Insulin Action and Its Disturbances in Disease - part 1 ppsx

Insulin Resistance

Insulin Resistance
Insulin Action and Its Disturbances in Disease
Editors
Sudhesh Kumar
Unit for Diabetes and Metabolism, Warwick Medical School, University of
Warwick, Coventry CV4 7AL, UK
Stephen O’Rahilly
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes
Hospital, Hill Road, Cambridge CB2 2QQ, UK
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Library of Congress Cataloging-in-Publication Data
Insulin resistance : insulin action and its disturbances in disease / editors, Sudhesh
Kumar, Stephen O’Rahilly.
p. cm.
Includes bibliographical references and index.
ISBN 0-470-85008-6
1. Insulin resistance. I. Kumar, Sudhesh. II. O’Rahilly, S. (Stephen)
RC662.4.I556 2004
616.4

6207 – dc22
2004016888
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
ISBN 0-470-85008-6
Typeset in 10.5/13pt Times by Laserwords Private Limited, Chennai, India
Printed and bound in Germany
This book is printed on acid-free paper responsibly manufactured from sustainable forestry
in which at least two trees are planted for each one used for paper production.
Contents
Preface xi
List of Contributors xiii
1 The Insulin Receptor and Downstream Signalling 1

Ken Siddle
1.1 Introduction 1
1.2 Insulin receptor structure and function 2
1.3 Insulin receptor substrates 15
1.4 Downstream signalling pathways 23
1.5 The basis of insulin’s signalling specificity 37
1.6 Conclusion 38
References 39
2 Insulin-mediated Regulation of Glucose Metabolism 63
Daniel Konrad, Assaf Rudich and Amira Klip
2.1 Introduction 63
2.2 Insulin as a master regulator of whole body glucose disposal 63
2.3 Insulin-mediated regulation of glucose metabolic pathways 67
2.4 Glucose uptake into skeletal muscle – the rate-limiting step in glucose
metabolism 69
Acknowledgements 78
References 78
3 Insulin Action on Lipid Metabolism 87
Keith N. Frayn and Fredrik Karpe
3.1 Introduction: does insulin affect lipid metabolism? 87
3.2 Molecular mechanisms by which insulin regulates lipid metabolism 88
3.3 Insulin and lipolysis 89
3.4 Insulin, lipoprotein lipase and cellular fatty acid uptake 94
3.5 Co-ordinated regulation of fatty acid synthesis and ketogenesis 96
3.6 Insulin and cholesterol synthesis 97
3.7 Insulin effects on lipoprotein metabolism 98
Acknowledgement 99
References 99
vi CONTENTS
4 The Effect of Insulin on Protein Metabolism 105

Laura J. S. Greenlund and K. Sreekumaran Nair
4.1 Introduction 105
4.2 Molecular mechanisms of insulin’s effect on protein turnover 107
4.3 Measurement of protein metabolism (synthesis and breakdown or turnover)
in human subjects 111
4.4 Whole body and regional protein turnover 114
Acknowledgements 125
References 125
5 Genetically Modified Mouse Models of Insulin Resistance 133
Gema Medina-Gomez, Christopher Lelliott and
Antonio J. Vidal-Puig
5.1 Introduction 133
5.2 Genetic modification as a tool to dissect the mechanisms leading to insulin
resistance 134
5.3 Candidate genes involved in the mechanisms of insulin resistance 134
5.4 Insulin signalling network 136
5.5 Factors leading to insulin resistance 137
5.6 Defining the function of the insulin cascade molecules through global
knockouts 137
5.7 Double heterozygous mice as models of polygenic forms of diabetes 139
5.8 Defining tissue and/or organ relevance for the maintenance of insulin
sensitivity 140
5.9 Genetically modified mice to study modulators of insulin sensitivity 142
5.10 Lipodystrophy versus obesity, the insulin resistance paradox 143
5.11 Excess of nutrients as a cause of insulin resistance 147
5.12 PPARs, key mediators of nutritional-regulated gene expression and insulin
sensitivity 148
References 148
6 Insulin Resistance in Glucose Disposal and Production in Man
with Specific Reference to Metabolic Syndrome and Type 2

