B3 Adrenoreceptor (Taylor & Francis Series in Pharmaceutical Sciences)

The ␤3-Adrenoreceptor

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The ␤3-Adrenoreceptor

Edited by A.DONNY STROSBERG Cochin Institute for Molecular Genetics CNRS Unit of Molecular Immunopharmacology Paris, France

First published 2000 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, 2003. © 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 The ß3 adrenoreceptor/edited by A.Donny Strosberg. p. cm.—(Taylor & Francis series in pharmaceutical sciences) Includes bibliographical references and index. 1. Beta adrenoreceptors. 2. Adrenergic beta agonists. I. Strosberg, A.D. II. Series. QP364.7.B13 2000 612'.01575–dc21 00–023999 ISBN 0-203-48444-4 Master e-book ISBN

ISBN 0-203-79268-8 (Adobe eReader Format) ISBN 0-748-40804-5 (Print Edition)

Contents

List of figures List of tables Contributors Preface Abbreviations used in this book 1

x xii xiii xv xvii

Structure and Function of the ß3-Adrenoreceptor

1

A.Donny Strosberg and Cindy C.Gerhardt 1.1 1.2

1.3

1.4

1.5

1.6

1.7

Introduction The ß3AR gene 1.2.1 Cloning of the gene 1.2.2 Structure of the coding region 1.2.3 Structure of the promoter The ß3AR protein 1.3.1 Comparison with ß1- and ß2AR 1.3.2 Comparison of ß3AR from various species 1.3.3 Polymorphism Structure-function relationships in the ß3AR 1.4.1 The ligand binding site 1.4.2 The regions of interaction with the G proteins Signal transduction and biological functions 1.5.1 ß3AR-mediated activation of adenylyl cyclase 1.5.2 ß3AR-mediated activation of other signalling pathways 1.5.3 A role for the ß3AR in adipocyte differentiation Distribution of the ß3AR 1.6.1 Functional detection of the ß3AR 1.6.2 Immunodetection of the ß3AR protein 1.6.3 Detection of the ß3AR mRNA Concluding remarks Acknowledgements v

1 2 2 3 3 4 4 7 9 10 10 11 12 12 14 15 15 16 17 18 19 19

Contents

vi

2

Regulation of the ß3-Adrenoreceptor Signalling Efficacy

20

Michel Bouvier 2.1 2.2

2.3

2.4 2.5 2.6 2.7 2.8 3

Introduction 2.1.1 The ß-adrenoreceptors and their signalling pathways General concepts of G protein-coupled signalling regulation 2.2.1 Agonist-promoted receptor phosphorylation and receptor uncoupling 2.2.2 Receptor sequestration; a process involved in desensitization and resensitization 2.2.3 Agonist-promoted down-regulation The ß3-adrenoreceptor, a prototypic system to study subtype-specific regulation 2.3.1 Comparative analysis of the structural determinants of receptor regulation The ß3-adrenoreceptor is resistant to rapid agonist-promoted uncoupling and sequestration Cell type-specific down-regulation of the ß3-adrenoreceptor Use of chimeric ß3/ß2-adrenoreceptors to delineate regulatory motifs Potential physiological consequences of the relative resistance of the ß3-adrenoreceptor to desensitization Conclusions

Using Transgenic and Gene Knockout Techniques to Assess ß3Adrenoreceptor Function

20 21 22 22 24 26 27 27 29 30 33 34 35

36

Bradford B.Lowell, Vedrana S.Susulic, Danica Grujic and Moriko Ito 3.1 3.2

3.3

3.4

Introduction Mice lacking ß3AR 3.2.1 Phenotype of mice lacking ß3AR 3.2.2 Effects of ß3AR deficiency on catecholamine-mediated stimulation of adenylyl cyclase and lipolysis in adipocytes 3.2.3 Effects of ß3AR deficiency on in vivo effects of CL-316,243 Role of ß3AR on white versus brown adipocytes in mediating effects of ß3-selective agonists on energy expenditure, insulin secretion and food intake 3.3.1 Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on energy expenditure 3.3.2 Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on insulin secretion 3.3.3 Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on food intake Creation of mice which express human, but not murine, ß3AR

36 36 36 38 40

40 41 42 42 43

Contents

3.5

3.6

4

vii

Using ß3AR gene knockout mice to study ‘ß4AR’ activity 3.5.1 Evidence for the existence of ‘ß4AR’ 3.5.2 Assessment of ‘ß4AR’ activity in ß3AR gene knockout mice Conclusions Acknowledgements

ß3-Adrenoreceptor Ligands and the Pharmacology of the ß3-Adrenoreceptor

46 46 47 47 47

48

Jonathan R.S.Arch 4.1 4.2

4.3

4.4

4.5 5

Introduction Antagonists 4.2.1 ß1/2AR-selective antagonists 4.2.2 Non-selective and ß3AR-selective antagonists Agonists 4.3.1 ß1/2AR-selective agonists 4.3.2 Arylethanolamine ß3AR-selective agonists 4.3.3 Aryloxypropanolamine ß3AR agonists 4.3.4 Trimetoquinol and analogues 4.3.5 Metabolism and pharmacokinetics Idiosyncrasies of ß3AR pharmacology 4.4.1 Relative agonist potencies vary with the nature of the assay 4.4.2 Prediction of human tissue pharmacology from cloned receptor pharmacology Conclusions

The Native Human ß3-Adrenoreceptor

48 49 49 52 55 55 55 66 72 73 74 74 75 76 77

Peter Arner and Fredrik Lönnqvist 5.1 5.2 5.3

5.4 5.5 5.6 5.7

Introduction The human ß3AR gene Tissue expression of mRNA for ß3AR 5.3.1 White adipose tissue 5.3.2 Brown adipose tissue 5.3.3 Heart 5.3.4 Colon 5.3.5 Brain 5.3.6 Urinary bladder 5.3.7 Other tissues Is there a fourth ßAR? Structural variations in the native human ß3AR ß3AR as a therapeutic target Conclusions

77 77 78 78 80 81 81 81 81 82 82 83 84 85

Contents

viii

6

ß3-Adrenoreceptor-Mediated Responses in Heart and Vessels

87

Max Lafontan, Dominique Langin, Jean Galitzky, Michel Berlan, Chantal Gauthier and Geneviève Tavernier 6.1 6.2

6.3 6.4

6.5 6.6 7

Introduction ßAR subtypes in the heart 6.2.1 Functional ß3AR in the human heart 6.2.2 The putative ß4AR in cardiac tissue ß3AR-mediated vasodilatation ß3AR regulation of blood flow 6.4.1 Regulation of pancreatic islet blood flow 6.4.2 Regulation of gastric mucosal blood flow ß3AR-mediated cardiovascular effects Conclusions and future trends

ß3-Adrenoreceptors in Brown and White Adipocytes: Roles in Thermogenesis and Energy Balance

87 87 88 90 91 93 93 95 95 95

97

Jean Himms-Hagen 7.1 7.2

7.3

7.4

Introduction 97 Brown and white adipocytes and tissues 98 7.2.1 Functions of brown and white adipocytes 98 7.2.2 Definition and tissue distribution of brown and white adipocytes 99 7.2.3 Definition of BAT and WAT 101 7.2.4 Multiple UCPs in BAT 101 7.2.5 Adrenoreceptors in brown adipocytes 102 7.2.6 Control of brown adipocytes by the sympathetic nervous system and the hypothalamus 103 7.2.7 Central and peripheral effects of insulin in maintenance of thermogenic capacity of BAT 104 7.2.8 Control of blood flow in BAT by the sympathetic nervous system and the hypothalamus 105 Origin of brown adipocytes 105 7.3.1 Differentiation of brown adipocytes and mitochondriogenesis 105 7.3.2 Induction of hyperplasia of brown adipocytes in BAT 108 7.3.3 Expression of multiple UCPs in brown adipocytes: function and regulation 109 7.3.4 Induction of emergence of ‘ectopic’ brown adipocytes in WAT 109 7.3.5 Is there more than one type of brown adipocyte? 110 Physiological functions of brown adipose tissue 112 7.4.1 Role in thermoregulation in a cold environment 112 7.4.2 Role in food intake 113 7.4.3 Role in defence against obesity 113

Contents

7.5

7.6 7.7 8

ix

Secretions from brown adipocytes 7.5.1 Satiety factors 7.5.2 Other factors ß3ARs in brown and white adipocytes as a target for anti-obesity and anti-diabetes drugs Perspective

The Putative ‘ß4’-Adrenoreceptor and Other Atypical ß-Adrenoreceptors

115 115 116 117 119

120

A.Donny Strosberg and Jonathan R.S.Arch 8.1 8.2 8.3 8.4

Introduction Pharmacology of the ß4AR and other atypical ßARs A summary of ‘ß4’AR pharmacology The search for molecular evidence for atypical ßARs 8.4.1 The discovery of the ß3AR gene and other putative homologues 8.4.2 The discovery of a receptor which binds iodocyanopindolol and other ß3AR ligands 8.4.3 Non-ß3AR-like pharmacological properties of ß3AR proteins

