol. Chem., 267, 16904–16910. 88. Pak, Y., Paule, C.R., Bao, Y.-D., Huang, L.C. and Larner, J. (1993) Insulin stimulates the biosynthesis of chiro-inositol containing phospholipids in a rat ﬁbroblast line expressing the human insulin receptor. Proc. Natl. Acad. Sci. USA, 90, 7759–7763. 89. Pak, Y., Piccariello, T., Farese, R.V. and Larner, J. (1998) In vivo chiro-inositol metabolism in the rat: A defect in chiro-inositol synthesis from myo-inositol and an increased incorporation of chiro[3H]inositol into phospholipid in the type 2 diabetic Goto-Kakizaki (G.K.) Rat. Mol. Cells, 8, 301–309.

14.

GLUCAGON-LIKE PEPTIDE-1, EXENDIN AND INSULIN SENSITIVITY ANDREW A. YOUNG Amylin Pharmaceuticals Inc., 9373 Towne Centre Drive, San Diego, CA 92121, USA

INTRODUCTION Glucagon-like peptide-1 (GLP-1), secreted from intestinal mucosa in response to nutrients, has been proposed as a therapy for treatment of type 2 diabetes, type 1 diabetes and obesity. Exendin-4 from the salivary secretions of the Gila monster shares many biological actions with GLP-1, but has superior pharmaceutical properties. A part of the rationale proposed for use of this class of agent has been an insulin-sensitizing effect. This chapter reviews preclinical and clinical evidence for the proposition that GLP-1 (either exogenous peptide or the endogenous hormone), GLP-1 analogs, or exendin-4 may alter insulin sensitivity. Most data have been obtained using GLP-1 and exendin-4. This chapter will focus upon studies using those peptides. In addressing this proposition, it is necessary to understand or deﬁne what might be meant by “insulin sensitivity” and/or “insulin resistance.” In a broad whole-organism context, insulin sensitivity is a measure of the concentration of extracellular insulin required to exert a deﬁned insulin-dependent effect, all other factors being equal. Even with this broad deﬁnition, it is apparent that as many “insulin sensitivities” exist as do responses that depend upon its extracellular concentration. Hence, “insulin sensitivity” or “insulin resistance” could, for example, be quantiﬁed in relation to its effects to lower plasma glucose, promote peripheral glucose uptake, stimulate non-oxidative glucose disposal or muscle glycogen synthesis, to promote lipogenesis, to inhibit lipolysis, to inhibit gluconeogenesis, to promote renal sodium retention, to increase vascular conductance, to stimulate amino acid uptake, to inhibit islet hormone secretion, etc. The importance of tissue-speciﬁc insulin sensitivities may be recognized when we consider the etiopathogenesis of “insulin resistance syndrome” (Syndrome-X). This condition of reduced peripheral glucose disposal in the face of increasing adiposity is likely to reﬂect unequal insulin action in different tissues. That is, there may be a comparatively greater decrement in insulin’s effect to promote glucose storage while its effect to promote lipogenesis and inhibit lipolysis in fat may be comparatively maintained (Yki-Järvinen et al., 1987). Under these circumstances, hyperinsulinism stemming from excessive circulating carbohydrate (due to slowed rates of storage) will result in an excess of insulin secretion and, hence, insulin effect in comparatively insulin-sensitive fat. In relation to insulin’s multiple actions in the study of diabetes the insulin-dependent response most commonly considered is glucose disposal in peripheral tissues, and unless made otherwise explicit, glucose disposal should be considered the response being discussed. Effects of GLP-1 and exendin-4 on insulin sensitivity can be viewed from a wide perspective, wherein improvements in glycemic control could stem not only from direct changes in insulin-sensitive tissues, but could also include indirect effects secondary to better glycemic control resulting, for example, from ampliﬁed insulin secretion. The latter effect of improved 235

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glycemic control per se has been interpreted as a reversal of “glucose toxicity,” and is apparent with many antidiabetic therapies, including those not typically classed as “insulin sensitizing.” The end result is manifest as an apparent increase in insulin sensitivity in diverse study designs, ranging from humans to rodents. Thus, both true insulin-sensitizing effects and the phenomenon of reversal of glucose toxicity need to be taken into consideration when addressing effects speciﬁc to GLP-1, its analogs, and exendin-4, as is discussed below. Effects of GLP-1 and exendin-4 on insulin sensitivity can also be viewed more narrowly from the perspective of individual insulin-responsive tissues. From the point of view of nutrient storage, insulin-responsive tissues include only muscle, fat and liver. Direct effects of GLP-1 and exendin-4 on these tissues will be examined. Most data have been obtained using GLP-1 and exendin-4. This chapter will focus upon studies using these peptides.

BIOLOGY OF GLP-1 AND EXENDIN-4 GLP-1 GLP-1 is produced by the tissue-speciﬁc cleavage of proglucagon into several subpeptides in L-cells in the mucosa of ileum and other gut segments. A 37 amino acid fragment, proglucagon 72–107 was originally reported as a candidate hormone (Orskov et al., 1986), but it was subsequently recognized that the secreted biologically active peptide was shorter and amidated, corresponding to proglucagon 78–107 (Holst et al., 1987). Today, the term “GLP-1” generally refers to the molecule formerly named GLP-1 [7-36]amide (or truncated GLP-1), which is the predominant secreted form. Other cleavage products of proglucagon at this site include GLP-2 and glicentin (Orskov et al., 1986, 1989), while a distinct tissuespeciﬁc cleavage of proglucagon in pancreatic A-cells results in glucagon. GLP-1 is released into the circulation following meals, predominantly in response to fat and carbohydrate in the ileum (Göke et al., 1993b). Interest in developing GLP-1 as an antidiabetic agent followed its identiﬁcation as an insulin secretagogue (Habener, 1997; Mojsov et al., 1987; Holst et al., 1987) or insulinotropic agent which, unlike sulfonylurea drugs, stimulated insulin secretion only in the presence of normal or elevated plasma glucose concentrations (Weir et al., 1989; Göke et al., 1993c). The glucose dependence of its insulinotropic effect promised to confer some protection from the potential side effect of hypoglycemia. Unfortunately, GLP-1 exhibits a half-life of ~5 min in man (Orskov et al., 1993), principally due to its rapid degradation to GLP-1[9-36]NH2 by the enzyme, dipeptidyl peptidase IV (Hansen et al., 1999; Knudsen and Pridal, 1996). This rapid degradation of GLP-1 has impeded its development as an antidiabetic agent, and forced alternate approaches such as the development of more stable analogs (Knudsen et al., 1999; Deacon et al., 1997; Chou et al., 1997; Gallwitz et al., 2000) or the inhibition of dipeptidyl peptidase IV activity, to enhance the action of endogenous GLP-1 (Balkan et al., 1997; Deacon et al., 1998; Stockel-Maschek et al., 2000; Tang-Christensen et al., 1998; Niklasson et al., 1998; Demuth et al., 1993; Steinmetzer et al., 1993). Exendin-4 The identiﬁcation of the bioactivity of exendin began with the observation that venom from Gila monsters (see Figure 1) was active in pancreatic acinar cells (Raufman et al., 1982). This

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Figure 1 The Gila monster, Heloderma suspectum. (Photograph courtesy of Dr Mark Seward.)

activity was at least partly attributable to exendin-3 (in Heloderma horridum), a 39 amino acid peptide with some structural similarity to peptides in the VIP/secretin family (Raufman et al., 1991; Raufman, 1996). The truncated peptide, exendin[9-39] was shown to antagonize this activity (Raufman et al., 1991). Exendin-4, the homolog to exendin-3 obtained from Heloderma suspectum (Eng et al., 1992), was shown to bind to acinar cells at sites named at the time “exendin receptors” (Eng, 1992). GLP-1, which was 53% identical in sequence to the N-terminal part of exendin-4 (Rai et al., 1993), was also found to bind to the so-called exendin receptor (Raufman et al., 1992). The “exendin” receptor was eventually cloned and characterized and became known as the GLP-1 receptor (Thorens et al., 1993; Wheeler et al., 1993; Graziano et al., 1993). Full-length exendins were found to be GLP-1 receptor agonists, and truncated exendins were found to be antagonists at that receptor (Thorens et al., 1993; Göke et al., 1993a; Fehmann et al., 1994; Schepp et al., 1994). The [9-39] fragment of exendin, one of these antagonists, has often been mislabeled “exendin” in the literature, generating confusion with the full-length molecule. In the present chapter, “exendin” refers to the full length [1-39] sequence. Even though, in an N-terminal alignment, exendin-4 has 53% sequence identity with human GLP-1, it is not the Gila monster homolog of mammalian GLP-1. These peptides are the products of two distinct genes. Exendin-4 is found in the salivary glands, and not in the gut, of the Gila monster. In the gut of the lizard is found another peptide 83% identical to human GLP-1 (there being two genes) that instead appears to be the lizard homolog of GLP-1 (Chen and Drucker, 1997). This ﬁnding raises the question as to the physiological role of exendin in the Gila monster. First, although originally described as a component of the venom, there appears to be no distinction between venom and salivary glands in this lizard (Bogert and Martin del Campo, 1956). Thus, exendin may properly be described as a salivary secretion of the Gila monster. Second, given that the prey of the Gila monster is eggs and nestlings that are found, not hunted (Beck, 1989), and given that no animal deaths have resulted from its administration at any dose in any species, including dosing at 300,000 times the antidiabetic dose (Noveroske et al., 1999), it is unlikely that exendins serve as venoms in Gila monsters. Instead there are aspects of feeding behavior and economics of nutrient assimilation in this animal that might predict a role in signaling meal ingestion. With the idea that

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exendin-4 might act within the lizard, rather than acting upon its prey, plasma exendin concentrations were serially measured in Gila monsters during their ﬁrst meal after a fast of several months. Very high plasma concentrations were found within minutes of beginning chewing (Young et al., 1999b), as shown in Figure 2. The rapidity of elevation of plasma exendin-4 concentration suggested a direct secretion from salivary glands into the blood, and raised the possibility that the peptide had an endocrine role in the lizard. Gilas belong to a class of infrequently feeding animals in which it is often more energetically favorable to allow the gut to atrophy between meals and to rapidly regrow in response to meals (Secor and Diamond, 1998). It is possible that exendins secreted in response to chewing could be a feedforward signal that primed the gut to receive, digest and absorb an infrequent meal. While such a role of exendin as a salivary endocrine hormone remains to be afﬁrmed in Gila monsters, its trophic effects on gut-derived tissues in mammals (Zhou et al., 1999; Xu et al., 1999; Zhou and Egan, 2000; Stoffers et al., 2000a; Tourrel et al., 2001) is consistent with this role. Pharmacokinetics of GLP-1 and Exendin-4 The shortness of the duration in plasma and effect of GLP-1, as explained above, has impeded its development as an antidiabetic agent. In contrast, the effects of exendin-4 in diabetic mice were found to be prolonged (Eng, 1996). Sensitive 2-site assays speciﬁc for each, full-length exendin-4 and full-length GLP-1, have been recently developed and made commercially available, allowing the pharmacokinetics of each molecule to be compared (Petrella et al., 1999; Parkes et al., 2001; Baron, 2001). In contrast to GLP-1, exendin circulates predominantly as the intact molecule. Studies of its pharmacokinetics in rats functionally nephrectomized by ligation of the renal artery indicate that renal ﬁltration accounts for ~80% of its clearance (Chen et al., 1999). Exendin-4 clearance is only ~10% of that of GLP-1 (Parkes et al., 2001), which is rapidly degraded by proteases including dipeptidyl peptidase IV and neutral

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endopeptidase. Thus, via all routes and modes of administration, the presence of exendin-4 far outlasts that of GLP-1, as is shown for intravenous administration in Figure 3. The pharmaceutic beneﬁts of extended kinetics, such as those observed with exendin-4, are profound. For example, an 11-fold extension of t1/2 for exendin-4 vs. GLP-1 translates to a 211-fold (2048-fold) reduction in GLP-1 concentration in the same time that exendin-4 concentration is diminished by 50%. This calculation agrees well with the observed ~3000-fold GLP-1

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Figure 3 Decay of plasma concentrations of GLP-1 and exendin-4 after intravenous bolus injection (see Parkes et al., 2001).

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increase in glucose-lowering potency of exendin-4 vs. GLP-1 measured over 1 h in db/db mice (Young et al., 1998, 1999).

ACTIONS OF GLP-1 AND EXENDIN-4 General Antidiabetic Actions A considerable literature now exists supporting general antidiabetic effects of both GLP-1 (Byrne and Göke, 1996; Creutzfeldt et al., 1996; D’Alessio et al., 1994; Gutniak et al., 1992a; Juntti-Berggren et al., 1996; Nauck et al., 1993, 1998; Rachman et al., 1997; Schirra et al., 1998; Todd et al., 1997, 1998; Toft-Nielsen et al., 1999; Vella et al., 2000a; Willms et al., 1998) and exendin-4 (Kolterman et al., 1999; Buse et al., 2000; Dupre et al., 2001; Edwards et al., 2000, 2001; Fineman et al., 2000; Kolterman et al., 2000a; Levy et al., 2000a,b; Maksoud et al., 2000) in humans. These afﬁrm antidiabetic and glucose-lowering effects of GLP-1 (Ahren, 1995; Ahren and Pacini, 1999; Scrocchi et al., 1996), the GLP-1 analog NN2211 (Rolin et al., 2000) and exendin-4 (Eng, 1996; Greig et al., 1999; Young et al., 1998, 1999; Nolan et al., 2000; Baggio et al., 2000) identiﬁed in mice. GLP-1 (Aspedlund et al., 2000; Dachicourt et al., 1997; De Ore et al., 1997; Hendrick et al., 1993), GLP-1 analogs (Sturis et al., 2000) and exendin-4 (Aspedlund et al., 2000; Bhavsar et al., 2001; Gedulin et al., 2001; Greig et al., 1999; Xu et al., 1999; Young et al., 1999) are also effective in rats. Glucoselowering effects have also been reported in dogs for GLP-1 (Widmaier et al., 1991), in minipigs for a GLP-1 analog (Ribel et al., 2000), and in monkeys for exendin-4 (Bodkin et al., 1998; Young et al., 1999).

MECHANISM OF ANTIDIABETIC ACTIONS Insulinotropic Effects The ﬁrst described biological action of GLP-1, and that which prompted its investigation as an antidiabetic therapy (Habener, 1997), was its ampliﬁcation of nutrient-stimulated insulin secretion. This “insulinotropic” effect of GLP-1, its analogs, and exendin-4 has been observed in a number of systems, including isolated islets (Siegel et al., 1992; Zawalich et al., 1993; Holz et al., 1928; Hargrove et al., 1996; Parkes et al., 1998; Fridolf and Ahren, 1991; Sreenan et al., 2000), the isolated perfused pancreas (Fehmann et al., 1995; Masiello et al., 1995; Weir et al., 1989; Mojsov et al., 1987; Gutniak et al., 1996; Malhotra et al., 1992), or the whole organism, including rats (Hargrove et al., 1996; Dachicourt et al., 1997; Shen et al., 1998; Parkes et al., 2001), dogs (Kawai et al., 1990), minipigs (Ribel et al., 2000) and humans (Nauck et al., 1996; Porksen et al., 1997, 1998; Byrne et al., 1998; Edwards et al., 1998). The effect of GLP-1, its analogs, and exendin-4 to amplify insulin secretion is present only during euglycemia and hyperglycemia, and generally not during hypoglycemia, and hence has been described as a “glucose-dependent” insulinotropic effect (Habener, 1997; Weir et al., 1989; Heimesaat et al., 1999; Göke et al., 1993c; Parkes et al., 2001). The suppression of GLP-1- and exendin-stimulated insulin secretion at low glucose concentrations is a feature both, in isolated preparations (Weir et al., 1989; Göke et al., 1993c; Parkes et al., 2001) as well as in the intact individual (Parkes et al., 2001; Heimesaat et al., 1999). The glucose dependence of this insulinotropic effect has potential therapeutic advantages, in that it is predicted to

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reduce the risk of hypoglycemia. This is in contrast to effects of non-glucose-dependent insulin secretagogues, such as sulfonylurea agents (Hargrove et al., 1996). This potential advantage initially drove drug development efforts in the area of GLP-1 and its analogs. Although reactive hypoglycemia has been seen in the setting of a hyperglycemic clamp with both GLP-1 (Edwards et al., 1998) and exendin-4 (Egan et al., 1999) in non-diabetic (insulinsensitive) subjects, this is not a feature seen in subjects with type 2 diabetes (Vilsboll et al., 2001; Egan et al., 1999). Hypoglycemia has not been observed in patients with type 2 diabetes when GLP-1 or exendin-4 is administered outside the context of a hyperglycemic clamp, in subjects not treated with sulfonylurea insulin secretagogues. Glucagonostatic Effects Exaggerated secretion of glucagon, especially in response to protein-containing meals (Müller et al., 1970), has been implicated in the excess gluconeogenesis and propensity to ketoacidosis in diabetes characterized by lack of insulin (Unger, 1971). A proposed beneﬁt of GLP-1 therapy in diabetes is its glucagonostatic effect, the suppression of inappropriate glucagon secretion (Creutzfeldt et al., 1996; Ritzel et al., 1995). A glucagonostatic effect of exendin-4 has also been observed in type 2 diabetic animal models (Gedulin et al., 1999) and in human type 2 diabetes (Kolterman et al., 2000b), where the dose-response corresponded with exendin’s other antidiabetic actions. In the isolated perfused pancreas, exendin-4 inhibited the glucagon response to arginine in pancreata from normal rats, but not in insulin-deﬁcient (STZ) pancreata in which an insulinotropic effect was precluded by the absence of ␤-cells. It may thus be the case that the glucagonostatic effect of exendin-4 (and of GLP-1) is secondary to a paracrine effect of ␤-cell products to inhibit ␣-cell secretion (Rodriguez-Gallardo et al., 2000). Satiogenic Effects Centrally delivered GLP-1 acutely decreases food and/or water intake in rats and other species (Turton et al., 1996; Tang-Christensen et al., 1996; Navarro et al., 1996; Rodriguez de Fonseca et al., 1997; Furuse et al., 1997a,b; Donahey et al., 1998; Wang et al., 1998). Peripheral (Szanya et al., 2000) and intracerebroventricular exendin-4 evoked a similar effect, but with much greater potency (Bhavsar et al., 1998a,b). However, the role of GLP-1 as a physiologically relevant peripheral satiety agent has been disputed on the basis that the inhibition of food intake following ICV injection was associated with conditioned taste aversion (i.e. represented a “sickness” rather than a “satiety” response) ( Jensen et al., 1998; Thiele et al., 1997, 1998a; Van Dijk and Thiele, 1997). Others have discounted this argument by ﬁnding that the effect of GLP-1 to induce conditioned taste aversion is dissociable from effects to reduce food and water intake (McMahon and Wellman, 1997, 1998; Tang-Christensen et al., 1998). The concept of GLP-1 as an endogenous meal-related satiety signal has been questioned following observations that its acute anorectic effect rapidly faded (Donahey et al., 1998). The concept further suffered from the failure of peripherally administered (as opposed to ICV injected) GLP-1 to inhibit food intake in rats (Bhavsar et al., 1998a; Navarro et al., 1996; Tang-Christensen et al., 1996; Turton et al., 1996), and by the observation of no apparent change in body weight or ingestive behavior in GLP-1 receptor knockout mice (Scrocchi et al., 1996; Scrocchi and Drucker, 1998). On the other hand, a potential satiogenic role of circulating GLP-1 has been supported in humans in which peripherally-infused GLP-1 increased sensations of satiety and fullness, and

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decreased energy intake (Flint et al., 1998, 2001; Gutzwiller et al., 1999a,b). A physiologic role of endogenous GLP-1 was further supported by a report that exendin[9-39], a GLP-1 antagonist, enhanced food intake in non-fasted animals (Turton et al., 1996). This result supported the interpretation that the effect of the antagonist was a disinhibition of a tonic GLP-1 signal that was therefore physiologically relevant. The inference of physiological relevance of GLP-1 was further supported by an increase in body weight following continuous administration of exendin[9-39]. It is however worth mentioning that other workers have not been able to replicate an increase in food intake during treatment with exendin[9-39] (Thiele et al., 1998b; Bhavsar et al., unpublished). Changes in HbA1c over 6 weeks were highest in ad lib fed controls and the pair-fed controls, but decreased in exendin-4-treated rats. Changes in plasma glucose showed a similar pattern, and in the ﬁnal hour of a 3-h clamp protocol, glucose infusion rate in exendin-4-treated rats tended to be higher than in pair fed (105%) and ad lib fed (20%) controls, respectively (10.14 1.43, n 5; 8.46 0.87, n 4; 4.93 2.02 mg/kg/min, n 3). Another index of insulin sensitivity, plasma lactate concentration (discussed below), differed signiﬁcantly between treatment groups, and was lowest in exendin4-treated rats. Thus, exendin-4 treatment was associated with improvement in glycemic indices and in insulin sensitivity that was partly, but not fully, matched in controls fed the same amount of food. Because the energetics of urinary glucose loss can confound interpretation of experiments in which glycosuria is ameliorated, as in the example just cited, the experiment was repeated using non-diabetic Fatty Zucker (fa/fa) rats (Gedulin et al., unpublished). The effect of exendin-4 on whole-body insulin sensitivity, as measured by glucose infusion rate in a clamp, was even more striking; exendin-4 therapy increased whole-body insulin sensitivity by 2.39fold (10.25 vs. 4.29 mg/kg/min), while the effect of caloric restriction per se, in the pair-fed cohort, was a 1.8-fold increase (7.37 vs. 4.29 mg/kg/min). These results implied that improvements in metabolic control with exendin-4 in both Diabetic (ZDF) and non-diabetic (fa/fa) Zucker rats were only partly due to caloric restriction. Each study showed evidence for an insulin-sensitizing effect of chronic exendin-4 that was separate from an effect of caloric restriction. In a similar pair-feeding study using the GLP-1 analog, NN2211, only ~50% of the glycemic beneﬁt in ZDF rats could be accounted for by reduced food intake. That is, there was a glycemic beneﬁt that required accounting for by as-yet undeﬁned mechanisms that were separate from caloric restriction (Sturis et al., 2000).

Effects on Gastric Emptying GLP-1 has been shown to slow emptying of the stomach in rodents (Young et al., 1996) and in humans (Wettergren et al., 1993; Dupre et al., 1995, 1997; Willms et al., 1996; Schirra et al., 1997). The importance of this action in glucose control, vs. an insulinotropic effect, for example, was recognized when Dupre et al. (1997) described its post-prandial beneﬁt in insulindeﬁcient patients. The signiﬁcance of regulation of gastric emptying on glycemic excursions has also been afﬁrmed in insulin-replete subjects (Schirra et al., 1998; Nauck et al., 1997). Some authors propose that the gastric effect predominates in post-prandial glucose control (Nauck et al., 1997). In rats, exendin-4 is one of the most potent modulators of gastric emptying identiﬁed to the present ( Jodka et al., 1998). Its potency for this action in rodents is reﬂected also in human studies (Young et al., 1999a; Baron et al., 2000; Edwards et al., 2000, 2001; Kolterman et al., 2000b). The effects of both GLP-1 and exendin-4 on gastric emptying are glucose dependent, in that they are overridden during insulin-induced hypoglycemia ( Jodka et al., 2000).

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EVIDENCE OF INSULIN-SENSITIZING EFFECTS OF GLP-1 AND EXENDIN-4 Insulin sensitivity, insulin resistance or changes in those parameters, can be measured using a variety of techniques, including tolerance tests (glucose- and insulin-tolerance), glucoregulatory control system modeling (e.g. the “minimal model” (Bergman et al., 1985)), steady-state parametrization (glucose/insulin infusion), and various clamp protocols, particularly the hyperinsulinemic euglycemic clamp (DeFronzo et al., 1979). Several authors have studied potential effects of GLP-1 or exendin-4 on different measures of insulin sensitivity, including with glucose clamp protocols (Ahren et al., 1997; Ahren and Pacini, 1999; D’Alessio et al., 1994, 1995; Duvigneau et al., 2000; Egan et al., 1999; Giacca et al., 1997; Gutniak et al., 1992b; Hargrove et al., 1995, 1996; Jodka et al., 1999; Mizuno et al., 1997; Orskov et al., 1996; Ryan et al., 1998; Sandhu et al., 1999; Shalev et al., 1998; Todd et al., 1997, 1998; Wahlin-Boll et al., 1982; Toft-Nielsen et al., 1996; Vilsbøll et al., 1999; Young et al., 1999). Effects on insulin sensitivity of both acute and chronic administration have been explored. Acute Effects on Whole-Body Insulin Sensitivity Treatment with GLP-1 appears not to acutely alter whole-body insulin sensitivity in humans (Castellano et al., 1998; Ahren et al., 1997; Ryan et al., 1998; Vella et al., 2000b). These ﬁndings are consistent with observations of unchanged glucose infusion rate following acute dosing of exendin-4 during euglycemic hyperinsulinemic clamps in rats (Gedulin et al., unpublished). Chronic Effects on Whole-Body Insulin Sensitivity In contrast to the absence of an acute effect on indices of insulin sensitivity, chronic (4-week) administration of GLP-1 by osmotic pump into Otsuka rats resulted in a large (88%) increase in insulin sensitivity (Mizuno et al., 1997). Comparable dose-dependent improvements in glucose infusion rate in clamps were observed following 6 weeks of twice-daily treatment with exendin-4 in ZDF rats (Young et al., 1999), shown in Figure 4. Even more profound increases in whole-body insulin sensitivity were observed in nondiabetic Fatty Zucker ( fa/fa) rats in which insulin resistance was reﬂected by mild hyperglycemia, but in which plasma glucose concentrations were not sufﬁciently high to cause glycosuria and urinary caloric loss. Hyperinsulinemic euglycemic clamp studies (insulin ~1 nM) were performed after 6 weeks of twice-daily treatment (3 g) and 24 h after the last injection of exendin-4. Insulin sensitivity, measured as the mean glucose infusion rate required to maintain euglycemia from 60–180 min of the clamp, was 2.3-fold higher in the exendin-treated group than in saline-treated controls (10.31 0.56 vs. 4.43 0.76 mg/kg/min; p 0.001). Glucose infusion rate in a pair-fed group, fed the same quantity of food as was consumed by exendin-treated rats, was intermediate (7.91 0.56 mg/kg/min; p 0.05 vs. exendin; p 0.01 vs. saline controls) (Gedulin et al., unpublished). Thus chronic (but not acute) administration of exendin-4 and GLP-1 appears to be associated with increases in insulin sensitivity. These changes were at least as great as those reported with other therapies, including thiazolidinediones, biguanides, sulfonylureas and insulin itself. There is an association between elevations of plasma lactate concentration and insulin resistance. The reason for this association, apparent in humans (Lovejoy et al., 1992; Vettor et al., 1997) as well as rats, is unclear. Fat may be involved (Coppack et al., 1996). A major

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determinant of insulin resistance, the inability of muscle to store transported glucose as glycogen (Bogardus et al., 1984; Young et al., 1988; Ortmeyer et al., 1993), may be contributory, since transported glucose that is not stored and passes through the glycolytic pathway emerges as lactate if it cannot be oxidized. Studies in LA/N corpulent vs. lean rats using the lactate clamp (Rink et al., 1994; Gedulin et al., 1994) identiﬁed a ~4-fold higher lactate release (rather than reduced lactate clearance) to explain the hyperlactemia in insulin-resistant animals. Effects of 6 weeks exendin-4 therapy on fasting plasma lactate concentration are of interest in this regard. Plasma lactate concentration before and during the clamp procedure was

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Figure 6 Published relative changes in indices of insulin sensitivity in preclinical and clinical studies following chronic therapy with different classes of antidiabetic agents. Studies contributing to each bar are numbered as follows: 1. Boyd, K., Rogers, C., Boreham, C. et al. Diabetes Res 19, 69–76 (1992). 2. Schnack, C., Prager, R.J., Winkler, J. et al. Diabetes Care 12, 537–543 (1989). 3. Groop, L., Widen, E., Franssila-Kallunki, A. et al. Diabetologia 32, 599–605 (1989). 4. Jeng, C.Y., Hollenbeck, C.B., Wu, M.S. et al. Diabet Med 6, 303–308 (1989). 5. Leblanc, H., Thote, A., Chatellier, G. et al. Diabete Metab 16, 93–97 (1990). 6. Simpson, H.C., Sturley, R., Stirling, C.A. et al. Diabet Med 7, 143–147 (1990). 7. Matthews, D.R., Hosker, J.P. & Stratton, I. Diabetes Res Clin Pract 14 (Suppl 2), S53–S59 (1991). 8. Vestergaard, H., Weinreb, J.E., Rosen, A.S. et al. J Clin Endocrinol Metab 80, 270–275 (1995). 9. Suter, S.L., Nolan, J.J., Wallace, P. et al. Diabetes Care 15, 193–203 (1992). 10. Kraegen, E.W., James, D.E., Jenkins, A.B. et al. Metabolism 38, 1089–1093 (1989). 11. Lee, M.K. & Olefsky, J.M. Metabolism - Clinical and Experimental 44, 1166–1169 (1995). 12. Khoursheed, M., Miles, P.D., Gao, K.M. et al. Metabolism 44, 1489–1494 (1995). 13. O’Rourke, C.M., Davis, J.A., Saltiel, A.R. et al. Metabolism Clinical and Experimental 46, 192–198 (1997). 14. Orskov, L., Holst, J.J., Moller, J. et al. Diabetologia 39, 1227–1232 (1996). 15. Ahren, B., Larsson, H. & Holst, J.J. Journal of Clinical Endocrinology and Metabolism 82, 473–478 (1997). 16. Mizuno, A., Kuwajima, M., Ishida, K. et al. Metabolism 46, 745–749 (1997). 17. Maggs, D.G., Buchanan, T.A., Burant, C.F. et al. Ann Intern Med 128, 176–185 (1998). 18. Hirshman, M.F. & Horton, E.S. Endocrinology 126, 2407–2412 (1990). 19. Miles, P.D., Romeo, O.M., Higo, K. et al. Diabetes 46, 1678–1683 (1997). 20. Young AA, Gedulin BR, Bhavsar S et al. Diabetes 48, 1026–1034 (1999).

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dose-dependently reduced by prior treatment with exendin-4. This effect, representing up to a 42% reduction in mean lactate, appeared primarily due to a reduction in pre-clamp (basal) lactate concentration, such that values were lowered toward those more typical for normal insulin-sensitive rats (see Figure 5). General Effects of Antidiabetic Therapy on Measures of Insulin Sensitivity Insulin therapy alone increased insulin sensitivity 10–39% in humans (Groop et al., 1989; Boyd et al., 1992; Jeng et al., 1989). Thiazolidinediones are reported to increase insulin sensitivity between 3% and 55% (median 32%) (Khoursheed et al., 1995; O’Rourke et al., 1997; Lee and Olefsky, 1995; Suter et al., 1992; Kraegen et al., 1989; Maggs et al., 1998; Miles et al., 1997). Metformin is reported to increase it by 11% (Boyd et al., 1992) to 32% (Groop et al., 1989) while sulfonylureas range from no effect (Leblanc et al., 1990; Matthews et al., 1991) to a 42% increase (median 26%) (Hirshman and Horton, 1990; Boyd et al., 1992; Vestergaard et al., 1995; Simpson et al., 1990; Jeng et al., 1989). These literature ﬁndings are graphed in Figure 6. It thus appears that it may be difﬁcult to assign any effects that GLP-1, its analogs, or exendin-4 may have on measures of insulin sensitivity to a direct effect vs. an indirect effect, secondary to improvement in glycemic control. In an effort to distinguish between these possibilities, the literature regarding direct effects of these agents on insulin-sensitive tissues is thus reviewed.

DIRECT EFFECTS IN INSULIN-SENSITIVE TISSUES Several authors have studied direct effects of GLP-1 or exendin-4 in insulin-responsive tissues, including muscle (Alcantara et al., 1997; Freyse et al., 1999; Furnsinn et al., 1995; Hansen et al., 1998; Luque et al., 1997; Morales et al., 1997; O’Harte et al., 1997; Pittner et al., 2000; Puente et al., 1997; Sandhu et al., 1999; Valverde and Villanueva-Penacarrillo, 1996; Villanueva-Penacarrillo et al., 1994), fat (Egan et al., 1994; Marquez et al., 1997, 1998; Miki et al., 1996; Montrose-Raﬁzadeh et al., 1997a,b; Perea et al., 1997; Pittner et al., 2000; Puente et al., 1997; Valverde and Villanueva-Penacarrillo, 1996; Villanueva-Penacarrillo et al., 1999; Wang et al., 1997a), and liver (Alcantara et al., 1997; Idris et al., 2001; Lopez-Delgado et al., 1997, 1998; Marquez et al., 1998; Morales et al., 1997; Nakagawa et al., 1996; Puente et al., 1997; Valverde and Villanueva-Penacarrillo, 1996; Villanueva-Penacarrillo et al., 1999). Muscle Several publications describe a direct effect of GLP-1 to stimulate glucose incorporation into glycogen in skeletal muscle or muscle-derived tissue (e.g. myotubes); most of these publications are from one laboratory (Alcantara et al., 1997; Morales et al., 1997; Puente et al., 1997; Valverde and Villanueva-Penacarrillo, 1996; O’Harte et al., 1997; Luque et al., 1997). Other evidence from the same laboratory supports a direct effect of GLP-1 in skeletal muscle which results in a doubling of GLUT-4 expression (Puente et al., 1997), and in other insulinsensitive tissues, a change in inositol second messenger expression that mimics that observed with insulin (Villanueva-Penacarrillo et al., 1999). The increase in peripheral glucose disposal reported in dogs following acute GLP-1 infusion supports such an effect (Sandhu et al., 1999).