Diabetes 155
Henning Beck-Nielsen, Frank Alford and Ole Hother-Nielsen
6.1 Introduction 155
6.2 Measurement of insulin resistance 157
6.3 Insulin-resistant states 162
6.4 Conclusion and perspectives 171
References 172
7 Central Regulation of Peripheral Glucose Metabolism 179
Stanley M. Hileman and Christian Bjørbæk
7.1 Introduction 179
7.2 Counter-regulation of hypoglycaemia – role of the CNS 180
7.3 Brain regions involved in counter-regulation 182
7.4 Glucosensing neurons 184
CONTENTS vii
7.5 Central control of peripheral organs involved in glucoregulation 187
7.6 Additional afferent signals to the CNS regulating peripheral glucose
metabolism 189
7.7 Conclusions and future perspectives 194
Acknowledgements 196
References 196
8 Relationship between Fat Distribution and Insulin Resistance 207
Philip G. McTernan, Aresh Anwar and Sudhesh Kumar
8.1 Introduction 207
8.2 Fat and its distribution 207
8.3 Basis for variation in adipose tissue mass 209
8.4 Change in adipocyte phenotype with obesity 210
8.5 Obesity and its association with insulin resistance 210
8.6 Subcutaneous and visceral adipose tissue 211
8.7 The pathogenic significance of abdominal adipose tissue 211
8.8 Potential mechanisms linking central obesity to the metabolic syndrome 212

8.9 Randle hypothesis/glucose–fatty acid hypothesis 212
8.10 Alternatives to the Randle hypothesis 213
8.11 Ectopic fat storage: fat content in obesity 214
8.12 Adipose tissue as an endocrine organ 214
8.13 Plasminogen activator–inhibitor 1 215
8.14 Renin angiotensin system in adipose tissue 216
8.15 Visceral obesity and steroid hormone metabolism 217
8.16 Glucocorticoid metabolism and obesity 217
8.17 11β-hydroxysteroid dehydrogenase (11β-HSD) 218
8.18 Isoenzymes of 11β-HSD 218
8.19 11β-HSD and obesity 219
8.20 Sex steroid metabolism and obesity: oestrogen biosynthesis 220
8.21 Aromatase 220
8.22 Sex steroids and body fat 222
8.23 Summary 224
Acknowledgement 224
References 224
9PPARγ and Glucose Homeostasis 237
Robert K. Semple and Stephen O’Rahilly
9.1 Evidence from cell and rodent models 238
9.2 Insights from human studies 251
References 256
10 Adipokines and Insulin Resistance 269
Daniel K. Clarke and Vidya Mohamed-Ali
10.1 Obesity and insulin resistance 270
10.2 Adipokines implicated in insulin resistance 272
10.3 Conclusions 280
References 280
viii CONTENTS
11 Dietary Factors and Insulin Resistance 297

Jeremy Krebs and Susan Jebb
11.1 Introduction 297
11.2 The importance of body fatness 298
11.3 Specific dietary factors 302
11.4 Summary 310
References 311
12 Physical Activity and Insulin Resistance 317
Nicholas J. Wareham, Søren Brage, Paul W. Franks and
Rebecca A. Abbott
12.1 Introduction 317
12.2 Evidence from observational studies of the association between physical
activity and insulin resistance 318
12.3 Summary of findings from observational studies in adults 318
12.4 Summary of findings from observational studies in children and adolescents 340
12.5 Mechanisms underlying the association between physical activity and insulin
resistance 351
12.6 Trials of the effect of physical activity on insulin sensitivity in adults 353
12.7 Trials of the effect of physical activity on insulin sensitivity in children and
adolescents 374
12.8 Evidence of heterogeneity of the effect of physical inactivity on insulin
resistance in sub-groups of the population 375
12.9 Conclusions 385
References 386
13 Genetics of the Metabolic Syndrome 401
George Argyropoulos, Steven Smith and Claude Bouchard
13.1 Historical perspective 401
13.2 Pathophysiology 404
13.3 Genetic epidemiology 407
13.4 Monogenic disorders 411
13.5 Candidate genes 414

13.6 Genomic scans 426
13.7 Conclusions 427
References 427
14 Insulin Resistance and Dyslipidaemia 451
Benoˆıt Lamarche and Jean-Fran¸cois Mauger
14.1 Introduction 451
14.2 Historical notes 451
14.3 Obesity versus the insulin resistance syndrome 453
14.4 Hypertriglyceridaemia 453
14.5 Reduced HDL cholesterol concentrations 455
14.6 Small, dense LDL particles 457
14.7 LDL cholesterol levels versus LDL particle number 459
CONTENTS ix
14.8 Insulin resistance, dyslipidaemia and the risk of cardiovascular disease 460
14.9 Conclusions 461
References 461
15 Insulin Resistance, Hypertension and Endothelial Dysfunction 467
Stephen J. Cleland and John M. C. Connell
15.1 Introduction 467
15.2 Hyperinsulinaemia, insulin resistance and hypertension 467
15.3 Possible mechanisms linking insulin with blood pressure 468
15.4 Atherosclerosis and insulin resistance 469
15.5 Vascular endothelial dysfunction and mechanisms of atherothrombotic disease 469
15.6 Direct vascular action of insulin 471
15.7 What causes abnormal insulin signalling in metabolic and vascular tissues? 474
15.8 Summary and conclusions (Figure 15.8) 477
References 478
16 Insulin Resistance and Polycystic Ovary Syndrome 485
Neus Potau
16.1 Introduction 485