References Index

120 120 121 123 123 123 123 125 167

Figures

1.1 1.2 1.3 1.4 1.5 2.1 3.1 3.2 3.3 3.4 3.5 3.6 3.7 4.1 4.2 4.3 4.4 4.5 6.1 6.2

Primary structure of the human ß3AR Comparison of the amino acid sequences of human, monkey, bovine, dog, rat, mouse, hamster, and guinea-pig ß3ARs A composite image of the ß3AR ligand binding region Signal transduction pathways activated by the human ß3AR Immunofluorescent staining of ß3AR in infected Sf9 cells Primary sequences of the ß2AR and ß3AR and schematic representation of their proposed membrane topology The ß3AR gene, targeting vectors and recombinant allele Adenylyl cyclase activity and lipolysis in response to CL-316,243 and isoproterenol Regulation of lipolysis in mouse adipocytes (a working model) Transgenic tissue-specific re-expression of murine ß3AR in knockout mice Transgenic expression of human ß3AR in knockout mice RNase protection analysis of human (HU) ß3AR gene expression in multiple tissues In vivo effects of CGP 12,177 on O2 consumption Chemical structures of selective antagonists of ß1- and ß2ARs Chemical structures of non-selective and ß3AR-selective antagonists Chemical structures of phenylethanolamine agonists Chemical structures of aryloxypropanolamines Chemical structures of trimetoquinols Effects of BRL-37,344 and SR-58,611 on the twitch tension of human endomyocardial biopsies Effect of the intravenous (i.v.) infusion (from 0 to 10min) of CL-316,243 (0.4 nmol/kg/min) on cutaneous blood flow in normal dogs, and effect of the i.v. infusion of BRL-37,344 (0.4 nmol/kg/min) on cutaneous blood flow in normal dogs before and after bupranolol administration x

5 8 11 13 18 28 37 39 40 41 44 44 45 50 53 60 66 73 89

92

6.3

7.1 7.2

Plasma insulin concentrations and islet blood flow, a percentage of pancreatic blood flow, in rats under basal conditions, after perfusion of CL-316,243 alone or in combination with an injection of bupranolol or nadolol Diagrammatic representation of the principal features that distinguish a brown adipocyte from a white adipocyte Use of ß3AR agonists to promote oxidation of excess fat stores

xi

94 99 118

Tables

1.1 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.1 5.2 5.3 5.4 8.1

Properties of the three human ß-adrenoreceptors Affinities (pA2 or pKB) of ß1/2AR-selective antagonists for ßARs determined from functional studies using rodent tissues Affinities of ß1/2AR-selective antagonists for human and rodent cloned ßARs Affinities of non-selective and ß3AR-selective antagonists for human and rodent ßARs Potencies (pD2, i.e. pEC50 values) of ßAR agonists in rodent tissues Binding affinities and functional potencies of ßAR agonists in human cloned ßARs ß3AR selectivities of esters or amides and related acids in animal tissue and human cloned receptors Comparison of the effects of BRL-26830/28410 and Ro-16–8714 on rat right atrial rate and in vivo heart rate Clinical experience with first-generation ß3AR agonists Relative potencies of agonists at the human cloned ß3AR vary with assay Human tissues expressing ß3AR Modulation of ß3AR lipolytic function in white human fat cells Phenotypes influenced by the Trp64Arg polymorphism in human ß3AR Possible clinical use of ß3AR agents Comparison of ß3AR and ‘ß4’AR pharmacology

xii

6 51 51 54 56 57 64 65 71 74 78 79 83 85 121

Contributors

Jonathan R.S.Arch is Director of Vascular Biology, SmithKline Beecham Pharmaceuticals, Coldharbour Road, Harlow, Essex CM19 5AD, United Kingdom Peter Arner is at the Department of Medicine Karolinska Institutet Sjukhus, Huddinge University Hospital, S-141 Huddinge, Sweden Michel Berlan is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Michel Bouvier is at the University of Montreal, Faculty of Medicine, Department of Biochemistry, Montreal, Quebec H3C 3J7, Canada Jean Galitzky is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Chantal Gauthier is at the Laboratoire de Physiopathologie et Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96–01 Hôpital Hôtel-Dieu and Faculté des Sciences et Techniques, Université de Nantes, 44093 Nantes Cedex, France Cindy G.Gerhardt is at Unilever Research Vlaardingen, Olivier van Noortlaan 3133AT, Vlaardingen, The Netherlands Danica Grujic is at Transkaryotic Therapeutics, 195 Albany St., Cambridge, MA 02139, USA Jean Himms-Hagen is at the Department of Biochemistry, University of Ottawa, Health Science Center, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada Moriko Ito is at Novartis, Basel, Switzerland Max Lafontan is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Dominique Langin is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Frederik Lönnqvist is at the Department of Medicine Karolinska Institutet Sjukhus, Huddinge University Hospital, S-141 Huddinge, Sweden xiii

xiv

Contributors

Bradford B.Lowell is at the Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA A.Donny Strosberg is at the Institut Cochin de Génétique Moleculaire, Laboratoire d’ImmunoPharmacologie Moléculaire, CNRS UPR 0415 and Université de Paris VII, 22, rue Méchain, 75014 Paris, France Vedrana S.Susulic is at the Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA Geneviève Tavernier is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France

Preface

Fifteen years have now passed since the first pharmacological description of an ‘atypical’ ß-adrenoreceptor, and nearly ten since the corresponding gene, renamed ‘ß3’ adrenoreceptor (ß3AR) was actually cloned. Hundreds of peer-reviewed publications and tens of patents have appeared describing various properties of the ß3AR or its ligands. Thanks to the lipolytic activity of ß3AR ligands, several of these compounds have now entered clinical trials. Moreover, genotyping for the presence of a polymorphism in the ß3AR gene, possibly associated with increased risk of developing diabetes and/or obesity, has become one of the possible targets for pharmacogenomic approaches to treat these growing health problems. The present monograph is centred entirely on the ß3AR, and has been assembled by several of the leading investigators in the field. The first chapter, which I wrote with Cindy Gerhardt, provides a general overview on the structure, function and expression of the ß3AR. The regulation of the receptor is discussed by Michel Bouvier, whereas the biological consequences of knocking-out the ß3AR gene are reviewed by the group of Brad Lowell. Jon Arch, the leading author of one of the very first articles outlining the properties of an ‘atypical’ ßAR, gives a detailed description of the pharmacology of the ß3AR. Peter Arner and Fredrik Lönnqvist present the functional role of ß3AR in humans, while Max Lafontan and colleagues venture into the less known role of the ß3AR in vasodilatation. Finally, Jean Himms-Hagen, a specialist on brown adipose tissue, discusses the biology of this tissue, in which the ß3AR plays a crucial functional role. Although all authors place the emphasis on the ß3AR, they do it in the context of the ß1AR and ß2AR homologues, as well as the more recently described, albeit still putative, fourth ßAR, which is also discussed separately in a last chapter by Jon Arch and myself. This book attempts to present a clear and complete description of the ß3AR, prepared by the original researchers that have been involved in the work since the beginning of the ß3AR history. The reader will certainly enjoy sharing how much has been learned about this receptor during the past 15 years. A.Donny Strosberg

xv

Abbreviations

AC ADA ADD AR BASH BAT cAMP C/EBPß CHO CHW CRE CREBP1 CYP GPCR CPT EDRF ERK FABP FFA FGF GLUT G protein GPCR GRE GRK HEK-293 ISO LH LPL

adenylyl cyclase adenosine deaminase adipocyte determination differentiation factor adrenoreceptor (subtype indicated by Greek letter) brown adipocyte satiety hormone brown adipose tissue cyclic adenosine 3’5'-monophosphate CCAAT enhancer binding protein Chinese hamster ovary Chinese hamster fibroblasts cyclic AMP response element CRE binding protein 1 Cyanopindolol G protein-coupled receptor I carnitine palmitoyltransferase I endothelium-derived relaxing factor extracellular regulated kinase fatty acid binding protein (M, muscle type; A, adipose type, also known as aP2) free fatty acids fibroblast growth factor glucose transporter guanine-nucleotide binding protein G protein-coupled receptor glucocorticoid response element G protein-coupled receptor kinase human embryonic kidney cells isoproterenol lateral hypothalamus, luteinizing hormone lipoprotein lipase xvii

xviii

mRNA NA NGF PIA PGC-1 PCR PKA PKB PI3K PPARE PPAR? PTX PVN RAR RARE RT-PCR RXR TR TNFa TRE UCP UCP-DTA VMH WAT

Abbreviations

messenger ribonucleic acid noradrenaline nerve growth factor phenylisopropyladenosine PPAR gamma coactivator-1 polymerase chain reaction protein kinase A protein kinase B phosphatidylinositol-3 kinase PPARß response element peroxisome proliferator activated receptor gamma pertussis toxin paraventricular nucleus of the hypothalamus retinoic acid receptor retinoic acid response element reverse transcriptase-polymerase chain reaction retinoid X receptor thyroid hormone receptor tumour necrosis factor a thyroid response element uncoupling protein (isoform indicated by number) term used to describe transgenic mice expressing diphtheria toxin A chain under the control of the UCP-1 promoter ventromedial nucleus of the hypothalamus white adipose tissue