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In opposition to these ﬁndings, others report no direct effect, or minimal effect, of GLP-1 on glucose incorporation into glycogen in soleus strips (Furnsinn et al., 1995; Pittner et al., 2000; Hansen et al., 1998), and no direct effect of GLP-1 on glucose disposal, metabolic clearance rate, or conversion of alanine to glucose (Freyse et al., 1999). Similarly, as is shown in Figure 7, there was no detectible effect of exendin-4 on glycogen metabolism in isolated soleus muscle (Pittner et al., 2000). In support of these negative observations, no GLP-1 receptor mRNA has been detected in muscle, fat or liver (Yamato et al., 1997; Bullock et al., 1996; Wei and Mojsov, 1995; Dunphy et al., 1998). Fat At least four different groups have investigated the potential for direct effects of GLP-1, GLP-1 agonists and exendin-4 in fat cells (Egan et al., 1994; Marquez et al., 1997, 1998; Miki et al., 1996; Montrose-Raﬁzadeh et al., 1997a,b; Perea et al., 1997; Pittner et al., 2000; Puente et al., 1997; Valverde and Villanueva-Penacarrillo, 1996; Villanueva-Penacarrillo et al., 1999; Wang et al., 1997a). In 3T3-L1 model of adipocytes, GLP-1 augmented insulin-stimulated 2-deoxyglucose uptake (a non-metabolized marker of transport), lipogenesis (Egan et al., 1994), and GLUT-4

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protein levels (Wang et al., 1997a). Another laboratory also reported that GLP-1 induced an increment in 2-deoxyglucose uptake in isolated rat adipocytes, which was additive to that of insulin. Furthermore, in rat fat, GLP-1 provoked a rise in glycogen synthesis, glucose oxidation and utilization and lipogenesis, the increments being lower than those obtained with insulin. These data were reported as supporting the idea that GLP-1 exerts insulin-like effects on glucose metabolism in rat adipose tissue (Perea et al., 1997). Both GLP-1 and insulin were reported to normalize the deﬁcit in GLUT-4 in fat cells in STZ-treated rats (Puente et al., 1997). In distinction to this reported insulinomimetic effect, another group working with isolated rat adipocytes reported that, although GLP-1 augmented insulin-stimulated glucose uptake, it had no effect of its own (Miki et al., 1996). In yet another study of isolated rat brown and white adipocytes, there was no effect of either GLP-1 or exendin-4 on lipogenesis or general cellular activity (as measured in a physiometer), either in the absence or presence of insulin stimulation (Pittner et al., 2000). That is, there was neither an insulinomimetic nor an insulin-potentiating effect of either GLP-1 or exendin-4, as shown in Figure 8. GLP-1 was reported to dose-dependently stimulate cAMP content in isolated human adipocytes, and to stimulate lipolysis, as evidenced by glycerol release (Marquez et al., 1997). In isolated normal rat adipocytes and hepatocytes, GLP-1 exerted a rapid decrease of radiolabeled glycosylphosphatidylinositol (GPI) in a manner similar to that of insulin, indicating GPI hydrolysis and the immediate short-lived generation of inositol phosphoglycans (IPGs). The authors proposed that IPGs could mediate direct GLP-1 actions in adipose tissue and liver (as well as in skeletal muscle) (Villanueva-Penacarrillo et al., 1999). Such an effect was further proposed to be mediated via GLP-1 receptors which were functionally distinct from those present in pancreatic ␤-cells (Marquez et al., 1998). In contrast to reported cAMP elevation in human adipocytes (Marquez et al., 1997), in the 3T3-L1 model of adipocytes, others reported that GLP-1 caused a decrease in intracellular cAMP levels (MontroseRaﬁzadeh et al., 1997a). Peptides with partial homology to GLP-1 such as GLP-2, GLP-1 [1-36], and glucagon also lowered cAMP levels in 3T3-L1 adipocytes. In addition, an antagonist of the pancreatic GLP-1 receptor, exendin[9-39], was reported to behave similarly to GLP-1 agonists and exendin-4 [1-39] in that it too decreased cAMP levels in 3T3-L1 adipocytes (Montrose-Raﬁzadeh et al., 1997b). This pharmacological proﬁle, distinct from

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that observed with ␤-cells, was held to indicate the presence of a receptor on fat cells that was different from the cloned GLP-1 receptor of Thorens et al. (1992). Thus in isolated adipocytes, in fully differentiated fat cell lines, and in skeletal muscle, there appears to be no present consensus on the existence or direction of effects of GLP-1, GLP-1 agonists or exendin-4. Liver Direct effects of GLP-1 in liver have been studied mainly by one laboratory (Alcantara et al., 1997; Lopez-Delgado et al., 1997, 1998; Marquez et al., 1998; Morales et al., 1997; Nakagawa et al., 1996; Puente et al., 1997; Valverde and Villanueva-Penacarrillo, 1996; Villanueva-Penacarrillo et al., 1999). GLP-1 (Lopez-Delgado et al., 1997, 1998) and exendin-4 (Alcantara et al., 1997) was reported to stimulate glycogen synthase a in hepatocytes, and exendin[9-39] to block such effects (Alcantara et al., 1997). Effects of GLP-1 in hepatocytes were accompanied by a rapid decrease of the radiolabeled GPIs – precursors of IPGs – in the same manner as insulin, indicating their hydrolysis (Marquez et al., 1998). Effects of diabetes on GLUT-1 and GLUT-2 transporter expression in liver were normalized following 3 days in vivo treatment with GLP-1 or insulin in osmotic pumps (Puente et al., 1997). The ﬁndings of direct effects of GLP-1 or exendin-4 in liver, as in other insulin-sensitive tissues, is controversial in view of several reports of absence of GLP-1 receptor mRNA in liver (Yamato et al., 1997; Bullock et al., 1996; Wei and Mojsov, 1995; Dunphy et al., 1998). A possible mechanism via which GLP-1 may inﬂuence hepatic function is via the autonomic nervous system. Nishizawa et al. (2000) having previously reported that the hepatic vagal nerve was receptive to intraportal GLP-1[7-36]amide (but not to non-insulinotropic full-length GLP-1[1-37]), examined effects of full-length exendin-4, and of exendin[9-39]. Periphysiological and pharmacological doses of GLP-1 delivered intraportally facilitated the afferent impulse discharge rate of the hepatic vagus in anesthetized rats. However, unexpectedly, intraportal injection of exendin-4 at these and even higher doses did not facilitate the afferents at all. Moreover, intraportal injection of exendin[9-39] at 100 times or more the molar dose of GLP-1, either 5 min before or 10 min after GLP-1 injection, failed to modify the GLP1-induced facilitation of the afferents. Again, the interpretation was that this action of GLP-1 involved a receptor that was distinct from that mediating the well-known humoral insulinotropic action.

EFFECTS ON ␤-CELL NEOGENESIS AND EFFECTS ON SECRETORY FUNCTION Both GLP-1 and exendin-4 have been shown to induce proliferation and differentiation of glucagon- and insulin-producing cells (Zhou et al., 1999; Egan et al., 1998). This work showing effects on ␤-cells is supported by the ﬁndings of others (Edvell and Lindstrom, 1999; Bernard et al., 1999; Hoersch et al., 2000; Seufert et al., 2000; Stoffers et al., 2000b; Perfetti et al., 2000; Prentki et al., 1999; Wang et al., 1999). Exendin-4 has been shown to have similar effects (Xu et al., 1999; Zhou et al., 1999; Perfetti et al., 2000; Tourrel et al., 2001). A separate consequence of chronic treatment with GLP-1, its analogs and exendin-4 is a restoration of nutrient-driven secretory function, or “glucose competence” (Holz et al., 1928; De Ore et al., 1997; Egan et al., 1997). That is, GLP-1 can reverse the age-dependent

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decline in ﬁrst-phase insulin secretion (Egan et al., 1997) and glucose tolerance (Wang et al., 1997b). Similarly, exendin-4 stimulates islet growth and improves glucose tolerance (Aspedlund et al., 2000). However, the extent to which such restoration of islet structure and function (vs. possible peripheral effects, for example) contributes to improvements in insulin sensitivity and glucose tolerance is presently uncertain. SUMMARY GLP-1, GLP-1 analogs and exendin-4 show promise in animal and clinical studies as agents that will promote glucose control. These effects can be attributed in the acute setting to several direct actions, including stimulation of insulin secretion, slowing of gastric emptying, inhibition of glucagon secretion, and a potential satiogenic effect. In a chronic setting there additionally appears to be an enhancement of insulin action, an “insulin-sensitizing” effect. The nature of this effect is controversial, with comparable groups of authors claiming and disclaiming direct effects in insulin-sensitive tissues. Effects on insulin sensitivity in animal models are incompletely explained by reduction in food intake. Improvements in insulin sensitivity following chronic administration of these agents may, at least in part, be a consequence of general improvement in glycemic control, since similar effects are observed with numerous antidiabetic agents. Finally, an effect of these agents to promote neogenesis, differentiation and to restore secretory function to islets has recently been identiﬁed. To what extent an improvement in ␤-cell function subsequent to the use of these agents contributes to an improvement in tissue response to insulin remains to be deﬁned. REFERENCES Ahren, B. (1995) Antidiabetogenic action of truncated glucagon-like peptide-1 in mice. Endocrine, 3, 367–369. Ahren, B., Larsson, H. and Holst, J.J. (1997) Effects of glucagon-like peptide-1 on islet function and insulin sensitivity in noninsulin-dependent diabetes mellitus. J. Clin. Endocrinol. Metab., 82, 473–478. Ahren, B. and Pacini, G. (1999) Dose-related effects of GLP-1 on insulin secretion, insulin sensitivity, and glucose effectiveness in mice. Am. J. Physiol., 277, E996–E1004. Alcantara, A.I., Morales, M., Delgado, E., Lopez-Delgado, M.I., Clemente, F., Luque, M.A., Malaisse, W.J., Valverde, I. and Villanueva-Penacarrillo, M.L. (1997) Exendin-4 agonist and exendin (9-39)amide antagonist of the GLP-1(7-36)amide effects in liver and muscle. Arch. Biochem. Biophys., 341, 1–7. Aspedlund, G., Egan, J.M., Slezak, L.A., Sritharan, K.C., Elahi, D. and Andersen, D.K. (2000) Glucagon-like peptide-1 and exendin-4 improve glucose tolerance and induce islet cell growth in diabetic rats. Diabetologia, 43, A144. Baggio, L., Adatia, F., Bock, T., Brubaker, P.L. and Drucker, D.J. (2000) Sustained expression of exendin-4 does not perturb glucose homeostasis, beta-cell mass, or food intake in metallothioneinpreproexendin transgenic mice. J. Biol. Chem., 275, 34471–34477. Balkan, B., Kwasnik, L., Miserendino, R., Mone, M., Hughes, T.E. and Li, X. (1997) Improved insulin secretion and oral glucose tolerance after in vivo inhibition of DPP-IV in obese Zucker rats. Diabetologia, 40, A131. Baron, A., Fineman, M., Young, A., Gaines, E. and Prickett, K. (2000) Synthetic exendin-4 (AC2993) reduces post-prandial glycemia, glucagon levels and slows gastric emptying in subjects with type 2 diabetes. Diabetologia, 43, A190 (abstract 730).

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Toft-Nielsen, M.B., Madsbad, S. and Holst, J.J. (1999) Continuous subcutaneous infusion of glucagonlike peptide 1 lowers plasma glucose and reduces appetite in type 2 diabetic patients. Diabetes Care, 22, 1137–1143. Tourrel, C., Bailbe, D., Meile, M.J., Kergoat, M. and Portha, B. (2001) Glucagon-like peptide-1 and exendin-4 stimulate beta-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes, 50, 1562–1570. Turton, M.D., O’Shea, D., Gunn, I., Beak, S.A., Edwards, C.M., Meeran, K., Choi, S.J., Taylor, G.M., Heath, M.M., Lambert, P.D., Wilding, J.P., Smith, D.M., Ghatei, M.A., Herbert, J. and Bloom, S.R. (1996) A role for glucagon-like peptide-1 in the central regulation of feeding [see comments]. Nature, 379, 69–72. Unger, R.H. (1971) Glucagon physiology and pathophysiology. N. Engl. J. Med., 285, 443–449. Valverde, I. and Villanueva-Penacarrillo, M.L. (1996) In vitro insulinomimetic effects of GLP-1 in liver, muscle and fat. Acta Physiol. Scand., 157, 359–360. Van Dijk, G. and Thiele, T.E. (1997) Glucagon-like peptide-1 and satiety. Nature, 385, 214. Vella, A., Shah, P., Basu, R., Basu, A., Holst, J.J. and Rizza R.A. (2000a) Effect of glucagon-like peptide 1(7-36) amide on glucose effectiveness and insulin action in people with type 2 diabetes. Diabetes, 49, 611–617. Vella, A., Shah, P., Basu, R., Basu, A., Holst, J.J. and Rizza, R.A. (2000b) Effect of glucagon-like peptide 1(7-36) amide on glucose effectiveness and insulin action in people with type 2 diabetes. Diabetes, 49, 611–617. Vestergaard, H., Weinreb, J.E., Rosen, A.S., Bjorbaek, C., Hansen, L., Pedersen, O. and Kahn, B.B. (1995) Sulfonylurea therapy improves glucose disposal without changing skeletal muscle GLUT4 levels in noninsulin-dependent diabetes mellitus subjects: A longitudinal study. J. Clin. Endocrinol. Metab., 80, 270–275. Vettor, R., Lombardi, A.M., Fabris, R., Pagano, C., Cusin, I., RohnerJeanrenaud, F., Federspil, G. and Jeanrenaud, B. (1997) Lactate infusion in anesthetized rats produces insulin resistance in heart and skeletal muscles. Metab. Clin. Exp., 46, 684–690. Villanueva-Penacarrillo, M.L., Alcantara, A.I., Clemente, F., Delgado, E. and Valverde, I. (1994) Potent glycogenic effect of GLP-1(7-36)amide in rat skeletal muscle. Diabetologia, 37, 1163–1166. Villanueva-Penacarrillo, M.L., Márquez, L., Luque, M.A., González, N. and Valverde, I. (1999) Effect of GLP-1 on liver and fat GPI/IPG system in diabetic states. Diabetologia, 42, A197. Vilsboll, T., Krarup, T., Madsbad, S. and Holst, J.J. (2001) No reactive hypoglycaemia in type 2 diabetic patients after subcutaneous administration of GLP-1 and intravenous glucose. Diabet. Med., 18, 144–149. Vilsbøll, T., Toft-Nielsen, M.-B., Krarup, T., Madsbad, S., Dinesen, B. and Holst, J.J. (1999) Evaluation of the “GLP-1 test” in patients with type 2 diabetes and healthy subjects. Diabetologia, 42, A153. Wahlin-Boll, E., Sartor, G., Melander, A. and Schersten, B. (1982) Impaired effect of sulfonylurea following increased dosage. Eur. J. Clin. Pharmacol., 22, 21–25. Wang, T.L., Edwards, G.L. and Baile, C.A. (1998) Glucagon-like peptide-1 (7-36) amide administered into the third cerebroventricle inhibits water intake in rats. Proc. Soc. Exp. Bio. Med., 219, 85–91. Wang, X., Cahill, C.M., Pineyro, M.A., Zhou, J., Doyle, M.E. and Egan, J.M. (1999) Glucagon-like peptide-1 regulates the beta cell transcription factor, PDX-1, in insulinoma cells. Endocrinology, 140, 4904–4907. Wang, Y., Kole, H.K., Montrose-Raﬁzadeh, C., Perfetti, R., Bernier, M. and Egan, J.M. (1997a) Regulation of glucose transporters and hexose uptake in 3T3-L1 adipocytes: Glucagon-like peptide-1 and insulin interactions. J. Mol. Endocrinol., 19, 241–248. Wang, Y.H., Perfetti, R., Greig, N.H., Holloway, H.W., DeOre, K.A., MontroseRaﬁzadeh, C., Elahi, D. and Egan, J.M. (1997b) Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats. J. Clin. Invest., 99, 2883–2889. Wei, Y. and Mojsov, S. (1995) Tissue-speciﬁc expression of the human receptor for glucagon-like peptide-I: Brain, heart and pancreatic forms have the same deduced amino acid sequences. FEBS Lett., 358, 219–224.

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Weir, G.C., Mojsov, S., Hendrick, G.K. and Habener, J.F. (1989) Glucagonlike peptide I (7-37) actions on endocrine pancreas. Diabetes, 38, 338–342. Wettergren, A., Schjoldager, B., Mortensen, P.E., Myhre, J., Christiansen, J. and Holst, J.J. (1993) Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in man. Dig. Dis. Sci., 38, 665–673. Wheeler, M.B., Lu, M., Dillon, J.S., Leng, X.H., Chen, C. and Boyd, A.E. 3d (1993) Functional expression of the rat glucagon-like peptide-I receptor, evidence for coupling to both adenylyl cyclase and phospholipase-C. Endocrinology, 133, 57–62. Widmaier, E.P., Shah, P.R. and Lee, G. (1991) Interactions between oxytocin, glucagon and glucose in normal and streptozotocin-induced diabetic rats. Regul. Pept., 34, 235–249. Willms, B., Idowu, K., Holst, J.J., Creutzfeldt, W. and Nauck, M.A. (1998) Overnight GLP-1 normalizes fasting but not daytime plasma glucose levels in NIDDM patients. Exp. Clin. Endocrinol. Diabetes, 106, 103–107. Willms, B., Werner, J., Holst, J.J., Orskov, C., Creutzfeldt, W. and Nauck, M.A. (1996) Gastric emptying glucose responses, and insulin secretion after a liquid test meal: Effects of exogenous glucagonlike peptide-1 (GLP-1)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients. J. Clin. Endocrinol. Metab., 81, 327–332. Xu, G., Stoffers, D.A., Habener, J.F. and Bonner-Weir, S. (1999) Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes, 48, 2270–2276. Yamato, E., Ikegami, H., Takekawa, K., Fujisawa, T., Nakagawa, Y., Hamada, Y., Ueda, H. and Ogihara, T. (1997) Tissue-speciﬁc and glucose-dependent expression of receptor genes for glucagon and glucagon-like peptide-1 (GLP-1). Horm. Metab. Res., 29, 56–59. Yki-Järvinen, H., Kubo, K., Zawadzki, J., Lillioja, S., Young, A., Abbott, W. and Foley, J.E. (1987) Dissociation of in vitro sensitivities of glucose transport and antilipolysis to insulin in NIDDM. Am. J. Physiol., 253, E300–E304. Young, A., Gottlieb, A., Fineman, M. and Kolterman, O. (1999a) Measurements of satiety and fullness following a sustacal challenge in type 2 diabetic subjects administered synthetic exendin-4 (AC2993). Diabetologia, 42, A40. Young, A., Petrella, E., Blase, E. and Seward, M. (1999b) Exendin-4 is a circulating meal-related peptide in the Gila Monster (Heloderma suspectum). Diabetes, 48, A425 (abstract 1880). Young, A.A., Bhavsar, S.P., LaChappell, R. and Watkins, J.J. (1998) Exendin-4 is more potent than GLP-1 lowering plasma glucose in diabetic mice. Diabetologia, 41, A176 (abstract 686). Young, A.A., Bogardus, C., Wolfe-Lopez, D. and Mott, D.M. (1988) Muscle glycogen synthesis and disposition of infused glucose in humans with reduced rates of insulin-mediated carbohydrate storage. Diabetes, 37, 303–308. Young, A.A., Gedulin, B.R., Bhavsar, S., Bodkin, N., Jodka, C., Hansen, B. and Denaro, M. (1999) Glucose-lowering and insulin-sensitizing actions of exendin-4: Studies in obese (ob/ob, db/db) diabetic mice, diabetic Fatty Zucker rats and diabetic rhesus monkeys (Macaca mulatta). Diabetes, 48, 1026–1034. Young, A.A., Gedulin, B.R. and Rink, T.J. (1996) Dose-responses for the slowing of gastric emptying in a rodent model by glucagon-like peptide (7-36)NH2, amylin, cholecystokinin, and other possible regulators of nutrient uptake. Metabolism, 45, 1–3. Zawalich, W.S., Zawalich, K.C. and Rasmussen, H. (1993) Inﬂuence of glucagon-like peptide-1 on beta cell responsiveness. Regul. Pept., 44, 277–283. Zhou, J. and Egan, J.M. (2000) Transcriptional factors involved in pancreatic duct Capan-1 cells differentiation into endocrine-phenotype cells. Program and Abstracts, 82nd Annual Endocrine Meeting 80. Zhou, J., Wang, X., Pineyro, M.A. and Egan, J.M. (1999) Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes, 48, 2358–2366.

15.

INSULIN RESISTANCE AND THE AUTONOMIC NERVOUS SYSTEM

L. PENICAUD, C. LELOUP, A. LORSIGNOL AND T. ALQUIER UMR 5018 UPS-CNRS, IFR 31, CHU Rangueil, 1 Avenue Jean Poulhés, 31403 Toulouse Cedex, France

INTRODUCTION The autonomic nervous system and insulin appear to be related by different ways. Indeed, insulin secretion is under the inﬂuence of both sympathetic and parasympathetic nerves. Conversely, there are numerous reports, although often contradictory, describing the inﬂuence of insulin on the activity of the sympathetic nervous system (SNS). The aim of this chapter will be to have a quick survey of these two aspects and ﬁnally to ascertain, in what way, changes in the activity of the autonomic nervous system, and more speciﬁcally, the sympathetic one can be involved in the development of human insulin resistance syndrome.

Control of Insulin Level (Secretion) by the Autonomic Nervous System The pancreas is richly innervated by ﬁbers that penetrate the organ following the main blood vessels, particularly, the pancreatic artery. Sympathetic (splanchnic nerve) and parasympathetic ﬁbers (vagus nerve) are directly connected to the islets of Langerhans (Helman et al., 1982; Woods and Porte, 1974). Presynaptic ﬁbers of the parasympathetic system make synapses inside the pancreas itself. Acetylcholine, that is, the neurotransmitter liberated by the postsynaptic ﬁbers when the vagus nerve is activated stimulates insulin secretion after binding to muscarinic receptor. Presynaptic ﬁbers of the splanchnic nerve make synapses at the level of the coeliac ganglia with noradrenergic ﬁbers. The stimulation of the splanchnic nerve results in an inhibition of insulin secretion after binding of norepinephrine to adrenoceptor of the alpha-2 subtype. It must be underlined that apart from these neurotransmitters, numerous peptides are present and liberated by nerve endings. These peptides have either a stimulating (VIP, CCK and GRP) or inhibiting (NPY, CRP and galanin) effect on insulin secretion (Ahren et al., 1986). The autonomic nervous system can also modulate insulin secretion indirectly via the regulation of pancreatic and islet blood ﬂow (Jansson and Hellerström, 1986). This later is one of the processes that ultimately participates in the control of plasma insulin level by modulating (1) the delivery of nutrients and/or other factors involved in the regulation of insulin secretion and (2) the dispersion of insulin itself in the blood. It has been shown in rats that islet blood ﬂow is increased after a glucose load and that increase is under the inﬂuence of both parasympathetic and sympathetic activity. Indeed, this enhanced islet blood ﬂow is abolished by vagotomy and by previous injection of alpha-2 adrenergic receptor ( Janssson and Hellerström, 1986; Atef et al., 1992, 1995). Although there are no direct evidences for a role of the autonomic nervous system in proliferation and differentiation of pancreatic endocrine cells, some data suggests such an inﬂuence. 263

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Thus, in normal rats submitted to a 48 h glucose infusion, we have been able to detect an increased islets DNA content and to show the presence of cells, near canal ducts, stained with insulin immunoreactivity (Thibault et al., 1994). Indeed this could be due to the well-known effect of glucose on beta cells proliferation (Bonner-Weir and Smith, 1994; Swenne, 1992). However, these rats present alterations in autonomic nervous system activity, namely, a decreased sympathetic one and an increased parasympathetic one (N’Guyen et al., 1994). This, which in view of the described effect of these changes on differentiation and proliferation in other tissues (adipose tissues, liver), could also be involved in beta cells growth.

Insulin Effects on Autonomic Nervous System Activity Areas of the brain that regulate a downward ﬂow of impulses toward the sympathetic and vagus nerves determine autonomic nervous system outﬂow. Numerous data are in support of the involvement of metabolic and hormonal factors in the mechanism permitting a feedback loop signaling the metabolic-related nutritional changes to the brain. Among these factors, a considerable amount of evidences, among which the pioneer works of Landsberg, Young and coll. (1985, 1989) has been marshaled in support of insulin as playing a crucial role. One of the ﬁrst indirect evidence was the demonstration that an insulin injection into the carotid artery of a dog increased blood pressure and that this effect was abolished by sympathetic blockade (Pereda et al., 1962). Then, the relationship between insulin and the SNS was studied at ﬁrst in the context of the physiologic response to food intake. Thus, it was demonstrated that fasting or overfeeding associated with hypo- or hyperinsulinemia suppresses or increases SNS activity respectively in rodents (Young and Landsberg, 1977a,b) and in human (O’Dea et al., 1982; Young et al., 1984). Further proofs came from studies in diabetic rats induced after streptozotocin, in which a decreased sympathetic outﬂow to brown adipose tissue (BAT) was described (Young et al., 1983). This was corrected by insulin treatment (Young et al., 1988). These results have been conﬁrmed in humans in which elevation of plasma insulin concentrations has been shown to stimulate sympathetic nerve activity (Rowe et al., 1981; Anderson et al., 1991; Arauz-Pacheco et al., 1996; Berne et al., 1992; Lembo et al., 1992; Scherrer et al., 1993). This effect is independent of a stimulation of carbohydrate metabolism (Vollenweider et al., 1993). Other studies using insulin infusions have demonstrated a direct relationship between this hormone and sympathetic activation by looking at thermogenesis in human. Thus, propanolol has been shown to block the increased metabolic rate that occurs during insulin infusion (Acheson et al., 1983). Although the mechanism by which insulin-induced sympathetic overactivity is not totally elucidated, numerous evidences suggest an action via the central nervous system. It is now well demonstrated that the ventromedial hypothalamus (VMH) is one of the main areas of the brain from which the SNS originates (Pénicaud et al., 1996, 2000; Shimazu, 1987, 1996, 1998; Steffens et al., 1988, 1991). The injection of glucose or insulin into this nucleus has been shown to modify sympathetic outﬂow to different tissues and in particular BAT (Sakaguchi and Bray, 1987a,b; Menendez and Atrens, 1991). When lesion of this area is performed such as after injection of gold-thioglucose in the mouse, the dietary modulation of SNS activity described above is no longer present (Young and Landsberg, 1980). Furthermore, third ventricle lesions abolished lumbar sympathetic responses to insulin in rats, and the administration of insulin into the third cerebral ventricle produces an increase in lumbar sympathetic outﬂow in the absence of changes in blood glucose or plasma insulin (Muntzel et al., 1994a,b).

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This effect of insulin on the brain has to be related to different observations. First, it has been demonstrated that insulin can modulate the ﬁring rate and hyperpolarization of neurons in the hypothalamus and particularly in the VMH (Oomura and Kita, 1981; Shapiro et al., 1991). At the cellular and molecular levels one has to underline that insulin receptors and insulin receptor substrate are present throughout the brain (Baskin et al., 1987, 1994). Insulin is involved in numerous biological effects in the brain among which modulation of the expression of neuropeptides, catecholamine neurotransmission, phosphoinositol turnover, regulation of food intake and thermogenesis (for review, see Schwartz et al., 1992, 2000). It has thus been proposed by Landsberg that insulin would modulate glucose metabolism in speciﬁc neurons in the VMH and then in turn SNS activity (Landsberg and Young, 1985; Landsberg, 1986). Although it is clearly admitted that brain glucose metabolism is not dependent of insulin, such a hypothesis cannot be totally excluded. Indeed, there are data in the literature demonstrating that glucose uptake can be modulated by insulin in some brain areas. Surprisingly, insulin induced a decreased glucose utilization (Grundstein et al., 1985; Namba et al., 1984; Marfaing et al., 1990). Furthermore, others have shown that Glut4, the insulindependent glucose transporter, is present in the brain and more speciﬁcally in neurons (Leloup et al., 1996; El Messari et al., 1998) The expression of this transporter is altered in conditions associated with changes in plasma insulin levels (Vannuci et al., Alquier et al., 2001). Autonomic Nervous System Activity and Insulin Action This is rather a controversial issue both within and between species. One of the ﬁrst demonstration of an important regulatory role played by the SNS in metabolic functions and more

BAT

Glucose utilization

Muscle

Sympathetic

Parasympathetic

Glycemia

Pancreas

Hepatic glucose production Insulin Liver

Figure 1 Schematic representation of the relationships between the brain and the periphery, concerning insulin secretion and action. Some brain areas participate in the control of the secretory activity of the pancreas as well as in glucose production by the liver and utilization by some insulin-sensitive tissue (BAT and skeletal muscles). The brain is informed in return both by insulin itself and glucose.

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speciﬁcally in insulin sensitivity came from the evidence that sympathetic activity was altered in animal models of obesity (Bray and York, 1979; Jeanrenaud, 1985; Pénicaud et al., 1996, 2000). In these models, the general agreement is that insulin resistance is associated with/or a consequence of a decreased sympathetic activity. This is further strengthened by the works of Shimazu and colleagues demonstrating a stimulating role of sympathetic innervation in glucose uptake by different tissues including BAT and skeletal muscles (Shimazu, 1996, 1998). However, other studies in rodents demonstrate that norepinephrine inhibits insulin-induced glucose utilization (Marfaing et al., 1991). In human subjects, the consensus is the opposite of that described in rodents. Thus, in normal subjects an enhanced sympathetic activation inhibits the insulin-induced glucose utilization and metabolism ( Jamerson et al., 1993; Lembo et al., 1994; Tappy et al., 1995). Norepinephrine as epinephrine can reduce insulin sensitivity acutely by stimulating hepatic glucose production and by blocking peripheral insulin-stimulated glucose uptake (Rizza et al., 1979; Sacca et al., 1980; Sherwin and Sacca, 1984). Such defects have been documented in human type II diabetes and hypertension. In conclusion, there is a feedback loop linking the brain and the periphery in terms of regulation of insulin secretion and probably action (Figure 1). Indeed, on one hand, some brain areas informed of the level of plasma insulin, probably by the hormone itself, can control insulin secretion by the beta cell of the pancreas via both parasympathetic and sympathetic nerves. On the other hand, the same brain areas principally through the sympathetic system, can modulate both hepatic glucose production and tissues glucose utilization, and thus modulate insulin action. Defects in this loop can be involved in metabolic abnormalities such as the X syndrome.

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Oomura,Y. and Kita, H. (1981) Insulin acting as a modulator of feeding through the hypothalamus. Diabetologia, 20, 290–298. Pénicaud, L., Cousin, B., Leloup, C., Atef, N., Casteilla, L. and Ktorza, A. (1996) Changes in autonomic nervous system activity and consecutive hyperinsulinemia: respective roles in the development of obesity in rodents. Diabetes Metab., 22, 15–24. Pénicaud, L., Cousin, B., Leloup, C., Lorsignol, A. and Casteilla, L. (2000) The autonomic nervous system, adipose tissue plasticity, and energy balance. Nutrition, 16, 903–908. Pereda, S., Eckstein, J.W. and Abboud, F.M. (1962) Cardiovascular responses to insulin in the absence of hypoglycemia. Am. J. Physiol., 202, 249–252. Reaven, G.M., Lithell, H. and Landsberg, L. (1996) Hypertension and associated abnormalities – the role of insulin resistance and the sympathoadrenal system. N. Engl. J. Med., 334, 374–381. Rizza, R., Haymond, M., Cryer, P. and Gerich, J. (1979) Differential effects of epinephrine on glucose production and disposal in normal man. Am. J. Physiol., 237, E356–E362. Rowe, J.W., Young, J.B., Minaker, K.L., Stevens, A.L., Pallota, J. and Landsberg, L. (1981) Effect of glucose and insulin infusion on sympathetic nervous system activity in normal man. Diabetes, 30, 219–225. Sacca, L., Morrone, G., Cicala, M (1980) Inﬂuence of epinephrine, norepinephrine and isoproterenol on glucose homeostasis in normal man. J. Clin. Invest., 50, 680–684. Sakaguchi, T. and Bray, G.A. (1987a) The effect of intrahypothalamic injections of glucose on sympathetic efferent ﬁring rate. Brain Res. Bull., 18, 591–595. Sakaguchi, T. and Bray, G.A. (1987b) Intrahypothalamic injection of insulin ﬁring rate of sympathetic nerves. Proc. Natl. Acad. Sci. USA, 84(7), 2012–2014. Scherrer, U., Vollenweider, L., Randin, D., Jéquier, E., Nicod P. and Tappy, L. (1993) Suppression of insulin induced sympathetic activation and vasodilatation by dexamethasone in humans. Circulation, 88, 388–394. Schwartz, M.W., Figlewicz, D.P., Baskin, D.G., Woods, S.C. and Porte, D. Jr. (1994) Insulin and the central regulation of energy balance. Endocr. Rev., 2, 109–113. Shapiro, E., Brown, S.D., Saltiel, A.R. and Schwartz, J.H. (1991) Short term action of insulin on aplysia neurons: generation of a possible novel modulation of ion channels. J. Neurobiol., 22, 55–62. Sherwin, R.S. and Sacca, L (1984) Effect of epinephrine on glucose metabolism in humans: contrbution of the liver. Am. J. Physiol., 247, E157–E165. Shimazu, T. (1987) Neuronal control of hepatic glucose metabolism. Diabetes Metab. Rev., 3, 185–206. Shimazu, T. (1996) Innervation of the liver and glucoregulation. Nutrition, 12, 65–66. Shimazu, T. (1998) Hypothalamic control of liver, muscle and adipose tissue metabolism. In Liver and Nervous System Häusinger, D. and Jungermann, K. eds), Kluwer Academic Press Publishers, Lancasters), pp. 118–133. Swenne I. (1992) Pancreatic beta-cell growth and diabetes mellitus. Diabetologia, 35, 193–201. Steffens, A.B., Scheurink, A.J.W., Luiten, P.G. and Bohus, B. (1988) Hypothalamic food intake regulating areas are involved in the homeostasis of blood glucose and plasma FFA levels. Physiol. Behav., 44, 581–589. Steffens, A.B., Strubbe, J.H., Balkan, B. and Scheurink, A.J.W. (1991) Neuroendocrine factors regulating blood glucose, plasma FFA and insulin in the development of obesity. Brain Res. Bull., 27, 505–510. Tappy, L., Girardet, K., Schwaller, N., Vollenweider, L., Jéquier, E., Nicod, P. et al. (1995) Metabolic effects of an increase of sympathetic activity in healthy humans. Int. J. Obes., 19, 419–422. Thibault, C., Saulnier, C., N’Guyen, J.M., Portha, B., Pénicaud, L. and Ktorza A. (1994) Recovery of the beta cell mass after superimposed hyperglycemia is not associated with improved pancreatic function in glucose intolerant rats. Diabetologia, 37(Suppl 1), A99. Verwaerde, P., Galinier, M., Forcade, J., Massabuau, P., Galitzky, J., Senard, J.M. et al. (1997) Autonomic nervous system abnormalities in the initial phase of insulin resistance syndrome. Value of the study of variability of cardiac rate and blood pressure on a model of nutritional obesity. Arch. Mal. Cœur. Vaiss., 90, 1151–1154.

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Vollenweider, L., Tappy, L., Owlya, R., Jéquier, E., Nicod, P. and Scherrer, U. (1995) Insulin-induced sympathetic activation and vasodilatation in skeletal muscle. Effects of insulin resistance in lean subjects. Diabetes, 44, 641–645. Vollenweider, L., Tappy, L., Randin, D., Schneiter, P., Jequier, E., Nicod, P. et al. (1993) Differential effects of hyperinsulinemia and carbohydrate metabolism on sympathetic nerve activity and muscle blood ﬂow in humans. J. Clin. Invest., 92, 147–154. Woods, S.C. and Porte, D. Jr. (1974) Neural control of the endocrine pancreas. Physiol. Rev., 54, 596–619. Xie, H. and Lautt, W.W. (1996) Insulin resistance of skeletal muscle produced by hepatic parasympathetic interruption. Am. J. Physiol., 270, E858–E863. Young, J.B. (1988) Effect of experimental hyperinsulinemia on sympathetic nervous system in the rat. Life Sci., 193–200. Young, J.B., Einhorn, D. and Landsberg, L. (1988) Decreased sympathetic activity in interscapular brown adipose tissue of streptozotocin-treated rats. Diabetes, 183(Suppl 1) 26. A.Young, J.B. and Landsberg, L. (1977a) Stimulation of the sympathetic nervous system during sucrose feeding. Nature, 269, 615–617. Young, J.B. and Landsberg, L. (1977b) Suppression of sympathetic nervous system during fasting. Science, 196, 1473–1475. Young, J.B. and Landsberg, L. (1980) Impaired suppression of sympathetic activity during fasting in the gold thioglucose-treated mouse. J. Clin. Invest., 65, 1086–1094. Young, J.B., Rosa, R.M. and Landsberg, L. (1984) Dissociation of sympathetic nervous system and adrenal medullary responses. Am. J. Physiol., 247, E35–E40.

16.