16.2 Definition of polycystic ovary syndrome (PCOS) and diagnostic criteria 486
16.3 Hyperandrogenism and hyperinsulinism 489
16.4 Assessment of insulin resistance in PCOS 491
16.5 Gene studies on PCOS 492
16.6 Premature pubarche, hyperinsulinism and PCOS 495
16.7 Treatment approach with antiandrogens 497
16.8 Treatment approach with insulin sensitizers (metformin) 498
16.9 Treatment approach with insulin sensitizers (thiazolidinediones) 501
16.10 Conclusion 502
References 502
17 Syndromes of Severe Insulin Resistance (SSIRs) 511
David Savage and Stephen O’Rahilly
17.1 Introduction 511
17.2 General biochemical and clinical features of severe insulin resistance 512
17.3 Classification of specific syndromes of insulin resistance 514
17.4 Primary disorders of insulin action 515
17.5 Lipodystrophic syndromes and a lipocentric approach to diabetes 518
17.6 Complex genetic syndromes associated with severe insulin resistance 525
17.7 Therapeutic options in the syndromes of severe insulin resistance 526
References 527
18 Therapeutic Strategies for Insulin Resistance 535
Harpal S. Randeva, Margaret Clarke and Sudhesh Kumar
18.1 Introduction 535
18.2 Obesity and insulin resistance 535
18.3 Management of obesity 537
x CONTENTS
18.4 Dietary management of obesity 539
18.5 Exercise and physical activity 540
18.6 Anti-obesity drugs 540
18.7 Surgical management of obesity 543

18.8 Pharmacological treatment of insulin resistance 544
18.9 Insulin sensitizers and cardiovascular risk factors 551
18.10 Conclusions 553
References 554
19 Drug Therapy for Insulin Resistance – a Look at the Future 561
Bei B. Zhang and David E. Moller
19.1 Introduction 561
19.2 Targeting molecules within the insulin signal transduction pathway 563
19.3 Targeting negative modulators of insulin signalling 567
19.4 Targeting obesity and insulin resistance 569
References 575
Index 587
Preface
Hormone resistance syndromes are typically thought of as rare, usually genetic,
disorders with a severe but relatively stereotyped clinical and biochemical pro-
file. While there are syndromes of severe insulin resistance that conform to
this description, defective insulin action is of much more pervasive biomedical
importance. Even moderate degrees of insulin resistance are closely linked to
a range of common diseases, including Type 2 diabetes, polycystic ovary syn-
drome, obesity and hypertension. Not surprisingly, in recent years, there has been
a tremendous increase in interest within the medical and scientific community
in understanding the causes, consequences and treatment of insulin resistance.
There are several reasons for this. Firstly, we are now witnessing a revolution
in unravelling the molecular mechanism of insulin action and in understanding
the molecular basis for the various syndromes associated with insulin resistance.
Secondly, we are now seeing a global epidemic of Type 2 diabetes that may
pose a major threat to international public health. Thirdly, the pharmaceutical
and biotechnology industries are investing heavily in the development of new
drugs that can improve insulin action. Therefore, we believe that the publication
of this book is timely.

There is considerable literature available on the subject of insulin resistance. A
recent search on Medline revealed more than 20,000 articles on this subject. This
information is readily accessible and one might argue that a book such as this
one might become outdated as soon as it is published! One guiding principle for
this book was, therefore, to bring to the reader not only a synthesis of important
information, but also the wisdom of leading researchers and clinicians who are
recognised as leaders in their own fields.
Each chapter stands independently and is written by one or more experts on
the subject. The book is divided into five sections with a total of 19 chapters.
Section 1 reviews our current understanding of the normal biology of insulin
action and separate chapters cover insulin action in relation to glucose, lipid and
protein metabolism. Section 2 explores the pathophysiological mechanisms of
insulin resistance, with discussion of the effects of glucose disposal in humans
and in animal models. It also reviews the central regulation of energy metabolism
and its perturbation, as well as the relationship between fat distribution and
insulin action and the role of the nuclear hormone receptor PPARγ in glucose
metabolism. Finally, there is a chapter discussing the role of adipose tissue-
secreted products in causing insulin resistance. Section 3 examines the role of
xii PREFACE
genetic and environmental factors that result in insulin resistance, including the
effects of dietary factors and physical inactivity. The genetic basis of syndrome
X, a common disorder associated with insulin resistance, is described. Section 4
discusses the relationship between insulin resistance and common diseases such
as dyslipidemia, hypertension and polycystic ovary syndrome. Finally, Section 5
reviews the clinical management of insulin resistance, covering the many syn-
dromes of severe insulin resistance, currently available therapeutic approaches
and possible future options for drug therapy for this condition.
Although the book aims to provide comprehensive coverage of the subject,
there are some obvious omissions, for example, the relationship between insulin
resistance and Type 2 diabetes. Whilst this relationship is alluded to in many