1

Structure and Function of the ß3-Adrenoreceptor A.DONNY STROSBERG 1 AND CINDY C.GERHARDT2 Institut Cochin de Génétique Moléculaire—Laboratoire d’Immuno-Pharmacologie Moléculaire, CNRS UPR 0415 and Université de Paris VII, 22, rue Méchain, 75014 Paris, France 2 Unilever Research Vlaardingen, Biotechnologie A1260, Oliver van Noortlaan 120–3133AT, Vlaardingen, The Netherlands 1

1.1

Introduction

The neuroendocrine, metabolic and other effects of catecholamines such as adrenaline and noradrenaline are mediated by a family of membrane-bound intrinsic proteins known as the ‘adrenergic’ receptors, more recently renamed as ‘adrenoreceptors’ or ‘adrenoceptors’. The natural biogenic amines modulate various cellular signal transduction mechanisms by binding to these receptors and thus activating GTP-binding regulatory G proteins which, in turn, stimulate or inhibit effectors such as adenylate cyclase, phospholipase C, other enzymes or even ion channels. The adrenoreceptors share general structural features with members of a very large family of single polypeptide chain membrane receptors all known to display seven transmembrane hydrophobic regions, and all presumed to be coupled to G proteins. This family includes receptors for other biogenic amines such as dopamine, serotonin, acetylcholine, other small ligands, peptides and even sensory stimuli such as light, olfactory or gustatory substances. It is one of the receptors for light, rhodopsin, which was the first one to be characterized in great detail both in terms of structure, and in terms of coupling to transducin and, through this G protein, to the effector: phosphodiesterase (for reviews, see Strosberg, 1993; Trends in Pharmacological Science, Annual Supplement 1998, 9th edition, S.P.H.Alexander and J.A.Peters, Eds). From the outset, adrenoreceptors (AR) were subdivided into
AR and ßAR subclasses, on the basis of relative potency for catecholamines and isoproterenol, which has no effect on
AR. Later, these receptors were further subdivided into
1AR,
2AR subclasses, whereas ßAR were subdivided into ß1AR and ß2AR (Lands et al., 1967a,b). Today, nine subtypes have been recognized:
1A,B,C,
2A,B,C, ß1, ß2, ß3 subdivisions of ßAR. Subdivision of ßAR into ß1AR and ß2AR was made on the following basis: ß1AR is equally sensitive to noradrenaline and adrenaline, and ß2AR is more sensitive to adrenaline (Lands et al., 1967a,b). Convincing pharmacological evidence for the existence of additional, or atypical, ßARs stems from the early 1980s (Arch et al., 1984a; Wilson et al., 1984; see Chapter 4). Five years later, a gene encoding what was designated as the ß3AR was cloned 1

Strosberg and Gerhardt

2

(Emorine et al., 1989) by low stringency-screening of a human genomic library with a ß2AR probe. Homologous ß3AR were cloned subsequently from rodents and other mammals. Pharmacological characterization of these ß3AR have however not completely reconciled all the data on atypical ßAR, and periodically new studies describe atypical non-ß3AR or ß4AR. For the sake of clarity, we discuss these ß4AR and possibly other atypical ßAR in Chapter 8. The availability of the sequence of the ß3AR for several different species, has considerably increased our knowledge about this receptor. Heterologous expression of the ß3AR in cell lines that do not express ß1AR or ß2ARs, has facilitated pharmacological studies and revealed interesting species-related differences. Studies using mutated or chimeric ß3ARs have pinpointed the regions involved in ligand recognition and signal transduction. In addition, molecular biological tools were used to localize transcripts encoding ß3ARs in different tissues. In this chapter, we provide an introductory overview on the ß3AR that is strongly orientated on molecular biological studies. We describe the structure of the gene and its promoter, and discuss interesting features in the ß 3 AR protein. ß 3 AR-specific characteristics, either in comparison with the ß1AR and the ß2AR, or with species homologues, are also outlined. We then summarize the current knowledge on structurefunction relationships of the ß3AR. The signals that are activated in response to ß3AR activation, and the biological role that these may play are subsequently described. We finish this chapter with a description of the distribution of the ß 3AR. Wherever appropriate, we refer to further discussions in the other chapters of this volume.

1.2

The ß3AR gene

The development of alprenolol-agarose based affinity chromatography, used first for the purification of the turkey ßAR (Vauquelin et al., 1977, 1979), led to the isolation of the hamster ß2AR and the partial sequencing of several cyanogen bromide-fragmented peptides by Benovic et al. (1984). Screening of a hamster genomic library with oligonucleotides corresponding to these peptide sequences led to the isolation of a single continous open reading frame encoding the hamster ß2AR (Dixon et al., 1986). Analysis of the corresponding predicted protein sequence revealed a significant similarity with the previously described bovine rhodopsin, as well as with bacterio-rhodopsin, especially in terms of the presence of seven hydrophobic stretches each of about 25 amino acid residues. The homology with the rhodopsins, that are coupled to the G protein transducin, prompted the hypothesis that other G protein-coupled receptors might have similar structural properties. Indeed, soon after the cloning of hamster ß2AR (Dixon et al., 1986), the ß1AR-like ßAR from turkey (Yarden et al., 1986) and the human ß1AR (Frielle et al., 1987) were cloned, and confirmed a general topology of G protein-coupled receptors.

1.2.1 Cloning of the gene The human ß3AR was isolated by screening a human genomic library with the entire coding sequences of the genes encoding the turkey ß 1AR and human ß2AR used as probes (Emorine et al., 1989). Upon expression of the open reading frame in CHO

Structure and function of the ß 3-adrenoreceptor

3

cells, it transpired that this third human ßAR subtype, designated as ‘ß 3 AR’, exhibited a number of the pharmacological properties previously described for ‘atypical’ ßARs mainly expressed in adipose tissues of rodents (Arch et al., 1984a; see Chapter 4). From that moment on, this adipocyte-specific atypical ßAR became gradually but widely accepted as the ß3AR. Brad Lowell in Chapter 3, and Strosberg and Arch in Chapter 8 discuss the possible existence of yet more atypical receptors, including the ß 4AR.

1.2.2

Structure of the coding region

The coding region of the human ß3AR gene contains 1224 nucleotides which putatively encode a 408-amino acid residues protein (Emorine et al., 1989; van Spronsen et al., 1993). Comparison of the amino acid sequences predicted from the genomic nucleotide sequences and from the corresponding cDNA sequences revealed however unexpected differences in the length and sequence of the carboxy-terminal regions of the ß3 receptors. Indeed, in contrast to the homologous ß1- and ß2ARs, the ß3AR-coding region was found to be present on two different exons, both in man as in mouse and rat (Bensaid et al., 1993; Granneman et al., 1993; van Spronsen et al., 1993). A first exon of 1.4kb contains the coding sequence for the first 402 and 388 amino acid residues in man and mouse or rat, respectively. In the human ß3AR gene, a second exon of 700 bp contains the sequence coding for the six carboxy-terminal residues of the receptor and the entire mRNA 3' untranslated region. In mouse and rat, a second exon of 68 bp codes for the 12 carboxy-terminal residues of the receptor, while a third exon contains the 3' untranslated region of the ß3AR mRNA. Until today, no good evidence existed for distinct functional roles for differential splice forms generating different protein variants. However, different mRNA lengths exist due to the use of widely separated promoters (Granneman and Lahners, 1994) and/ or the use of alternate acceptor splice sites in exon 3 in rodents (van Spronsen et al., 1993). In addition, human ß3AR transcripts with different 3' untranslated regions are produced by continuation of transcription beyond termination signals (van Spronsen et al., 1993) or result from the use of alternative polyadenylation signals (Granneman and Lahners, 1994).