HYPOTHALAMIC ROLE IN THE INSULIN RESISTANCE SYNDROME ANTHONY H. CINCOTTA

Domani Sciences LLC, 1334 Main Road, Tiverton, R.I. 02878, USA (Email: [email protected])

PREFACE At the outset I must logically concede that in the span of a single chapter, it is not possible to aptly review the existing common fund of knowledge relating to hypothalamic control of insulin resistance. I have therefore chosen to discuss a few choice groups of “neuronal circuits” within the hypothalamus that have profound inﬂuences on peripheral metabolism, are generally poorly recognized, and are not primarily focused on feeding behavior per se. Even within the self-imposed conﬁnes of this outline, the complexity of the brain and the complications of the insulin resistance syndrome prevent the composition of a ﬁnished story. Nonetheless, the presented material offers new insights and perspectives into the pathophysiology of the insulin resistance syndrome that hopefully will trigger new approaches to scientiﬁc endeavor by investigators in this ﬁeld. A primary tenet of this chapter is that the insulin resistance syndrome need not be the result solely of genetic “defects” but contrariwise evolved as a survival strategy in an environment of (annual) cyclic food availability and quality. Evolution selected for neuronal plasticity within the central nervous system (CNS), particularly within the hypothalamus, that allows for both the natural development and reversal of the syndrome. INTRODUCTION – EVOLUTION OF THE INSULIN RESISTANCE SYNDROME In vertebrates, the hypothalamus is the main CNS control center for the regulation of peripheral metabolism (Frohman, 1979; Torii et al., 1998). Fundamental roles for the hypothalamic regulation of physiologic activities have been preserved over 400 million years and are strikingly similar among vertebrates from teleosts to mammals despite their subtle to moderate differences in hypothalamic macro and micro neuroanatomy (Morgane, 1969; Lowry and Scott, 1975). This review will focus on the neuronal circuitry within the hypothalamus of mammals that functions to modulate peripheral metabolism, including the development and reversal of the insulin resistance syndrome. However, a true appreciation of hypothalamic organization of metabolism cannot be attained without an understanding of evolutionary natural selection forces that sculpted the functionality of this regulatory center. The earth’s rotation on its axis and its revolution around the sun produce respective 24 h and 365 day cyclic changes in the environment, under which conditions, life developed on the planet. Life forms, from prokaryotes to complex multicellular organisms such as humans, evolved biochemical mechanisms to predict such daily and annual changes in the environment (reviewed in Zordan et al., 2000) that thereby increased survival and as such were selected for by natural selection processes. These biochemical mechanisms comprise circadian rhythms: cyclic biological events that persist in constant environmental conditions with periods that approximate 24 h. Circadian 271

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rhythm, the basic unit of temporal organization of cellular biochemistry, is as old as life itself, being identiﬁable in all known life forms (Zordan et al., 2000). Although the phase and amplitude of cellular circadian rhythms can be “adjusted” by exogenous cues, they are endogenous in nature (i.e. their expression is not dependent on or driven by external sources). In multicellular organisms such as mammals, it is the important role of the neuroendocrine system to synchronize cellular rhythms for the construction of tissue and organismal level rhythmic activities so that the animal is temporally organized internally and synchronized with its cyclic environment. Moreover, circadian events within the neuroendocrine system itself and its control center, the hypothalamus, are integral components of a regulatory system manifesting seasonal changes in physiology among vertebrates in the wild, including the annual cycle of metabolism (reviewed in Meier and Cincotta, 1996). As a simple example of the importance of this temporal organization in metabolism, in seasonally obese Syrian hamsters the daily peak of plasma insulin level coincides with the daily peak of lipogenic responsiveness to insulin in the liver and consequently fat synthesis is maximized whereas in seasonally lean hamsters the daily peak of plasma insulin is several hours out of phase with the lipogenic response rhythm peak and very low insulin levels occur during the lipogenic interval of the day (Cincotta and Meier, 1984; deSouza and Meier, 1987; Cincotta et al., 1991). Details of seasonality and the fundamental aspects of circadian rhythms involved in seasonality, especially as it relates to the present topic, are fully described elsewhere (Meier and Cincotta, 1996), however a concise review is necessary here. An enormous amount can be learned of the insulin resistance syndrome by studying circumstances of its natural evolution and expression in the wild. The thrifty gene hypothesis, which proffers the existence of a genome in prehistoric man that facilitates the development of obesity during periods of food abundance as a mechanism to prepare for survival during naturally occurring long periods of low food availability, was ﬁrst proposed by Neel (1962) to explain the increased incidence of obesity and diabetes in afﬂuent, “westernized” societies. The contention is that the insulin resistance syndrome results from activation of the thrifty genome in a “westernized” environment via excesses in dietary fat and caloric consumption. However, fundamental components of this basic concept are seated in the observation of seasonal fattening prevalent among numerous vertebrate species in the wild. And, the scientiﬁc postulate that this seasonal fattening mechanism evolved to support survival over ensuing long periods (months, i.e. season) of extreme low food availability is well established (reviewed in Meier and Cincotta, 1996). Prehistoric man was subjected to the same seasonal pressures of low food availability as vertebrates in the wild were and are today. The so called “thrifty genome” is very old and evolved in vertebrates over hundreds of millions of years as part of an annual cycle of metabolism. This point cannot be overemphasized. Since the hypothalamus and circadian rhythms play dominant roles in the expression of the annual cycle among all vertebrate classes, it follows that they are integrally involved in the physiologic development of the thrifty phenotype, including the insulin resistance syndrome. Before we begin discussing speciﬁc neuronal events within the hypothalamus, critical in the development of the insulin resistance syndrome, it is worth reviewing the salient features of the annual cycle of its expression so well preserved over the evolution of all vertebrate classes. Seasonal fattening in vertebrates is coupled to hyperinsulinemia, insulin resistance, glucose intolerance, and hyperlipidemia (Cincotta and Meier, 1991; Florant and Bauman, 1984; Meier and Cincotta, 1996; Melnyk et al., 1983) in other words the insulin resistance syndrome. This seasonal obesity is associated with increased lipid mobilization and oxidation as well as increased hepatic gluconeogenesis. During the ensuing long period (season) of low food availability, increased utilization of fat as an energy source reduces insulin-mediated

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glucose disposal in muscle and stimulates hepatic glucose production thus channeling endogenous glucose from fat utilizing tissues to glucose requiring tissues such as the brain. Consequently, animals can survive months of starvation with no problem. This seasonal insulin resistance syndrome evolved as a survival strategy, selected for, not against, by natural selection. Individuals not expressing the thrifty genome died out. Indeed, modern day observations of animals in the wild indicate that all or nearly all individuals within any particular group of seasonal vertebrates express the insulin resistance syndrome at the appropriate time of year. There are two subtle but important points here. First, the seasonal insulin resistance syndrome is not the consequence of seasonal mutations but rather seasonal changes in “normal” gene expression. Polymorphisms within the organismal pool of metabolic genes that may predispose to the syndrome need not be required for its development. Second, the syndrome resolves itself at the end of the season (as part of the annual cycle), indicating that the system is reversible or better put, malleable. A search for the understanding of the nature of the syndrome need not be centered on defects so much as on regulation of differential gene expression governing the manifestation of the syndrome: the role of circadian hypothalamic activities. (Obviously, polymorphisms conveying “susceptibility” to the syndrome that reside within this regulatory system will be particularly potent in doing so.) If this timing mechanism is preserved in all vertebrate species including those that do not express seasonal fattening such as humans, as all available evidence suggests (reviewed in Meier and Cincotta, 1996; and in Mercer, 1998), then it follows that all members of any particular vertebrate species may be capable of developing the syndrome. What this means in a strict sense is that all humans may carry “thrifty” genes as appears to be the case for members among other vertebrate species through which the syndrome evolved. The expression of these thrifty genes in seasonal animals encompasses some remarkable physiologic characteristics yielding clues of underlying hypothalamic function. In seasonal animals, body fat level does not simply cycle up and down but rather is defended at very speciﬁc set points during the year. And, during the fat utilization season, animals do not feed even when presented with highly palatable foods until body fat has been reduced to a speciﬁc level (Mrosovsky, 1984). That is, an endogenous regulatory system is selecting fat as a fuel source at speciﬁc times of year. Furthermore, seasonal fattening is not always associated with seasonal hyperphagia or reduced energy expenditure (Dark, 1984). For migratory species, body fat level and energy expenditure are both coincidentally at their greatest during the migration compared to intermigratory periods when food availability is much greater, energy expenditure much less, and body fat level greatly reduced (Meier and Russo, 1984). Another feature of seasonal metabolism is the ability to channel substrates into different metabolic pathways at different times of year. For example, Syrian hamsters go through a dramatic annual cycle of body fat store level without an alteration in daily food consumption. During the fattening season, substrates are channeled into lipogenesis and away from protein turnover. During the fat utilization period, protein turnover increases dramatically associated with growth, reproductive recrudescence, and/or pregnancy and lactation (Cincotta et al., 1993). Such exemplary observations of seasonal metabolism, pervasive among seasonal species of all vertebrate classes, whether migratory, hibernatory, or over wintering, argue for the presence of a central metabolistat: a neuronal organization regulating peripheral metabolism and having adjustable set points inﬂuenced by external and internal (feedback) stimuli. It must be appreciated that the survival advantage conferred by the thrifty genome regulated by this metabolistat is not merely the ability to fatten, but more accurately, the ability to fatten at precisely the right time (of year). As such, the metabolistat must contain an exquisite timing mechanism as one of its inextricable components. A major portion of this metabolistat resides within the hypothalamus that in

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turn contains the suprachiasmatic nuclei (SCN), the mammalian circadian pacemaker system that is pivotal in the temporal organization of circadian events regulating organismal physiology, including metabolism (Moore, 1995). Let us next review the chrono-physiology of the annual cycle before examining these hypothalamic activities. A common misnomer concerning annual cycles of biologic events is that they are merely driven by changes in the environment especially photoperiod, however such is not the case. Rather, annual cycles in vertebrates are the net result of an interaction between photoperiodism and seasonality (Meier and Russo, 1984). Photoperiodism is the differential physiologic responsiveness to daily photoperiods of different length and seasonality is the changing annual responsiveness to the same photoperiod (Elliot, 1976). For example, in laboratory housed female Syrian hamsters, a shift to short daily photoperiods (less than 10 h in length; e.g. winter) following 10 weeks of long daily photoperiods (more than 14 h in length; e.g. summer– autumn) induces the insulin resistance syndrome (photoperiodic response). However, after about 20 weeks on such short daily photoperiods (e.g. spring), the animals respond entirely differently to the same photoperiod and the syndrome disappears without changes in food consumption (seasonal response) (Bartness and Wade, 1984; Cincotta and Meier, 1995). How does this happen? Results from a multitude of studies from different laboratories, primarily that of Meier et al., (1971) (Meier and Russo, 1984; Meier and Wilson, 1984; Meier and Cincotta, 1996; Temporal interaction of neural oscillations A and B produce:

B Short day metabolic set point A

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Figure 1 An internal coincidence model for photoperiodism. Metabolic conditions (e.g. increases or decreases in body fat stores) in photoperiodic animals are determined by the phase relationship of two circadian neuroendocrine oscillations. One oscillation (A) is directly entrained by the daily photoperiod (e.g. light onset). The second oscillation (B) is loosely coupled with oscillation A in different phase relationships as a function of day length (i.e. whether or not light coincides with a reactive [i.e. sensitive] phase of oscillation A). (Reproduced from Diabetes Reviews, 4, 464–487.)

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One seasonal condition (e.g. scotosensitive) – obese B

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Figure 2 Temporal synergism model for seasonality. Although day lengths may be similar at two seasons, metabolic responsiveness to the day lengths are dissimilar. Phase relationships between rhythms (e.g. plasma hormones and suprachiasmatic neurotransmitters) differ at the two seasons even when the photoperiod is similar. Phase relationships of neural and hormonal rhythms may be altered by treatments with hormones and drugs that alter metabolic conditions and simulate seasonal differences. (Reproduced from Diabetes Reviews, 4, 464–487.)

Meier, 1993), have led to the construct that the annual cycle of physiologic events in vertebrates is a function of changing temporal interactions of (at least two) circadian neural oscillations within the CNS. It has been established that circadian rhythms play central roles in both photoperiodism and seasonality. Regarding photoperiodism, the daily photophase sets a rhythm of photosensitivity to light so that a sensitive interval occurs 12–24 h after the daily onset of light. If light is present during this sensitive interval it acts as an inducer of the photoperiodic effect. According to the internal coincidence model of photoperiodism, physiologic conditions in photoperiodic animals are determined by the phase relation of two circadian neural oscillations. The daily onset of light sets the phase of one circadian oscillation and if light is present later in the day (e.g. 14 h later) during the sensitive or reactive phase of this oscillation, a second neural oscillation is coupled to it in a speciﬁc temporal relation. If light is not present later in the day (e.g. short daily photoperiods of 10 h) then the second oscillation assumes a different phase relation to the ﬁrst light sensitive neural oscillation (Moore-Ede and Moline, 1985). Regarding seasonality, a circannual clock directs a shift in the phase relation of these oscillators without any change in the photoperiod. Evidence for this comes from observations of changes in peripheral hormone rhythms, believed to be expressions of these oscillations as well as from changes in rhythms of neurotransmitter measurements within the SCN in animals held on constant photoperiods at different times of year (Meier, 1993) (Figures 1–3). These two circadian neural oscillations are believed to interact with or reside within the SCN (see below). In turn, the SCN strongly modulate activities in the ventromedial hypothalamus

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Figure 3 Circadian organization in the annual cycle of the Syrian hamster. Seasonal changes in body fats stores are consequences of changes in day length (i.e. photoperiodism) coupled with seasonal alterations in responsiveness to day length (seasonality: scotosensitive and scotorefractory responsive conditions). Scotosensitive female hamsters become fat in response to the short day lengths that occur naturally during the fall. After ~20 weeks of short day lengths, the hamsters become scotorefractory to the stimulatory effects of short day lengths (i.e. long dark periods) so that body fat stores are depleted and remain reduced in hamsters retained on short day lengths. Long day lengths for 8–10 weeks, occurring naturally during summer, are required to reinitiate scotosensitivity to short day lengths. Day length coupled with seasonality establishes the phase relationships of two or more circadian pacemaker oscillations which in turn are thought to produce low (A) or high (B) fat stores by way of a temporal synergism of their circadian expressions. (Reproduced from Diabetes Reviews, 4, 464–487.)

(VMH), lateral hypothalamus (LH), dorsomedial hypothalamus (DMH), paraventricular nuclei (PVN), and other hypothalamic and extra-hypothalamic nuclei regulating peripheral metabolism (Moore, 1995; Nagai and Nakagawa, 1992). Circadian neuronal events within the SCN, acting through these CNS metabolic centers, produce circadian rhythms of stimulus (e.g. insulin) and target tissue response (e.g. hepatic lipogenic reposes to insulin) rhythms in the periphery that interact temporally to produce organismal physiologic states, such as body fat store level. The phase angle between the stimulus and response rhythm governs the net effect as described above for fattening in the Syrian hamster. A multitude of such stimulus and response rhythms are ultimately integrated to produce an overall metabolic status (reviewed in detail in Meier and Cincotta, 1996). An important point here is that circadian activities regulate metabolic states not generally viewed in circadian terms, such as level of obesity, degree of insulin resistance or glucose intolerance, magnitude of hyperlipidemia, etc. A second important point is that the SCN receive input from not only the eye via the optic chiasm, but also from non-photic centers throughout the CNS including the intergeniculate leaﬂet, other hypothalamic nuclei, the peripheral neuroendocrine system and humoral factors, including metabolites (and therefore food itself ). As such, physiologic (or pharmacologic) modulation of this circadian metabolic control center need not itself be circadian at all. Therefore, neither inputs (e.g. food quality) to nor the organismal level output (e.g. expression of metabolic

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status – insulin sensitive or resistant) from this circadian regulatory center for peripheral metabolism are necessarily circadian in nature (Figure 4). This may explain why many investigators in the ﬁeld of metabolic physiology have failed to appreciate the important role of circadian neuroendocrine activities in the regulation of metabolism. However, an excellent example of this physiologic organization is the observation that the annual cycle of physiologic activities, although regulated by circadian rhythms and endogenous in nature, can be inﬂuenced by the quality of foods consumed at different seasons. Food quality (molecular constituents of food) for foraging animals changes seasonally with the life cycles of ﬂora and fauna prey. This annual cycle of food quality interacts with and upon the above-described circadian neural oscillators to modulate the CNS regulation of the annual cycle (Meier and Cincotta, 1996). The expression of the thrifty genome evolved under such circumstances, and therefore, is likely regulated Hypothalamic circadian pacemaker (e.g. mammalian SCN) oscillation I

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Figure 4 Circadian metabolistat and blueprint for regulation of metabolism. The metabolistat is thought to have circadian components that include the primary circadian pacemaker (i.e. mammalian SCN) and a hierarchy of secondary (e.g. VMH) centers. The set point of the metabolistat is determined in many vertebrate species by variations in day length and ambient temperature (especially ectothermic vertebrates) to produce seasonally appropriate changes in metabolic conditions (e.g. migratory and overwintering fattening). The set point is adjusted by altering the phase relationships of at least two major pacemaker oscillations. Each of the pacemaker oscillations in turn entrains separate neural and hormonal circadian expressions (e.g. autonomic activities, pituitary and pancreatic hormones). Accordingly, alteration of the phase relationships of pacemaker rhythms alters the phase relationships of numerous regulatory rhythms that synergize temporally at the cellular level to produce a wide spectrum and gradation of physiological conditions. This ancient circadian system is thought to persist in humans and to have important organizational functions. Deterioration of this circadian system, as in aging, is thought to be the basis of some age-related diseases (e.g. NIDDM). Adjustments of the circadian metabolistat, as with bromocriptine and other neurotransmitter-affecting drugs, may be an effective treatment aimed at the cause rather than the symptoms of many disorders. (Reproduced from Diabetes Reviews, 4, 464–487.)

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by this interaction of food quality with an endogenous circadian neuroendocrine control system for metabolism (discussed later in this chapter). Let us now more closely examine the SCN regulation of metabolism.

HYPOTHALAMIC SCN INVOLVEMENT IN THE INSULIN RESISTANCE SYNDROME The ﬁrst evidence that interactions of circadian activities within the hypothalamic SCN modulate metabolism, including the expression of the thrifty phenotype, came indirectly from observations that the circadian rhythms of plasma hormone levels critical in the regulation of metabolism, particularly prolactin and cortisol, differed in seasonally lean and obese animals (Meier, 1972). The expressions of these hormone rhythms are governed to a large extent by the SCN (Hastings et al., 1995; Buijs et al., 1999; Mai and Pan, 1995). Merely simulating the circadian pattern of plasma prolactin and cortisol of a particular season by injection of the hormones at the appropriate times of day in animals held under constant light conditions produce the full physiology of that particular season, irrespective of the actual time of year when the study was conducted. That is, injections of prolactin and cortisol into lean animals at the times of day they peak in the blood of obese animals produced obese animals, while injections of the same hormones into obese animals at the time of day they peak in lean animals produced lean animals (Meier, 1972; Meier and Burns, 1976; Meier et al., 1971; Spieler et al., 1978). This rather intriguing phenomenon needs to be carefully contemplated and fully appreciated if an understanding of neuroendocrine regulation of metabolism is being sought. Moreover, the effects of these timed hormone treatments were not subtle in magnitude, altering fat store levels by 2–5-fold. And, these effects were demonstrable among species of all the major vertebrate classes. Although prolactin and cortisol have direct effects on peripheral metabolic tissues, it was postulated that their time of day dependent metabolic effects were somehow via the CNS inasmuch as their timed injections inﬂuenced other physiologic parameters of the annual cycle such as reproduction and behavior (Meier, 1993) and the effects of these timed hormone injections persisted long after the termination of treatment (Meier et al., 1981; Cincotta et al., 1989). Such long-term inﬂuences of timed prolactin and corticosteroid treatment on metabolism were demonstrable in non-seasonal as well as seasonal animals, preventing the age associated development of insulin resistance in rats for several months after the termination of a 10-day treatment (Cincotta et al., 1993). It was suspected that the neuronal targets for prolactin and cortisol may be dopaminergic and serotonergic neurons, respectively. Prolactin stimulates dopamine synthesis by increasing the amount and activity of tyrosine hydroxylase, a rate-limiting enzyme for dopamine synthesis (Moore et al., 1980). Similarly, cortisol stimulates the synthesis of serotonin by increasing the activity of tryptophan hydroxylase activity (Telegdy and Vermes, 1975), which converts tryptophan to 5-hydroxytryptophan (5-HTP). To test this hypothesis, Meier et al. (1971) designed experiments wherein different groups of animals were injected daily with 5-HTP (precursor for serotonin that crosses the blood brain barrier) and L-DOPA (precursor for dopamine that crosses the blood brain barrier) at one of several different time interval relations (i.e. at 0, 4, 8, 12, 16, or 20 h apart) for 10 days while held on constant light conditions (Miller and Meier, 1982; Emata et al., 1985; Wilson and Meier 1989). Injections of 5-HTP and L-DOPA could induce the physiologic characteristics of each different season as a function of the daily interval between their injections in vertebrate species from ﬁsh to mammals. These ﬁndings strongly suggest that central circadian dopaminergic and serotonergic activities each impinge upon and modulate the phase of circadian

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neural oscillations directly regulating peripheral metabolism (including the development and reversal of the insulin resistance syndrome) as a function of the time of day of their activity. Alternatively, dopaminergic and serotonergic activities may actually comprise the two neural oscillators regulating metabolism. In either case, where is this happening in the brain? The most logical place to begin looking for this center is the SCN. SCN physiology is exceedingly complex and a science unto itself and beyond the focus of this chapter. However, the metabolic physiologist interested in hypothalamic involvement in the insulin resistance syndrome must ﬁrst be aware of some basic features of these nuclei to fully appreciate their role in this syndrome. These features relevant to the present discussion may be brieﬂy summarized as follows. As mentioned above, the SCN are the seat of the primary mammalian circadian pacemaker system, responsible for the orchestration of organismal rhythms of physiology and behavior (Moore, 1995). Circadian rhythmicity of neuronal activity of these nuclei is maintained following knife cuts isolating them from all other neuronal input, indicating that they act as an independent oscillator system (Inouye and Kawamura, 1982). And, SCN explants and cell preparations maintain rhythmicity in vitro (Green and Gillette, 1982; Earnest et al., 1999). Ablation of the SCN results in the loss of most physiologic (e.g. metabolic, reproductive, endocrine, immunologic) and behavioral circadian rhythms in vivo (reviewed in Pickard and Turek, 1983). Transplantation of SCN from donor to SCN-ablated host restores physiologic rhythmicity of the host with a circadian timing characteristic of the donor (Ralph et al., 1990). Evidence from different lines of investigation indicates that the SCN comprise multiple circadian oscillator systems that can function independently with different phases. Moreover, different oscillators appear to be anatomically distinct within the SCN (Welsh et al., 1995; Zlomanczuk et al., 1991; Harrington et al., 1994; Pickard and Turek, 1983). These different neuronal oscillations may direct different physiologic rhythms that interact at various levels to produce an overall organismal physiologic character, a part of which would be body fat level, as will be discussed. Although circadian oscillator activity is intrinsic to the SCN, the circadian character (e.g. phase, amplitude, period) of these neuronal activities may be modulated by external and internal cues. The SCN receive photic information from the retino-hypothalamic tract and the intergeniculate leaﬂet that modulate its activity (Treep et al., 1995), however several nonphotic neuronal stimuli can also function in this capacity (Mrosovsky, 1991; Reebs and Mrosovsky, 1989; Zhang and Rusak, 1989). The SCN receive input from several CNS centers (hypothalamic and extra-hypothalamic) not connected to the eye (Pickard, 1982; Legoratti-Sanchez et al., 1988; Meyer-Bernstein and Morin, 1996). The list of molecules (endogenous and exogenous) known to inﬂuence SCN activities is growing and includes a wide variety of neurotransmitters and neuropeptides, including neuropeptide Y (NPY) (Mai and Pan, 1995; Moore, 1993; van den Pol, 1993; Ding et al., 1994; Janik et al., 1995; van den Pol and Tsujimoto, 1985; Piggins et al., 1995). The important point here is that SCN activities governing peripheral metabolism can be altered by nonphotic cues including food-derived metabolites such as tryptophan (Glass et al., 1995). Among these modiﬁers of SCN activities, those relevant to this discussion include prolactin, corticosteroid hormone, dopamine and serotonin. A multitude of studies have now demonstrated integral roles for SCN serotonin (intrinsic and from the raphe nuclei) in the regulation of oscillator functions therein (Kohler et al., 1999; Horikawa et al., 2000; Szafarczyk et al., 1981; Williams et al., 1983). Pulsed serotonin or serotonin-agonist administration (systemically or directly to the SCN) can phase-shift SCN circadian neuronal activity translating into alterations in animal locomotor activity and neuroendocrine rhythms, including the rhythm of plasma corticosterone, a key player in the insulin resistance syndrome (see below). Similarly, dopamine has been shown to inﬂuence the character of SCN activity (Yamada and Martin-Iverson, 1991).

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What evidence is there that the SCN regulates metabolism? In nocturnal rodents typically used in metabolic research such as rats, mice, and hamsters, circadian rhythms of feeding, macronutrient selection, energy expenditure, and intermediary metabolism including lipid and glucose ﬂuxes are very marked (reviewed in detail in Nagai and Nakagawa, 1992). Generally, during the fasting portion of the day (light period) sympathetic and endocrine activities are increased which drive increases in lipolytic and hepatic glucose output (HGO) functions. At this time, whole body fat oxidation is increased and glucose storage and oxidation decreased while increased HGO supports the glucose requiring tissues such as the brain. Contrariwise, during the feeding period (dark phase) parasympathetic and endocrine processes are increased that support lipogenesis and glucose storage. For example, the rise in plasma corticosteroid just before the onset of dark stimulates NPY release at the PVN that coincides temporally with the daily peak in PVN sensitivity to NPY and adrenergic ␣2 receptor number to induce feeding and increased insulin secretion (Bhakthavasalam and Leibowitz, 1986; Akabayashi et al., 1994). Inasmuch as interest has centered around the inﬂuence of insulin on feeding, it is noteworthy that insulin administration to the SCN in the light period stimulates feeding yet during the dark inhibits it. And, insulin binding to the SCN is increased in the light versus dark period of the day (Nagai and Nakagawa, 1992). Such a physiologic construct may support feeding during the dark when insulin levels are high and allow for the curtailment of feeding at the onset of light when SCN insulin sensitivity is increasing. To digress for a moment, this circadian regulation of metabolism is, in effect, a microcosm of the organization of the annual cycle of metabolism, respecting periods of feeding and starvation. During different points in the annual cycle of metabolism, the daily peaks of sympatheticneuroendocrine and parasympathetic-neuroendocrine activities are adjusted to different set points. As animals make the transition from lean, insulin sensitive to obese, insulin resistant the parasympathetic to sympathetic activity ratio increases (to support fattening) to be followed very rapidly by an increase in sympathetic activity (to support fat mobilization and utilization) so that the parasympathetic : sympathetic activity ratio is re-established at a different higher level (Figure 5). That is, in the obese, insulin resistant state both parasympathetic and sympathetic activities are proportionately increased and a new steady state of metabolism is established. This autonomic organization is common among a wide variety of obese insulin resistant animal models and will be discussed in detail later. All of the circadian metabolic activities described above as well as the daily rhythms of insulin, glucagon, corticosteroids, and several other metabolic hormones are lost following ablation of the SCN (reviewed in Nagai and Nakagawa, 1992). A major clue as to the neuronal circuitry by which the SCN exerts its inﬂuence on peripheral glucose and lipid metabolism comes from studies of circadian metabolic responses to intracranial injections of 2-deoxy-glucose. 2-deoxy-glucose cannot be metabolized to generate ATP and its overabundance in the extracellular space confers a hypoglycemic environment to neurons. Intracranial injections of 2-deoxy-glucose induce a rapid increase in plasma glucagon, glucose, and free fatty acids (FFA) as well as feeding. However, the hyperglycemic, lipolytic, and hyperphagic counter-regulatory response to intracranial 2-deoxy-glucose is time of day dependent in nocturnal rodents, being predominately expressed during the light period of the day. And, SCN lesions abolish this circadian CNS response to 2-deoxy-glucose (Nagai and Nakagawa, 1992). That is, following SCN lesion there is now essentially no metabolic response to intracranial 2-deoxy-glucose at any time of day! Remember that during the light period in nocturnal rodents, sympathetic functions drive mobilization of lipid and glucose for utilization during this fasting period and parasympathetic activities drive anabolic activities during the dark (feeding) phase of the day. The SCN modulate the amplitude of sympathetic functions during

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C

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Figure 5 Schematic of the daily variations in sympathetic (solid line) and parasympathetic (dashed line) activities of nocturnal seasonal mammals as they transition from the lean, insulin sensitive (A) through the initiation of the fattening, insulin resistant (B) to the newly established obese, insulin resistant steady state (C).

the light period via connections to the VMH, PVN, and locus coeruleus (LC), all integrated nervous centers for sympathetic outﬂow to the periphery (Nagai and Nakagawa, 1992; Legoratti-Sanchez et al., 1988). Likewise, the SCN modulate the amplitude of parasympathetic activity during the dark period of the day via connections to the LH and vagus nerve (Nagai and Nakagawa, 1992). Therefore, the seasonal changes in sympathetic and parasympathetic amplitudes, described above, that inﬂuence the seasonal development and regression of the obese insulin resistant state are regulated to a large extent by the SCN. Available information at this time indicates important roles for SCN interactions particularly with the VMH and PVN in the modulation of peripheral metabolism. The exact neurochemistry within the VMH and PVN pivotal in the development of the insulin resistant syndrome that is being modulated by the SCN will be reviewed later. First, let us ﬁnish our review of SCN functions governing metabolism by moving from neuronal activities to neurochemical investigations therein. The 5HTP and L-DOPA experiments relating to seasonality described above suggest that circadian dopamine and serotonin activities are interacting temporally to inﬂuence seasonal metabolism, including the expression of the insulin resistance syndrome. Also, as just described, the probable site of this interaction is the SCN where such treatment can set the phase of neural oscillators, the temporal interactions of which may regulate peripheral metabolism. If this is the case, then the endogenous rhythms of dopaminergic and serotonergic activities at the SCN should vary between insulin resistant and sensitive animals. And, one should be able to induce the insulin resistant or sensitive states by mimicking the SCN dopamine and/or serotonin rhythms observed in insulin resistant or sensitive animals, respectively. All available experimental evidence conﬁrms these suppositions. Syrian hamsters shift from glucose tolerant to intolerant without any change in photoperiod, food consumption, or body weight within a time frame of a few weeks at the appropriate time of year (Cincotta et al., 1991). During this transitional time period, studies were undertaken by our laboratory

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wherein animals of similar body weight were selected that were either glucose tolerant (just before the shift) or intolerant (just after the shift) and the in vivo daily variations in dopamine and serotonin metabolism at the SCN were determined. It was found that, indeed, there was a marked difference in the daily variations of these monoamine metabolites at the SCN between glucose tolerant and intolerant animals. The prominent and coincident nocturnal peaks of SCN extracellular dopamine and serotonin metabolites in glucose tolerant animals were absent in glucose intolerant animals (Luo et al., 1999a). It would be of interest to determine if selectively reducing the nocturnal monoamine activities at the SCN in glucose tolerant animals would induce glucose intolerance. In fact, selective destruction of dopaminergic input to the SCN, via the use of site-speciﬁc neurotoxin, to mimic the low dopaminergic activity present in glucose intolerant animals produces a severe hyperinsulinemic, insulin resistant, and glucose intolerant state within just a couple of weeks (Luo et al., 1997). This response is later followed by the development of obesity. The anatomical origin of the dopaminergic neurons projecting to the SCN that are involved in this response is currently unknown, however dopamine producing neurons have been identiﬁed just outside the SCN suggesting that local dopaminergic neurons inﬂuence SCN function (Novak and Nunez, 1998). A major point here is that a very small group of neurons has a dramatic inﬂuence on peripheral metabolism. Likewise, a selective destruction of serotonergic neurons at the SCN of glucose tolerant hamsters also produces a severe glucose intolerant state (Cincotta et al., unpublished data). These serotonergic neurons may arise from the raphe nuclei or within the SCN itself. In accordance with these observations, bromocriptine (sympatholytic, dopaminergic agonist with serotonergic modulator activity) treatment that completely abolishes the obese, insulin resistant condition in seasonal hamsters also re-establishes the nocturnal peak of SCN serotonergic activity in these animals to mimic that of seasonally lean insulin sensitive animals (Luo et al., 2000). Furthermore, in leptin deﬁcient ob/ob mice the normal circadian expression of c-fos and per genes, that are integral parts of cellular clock mechanisms within SCN neurons, is markedly deranged relative to normal lean mice. And, dopaminergic treatment restores these SCN biochemical clock activities to normal while also normalizing metabolism in these ob/ob mice ( Jakubowski et al., 1998; Bina et al., 1999; Scislowski et al., 1999). Inasmuch as circadian neuroendocrine regulation of metabolism may not be familiar to many readers, let us ﬁnish this discussion on SCN functions in the modulation of metabolism by reviewing the main points presented thus far. ●

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The thrifty genome evolved in a cyclic environment where food quality and quantity varied seasonally. The thrifty genome is comprised of circadian components, changing temporal interactions of which allow for the seasonal expression of the obese, insulin resistant condition at precisely the appropriate time of year enhancing survival during very long periods of ensuing low food availability. Circadian activities within the pacemaker, the SCN, modulate parasympathetic, sympathetic, and endocrine functions governing peripheral metabolism. Temporal interactions of (at least two) circadian neural oscillators within the SCN regulate the annual cycle of metabolism in vertebrates as a function of their phase relation (Figure 4). These SCN neural oscillators are themselves comprised of or modulated by dopaminergic and serotonergic activities. The daily rhythms of SCN dopaminergic and serotonergic activities vary in insulin resistant vs. sensitive animals.

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Mimicking the phase relation of dopaminergic and serotonergic activities at the SCN observed in insulin resistant or sensitive animals by direct SCN modiﬁcations or systemic precursor treatments produces sensitive or resistant animals, respectively. Alterations of SCN activities are associated with insulin resistant states in seasonal as well as non-seasonal animals such as aging rats and ob/ob mice. Complete electrolytic ablation of the SCN induces obesity and shortens the life span of rodents. Transplantation of fetal SCN into older adults extends their life span (Nagai and Nakagawa, 1992; Hurd and Ralph, 1998; Viswanathan and Davis, 1995).

NEUROCHEMISTRY OF VMH NEURONS INVOLVED IN THE INSULIN RESISTANCE SYNDROME What hypothalamic centers might the SCN communicate with to regulate peripheral glucose metabolism and insulin sensitivity? A large clue to this answer may be derived from the ﬁndings described earlier that the circadian rhythm of 2-deoxyglucose induced hyperglycemia is abolished following SCN lesion. It has been established that the VMH contains glucose sensing neurons able to induce counter-regulatory responses to systemic and local hypoglycemia including increased sympathetic activity and glucagon secretion to increase plasma glucose and FFA levels (Borg et al., 1995). The implication here is that the described SCN regulated responses to hypoglycemia are mediated via the VMH inasmuch as the SCN communicates with the VMH via direct and indirect connections. Based on these ﬁndings, our laboratory decided to investigate the possibility that SCN driven seasonal changes in insulin sensitivity may involve neurochemical changes within the VMH. Our initial focus was on VMH monoamine metabolism since it had been demonstrated that acute norepinephrine (NE) administration to the VMH induced a rapid simultaneous rise in plasma glucose, FFA, glucagon, and insulin in part via the sympathetic nervous system (SNS) (De Jong et al., 1977; Steffens et al., 1984); a neuroendocrine situation characteristic of insulin resistant/diabetic states (Unger, 1995). Moreover, serotonergic activities within the VMH function to potentiate these sympathetic responses to NE (Suzuki et al., 1995; reviewed in Luo et al., 1998). The in vivo microdialysate 24-h levels of VMH extracellular NE and serotonin metabolites, indicators of neurotransmitter release, were assessed in seasonally insulin resistant and sensitive animals. It was found that the 24-h levels of VMH NE and serotonin metabolites were much higher in seasonally resistant vs. sensitive animals (Luo et al., 1998). Increases in VMH extracellular NE metabolites were also observed in obese vs. lean Zucker rats (Luo et al., 1996) and in adult offspring of malnutritioned or insulin treated pregnant rats ( Jones et al., 1995). Also, the extracted levels of VMH NE are much higher in leptin deﬁcient ob/ob and leptin unresponsive db/db mice than in their normal counterparts (Garris, 1995; Oltmans, 1983). The important underlying reality is that increased VMH noradrenergic activity and/or level is consistently observed in a wide variety of insulin resistant animal models. The next important step would be to examine if chronic elevation of VMH NE level in normal animals via chronic microinfusion would induce the insulin resistant state to establish a cause–effect relationship between this VMH neurochemistry and the insulin resistance syndrome. In fact, chronic infusion (for only 14 days) of NE into the VMH of normal animals induces a profound insulin resistant state with major physiologic and biochemical hallmarks of the syndrome without inducing hyperphagia (Cincotta et al., 2000; Luo et al., 1999a). These physiologic changes are worth reviewing at this time since many coincident events induced by VMH NE infusion, although characteristic of the syndrome, may not be well appreciated as having a common origin.