places, we have not devoted a full chapter to it as there are several excellent
recent reviews on the subject.
The book is intended mainly for a specialist readership, although it may prove
to be a useful resource for a wide variety of scientists, clinicians and postgraduate
students with an interest in any of the related conditions. We hope that regardless
of your background as a physician, medical researcher or scientist, you will
find this book appropriate for your needs. Finally, all contributing authors have
produced outstanding chapters that reflect their expertise and wisdom and spared
their valuable time despite tremendous pressures from competing obligations.
We wish to thank them all for their support, hard work and friendship.
Sudhesh Kumar
Stephen O’Rahilly
August 2004
List of Contributors
Rebecca A. Abbott, MRC Epidemiology Unit, Strangeways Research
Laboratory, Worts Causeway, Cambridge CB1 8RN, UK
Frank Alford, St. Vincent’s Hospital, Melbourne, Endocrine Unit, 41 Fitzroy
Parade, Fitzroy, Victoria 3065, Australia
Aresh Anwar, University Hospitals of Coventry and Warwickshire, Walsgrave
Hospital, Clifford Bridge Road, Coventry CV2 2DX, UK
George Argyropoulos, Pennington Biomedical Research Center, 6400 Perkins
Road, Baton Rouge, LA 70808, USA
Henning Beck-Nielsen, Odense University Hospital, Department of
Endocrinology, Kloevervaenget 64, 5000 Odense C, Denmark
Christian Bjørbæk, Division of Endocrinology, Beth Israel Deaconess
Medical Center Research North, 330 Brookline Avenue, Boston, MA
02215, USA
Claude Bouchard, Pennington Biomedical Research Center, 6400 Perkins
Road, Baton Rouge, LA 70808, USA
Søren Brage, MRC Epidemiology Unit, Strangeways Research Laboratory,

Worts Causeway, Cambridge CB1 8RN, UK
Daniel K. Clarke, Adipokines and Metabolism Research Group, Department
of Medicine, University College London, 48 Riding House Street, London
W1W 7EY, UK
Margaret Clarke, Heartlands and Solihull NHS Trust, Birmingham B19
9RA, UK
Stephen J. Cleland, Department of Medicine and Therapeutics, University of
Glasgow, Glasgow G11 6NT, UK
John M. C. Connell, Division of Cardiovascular and Medical Sciences,
University of Glasgow, Glasgow G11 6NT, UK
Paul W. Franks, MRC Epidemiology Unit, Strangeways Research
Laboratory, Worts Causeway, Cambridge CB1 8RN, UK
xiv LIST OF CONTRIBUTORS
Keith N. Frayn, Oxford Centre for Diabetes, Endocrinology and Metabolism,
Churchill Hospital, Oxford OX3 7LJ, UK
Laura J. S. Greenlund, Department of Endocrinology, Mayo Clinic, 200 First
Street SW, Rochester, MN 55905, USA
Stanley M. Hileman, Department of Physiology and Pharmacology, West
Virginia University, Morgantown, WV 26506, USA
Ole Hother-Nielsen, Odense University Hospital, Department of
Endocrinology, Sdr. Boulevard 29, 5000 Odense C, Denmark
Susan Jebb, MRC Human Nutrition Research, Elsie Widdowson Laboratory,
Fulbourn Road, Cambridge CB1 9NL, UK
Fredrik Karpe, Oxford Centre for Diabetes, Endocrinology and Metabolism,
Churchill Hospital, Oxford OX3 7LJ, UK
Amira Klip, Programme in Cell Biology, The Hospital for Sick Children, 555
University Avenue, Toronto, ON, M5G 1X8, Canada
Daniel Konrad, Programme in Cell Biology, The Hospital for Sick Children,
555 University Avenue, Toronto, ON, M5G 1X8, Canada
Jeremy Krebs, Wellington Clinical School of Medicine, University of Otago,

P.O. Box 7343, Wellington South, New Zealand
Sudhesh Kumar, Unit for Diabetes and Metabolism, Warwick Medical
School, University of Warwick, Coventry CV4 7AL, UK
Beno
ˆ
ıt Lamarche, Institute on Nutraceuticals and Functional Foods, 2440
Boulevard Hochelaga, Laval University, Quebec, G1K 7P4, Canada
Christopher Lelliott, Department of Clinical Biochemistry and Metabolic
Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 2QR, UK
Jean-Fran¸cois Mauger, Institute on Nutraceuticals and Functional Foods,
2440 Boulevard Hochelaga, Laval University, Quebec, G1K 7P4, Canada
Philip G. McTernan, Unit for Diabetes and Metabolism, Warwick Medical
School, University of Warwick, Coventry CV4 7AL, UK
Gema Medina-Gomez, Department of Clinical Biochemistry and Metabolic
Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 2QR, UK
Vidya Mohamed-Ali, Adipokines and Metabolism Research Group,
Department of Medicine, University College London, 48 Riding House Street,
London W1W 7EY, UK
LIST OF CONTRIBUTORS xv
David E. Moller, Departments of Molecular Endocrinology and Metabolic
Disorders, Merck Research Laboratories, Rahway, NJ 07065, USA
K. Sreekumaran Nair, Department of Endocrinology, Mayo Clinic, 200 First
Street SW, Rochester, MN 55905, USA
Stephen O’Rahilly, Department of Clinical Biochemistry and Medicine,
University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge
CB2 2QQ, UK
Neus Potau, Hormonal Laboratory, Hospital Matemo-Infantil Vail d’Hebron,
Passeig Vail d’Hebron, 119–129, 08035 Barcelona, Spain