1.2.3

Structure of the promoter

The study of the promoters of the human and murine ß3AR genes led to the identification of several potentially important sequences. Transcription of the rat ß3AR starts mainly at position -161 relative to translation initiation ATG, whereas minor transcription begins around -123 and -109. Interestingly, almost all of the (few) ß3AR messenger molecules present in the rat gastric fundus were found to start at the -123 and -109 sites (Granneman and Lahners, 1994). In human brown fat and neuroepithelioma cells, the vast majority (80%) of ß3AR transcripts begin at multiple sites around -130 (Granneman and Lahners, 1994). Motifs potentially implicated in heterologous regulation of the murine ß3AR expression by glucocorticoids (Fève et al., 1992), butyrate (Krief et al., 1993), phorbol esters (Fève et al., 1995; El Hadri et al., 1998) and insulin (Fève et al., 1994; El Hadri et al., 1998) were identified upstream of the cap sites. Four potential cyclic AMP response elements (CREs) exist in the 5' flanking region

Strosberg and Gerhardt

4

of the human ß3AR gene. Three of these CREs were reported to contribute to the agonistinduced up-regulation of the human ß 3 gene expression (Thomas et al., 1992). Interestingly, no CRE was identified in the rodent ß3AR gene, which, in contrast to the human gene, was shown to be down-regulated by agonist treatment (Granneman and Lahners, 1992, 1994; Granneman, 1995).

1.3

The ß3AR protein

The human ß3AR, as well as the ß1- and ß2ARs, belongs to the now well-established superfamily of G protein-coupled receptors. As such, it is composed of a single, 408 amino acids long peptide chain which is thought to traverse the membrane seven times. Each of those transmembrane (TM) regions is constituted of a hydrophobic stretch of about 22 to 28 residues (Figure 1.1). The extracellular, amino-terminal region of G protein-coupled receptors is of variable length and contains almost always glycosylation sites. Amino acid sequencing of the amino-terminal region of the ß3AR, immunopurified from solubilized membranes of CHO cells overexpressing the ß3AR, confirmed the sequence predicted on the basis of the DNA sequence (Guillaume et al., 1994). The Asn residue at position 7, a putative target for Asn-linked glycosylation (Asn-Ser-Ser), could not be identified in the amino acid sequence, suggesting that it is indeed glycosylated. The intracellular domains of G protein-coupled receptors often contain consensus phosphorylation sites. Receptor phosphorylation by protein kinase A or C (PKA/PKC) or G protein-coupled receptor kinase (GRK; formerly named ß-adrenergic receptor kinase, ßARK), underlies receptor desensitization by regulating for example G protein coupling or receptor internalization. Interestingly, the intracellular domains of the ß3AR do not contain any potential sites for phosphorylation by either PKA or PKC, and contain only one putative phosphorylation site for GRK (compared with eight in the ß2AR). Consequently, the ß3AR (in contrast to the ß2AR) is resistant to rapid agonist-promoted desensitization, both in transfected cells (Liggett et al., 1993; Nantel et al., 1993) and in rat adipocytes (Granneman et al., 1992) that express ß 3ARs in an endogenous environment. The regulation of ß3AR-mediated signalling is discussed in more detail in Chapter 2.

1.3.1

Comparison with ß1- and ß2AR

Although the three ßAR subtypes share a certain functional homology (e.g. their stimulation leads to the activation of adenylyl cyclase), they all play their own specific physiological role, as summarized in Table 1.1. Indeed, the structural homology between the ß1AR, ß2AR and ß3AR is restricted; the subtypes differ in length both at the N-terminus and at the C-terminus, and the number of residues conserved between the three receptors is limited. As within all members of the superfamily of G protein-coupled receptors, the conserved residues are almost exclusively restricted to the seven TM segments and the membrane-proximal regions of the intracellular loops (Figure 1.1). Site-directed mutagenesis (Guan et al., 1995; Gros et al., 1998) and studies using chimeric ß2/ß3ARs (Nantel et al., 1993, 1994; Jockers et al., 1996) support the conclusion that the conserved parts are involved in ligand binding and G

Figure 1.1 Primary structurte of the human ß3AR. The sequences are represented in the one-letter code for amino acids. The single polypeeptide chain is arranged according to the model for rhodopsin. The disulphide bond essential for Cys110 and Cys189 activity is represented by -S-S-. The two N-glycosylation sites in the amino-terminal portion of the protien are indicated by Y. palmitoylated Cys360 residue in the N-terminus of the i4 loop is indicated by a. Residues in black cicles are common to the three ßAR subtypes. The underlined sequence RSSPAQPRLCQRLDG corresponds to the synthetic peptyde which was used to obtain the anti C-terminal antibodies reacting with the whole ß3AR.

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Table 1.1 Properties of the three human ß-adrenoreceptors

CRE, cyclic AMP response element; GCE, glucocorticoid response element. *CL-316,243 is most selective for rodent ß3AR. Merck L-755,507 is most selective for human ß3AR (Parmee et al., 1998) **Bupranolol is not selective, but is the strongest known ß3AR antagonist. SR-59,230A is not selective but has been recognized as a good antagonist for human ß3AR expressed in several tissues, but not in model systems (De Ponti et al., 1996; Nisoli et al., 1996a)

protein interaction, respectively. The ß3AR possesses a number of pharmacological properties that distinguish it from the ß1AR and ß2AR. It has a higher affinity for noradrenaline than for adrenaline, while the ß2AR has a higher affinity for adrenaline, and the ß1AR binds both ligands equally well. In addition, several ligands that act as antagonists on ß1ARs and ß2ARs, act as agonists (e.g. bucindolol, ICI-201651, carazolol, CL-316,243) or partial agonists (e.g. CGP-12177, (cyano-)pindolol) on the ß3AR. For a detailed description of the pharmacological profile of the ß3AR, the reader is referred to Chapter 4. Recent studies by Granneman et al. (1998) using chimeric and mutant rat ß1and ß3ARs have indicated the presence of (at least partially) distinct binding sites for ß3AR-selective phenylethanolamines and catecholamines in the seventh TM domain. The findings on structure-function relationships of the ß3AR are discussed in more detail in Section 1.4. G protein-coupled receptors often contain cysteine residues in their second and third extracellular loops that can form disulphide bonds to stabilize the receptor conformation. All three ßAR subtypes contain one cysteine residue in their second extracellular loop, and three cysteine residues in their third extracellular loop. As was shown for the ß1- and the ß2AR, the cysteine residues in the human ß3AR are likely to

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form a functional disulphide bond. Reduction of the receptor by dithiothreitol inactivates 45% of the [125I] ICYP binding sites, while preincubation with certain ligands protects the receptor from dithiothreitol-induced inactivation (Méjean-Galzi et al., 1995). Interestingly, this protection was not induced by the agonists adrenaline, isoproterenol or BRL-37,344, while it was induced by the agonist carazolol as well as by the antagonists CGP-20712A and ICI-118551. In this respect, the human ß3AR differs from the human ß2AR, which was (in the same study) found to be protected by all ligands. Whereas the ß2AR contains two PKA target sites in its third intracellular domain and its C-terminus, none is observed in the ß3AR. Similarly, whereas the ß2AR has eight potential GRK sites in its C-terminus, the ß3AR has only one. Furthermore, the ß 2AR possesses two PKC sites and tyrosine residues involved in heterologous desensitization (Bouvier et al., 1991) and down-regulation (Valiquette et al., 1990, 1993), respectively, that are absent in the ß3AR. As described above, and discussed in more detail in Chapter 2, this has clear consequences for the desensitization properties of the ß3AR. A very low degree of homology is found in the distal C-terminal region, either within ßAR subtypes or within ß3AR species variants, and it remains to be determined whether this domain exerts a specific function. No functional differences have been found upon expression of ß3AR receptors with or without the last six (in man) or twelve (in rodent) C-terminal residues (Granneman et al., 1993; Nantel et al., 1993, 1994). Interestingly, it was reported recently that the last few residues of the C-terminal domain of the ß2AR interact directly, independently of G proteins, with a Na+/H+ exchanger regulatory factor (Hall et al., 1998). However, since this sequence is very different in the ß3AR, a similar interaction seems unlikely.