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First of all, as referenced above, VMH NE activity stimulates speciﬁc sympathetic functions activating HGO. VMH NE activity induces glucagon release and increases liver glycogenolysis and gluconeogenesis (Shimazu, 1987). Also, such VMH NE action potentiates an increase in basal and adrenergic stimulated lipolysis from white adipose tissue (WAT) (De Jong et al., 1977; Cincotta et al., 2000). Consequently, increases in VMH NE activity increase plasma FFA and glucose levels. The chronic increase in plasma FFA, in and of itself, can potentiate increased HGO, plasma triglyceride levels, insulin resistance in liver and muscle, and ␤ cell dysfunction characteristic of the syndrome (Unger, 1995). Glucose and insulin levels during a glucose tolerance test of VMH NE infused animals are greatly increased relative to sham controls. And, such treatment also induces a marked basal hyperinsulinemia. How can increased VMH NE activity induce hyperinsulinemia if sympathetic stimulation is a potent inhibitor of glucose stimulated insulin release from the ␤ cell? Sympathetic hyperinnervation to the pancreatic ␤ cell can induce ␤ cell desensitization to (such inhibitory) ␣-adrenergic stimuli (Grodsky et al., 1997). This occurrence, coupled with a VMH NE induced stimulation of vagal tone to the pancreas (Steffens et al., 1984) and induced glucagon secretion may provide additional stimuli beyond increased plasma FFA and glucose levels accounting for the sustained increased insulin secretion observed following acute and chronic VMH NE administration (Liang et al., 1999). Respecting energy balance, VMH NE infusion stimulates dramatic (3–5-fold) increases in adiposity without inducing changes in body weight or food consumption. During the ﬁrst few days of infusion, adipose lipogenesis increases over 4-fold and adipose lipolysis also increases at this time by 2-fold. Therefore, a unique physiologic situation is created wherein eventhough lipid mobilization is increased, fat deposition still occurs. Determinations of respiratory quotient (RQ) over this period reveal a lipogenic state with RQs above 1.0. And, plasma FFA levels are decreased (and triglycerides increased) relative to controls due to increased liver re-esteriﬁcation. With increasing time beyond 2 weeks, whole body RQ drops below control values of 1.0 and plasma FFA levels now increase. This indicates an increase in fat oxidation to match the increased synthetic rate and consequently fat increases are halted and maintained thereafter at a new higher steady state (i.e. simultaneously increased and balanced lipogenic and lipolytic/oxidative activities). During this time period of rise in plasma FFA level, glucose intolerance emerges likely as a culmination of all the preceding physiologic alterations described thus far. Also during this time, brown fat adiposity increases and there is a reduction in brown fat thermogenesis as indicated by a reduction in whole body energy expenditure and heat production (Cincotta et al., 2000). This may explain in part the dramatic increase in white adipose fat level without a concurrent increase in food consumption following this VMH NE treatment. The increases in lipogenesis may be driven in part by the hyperinsulinemia, however what causes the decrease in brown fat activity following VMH NE infusion? Iontophoretic application of NE into the VMH inhibits neuronal activity therein and this effect may be mediated in part via GABA (Kraszewski and Cincotta, 2000; Beverly et al., 1998). In accordance with this and the above discussion, GABA release is increased in the VMH of obese, insulin resistant animal models (Spector et al., 1996a). Glutamate application to the VMH stimulates sympathetic ﬁbers innervating the brown fat and results in increased thermogenic activity (Amir, 1990; Yoshimatsu et al., 1993). NE application to the VMH inhibits glutamate evoked neuronal activity therein. More importantly, VMH neurons within obese, insulin resistant ob/ob mice are much more sensitive to NE induced inhibition of glutamate evoked neuronal activity than those within lean mice (i.e. lower threshold and greater duration of electrophysiologic response to NE in ob/ob vs. lean mice) (Kraszewski and Cincotta, 2000). This very likely may contribute to the low thermogenic activity of brown fat

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in ob/ob mice and other models of obesity/insulin resistance. There are two intriguing and critical points to be appreciated here. First, VMH NE activity can simultaneously inhibit sympathetic activation of brown fat thermogenesis and stimulate sympathetic activation of HGO and white adipose lipolysis. Remember, this same VMH NE activity can increase glucose stimulated insulin secretion by activation of vagal connections to the pancreas and also via a desensitization of ␤ cell responsiveness to resultant increased sympathetic stimuli. Furthermore, VMH NE infusion increases circulating NE and epinephrine (E) levels while decreasing brown fat thermogenesis (Cincotta et al., 2000b). This suggests that such infusion not only decreases sympathetic stimulation of brown fat via VMH neurons but also desensitizes the brown fat to circulating NE. As a consequence of these functions, increased VMH NE activity results in stimulation of speciﬁc parasympathetic and sympathetic activities as well as inhibition and deactivation of other sympathetic functions to potentiate the insulin resistance syndrome (Figure 6). In this regard, unilateral VMH NE infusion creates a physiology similar to that resulting from bilateral VMH lesions except that NE infusion does not produce hyperphagia (Penicaud et al., 1989). Second, both VMH NE release and postsynaptic responsiveness to NE may be elevated in the obese, insulin resistant state, a physiologic condition not “normally” encountered. In normal physiologic states, neuronal presynaptic NE release rate is inversely coupled to postsynaptic receptor responsiveness (Cooper et al., 1991). The inference here is that this simultaneously increased VMH NE release rate and postsynaptic responsiveness is not “abnormal” but rather exemplary of “normal” neurophysiologic plasticity potentiating the insulin resistance syndrome, a naturally evolved condition that cycles annually among animals in the wild. How this annual “switch” takes place is obviously well worth investigating. Support for this simultaneously increased noradrenergic stimulus and response system comes from studies in female ob/ob mice and obese humans. Female ob/ob mice have increased VMH NE activity

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Figure 6 Metabolic consequences of increased VMH NE activity. SNS: sympathetic nervous system, HGO: hepatic glucose output; WAT: white adipose tissue; FFA: free fatty acids; NE: norepinephrine; E: epinephrine; PSNS: parasymapathetic nervous system; BF: brown fat; stimulation, inhibition.

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levels of daily urinary noradrenergic metabolite and physiologic responsiveness to NE (Leigh et al., 1992; Liang et al., 1999) and obese humans exhibit increased sympathetic tone and increased responsiveness to the lipolytic effects of NE in visceral fat (Lonnquist et al., 1994; Scherrer et al., 1996; Grassi et al., 1996; Hogikyan et al., 1999). To further explore the nature of this apparent simultaneously increased NE stimulus–response system in the VMH, we analyzed the adrenergic receptor proﬁle of discrete hypothalamic nuclei in lean and ob/ob mice. It was found that ␣1, ␤1, and ␤2 ligand binding was in fact increased in the VMH, LH, and PVN of ob/ob vs. lean mice (Boundy and Cincotta, 2000). Furthermore, the ␣2A ligand binding was decreased in the VMH and DMH of ob/ob mice relative to lean controls. The increased VMH ␣1 and ␤ binding capacities can support the increased electrophysiologic responsiveness to NE observed in ob/ob mice. And, decreased VMH ␣2A binding can potentiate an increase in NE release rate in these obese mice. In the hypothalamus, presynaptic ␣2 receptors are ␣2A and NE stimulation of ␣2A receptors is a primary stimulus for inhibition of NE release (Sperlagh et al., 1998). Therefore the hypothalamic noradrenergic receptor proﬁle supports the neurophysiologic state … concurrent increased NE release and postsynaptic responsiveness in the VMH of insulin resistant animals. This VMH receptor proﬁle in the insulin resistant condition has important ramiﬁcations for whole body glucose homeostasis. The VMH is a glucose sensor, able to induce, via NE release and subsequent stimulation of the SNS, a rapid rise in blood glucose in response to systemic or local hypoglycemia (Borg et al., 1995; Shimazu, 1987; Steffens et al., 1984). Hypoglycemia decreases medial hypothalamic ␣2 binding thereby increasing NE release to induce the counter-regulatory response (Chafetz et al., 1986). In normal animals, however, local VMH hyperglycemia blocks this response to systemic hypoglycemia (Borg et al., 1997) and there is a positive correlation between the plasma glucose level and the medial ␣2 binding capacity. As such, in normal animals, high glucose levels turn off VMH NE drive for increased HGO. In insulin resistant states associated with hyperglycemia this is not the case and this is a very important point. For instance, the hyperglycemia of ob/ob mice is coupled to an increased VMH NE level, postsynaptic noradrenergic ␣1 and ␤ binding, and decreased ␣2A binding; all potentiating increased HGO. The set-point for VMH “sensing” of hypoglycemia essentially is raised in insulin resistant states and in ob/ob mice has been raised into the hyperglycemic range (i.e. the VMH neurophysiology of ob/ob mice, even at plasma glucose levels well above 250 mg/dl, is as that of a normal animal constantly sensing and responding to hypoglycemia). Likewise, diet-induced obesity prone rats have a very similar loss of normal VMH ␣2 adrenergic receptor and neuronal responsiveness to glucose predisposing to VMH NE driven increases in plasma glucose (Levin and Planas, 1993; Levin et al., 1998). The decreased ␣2 binding in the DMH of ob/ob mice as well can facilitate SNS activity to increase HGO (Bernardis and Bellinger, 1998). This “adjustable” hypothalamic set point for stimulation of HGO appears to be positively correlated with the hypothalamic NE to dopamine ratio and this will be discussed later. What are the stimuli for these neurophysiologic changes in the VMH (and DMH) potentiating increased NE action and thereby the insulin resistance syndrome? One such stimulus appears to be insulin. Hyperinsulinemia reduces medial hypothalamic ␣2 binding (Levin et al., 1998). And, we have demonstrated that a hyperinsulinemic–euglycemic clamp in normal rats induces sustained VMH release of NE (Cincotta et al., 2000a). Whether this insulin action is direct or indirect is not known, however, it has been shown that direct application of insulin to hypothalamic preps in vitro does stimulate NE release (Boyd et al., 1986; Sauter et al., 1983). Therefore, hyperinsulinemia arising from any exogenous or endogenous source(s) may feedback positively to sustain the hyperinsulinemic condition by way of increasing VMH NE activity. In this context, it may be very germane that diet-induced obesity prone rats can be identiﬁed prospectively by a strong sympathetic response to intravenous glucose infusion.

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Rats that exhibit a sharp rise in plasma NE in response to glucose infusion go on to become obese in response to a sucrose-fat diet whereas those that do not show this response to glucose do not become obese on the same diet (Levin and Sullivan, 1987). In line with the above described ﬁndings, a possible contributory cause for this prospective identiﬁcation of dietinduced obesity prone rats may be an increased sensitivity to hyperinsulinemia (induced by the diet) by the medial hypothalamus (i.e. increased NE action therein) and prospectively identiﬁed by the glucose challenge associated hyperinsulinemia. How this adjustable set-point for VMH sensitivity to hyperinsulinemia is coupled to that for plasma glucose level described above is presently unknown and needs to be delineated. However, all available evidence suggests that an increased sensitivity to the VMH NE activating effects of insulin in turn may raise the VMH set-point for glucose stimulation of these same VMH NE activities (i.e. classic VMH counter-regulatory response to hypoglycemia at high(er) plasma glucose levels). Insulin, by inducing plasma amino acid ﬂux into the peripheral issues, shunts protein-bound tryptophan to the brain, where it now outcompetes other neutral amino acids for transport into neurons. As such, hyperinsulinemia can increase CNS serotonin level that in turn potentiates NE effects in the VMH (Lou et al., 1998, 1999b). Thus, in insulin resistant states the euglycemic– hyperinsulinemic or hyperglycemic–hyperinsulinemic conditions feedback on the medial hypothalamus (and likely elsewhere) to maintain the condition (Figure 7) and conceivably hyperinsulinemia could contribute to the initiation of the insulin resistant state in part through such a central mechanism. The VMH is a primary central target for leptin, mediating many of its peripheral effects on metabolism. Leptin infusion to the VMH reduces weight gain, and increases glucose disposal in muscle ( Jacob et al., 1997; Minokoshi et al., 1999). Furthermore, leptin stimulates VMH neuronal activity (Elmquist et al., 1997; Shiraishi et al., 1999). These leptin effects are essentially the opposite of those induced by NE infusion into the VMH. And, central (likely VMH) leptin activates symapthetic input to brown fat to increase thermogenesis (Haynes et al., 1999; Elias et al., 1998) (again opposite of VMH NE effects). However, chronic NE infusion into the VMH itself induces a rapid and prolonged hyperleptinemia and leptin resistance respecting lipid and glucose metabolism. The marked immediate and sustained rise in plasma leptin following chronic VMH NE infusion does not block the subsequent fattening or glucose intolerance (Cincotta et al., 2000). In agreement with these ﬁndings is the observation that hyperleptinemia resulting from VMH lesion does not prevent the VMH-lesion-induced +

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Figure 7 Positive feedback loop between hyperinsulinemia and increased VMH NE activity via (a) direct action on noradrenergic neurons and (b) stimulation of CNS serotonin production.

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obesity (Dube et al., 1999). Moreover, central leptin activates sympathetic stimulation of vascular tone (Dunbar et al., 1997; Dunbar and Lu, 1999) and VMH NE induced hyperleptinemia may or may not, depending upon extenuating circumstances, resultantly mediate or augment this leptin activity (i.e. no leptin resistance in this regard) (Haynes et al., 1997; Hirose et al., 1998; Aizawa-Abe et al., 2000; Haynes, 2000). For example, plasma NE and E levels are elevated following NE or leptin infusion into the VMH (Cincotta et al., 2000b; Satoh et al., 1999). Such a physiologic scenario may support the hypertensive condition associated with the insulin resistance syndrome (Figure 8) (see also below). Interactive counter-regulation of VMH NE and leptin functions appear to be at a crossroads in the hypothalamic modulation of the insulin resistance syndrome and more work is warranted in this area. In fact, leptin does block depolarization-induced NE release from hypothalamic synaptosomes (Brunetti et al., 1999). Where do these neuronal NE afferents to the VMH arise from? Available evidence suggests that ascending noradrenergic ﬁbers from the brain stem (primarily the periaqueductal gray (PAG) and locus coeruleus (LC) may innervate the VMH (Li et al., 1991). Moreover, the LC receives input from the SCN and CRH containing ﬁbers from the hypothalamus (in turn regulated by the SCN) (Legoratti-Sanchez et al., 1988). Therefore, the SCN may direct NE release from the VMH via the LC, PVN CRH neurons, and via direct connections to the VMH to regulate the above described VMH-driven physiologic activities. VMH efferents may communicate with SNS centers in the brain stem via the PAG, DMH and PVN (Luiten et al., 1987). As stated above, chronic VMH NE infusion results in plasma increases in NE and E. However, interactions of VMH neurons with the PAG can also regulate parasympathetic stimulation for insulin release as observed following NE administration to the VMH. The neurophysiologic “blueprint” of these VMH efferent pathways in normal vs. insulin resistant states is deserving of further investigation. From the above discussion it can be appreciated that VMH NE may (a) inhibit glutamate stimulation of sympathetic activation of brown fat thermogenesis, (b) stimulate sympathetic drive for increased HGO and white adipose ed

k oc bl n itio e) ib anc h in ist s al m n re r o i t N ep (l

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Figure 8 Relation between VMH NE activity, hyperleptinemia, insulin resistance, and hypertension.

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lipolysis, and (c) desensitize pancreatic ␤ cell responsiveness to hyperstimulation of sympathetic input. Increases in noradrenergic activities in other hypothalamic centers can also potentiate the insulin resistance syndrome. For example, the increased ␣1 and ␤ binding in the LH of ob/ob mice can stimulate insulin release via vagal activation (Steffens et al., 1984). Moreover, such increased adrenergic binding in the PVN can induce corticotropin releasing hormone (CRH) release (Plotsky, 1987), an important topic relative to insulin resistance to be discussed next.

HYPOTHALAMIC NPY AND CRH INTERACTIONS IN RELATION TO THE INSULIN RESISTANCE SYNDROME It has been established for many years now that neuropeptide Y (NPY) injection into the PVN, VMH, DMH, and perifornical hypothalamus can induce hyperphagia, most prominently so, at the perifornical hypothalamus (Jolicoeur et al., 1995). NPY containing neurons of the arcuate nucleus (AN) within the hypothalamus innervate the PVN and are believed to mediate many of the NPY effects on feeding. And, diet induced obesity in mice greatly increases NPY expression in the VMH and DMH believed to thereby potentiate leptin resistance (Guan et al., 1998). However, several studies have conﬁrmed that NPY is not necessary for the development of diet, chemical or genetic-induced obesity associated with leptin resistance (Hollopeter et al., 1998). Intracerebroventricular NPY administration also inﬂuences endocrine functions primarily via the PVN, DMH, and VMH resulting in hyperinsulinemia and lipogenesis even in animals pair-fed to control levels (Zarjevski et al., 1993; Marks et al., 1996). This hyperinsulinemia is mediated via stimulation of parasympathetic vagal efferents to the ␤ cell (Sainsbury et al., 1997). Consequently, NPY can have potent effects on neuroendocrine activities regulating metabolism independent of its inﬂuence on feeding behavior. NPY (from the intergeniculate leaﬂet) is a nonphotic modulator of SCN circadian activity as discussed above. Therefore, it can have a major impact on the temporal organization of neuroendocrine events modulated by the SCN that regulate peripheral metabolism as outlined above. Moreover, intergeniculate leaﬂet and SCN NPY levels are markedly elevated in ob/ob vs. lean mice and are reduced to normal levels by dopamine agonist treatment which also normalizes SCN circadian c-fos expression and the metabolic syndrome in these animals (Bina et al., 1999; Bina and Cincotta, 2000; Scislowski et al., 1999). Furthermore, NPY acting at the PVN can have a marked effect on insulin resistance via inﬂuencing the Hypothalamo–Pituitary–Adrenal (HPA) axis. NPY release from terminals within the PVN stimulate CRH release (especially in the presence of NE), ultimately increasing plasma levels of corticosteroid (Haas and George, 1987; Wahlestedt et al., 1987). Chronic corticosteroid treatment induces insulin resistance (Shimomura et al., 1987) and many insulin resistant species including humans have elevated plasma levels of corticosteroids, (King et al., 1983; Bjorntorp et al., 1999). Corticosteroids feedback to stimulate NPY release at the PVN and possibly other hypothalamic areas as well (Akabayashi et al., 1994). And, adrenalectomy inhibits the metabolic effects of NPY (Stanley et al., 1989). This positive feedback loop between hypothalamic NPY and plasma corticosteriods can best be maintained if the normal negative feedback of corticosteroids on PVN CRH release and the normal CRH inhibition of NPY release are both attenuated. In fact, these circumstances do occur in insulin resistant states. A reduction in PVN glucocorticoid receptor II, that mediates the negative feedback effect of corticosteroid on CRH release, is observed in animals and humans with the metabolic syndrome (Tsai and Romsos, 1991; Bjorntorp and Rosmond, 2000). Also, stress can induce

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the same desensitization of hypothalamic neurons to the negative feedback effects of corticosteroid hormone and result in stimulation of CRH secretion (Young et al., 1990) and this can have important consequences for the insulin resistance syndrome (see below). Although the association of increased NPY with the metabolic syndrome is well delineated, that of increased CRH activity with the syndrome has been less appreciated. Many people associate CRH activity with inhibition of feeding and this is certainly the case. However, this effect is mediated via very speciﬁc receptors in the VMH and is dissociated from CRH neuronal projections from the PVN to the median eminence (ME) regulating ACTH secretion and to the DMH activating the SNS (Nishiyama et al., 1999; Brown, 1986). This is a key point. CRH plays a role in feeding behavior, however, its role in neuroendocrine regulation of glucose and lipid metabolism is vital. Furthermore, these distinct CRH activities are differentially regulated. CRH activates DMH neurons that in turn stimulate the SNS to induce increases in HGO and adipose lipolysis (Bernardis and Bellinger, 1998; Gunion et al., 1988). Chronic CRH stimulation of corticosteroid secretion via ACTH can induce an insulin resistant condition without necessarily inﬂuencing feeding (Jeanrenaud, 1994). Importantly, obese, insulin resistant animals express increased CRH levels at the ME and DMH (Bina and Cincotta, 2000; Bestetti et al., 1990). Furthermore, under stress conditions, hypothalamic release of and/or sensitivity to CRH is greatly increased in insulin resistant vs. sensitive animals leading to hyperglycemia speciﬁcally via SNS activation (Bina et al., 1998; Timofeeva and Richard, 1997). Therefore, increased CRH release can act as a double edge sword to potentiate insulin resistance through the HPA axis and the SNS. As such, the classic yin–yang interpretation of NPY–CRH interactions in metabolism regulation, based on respective stimulation–inhibition of feeding is somewhat misleading. It appears that NPY and CRH can act in concert not in opposition to potentiate the insulin resistance syndrome. NPY stimulates parasympathetic activities to increase insulin release and lipogenesis (and decreases speciﬁc sympathetic activities to reduce brown fat thermogenesis and energy expenditure) while CRH increases sympathetic and HPA functions stimulating HGO, white adipose lipolysis, and insulin resistance. Again, these neuropeptide functions direct a simultaneous increase in both parasympathetic (NPY) and sympathetic (CRH) activities to potentiate the insulin resistance syndrome. How does leptin ﬁt into this picture? Leptin interactions with the NPY–CRH system have received much attention in the past few years, primarily centered on aspects of feeding behavior and energy balance. An attempt at understanding this complex neuroendocrine interplay must begin with an appreciation of the animal model system within which these interaction studies are performed. Let us consider these interactions in normal animals ﬁrst. In normal animals, leptin is a potent inhibitor of arcuate NPY synthesis and secretion, thereby contributing to its inhibitory effect on feeding. However, the leptin effects on CRH and the HPA axis are much more complicated. Leptin stimulates PVN CRH synthesis as well as CRH 2␣ receptor expression in the VMH, responsible for the anorectic effects of CRH (Nishiyama et al., 1999). It has also been shown that the SNS stimulatory effects of leptin may be mediated in part via a stimulation of CRH (Okamoto et al., 2000). Leptin, via CRH can also stimulate the HPA axis (Morimoto et al., 2000). However, during stress, prolonged fasting or starvation, leptin levels decline and are coupled with a rise in both NPY and plasma corticosterone that can be partially restored by leptin treatment (Heiman et al., 1997; Ahima et al., 1996). That is, the normal stimulatory effects of leptin on CRH and the HPA axis are reversed (now inhibitory) in starvation or stress conditions. This stress-associated rise in CRH and the HPA axis is the result of consequent changes in several neuromodulators and not solely of leptin itself. Leptin may partially restore the “normal” HPA condition by inﬂuencing some of these neuromodulators. For example,

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leptin reduces the rise in NPY induced by starvation and NPY is a strong stimulus for CRH release. The circadian rhythm of plasma leptin is out of phase with that of corticosteroid hormone in rodents and humans (Ahima et al., 1998; Casanueva and Dieguez, 1999). This temporal relation is a part of a larger circadian organization governing metabolism and feeding. Plasma leptin level generally peaks near the end of the feeding cycle coupled with the onset of fasting and SNS activity to potentiate HGO and lipid mobilization/oxidation. Contrariwise, plasma corticosteroid level peaks before the onset of daily feeding and is in part responsible for the onset of feeding as discussed earlier. Dose-response studies suggest that under normal conditions, the neuronal NPY response to leptin is more sensitive than is the neuronal CRH response (Ahima et al., 1999) and this may have important consequences for the hyperleptimemic state and the insulin resistance syndrome (see below). In the obese, insulin resistant state, the neuroendocrine organization of the leptin–NPY– CRH axis is deranged. Let us ﬁrst consider the circumstance of loss of leptin signaling as in prominently studied genetic models of the obese, insulin resistant state, and then the more relevant situation to human pathology of leptin resistance. The complete lack of leptin or leptin action such as in ob/ob or db/db mice, respectively induces a severe hyperphagic and obese, insulin resistant state. In these situations, hypothalamic NPY levels and activities are dramatically elevated as are those of NE. Consequently, this provides a strong stimulus for CRH secretion and thereby activation of the HPA and SNS axes that are all also elevated in these conditions of absent leptin signaling (Shimomura et al., 1987; Liang et al., 1999). Second, because of the prolonged lack of leptin signal, a starvation stress response ensues that again increases CRH (Huang et al., 1998). The increase in CRH in these hyperphagic conditions may seem at odds with its inhibitory role in feeding, however, in stress states the VMH response is desensitized to the inhibitory effect of CRH on feeding (Makino et al., 1999). Consequently, a loss of leptin signaling is associated with concurrent elevations of NPY and CRH, which can potentiate increases in PSNS and SNS activities, respectively. Leptin injections to leptin deﬁcient ob/ob mice reduce the hypothalamic NPY, and (possibly thereby) CRH, and HPA activities (Huang et al., 1998). In obese, insulin resistant conditions associated with leptin resistance, where no genetic loss of leptin signaling is present, leptin levels are generally elevated (although in reality, correlation studies demonstrate a wide range of leptin levels for any given body fat store level) (Considine et al., 1996). The hyperleptinemic–leptin resistant state can desensitize the NPY response to leptin as is observed in Ay/a mice (Kesterson et al., 1997), given an intact VMH (Dube et al., 1999). This can produce a rise of NPY activity and potentiate hyperinsulinemia and feeding. However, since the CRH secretory response to leptin is less sensitive than is that of NPY (i.e. rightward shift in the dose–response curve for leptin induced CRH vs. NPY secretion), leptin induced CRH secretion may not be desensitized by the hyperleptinemia. Therefore, hyperleptinemia of the metabolic syndrome can be coupled to increased HPA axis (and normal to elevated NPY) activities (Bjorntorp et al., 1999; Bjorntorp and Rosmond, 2000). Again, this hyperleptinemia-induced CRH secretion can potentiate a rise in SNS activity (Okamoto et al., 2000) and preliminary evidence does indicate a positive correlation between obesity, leptin level, and SNS activity in humans (Snitker et al., 1997) (Figure 8). Whether or not VMH CRH activity to inhibit feeding is desensitized under hyperleptinemic conditions is unknown, but clearly there is no appropriate inhibition of feeding in these obese, hyperleptinemic states. Herein lies another important aberration from the metabolic norm (Bray, 2000) typifying the insulin resistance syndrome: increased SNS activity coupled with normal or increased feeding activity. Now, remember that CRH neurons of the PVN are under the very dominant regulation of the SCN (where our review began) and plasma corticosteroid exhibits a pronounced circadian

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rhythm that feeds back on the SCN to regulate metabolism. As mentioned above, in nocturnal rodents such as rats and mice, corticosteroid peaks just before the onset of the feeding/ lipogenic (activity) phase to stimulate NPY release in the hypothalamus. The circadian rhythm of responsiveness to NPY in turn also peaks at the onset of the feeding/lipogenic phase of the day so that parasympathetic–anabolic activities such as insulin stimulated lipogenesis are synchronized with the feeding cycle. The plasma leptin rhythm peaks to initiate SNS catabolic activities during the normal fasting period of the day (Chou and Boozer, 2000; Ahima et al., 1998; Casanueva and Dieguez, 1999; Sinha et al., 1996), after the duration of which a rise in HPA activity begins another cycle in sync with the photoperiod (Figure 9). An important point to recognize is that the SCN also regulates the amplitudes of these rhythms as well as their phase. In other words, SCN input to hypothalamic centers can inﬂuence how much NPY is released or how strong the parasympathetic or CRH responses to NYP are, etc. Our review thus far has led to a hypothalamic neurophysiologic framework of the insulin resistance syndrome comprised of strong circadian components within the SCN that function to shift hypothalamic functionality whereby among other things, VMH and DMH noradrenergic, PVN and DMH CRH, and arcuate NPY activities are potentiated. This new neuroendocrine organization of metabolism induces a state wherein PSNS, SNS, and HPA activities are simultaneously stimulated at appropriate times of the day. This situation produces a metabolic condition characterized by decreased thermogenesis, increased lipogenesis, lipolysis, and HGO. Increased FFA transport to liver induces resistance to insulin suppression of HGO and increased lipid deposition in muscle reduces insulin stimulated glucose disposal therein (Unger, 1995). Hyperinsulinemia induced by increased noradrenergic VMH and LH activities supports fattening and lipid deposition (from FFA) in muscle and ␤ cells potentiating insulin resistance and ␤ cell dysfunction, respectively (Unger, 1995). The precise neuronal circuitry whereby the SCN produces these changes in VMH, LH, DMH, PVN, and arcuate neurophysiology to potentiate the insulin resistance syndrome is not completely delineated and is the subject of continued investigation by our laboratory. Furthermore, clearly other hypothalamic functions are involved in insulin resistance however, their review is beyond the scope of this chapter. The ﬁnding and conclusion that increased HPA and SNS activities contribute to the insulin resistance syndrome has recently gained new support from several laboratories conﬁrming

PVN NPY release PVN NPY responsiveness PVN NE 2 adrenoceptor binding Plasma leptin

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Figure 9 Daily variations in neueroendocrine factors regulating metabolism. Arrows represent the daily peak in stimulus or response rhythms. The gray bar represents the dark phase of the day.

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these relations (Bjorntorp et al., 1999; Bjorntorp and Rosmond, 2000; Weaver et al., 1993). Particularly, the work of Bjorntorp et al., 1999 has identiﬁed neuroendocrine abnormalities highlighted by loss of normal diurnal cortisol secretion associated with a hyperactive HPA axis, decreased growth hormone (GH) and sex steroid secretion and increased SNS activity in humans with the insulin resistance syndrome (Bjorntorp et al., 1999; Bjorntorp and Rosmond, 2000). Although the emphasis of the ﬁndings was on the level of HPA activity and the inﬂuence of this neuroendocrine proﬁle on insulin resistance syndrome, there are other equally important aspects of these observations. First, a variety of external stress factors were associated with the syndrome. Recall that stress can desensitize the VMH to the anorectic effects of CRH and PVN CRH neurons to the negative feedback effects of corticosteroid. Therefore, the HPA axis may become chronically activated. However, an important point here is that the increased HPA activity starts with increased CRH secretion that can also stimulate the SNS via the DMH leading to insulin resistance (Bina and Cincotta, 2000). In agreement with such ﬁndings in humans, stress induced – CRH mediated hyperglycemia is much greater in animals with the insulin resistance syndrome and this stress effect can be completely blocked by sympathetic ganglion blockers (Bina et al., 1998). Second, the circadian rhythm of plasma cortisol in humans is deranged in the syndrome. Studies from other laboratories have demonstrated aberrations in circadian rhythms of several neuroendocrine factors, including growth hormone, prolactin and cortisol in obese/type 2 diabetic humans (Cupinschi et al., 1978; Ferrari et al., 1986; Seki and Nagata, 1991; Johnston et al., 1980). As indicated at the outset of this chapter, it has been demonstrated over thirty years ago that the phase relations between the circadian rhythms of prolactin and cortisol differ in seasonally obese, insulin resistant vs. lean, insulin sensitive vertebrates. And, mimicking the daily phase relation of these plasma hormone rhythms associated with each metabolic condition (by injection at the appropriate times of day) produces the associated metabolic state. Therefore, high cortisol can induce and reverse the insulin resistance syndrome dependent upon its circadian variation in relation to other circadian neuroendocrine activities. Furthermore, the effects of such timed hormone administrations persist long after the termination of treatment and can be demonstrated in seasonal and nonseasonal animals alike. This suggests that the hormones are feeding back centrally to affect the phase relation of (at least two) circadian oscillations regulating metabolism. Each central (SCN) oscillation has many neural and hormonal circadian expressions, including the rhythms of corticosteroid hormone and prolactin that regulate metabolism. Therefore, metabolic conditions are the consequences of temporal circadian interactions among many neuroendocrine factors and not of corticosteroid hormone and prolactin alone (Figure 4). Inasmuch as the circadian rhythms of plasma cortisol and prolactin are tightly regulated by the SCN, the observation of a deranged circadian rhythm of plasma cortisol and prolactin in insulin resistant humans coupled to the animal studies just described suggest important roles for the SCN and circadian organization in the regulation of human metabolism.

ALTERED VMH-AUTONOMIC CONTROL OF SYMPATHETIC TONE The relation between the insulin resistance syndrome along with its pathologic correlates of obesity and type 2 diabetes and sympathetic tone has long been controversial. Individual studies have identiﬁed increased, decreased, or no change in SNS activity in obese, insulin resistant states (Young and Macdonald, 1992). However, as clearly pointed out above and described elsewhere (Young, 1992), sympathetic activities are not universally regulated by the CNS or the hypothalamus. In many studies of humans and different animal models with the obesity or

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the insulin resistance syndrome, sympathetic tone as measured by muscle sympathetic activity, plasma NE level, or urine NE metabolite level is increased (Scherrer et al., 1996; Grassi et al., 1996). Inasmuch as hyperinsulinemia can be a potent stimulus for increased sympathetic tone (Scherrer and Sarotri, 1997), the hyperinsulinemia of the syndrome may account for the hypertension commonly associated with it (Landsberg, 1996; Brands et al., 1995; Reaven et al., 1996). Since the VMH and DMH are central relay centers for stimulation of speciﬁc branches of the SNS including those regulating vascular tone and heart rate, it is likely that the associations between hyperinsulinemia and increased sympathetic tone involve actions of insulin directly or indirectly upon the medial hypothalamus (Muntzel et al., 1995). The increase in VMH NE release induced by hyperinsulinemia (Cincotta et al., 2000a) causes increases in circulating NE and E (Cincotta et al., 2000b) that can contribute to sympathetic vascular effects. Insulin acting at glucose sensitive neurons within the VMH is believed to cause inhibition of an inhibitory pathway to tonically active SNS centers in the brain stem thereby causing disinhibition of these neurons and activation of the SNS (Landsberg, 1996). The insulin-induced VMH inhibition described here may well relate to the insulin-induced VMH NE release that inhibits neuronal activity in the VMH (Kraszewski and Cincotta, 2000). The interactions between insulin and NE at the VMH to modulate this center’s pressor activities may also involve leptin. Hyperinsulinemia induces VMH NE release, chronic increases in which induce hyperleptinemia (Cincotta et al., 2000b). Leptin stimulates the SNS, particularly its pressor effects (Dunbar et al., 1997; Dunbar and Lu, 1999), and this hypothalamic pathway may somehow be involved in the association of hypertension with obesity and obese type 2 diabetes (Figure 8). As mentioned above, both increased plasma leptin and SNS activity have been associated with obesity (Snitker et al., 1997). The circuitry between VMH NE activity and activation of sympathetic activities modulating vascular tone has not been delineated. However, the VMH is a hypothalamic center potentiating peripheral pressor effects (Takenaka et al., 1996).

HYPOTHALAMIC MELANOCORTIN 4 RECEPTOR Dominant mutations of the agouti gene, typically involved in coat color regulation in rodents, produce a phenotype of insulin resistance, obesity, hyperleptinemia, and moderate hyperphagia. (reviewed in Moussa and Claycombe, 1999). Agouti antagonizes melanocortin receptor 4 (MC4-R) that is widely distributed in the brain and hypothalamus. Activation of the MC4-R via i.c.v. injection of agonist (MTII) inhibits feeding in mice carrying the dominant agouti mutation (Ay/a mice), ob/ob mice and mice injected with NPY (Fan et al., 1997). And, disruption of the MC4-R results in hyperphagia, obesity, and elevated hypothalamic NPY levels (Huszar et al., 1997). The endogenous agonist, ␣-melanocyte stimulating hormone (␣-MSH) and antagonist, agouti related protein (AgRP) compete for binding to hypothalamic MC4-R to inhibit or stimulate feeding, respectively (Moussa and Claycombe, 1999; Li et al., 2000). ␣-MSH is derived from the precursor gene, pro-piomelanocortin (POMC) that in turn is expressed in the arcuate nucleus of the hypothalamus and medial basal hypothalamus. Leptin binding to POMC neurons in the arcuate and medial basal hypothalamus induces POMC expression (Korner et al., 1999). POMC neurons project from the arcuate and possibly the MBH to the PVN where ␣-MSH is believed to regulate (inhibit) food consumption in part by blocking NPY release (King et al., 2000). And, arcuate POMC and PVN ␣-MSH expressions are reduced in obese animals with deﬁcient leptin signaling (Kim et al., 2000). Studies by Boston et al. (1997) suggest that central effects of leptin are attenuated by antagonism of MC4-R. Therefore, leptin, by stimulating ␣-MSH and inhibiting NPY and AgRp

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(Ziotopoulou et al., 2000) within the hypothalamus, may act to normalize the obese, insulin resistant condition. Leptin stimulation of the SNS may also be in part via ␣-MSH (Dunbar and Lu, 1999). Adrenalectomy of ob/ob mice with elevated HPA activity also normalizes AgRP and POMC levels in these mice, suggesting a role for HPA hyperactivity in MC4-R pathway alterations supporting the syndrome (Makimura et al., 2000). As mentioned above, the SCN send strong efferent connections to the AN and PVN which contain the leptin–MC4-R and leptin–NPY pathways. As such, circadian organization of these leptin activities (Chou and Boozer, 2000; Sinha et al., 1996) is likely central to their composite output signaling to peripheral metabolism.