Harpal S. Randeva, Molecular Medicine Research Group, Biomedical
Research Institute, Biological Sciences, University of Warwick, CV4 7AL, UK
Assaf Rudich, Programme in Cell Biology, The Hospital for Sick Children,
555 University Avenue, Toronto, ON, M5G 1X8, Canada
David Savage, Department of Clinical Biochemistry and Medicine, University
of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK
Robert K. Semple, Department of Clinical Biochemistry, University of
Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QR, UK
Ken Siddle, Department of Clinical Biochemistry, University of Cambridge,
Addenbrooke’s Hospital (Box 232), Hills Road, Cambridge CB2 2QR, UK
Stephen Smith, Pennington Biomedical Research Center, 6400 Perkins Road,
Baton Rouge, LA 70808, USA
Antonio J. Vidal-Puig, Department of Clinical Biochemistry and Metabolic
Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 2QR, UK
Nicholas J. Wareham, MRC Epidemiology Unit, Strangeways Research
Laboratory, Worts Causeway, Cambridge CB1 8RN, UK
Bei B. Zhang, R80W180, Merck Research Laboratories, P.O. Box 2000, 126
E. Lincoln Avenue, Rahway, NJ 07065, USA

1
The Insulin Receptor
and Downstream Signalling
Ken Siddle
1.1 Introduction
Insulin regulates diverse physiological processes in mammals, including mem-
brane transport, intermediary metabolism and cell growth and differentiation.
These actions involve rapid effects on subcellular membrane traffic, enzyme
activity and protein synthesis as well as longer term actions on gene tran-
scription. The most conspicuous metabolic effects of insulin are associated

with skeletal muscle, adipose tissue and liver
1
but its physiologically impor-
tant actions are by no means confined to such tissues, as evidenced by the
phenotypes of mice with tissue-specific knockout of insulin receptor in brain,
pancreatic β-cells or endothelia.
2
Insulin signalling pathways have also been
implicated in accelerating the ageing process.
3, 4
Understanding of the signalling pathways by which the insulin receptor is
able to influence so many and such diverse cellular targets is still far from
complete, although the last 20 years have seen major advances. A surprising
feature is that to date the only signalling component known to be unique to
insulin action is the insulin receptor (IR) itself, which is widely expressed in
mammalian cells, although levels vary greatly between cell types. The IR binds
insulin with high affinity and specificity, and transmits a signal to the cytosol
via its intrinsic tyrosine-specific protein kinase activity. This phosphorylates a
number of intracellular substrates, most especially the so-called insulin receptor
substrates (IRSs), which recruit and activate an array of signalling proteins con-
taining Src homology-2 (SH2) domains. Two signals have been shown to play
major roles in insulin action, namely those transmitted by the enzyme phospho-
inositide 3-kinase (PI 3-kinase), which generates PtdIns(3,4,5)tris-phosphate at
Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly
 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6
2 THE INSULIN RECEPTOR AND DOWNSTREAM SIGNALLING
the cytosolic face of membranes, and the guanine nucleotide exchange factor
Grb2/Sos, which activates the small G-protein Ras. These act as switch mech-
anisms to change the ‘currency’ of signalling from tyrosine phosphorylation to
serine/threonine phosphorylation of target proteins. However, these signals, and