1.3.2

Comparison of ß3AR from various species

Today, the sequence of the ß3AR has been determined in human (Emorine et al., 1989), mouse (Nahmias et al., 1991), rat (Granneman et al., 1991), bovine (Piétri-Rouxel et al., 1995), guinea-pig (Atgié et al., 1996), monkey (Walston et al., 1997), dog (Lenzen et al., 1998) and hamster (S.Baude et al., unpublished results). The degree of amino acid sequence homology (Figure 1.2) between the ß3ARs from these species is considerably higher (80% to 90%) than that calculated between different subtypes (40% to 50%), a feature generally observed for receptor subtype homologies across species. Several ‘ß3-specific’ residues are shared between all partial or complete ß3 sequences analysed so far, and are not found in ß1 or ß2AR sequences (Figure 1.2). Furthermore, the human, monkey, bovine and dog ß3AR are closer to each other than to any of the rodent (rat, mouse, hamster) sequences, in particular in TM1, where a (ValLeu-Ala) deletion is observed in the rat, mouse and hamster, but not in the larger mammals. When the human ß3AR is compared with its animal counterparts, several residues are found to be unique to man (Figure 1.2). For instance, in front of TM4 and TM6 one finds a cysteine residue in the human ß3AR, while in bovine, dog, mouse, rat, guineapig and hamster and monkey, an arginine residue is present. It remains to be elucidated whether these cysteine residues play a specific role in the human ß3AR. Considerable differences in ligand binding and adenylyl cyclase activation constants

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Figure 1.2 Comparison of the amino acid sequences of human, monkey, bovine, dog, rat, mouse, hamster, and guinea-pig ß3ARs. To maximize homologies, gaps represented by empty spaces have been introduced in four sequences. The seven presumed transmembrane domains (TM1–TM7) are boxed and are separated by extracellular (E1 to E4) and intracellular (I1 to I4) loops. In the aminoterminal region, consensus sequences for asparagine-linked glycosylation site (N-X-S) are highlighted by bars. The presumed palmitoylated Cys361 is noted with a grey box. Residues marked with a dot are common to human ß1- and ß2-adrenoreceptors and to all ß3ARs analysed so far.

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have been reported when a variety of ß3AR agonists were evaluated on different species variants of the ß3AR expressed in CHO cells. For example, BRL-37,344 and CL-316,243 have much higher efficacies on rodent ß3AR than on human ß3AR. Also bupranolol, a non-selective ßAR antagonist on human and rodent ß3ARs, was found to be a partial agonist on the bovine ß3AR (Piétri-Rouxel et al., 1995). Similarly, propranolol acts as an antagonist on the mouse ß3AR, while it has partial agonist activity on the human receptor (Blin et al., 1993). Carazolol has a ten-fold higher affinity for the human ß3AR compared with the mouse ß3AR; however it has the same efficacy and potency to stimulate adenylyl cyclase, maybe because the mouse ß3AR couples to Gs (G-protein) more efficiently (Blin et al., 1994). Indeed, many reports have shown that stimulation of the human ß3AR in adipocytes stimulates adenylyl cyclase activity less efficiently than does the rodent ß3AR. It was shown recently that stimulation of the dog ß3AR hardly increases intracellular cAMP concentrations upon expression in hamster CHO/K1 cells, while it does efficiently activate adenylyl cyclase in human HEK 293 cells. Apparently, the coupling of the hamster Gs to the canine ß3AR is highly sensitive to the cellular background.

1.3.3 Polymorphism In the human ß3AR, a tryptophan residue is present at position 64 (just outside TM1), whereas in all other species an arginine (or a cysteine in guinea-pig) is found (Atgié et al., 1996). Interestingly, a polymorphism has been described (Clément et al., 1995; Walston et al., 1995; Widen et al., 1995; Strosberg and Piétri-Rouxel, 1997) that is present in about 10% to 50% of the population, and that ‘restores’ the arginine residue present in most animals. In some groups of patients, as in the initially described Pima Indians (Walston et al., 1995) but also in Japanese, Swedish, Finnish or French obese individuals, this polymorphism was found to be associated with morbid obesity and insulin resistance. However, in other cohorts no correlation could be found (for reviews, see Strosberg, 1997; Allison et al., 1998; Fujisawa et al., 1998). An increasingly likely explanation for the apparent discrepancies between the various studies could be found in the association between various alleles if different genes are implicated in the genetic control of metabolism (Strosberg, 1997). It was thus observed that the human population displays at least two alleles of UCP 1 gene, one in 65%, the other in 35% of individuals. Homozygotes for the less abundant allele seem to have an increased tendency to accumulate weight (Ricquier and Bouillaud, 1997; Gagnon, J. et al., 1998b). Individuals that bear both the Arg64 allele and the A-3826G region allele of UCP 1 have an even higher tendency towards obesity. Proenza et al. (2000) have now started to investigate various sets of alleles, to uncover genotypes predisposing humans to obesity. The recent discovery of polymorphisms in gene for MC4 or PPARg (Ristow et al., 1998) suggests that more genotypes will be uncovered in the future. The massive search for Single Nucleotide Polymorphism or ‘SNIPs’ by the pharmaceutical and biotechnology industry will soon replace the gene-by-gene approach followed so far by academic laboratories, and should therefore help define obesity-associated genotypes. Today, it is still unknown how the Arg64 mutation exactly affects adipocyte biology. To evaluate the effect of the tryptophan?arginine (W64R) substitution on receptor function in vitro, the ß3AR-Arg64 was expressed at various levels in model cells. Compared with the ß3AR-Arg64, no difference was observed in ligand binding or

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adenylyl cyclase activation constants (Candelore et al., 1996; Piétri-Rouxel et al., 1997). However, a consistent reduction was observed in the maximal agonist-stimulated cAMP concentration, both in CHO and in HEK293 cells (Piétri-Rouxel et al., 1997). The effect of the mutation on ERK kinase activity (Gerhardt et al., 1999) is currently being investigated. Lipolytic activity of the ß3AR in isolated visceral fat cells of heterozygote ß3AR-Arg64 carriers was not found to be significantly different from that of ß3AR-Trp64 controls in a first study by Li et al. (1996). A more recent similar study performed by the same group (Hoffstedt et al., 1997) on a larger number of the same Swedish population has however correlated the occurrence of the Arg64 allele with a ten-fold reduction of lipolysis in omental adipose tissue. In contrast, in homozygous individuals of the Pima Indian population, no differences were found in terms of in vivo lipolysis of subcutaneous adipose tissue (Snitker et al., 1997).

1.4

1.4.1

Structure-function relationships in the ß3AR

The ligand binding site

The putative ligand binding site of the ß3AR has been described on the basis of computer modelling combined with data obtained by site-directed mutagenesis and photoaffinity labelling of the ß3AR, the ß2AR or other related receptors (Figure 1.3). The following residues are considered to be most important for ligand binding: 1 Asp117 in TM3 is highly conserved in all G protein-coupled receptors for biogenic amines, and it was found to be essential for binding of these ligands. The acidic sidechain most likely forms a salt bridge with the basic group of the ligand. Indeed, substitution of this residue by a leucine residue in the human ß3AR completely suppresses agonist binding (Gros et al., 1998). 2 Ser169 in TM4 is thought by certain authors to form a hydrogen bond with the hydroxyl group of the ethanolamine side-chain (Strader et al., 1989), whereas others contest a role for this residue. 3 Ser209 and Ser212 in TM5 are also found (or substituted by Thr) in many biogenic amine receptors, and are thought to form hydrogen bonds with the hydroxyl groups of the catechol moiety. 4 Phe309 in TM6 is thought to be involved in a hydrophobic interaction with the aromatic ring of the catecholamine. Beside these residues, many other residues may play more subtle roles in the interactions between the ß3AR and (certain of) its ligands. Recent results produced by Granneman et al. (1998) have thus pinpointed two residues in TM7 to be critical for receptor activation by ß 3 AR agonists such as BRL-37,344 and CL-316,243. Replacement of Phe350 and Phe351 in TM7 of the ß1AR for Ala and Leu residues, normally present in the ß3AR, is sufficient to allow activation of the mutated ß1AR by ß3AR agonists. These residues seem to be specifically involved in binding of the ß3AR agonists, since neither the activity of catecholamines, nor the antagonism by propranolol, were affected by the substitutions. It is still not understood why several ß1/ß2AR antagonists (e.g. ICI-201651, carazolol,

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Figure 1.3 A composite image of the ß3AR ligand binding region. Proposed interactions in the ligand binding region of the ßAR viewed from the outside of the cell. The ligand noradrenaline is shown surrounded by several of the amino acids involved in agonist binding. These are Asp117 in TM3, Ser209 and Ser212 in TM5, and Tyr336 in TM7. The essential disulphide bond (-SS-) linking Cys110 (E2) and Cys189 (E3) is also represented. Asp83 (TM2), not represented here, is likely to be more important for signal transmission to Gs than for actual ligand binding, in which it is nevertheless involved.

CL-316,243, CGP-12177) behave as ß3AR agonists. Based on computer modelling studies, the ligand binding site of the ß3AR contains less bulky amino acid residues than that of the ß1/ß2ARs, and could therefore could more easily accommodate the larger ß1/ ß2AR antagonists (Blin et al., 1993, 1994, 1995; Strosberg et al., 1993). However, substitution of the small Gly53 for a bulky Phe (present at this position in the human ß2AR) in the human ß3AR turned out not to be sufficient to convert the ß3 agonists into antagonists (Gros et al., 1998). In addition, the molecular basis for the pharmacological differences between human and rodent ß3ARs is not yet known. The Val-Leu-Ala tripeptide, present in rodent ß3ARs but absent in the human or other large mammalian ß3AR, does not seem to be involved in this species-specific difference (Gros et al., 1998).