HYPOTHALAMIC DOPAMINE : NOREPINEHRINE ACTIVITY RATIO IN THE INSULIN RESISTANCE SYNDROME Hypothalamic NE levels are elevated in a wide variety of insulin resistant animal models as described above. Increased noradrenergic activities at various hypothalamic centers can initiate physiologic functions that potentiate the insulin resistance syndrome. As already outlined in this chapter, increased VMH noradrenergic activity in and of itself, can induce the full blown insulin resistance syndrome. However, increased noradrenergic activity in the LH can potentiate hyperinsulinemia (Steffens et al., 1984), in the PVN can stimulate feeding (Lichtenstein et al., 1984) as well as CRH release (Plotsky, 1987; Bina and Cincotta, 2000) potentiating increases in HPA and SNS activities leading to increases in HGO and lipolysis, and in the DMH can stimulate SNS activities leading to increases in HGO and lipolysis (Bernardis and Bellinger, 1998). NE can potentiate or inhibit hypertension depending upon the hypothalamic site of action (Oparil et al., 1996; Pacak et al., 1993). Dopamine, via D2 receptor sites acts to markedly reduce noradrenergic turnover and presynaptic noradrenergic release within the CNS, including the hypothalamus (Misu et al., 1985; Wichmann and Starke, 1988; Jackisch et al., 1985). And, under the appropriate circumstances, this effect may also be elicited by dopamine D1 receptors ( Jackisch et al., 1985). Dopamine D1 receptor activation can augment various neuronal responses to D2 receptor stimulation (Cooper and Al-Naser, 1993). Intracerebroventricular administration of bromocriptine, a potent dopaminergic sympatholytic agent (that stimulates dopaminergic and inhibits noradrenergic neuronal activity), normalizes hyperinsulinemia, insulin resistance, and glucose intolerance in Syrian hamsters (Luo et al., 1998) as does its systemic administration (Cincotta et al., 1993; Cincotta and Meier, 1995). Systemic administration of bromocriptine improves glycemic control and hyperlipidemia in obese nondiabetic and type 2 diabetic humans without inﬂuencing body weight or food consumption (Pijl et al., 2000; Kamath et al., 1997; Cincotta et al., 1999). In insulin resistant hamsters, such bromocripitne treatment has been shown to normalize the circadian rhythm of serotonin turnover in the SCN as well as reduce the classic elevation of VMH NE activity; factors contributing to the development of the syndrome as discussed herein (Luo et al., 2000; Luo et al., 1998). Co-administration of bromocriptine and SKF38393, a dopamine D1 agonist, markedly improves hyperglycemia, hyperlipidemia, hyperinsulinemia, ␤ cell dysfunction, obesity and hyperphagia in ob/ob, db/db and Ay mice (Scislowski et al., 1999; Zhang et al., 1999; Cincotta et al., 1998; Liang et al., 1998a,b). However, these effects of the D1D2 agonist treatment cannot at all be achieved by pair-feeding of control animals to match the reduced caloric intake induced by the treatment. Thus, dopamine improves the syndrome by means other than reducing food intake. Such dopamine treatment reduces elevated VMH responsiveness to NE, arcuate NPY level, and PVN and DMH CRH level in

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ob/ob mice (all known to potentiate the syndrome as described herein) to levels observed in normal lean mice (Kraszewski and Cincotta, 2000; Bina and Cincotta, 2000). Furthermore, dopamine D1D2 agonist treatment normalizes alterations in SCN neurochemistry that are coupled to the insulin resistance syndrome (Bina and Cincotta, 2000; Bina et al., 1999). Dopamine is also a potent inhibitor of arcuate NPY gene expression, perifornical hypothalamic responsiveness to NPY and stimulator of arcuate POMC mRNA expression (Gillard et al., 1993; Tong and Pelletier, 1992; Pelletier and Simard, 1991). These ﬁndings in these models suggest that dopamine is an important regulator of metabolism that is independent of or down stream from the leptin – NPY/MC4-R pathways. Beyond NPY and POMC, dopamine shares other postsynaptic response proteins with leptin, such as CRH, and CART that function in regulation of autonomic control of metabolism and feeding behavior (see above; Trayhurn et al., 1999). Hypothalamic dopamine levels are reduced in all these animal models of the insulin resistance syndrome (Oltmans, 1983; Shimizu et al., 1992; Garris, 1995). Recent ﬁndings suggest that hypothalamic dopamine is also reduced in obese women (Ferreira et al., 1998). In good agreement with these ﬁndings, central acting dopamine antagonists as a class, used in the treatment of psychotic disorders, are well known to induce obesity (Bhavnani and Levin, 1996; Sachs and Guille, 1999). Furthermore, there is a strong association between plasma leptin level and cerebrospinal ﬂuid dopamine concentrations in normal humans (Hagan et al., 1999). It appears that the hypothalamic dopamine to noradrenaline activity ratio is reduced in the insulin resistance syndrome and that reversing this relation (increasing dopaminergic and decreasing noradrenergic tone) reverses major physiologic components of the syndrome. Dopamine beta-hydroxylase (DBH) converts dopamine into noradrenaline and DBH inhibitors (that increase dopamine and decrease noradrenaline levels in neurons) improve insulin resistance and glucose intolerance in Syrian hamsters (Cincotta et al., 1998). Interestingly, DBH knockout mice exhibit an increase in energy expenditure despite a systemic inability to synthesize noradrenaline (Thomas and Palmiter, 1997) as is observed in ob/ob mice following dopamine agonist treatment (Scislowski et al., 1999). CNS (including hypothalamic) dopamine levels decrease while insulin resistance increases with aging (Lai et al., 1987; Reymond and Porter, 1981). And, CNS dopamine levels are increased by moderate calorie restriction that improves insulin resistance (Levin et al., 1981; Friedman et al., 1973). The speciﬁcs of the hypothalamic dopamine–noradrenaline neuronal anatomic and physiologic interactions critical in the regulation of metabolism are as yet poorly deﬁned. However, research of seasonal animals strongly suggests that shifts in the temporal organization of circadian (dopaminergic and serotonergic) activities at the SCN function to increase or decrease the dopamine : noradrenaline activity ratio in other hypothalamic centers of insulin sensitive and resistant animals, respectively (Figure 10). Treatment of insulin resistant animals with dopamine agonists that normalizes multiple insulin resistance syndrome-associated alterations in SCN circadian neuronal activity as well as peripheral circadian neuroendocrine parameters including the circadian rhythm of plasma corticosteroid also normalizes the syndrome itself (Liang et al., 1998; Cincotta et al., 1993; Tozzo et al., 1997). Such ﬁndings are in good agreement with the results reported by Bjorntorp et al. (1999) described above. How might the seasonal changes in hypothalamic dopamine/NE activity ratio occur to drive the seasonal cycle of insulin resistance so pervasive among vertebrates in the wild? Although speculative at this time, one possible mechanism may involve the physiologic phenomenon of neuronal plasticity. Neuronal plasticity, the morphological, and neurochemical restructuring of existing neuronal communications, is now known to be a very active process within the CNS (Thompson, 2000; Paulsen and Sejnowski, 2000; Shiosaka and Yoshida, 2000; Luscher et al., 2000; Fortune and Rose, 2000) and studies link this plasticity to circadian physiology and seasonal

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changes in behavior (Tramontin and Brenowitz, 2000; Cirelli and Tononi, 2000). A variety of neurotrophic factors, particularly brain derived neurotrophic factor (BDNF) are involved in neuronal differentiation (Lindsay et al., 1994; Kirschenbaum and Goldman, 1995). BDNF is a CNS peptide that is a potent neurotrophic factor able to stimulate neuronal growth and maturation in cultured adult progenitor tissue and plays a critical role in neuronal plasticity (Goldman et al., 1997; Pincus et al., 1999). Although BDNF can inﬂuence a variety of neuronal types, its most substantial effect relates to the potentiation of dopaminergic neuronal activity (reviewed in Altar et al., 1995; Studer et al., 1995; Cellerino et al., 1998). BDNF supports the growth, arborization, and survival of dopaminergic neurons in culture, stimulating increases of dopaminergic neuron number and dopamine content by 3–7-fold. And, it attenuates neurotoxic effects directed against dopaminergic neurons. BDNF also markedly increases the ﬁring and dopamine turnover rates of dopaminergic neurons in vivo. There appears to be a positive feedback loop of some sort between dopamine and neurotrophic factors including BDNF since dopamine can induce their expression in the CNS (Inoue et al., 1997; Ohta et al., 2000; Angelucci et al., 2000). Finally, BDNF (and other neurotrophins) has been shown to stimulate the growth and arborization of dopaminergic neurites within the hypothalamus (Loudes et al., 1999). Interestingly, systemic treatment with BDNF results in dramatic improvements in the insulin resistance syndrome in db/db mice (Tonra et al., 1999; Ono et al., 1997, 2000). Furthermore, the BDNF effects on hyperglycemia persist after the termination of the treatment, suggesting a resetting of central activities regulating metabolism similar to that observed SCN

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Figure 10 Association between the phase relation of two SCN circadian neuronal oscillations (a) (e.g. serotonin) and (b) (e.g. dopamine), hypothalamic dopamine/NE activity ratio, and peripheral metabolism.

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with timed daily injections of 5HTP and L-DOPA or dopamine D1D2 agonist treatment (Scislowski et al., 1998). Given such strong associations of decreased dopaminergic function with the insulin resistance syndrome, it would be well worth investigating the possibility that these beneﬁcial BDNF (and other neurotrophin) effects on the syndrome are mediated via activiation of (hypothalamic) dopaminergic systems. The leptin-like effects of dopamine agonist treatment in leptin deﬁcient, leptin receptor deﬁcient and leptin resistant mice may also involve a positive feedback loop with such neurotrophins that share receptor similarities with the leptin receptor (Gloaguen et al., 1997). A second link between central neuronal plasticity and regulation of the insulin resistance syndrome comes by way of nitric oxide (NO). NO is a key modulator of neuronal plasticity (reviewed in Kara and Freidlander, 1998; Wang et al., 1997) and central administration of inhibitors of NO synthesis induce insulin resistance (Shankar et al., 1998). NO is known to inhibit noradrenaline synthesis and stimulate dopamine synthesis in part by inhibiting dopamine beta hydroxylase activity (Zhou et al., 2000). Consequently, NO may alter (increase) the dopamine/noradrenaline neuronal activity ratio and thereby inﬂuence (reduce) insulin resistance in the periphery. Once again, this neuronal plasticity involved in the (hypothalamic) regulation of metabolism, evident in seasonal animals, must somehow be guided by SCN clock components and NO itself is also a strong modulator of SCN circadian activity (Ding et al., 1994). Ascertaining precisely how modulators of SCN functions thereby regulate neuronal plasticity governing metabolic status will likely lead to a major advance in our understanding of hypothalamic regulation of the insulin resistance syndrome. Alterations in the hypothalamic dopamine : noradrenaline activity ratio clearly regulate peripheral metabolism as discussed herein. Although not completely elucidated at the present time, the conceptual paradigm of central neuronal plasticity participating in the oscillation between insulin sensitive and resistant states via modulating alterations in the dopamine : noradrenaline activity ratio is building a substantial and corroborative body of scientiﬁc evidence as outlined above. External modulators of this plasticity may include food quality. However, what relevant information exists relating to diet-induced insulin resistance and hypothalamic function?

FOOD QUALITY INFLUENCES ON HYPOTHALAMIC REGULATION OF INSULIN SENSITIVITY High simple sugar (e.g. sucrose) and saturated fat feeding can induce the insulin resistance syndrome (Storlien et al., 1988; Ahren and Scheurink, 1998; Harte et al., 1999; Hallfrisch et al., 1981; Maron et al., 1991; Marshall et al., 1997). Such dietary manipulations are well known to stimulate the SNS (Schwartz et al., 1983; Young et al., 1994). Since SNS activity is regulated centrally, this diet may impact central centers modulating SNS function. A high sucrose – saturated fat diet induces hyperinsulinemia (reviewed in Landsberg, 1986) and we have reviewed the inﬂuence of insulin to stimulate VMH NE release along with its subsequent pathophysiologic consequences, including activation of branches of the SNS, leading to development of the syndrome. Recall that NE inhibits neuronal activity within the VMH (leading to activation of speciﬁc branches of the SNS) and this responsiveness to NE is more pronounced in obese, insulin resistant vs. lean, insulin sensitive animals (Kraszewski and Cincotta, 2000). Consistent with saturated fat-induced stimulation of the SNS, high saturated (but not unsaturated) fat diets reduce c-fos immunoreactivity (an index of neuronal activity) within VMH neurons (Wang et al., 1999) and this may be mediated by NE release therein. Collectively, these data suggest that saturated fat may inﬂuence VMH activities to potentiate

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the syndrome directly and/or indirectly (through increased insulin secretion). Moreover, such saturated fat feeding increases c-fos immunoreactivity in the PVN (Wang et al., 1999). And, increased PVN activities as discussed, stimulate HPA and SNS activities that potentiate the syndrome in animals and humans. As discussed herein, the set-point for hypothalamic responsiveness to the high sucrose – fat diet may vary from animal to animal. Those animals most sensitive to the syndrome-inducing effects of this diet exhibit a pronounced increase in circulating NE in response to glucose infusion (Levin and Sullivan, 1987) that may be mediated via the VMH and PVN as reviewed above. Interestingly, the normal functional Magnetic Resonance Imaging signal in the VMH and PVN following glucose ingestion is markedly attenuated in obese vs. lean humans (Matsuda et al., 1999). High fat diets also induce hyperleptinemia (possibly in part via increased VMH NE activity) that can contribute to increased sympathetic tone as discussed above. Low protein diets also potentiate the activation of the SNS and this may also be mediated via the VMH (Spector et al., 1996b; Young et al., 1985). Although much more work is needed in the area of nutritional regulation of hypothalamic functions governing metabolism, the existing database indicates that high simple sugar, saturated fat–low protein diets can be potent stimuli for the induction of hypothalamic functions stimulating the insulin resistance syndrome. Because the hypothalamic modulators of peripheral metabolism can be maintained to sustain the syndrome by feedback from metabolic and neuroendocrine activities generated by them (e.g. hyperinsulinemia), this central inﬂuence of the diet can have long-lasting devastating consequences. Contrariwise, the appropriate diet may stave off the deleterious effects of aging and stress on central regulation of metabolism and thereby support a prolonged insulin sensitive condition.

SUMMARY The transition from the lean, insulin sensitive to obese, insulin resistant state is orchestrated by a myriad of temporally organized CNS (particularly hypothalamic) events that likely involve neuronal plasticity mechanisms. Vertebrates evolved an exquisite mechanism for inducing the insulin resistance syndrome thus allowing survival of very long periods (seasons) of low food availability. The survival strategy is not merely the ability to induce the syndrome but rather to induce it at precisely the appropriate time of year. Changing temporal interactions of circadian neural oscillations within the SCN function to generate this annual cycle of metabolism. These circadian oscillations modulate the activities of other hypothalamic nuclei governing metabolism. Circadian neuroendocrine activities function or organize systemic metabolic biochemistry so that metabolic events within the organism are synchronized with the daily cyclic environment of food availability and locomotor activity. Parasympathetic–anabolic activities coincide with the feeding period of the day whereas sympathetic–catabolic activities are coupled to the fasting portion of the day. Changes in hypothalamic activities inducing the syndrome stimulate coincident increases in parasympathetic and sympathetic (and HPA) functions to establish a new steady state of metabolism. This syndrome is abolished when this hypothalamic circadian neural organization potentiating it is “normalized” and such events occur naturally as part of the endogenous annual cycle of metabolism in vertebrates that is over 400 million years old. This neuronal plasticity of hypothalamic events can be modiﬁed by exogenous cues such as food quality and once “reset” may be maintained long term by internal feedback mechanisms. No genetic defects are required for the genesis of this syndrome. However, polymorphisms within key genetic elements of this hypothalamic control system that predispose to the syndrome would be particularly dominant.

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Some key hypothalamic elements in the development of the insulin resistance syndrome that are modiﬁed by SCN clock mechanisms include: (1) increased VMH NE activity potentiated by hyperinsuliinemia, (2) simultaneous increases in NPY and CRH activities governing parasympathetic and sympathetic/HPA functions, respectively, (3) resistance to leptin effects modulating metabolism with a preservation of its ability to stimulate sympathetic activation of vascular tone, and (4) a general increase in the noradrenaline to dopamine activity ratio. The metabolic inﬂuences of these hypothalamic alterations reviewed herein produce a unique physiology wherein sympathetic activities to brown fat are inhibited, to liver, WAT and the vasculature are increased, and to the pancreatic islets are desensitized by the ␤ cell. The hypothalamic “set-point” for SNS counter-regulatory responsiveness to hypoglycemia is raised and HGO is increased in the face of euglycemia or hyperglycemia. An induced concurrent hyperinsulinemia and hyperleptinemia feedback to maintain this hypothalamic organization. These hypothalamic activities can be “reset” by neurotransmitter, hormonal or food substrate administrations that shift circadian activities within the SCN and alter hypothalamic activities governing peripheral metabolism. The widespread expression of the “thrifty” obese/type 2 diabetic phenotype among individuals within agrural cultures that undergo an abrupt switch to the “westernized” diet may be the result of such food quality inﬂuences on hypothalamic centers regulating metabolism as described herein. Future research into the hypothalamic mechanisms regulating peripheral metabolism will yield new approaches of treating the insulin resistance syndrome, using the natural evolution of hypothalamic function as a guide. REFERENCES Ahima, R.S., Prabakaran, D. and Flier, J.S. (1998) Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function. Journal of Clinical Investigation, 101, 1020–1027. Ahima, R.S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier, E. et al. (1996) Role of leptin in the neuroendocrine response to fasting. Nature, 382, 250–252. Ahren, B. and Scheurink, A.J. (1998) Marked hyperleptinemia after high-fat diet associated with severe glucose intolerance in mice. European Journal of Endocrinology, 139, 461–467. Aizawa-Abe, M., Ogawa, Y., Masuzaki, H., Ebihara, K., Satoh, N., Iwai, H. et al. (2000) Pathophysiologic role of leptin in obesity-related hypertension. Journal of Clinical Investigation, 105, 1243–1252. Akabayashi A., Watanabe, Y., Wahlestedt, C., McEwen, B.S., Paez, X. and Leibowitz, S.F. (1994) Hypothalamic neuropeptide Y, ts gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats. Brain Research, 665, 201–212. Altar, C.A., Alderson, R.F., Anders, K.D., Hyman, C., Wiegand, S.J. and Lindsay, R.M. (1995) Potential therapeutic use of BDNF or NT-4/5 in Parkinson’s and Alzheimer’s diseases. In I. Hanin et al. (eds), Alzheimer’s and Parkinson’s Diseases. Plenum Press, New York, pp. 611–619. Amir, S. (1990) Intra-ventromedial hypothalamic injection of glutamate stimulates brown adipose tissue thermogenesis in the rat. Brain Research, 511, 341–344. Angelucci, F., Mathe, A.A. and Aloe, L. (2000) Brain-derived neurotrophic factor and tyrosine kinase receptor TrkB in rat brain are signiﬁcantly altered after haloperidol and risperidone administration. Journal of Neuroscience Research, 60, 783–794. Bartness, T.J. and Wade G.N. (1984) Photoperiodic control of body weight and energy metabolism in Syrian hamsters (Mesocricetus auratus): role of pineal gland, melatonin, gonads and diet. Endocrinology, 114, 492–498. Bernardis, L.L. and Bellinger, L.L. (1998) The dorsomedial hypothalamic nucleus revisited: 1998 update. Proceeding of the Society of Experimental Biology and Medicine, 218, 284–306. Bestetti, G.E., Abramo, F., Guillaume-Gentil, C., Rohner-Jeanrenaud, F., Jeanrenaud, B. and Rossi, G.L. (1990) Changes in the hypothalamo–pituitary–adrenal axis of genetically obese fa/fa rats: a structural, immunochemical, and morphometrical study. Endocrinology, 126, 1880–1886.

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17.

INSULIN RESISTANCE – EMERGING THERAPIES FOR AFFECTED SITES JULIE S. MOYERS AND JOSÉ F. CARO Endocrine Research, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285

INTRODUCTION Glucose homeostasis relies on adequate and coordinated stimulation of insulin secretion from beta cells, insulin-stimulated glucose uptake in the periphery (primarily muscle and also fat) and insulin-stimulated suppression of hepatic glucose output. Insulin resistance is characterized as a failure or attenuation of insulin action in response to normal or elevated levels of insulin. Though the presence of insulin resistance does not imply a certainty for progression to type 2 diabetes, insulin resistance in combination with a cluster of physiological abnormalities, including central obesity, impaired glucose tolerance and a number of risk factors for cardiovascular disease (hypertension, atherosclerosis, dyslipidemia) comprises “syndrome X” or “insulin-resistance syndrome” and plays a critical role in the pathogenesis of type 2 diabetes (Reaven, 1995). Initially, insulin resistance is compensated by an increased production of insulin. Progressive beta cell failure combined with defects in insulin action eventually result in the hyperglycemic condition of type 2 diabetes. An estimated 150 million people worldwide have type 2 diabetes. This number is increasing at a rapid rate and is expected to reach 300 million by 2025, as estimated by the World Health Organization. The increasing prevalence of obesity has contributed to this trend. Given the difﬁculties in management of type 2 diabetes and the long-term complications and economic impact associated with the disease, it is apparent that efforts to develop drug therapy strategies to treat insulin resistance and to prevent the progression of type 2 diabetes are critical. An understanding of the regulation of insulin action at the molecular and cellular level is key to discovering the mechanisms of insulin resistance that underlie type 2 diabetes. The translation of that knowledge into rodent models of insulin resistance and diabetes has been a powerful approach to provide important insights for therapeutic strategies to overcome defects in insulin action. The insulin signaling cascade is a complex network transmitting signals from the cell surface to the nucleus. For the sake of focus, we will limit our discussion to insulin signaling events at or proximal downstream of the receptor. This chapter will focus on (1) an overview of the regulation of insulin action at the biochemical and cellular level; (2) insights gained from transgenic animal models with defects in proximal insulin signaling and (3) discussion of therapeutic strategies to treat insulin resistance at the cellular level, with two examples of emerging strategies to increase insulin sensitivity.

Address correspondence to: Julie S. Moyers, Ph.D., Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285. Tel.: 317-277-4006; Fax: 317-276-6510; Email: [email protected] 313

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OVERVIEW OF REGULATORS OF INSULIN SIGNALING Key Players Insulin binds to the extracellular ␣-subunit of the insulin receptor leading to activation of the ␤-subunit tyrosine kinase domain and autophosphorylation of the heterotetrameric receptor on several tyrosine residues (Cheatham and Kahn, 1995). These phosphotyrosine residues provide protein–protein interaction sites for several signaling molecules that bind and serve as substrates for phosphorylation by the receptor tyrosine kinase. Figure 1 illustrates some of the key signaling molecules in insulin action proximal to the insulin receptor. Several receptor substrates have been identiﬁed and studied as messengers that carry out the pleiotropic signaling cascades that emanate following hormone binding. These include the insulin receptor substrate (IRS) family of proteins (IRS-1, IRS-2, IRS-3 and IRS-4), SHC and SHP-2 (Myers and White, 1996; Sun et al., 1995; Lavan et al., 1997a,b; Yamauchi et al., 1995). Additionally, STAT5 has been shown to bind and serve as a substrate for the insulin receptor (Storz et al., 1999). IRS-1 is the best characterized IRS and contains approximately twenty potential phosphotyrosine residues. IRS-1 possesses no intrinsic enzyme activity but contains numerous sites Insulin α

Insulin receptor P P P

P P P

Active

α

(–) Tyrosine phosphatases

Inactive (–)

Serine kinases

Substrates

SHC

(–) Degradation

IRS

P

P

P

Grb-2

Grb-2 SHP-2

SOS

SOS

IRS

P

PI3K

Effectors

p70

PKB

PKC

ras Glut4

Insulin action

Translocation

Cell growth, anti-apoptosis, DNA synthesis, glucose metabolism

Figure 1 Schematic diagram of key molecules involved in signaling proximal to the insulin receptor. The molecules and discussion of points of regulation are described in the text.

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for protein–protein or protein–lipid interactions that mediate signaling in response to insulin. The pleckstrin–homology (PH) and phosphotyrosine-binding (PTB) domains are contained within the N-terminus of IRS-1 and mediate interaction with the insulin receptor via direct binding and interaction with membrane phospholipids. Numerous potential sites of tyrosine phosphorylation exist throughout the remainder of the IRS-1 molecule. Of these, several have been shown to be phosphorylated in response to insulin and serve as binding sites for Src homology 2 (SH2) domain-containing downstream signaling molecules, including the regulatory subunits of phosphatidylinositol 3-kinase (PI3K), Grb2 and SHP-2. PI3K is a lipid 3 kinase that phosphorylates phosphatidylinositol (PI)-4,5-P2 and other lipid substrates to produce PI-3,4,5-P3 in response to insulin. This lipid product is thought to be a key intermediate in the pathway involving the serine kinases, PKB/Akt, protein kinase C zeta/lambda and others, leading to insulin-stimulated Glut4 transporter translocation and glucose uptake (Toker, 2000). PI3K also signals through p70 S6 kinase and mTOR to mediate protein synthesis. Thus, PI3K may be a central mediator of insulin metabolic effects. Grb2 is an adapter molecule that binds IRS proteins and signals through the Ras and MAP kinase mitogenic pathway. SHP-2 is a tyrosine phosphatase that appears to play a positive role in mitogenic signaling by insulin and other growth factors, though it has also been suggested to dephosphorylate IRS-1, perhaps in a negative feedback manner (Myers et al., 1998). Thus, divergent insulin signaling pathways mediate the pleiotropic effects of insulin to modulate glucose transport, glycogen synthesis, protein synthesis, lipid synthesis, anti-apoptosis and gene expression. Regulation of Insulin Signaling Knowledge of the key players and mechanisms of regulation of insulin signaling events provides the foundation for development of therapeutic approaches to treat insulin resistance. Though not fully understood, it is clear that regulation of insulin action occurs at many levels, including the coordination of phosphorylation and dephosphorylation events by kinases and phosphatases, protein degradation, intracellular localization and protein expression. Phosphorylation/dephosphorylation Tyrosine phosphorylation and activation of the insulin receptor is a rapid event, occurring within minutes after hormone binding. Receptor phosphorylation is transient even in the continued presence of insulin. Following acute insulin stimulation and withdrawal, receptor dephosphorylation takes place much more rapidly (Bernier et al., 1994). Several protein tyrosine phosphatases may be involved in this process, including PTP1B and LAR (Goldstein et al., 1998). In the case of PTP1B, in vitro studies indicate that PTP1B binds to and dephosphorylates the insulin receptor and IRS-1 (Seely et al., 1996; Ahmad et al., 1995; Goldstein et al., 2000). Neutralizing antibodies and overexpression of dominant-negative constructs indicate that PTP1B can negatively regulate signaling, resulting in suppression of insulinstimulated glycogen synthesis and Glut4 translocation (Chen et al., 1997). Consistent with a role for PTP1B as an inhibitor of insulin action, genetic disruption of PTP1B in vivo results in increased insulin sensitivity and a resistance to weight gain (below). Thus, alleviation of negative regulatory mechanisms of insulin signaling may provide a mechanism for treatment of insulin-resistance. Future studies of genetic crosses between insulin-resistant animal models and PTP1B knockout mice, for example, will hopefully support this idea.

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Similar to the receptor, the tyrosine phosphorylation of IRS-1 is rapid and transient, correlating with associated PI3K- activity in L6 cells and rat skeletal muscle (Ogihara et al., 1997). In comparison to IRS-1, dephosphorylation of IRS-2 appears to occur more rapidly and is prolonged by vanadate, suggesting that down-regulation of tyrosine phosphorylation of these related proteins occurs, at least in part, via different, as yet unidentiﬁed tyrosine phosphatases. These studies suggest that inhibitors of tyrosine phosphatases may potentiate or prolong insulin action and alleviate insulin resistance. Indeed, cell-based and in vivo studies with the general tyrosine phosphatase inhibitor, vanadyl sulfate, are encouraging in this regard (Goldﬁne et al., 2000). Speciﬁcity, however, is a key challenge since PTP1B and LAR, for example, have been implicated in signaling through other growth factor receptors. Recent attention has been focused on serine/threonine phosphorylation of the insulin receptor and IRS proteins as a mechanism of insulin resistance. One key mediator of impaired insulin action in obesity is TNF␣ which is overexpressed in adipose tissue of obese insulinresistant rodents and in the muscle and adipose tissue of obese, insulin-resistant humans (Hotamisligil, 1999). TNF␣ treatment of cultured cells results in inhibition of insulin signaling. Loss of TNF␣ function by targeted knockout improves insulin sensitivity in a diet-induced obesity model and in the ob/ob genetic insulin-resistant obese model (Uysal et al., 1997). In an initial study, administration of a single dose of humanized TNF␣-neutralizing antibody had no effect on insulin sensitivity in obese diabetic patients (Ofei et al., 1996). Further studies are needed to address the potential efﬁcacy of agents that suppress TNF␣ action. Studies suggest that the mechanism by which TNF␣ elicits its adverse effects on insulin signaling is by serine phosphorylation of IRS-1 proteins on Ser307, rendering them poor substrates and inhibitors of the insulin receptor kinase (Rui et al., 2001). Inhibitory serine phosphorylation may be invoked in other models of insulin resistance. Insulin itself induces phosphorylation of IRS-1 on Ser307 in a mechanism distinct from that of TNF␣, suggesting that serine phosphorylation may be a feedback regulatory mechanism or a mechanism of insulin resistance in the chronic hyperinsulinemic state (Rui et al., 2001). Several serine kinases have been implicated as negative regulators of IRS proteins. These include JNK, MAPK, PKC isoforms and GSK-3 (Aguirre et al., 2000; Ravichandran et al., 2001). Thus, negative regulation of insulin signaling may involve complex feedback loops involving some of the same key players that propagate insulin signals. Signaling at the level of PI3K illustrates another site for regulation of insulin action by phosphorylation/dephosphorylation mechanisms, in this case by lipid kinase and phosphatases. The 3 lipid phosphatase, PTEN, and the 5 lipid phosphatases, SHIP1 and SHIP2, have been implicated as negative regulators of PI3K signaling in response to insulin, growth factors or cytokines. A homozygous deﬁciency in SHIP2 in mice results in increased insulin sensitivity and severe hypoglycemia resulting in neonatal death (Clement et al., 2001). Heterozygous knockouts are viable and exhibit increased glucose tolerance and augmented insulin sensitivity in skeletal muscle and liver. Thus, regulation of insulin signaling by the interplay of protein kinases and phosphatases represents a possible area for therapeutic intervention to alleviate insulin resistance. Future studies will hopefully reveal candidate molecules that are speciﬁc to the insulin signaling cascade.

Protein expression and stability In addition to reduced insulin signaling capacity as a mechanism of insulin resistance, impaired insulin action can also result from a quantitative decrease in the expression of key

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signaling molecules. In cellular models of insulin resistance, chronic insulin or TNF␣ treatment results in decreased IRS-1 expression (Saad et al., 1995; Stephens et al., 1997). Treatment of 3T3-L1 cells with TNF␣ also decreases Glut4 expression. IRS-1 expression is blunted in the liver and skeletal muscle of several rodent models of insulin resistance and, in ob/ob mice, differential regulation of the expression of PI3K regulatory subunits is observed (Saad et al., 1992; Kerouz et al., 1997; Anai et al., 1998). In humans, reduced IRS-1 protein levels have been observed in isolated adipocytes from type 2 diabetics while IRS-2 expression was unchanged (Rondinone et al., 1997). In another study of two groups at risk for type 2 diabetes (one with ﬁrst-degree relatives with diabetes and another obese group), low IRS-1 gene expression correlated with insulin resistance (Carvalho et al., 1999). In addition to regulation of gene expression, IRS-1 is targeted for degradation via proteosomes in a PI3K-dependent pathway following chronic insulin treatment of CHO cells (Sun et al., 1999). Interestingly, IRS-2 was resistant to this effect, suggesting differing modes of negative regulation for these proteins. Also, in 3T3-L1 cells, chronic insulin has been shown to induce rapamycin-sensitive serine/threonine phosphorylation of IRS-1 resulting in IRS-1 degradation (Pederson et al., 2001). Together these studies suggest that down-regulation of IRS-1 or other key insulin signaling molecules, whether by gene regulation or protein stability, may be a mechanism of insulin resistance and alleviation of this effect may be of therapeutic value. Genetic mutations Are genetic mutations in insulin signaling molecules a cause of insulin resistance and diabetes? Genetic defects correlating with decreased biochemical function have been found in insulin, the insulin receptor and IRS-1 proteins in humans and polymorphisms in IRS-2, IRS-4 and the p85 subunit of PI3K have also been identiﬁed. Their rare occurrence suggests that these mutations are unlikely to represent a general mechanism of genetic susceptibility for type 2 diabetes. Mutations in the insulin receptor, for example, are estimated to exist in 1–2% of the diabetic population and do not explain the prevalence of the deﬁciency of insulin receptor kinase activity in type 2 diabetes (Caro, 1996). Mutations in IRS-1 are more prevalent, with the G972R polymorphism being the best characterized to date. This mutation was found to be associated with type 2 diabetes in a Caucasian population and, in cultured cells, interferes with IRS-1 binding to the p85 subunit of PI3K (Almind et al., 1996). More recent studies in a Japanese population and in ﬁrst degree relatives of patients with type 2 diabetes indicate that this mutation does not predict insulin resistance in these groups (Ito et al., 1999; Koch et al., 1999). Thus, IRS-1 mutations, along with mutations in the related IRS-2 and IRS-4 genes identiﬁed to date, do not widely predict insulin resistance. It is likely that the range of mutations in various signaling molecules is not yet fully appreciated. We shall await further studies of these genes or the identiﬁcation of other prominent diabetes genes. Obesity Obese humans, with and without diabetes, are insulin resistant. Insulin receptor expression and activity and IRS-1 expression are reduced in skeletal muscle and adipocytes of obese subjects (Caro, 1996). In Zucker fatty rats and ob/ob mice, the expression of IRS-1 and IRS-2 are down-regulated in liver and muscle (Anai et al., 1998; Kerouz et al., 1997). Insulin-stimulated IRS-associated PI3K activity is blunted in both rodent models and this is associated with

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changes in the expression of the regulatory subunits of PI3K. The mechanism by which increased adiposity is linked to insulin resistance is not clear. Factors secreted by adipose tissue have been speculated to mediate this effect, such as release of free fatty acids or TNF␣, that may impair insulin signaling by serine phosphorylation of IRS proteins. Recently, resistin was identiﬁed as a candidate inhibitory protein secreted by adipose. Resistin levels are elevated in obese rodent models and expression of resistin is suppressed in cultured 3T3-L1 adipocytes by antidiabetic thiazolidinedione treatment (Steppan et al., 2001). Resistin impairs insulin action and glucose tolerance in normal mice and suppresses insulin-stimulated glucose uptake in cultured adipocytes. Thus, targeting of such proteins for therapeutic modulation may represent an approach for the treatment of insulin resistance.

KNOCKOUT ANIMAL MODELS OF INSULIN RESISTANCE A number of transgenic animal models lend to our understanding of the role of signaling proteins in normal insulin action, insulin resistance and diabetes. Though they do not mimic the human disease per se, these models have served to validate some of the plethora of signaling molecules that could serve as targets for therapies to treat insulin resistance in humans. We will focus on transgenic knockout models with gene alterations at or proximal to the insulin receptor. The reader is referred to other chapters of this book for discussion of other important rodent models associated with insulin resistance, including models of obesity and hypertension. Insulin Receptor The critical importance of the insulin receptor in maintenance of glucose homeostasis in peripheral tissues is observed in mice with a homozygous null mutation for the receptor. Insulin receptor knockouts exhibit severe insulin resistance and die from ketoacidosis 3–7 days after birth and heterozygous mice exhibit insulin resistance (Accili et al., 1996; Joshi et al., 1996). A recent series of knockout studies has probed the effect of localized insulin resistance by tissue-speciﬁc ablation of insulin receptor expression. A summary of phenotypes of several insulin-resistant animal models, generated by knocking out the insulin receptor, is presented in Table 1. These studies have yielded both expected and unexpected ﬁndings. In contrast to what one might have expected, given that muscle accounts for approximately 80% of glucose disposal, mice lacking insulin receptor expression in skeletal muscle and heart (MIRKO) exhibited normal insulin and glucose levels despite impaired insulin signaling in muscle tissue (Bruning et al., 1998). Altered fat metabolism in these animals was evidenced by elevated fat pad mass, elevated serum triglyceride and free fatty acid levels, conditions of syndrome X. This was associated with a compensatory 3-fold increase in insulin-stimulated glucose uptake in adipose tissue, revealing a redistribution of glucose to this tissue (Kim et al., 2000). In hyperinsulinemic-euglycemic clamp studies, insulin-stimulated whole-body glucose uptake and glycogen synthesis were blunted by 45% and 80%, respectively and whole-body glucose disposal was reduced 45% in MIRKO vs. control mice. The mild phenotype of MIRKO mice challenges the view of muscle as a primary site of insulin resistance and contrasts with the severe insulin resistance observed in mice lacking insulin signaling in liver (LIRKO; Michael et al., 2000). LIRKO mice are glucose intolerant

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Knockout animal models of insulin resistance – insulin receptor knockouts.