the downstream signalling cascades involving protein kinase B and mitogen-
activated protein kinases (MAPKs), have been implicated in the actions of a
wide variety of hormones and growth factors as well as specific actions of
insulin. This chapter will focus particularly on the IR and its substrates, and
consider more briefly what is known about downstream signalling pathways,
which have been reviewed in detail elsewhere.
5–9
1.2 Insulin receptor structure and function
The insulin receptor family
The IR is a large, heterotetrameric, transmembrane glycoprotein containing two
types of subunit, designated α (M
r
140 kDa) and β (95 kDa), linked by disul-
phide bonds in a β–α–α–β configuration. The principal members of the IR
family of receptor tyrosine kinases are represented in Figure 1.1, together with
their high affinity ligands. It is possible that IRR may also form hybrids with IR,
although because of the very restricted distribution of IRR these are unlikely to
be of major significance. It is likely that the two isoforms of IR will also form
heterodimers, although this has recently been questioned.
22
It is assembled from
a single polypeptide pro-receptor, by dimerization, proteolytic cleavage and gly-
cosylation within the endoplasmic reticulum and Golgi apparatus, before traffick-
ing of mature receptor to the plasma membrane. The IR was initially defined by
radioligand binding studies, which provided information on affinity, specificity
and tissue distribution. It was shown to bind insulin with high (sub-nanomolar)
affinity, marked pH dependence (decreased affinity even at mildly acid pH) and
unexpectedly complex kinetics (manifested as negative co-operativity).
10
The

first real insight into signalling mechanisms came with the demonstration that
the receptor possessed intrinsic, tyrosine-specific protein kinase activity that was
stimulated by insulin binding.
11
Soon afterwards, cloning of the pro-receptor
cDNA
12, 13
and the receptor gene
14
opened the door to analysis of receptor
structure and function, which is now understood in considerable detail.
15–18
The IR gene consists of 22 coding exons spanning 120 kilobases on chro-
mosome 19p13.2. Exon 11, of just 36 nts, is subject to alternative splicing,
resulting in the generation of two isoforms designated IR-A (Ex 11−) and IR-B
(Ex 11+), which differ in sequence by 12 amino acids at the carboxyl-terminus
of the α-subunit (the numbering used here includes the exon 11 sequence). The
relative proportions of the two isoforms differ between tissues, IR-A predom-
inating in brain and IR-B in liver, while both are found in similar amounts in
skeletal muscle and placenta.
19, 20
The isoforms differ modestly but significantly
INSULIN RECEPTOR STRUCTURE AND FUNCTION 3
Extracellular
Intracellular
High affinity
ligands
IR-A
SS
SS

S
S
S
S
Insulin, IGF-II
IR-B
SS
SS
S
S
S
S
Insulin
Type 1 IGFR
SS
SS
S
S
S
S
IGF-I, IGF-II
IRR
SS
SS
S
S
S
S
–
IR/IGFR hybrid

SS
SS
S
S
S
S
IGF-I, IGF-II
αα
ββ
Figure 1.1
The insulin receptor family
4 THE INSULIN RECEPTOR AND DOWNSTREAM SIGNALLING
with respect to their binding affinity for insulin and IGFs, and this is perhaps not
surprising in view of the proximity of the variable sequence to a known major
binding epitope at the α-subunit carboxyl-terminus. More controversially, it has
been suggested that the short peptide sequence encoded by exon 11 also acts as
a sorting signal, causing the isoforms to localize to different plasma membrane
microdomains from which they activate distinct signalling cascades.
21, 22
The IR
gene is transcribed as mRNAs of 7–11 kilobases, which include substantial 5

-
and 3

-untranslated regions either side of the coding sequence. (NCBI database
references for complete IR (InsR) cDNA and protein sequences are human NM
000208, mouse NM 010568 and rat NM 017071). The deduced sequence of the
human IR precursor contains 1382 (or 1370) amino acids, including a signal
sequence of 27 amino acids which is absent from mature receptor. A tetrabasic

RKRR motif marks the site of proteolytic cleavage to generate the α- and β-
subunits, of 731 (719) and 620 amino acids respectively. In the mature receptor
the α-subunit is wholly extracellular and contains the ligand binding site, while
the β-subunit contains a single predicted membrane-spanning segment and an
intracellular tyrosine kinase domain.
The extracellular portion of IR is heavily glycosylated, and some glycosyla-
tion is essential for normal receptor function.
23
Disulphides between α-subunits
are contributed by Cys524 and Cys682/3/5,
24
and can readily be reduced in vitro
to generate half-receptors that bind insulin with decreased affinity. In contrast
the α–β disulphide between Cys647 and Cys872 can be reduced only under
denaturing conditions. Experimental perturbation of glycosylation,
24
proteolytic
cleavage
25
and disulphide bonding
26
can profoundly affect receptor function.
There is no compelling evidence that these processes are modulated under
physiological conditions in vivo, although it remains possible that there are cir-
cumstances where this does occur.
Just as insulin is structurally related to the insulin-like growth factors, so
the IR is similar in structure and function to the type 1 IGF receptor (IGFR),
with which it shares approx 60 per cent amino acid sequence identity.
27
(The