1.4.2

The regions of interaction with the G proteins

As is now known from a number of mutagenesis studies on different G protein-coupled receptors, the site of receptor-G protein coupling is situated in the membrane-proximal regions of the second and third intracellular loops, as well as in the C-terminal domain of the receptor. Deletion mutagenesis of the third intracellular loop of the ß3AR confirmed that the ß3AR constitutes no exception: deletion of eight amino-terminal and 13 carboxy-terminal

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residues from the
s subunit of the Gs protein led to functional uncoupling of the human receptor ß3AR from adenylyl cyclase (Guan et al., 1995). The site of interaction with the G protein seems to be dictated by its position rather than by its primary amino acid sequence. For example, the G protein-binding site of the ß2AR is located at the same position as in the ß3AR, but the sequence homology in these regions is only 63% at the amino-terminal and 31% at the carboxy-terminal end of the third intracellular loop. The different primary sequences may allow the receptor to couple preferentially to different subtypes of Gs protein subunits, although today no data are available on the ß 3AR specificity concerning certain combinations of Ga /ß/ g subunits. In addition to a direct, physical interaction between the receptor and a G protein, an independent process of G protein activation is necessary. It has been suggested that Asn312 in TM6 of the human ß3AR plays a role in activation of adenylyl cyclase by certain agonists (Gros et al., 1998). Thus, whereas no differences were observed in intrinsic activities of noradrenaline or CGP-12177, nadolol and tertatolol (ß3AR agonists and ß1/2AR antagonists) displayed a decreased intrinsic activity for the mutant receptor, without any change in binding affinity. With the increasing evidence of functional coupling between the ß 3AR and Gi or Go proteins (Gerhardt et al., 1999) it now becomes necessary also to investigate sites of interaction of the receptor with these other G proteins. This has not yet been attempted.

1.5

Signal transduction and biological functions

Like the ß1- and ß2ARs, the ß3AR preferentially couples to Gs in order to stimulate adenylyl cyclase, PKA and subsequent target proteins such as the hormone-sensitive lipase in adipocytes. Treatment with ß3AR agonists leads to increased concentrations of intracellular cAMP, both in transfected cell lines as well as in tissues that endogenously express ß3ARs. Below, we present a short overview on the Gs-mediated signalling by the ß3AR. For a more detailed description of this subject, the reader is referred to Chapter 2. More recently, it has become clear that the ß3AR may also modulate the activity of other signalling pathways. For example, it can lead to changes in intracellular calcium concentration, and it can modulate the activity of MAPK and PI3K (Figure 1.4). We discuss these findings in more detail, and place them in the context of the role that the ß3AR may play in adipocyte differentiation.

1.5.1 ß3AR-mediated activation of adenylyl cyclase In rodent adipocytes, ß3AR agonists potently activate adenylyl cyclase, PKA and hormone-sensitive lipase, resulting in lipolysis (e.g. Shih and Taberner, 1995). cAMP-activated lipolysis via the ß 3AR has also been well characterized in the murine 3T3-F442A pre-adipocyte cell line (Fève et al., 1991). In addition to the lipolytic activity of ß3AR agonists, these agents inhibit insulin-stimulated glucose transport. This inhibition results from a cAMP-dependent phosphorylation and subsequent loss of intrinsic acitivity of GLUT4 transporters (Carpéné et al., 1993a,b).

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Figure 1.4 Signal transduction pathways activated by the human ß3AR. Schematic model for intracellular signalling pathways activated upon stimulation of the ß3AR. The model is based upon studies with CHO cells, stably expressing human ß3AR, in which a coupling of the ß3AR to both Gs and Gi was established. Biological effectors and consequences that play an important role in adipocyte biology (lipolysis, thermogenesis, adipogenesis) are tentatively included into the scheme.

Stimulation with ß3AR agonist can also lead to changes in gene expression via the PKA-mediated phosphorylation of CREB. In addition to the well-documented transcriptional activation of UCP-1 expression (e.g. Champigny and Ricquier, 1996) the expression of other genes such as LPL (Kuusela et al., 1997a,b), members of the C/EBP family (Rehnmark et al., 1993) or a1ARs is up-regulated by ß3AR agonists. Stimulation of the ß3AR can furthermore lead to inhibition of gene expression, as for example the satiety hormone, leptin (Trayhurn et al., 1996). In contrast to the efficient coupling of the ß3AR to adenylyl cyclase in rodents, diverging results have been obtained on functional effects of ß3AR agonists in human tissues. Even when ß3AR is present (as determined by RT-PCR and binding studies), the increase in cAMP and the stimulation of lipolysis may be quite small (Zilberfarb et al., 1997). As outlined below, increasing evidence reveals the coupling of the ß3AR to other G proteins. It remains to be determined whether the human ß3AR, in contrast to its rodent homologue, couples preferentially to these ‘new pathways’. Interestingly, upon heterologous expression in either rodent or human cells, the human ß3AR efficiently couples to adenylyl cyclase. This may be due to different expression levels of the receptor, increased presence of G protein (subtypes), or adenylyl cyclase (subtypes) in fibroblasts as compared with adipocytes. Expression of the ß3AR in transfected model cells has enabled detailed functional pharmacological analyses, without interference from ß 1AR or ß 2AR. The ß 3ARmediated increase in cAMP concentration can be blocked by non-selective ßAR antagonists such as bupranolol, or by high concentrations of ß1- or ß 2-selective antagonists such as ICI-118,551 or CGP-20712A. SR-59230A was introduced as a selective ß3AR antagonist, but although this agent efficiently blocks endogenously

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expressed ß3AR (Nisoli et al., 1996a,b), it was found to act as a partial agonist in transfected cell lines (Strosberg and Piétri-Rouxel, 1997). Clearly, care should be taken when transfected cell systems are used to determine agonist/antagonist properties. Not only the number of receptors expressed, but also the assay methodology may change the ligand potency and intrinsic activity. Indeed, it has been shown that with increasing numbers of ß3AR expressed in CHO cells, the potency and intrinsic activity of a range of agonists also increased (Wilson et al., 1996). Moreover, the potency for example of noradrenaline was found to depend strongly on how the activity of adenylyl cyclase was determined.

1.5.2 ß3AR-mediated activation of other signalling pathways Several reports have described ß3AR-mediated responses that are sensitive to pertussis toxin (PTX), suggesting that this receptor can also couple to G proteins of the Gi/o class. Chaudry et al. (1992) thus described the simultaneous coupling of the ß3AR (but not the ß1AR) to either Gs or Gi in rat adipocytes. In intact adipocytes, PTX treatment significantly increased BRL-37,344-stimulated cAMP accumulation, indicating that Gi activation limits ß3AR-stimulated cAMP accumulation. Also in isolated membranes, concentrations of GTP that have been shown to activate Gi proteins, inhibited the ß3AR(but not the ß1AR-) mediated stimulation of adenylyl cyclase, again suggesting that ß3AR can interact with Gi proteins. In addition, Gauthier et al. (1996) described that treatment of isolated preparations of human ventricle with ß3AR agonists results in PTX-sensitive negative inotropic effects and reduction of the amplitude and duration of action potentials. In a followup of this study, it was shown that the negative inotropic effect was mediated via the increase in the production of nitric oxide and cGMP, indicating a ß3AR-mediated (and PTX-sensitive) activation of nitric oxide synthase in the human ventricle (Gauthier et al., 1998). It has also been reported (Seydoux et al., 1996) that isoproterenol induces an increase in intracellular calcium concentration via stimulation of both ß1-, ß2- and ß3ARs in human white adipocytes. However, this work did not address the question of the underlying mechanism of the coupling of the ß3AR to Gs, Gi/o or Gq. The pharmacological properties of the human ß3AR have been found to vary with the coupled G protein. The order of potency and intrinsic activities of both natural ligands, noradrenaline and adrenaline, is inverted between the G s -mediated activation of adenylyl cyclase, and the G i/o-mediated activation of extracellular regulated kinase (ERK)1/2 (Gerhardt et al., 1999; see also below). In addition, BRL37,344 and propranolol act as agonists in the stimulation of adenylyl cyclase, but as antagonists in the activation of ERK1/2. As was already mentioned above, these results highlight the fact that pharmacological profiles may be highly dependent not only on cellular background, but also on the signal that follows receptor activation. Although PTX similarly inhibits G
i- and G
o-subunits, and thus cannot be used to discriminate between these two G proteins, indirect evidence exists that the ß3AR couples to G o rather than to G i. In fact, preincubation with PTX of the ß 3ARexpressing CHO cells which display a functional PTX-sensitive coupling (Gerhardt et al., 1999) does not influence the positive coupling to adenylyl cyclase (PiétriRouxel et al., 1997).