Knockout model

Phenotype

Reference

Insulin receptor/

Severe insulin resistance, die of ketoacidosis at 3–7 days Slight insulin resistance

Accili et al., 1996; Joshi et al., 1996 Accili et al., 1996; Joshi et al., 1996 Bruning et al., 1998

Insulin receptor/ Muscle-speciﬁc insulin receptor knockout Liver-speciﬁc insulin receptor knockout Beta cell-speciﬁc insulin receptor knockout CNS-speciﬁc insulin receptor knockout

Normal glucose disposal, altered fat metabolism (elevated fat pad mass, elevated serum TG, FFA), compensation by adipose Insulin resistant, liver hypoplasia, islet hyperplasia, FFA and TG decreased 30–50%, fasting hyperglycemia Impaired insulin secretion, progressive glucose intolerance Mild insulin resistance, diet-induced obesity, reproductive defects

Michael et al., 2000

Kulkarni et al., 1999 Bruning et al., 2000

despite marked hyperinsulinemia and islet hyperplasia. Insulin fails to suppress hepatic glucose production and expression of phosphoenolpyruvate carboxykinase (PEPCK), consistent with insulin resistance in the liver. Interestingly, the phenotype of these mice improves with age and blood glucose levels return to normal at 4 months of age, though the mice remain insulin resistant. Thus, an isolated defect of insulin action in the liver in this model results in severe defects in whole-body glucose homeostasis. Additionally, insulin resistance in the brain and central nervous system may contribute to secondary insulin resistance in the periphery. Mice with a neuronal-speciﬁc knockout of the insulin receptor develop diet-induced obesity and mild insulin resistance (Bruning et al., 2000). Interestingly, this phenotype is almost opposite that of knockout animals lacking PTP1B that exhibit enhanced insulin sensitivity and resistance to diet-induced obesity in comparison to controls. Perhaps the altered regulation of weight gain in the PTP1B knockout animals is due to increased insulin signaling in the central nervous system. The phenotype of mice lacking insulin receptor expression in pancreatic beta cells illustrates a previously unappreciated role of the insulin receptor in these cells and suggests that the pancreas is also an important site for insulin resistance. ␤IRKO mice exhibit a defect in ﬁrst-phase insulin secretion in response to glucose and impaired glucose tolerance (Kulkarni et al., 1999). These ﬁndings emphasize that insulin resistance conferred by defects in insulin action is not just a malady of classic target tissues (muscle, fat and liver) but may involve a defect in autocrine insulin signaling in the beta cell important for glucose sensing and may contribute to progressive loss of insulin secretion in type 2 diabetes. Thus, targeting defects in insulin action at this tissue site, as well as the periphery, may prove useful for the treatment of insulin resistance and type 2 diabetes. IRS proteins Knockout mice lacking IRS-1 and IRS-2 establish the important distinct physiological roles for these insulin signaling proteins in the peripheral metabolic response to insulin in classic target tissues, as well as in beta cell development or survival. The phenotypes of mice with genetic disruption of these molecules are summarized in Table 2. Mice with a homozygous

320 Table 2

JULIE S. MOYERS AND JOSÉ F. CARO Knockout models of insulin resistance – targeting downstream insulin signaling molecules.

Knockout model

Phenotype

Reference

IRS-1 /

Mild insulin resistance, 40% growth retardation, beta cell hyperplasia

Araki et al., 1994; Tamemoto et al., 1994; Patti et al., 1995 Withers et al., 1998

IRS-2 /

Severe diabetes at 8–10 weeks of age, reduced beta cell mass, leading to death IRS-4 / Mild glucose intolerance, 10% reduced size IRS-1/ IRS-2/ Diabetic at 2–3 weeks of age IRS-1 / IRS-2 / Mild glucose intolerance IRS-1 / IRS-2 / Mild glucose intolerance IR/ IRS-1/ Insulin resistance in skeletal muscle and liver, hyperinsulinemia, beta cell hyperplasia, 20–40% diabetic IR/ IRS-2/ Insulin resistance in liver, mild in skeletal muscle, mild beta cell hyperplasia, 17% diabetic IR/ IRS-1/ IRS-2/ and muscle, beta cell Kido et al., 2000 hyperplasia, 40% diabetic Glut4 knockout/ Mild insulin resistance, reduced adipose, cardiac hypertrophy Glut4 knockout/ Insulin resistance phenotype more severe than Glut4/ Glut4 muscle-speciﬁc Insulin resistance and glucose intolerance knockout Glut4 fat-speciﬁc knockout Insulin resistance in adipose, muscle and liver, glucose intolerant hyperinsulinemia

Fantin et al., 2000 Withers et al., 1999 Withers et al., 1999 Withers et al., 1999 Bruning et al., 1997; Kido et al., 2000 Kido et al., 2000 Severe resistance in liver

Katz et al., 1995 Rossetti et al., 1997 Zisman et al., 2000 Abel et al., 2001

null mutation in IRS-1 exhibit mild insulin resistance, hyperinsulinemia associated with increased beta cell mass and retarded growth (Araki et al., 1994; Tamemoto et al., 1994). The main site of resistance in these animals appears to be skeletal muscle, though adenovirusmediated restoration of IRS-1 expression to near normal levels in liver restores insulin sensitivity in IRS-1-deﬁcient mice (Ueki et al., 2000), indicating a signiﬁcant contribution of liver to glucose homeostasis in this rodent model. The relatively mild phenotype of IRS-1 knockouts is partially attributed to compensation by IRS-2. In contrast to IRS-1 knockouts, insulin resistance and progressive development of diabetes is seen in mice lacking IRS-2. Homozygous loss of IRS-2 results in reduced beta cell mass. Over time, loss of beta cell compensation, together with peripheral insulin resistance, leads to hyperglycemia (Withers et al., 1998). This model, together with the ␤IRKO model, present the insulin receptor and IRS-2 proteins as determinants of beta cell function and likely important contributors to beta cell compensation in addition to the important role that they play in peripheral insulin sensitivity. Recent data assessing rates of glucose and glycerol turnover under basal and hyperinsulinemic-euglycemic clamp conditions compared the phenotype of IRS-1/ and IRS-2/ knockouts. Insulin resistance in IRS-1/ mice was attributed largely to peripheral insulin resistance in muscle (Previs et al., 2000). Insulin resistance in IRS-2 knockout mice was most pronounced in liver with some effect on insulin-stimulated glycogen synthesis in muscle and lipolysis in adipose tissue. Thus, insulin resistance is manifested by different means in mice lacking these two important, related signaling molecules. The important role of IRS-2 in the liver was also noted in two other animal models of insulin resistance, the

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lipodystrophic SREBP-1c mice that express an active form of SREBP-1c in adipose and the ob/ob obese, leptin deﬁcient mice. In response to chronically elevated insulin levels, IRS-2 mRNA was down-regulated in the liver but not skeletal muscle, while IRS-1 was unaffected (Shimomura et al., 2000). IRS-2 deﬁciency leads to insulin resistance and failure to suppress hepatic gluconeogenesis but not insulin-stimulated lipogenesis, resulting in a vicious cycle of aggravated hyperinsulinemia and insulin resistance.

Additional insulin signaling molecules We have focused attention on insulin-resistant animal model with defects at the level of the insulin receptor or IRS proteins. It is important to note, though, that knockout models with other insulin signaling defects have revealed important regulators of glucose homeostasis and have expanded our knowledge for potential therapeutic approaches to overcome insulin signaling defects. One interesting example is the recent report of mice lacking the Glut4 glucose transporter in adipose tissue (Abel et al., 2001). These mice exhibit insulin resistance in adipose in addition to secondary insulin resistance in muscle and liver. Future identiﬁcation of the underlying mechanism for this, such as factors secreted by adipose, may reveal novel regulators of insulin sensitivity. In addition to genetically engineered mice, animals with nutritionally induced insulin resistance and type 2 diabetes have shed light on other important signaling molecules. Of note is the desert rodent, Psammomys obesus, which, on a high energy diet, exhibits progressive insulin resistance and diabetes (Ikeda et al., 2001). In comparison to a diabetes-resistant line, elevated levels of PKC isoforms (␣, ␧ and ␨) are seen in skeletal muscle of diabetic animals. PKC␧ is overexpressed even in the prediabetic state of diabetes prone animals and is associated with the plasma membrane, indicative of increased activity. Increased levels of PKC␧ or other isoforms may result in suppression of insulin signaling through inhibitory phosphorylation of IR or IRS proteins. Aside from data in nutritional models of diabetes, other studies implicate roles for PKC isoforms in insulin resistance and diabetes. In humans, PKC overexpression has been observed in the liver of type 2 diabetes, suggesting a role in the progression of the disease state (Considine et al., 1995). Additionally, some PKC isoforms play positive roles in insulin signaling. The atypical PKC␨ isoform expressed via adenovirus in the muscle of Zucker rats results in elevated glucose transport activity (Etgen et al., 1999). Thus, several PKC isoforms may represent targets for therapeutic approaches to treat insulin resistance and diabetes.

Polygenic models to mimic human disease The generation and study of mice with speciﬁc gene mutations in insulin signal transduction molecules has provided important insights into the role of these proteins and the interplay of multiple tissues in insulin resistance. Insulin resistance and type 2 diabetes are likely, for the most part, to be polygenic in nature and the generation of rodent models with deﬁned multiple lesions will no doubt further our understanding of the consequences of combined defects in insulin action. An example of this is the combined heterozygosity of insulin receptor, IRS-1 and IRS-2 knockout mice (Table 2). The combined defects of mice heterozygous for insulin receptor, IRS-1 and IRS-2 null alleles result in synergistic effects on insulin resistance (Kido et al., 2000).

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Knockout models of increased insulin sensitivity Genetic disruption of negative regulators of insulin signaling offers a means of further validation of these molecules as therapeutic targets. Two examples of this are mice lacking the PTP1B phosphatase or the SHIP2 lipid phosphatase (Table 3). Mice lacking PTP1B exhibit enhanced insulin sensitivity and a resistance to diet-induced weight gain (Elchebly et al., 1999). Studies in isolated tissues indicate that the main sites of enhanced sensitivity are muscle and liver where insulin receptor tyrosine phosphorylation is augmented. These results suggest that PTP1B may be a viable target for treatments for insulin resistance and possibly obesity. The resistance to diet-induced obesity in these mice was a surprising phenotype and future studies should reveal additional insights into the underlying mechanism. PTP1B knockout mice exhibit decreased adiposity correlating with increased energy expenditure (Klaman et al., 2000). Perhaps this reﬂects a role for PTP1B in signaling through the leptin receptor or other undeﬁned receptor pathways that regulate energy balance and body weight or that this occurs through the insulin signaling pathway itself, as suggested by the propensity for diet-induced obesity seen in mice lacking insulin receptor expression in the brain (above). Evidence indicates that the effect is, atleast in part, mediated by PTP1B effects on leptin signaling through Jak2, resulting in enhanced leptin sensitivity in mice lacking PTP1B (Cheng et al., 2002; Zabolotny et al., 2002). Increased insulin sensitivity is also seen in mice lacking the SHIP2 lipid phosphatase that is believed to act in opposition to PI3K. Homozygous loss of SHIP2 results in perinatal death associated with severe hypoglycemia (Clement et al., 2001). Heterozygous animals are viable and exhibit enhanced insulin sensitivity in glucose and insulin tolerance tests in comparison to controls. Increased Glut4 translocation and modest elevation of glycogen synthesis are observed in isolated muscle in response to insulin. Enhanced sensitivity in liver was evidenced by decreased expression of gluconeogenic enzymes in SHIP2/ mice. Illustration of the complicated regulatory mechanisms of insulin signaling is exempliﬁed by knockout of the regulatory subunits of PI3K. Mice lacking the p85␣ or the p85, p55 and p50 subunits of PI3K exhibit increased insulin sensitivity (Terauchi et al., 1999; Fruman et al., 2000). These ﬁndings shed new light on p85␣ as an inhibitory regulator of insulin signaling and potential therapeutic target. In summary, studies of rodent models with genetic defects in insulin signaling molecules emphasize the complexity of insulin resistance and type 2 diabetes as a multitissue and multigenic disorder. We have focused on models with defects at or proximal to the insulin receptor to illustrate these points. The development of additional models of insulin resistance or insulin sensitivity will no doubt add to our understanding of these complex issues. Table 3

Knockout models with increased insulin sensitivity – targeting proximal insulin signaling molecules.

Knockout model

Phenotype

Reference

PTP1B knockout

Increased insulin sensitivity, resistance to diet-induced obesity, increased energy expenditure Severe hypoglycemia, lethal Increased insulin sensitivity Increased insulin sensitivity, upregulation of p55 and p50 Lethal

Eschelby et al., 1998; Klaman et al., 2000 Clement et al., 2001 Clement et al., 2001 Terauchi et al., 1999 Fruman et al., 2000

Increased insulin sensitivity

Fruman et al., 2000

SHIP2 knockout/ SHIP2 knockout/ PI3K p85 regulatory subunit PI3K p85, p55, p50 regulatory subunits/ PI3K p85, p55, p50 regulatory subunits/

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THERAPEUTIC STRATEGIES TO TREAT INSULIN RESISTANCE Diabetes as a disease has been known for centuries and insulin was discovered by Banting and Best in 1921. Why is it that deﬁnitive, effective therapeutic strategies have eluded us? One issue is that type 2 diabetes is a progressive disease and the primary sites are not clear. In humans, insulin resistance and diabetes are associated with complex disorders of varying severity, including obesity, dislipidemia and cardiovascular abnormalities. Aside from the human condition, the complexity of phenotypes seen even in insulin-resistant animal models with deﬁned lesions underscores the complexity of the factors involved in insulin resistance that underlies type 2 diabetes. Of consequence is the difﬁculty in designing therapeutic strategies to effectively overcome these defects. Another challenge is the multiplicity of genetic and environmental factors of underlying etiology, such that one “cure-all” is in all likelihood impossible. Strategies to treat multiple lesions or to treat early stages of the disease progress may be effective. Our ever-increasing understanding of the complexity of insulin signaling should pave the way for the continued emergence of validated targets of therapeutic potential to treat insulin resistance. Why the need to validate proteins of interest? Drug development is an inherently difﬁcult process. Even with a validated target, the statistics do not bode well. It has been estimated that the development of a drug in the United States takes an average of nearly ﬁfteen years and an average 880 million US dollars (Dimasi et al., 1995; Tollman et al., 2001). Nonetheless, progress over even the last several years in the area of insulin signaling has been immense. A deeper understanding of critical regulators of insulin action sets the stage for the identiﬁcation of novel therapeutic interventions.

Targeting Insulin Action This chapter has addressed some points of regulation of insulin signaling and has addressed the inﬂuence of alterations in some key signaling molecules on insulin resistance. It is clear that insulin resistance can be modiﬁed by factors at many steps of the insulin signaling cascade. Where in the many levels of signaling should we focus our attention for targets for effective therapeutic intervention? Will appropriate modiﬁcation at any of the many steps in the insulin signaling cascade be effective? There are a number of factors to consider. First, we must remember that normal insulin action involves a complex balance of positive and negative regulation. While we hope to overcome or correct the defects that cause insulin resistance, we must devise strategies that will be efﬁcacious and not otherwise detrimental. For example, targeting of molecules downstream from the insulin receptor could bypass needed regulatory signals elicited by upstream molecules and cause an imbalance that could exacerbate rather than correct the problem. Another issue involves the potential targeting of insulin signaling at a point either upstream or downstream of a rate-limiting signaling step or disease-associated defect in the cascade. Skeletal muscle glucose transport is thought to be the rate-limiting step in normal glucose metabolism and overexpression of Glut4 in the muscle and fat of mice increases insulin-stimulated whole-body glucose disposal (Ren et al., 1995). Will enhancement of upstream signaling elicit a feedforward mechanism? Data from knockout mice, PTP1B as an example, suggest that signaling, at least of normal insulin action, can be augmented by such an approach. Will targeting downstream be effective? Data would suggest that it probably would since an inhibitor of GSK-3 has been shown to stimulate glycogen synthesis in human liver cells and induce gene expression in 293HEK cells (Coghlan et al.,

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2000). These are complex issues and, at least to some degree, are speculations. Emerging knowledge will continue to clarify these and other questions regarding targeting of the insulin signaling pathway.

Emerging Therapies for the Treatment of Insulin Resistance The oral biguanides (such as metformin) and oral thiazolidinediones (such as pioglitazone, rosiglitazone and troglitazone) have been shown to have insulin sensitizing properties and have been used as therapeutic agents to treat diabetes (Cusi and Defronzo, 1998; Olefsky and Saltiel, 2000). We will not discuss these in detail but, rather, will focus on emerging approaches to increase insulin sensitivity by targeting signaling molecules at or proximal to the insulin receptor. The identiﬁcation of regulators of insulin signaling combined with lessons from transgenic rodent models allows us to make some rational speculations about future therapies to treat insulin resistance. Hopefully these and other targets for therapeutic intervention will someday prove to be more than hypotheses. While this section is focused on emerging therapies to treat insulin resistance by augmentation of the insulin signaling pathway, it is worth noting that other mechanisms to improve glycemic control, such as treatments for obesity, may indirectly improve insulin resistance. Here we will present two examples of efforts based on validated targets in the proximal insulin signaling cascade, the insulin receptor itself and the PTP1B phosphatase.

Insulin receptor activators Will compounds that activate the insulin receptor be effective therapies in patients who are insulin resistant? Studies in humans have shown that the insulin receptor kinase activity is down-regulated in skeletal muscle, adipocytes, hepatocytes and circulating blood cells of type 2 diabetics and that this is not explained by a wide prevalence of genetic mutations in the ␤ subunit (Caro, 1996). Furthermore, this effect is reversed upon weight loss in obese patients with type 2 diabetes. While the nature of this defect preventing insulin activation of the receptor is not fully understood, it is conceivable that agents distinct from insulin that activate the receptor kinase could be effective in these patients. The possibility of identifying compounds other than insulin that directly activate the insulin receptor has long appeared remote. Recently though, Zhang et al. (1999) have reported the identiﬁcation of a non-peptide small molecule fungal metabolite that activates the insulin receptor in in vitro assays, in cultured cells and in vivo (Zhang et al., 1999). The compound was identiﬁed in a cell-based screening assay designed to detect phosphorylation of overexpressed insulin receptor protein. The compound stimulated activity of a recombinant insulin receptor kinase domain in vitro, indicating that it acts on the intracellular ␤ subunit of the receptor in a mechanism distinct from that of insulin that binds to the extracellular ␣ subunit. In cells, the compound activated insulin receptor tyrosine kinase activity toward an exogenous substrate in a dose-dependent manner (Figure 2). With low concentrations of compound, insulin-stimulated receptor kinase activity was augmented, indicating that the molecule can also act as an insulin sensitizer (Figure 2). The compound exhibited selectivity vs. the insulin-like growth factor receptor, epidermal growth factor receptor and platelet-derived growth factor receptor when tested in cells and did not inhibit tyrosine phosphatases in vitro. Furthermore, the compound was shown to activate downstream insulin signaling pathway through PI3K and Akt in cultured cells and to stimulate glucose uptake in isolated rat adipocytes and murine skeletal muscle (Figure 3). In in vivo studies, oral administration resulted in both acute and long-term glucose lowering in db/db mice

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A H N

O

HO

HO

OH

OH O

N H

IRTK activity (% of control)

140

O

N H

L-783,281

B

H N

O

L-767,827

L-783,281

120

L-767,827

100 80 60 40 20 0 0

IRTK activity (% of control)

C

10–6

10–5 Concentration (M)

200

Control

150

L-783,281 (2 µM)

10–4

L-783,281 (0.6 µM)

100

50

0

10–10

10–9

10–8

10–7

10–6

Insulin (M)

Figure 2 Effects of small molecule insulin mimetics on insulin receptor tyrosine kinase activity in CHO-IR cells. (A) Structures and (B and C) effects on insulin receptor tyrosine kinase activity. Cells were serum-starved before treatment with test compounds or insulin in the presence of 0.1% dimethyl sulfoxide (DMSO) in the medium for 20 min at 37 C. Insulin receptor activity in immunoprecipitates is expressed as a percentage of the maximal activity achieved with 100 nM insulin using poly (Glu : Tyr) as substrate. (Reproduced with permission from Zhang, B. et al. (1999) Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science, 284, 974–977. Copyright 1999 American Association for the Advancement of Science.)

(Figure 4). The compound was also effective in lowering blood glucose levels in insulin-resistant ob/ob mice during a glucose tolerance test and lowered insulin levels following acute administration. Based on this structure, this group and colleagues have synthesized and tested additional compounds and have reported an additional insulin mimetic compound with improved potency

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JULIE S. MOYERS AND JOSÉ F. CARO 120 Pl3-kinase activity (% of control)

A

100 80 60 40 20 0 10–6 10–5 L-783,281 (M)

0

B

10–4

281 (µM) 0

1

3 In

D

2000 1500

*

1000 500 0

0 L-783,281

Glucose uptake (µmole/ml/hour)

C

C-14-glucose uptake (cpm)

p-Akt

7.5

*

5 2.5 0

0 0.2 2 L-783,281 (µM)

Figure 3 Activation of the insulin signaling pathway in cells treated with a small molecule insulin receptor activator. (A) Activation of PI3K. CHO-IR cells were untreated or treated with compound or 100 nM insulin for 20 min. PI3K activity in anti-phosphotyrosine immunoprecipitates is expressed as a percentage of the 100 nM insulin control. (B) Phosphorylation of Akt (PKB) in cells treated with compound or 10 nM insulin for 20 min. (C) Glucose uptake in adipocytes from male Wistar rats following treatment of cells with compound. (D) Glucose uptake in intact soleus muscle from lean (C57BL6) mice. (Reproduced with permission from Zhang, B. et al. (1999) Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science, 284, 974–977. Copyright 1999 American Association for the Advancement of Science.)

(Qureshi et al., 2000; Liu et al., 2000). This compound augments insulin-stimulated glucose lowering in a streptozotocin-induced diabetic mouse model, and lowers blood glucose during a glucose tolerance test in Harlan Sprague–Dawley rats. Thus, these ﬁndings have not only provided tools for further study and development, they have demonstrated that the feasibility of identifying and developing novel, orally active activators of the insulin receptor as therapeutics may not be so elusive. Insulin sensitizers Biochemical, cellular and animal studies have provided validation that inhibition of negative regulators of the insulin signaling cascade may be of therapeutic value. Vanadate and vanadium-like compounds have received much attention for their potential use as inhibitors of tyrosine phosphatases that negatively regulate insulin action, though they may have other

200 100 1

2 3 Time (h)

100

4

83

D Blood glucose (mg/dl)

B

100 50 h

0

250 200 150

–3

0

350 300

0h 30 m 60 m 90 m 12 0m 18 0m

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Plasma insulin (ng/ml)

Blood glucose (mg/dl)

A

327

,28 1, p.o . G lu c 0.3 ose g/k g, i.p.

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450 350 250 150 50

0

3 Day

7

75 Predosing 4h

50 25 0 Vehicle L-783,281

Figure 4 Antidiabetic efﬁcacy of orally administered small molecule insulin receptor activator. (A) Acute glucose lowering in 9-week-old db/db mice. (●) vehicle, (嘷) 5 mg/kg compound, (□) 25 mg/kg compound. (B) Glucose lowering in db/db mice after long-term dosing. (●) vehicle, (嘷) 5 mg/kgday, (□) 20 mg/kg/day, (▲) lean untreated control. (C) Glucose tolerance test in 12-week-old ob/ob mice. (●) vehicle, (嘷) 5 mg/kg compound, (□) 20 mg/kg compound, (▲) lean control. (D) Plasma insulin levels in ob/ob mice before and 4 h after single oral dosing of compound. (Reproduced with permission from Zhang, B. et al., 1999, Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science, 284, 974–977. Copyright 1999 American Association for the Advancement of Science.)

actions and their exact mechanism of enhancing insulin metabolic effects is not fully understood (Cam et al., 2000). Vanadate stimulates insulin receptor phosphorylation and activity, glucose uptake and glycogen synthase in cultured cells and in adipose, muscle and liver tissues. Oral vanadium salts alleviate hyperglycemia in rodent genetic models of insulin resistance and diabetes and in Psammomys obesus, a model of nutritionally induced insulin resistance (Goldﬁne, 1995; reviewed in Badmaev et al. 1999; Shafrir et al., 2001). Studies in humans have been less consistent and progress has been limited perhaps in part by issues of potential tissue accumulation and limited therapeutic index. One potential mechanism by which vanadate exerts its insulin sensitizing properties is by general inhibition of tyrosine phosphatases such as PTP1B or LAR. Speciﬁc targeting of inhibitory phosphatases is a promising strategy for the development of insulin sensitizers. Progress in this area was recently reported by Wrobel et al. (1999) for the identiﬁcation of PTP1B small molecule inhibitors that exert glucose lowering effects in ob/ob mice. Several compounds were identiﬁed that inhibit PTP1B activity toward an insulin receptor-based phospho-peptide substrate with sub-micromolar IC50 in vitro and the most potent compound (number 8) was competitive with substrate (Table 4). Two compounds, numbers 4 and 7, exhibited selectivity for PTP1B in vitro vs. several other tyrosine phosphatases tested, with a range of 1.5–160-fold selectivity observed vs. the various phosphatases tested. Thus, it is

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Table 4 Compounds that inhibit PTP1B activity toward an insulin receptor-based phospho-peptide substrate with Sub-micromolar IC50. Compound

3 4 5 6

384 386 145 61

7

83

8 Ciglitazoneg Troglitazoneg a

b c

d e f g

hPTP1B IC50 (nM) a

11

Dose b (mg/kg/day)

100 100 90 75 50 25 10 25 10 5 1 (ip) 100 100 200

4-day ob/ob mouse c (% decrease) Glucose d

Insulin d

e

e

23

61

e

e

50 35 26 6 42 29

85 71 72

e

38

e

86 69 56 f

e

e

43 34

39 e

Substrate is the triphosphorylated peptide TRDI(P)YETD(P)Y(P)YRK. The reported IC50 values are from direct regression curve analyses that are signiﬁcant (p 0.05). All doses are p.o. unless otherwise indicated. Plasma glucose and insulin values of 200–350 mg/dL and 300–775 IU/mL, respectively, in untreated mice. (Reproduced with permission from Wrobel et al. (1999) PTP1B inhibition and antihyperglycemic activity in the ob/ob mouse model of novel 11-arylbenzo[b] naphtho[2,3-d]furans and 11-arylbenzo[b] naphtho [2,3-d] thiophenes. Journal of Medicinal Chemistry, 42, 3199–3202. Copyright 1999 Amer. Chem. Soc.) All values from drug-treated mice, other than d, are signiﬁcant vs. vehicle-treated mice (p 0.05). Less than 15% decrease at dose tested. Not determined. Reference standard.

possible to achieve selectivity for inhibition amongst tyrosine phosphatases, though their catalytic domains have approximately 35% conserved identity. Analysis of several compounds in vivo demonstrated oral efﬁcacy following 4-day treatment of ob/ob mice where blood glucose and insulin levels were reduced. Additionally, members of this group and colleagues have reported the identiﬁcation of other compounds that inhibit PTP1B in vitro, correlating with in vivo efﬁcacy in both db/db and ob/ob models (Malamas et al., 2000a,b). Additional efﬁcacy and pharmacodynamic studies in vivo, as well as mechanistic studies in cells or isolated tissues will lend to our understanding of the potential therapeutic uses of these compounds. Given the phenotypes of increased insulin sensitivity and resistance to weight gain coupled with increased energy expenditure in mice lacking PTP1B, it is interesting to speculate that PTP1B inhibitors might also prove useful for the treatment of obesity. SUMMARY The study of insulin action at the biochemical, cellular and in vivo rodent model level is important since insulin resistance is a powerful predictor of the development of type 2 diabetes. We have reviewed the insulin signaling pathway and focused on a few of the multitude of signaling

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molecules and mechanisms involved in the regulation of insulin action that could serve as therapeutic targets. We have focused on several knockout models of insulin resistance or increased insulin sensitivity that illustrate the complex physiology of glucose homeostasis, even in a rodent model of deﬁned lesions. With acknowledgment that some degree of caution needs to be taken in extrapolating rodent physiology to the pathophysiology of the human condition, these and other rodent models have provided insights and encouragement for the development of therapeutic agents to combat insulin resistance and type 2 diabetes. Emerging progress in the area of insulin mimetics and insulin sensitizers are examples. We face many challenges on the road from target to drug. What will the future bring? We will likely identify additional novel regulators of insulin signaling, including modulators of gene expression, protein stability and feedback signaling mechanisms. Genetic approaches may identify novel genes linked to insulin resistance and diabetes. Additional rodent models will serve to validate new targets. We gain momentum with ever-increasing understanding of new targets and new approaches to allow identiﬁcation of small molecule modulators or other techniques for new therapies. REFERENCES Abel, E.D., Peroni, O., Kim, J.K., Kim, Y.B., Boss, O., Hadro, E., Minnemann, T., Shulman, G.I. and Kahn, B.B. (2001) Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver. Nature, 409, 729–733. Accili, D., Drago, J., Lee, E.J., Johnson, M.D., Cool, M.H., Salvatore, P., Asico, L.D., Jose, P.A., Taylor, S.I. and Westphal, H. (1996) Early neonatal death in mich homozygous for a null allele of the insulin receptor gene. Nat. Genet., 12, 106–109. Aguirre, V., Uchida, T., Yenush, L., Davis, R. and White, M.F. (2000) The c-Jun NH2-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser (307). J. Biol. Chem., 275, 9047–9054. Ahmad, F., Li, P.M., Meyerovitch, J. and Goldstein, B.J. (1995) Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway. J. Biol. Chem., 270, 20503–20508. Almind, K., Inoue, G., Pedersen, O. and Kahn, C.R. (1996) A common amino acid polymorphism in insulin receptor substrate-1 causes impaired insulin signaling – evidence from transfection studies. J. Clin. Invest., 97, 2569–2575. Anai, M., Funaki, M., Ogihara, T., Terasaki, J., Inukai, K., Katagiri, H., Fukushima, Y., Yazaki, Y., Kikuchi, M., Oka, Y. and Asano, T. (1998) Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes, 47, 13–23. Araki, E., Lipes, M.A., Patti, M.E., Bruning, J.C., Haag, B., Johnson, R.S. and Kahn, C.R. (1994) Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature, 372, 186–190. Badmaev, V., Prakash, S. and Majeed, M. (1999) Vanadium: A review of its potential role in the ﬁght against diabetes. J. Altern. Complement. Med., 5, 273–291. Bernier, M., Liotta, A.S., Kole, H.K., Shock, D.D. and Roth, J. (1994) Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells. Biochemistry, 33, 4343–4351. Bruning, J.C., Michael, M.D., Winnay, J.N., Hayashi, T., Horsch, D., Accili, D., Goodyear, L.J. and Kahn, C.R. (1998) A muscle-speciﬁc insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol. Cell, 2, 559–569. Bruning, J.C., Gautam, D., Burks, D.J., Gillette, J., Schubert, M., Orban, P.C., Klein, R., Krone, W., Muller-Wieland, D. and Kahn, C.R. (2000) Role of brain insulin receptor in control of body weight and reproduction. Science, 289, 2122–2125.

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Part 3

Aging

18.

EFFECT OF AGE ON THE EMERGENCE OF INSULIN RESISTANCE NIR BARZILAI AND ILAN GABRIELY

Department of Medicine, Divisions of Geriatrics, Endocrinology, and the Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA

INTRODUCTION Insulin sensitivity declines with aging, and manifests itself with a progressive increase in fasting and postprandial plasma insulin concentrations (Reaven and Reaven 1985; Davidson, 1979; Weingard et al., 1990). Typically in humans, plasma glucose levels are maintained at a normal range during fasting and postprandially; however, higher insulin levels are required to appropriately regulate endogenous glucose production and to avoid hyperglycemia (Fraze et al., 1987; Meneilly et al., 1987; Pagano et al., 1984; Robert et al., 1982; Jackson et al., 1988). As reviewed in other chapters, insulin resistance may further develop into a glucose intolerance state and type 2 diabetes mellitus (DM2). This is a process that is accelerated with aging; the incidence of DM2 reaching 30% in some older populations. Decline in insulin action is manifested in most animal models. In this chapter we will review the evidence for the decline in insulin action throughout aging in the obese rodent model and the beneﬁcial effects of caloric restriction (CR), suggesting that increased fat mass (FM) may play an important role in insulin resistance. Recent evidence suggests a cause–effect relationship between increased visceral fat (VF) and decreased insulin action. Such alterations in body fat distribution and in insulin action are modulated in part by the fat-derived peptide leptin, a process that fails with obesity and aging. A variety of other fat-derived peptides are over-expressed and released in excess in obese animals. Such peptides are regulated by nutrients and may have a pathophysiological role in the development of age-related disease, and possibly in longevity. Each section will begin by describing our knowledge on insulin action in aging humans, followed by examples learned from animal models, allowing for a more profound understanding.

AGING IS ASSOCIATED WITH INCREASE IN FM AND INSULIN RESISTANCE The epidemic of obesity affects approximately half of the US population over age 50 (Kuczmarski et al., 1994). Increased FM typically occurs between the third and seventh decades of life; thereafter FM may increase, remain unchanged, or decrease (Andres et al., 1985;

Address correspondence to: Nir Barzilai, Divisions of Geriatrics and Endocrinology, Department of Medicine, Belfer Bld. #701, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Tel.: (718)-430-3312; Fax: (718)-430-8557; Email: [email protected] 337

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Old

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Figure 1 MRI scans of the abdomen in young, old and old CR rats. Cross-sectional cut above the level of the pelvis. The white color depicts fat tissue. The old ad libitum-fed rat has signiﬁcantly more fat tissue (visceral and SC), compared to the young animal. By contrast, in the old CR animal there was a marked reduction in both visceral and SC fat stores.

Cohn et al., 1980; Borkan et al., 1983; Poehlman et al., 1995). Abdominal obesity in humans is due to increased VF, deﬁned as the sum of fat depots inside the abdominal cavity. CT studies in men and women of all ages revealed the sites of accumulation and a signiﬁcant inverse correlation between the ratio of subcutaneous (SC) to VF with age (Enzi et al., 1986). Numerous studies in subjects of all ages have demonstrated that increased VF is associated with insulin resistance (Kissebah, 1996; Bjorntrop, 1991) and the development of DM2 (Larson, 1992). Moreover, increased VF is an independent risk factor for the development of coronary artery disease (Prineas et al., 1993), stroke (Larson, 1992), and death (Folsom et al., 1993) in prospective studies in men and women. Thus, both total FM and VF are increased with aging, and may induce early mortality. Aging ad libitum fed rats develop increased FM, with a disproportionate increase in VF, demonstrating similarity to humans aging (Figure 1), (Barzilai et al., 1995; Banerjee et al., 1997; Gupta et al., 2000a). Importantly, age-related changes in fat distribution are manifested between 8 and 20 months old (mo) rats, which are matched for lean body mass (LBM) (Gupta et al., 2000b). The similarities in fat and its distribution with aging between humans and rats allow one to engage in the comparative biology of this aging model.

PERIPHERAL INSULIN SENSITIVITY WITH AGING Increased FM and VF are associated with peripheral insulin resistance in aging. A large multicenter study has demonstrated that FM, and not age, is associated with decreased peripheral insulin sensitivity. Additionally, obese subjects with similar body mass index who had more VF had more severe insulin resistance measured by leg balance studies (Ferrannini et al., 1996) or by the clamp technique (Peiris et al., 1988) than subjects with less VF. Indeed, VF accounted for much of the variance in insulin sensitivity in women (Carey et al., 1996) and in lean diabetic African Americans (Banerji et al., 1995). VF affected insulin sensitivity in healthy older men the most, followed by obesity and VO2max (Coon et al., 1992). These studies in humans, however, should be cautiously interpreted because any increase or a decrease in VF is commonly associated with a parallel change in FM, making the cause–effect relationship between increased VF to insulin sensitivity more difﬁcult. Animal models are homogenous in their food intake, energy expenditure, and changes in body weight throughout aging. However, the gold standard technique to measure insulin

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sensitivity, that is, insulin clamp, was developed in humans and much later introduced to study animal models. Several groups have perfected this technique in rats and mice, and studied animals that are chronically catheterized, awake, and unstressed, also obtaining tissues at the end of the study for in vitro analysis. Insulin clamps employed in aging rats demonstrated that with increased FM and VF they became insulin resistant (Masoro, 1980). Since the process of aging is associated with both increases in VF and insulin resistance, it is important to clarify what is the effect of aging per se (independently from VF) on insulin sensitivity. Indeed, any effort to study insulin action has to be viewed relative to adiposity. Rats that were fed ad libitum and examined at the age of 2–18 months showed a signiﬁcant (~3-fold) gradual increase in FM, with a high correlation between body weight and epididymal fat (VF), and FM and epididymal fat (Barzilai et al., 1998). However, the rate of insulin-stimulated glucose uptake decreased signiﬁcantly between ages 2 months and 4 months with no further change in the older rats. This suggests that with increased FM, insulin resistance may be maximal at a relatively early age. To further differentiate between aging and obesity, a caloric restricted (CR) model was used. This animal model has been shown to live longer, but this effect has been traditionally attributed to a decrease in nutrients but not in fat. In order to correlate between FM and insulin action with aging, 18 months old rats were caloric restricted (55% food intake) and examined for peripheral insulin sensitivity using the hyperinsulinemic clamp. The results have demonstrated that once FM was reduced to below 13% of their body weight a dramatic improvement in insulin responsiveness was noted, which paralleled with the degree of insulin responsiveness seen in young rats (Figure 2), (Gupta et al., 2000b). Thus, the effect of visceral and total FM on peripheral insulin action is saturable, and because this saturable effect occurred at a relatively young age and did not change with aging, it suggests that aging

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Figure 2 The effects of aging on insulin-mediated glucose uptake in ad libitum fed and chronically CR rats. Glucose uptake was markedly decreased after puberty and did not decrease with aging after late adulthood, when expressed in terms of LBM. CR restored insulin action when FM was decreased to below 13–14% of body weight. When tight CR was applied and FM decreased to 13%, glucose uptake increased dramatically. Values represent the mean – standard error.