type 2 IGF receptor is an unrelated protein,
28
which is not thought to have any
signalling function but may have a role in clearance of IGF from the circula-
tion.) Like the IR, the IGFR is very widely expressed, albeit at different levels.
There is significant expression of IGFR in skeletal muscle, but very low levels
in hepatocytes and adipocytes. The very different biological roles of IR and
IGFR are emphasized by the distinct phenotypes of mouse knockout models.
Mice lacking IR exhibit only slight (10 per cent) growth retardation at birth, but
die within days as a result of uncontrolled hyperglycaemia and ketoacidosis,
2
although lack of IR causes more severe growth retardation in humans. In con-
trast, mice lacking IGFR are severely growth deficient (approximately 45 per
cent of normal size) and developmentally retarded and die at birth of respira-
tory failure.
29
However, the functions of the two receptors are not completely
INSULIN RECEPTOR STRUCTURE AND FUNCTION 5
distinct as shown by the efficacy of IGF-I in reducing hyperglycaemia in human
subjects lacking functional IR.
30
A third member of the IR/IGFR family is the insulin-receptor-related receptor
(IRR).
31
Although this has a similar degree of homology to IR and IGFR as these
receptors do to each other, the IRR does not bind either insulin or IGFs,
32
and no
ligand has yet been identified for this receptor. Expression of IRR is much more
restricted than that of IR and IGFR, but it is found in kidney, neural tissue, stom-

ach and pancreatic beta cells. Mice lacking IRR appear phenotypically normal,
33
although there is evidence that, along with other members of the IR family, IRR
contributes in a non-redundant fashion to testicular development in mice.
34
Insulin binds to IGFR with low affinity, which would not be sufficient to
permit significant activation by insulin in vivo under normal physiological condi-
tions, but could become important under pathological conditions associated with
hyperinsulinaemia. Indeed, it has been suggested that such ‘specificity spillover’
might contribute to features of insulin resistance syndromes such as acanthosis
nigricans and polycystic ovaries
35, 36
and it may well be responsible for effects
of insulin on growth of cultured cells. The converse phenomenon, stimulation
of the IR by IGFs, may be of greater physiological significance. Early studies of
ligand specificity, which gave rise to the notion that IGFs bound only with low
affinity to IR, commonly used rat liver as a source of receptors and such studies
reflected the properties of the IR-B isoform. In fact, although the isoforms differ
only slightly in affinity for insulin itself, the A isoform has substantially higher
affinity for IGFs, particularly IGF-II, than the B-isoform.
37, 38
Indeed the affin-
ity of IR-A for IGF-II is comparable to that of the type 1 IGFR, and it appears
that IR-A makes a significant contribution to mediating biological activity of
IGF-II, both in vivo and in vitro.
39, 40
When IR and IGFR are expressed in the same cells, they are can form hybrid
structures containing an insulin half-receptor, disulphide linked to an IGF half-
receptor (Figure 1.1).
41–43

Surprisingly heterodimerization of proreceptors to
form hybrids seems to occur with similar efficiency to homodimerization to form
classical receptors, so the proportion of receptors existing as hybrids is largely
a reflection of the relative expression levels of the individual receptors.
43–45
Hybrid receptors thus occur commonly in vivo, and in tissues such as heart
and skeletal muscle, where IR is expressed at higher levels than IGFR, hybrids
account for the majority of high affinity ‘IGF receptors’.
44
Conversely, when
IGFR is in excess, as in fibroblasts, the majority of IR is drawn into hybrids.
It remains possible that mechanisms exist that promote or inhibit assembly of
hybrid receptors but these have not been demonstrated. Hybrid receptors bind
IGF with high affinity, comparable to classical type 1 IGFR, and would there-
fore be expected to contribute significantly to mediating IGF actions in vivo.
However, hybrids bind insulin with relatively low affinity, especially those incor-
porating the IR-B isoform,
46, 47
and are unlikely to contribute significantly to
6 THE INSULIN RECEPTOR AND DOWNSTREAM SIGNALLING
insulin signalling at physiological insulin concentrations. In fact if IR are incor-
porated into hybrids this would be expected to decrease cellular sensitivity to
insulin, and it has been suggested that an increase in the proportion of hybrid
receptors in skeletal muscle of obese and diabetic subjects may contribute to
insulin resistance.
48, 49
However, in classical insulin target tissues such as liver,
fat and muscle the proportion of IR in hybrids is always likely to be small and
the potential for changes in IGFR expression to influence insulin sensitivity must
be correspondingly slight. It is unclear whether hybrid receptors have unique