Structure and function of the ß 3-adrenoreceptor

1.5.3

15

A role for the ß3AR in adipocyte differentiation

In addition to the above-mentioned cases, in which the ß 3 AR is expressed endogenously, the human ß3AR expressed in CHO cells can also couple to both Gs and Gi/o (Gerhardt et al., 1999). In these cells, the coupling of the ß3AR to Gi/o leads to the activation of PI3K, and, subsequently, to the activation of protein kinase B (PKB) and ERK1/2 (Figure 1.4). The ß3AR-mediated activation of ERK 1/2 and PKB has also been observed in cells that express the ß3AR endogenously, albeit by different mechanisms. Shimizu et al. (1997) reported that stimulation of the ß3AR (with BRL-37,344) expressed in rat adipocytes, leads to activation of ERK 1/2, but in a way mediated by the increase in cAMP, and independent of PI3K. In addition, it has been shown that ß3AR agonists can activate PKB in rat epididymal fat cells. The mechanism underlying these effects remains to be elucidated, but it seems to be independent of PI3K and cAMP accumulation (Moule et al., 1997). Since the effect of PTX was not investigated, no conclusions can be drawn about the involvement of Gi/o proteins. The activities of ERK1/2 (Sale et al., 1995), as well as of PI3K (Christoffersen et al., 1998) and PKB (Magun et al., 1996), play a crucial role in adipogenesis. Therefore, the ß3AR-mediated activation of ERK 1/2 and PKB may well form the basis for the frequent observations that treatment with ß3AR agonists, in vitro as well as in vivo, results in considerable expansion of brown adipocyte populations. For example, chronic treatment of rats with ß3 agonists leads to the appearance of brown adipocytes in white fat depots, leading to an increase in resting metabolic rate in these cells (Arbeeny et al., 1995; de Souza et al., 1997; Ghorbani et al., 1997a). Similarly, in man, brown fat reappears around catecholamine-secreting phaeochromocytoma tumours. Also in primary cultures of mouse brown pre-adipocytes, differentiation is stimulated by activation of the ß3AR (Bronnikov et al., 1992). Interestingly, the biological consequences of ß3AR stimulation are quite opposite in BAT and in WAT. For example, the positive interaction between the ß3AR and the adipocyte differentiation machinery seems to be restricted to brown adipose tissue (BAT), since in white adipose tissue (WAT) stimulation of the ß3AR actually leads to a decrease in the number of adipocytes (e.g. Cousin et al., 1993; de Souza et al., 1997; Ghorbany et al., 1997a). Although the molecular mechanism for this difference remains to be elucidated, it is remarkable that, also in contrast to BAT, ß3AR stimulation in WAT has been shown to inhibit the activity of PI3K (Ohsaka et al., 1997) and ERK 1/2 (Sevetson et al., 1993), both in a cAMP-dependent way. Effects of adrenergic stimulation on adipocyte proliferation and differentiation are discussed further in Chapter 7.

1.6 Distribution of the ß3AR In contrast to the ß1- and ß2ARs which are distributed all over the body, the ß3ARs appears to have a rather restricted pattern of expression (see also Chapter 5). Most studies have concentrated on the functional presence of atypical (non-ß1ARs, non-ß2AR) ßARs in (brown and white) adipose tissue, where they regulate lipolysis and thermogenesis, and in the gastrointestinal tract, where they regulate gut motility via smooth muscle relaxation. Since the cloning of the murine ß3AR (Nahmias et al., 1991), it has become possible to identify the atypical ßARs by molecular means, and it is now

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widely accepted that at least some of these receptors in adipocytes and gut are indeed ß3AR. However, the presence of atypical ßARs does not always correlate with the presence of ß3AR messenger RNA (see also Chapter 8). Moreover, their pharmacological profiles do not always overlap. Indeed, pharmacological evidence now exists for the presence of a fourth ßAR. Although molecular proof for the existence of such ß4AR is still missing, functional data are accumulating. The pharmacological characteristics of ß4ARs are described in detail in Chapter 3 and 8. Here, we describe in brief the regional distribution of the ß3AR, as assessed by means of pharmacological and functional responses, immunolocalization of the protein, and detection of mRNA.

1.6.1 Functional detection of the ß3AR A large number of reports have characterized the presence of atypical ßARs by functional effects, mainly on metabolism, smooth muscle relaxation and vasodilatation. These atypical receptors are activated by selective ß3AR agonists such as CGP-12177 and BRL-37,344, and cannot be blocked by ßAR antagonists used in concentrations which completely inhibit the ß1- and the ß2AR. Many reports describe atypical ßARs present in murine (brown as well as white) adipocytes, where they activate adenylyl cyclase and couple functionally to lipolysis via the activation of hormone-sensitive lipase. In general, the pharmacological profile of the atypical adipocyte ßAR correlates well with that of the ß3AR, and ß3AR messenger RNAs can be easily detected (see below). In man, the lipolytic activity of the ß3AR is less evident. On one hand, this may be due to the fact that strong and selective human- (in contrast to rodent) ß3AR agonists are still not widely available (e.g. Hoffstedt et al., 1996a,b), despite now being described by pharmaceutical companies (see Chapter 4). In addition, many reports have now indicated clear differences in ß3AR responses in different human fat depots. In man, ß3ARs are expressed at lower levels in subcutaneous fat than in omental and mesenteric fat, and their lipolytic activity is limited in subcutaneous fat (e.g. Arner, 1995; Hoffstedt et al., 1995; Barbe et al., 1996; Tavernier et al., 1996; van Harmelen et al., 1997) (see Chapter 5). Atypical ßARs have furthermore been localized to the gastrointestinal tract, namely in the colon and ileum, where they induce smooth muscle relaxation both in rodents (De Ponti et al., 1995; Molenaar et al., 1997a,b) and in man (De Ponti et al., 1996; Roberts et al., 1997; Bardou et al., 1998). As described below, the identification of these receptors as ß 3 ARs in the gastrointestinal tract has been confirmed by immunohistochemical and RT-PCR experiments. In addition to the atypical ßARs present in adipocytes and gut, several reports discuss ‘ß3’ AR-mediated effects on the ventricle of the heart (Gauthier et al., 1996), pulmonary vasodilatation (Dumas et al., 1998; Tamaoki et al., 1998), relaxation of oesophageal sphincter (de Boer et al., 1995; Oriowo et al., 1998), urinary bladder (Oshita et al., 1997) and bile duct (de Ponti et al., 1995). Furthermore, effects of atypical ßARs on insulin secretion (Atef et al., 1996), gastrin-and somatostatin secretion in the antrum (Levasseur et al., 1997), and glucose metabolism in skeletal muscle (Challis et al., 1988; Liu et al., 1996a,b) have been reported. However, the molecular nature of these receptors remains controversial. BRL-37,344

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does not stimulate adenylyl cyclase in rat soleus muscle (Roberts and Summers, 1998), and only very low levels of ß3AR messengers were detected in skeletal muscle, where adipsin transcripts were also found, suggesting the co-existence of adipocytes (Evans et al., 1996). In RT-PCR experiments on heart, no ß3AR transcripts could be detected at all (Evans et al., 1996), or only together with UCP-1 messengers (Krief et al., 1993), again suggesting a ‘contamination’ with adipocytes. On the other hand, Gauthier et al. (1996) have provided not only pharmacological, but also molecular evidence, for the presence of a ß3AR (but the absence of adipocyte-specific hormone-sensitive lipase) in human heart ventricle cells. For a more complete description of the atypical ßARs present in heart and vessels, the reader is referred to Chapter 8. Only a limited number of reports exist that have used autoradiographic techniques in order to localize atypical/ß3ARs. Binding sites have been localized in the rat ileum (Roberts et al., 1995) and colon (Sugasawa et al., 1997). However, these sites may not represent ß3ARs, since they are resistant to blockade by propranolol. Although ß3AR agonists induce smooth muscle contraction in these tissues, and ß3AR messenger RNA has been detected, different cyanopindolol binding sites may co-exist in ileum (Hoey et al., 1996a; Sugasawa et al., 1997).

1.6.2

Immunodetection of the ß3AR protein

Raising and characterization of antibodies Sixteen different synthetic peptides, corresponding to the (presumed) extra-or intracellular parts of the receptor have been used to generate antibodies in rabbits (Guillaume et al., 1994). Only seven of these peptides have yielded antibodies that reacted with the whole ß3AR protein, expressed in CHO cells. Of these seven, only one peptide (‘p12’; corresponding to the C-terminal region of the ß 3AR), generated antibody titres consistently sufficient for further experimentaion. On immunoblots of membranes of cells transfected with the ß3AR gene, p12 recognized a single dominant protein of about 70 kDa, which could be displaced by preincubation with the peptide used for immunization. No cross-reactivity was seen with ß1AR or ß2AR. It was furthermore shown that p12 could be used to immunopurify the ß3AR heterologously expressed in CHO cells, E.coli or baculovirus-infected Sf9 insect cells (Guillaume et al., 1994).