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per se is not associated with further decrease in peripheral insulin sensitivity. While one advantage of the animal model is the availability of tissues, studies failed to demonstrate persistent deﬁciency in the insulin-signaling pathway with aging. While age-associated alterations in insulin and IGF-1 receptor binding, glucose transporter levels in cardiac and skeletal muscle glucose transporters, and PI3-kinase protein contents were demonstrated in C57BL/6 mouse (Roubenoff, 1997) and rats (Hotamisligil et al., 1993), these were generally not adjusted for the effects of obesity. In fact, CR increases insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 in rat skeletal muscle. Interestingly, when CR rats are studied in different stages of adult life, alterations in in vivo insulin sensitivity cannot be demonstrated with aging (Gupta et al., 2000b). Thus, FM accumulation, rather than aging, may account for much of the age-dependent insulin resistance.

HEPATIC INSULIN SENSITIVITY WITH AGING Increased FM and VF are associated also with a progressive decline in the ability of insulin to suppress hepatic glucose production (HGP). While peripheral insulin sensitivity determines the ability to increase glucose uptake after meals, hepatic insulin sensitivity modulates glucose homeostasis for the rest of the day. The importance of the liver is exempliﬁed in diabetes, where morning hyperglycemia is determined by excess HGP, whether it is due to lack of insulin (DM1) or due to hepatic insulin resistance (DM2) (DeFronzo, 1992). While HGP is unchanged with aging, it occurs in the presence of increasing plasma insulin and glucose levels (Rowe et al., 1983). This suggests that the aging liver is resistant to the inhibitory effects of insulin on HGP. While various studies have demonstrated normal suppression of HGP by insulin in populations with normal fasting plasma glucose, plasma insulin levels, and normal weights, HGP suppression was delayed in elderly subjects (Jackson et al., 1988). When HGP was examined in lean and obese subjects, there was an inverse correlation between the ability of insulin to suppress HGP and body weight. VF may play even more of a determinate role on the liver than on the muscle, where HGP during hyperinsulinemia was increased ~4-fold in subjects with abdominal obesity (Carey et al., 1996). Similarly, a 2–3-fold increase in portal insulin levels was needed to suppress HGP in women with abdominal obesity as compared with lower body obesity (Peiris et al., 1988). Thus, increased FM and VF correlates with decreased hepatic insulin action. Aging rats develop impairment in the ability of insulin to suppress HGP and a higher insulin concentration is needed to maintain basal HGP (Barzilai et al., 1996). CR of aging rats can help to determine the relative role of increased VF on insulin action. In old rats (18 months), both FM and VF were decreased by CR to 33% of the levels observed in old ad libitum fed control. However, this intervention dissociated between VF and FM because the old CR animals had ~50% more FM but 33% less VF compared to the young controls. While fasting plasma glucose concentrations and the basal HGP were similar in all groups, the insulin infusion rates needed to clamp the plasma glucose at basal levels (hepatic pancreatic clamp) were 2-fold higher in the old controls and 3-fold lower in the old CR rats compared to young controls. Thus, the dissociation between VF and FM carried to insulin action, where excess VF seemed to determine hepatic insulin resistance. This study further supported the notion that the ability of insulin to modulate HGP is impaired by fat accumulation but not with aging per se. A decrease in VF uniformly leads to a marked increase in hepatic insulin action in young and old rats, so that hepatic insulin insensitivity is not demonstrated in aging CR animals (Gupta et al., 2000a).

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Figure 3 (A) VF (epididymal, perinephric, and mesenteric fat) and (B) Insulin infusion rate (IIR) during a hepatic – pancreatic clamp. Studies were performed in rats whose VF was removed (VF) and in sham-operated control rats (VF). *p 0.001 vs. VF.

REMOVAL AND DECREASE OF VF While removal of VF cannot be performed in humans because of the ﬁne network of blood vessels and nerves in omental fat that feeds the intestines, rodent models do provide an opportunity to test whether removal of VF modulates insulin action. To directly examine whether VF modulates hepatic insulin action, moderately obese Sprague–Dawley rats were randomized either to surgical removal of the epididymal and perinephric fat pads (VF), or to shamoperation (VF). Total VF in VF (due to unaltered mesenteric fat) was ~20% of that of VF (Barzilai et al., 1999). However, total FM was not signiﬁcantly different between the groups because the VF that was removed accounted for only ~10% of the total fat. The rates of insulin infusion required to maintain plasma glucose levels and basal HGP in the presence of hepatic–pancreatic clamp were ~50% decreased in VF compared with VF, and so were plasma insulin levels (Figure 3), (p 0.001). In spite of this difference in basal insulin, hepatic insulin sensitivity in VF rats decreases the expression of insulin-regulating hepatic genes such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxikinase (PEPCK). This study provides the best cause–effect evidence to demonstrate that VF is a potent modulator of insulin action. Similar decreases in VF were generated in rats chronically treated with ␤3-adrenoreceptor agonist (␤3) and CR, compared with control rats. These interventions were designated to decrease VF similarly, independent of changes in FM (Barzilai et al., 1999). While VF was similar, total FM was increased in the ␤3 treated group compared with the CR. Hepatic insulin sensitivity was increased ~3 fold in CR and ␤3 treated animals in comparison with the controls, suggesting that the reduction in VF rather total FM improved insulin action.

FAT AS A BIOLOGICALLY ACTIVE TISSUE In the last several years various peptides have been identiﬁed and shown to have the following characteristics: they are produced either primarily by fat tissues or also by other organs, are secreted and measured in the plasma, and have a distinct action on other tissues or organs.

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Some of these fat derived peptides (e.g. TNF, leptin) play a key role in insulin action and carbo-hydrate metabolism. Both angiotensinogen (AGT) and plasminogen activator inhibitor-1 (PAI-1) are secreted from fat, their expression is increased in obesity, and they are associated with morbidity and deaths from acute coronary events or stroke if untreated in humans. Another example of a fat-derived protein, Acrp30, which is similar to c1q, is produced exclusively in adipocytes, has high plasma levels measured in g/ml range, binds preferentially to muscle, has a cytokine tertiary structure and is regulated both acutely and chronically by insulin (Scherer et al., 1995). In addition to cytokines, complement factors (D, C3, B) are also secreted by fat tissue and have a role in controlling immune responses (Sherry and Cerami, 1988; White et al., 1992). Increased expression of AGT, PAI-1, cytokines, complement, and other acute phase reactants may have a role in immunological, hematological, vascular, and other functions. However, the modulation of synthesis and release of these peptides is greatly determined by age, percent body fat and ultimately by insulin sensitivity. On the other hand, some of these fat-derived peptides may modulate insulin action themselves. TNF-␣ Plasma levels of TNF-␣, previously known as lymphotoxin and cachectin, are increased in acute and chronic inﬂammatory and infectious diseases, as well as in cancer (Sherry and Cerami, 1988; Tracey et al., 1989). TNF-␣ was shown to be associated with, and probably mediates, weight loss, muscle wasting, increased urinary nitrogen excretion, as well as synthesis of acute phase proteins, changes that also occur with aging. However, in the basal state, TNF-␣ is believed to be derived from adipocytes, and its plasma levels are directly correlated with FM in animals and humans (Hotamisligil et al., 1995). Evidence suggests that fat-derived TNF-␣ also directly involved in the development of insulin resistance in obesity. In vitro studies have demonstrated that TNF-␣ decreases the phosphorylation of the insulin receptor tyrosine, insulin receptor substrate-1, and down regulates the GLUT-4 mRNA (Kanety et al., 1995; Feinstein et al., 1993). While plasma levels of TNF-␣ (in the range of pg/ml), seem physiologically irrelevant for TNF-␣ action (effects at ng/ml), in vivo studies have shown improvement of insulin action by TNF-␣ neutralizing agents. Thus, TNF-␣ may have a paracrine effect, a hypothesis that is supported by several genetic manipulations (knock-out) of both TNF-␣ and its receptor gene in mice (Ventre et al., 1997; Uysal et al., 1997). In these studies, while FM and body weight increased with aging, insulin sensitivity and glucose tolerance were improved. Interestingly, VF extraction resulted in a marked decrease in the expression of TNF-␣ in SC fat, supporting the notion that decreased expression of TNF-␣ increases insulin sensitivity that is observed with this manipulation. While evidence suggests that TNF-␣ may modulate insulin action, it is possible that a decrease in plasma levels of TNF-␣ plays a role in preventing sarcopenia, a variety of immunological functions, or other functions associated with aging, which are reversed by CR. Leptin Leptin (Lep) is a peptide expressed and secreted from adipose tissue that acts through a receptor in the hypothalamus, and may have peripheral actions as well (Zhang et al., 1994; Flier, 1997). The ﬁrst described mechanism of action of Lep in the hypothalamus was to decrease food intake through inhibition of neuropeptide-Y. Human Lep levels are directly correlated with body weight and FM, and the plasma levels (measured at ng/ml) are clinically relevant to

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the demonstrated action of this peptide (Considine et al., 1996). Thus, plasma leptin levels closely follow the increase in plasma insulin levels, and insulin resistance. Lep administration to obese mice models leads to marked improvement in glucose tolerance (Halaas et al., 1997). Rodents with a marked deﬁciency in Lep function, such as the ob/ob and db/db mice and the Zucker fa/fa rats, are extremely resistant to insulin action and develop diabetes mellitus later in life. In fact, administration of exogenous Lep in ob/ob mice resulted in a marked decrease in both plasma glucose and insulin concentrations (Halaas et al., 1995). Since these decreases were greater in Lep-treated mice than in pair-fed (PF) control mice (Pelleymounter et al., 1995), it has been proposed that Lep may directly (through the insulin signaling pathway) improve in vivo insulin action. In cultured myotubes, Lep activates JAK-2, which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase (Kellerer et al., 1997). Short-term administration of Lep (48 h) in normal rats, increased insulin sensitivity during hyperinsulinemia at clamped glucose. These effects did not appear to be dependent on altered body weight (Sivitz et al., 1997). Most importantly, Lep decreased VF and increased insulin action on the stimulation of glucose uptake. These effects were independent of the changes in fat distribution. In parallel, Lep markedly enhanced insulin’s inhibition of HGP to ~20% of the levels observed in AL and of PF rats with chronic infusion (Figure 4), (Barzilai et al., 1997), and even after 6 h of infusion, before apparent changes in FM (Rossetti et al., 1997). Thus, while it seems that Lep affects peripheral and hepatic insulin action directly, it also acts through its effect on FM and its distribution. Unfortunately, obesity and aging are associated with leptin resistance. Fat and the syndrome of insulin resistance Insulin resistance is frequently associated with hypertension, dyslipidemia, and ﬁbrinolytic alterations (the metabolic syndrome) that represent major risk factors for atherosclerotic cardiovascular disease (Shimokata et al., 1989). Two of the fat derived peptides (AGT and PAI-1) have been demonstrated to be over-expressed in obesity and with insulin stimulation. It was recently reported that in obesity or insulin resistance, insulin looses its capacity to suppress fat

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Figure 4 (A) Effect of leptin administration on total abdominal (visceral) fat. Total VF was markedly decreased by the 8th day of leptin administration (LEP) compared with controls ad libitum fed rats (C-AL) and controls PF rats (C-PF). (B) When HGP was examined using an insulin clamp, HGP was signiﬁcantly inhibited in the LEP compared to the C-AL and C-PF. *p 0.01 vs. all.

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Figure 5 (A) Hyperinsulinemia and PAI-1 gene expression in lean and obese rats. Insulin infusion rate was 3 mU/kg/min, plasma glucose was clamped at euglycemia for 3 h *p 0.001 vs. Insulin , (B) Hyperinsulinemia and AGT gene expression in lean and obese rats. Insulin infusion rate was 3 mU/kg/min, plasma glucose was clamped at euglycemia for 3 h. Baseline saline infusion. *p 0.01 vs. lean for AGT gene expression.

AGT gene expression, which possibly contributes to the increased prevalence of hypertension in this condition. Similarly, hyperinsulinemia and hyperglycemia induce a marked activation of adipocyte PAI-1 gene expression and activity, an effect that is several fold ampliﬁed in obesity and insulin resistance (Figure 5). Thus, increased PAI-1 activity in obesity may contribute to the decreased ﬁbrinolytic activity seen in this condition. Taken together, these examples demonstrate the importance of chronic increase in FM and insulin insensitivity not only in regard to carbohydrate metabolism but also in regard to various complex metabolic alterations, which result in the development of most common, but life threatening, diseases.

IN SUMMARY Rodents in the wild are hungry and run great distances to seek food. In contrast, rats in the laboratory are caged and inactive, fed ad libitum, and they become fat. This situation in rodents mimics lean humans, who may be exercising or eating less, and obese humans who are sedentary and eat ad libitum. Lean humans have a markedly decreased risk of death from all causes, compared with markedly obese humans. Animal models that are CR and lean also live markedly longer. This paradigm serves an excellent model to examine the cause–effect relationship between fat accretion, insulin resistance, and longevity. Because insulin resistance in humans is dependent also on fat-distribution, and on biological risk factors for inﬂammation and coronary disease, animal models can be used in vivo and in vitro to delineate the role of fat in insulin action and longevity. In this chapter we reviewed the evidence for the decline in insulin action throughout aging in obese but not in lean rodent models. Evidence suggests a cause–effect relationship between increased VF and decreased insulin action. Such alterations in body fat distribution and in

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insulin action are modulated in part by leptin, the action of which fails with obesity and aging. Finally, because fat and bone marrow are derived from similar stem cells and secrete similar proteins, their expression and secretion is increased when FM becomes hyperplastic, as it is in obese models. Such peptides are regulated by nutrients, and may have a pathophysiological role in the development of age-related diseases, and possibly in lifespan for humans and rodents. Similarities between human and rodent obesity allows for this model to serve as a platform for new metabolic discoveries related to metabolism and longevity.

ACKNOWLEDGMENT The authors wish to thank Ms. Rachel S. Berger for her signiﬁcant input. Dr. Barzilai is the Director of the Institute for Aging research at Albert Einstein College of medicine, and his work on animal models is supported by grants from the National Institutes of Health (RO1-AG18381).

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19. POSTNATAL AND ADULT INSULIN SENSITIVITY AND METABOLISM IN PROGENY OF NUTRITIONALLY COMPROMISED MOTHERS CLIVE J. PETRY, SUSAN E. OZANNE AND C. NICHOLAS HALES Clinical Biochemistry Department, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge, UK

SUMMARY The thrifty phenotype hypothesis has provided a conceptual framework that can be tested using animal models to investigate mechanisms behind the observations in humans that fetal growth restriction is associated with the future development of diabetes and the metabolic syndrome. The most widely studied model involves looking at offspring of rat dams who were protein restricted during pregnancy (and sometimes lactation). Offspring from such pregnancies are growth restricted at birth. In young adult life, male offspring tend to have increased whole body sensitivity to insulin, along with good glucose tolerance. Parallel with this, a number of tissues including liver, skeletal muscle and adipocytes have increased insulin receptor expression. As the animals age, however, the low protein offspring undergo a metabolic shift such that they become hyperglycaemic and hyperinsulinaemic. If the protein-reduced diet is supplemented with methionine, the offspring show consistently raised blood pressures. In one study, effects associated with both dietary-induced obesity and early protein restriction were linked (in an additive fashion) to hypertension in female rats. The maternal low protein rat model is therefore able to reproduce many of the ﬁndings observed in humans which are linked with restricted fetal growth.

INTRODUCTION Since the publication of the ﬁrst study showing the link between low birth weight and the future development of type 2 diabetes mellitus or impaired glucose tolerance (Hales et al., 1991), more than twenty studies have found a statistical link between some index of restricted fetal growth and future glucose intolerance or insulin resistance. The initial study involved oral glucose tolerance tests in 64-year-old men who were born in Hertfordshire, England and for whom birth weight records (and weight at 1 year of age) were available. Based on World Health Organization criteria at the time of publication, those men who were born with the lowest birth weights were more than six times as likely to have either diabetes or impaired glucose

Address correspondence to: C.N. Hales, Clinical Biochemistry Department, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge UK . Tel.: 44 1223 336787; Fax: 44 1223 330598; Email: [email protected] 349

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tolerance at age 64 compared with those with the highest birth weights. However, the increased risk was not just associated with the lowest birth weights. Instead there was a continuous distribution of gradually increasing risk for glucose intolerance as the birth weights decreased.

THE THRIFTY PHENOTYPE HYPOTHESIS The ‘thrifty phenotype hypothesis’ (Hales and Barker, 1992) was proposed as a conceptual framework that could be used to test and investigate mechanisms that link restricted fetal growth with forms of glucose intolerance, including type 2 diabetes and the metabolic syndrome. It was proposed that fetal (and possibly early postnatal) malnutrition was central to the whole process. Evidence in support of this concept was subsequently found by investigating effects in people who were exposed to relatively short periods of severe famine in utero caused by enemy occupation and blockade during war. It was found that plasma glucose concentrations, 2 h after a standard glucose load, were raised in people who were born around the time of the Dutch Hunger Winter of 1944–45 (Ravelli et al., 1998). People whose mothers were exposed to the famine during the second to third trimesters of pregnancy had higher glucoses than those people whose mothers were exposed only during the ﬁrst trimester or who were not exposed to the famine at all. The highest glucoses of all, however, were found in people who were born as thin babies to mothers with low body weights and who then became obese in adult life. It was proposed in the thrifty phenotype hypothesis that adaptations to fetal malnutrition, designed to prolong life in the short- and long-term when nutrition is poor, become detrimental to health when nutrition is adequate or even excessive. Elements in adult life were foreseen as being able to modulate the risk established in utero, such that factors associated with ageing, the development of obesity and a sedentary lifestyle increase the risk of both developing a disease phenotype and of developing it at an earlier age. This interaction is complicated by the possibility that fetal malnutrition itself may be associated with the development of obesity (Hales et al., 1991; Law et al., 1992; Ravelli et al., 1999), that is, being metabolically ‘thrifty’ may promote fat deposition when nutrition is good. One of the most important adaptations that a developing fetus makes to malnutrition appears to be the targeting of blood ﬂow, and therefore nutrients, towards the brain and away from more visceral organs. This leads to a relative ‘sparing’ of brain weight at the expense of weights of other organs (Winick and Noble, 1966; Desai et al., 1996). Of particular importance to the future development of type 2 diabetes and the metabolic syndrome is the fact that by 1 year of age the human islets of Langerhans have almost a complete complement of pancreatic ␤-cells (Rahier et al., 1981). Therefore, a diversion of nutrients away from pancreatic islets (towards the brain) in response to malnutrition at this critical stage of development may, in the long-term, impair the ability of the pancreas to mount an adequate insulin response to counteract any age-associated insulin resistance (Hales and Barker, 1992). Following on from this, insulin tolerance tests performed in 50-year-old people in Preston, England, showed that there was an inverse relationship between insulin sensitivity and the ponderal index at birth (meaning that thinner babies were less insulin sensitive ﬁfty years later) (Phillips et al., 1994). This suggests that in responding to the need for survival when malnourished, the developing fetus lays open the possibility of developing both insulin deﬁciency and resistance, the two prerequisites for the development of type 2 diabetes (Ferrannini, 1998). A number of factors can contribute towards fetal malnutrition, so several different animal models have been developed to try and simulate such situations. In this way the thrifty

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phenotype hypothesis has provided a setting in which potential mechanisms linking restricted fetal growth with subsequent disease have been tested. When the hypothesis was published (Hales and Barker, 1992), attention was drawn to potential long-term effects associated with maternal and early protein deprivation. This was due to protein’s role both in growth and development in utero and its relative high price as a foodstuff for the poorest communities where type 2 diabetes is most prevalent (Hales et al., 1997). The most widely studied animal models, therefore, have been ones looking at offspring of rats fed a restricted protein diet during pregnancy alone, during pregnancy and lactation, or a combination of maternal protein restriction during pregnancy and lactation with the restriction continuing after weaning up to a period of around seventy days of age (adolescence). The bulk of this chapter is therefore dedicated to reviewing the metabolic changes observed in the maternal low protein rat model, before comparing these to changes observed in other models.

MATERNAL LOW PROTEIN RAT MODEL In an attempt to differentiate effects associated with maternal protein restriction from those associated with a more global maternal calorie restriction, Snoeck et al. (1990) developed a model where the experimental group were offspring of rat dams fed a reduced protein diet that was rendered isocaloric with the control diet by increasing its carbohydrate content. After weaning, the offspring from such pregnancies and from control dams were fed the same diet which contains adequate amounts of protein. Clearly, using this model, any associated effects could, in theory, result from the increased carbohydrate content rather than the reduced protein content of the maternal diet. However, since its use was designed to look at the phenotype of offspring exposed to a relatively speciﬁc nutritional deﬁciency, this is considered preferable to use of a diet where a protein calorie deﬁcit is not substituted and, therefore, a more general calorie malnutrition ensues. Initial ﬁndings using the maternal low protein rat model were that as well as being growth restricted, fetuses from such pregnancies showed a number of differences from controls in their endocrine pancreases, including reduced pancreatic ␤-cell proliferation, islet size and islet vascularization (Snoeck et al., 1990). Their islets also showed a depressed insulin response to various secretagogues in vitro (Dahri et al., 1991). Subsequent studies have shown that in neonatal life these offspring have increased rates of pancreatic ␤-cell apoptosis, along with decreased proliferation and an increased G1 phase of the cell cycle (Petrik et al., 1999). Morphological studies have revealed that offspring of pregnancies where the maternal protein intake was restricted during both pregnancy and lactation had reduced numbers of pancreatic ␤-cells but increased ␣-cells just prior to weaning (Berney et al., 1997). Studies of pancreatic hormone contents of female low protein rats where the protein restriction was extended to seventy days of age, showed a decreased insulin content and increased glucagon content in adult life (Petry et al., 2000b). Similar rat offspring have also been shown to exhibit decreased pancreatic and speciﬁcally islet blood ﬂow, with the ﬂow increasing during a glucose challenge (Iglesias-Barreira et al., 1996). Such alterations in the pancreas could, therefore, render the animals susceptible to changes in glucose tolerance, particularly if the animals become insulin resistant later in life. Evidence that the maternal low protein rat model is useful for testing and reﬁning the thrifty phenotype hypothesis (Hales and Barker, 1992) has come from measurements of plasma catecholamine concentrations. Three-month-old male low protein rat offspring were bled in the fed-state and after 24 h of starvation for the measurement of plasma adrenaline and

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noradrenaline concentrations (Petry et al., 2000a). When fed, the low protein offspring were found to have raised adrenaline and noradrenaline concentrations. With starvation, however, the circulating catecholamine concentrations in the controls rose, but remained essentially unchanged in the low protein offspring so that the differences in concentrations disappeared. The metabolism of the low protein offspring, therefore, appears to operate as if it were in a period of starvation even when feeding is adequate (as may be predicted by the thrifty phenotype hypothesis (Hales and Barker, 1992)).

GLUCOSE TOLERANCE AND WHOLE BODY INSULIN SENSITIVITY The difference in glucose tolerance and insulin sensitivity between low protein offspring and controls appears to be very age dependent. At 6 weeks of age, 3 weeks after they had been weaned onto standard laboratory chow, offspring from protein restricted dams were actually more glucose tolerant than the controls (Shepherd et al., 1997). This improvement was evident whether the maternal protein restriction was during pregnancy alone or during pregnancy and lactation. In young adult life the glucose tolerance of various low protein rat offspring have been reported to be either better than (Hales et al., 1996; Holness, 1996b; Petry et al., 2000b) or equivalent to (Holness, 1996a; Wilson and Hughes, 1997) that of controls. Circulating insulin concentrations are lower at this stage (Hales et al., 1996; Ozanne et al., 1998b; Petry et al., 2000b). This fact, combined with the requirement for a faster glucose infusion rate to maintain euglycaemia in hyperinsulinaemic clamps (Holness, 1996a), suggests that these animals are more insulin sensitive than controls at this age. Consistent with this they also have lower circulating triglyceride concentrations (Lucas et al., 1996; Ozanne et al., 1998b). By middle age, however, this difference in glucose tolerance seems to disappear, with low protein rat offspring having glucose tolerances which are indistinguishable from those of controls (Langley et al., 1994; Hales et al., 1996; Petry et al., 1997b). After middle age a metabolic shift seems to take place in the low protein rat offspring. At 15–16 months of age, after a more rapid deterioration in their glucose tolerance (Hales et al., 1996; Petry and Hales, 1999), plasma glucose concentrations at certain time points in intraperitoneal glucose tolerance tests were signiﬁcantly higher than those of controls (Hales et al., 1996). In male rats this was associated with hyperinsulinaemia, suggesting a degree of whole body insulin resistance. In female rats, where the trends towards differences in glucose tolerance were still evident although slightly less strong, differences in circulating insulin concentrations in intra-peritoneal glucose tolerance tests suggested more of a relative insulin deﬁciency. Until recently, frank diabetes had never been observed in low protein offspring. Preliminary experiments performed in male low protein offspring who were around 18 months of age (i.e. older than the average lifespan in such animals, Hales et al., 1996), however, found that their fasting plasma glucose concentrations were higher than that deﬁned by the World Health Organization (Alberti and Zimmet, 1998) as being diagnostic for diabetes in humans (Petry et al., 2001). In intravenous glucose tolerance tests both circulating glucose and insulin concentrations were higher in low protein offspring than in controls, and the magnitude of the difference was larger than had previously been observed in 15-month-old animals. Changes in glucose and insulin concentrations in male low protein offspring relative to those of controls, therefore, would tend to show an initial enhanced insulin sensitivity followed by a rapid deterioration associated in some way with the ageing process. Investigating mechanisms behind this process have largely involved in vitro tissue studies.

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HEPATIC INSULIN SENSITIVITY Lower plasma glucoses in young low protein offspring may, at least in part, be explained by the ﬁnding of relative resistance to the ability of glucagon to stimulate their hepatic glucose output (Ozanne et al., 1996a). A ﬁve-fold reduction in hepatic glucagon receptor expression was observed, along with increased hepatic insulin receptor expression, both of which probably contributed to this functional resistance. Such livers also showed an increased internalization of insulin into hepatocyte endosomes and release of insulin degradation products. There was a biphasic response with respect to time in terms of hepatic glucose output to insulin in ex vivo perfusion studies, with an initial stimulation followed by the expected inhibition. Overall, perfusing in the presence of physiological glucose concentrations, young low protein offspring have been shown to have a reduced hepatic glucose output (Ozanne et al., 1996a) and a reduced endogenous glucose production during euglycaemic clamps (Holness, 1996a). Such ﬁndings are consistent with the lower fasting blood glucose concentrations found in young adult life (Hales et al., 1996). Other alterations observed in the livers of low protein rat offspring include the presence of larger hepatic lobules than those of controls (Burns et al., 1997). There is also a reduction in the expression of ␣-, ␤- and ␥-ﬁbrinogen genes (Zhang et al., 1997), ﬁbrinogen protein, glucocorticoid receptor mRNA and glucocorticoid receptor binding afﬁnity that is restricted to the left lobe of the liver (Zhang and Byrne, 2000). In addition, hepatic microsome ␦5-desaturase activities were reduced in these rats and these activities were inversely correlated with fasting insulin concentrations (unlike in controls). Such a ﬁnding has led to speculation that activity of this enzyme may have an inﬂuence on or may be inﬂuenced by changes in insulin sensitivity in these animals (Ozanne et al., 1998a).

ADIPOCYTE INSULIN SENSITIVITY In mesenteric adipocytes from young adult low protein rat offspring, euglycaemic hyperinsulinaemia has been shown to cause an enhanced glucose utilization (Holness, 1996a). Consistent with this (and similar to the observations in the liver) insulin receptor expression was found to be increased in epididymal (Shepherd et al., 1997; Ozanne et al., 1997) and intra-abdominal but not subcutaneous (Ozanne et al., 2000) adipocytes from these rats. Also basal glucose uptakes were higher in their adipocytes (Ozanne et al., 1997, 1999, 2000). However, while insulin did stimulate glucose uptakes in adipocytes from both low protein offspring and controls, the magnitude of the stimulation, in terms of fold stimulation, was smaller in the low protein adipocytes such that in absolute terms rates of uptake were similar. These ﬁndings may reﬂect the reaching of ‘maximal’ adipocyte glucose uptakes (not distinguishable from maximal values observed in controls). Consistent with this, there were no detectable differences in GLUT4 protein expression between adipocytes from low protein offspring and those from control animals from three different fat depots (Ozanne et al., 2000). Studies investigating lipolytic rates in adipocytes from low protein rat offspring have revealed a degree of insulin resistance even at a time when these animals appear to have increased whole body insulin sensitivity (Ozanne et al., 1999, 2000). Basal lipolytic rates were similar in adipocytes from low protein offspring and controls using epididymal (Ozanne et al., 1999), intra-abdominal and subcutaneous adipocytes (Ozanne et al., 2000). The magnitude of stimulation by the catecholamine isoproterenol (and noradrenaline (Holness and Sugden, 1999)), however, was larger in adipocytes from low protein offspring (Ozanne et al., 1999),

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irrespective of the fat depot from which the adipocytes were isolated (Ozanne et al., 2000). Consistent with this fact, isolated adipocytes from these animals were shown to have increased ␤-adrenergic receptor expression in the different fat depots (Petry et al., 2000a). The presence of a selective resistance to the metabolic actions of insulin was suggested by the ﬁnding that the magnitude of the inhibition of the catecholamine-stimulated lipolysis by insulin was smaller in epididymal and intra-abdominal adipocytes from low protein offspring (Ozanne et al., 1999, 2000). The exact mechanism of this resistance is unknown, but clues may be suggested by alterations in expression and activities of various components of the insulin signalling pathway. First, adipocytes isolated from young low protein offspring were shown to have signiﬁcantly higher insulin receptor substrate-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activities, both in the basal state and after insulin-stimulation (Ozanne et al., 1997). In addition they exhibited higher p85-associated PI 3-kinase activities. This may reﬂect the fact that while there were no differences in expressions of p110␣ or p85 PI 3-kinase subunits, there was a six-fold reduction in their expression of the p110␤ subunit (Ozanne et al., 1997). Downstream of PI 3-kinase in the insulin signalling cascade, protein kinase B activities were found to be higher in adipocytes from low protein offspring in both the basal state and when stimulated with insulin (Ozanne et al., 1999). As insulin showed a reduced ability to inhibit catecholamine-stimulated lipolysis in adipocytes from low protein offspring, despite them having these higher protein kinase B activities, the activity of the p110␤ subunit of PI 3-kinase may, therefore, limit insulin’s anti-lipolytic activity. In epididymal adipocytes from older male low protein offspring some of the metabolic changes observed in young adult life remained evident, while others differed (Ozanne et al., 2001). The rats used for these studies were 15 months of age, an age when there is demonstrable mild impaired glucose tolerance associated with hyperinsulinaemia (Hales et al., 1996). As at 3 months of age, adipocytes from 15 month old low protein offspring had increased basal glucose uptakes relative to those of controls (Ozanne et al., 2001). However, unlike what was observed with control adipocytes, insulin failed to stimulate glucose uptake. In vivo, at this age, insulin-independent glucose uptake (i.e. glucose effectiveness), rather than mechanisms relating to stimulation by insulin, may therefore be more important in low protein offspring for uptake of glucose into adipocytes. At 15 months of age insulin also showed a reduced ability to inhibit isoproterenol-stimulated lipolysis, after rates of basal and isoproterenolstimulated lipolysis appeared to be similar in adipocytes from control and low protein offspring. The overall reduced action of insulin in these adipocytes from 15-month-old male low protein offspring was not associated with apparent alterations in insulin receptor expression (unlike what was observed in younger animals) or insulin receptor tyrosine phosphorylation activity. However, the reduction in insulin action was associated with reduced PI 3-kinase and protein kinase B activation (Ozanne et al., 2001). In contrast activities of both of these enzymes had been raised at 3 months of age (Ozanne et al., 1997, 1999) when whole body insulin sensitivity was improved relative to that of controls (Hales et al., 1996; Holness, 1996a). The association of the p110␤ subunit with the p85 subunit of PI 3-kinase was reduced in a similar fashion to that observed at 3 months of age (Ozanne et al., 1997). This clearly predates the shift in glucose tolerance and whole body insulin sensitivity of the animals, and therefore it is tempting to speculate that reduced p110␤-associated PI 3-kinase activity may be involved in the process that caused the metabolic shifts that occurred in the low protein adipocyte and ultimately the whole animal in old age. Overall, at present, associations rather than causality of decreases in adipocyte insulin sensitivity with decreases in glucose tolerance and whole body insulin sensitivity are established. However, the progression from

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improved glucose tolerance when young to frank diabetes in old age, established in very early life (Petry et al., 2001) may well, at least partially, be driven by changes in adipocyte insulin sensitivity.

MUSCLE INSULIN SENSITIVITY Like in other insulin sensitive tissues, the majority of studies of muscle insulin sensitivity in low protein rat offspring have used young adult animals. Here, in tibialis anterior muscles, like in other insulin sensitive tissues at this age, low protein offspring were shown to have increased insulin receptor expression (Ozanne et al., 1996b). This was associated with increased insulinstimulated glucose uptake and a greater fractional presence of GLUT4 protein in the plasma membranes (without any detectable difference in total GLUT4 protein). Given the importance of skeletal muscle in determining glucose tolerance (De Fronzo, 1988), such alterations are likely to have made a signiﬁcant contribution to the improvement in glucose tolerance observed in young adult life (Hales et al., 1996). In vivo oxidative skeletal muscles of low protein offspring were shown to have enhanced glucose utilization (deﬁned in terms of glucose uptake and phosphorylation) when stimulated with euglycaemic hyperinsulinaemia (Holness, 1996a). It remains to be determined whether these alterations in muscular insulin action change with age, as has been observed with adipocyte insulin action (Ozanne et al., 2001), circulating insulin concentrations and glucose tolerance (Hales et al., 1996).

BLOOD PRESSURE The precise molecular entities giving rise to the factors that cluster as the metabolic syndrome in humans remain unresolved. Whilst most factors appear to be related to insulin resistance in some way (Reaven, 1988), the mechanism that links insulin resistance with hypertension, in particular, continues to be controversial (Voiculescu et al., 1997). In humans restricted fetal growth remains the factor with the highest associated risk for the future development of the metabolic syndrome (Barker et al., 1993). The maternal low protein rat model has, therefore, been used to try and deﬁne more precisely the factors that give rise to both reduced insulin sensitivity and increased blood pressure. What has been discovered is that variations in the actual low protein diets used (such as differences in protein content and source and differences in other constituents and length of exposure) have important inﬂuences on the blood pressure of the offspring of the dams who were protein restricted. One diet (containing 9% (w/w) protein and supplemented with methionine) consistently produced hypertension in the offspring (Langley and Jackson, 1994; Langley-Evans, 2000). In contrast another diet (containing 8% (w/w) protein but without methionine supplementation) caused either no change or a slight lowering of blood pressure in the offspring (Lucas et al., 1996; Langley-Evans, 2000; Petry et al., 2000a). These studies have used indirect tail cuff procedures to measure the blood pressures of the rats, and due to potential stress artefacts, criticisms have been raised about the validity of their ﬁndings. However, radiotelemetry, a procedure which is not associated with restraint stress, has also been used to show mild hypertension in the offspring of dams fed a low protein diet with methionine supplementation (Tonkiss et al., 1998). Where hypertension has been observed, its mechanism has been suggested to include impaired nephrogenesis and a hyperactive rennin–angiotensin system (Langley-Evans et al., 1999; Woods, 2000) associated with an early increased exposure to glucocorticoids (Langley-Evans et al., 1996).

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Obesity has been induced in female low protein offspring by feeding them a highly palatable, cafeteria-style diet (Petry et al., 1997a), as other dietary-induced changes in insulin sensitivity were not associated with large increases in body weight (Holness, 1996b; Wilson and Hughes, 1997; Holness and Sugden, 1999). After maternal and post-weaning protein restriction until 70 days of age and 10 months of cafeteria-feeding, the early protein restriction and the cafeteria-feeding were both associated with rises in systolic blood pressures (Petry et al., 1997b). These rises were independent of each other and additive, so that the highest systolic blood pressures were found in cafeteria-fed animals who were protein restricted in early life and the lowest pressures were found in chow-fed controls. Such changes are reminiscent of effects associated with low birth weight and current obesity observed in humans (Barker et al., 1989). At 1 year of age the pattern of glucose tolerances were similar to those observed earlier in life (Petry et al., 2000b); so in comparison to chow-fed controls the cafeteria-fed low protein offspring had hypertension, impaired glucose tolerance and hypertriglyceridaemia (Petry et al., 1997b). Such changes are obviously similar to those observed in humans with the metabolic syndrome, but tended to be more associated with the cafeteria-feeding than with the early protein restriction.