signalling properties, which might influence the nature of cellular responses. It
would be expected that binding of either insulin or IGF would lead to activation
of tyrosine kinase activity within both β-subunits of hybrids.
50
As discussed
below, the signalling competencies of IR and IGFR are very similar but proba-
bly not identical. In this context, hybrids might in principle have the signalling
properties of both IR and IGFR, or even additional novel properties reflecting
synergy between the individual half-receptors.
The IR extracellular domain: ligand binding
Apart from the intrinsic interest of unravelling the molecular basis of lig-
and binding and the mechanism of receptor activation, understanding of lig-
and–receptor interactions could facilitate the design of insulin mimetics with
therapeutic potential. However, the large size of the IR has presented a con-
siderable analytical challenge. Molecular modelling based on sequence analysis
predicts that the extracellular portion of each half-receptor contains six dis-
tinct structural domains, while three intracellular domains are recognized.
51
The N-terminal, membrane-distal half of the extracellular receptor contains
two β-helical L domains flanking a cysteine-rich (CR) region (Figure 1.2). The
structural domains of the IR are shown in Figure 1.2: L1 and L2 are β-helical
domains; CR is the cysteine-rich domain; Fn0, Fn1 and Fn2 are fibronectin type
III repeats; the inserted region within Fn1 includes the site of cleavage between
α- and β-subunits; JM is the juxtamembrane region; TK is the tyrosine kinase
domain; CT is the carboxyl-terminal domain. Positions of inter-subunit disul-
phide links and ligand binding epitopes are as indicated. The corresponding
portion of the IGFR, expressed as a recombinant protein, has been crystal-
lized and its structure has been determined.
52
This reveals the L domains and

disulphide-bonded modules of the CR domain surrounding a putative ligand-
binding cavity (although this IGFR fragment does not itself bind IGFs). The
orientation of the L domains within the crystal may not be the same as in native
receptor, and of course differences in conformation between IR and IGFR might
contribute to binding specificity. However, it is safe to assume that the struc-
tures of the L1/CR/L2 domains of the IR are similar to those of the IGFR. The
remaining extracellular portion of both IR and IGFR is believed to consist of
three fibronectin type III domains, each folded as a seven-stranded β-sandwich.
INSULIN RECEPTOR STRUCTURE AND FUNCTION 7
SS
S
S
L1 CR L2 Fn0 Fn1/insert Fn2 JM TK CT
S
S
S
S
Fn1′ Fn1′′
Ligand binding epitopes
Extracellular Intracellular
Figure 1.2 Insulin receptor structural domains
8 THE INSULIN RECEPTOR AND DOWNSTREAM SIGNALLING
However, the limitations of theoretical modelling are illustrated by the fact
that different groups who predicted structures for the first such domain (usu-
ally referred to as the Fn0 domain) assigned different β-strands within the FnIII
fold,
53, 54
and therefore proposed different positions for the inter-subunit disul-
phide bond formed by Cys524. The central FnIII domain (usually referred to as
Fn1) contains a large inserted region as a loop between β-strands, for which no

particular secondary or tertiary structure is predicted. In the middle of this are
the sequence encoded by the alternatively spliced Exon 11 and the site of cleav-
age between α- and β-subunits. The Fn1 domain is therefore assembled from
sequences within the C-terminal region of the α-subunit and the N-terminal
region of the β-subunit, so that the α- and β-subunits are not readily dissociated
as independent proteins but are held together by strong non-covalent forces as
well as disulphide bonds.
The residues in insulin that are important for receptor binding have been inten-
sively studied by comparing the properties of insulins from different species,
by chemical modification and by mutational analysis and alanine scanning.
10, 55
These studies indicate that two surfaces of the insulin molecule are impor-
tant for receptor binding.
15
The ‘classical’ binding surface contains a number of
hydrophobic residues (including B24Phe and B25Phe), while the second binding
surface is more polar. Both surfaces are essentially ‘conformational’ in nature
and include residues from disparate regions of primary sequence. A screen of
large, random, phage-displayed peptide libraries has identified novel peptides
that bind to the IR at or close to the insulin binding site and presumably mimic
critical aspects of the insulin surface.
56
Indeed derivatives of these peptides
function as insulin mimetics and activate the receptor.
57
It remains to be seen
whether it will be possible to model non-peptide mimetics using information
derived from the study of such peptides.
The task of identifying residues in the IR/IGFR that contact ligand is more
difficult, given the very large size of the receptors. The problem has been

approached by cross-linking insulin analogues, constructing IR/IGFR chimeras,
and by mutational analysis and alanine scanning (as reviewed in 9, 15 and
16). Four distinct binding epitopes have been identified, within the L1, CR, L2
and Fn1 insert domains. Residues in the L1 and L2 domains of IR, especially
Phe39, are important for insulin specificity, although IGFR specificity for IGF-1
is more dependent on residues in the CR domain. These putative binding epi-
topes flank the cavity enclosed by the L1, CR and L2 domains in the crystal
structure of the IGFR fragment described above, and this cavity is of appro-
priate dimensions to accommodate a molecule of ligand.
16
Although neither
this IGFR fragment nor the corresponding IR fragment bind ligand, addition to
either construct of the fourth binding epitope, a short peptide sequence from
the α-subunit carboxyl-terminus, confers ligand binding of moderate affinity.
58
Remarkably, this peptide confers binding ability on N-terminal fragments not
only when fused directly but even when added as a free peptide.
59
Mutational