OTHER MODELS While feeding rats protein restricted diets has produced many of the changes which are relevant to the thrifty phenotype hypothesis (Hales and Barker, 1992) in their offspring, protein restriction is not the only factor, nutritional or otherwise, that causes fetal malnutrition and subsequent disease. Other potential factors have generally not been as widely investigated, and therefore neither has the question of whether different causes of the growth restriction produce metabolic changes through similar or different mechanisms. The one exception to this is increased glucocorticoid exposure in fetal life, which has been reported to be evident in fetuses of pregnant rats fed a protein restricted diet that was supplemented with methionine (LangleyEvans et al., 1996). This has been shown through pharmacological manipulations of fetal glucocorticoid exposure to lead to reduced birth weight, hypertension (Lindsay et al., 1996a) and impaired glucose tolerance but not frank diabetes (Lindsay et al., 1996b) in the offspring (reviewed by Seckl et al., 1999). Other nutritional factors that have been investigated to assess effects on the offspring include restricting iron intake in pregnant rats, which led to reduced birth weights, altered cardiovascular development and increased blood pressure by 40 days of age in the offspring (Crowe et al., 1995). A similar model produced offspring that were hypertensive at 3 months of age (Lewis et al., 2001). More widely studied have been the effects of a generalized malnutrition of a pregnant rat on its offspring. Eight-week-old offspring of rats who had been fed half the amount of food as matched controls during the last trimester of pregnancy, showed no deﬁcits in glucose tolerance, glucose utilization or glucose production (Bertin et al., 1999). In contrast, while offspring of rats fed an isocaloric low protein diet during the same period showed broadly similar results, they also exhibited reduced pancreatic insulin contents and ␤-cell masses. It has also been demonstrated that underfeeding the pregnant rats during the ﬁrst two weeks of pregnancy does not alter either insulin secretion or action in the young offspring (Portha et al., 1995). Increasing the period of exposure to maternal malnutrition from day 15 of pregnancy to the time of weaning was shown to be associated with impaired pancreatic ␤-cell development (Garofano et al., 1998) and at 1 year of age impaired glucose tolerance and hypoinsulinaemia (Garofano et al., 1999). In 8-month-old female offspring

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exposed to this regime, there was a failure to mount the usual changes in the endocrine pancreas to pregnancy (Blondeau et al., 1999). A more severe maternal food restriction, to 30% of that of controls, has been shown to lead to prolonged hypertension (Woodall et al., 1996) and hyperphagia, raised fasting insulin concentrations and obesity in adult offspring (Vickers et al., 2000). Experimental fetal malnutrition has also been induced in rats by ligating one or both maternal uterine arteries during late pregnancy. Such manipulations caused a marked reduction in fetal body weights and a long-term increase in brain GLUT1 expression (Sadiq et al., 1999). This may reﬂect the shift of metabolism proposed in the thrifty phenotype hypothesis to preserve brain growth and metabolism at the potential expense of that of other organs (Hales and Barker, 1992). Uterine artery ligations also caused neonatal changes in hepatic redox potential (Lane et al., 1996), as well as alterations in fetal and juvenile skeletal muscle mitochondrial redox potential (Lane et al., 1998). 3–4-month-old female offspring from such manipulations have been shown to have increased sympathetic nervous system activity, increased fasting blood glucose concentrations and higher glucose concentrations in response to a glucose load ( Jansson and Lambert, 1999).

CONCLUDING REMARKS The thrifty phenotype hypothesis (Hales and Barker, 1992) has proved a useful conceptual and mechanistic framework to test potential mechanisms behind the now well established link between indices of restricted fetal (and possibly early postnatal) growth and subsequent disease, such as type 2 diabetes and the metabolic syndrome. The most widely studied model involves feeding a pregnant rat a diet that is deﬁcient in protein, and then investigating possible effects in their offspring. The relevance of such models to human disease has been questioned due to the multiparous nature of rat reproduction and the suggested necessity for fetal growth restraint because of this (Ong and Dunger, 2000). Such suggestions, however, fail to account for the fact that as fetal growth can never be allowed to totally outgrow the maximum amount of physical space that a mother is able to provide for the fetus and placenta, there must always be some degree of restraint even in uniparous pregnancies. The concept of ‘fetal restraint’ is therefore relative whatever the species in question. In the maternal low protein rat model, offspring of rats whose growth was severely restricted in fetal life due to poor maternal nutrition and the multiparous pregnancy are simply compared to offspring whose growth in fetal life was only restricted in response to the multiparous pregnancy. In addition it is quite remarkable how closely the maternal low protein model in rats has paralleled in the offspring, the links found in humans between indices of restricted fetal growth and subsequent disease. Variations of the maternal protein restriction model in rats have now successfully modelled all the major components of the thrifty phenotype hypothesis including restricted fetal growth and the subsequent development of diabetes and hypertension (along with hypertriglyceridaemia when combined with dietary-induced obesity). In addition, factors in adult life, such as the development of obesity, have been shown to be able to interact with effects associated with the early growth restriction to increase risk. These ﬁndings, therefore, add to studies of human twins, which have generally shown that the twin who was most growth-restricted at birth has a higher risk of diabetes (Poulsen et al., 1997; Bo et al., 2000) and hypertension (Levine et al., 1994; Dwyer et al., 1999; Poulter et al., 1999). Even the one major study in twins which did not ﬁnd this link (Baird et al., 2001) concluded that shared genetic determinants for fetal growth and adult disease are not likely to be prevalent or powerful. All these

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observations would suggest that the link between restricted fetal growth and subsequent disease can occur independently of a major genetic inﬂuence. This is important, since such non-genetic mechanisms may be more amenable to treatment prior to the onset of disease in the future. The fact that many of the ﬁndings in humans in this area have now been modelled in rats with maternal protein restriction suggests that in turn important consideration should be given to observations in this model that have not so far been found in humans. Of particular importance in this area may be the observed alterations in the insulin signalling pathways which predate the development of adult disease and do not appear to alter once the disease is established. As well as providing details of molecular mechanisms, such ﬁndings might in future provide markers that may be able to be used to predict those individuals at high risk of future disease and may even provide targets for pharmacological therapies.

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INDEX abdominal obesity 59; in humans 338 acanthosis nigricans 148 acetylcholine (ACh) 74, 263 ACRP30 135, 137, 342 adenylyl cyclase activity 78–80 adipocytes 342; enzyme activity 214; hyperplasia and hypertrophy 75; insulin action in 12; insulin sensitivity 353–4 adipocytokines 101 adipogenesis 162 adiponectin 98, 101–4 adipose body depots 199–201 adipose tissue 134–5; -Ars 77–81; cytokines 136; development 108; glucose uptake 135–6; in insulin resistance 110; role as a storage depot for energy 188 adipsin 135 African Americans 73, 80, 338 age 108; emergence of insulin resistance 337 aging: effects on insulin-mediated glucose uptake 339; peripheral insulin sensitivity 338–40; process 108, 339; and stress 299 agouti gene 294 agouti-related protein (AGRP) 189 Ahren, B. 76 Akt 126–7, 160; activity 151–3; defects in 152; in humans with type 2 diabetes 152 Akt/protein kinase B (PKB)150–3 alkaline phosphatase treatment 27 allosteric effectors 4, 218 angiotensinogen (AGT) 342 angiotensin II antagonist 54 animal models: for diabetes 8; of GDM 22–3; of human disease 179; of obesity 77 annual cycle of metabolism 272 anovulation, chronic 225 apolipoproteins 105 apoptosis 111 arcuate nucleus (AN) 289 Arnott, C.H. 44 Ashkenazie Jews 216 ATB-BMPA photolabelling method 155 atherosclerosis 59, 64, 225; spontaneous development of 61 ATP-binding cassette A1 protein (ABCA1) 108 Australian aborigines 175 autocrine insulin signaling 319 autonomic nervous system activity and insulin action 265–6 B6 mice see C57BL/6J mice Bandyopadhyay, D. 40

Banting 323 Bar Harbor, M.E. 23 baroreﬂex 54 beacon gene 180; effects in Psammomys obesus 181; obesity and type 2 diabetes 180 Beck-Nielsen, H. 218 Bergman’s minimal model method 221 Best 323 3AR mRNA levels 80 -galactoside residues 110 bezaﬁbrate 204 Biacore 181 biguanides 243 birth weight, low 349 Bjorntorp, P. 73 blood: glucose levels 125; pressure 355 Boesch, C. 198 Boston, B.A. 294 brain: derived enurotrophic factors (BDNF) 297; insulin effect on 265; and periphery 265–6; weight 350 bromocriptine 295 brown adipose tissue (BAT) 77 Brownlow, B.S. 75 Burns, T.L. 75 C57BL/6J mice: abnormal pancreatic function 76; diabetic tissues of 14; diet-induced obesity 74; feed efﬁciency of 74–5; hypertension 75–6; model 12, 73; strain 23 C57BLKS-Leprdb/+ mice 21, 23–4; impact on fetus 21 cachectin 342 caloric restriction (CR) 89; beneﬁcial effects 337 Calpain 10 178 c-AMP-dependent protein kinases (PKA) 3 Candelore, M.R. 81 candesartan cilexetil (TCV-116) 54 carbohydrate metabolism 175, 264, 344 cardiac hypertrophy 128, 133 cardiovascular disease 59, 61, 74, 109 catecholamines 77 Caucasian population 317 cDNA: microarrays 178; probes 179; technology 179 cell growth 111; and differentiation 147 cellular circadian rhythms 272 central nervous system (CNS) 271; control center 271; dopamine levels 296 ceramides 202 Cercopithecus aethiops (African green monkey) 91 Chen, H. 40 childhood obesity 22 363

364

INDEX

chiro-inositol: deﬁcit, role in insulin resistance 218–22; and myo-inositol, urinary excretion of 220 Chlorisondamine 76 CHO-IR cells 325 cholesterol 109; esters 63; metabolism 59, 109 chylomicrons 201 circadian metabolic activities 280 circadian neural oscillations 275 circadian neuroendocrine activities 277, 282 cocaine and amphetamine regulated transcript (CART) 189 Collins, S. 77–8 coronary artery disease 338 corticosteroids 279–80, 289 corticotropin releasing hormone (CRH) 289 C-peptide 90; molecules 90–1; sequence 91 Craig, J.W. 217 D-chiro-inositol (DCI) 224; administration to animals 222, 225; clinical trials 222–5; insulin mediator preparation 218–19; and insulin resistance 211; phosphoglycan, action of insulin 228; repletion therapy 222–5 diabetes: developing countries 175; development of 103; gene for 23; genetic basis 176; health care costs 175; obesity and hypertension 176 diabetes-related mortality 175 diazoxide (Dz) 78, 81–2; treated animals 82 dietary fat: content 74, 298; fat subtype 197 differential display polymerase chain reaction (ddPCR) 178–80 dipeptidyl peptidase IV 236 Dobbins, R.L. 198 dopamine 279, 297 dopamine beta-hydroxylase (DBH) 296 dorsomedial hypothalamus (DMH) 276 Dutch Hunger Winter of 1944-45 350 dyslipidemia 51, 69, 189, 195, 225, 343 EGF signaling 42 Elchebly, M. 41–2 endopeptidase: PC2 91; PC3 91 Escherechia coli 180 estradiol 66 estrogen receptors 105; modulator 66 etoﬁbrate 204 etomoxir 201 euglycemia 240 euglycemic hyperinsulinemia 353 European Prospective Investigation into Cancer and Nutrition 73 exendin: and insulin sensitivity 235; receptor 237 Exendin-4 235–7; glucose-lowering potency 240; pharmaceutic beneﬁts of 239 fat: as a biologically active tissue 341–4; derived peptides 342; feeding and muscle fat

deposition 195; glycogen synthase 13; metabolism 318; oxidation 284; and syndrome of insulin resistance 343; tissue of diabetic animals 14 fat mass (FM) 108, 182, 337, 344 fatty acid: composition 202–3; elevation, acute systemic 196; metabolism of 199; metabolites and insulin action 201–3; muscle uptake of 196; synthesis, genes regulating 190; tracer, non-metabolizable 200; transporter (CD36) 197; unesteriﬁed 68; utilization 200 fenoﬁbrate 105–6 fetal imprinting 21–3 fetal macrosomia 26; mechanisms 30 fetal malnutrition 350 ﬁbrate and glitizone drug activities 108 ﬁbrinolytic alterations 343 Finnish families 216 FKHR See Forkhead homologue to rhabdomyosarcoma Fluorescence and Bioluminescence Resonance Energy Transfer 181 Forkhead homologue to rhabdomyosarcoma (FKHR) 98 free fatty acids (FFA) 176; availability 197; uptake into muscle 200 fructose-6-phosphate aminotransferase (GFAT) 165 G6P see glucose 6 phosphate galectin-12 110–11 Gazzaniga, J.M. 75 Genbank 110 genes: expression of fat weight 104; and “gene knockout” technology 125 genetic basis for insulin resistance 216–17 gestational diabetes mellitus (GDM) 21–2; development of 25; effects on fetus 22; leptin administration and insulin resistance in 29; women with 24 Gila monster (Heloderma suspectum): plasma levels of exendin-4 238; salivary secretion of 235, 237 GLP-1: biological action of 240; effects of gastric emptying 242; physiologic role of endogenous 242; satiogenic role of 241; whole-body insulin sensitivity 243 GLP-1 and exendin-4: antidiabetic actions 240–2; -cell neogenesis 249–50; biology of 236–40; direct effects in fat 247–9; general antidiabetic actions 240; glucagonostatic effects 241; insulinotropic effects 240–1; insulin-sensitive tissues, direct effects in 246–9; insulin sensitivity 236; insulin-sensitizing effects 243; pharmacokinetics of 238–40; satiogenic effects 241–2; secretory function 249–50; whole-body insulin sensitivity 243–6 glucagon 280 glucagon-like peptide-1 and insulin sensitivity 235

INDEX

glucocorticoids 25, 105, 356 glucosamine 165, 215; infusion 152 glucose 6 phosphate (G6P) 4, 5, 7–8, 13–14, 97, 202; binding afﬁnity and GS activity 96 glucose: competence 249; disposal 212, 228; homeostasis 126, 131, 138, 313, 318; and insulin levels in non-diabetic patients 73; and insulin tolerance tests 42; intolerance 51, 225, 272; and lipid metabolism, abnormalities of 52; metabolism 44, 81, 137; oxidation and utilization 248; partitioning 138; sensor 286; tolerance and whole body insulin sensitivity 352; toxicity, reversal of 236; transport 9, 131, 151, 153, 160; transporter family, new members of 127; utilization 52, 265; utilizing pathways 95 glucose-insulin metabolism 60 glucose transporter 1(GLUT1) 165; protein content 137 glucose transporter 4 (GLUT4) 9, 40, 85, 108, 126–7, 154, 165; ablation, whole body effects of 129–30; adipose speciﬁc reduction 135; effect of manipulation 130–3; effects of genetic alterations 125; elimination 131; expression 95, 130; independence transporter system 131; insulin-sensitive 147; knockout models 138; levels 134; mediated glucose uptake 154–8; mRNA 342; null adipocytes 135; null adipose tissue 137; null hearts 133–4; null mice 127, 136, 139; null muscle 133; overexpression 29, 323; protein expression 353; regulation of 155–8; trafﬁc 163; translocation 27, 150, 152, 154–8, 315; vesicles 158–62; vesicle trafﬁcking 137 GLUT4/ adipocytes: insulin signaling 137–8 GLUT4 null and GLUT4/ knockout mice: cardiac proﬁle 133–4; general phenotype 128–9 glucose transporter-like molecules, new 138 glucose transporters 127 glutamine 165 glycogen: content in fat tissue 12; defects 14; incorporation of glucose 11; in liver 11; metabolism 12; and protein synthesis 147 glycogen synthase (GS) 226; a activity 166; activation 211, 216; activity 13–14; in diabetic fat tissue 12; in diabetic liver 9–12; in diabetic muscle 6–9; maximal catalytic activity 95; in muscle liver and fat 12–14 glycogen synthase kinase-3 (GSK-3) 3–5, 7, 13, 323; activation by insulin 6; allosteric activation of 5; compartmentalization 10; defects in 7; dephosphorylation of 4; inhibition of 4; regulation of 3, 6 glycogen synthesis 131, 248; human liver cells 324; rates 134; reduction 7 glycolysis 131

365

glycosylphosphatidylinositol (GPI) 248 Goto Kakizaki (G/K) rat model 152, 216 GPI-phospholipid 226 growth factor-binding protein-1 (IGFBP-1) gene 98 growth factors 126 5HTP and L-DOPA experiments 281 Henry, R.B. 217 hepatic glucose output (HGO) 280; suppression by insulin 11 hepatic glucose production (HGP) 10, 52, 340; during hyperinsulinemia 340 hepatic glycogen 10–11 hepatic insulin sensitivity 353; with aging 340 hepatocytes 10 hexosamine pathway 163, 165, 188 hexose, phosphorylated 218 high density lipoprotein-cholesterol (HDL-C) 109; plasma concentrations 105 Hispanic and non-Hispanic whites 73 Hotta, K. 101, 103, 110 HPA and SNS activities 292 HPLC methodology 199 Huang, L.C. 218 human adipocytes 82, 248 human growth hormone variant gene (hGH-V) 29 human insulin receptor 92 hyperandrogenism 225 hypercholesterolemia 50 hyperglycemia 7, 74, 130, 147, 163, 166, 175, 215, 240, 293, 320; activation of GS by 229; maternal 26; transient 59 hyperinsulinemia 7, 21, 51, 59, 61, 73, 78, 81–2, 130, 163, 166, 272, 286, 298, 319–20; fetal 22; and increased VMH NE activity 287; and PAI-1 gene expression 344 hyperinsulinism 235 hyperlactemia 244 hyperleptinemia 189, 287, 299 hyperlipidemia 73, 176, 215, 272 hyperphagia 74–5, 187, 294 hypertension 51–5, 69, 73, 195, 225, 343, 355; and insulin resistance 51 hypertriglyceridemia 59 hypertrophy, models of 134 hypoglycemia 286 hypothalamic melanocortin 4 receptor 294–5 hypothalamic NPY: and CRH interactions 289–93; levels 294 Hypothalamo-Pituitary-Adrenal (HPA) axis 289 hypothalamus 182, 271; dopamine, norepinephrine activity ratio in insulin resistance syndrome 295–8; modulation of insulin resistance syndrome 288; neurophysiologic framework of insulin

366

INDEX

hypothalamus (Continued) resistance syndrome 292; regulation of insulin sensitivity 298–9; role in insulin resistance syndrome 271; SCN involvement in insulin resistance syndrome 278–83 hypotriglyceridemic drug 62 hypoxia 131 impaired glucose tolerance (IGT) 22 inositol phosphoglycans (IPGs) 211, 219, 248 inositols: ring 150; urinary excretion 219–20 insulin action: critical regulators of 323; and lipid accumulation 201; and secretion, defects in 175; targeting 323 insulin: challenge test 25; daily rhythms of 280 insulin dose-response: of adipocyte GS activity state 213; of adipocyte PDH activity state 213; of carbohydrate storage 212; of GS activity state 212 insulin: effects on autonomic nervous system activity 264–5; exocytic process 25; level (secretion) 263; levels and coronary artery disease 73 insulin-induced glucose utilization 266 insulin-induced sympathetic overactivity 264 insulin-induced vasodilation 55 insulin-induced VMH inhibition 294 insulin-like growth factor (IGF) 129, 149 insulin-mediated activation of GS 198 insulin mimetic compound 326 insulin receptor (IR) 38, 125, 148, 318; activators 324; subunit (IR) 138; complex 92; exon 11 splice variant 93–4; gene, animal models of 148; and IRS in insulin resistance 149; knockouts 319; ligand-binding -subunits 148; mutations 148; neuron-speciﬁc disruption of 126; phosphorylation 38; signaling 39, 190; signaling proximal to 314; and its splice variants 92–4; transmembrane -subunits 148 IRS isoform-encoding genes 149 insulin receptor (IR) knockout mice: including -cell (IRKO) 126; including liver (LIRKO) 126; including muscle (MIRKO) 126, 148, 318; including neuron (NIRKO) 126 insulin receptor substrate (IRS) family of proteins (IRS-1, IRS-2, IRS-3, IRS-4) 95, 126, 148, 314, 319–21 insulin receptor substrate-1 (IRS-1) 148, 165, 316, 319; adenovirus-mediated restoration of 320; hyperphosphorylation of 161; knockout mice 149; molecules 160; phosphorylation 27, 43, 150; polymorphisms of IRS-1 149; tyrosine dephosphorylation 40 insulin receptor substrate-2 (IRS-2) 148, 316, 319; knockout mice 149; role in liver 320 insulin receptor substrate-3 (IRS-3) 148

insulin receptor substrate-4 (IRS-4) 148 insulin receptor tyrosine kinase (IRTK) activity 26, 325 insulin regulated aminopeptidase (IRAP) translocation 137 insulin regulated glucose uptake 134 insulin release, glucose stimulated 76 insulin resistance 59, 189, 198, 272; age-related 155; genetically determined 97; and autonomic nervous system 263; in C57BLKS-Leprdb/+ mice 26–8; in carbohydrate metabolism 211, 214; characteristics 211–18; determinant of 244; development of 74; emerging therapies 313, 324; in GDM, mechanisms 30; of glucose transport 163; glucose uptake 148, 159; insulin resistance, type A syndrome 148; mechanisms 125; models 151–2; as multilayered 215; muscle lipid oversupply hypothesis of 198, 200; natural history and background of 89–90; of non-oxidative glucose metabolism 217–18; non-reversible and genetic component 215–16; and obesity 75, 89, 110; pathogenesis of 166; peripheral 63; in pregnancy 21; syndrome 3; therapeutic strategies 323–8; treating in C57BLKS-Leprdb/+ mice 29–30; in vitro models 162–6; in Wistar fatty rat 52 insulin resistance syndrome 3, 51, 73, 81, 195, 201, 224–5, 266, 271, 313; etiopathogenesis of 235; neurochemistry of VMH neurons in 283–9 insulin resistant animal models 283; disease states and type 2 diabetes, therapeutics 44; liver 10; muscle 147; non-human primate 106; role in CNS 189 insulin secretagogue 236; secretion 24–6, 265 insulin-sensitive cell types 38 insulin-sensitive tissues 126, 130 insulin sensitivity 135–6, 221; antidiabetic therapy 246; changes in indices 245; drugs that increase 14; ponderal index at birth 350 insulin sensitizers 52, 324, 326–8 insulin signal: defect 217–18; mechanism 97; transduction 28 insulin signaling 99; cascade 125, 127, 202, 313; complexity of 323; components 41, 163; and down stream effects 95–8; effector 218; genetic disruption of negative regulators 322; identiﬁcation of regulators 324; modulators of 158; molecules 147, 321; pathway 165, 322, 326; proteins 148–54; regulation of 36, 314–18, 322 insulin-stimulated glucose metabolism 212 insulin-stimulated glucose uptake: mechanisms 157; model of 158 insulin-stimulated lipogenesis 166 insulin: stimulation 137; tolerance 128 interleukin-6 196

INDEX

intracerebroventricular (ICV) beacon administration 181–2 intramyocellular lipid 196, 200 islet hyperplasia 319 isoproterenol 133 Jackson Laboratories 23 JAK/STAT signaling pathway 99 Japan 221 JCR LA-cp rats 63; background 60; cardiovascular disease process in 69; genetic defect 61; insulin-resistant, atherosclerosisprone 59; metabolism 61; plasma leptin concentration as a function of age 62; vascular function 64–8 K-ATP channel agonist 78, 81–2 Kaufmann, R.C. 23 Kennedy, B.P. 41 Klaman, L.D. 41 knockout animal models: of insulin resistance 318–22; insulin sensitivity 322 Kolterman, O.G. 214 Kulas, D.T. 38 L6 muscle cells 155 Lammers, R. 40 Landsberg, L. 265 Larner, J. 218 lateral hypothalamus (LH) 276 Lawrence, J.C. 217 LCACoAs 198–9, 202 Lee, G.H. 76 leprechaunism 148 leptin (Lep) 30, 76, 103, 135, 287, 342–3, 337; action 100; administration 343; central effects 294; and CNS effects 189; expression of 188; insensitivity 77; and insulin 188–9; and insulin resistance in rodent models 187; and leptin receptor 98–101; and lipodystrophy 190; messenger RNA expression levels of 99; NPY–CRH system 290–1; and the periphery 189–90; receptor 25, 99, 101, 189–90; resistance 30, 179, 287, 291 leptin-deﬁciency, genetic models of 76 leptin-resistance 77 leukocyte antigen-related (LAR) 35, 38–9, 44, 315–16 leukocyte antigen-related phosphatase (LRP) 35, 38, 160; regulator of c-Src activity in 3T3-L1 adipocytes 44; (RPTP- ) 44 Lin, K.C. 76 lipid: accumulation 199–200; availability 196; and cholesterol related risk factors 109; and glucose metabolism 99; lowering agents 204; metabolism and insulin resistance, overview of 195–6; metabolites 201

367

lipoatrophy 190 lipogenesis 147, 248 lipolysis 77 lipoprotein particle size distribution 109 liver: direct effects of GLP-1 249; inhibition of phosphorylase 9; major roles of 9 Lox/Lox genotype 131 lymphotoxin 342 Macaca fascicularis (crab-eating macaque) 91 Macaca mulatta (rhesus monkey) 91 macronutrient availability 197 Maegawa, H. 43 Maffeis, C. 75 mammalian circadian pacemaker system 274, 279 maternal low protein rat model 351–2, 355, 357 matrix metalloproteinases (MMPs) 65; MMP-2 68 Maturity Onset Diabetes of the Young (MODY) 176 MEDICA 16 62 Meier, A.H. 274 melanocortin 4 receptor (MC4R) 189, 294 metabolic insulin resistance 215 metabolic syndrome: in humans 73, 355 metabolistat resides 273 metabolite 6 metformin 246, 324 Mexican American 178, 216 migratory species 273 Mills, E. 75 MIRKO mice see IR knockout mice mitogenic action 55 MMP activity 66 molecular biology, techniques of 187 molecular cloning techniques 187 monkey: amino acid sequence 92; and human preproinsulin, amino acid sequence comparison of 91 Murase, K. 52 muscle: direct effect of GLP-1 246; fatty acid oxidation, rate of 195; ﬁber composition 155; glycogen synthase, phosphorylation sites of 4; insulin resistance 130, 196; insulin sensitivity 355; lipid accumulation 195–6, 198, 203–4; malonyl CoA 200; triglyceride accumulation 200; triglyceride content and insulin resistance 197–9 myocardial infarcts 68 myocardial ischemia 61 myo-inositol 219; to chiro-inositol, conversion of 226; defect in conversion of 226–8 myosin 133–4 naïve type 2 diabetic subjects 222, 224 National Institutes of Health 60 Neel, J.V. 272

368

INDEX

Nestler, J.E. 225 neuronal NE afferents 288 neuronal plasticity 296 neuropeptide Y (NPY) 279, 289, 342 neurotrophic factors 297 NIDDM susceptibility gene 127 Nishizawa, M. 249 nitric oxide (NO) 298; metabolism 64; release 55 nitric oxide (NO)-mediated relations of arterial vessels 64 non-diabetic women, muscle biopsy study on 198 non-peptide small molecule fungal metabolite 324 nonreceptor-type enzymes 36–7 non-receptor type PTPase 38 noradrenergic activities 289 noradrenergic contractile response 64 norepinephrine (NE) 263, 283 nuclear hormone: family of transcription factors 106; receptors 202 nuclear magnetic resonance spectroscopy (NMR), 13 C studies 6 nuclear receptors 90, 104–9 obesity 8, 51, 74, 103, 195, 294, 317, 356; and diabetes 101, 272; genetic and dietary models of 78; and insulin resistance 12; and type 2 diabetes 178–9; in western societies 59, 176 Olefsky, J.M. 214 omega-3 fats 197 Oron, Y. 218 Ortmeyer, H.K. 97 p38 mitogen-activated protein (MAP) kinase 155; signals 157 p85 -PI 3-kinase 27 palmitate, effects on insulin signaling 202 palmitoyl CoA 202 pancreatic beta cells 319; failure 175 paraventricular nuclei (PVN) 276 peroxisome proliferator-activated receptor- (PPAR- ) 105–6, 202; agonists 203–4 peroxisome proliferator-activated receptor- (PPAR-) 108–9; agonists 109 peroxisome proliferator-activated receptor- (PPAR-) 106–8, 161–2, 202; agonists 52, 203; native ligands for 107 peroxisome proliferator-activated receptor-1 (PPAR-1) 107–8 peroxisome proliferator-activated receptor-2 (PPAR-2) 107–8 peroxisome proliferator activated receptors (PPARs) 90, 105–6, 202; agonist drug treatment 197 phenylephrine (PE) 64 Phlorizin treatment 152 phosphatidylinositol 3-kinase (PI3K) 126, 149–50, 315, 354; activation by insulin 149;

activity 150–1; and Akt activity 165; signals 157 phosphatidylinositol 3,4,5-trisphosphate (PIP3), cell permeant 157 phosphatidylinositol 3,4,5-trisphosphate-acetoxymethyl ester (PIP3-AM) 150 phosphoenolpyruvate carboxykinase (PEPCK) 319 phosphorylation/dephosphorylation 315 phosphotyrosine-binding (PTB) domain 149 phosphotyrosine phosphatases 158–60 photoperiodism 274 Pima Indians 76, 80, 176, 178, 211, 219 pioglitazone 52–3, 324 pituitary growth hormone (GH) 29 PKC-mediated signaling events 161 placental growth hormone (hPGH) 28–9 placental lactogen (hPL) 25, 28 plasma: adiponectin, analysis of 102; arterial natriuretic peptide (ANP) 55; catecholamine concentrations 351; cell differentiation factor-1 (PC-1) 162; cortisol and prolactin, 293; free fatty acids 136; glucose concentrations 24; glucose levels 337; insulin concentrations 25, 264; insulin levels 68; lactate concentration 243–4; leptin 29, 98–9, 101; NE level 294; triglyceride (TG) 105, 201 plasminogen activator inhibitor-1 (PAI-1) 64, 342 pleckstrin–homology (PH) and phosphotyrosine-binding (PTB) domains 315 pleiotropic effects of insulin 315 polycystic ovary syndrome (PCOS) 211, 215, 225 preproinsulin gene expression 24 progesterone receptor 105, 107 proglucagon 236 proinsulin 90–1 prolactin 25 pro-piomelanocortin (POMC) 189, 294 protein: expression and stability 316–17; glycosylation 165; kinase B/Akt 126 protein kinase C (PKC): activation 27, 163, 203; epsilon 202; family 153, 202; inhibitors 161; isoforms 28, 154, 160, 321; theta 202 protein kinase C-/ 153–4 protein–protein interactions 181; sites 314 protein tyrosine-kinase activity 92 protein-tyrosine phosphatases (PTPases) 36, 44, 315; activity 37–8; bacterially-expressed 40; cellular insulin action 35–6; inhibitors 36; insulin action pathway 37–8; in insulinsensitive tissues 44; loss of 39; in signal transduction 44; signature sequence motif 36; subcellular localization 38; substrate speciﬁcity 38; superfamily of enzymes 36–8; tissue distribution 37–8

INDEX

proton magnetic resonance spectroscopy (MRS) 198 Psammomys obesus (Israeli Sand Rat) 179, 321 PTP1B 35, 38, 40–2, 44, 160, 315–16, 319, 322–3; activity 328 PTP1B-deﬁcient mice 42 pyruvate dehydrogenase (PDH) 201, 212 Rabin, D.U. 43 Randle: cycle 198, 201; glucose-fatty acid cycle 204 Randle, P.J. 201 reactive oxygen species (ROS): oxidative damage by 166; production 163 Reaven, G.H. 73, 81 Rebuffe-Scrive, M. 75 receptor: activity 107; autophosphorylation 36 receptor-type enzymes 36–7 recombinant human leptin-IgG 30 regulation of insulin: molecular and cellular level 313; secretion 266 renin angiotensin system 53–4 repletion therapy, rationale for 226–8 representational difference analysis (RDA) 178–9 resistin 176, 178, 188, 196, 203, 318 retino-hypothalamic tract 279 retinoid receptors RAR and RXR 105 rhesus monkeys 89–90 rosiglitazone 203, 324 sarcopenia 342 seasonal fattening in vertebrates 272 seasonal insulin resistance syndrome 273 serine kinase activity 28, 149 serine phosphorylation of IRS-1 149 serine/threonine kinase 150 serotonin 278, 297 Ser/Thr phosphorylation of IRS-1 28 serum: free testosterone concentration 225; glucose and insulin concentrations 41; whole lipid concentrations 63 SHIP1 316 SHP-2 35, 42–4, 314, 316, 322 SH-PTP2 and insulin receptors 43 signal transduction pathways 43, 97 skeletal muscle 155; insulin resistance 195; of type 2 diabetic patients 148 SNAP-23 152 SNARE complex 152 Snoeck, A. 351 sortilin localization 137 Sprague–Dawley rat 61, 63 SREBP-1c model, leptin treatment of 191 STAT5 314 streptozotocin (STZ) Stunkard, A.J. 75 sulfonylureas 243, 246 superoxide anions 166

369

Surwit, R.S. 75 Suzuki, H. 54 sympathetic nervous system (SNS) 53–4, 263 syndrome X see insulin resistance syndrome syntaxin-4 152 syprachiasmatic nuclei (SCN) 274, 289; ablation of 280; circadian neuronal oscillations 297; regulation of metabolism 278; serotonin 279 Syrian hamsters 273, 276, 281, 295 systolic blood pressure: and hypoglycemic activity 54; and plasma insulin level 54 Szczepaniak, L.S. 198 3T3-L1 adipocytes 152, 155, 247 thermogenesis 77 thiazolidinediones (TZDs) 103, 243, 246; antidiabetic 318; drugs 106; oral 324 thrifty gene 272; in seasonal animals 273 thrifty genome 282; expression of 277 thrifty phenotype hypothesis 349–51, 356–7 thrombin 66 thyroid hormone receptors 105 TNE- 101, 135, 196, 203, 316, 342 triglycerides 62; accumulation 190; levels 224; measurement of 199; synthesis 63 troglitazone 111, 324 troponin 133; isoforms of (TnI and TnT) 134 tryptophan 279; hydroxylase activity 278 tumor necrosis factor- 176 TUNEL analysis of cells 111 type 1 diabetes 59, 69, 147, 175 type 2 diabetes 3, 6, 8, 69, 73, 76, 81, 105, 134, 147, 154, 163, 166, 175–7, 235, 313; genes for 175–6, 178; human subjects 157, 211; loss of insulin secretion in 319; mellitus 51, 195, 349 tyrosine phosphatases 328; inhibitors of 316 tyrosine phosphorylation 35–6, 38, 42, 138, 315–16 UCP1 82 UCSNP-43 G/G genotype 178 UDP-GlcNAc 165 United States 89, 175, 323 University of Maryland School of Medicine 89 University of Virginia 219 urinary glucose loss 242 urinary norepinephrine excretion 54 urine NE metabolite level 294 vanadate 326–7 vanadium-like compounds 326 vascular smooth muscle cells: culture of 66; migration of 66; proliferation of 67 vasodilative dysfunction 55 ventromedial hypothalamus (VMH) 275–6, 293–4; hyperglycemia, local 286; NE activity 284–5, 288; neurophysiologic changes 286; and PVN, neurochemistry 281

370

visceral fat (VF) 337–8; mass 190; as a potent modulator of insulin action 341; removal and decrease of 341 vitamin D receptor 105 VLDL: hyperlipidemia 60; secretion 63; triglycerides 109, 201 Waxman, M. 75 Wells, A.M. 217 Westernized societies 73, 175, 201 Wexler, B.C. 60 white adipose tissue (WAT) 77; mass 79 Winegar, D.A. 105

INDEX

Wistar fatty rat 51–2, 63 women: insulin sensitivity of 200; with PCOS 222, 224 World Health Organization criteria 349 Wrobel, J. 327 Yen, T.T. 75 Yoshimoto, T. 55 Zabolotny, J.M. 39 Zhang, B. 324 Zucker Diabetic Fatty (ZDF/Gmi-fa) rat 51, 61, 152