Immunohistochemistry using the p12 antibody, raised against the C-terminal reeion of the ß3AR The p12 antibody was used to detect expression of the ß3AR in CHO cells and in baculovirus-infected Sf9 insect cells (Figure 1.5). When p12 was used to detect endogenously expressed ß3ARs, positive staining was achieved in subaxillary brown adipose tissue of a 34-year-old woman (S.Cinti et al., personal communication). Immunoreactivity was furthermore detected in the gallbladder of an obese patient (Guillaume et al., 1994). Recently, p12 was successfully used to immunolocalize the ß3AR to vascular and non-vascular smooth muscle in the human gastrointestinal tract. No staining was found in epithelia, vascular endothelial cells or ganglia (Anthony et al., 1998).

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Figure 1.5 Immunofluorescent staining of ß3AR in infected Sf9 cells. Labelling by anti-p12 of methanol-fixed infected Sf9 cells (ß3) infected by a baculovirus recombinant for the huß3AR; (WT) non-infected cells.

Western blot analysis of ileum and sigmoid colon membrane proteins revealed a band of 55 kDa, a size which could be consistent with a partially glycosylated ß3AR. Parallel Western blot experiments on murine ileum/colon did not show any immunoreactive bands, suggesting that the antibody does not cross-react with any murine proteins expressed in these tissues. Cross-reacting murine proteins have however been suggested to exist (S.Cinti, unpublished observations). When p12 was used to characterize the expression pattern of the human ß3AR in transgenic mice, knocked-out for murine ß3AR but expressing the human homologue, various tissues stained positive (see Chapter 3). However, this staining turned out to aspecific and due to a cross-reaction with at least two murine proteins containing a sequence that was partially homologous to the C-terminal region of the human ß 3 AR (J.L.Guillaume, unpublished observations).

1.6.3 Detection of the ß3AR mRNA Due to the generally low expression level of G protein-coupled receptors, PCR-based detection of ß3AR cDNA has been a method of choice to perform expression studies. In rat tissues, ß3AR mRNA was detected in WAT and BAT, smooth muscle of colon and ileum and, to a lesser extent, in stomach fundus and skeletal muscle (Evans et al., 1996). However, the last two tissues also expressed adipsin, suggesting the presence of adipocytes in these tissues. No expression was found in heart, lung and liver. Divergent results have been obtained concerning the expression of the ß3AR in human tissues (see also Chapter 5). Some studies, for example those of Krief et al. (1993), have indicated the abundant presence of ß3AR transcripts in infant perirenal BAT. In adults, in whom (almost) all fat tissue is composed of white adipocytes, ß3AR mRNA levels were high in deep fat such as perirenal and omental fat, but low in subcutaneous fat. Furthermore, ß3AR mRNA (but not UCP-1 mRNA) was highly expressed in gallbladder and weakly in colon. In contrast to these results, it has been reported (e.g. Deng et al., 1996) that human omental white adipocytes do not express

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ß3AR, and that in newborn BAT, they represent a minority (9%) of adrenoreceptors compared with the ß1- (28%) and ß2ARs (63%). Although these studies were made using Northern blot analysis, which is less sensitive than RT-PCR, binding studies using [3H] SB206606, done in parallel, confirmed the absence or low presence of ß3 ARs in WAT and BAT, respectively. Berkowitz et al. (1995) used the method of RNase protection without previous PCR amplification in order to detect expression of ß3AR in a variety of human tissues. ß3AR mRNA was detected in white fat, gallbladder and small intestine, and also in the stomach and prostate gland. A few reports have shown the presence of ß3AR mRNA. in brain (Rodriguez et al., 1995; Summers et al., 1995). Expression levels were higher in children than in adults (Rodriguez et al., 1995), and highest in the hippocampus, cerebral cortex and striatum, but lower in hypothalamus, brainstem and cerebellum (Summers et al., 1995). Interestingly, expression of the ß3AR in fat tissue is restricted to mature, fully differentiated adipocytes. For the murine pre-adipocyte cell line 3T3-F442A, it has been well documented that the ß3AR can be considered as a late differentiation marker (Fève et al., 1991; El Hadri et al., 1998). Likewise, pre-adipocytes of the human brown cell line PAZ-6 do not express detectable ß3AR before differentiation into adipocytes (Zilberfarb et al., 1997). In conclusion, these studies suggest that the ß3AR is most widely found to be expressed in white and brown adipose tissue, where its expression is restricted temporally (in mature adipocytes rather than in preadipocytes) and spatially (in omental rather than subcutaneous fat). Moreover, the evidence for the expression of the ß3AR in the gastrointestinal tract, gallbladder and pancreas is growing. The functional presence of ß3ARs in the brain and heart remains a matter of controversy (see Chapter 8).

1.7 Concluding remarks Even though the ß3AR has now been well studied at the gene and protein level, considerable work remains to be done to understand fully its mechanism of action. We still do not know why a number of ß1/ß2 antagonists behave as ß3AR agonists, nor do we understand species-specific differences. The molecular basis for the ability to activate different G proteins and hence different effector systems remains unexplained. In fact, we continue to avoid analysing the exact G ß combinations involved in the coupling of the three different ßAR subtypes or activation of any of the eight adenylyl cyclase subtypes or of the PI3 kinase. Finally, no effort has been made so far to identify other proteins capable of interacting with the ß3AR such as GRK, arrestin or the equivalents of proteins found to associate to receptors and discovered by the two-hybrid system in yeast technology. We hope to answer a few of these questions in the next decade of research on the ß3AR. a

g

Acknowledgements We wish to thank all the collaborators cited in our laboratory reference list who participated in completing the investigations discussed above.

2

Regulation of the ß3-Adrenoreceptor Signalling Efficacy MICHEL BOUVIER Université de Montréal, Faculté de Médecine, Département de Biochimie, Montréal (Québec), Canada H3C 3J7

2.1 Introduction One of the most fascinating features of G protein-coupled receptor signalling systems is their high degree of plasticity. This permits the cell to adapt to its environment, and may play a major role in the sorting and integration of the information detected at the receptor levels. ß-Adrenergic signalling has been shown to be the target of various regulatory processes. In particular, negative regulation and desensitization in response to sustained activation of its own or of other signalling pathways have been well documented. Alterations in such regulatory processes probably underlie certain pathological conditions related to hyper-or hyposensitivity. In particular, reduction in ß-adrenoreceptor (ßAR) density and responsiveness which accompany conditions such as hypertension, heart failure and obesity have often been attributed to processes similar to those involved in agonist-promoted desensitization. Changes in the responsiveness of the ßAR are also believed to contribute to the phenomenon of habituation and withdrawal syndrome associated with the use of ß-adrenergic agonists and antagonists in clinical settings. Since ßARs represent important pharmacological targets in the treatment of asthma, hypertension, angina pectoris, arrhythmia, heart failure and obesity, analysis of the mechanisms leading to alterations of the ßAR responsiveness may lead to the identification of new means to modulate receptor function more efficiently for therapeutic purpose. The original characterization of the mechanisms underlying regulation of the ßAR signalling efficacy was carried out mainly with the ß2AR, with the implicit assumption that similar—if not identical—processes would also be involved in the regulation of ß1and ß3AR responsiveness. Data gathered during the past five years or so invalidated this assumption. Indeed, subtype-specific regulatory processes have been delineated for the three receptors. In particular, the regulatory profile of the ß3AR was found to be dramatically different from that of the ß2AR, and the molecular basis of the differences have begun to be unravelled. In the following sections, the general concepts of G protein-coupled signalling and regulation—as they were found to apply to the ß-adrenoreceptors—will be reviewed, with special attention being given to the specificities of the ß3AR. 20

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2.1.1 The ß-adrenoreceptors and their signalling pathways ßARs play roles of primary importance in mediating responses through the central nervous system, the sympathetic branch of autonomic nervous system, and the neuroendocrine system. Their specific interaction with the endogenous catecholamines, adrenaline and noradrenaline, or with drugs represents a key step in regulating processes as diverse as neurotransmission, cellular metabolism, secretion, cellular differentiation and growth. The three subypes (ß1, ß2 and ß3) all couple via Gs to the stimulation of the adenylyl cyclase that promotes the formation of the second messenger cAMP. Whether or not the three subtypes display the same selectivity toward the four isoforms of G
s is, however, unknown. Similarly, the possibility that different Gßg-subunit complexes may selectively interact with a given receptor subtype, as has been suggested for other receptors (Kleuss et al., 1992, 1993), remains unexplored. This could have important consequences for the signalling efficacy of the ßAR subtypes, since the six known adenylyl cyclase isoforms are differentially regulated by distinct ßg subunit complexes (Federman et al., 1992; Clapham and Neer, 1993). Stimulation of adenylyl cyclase activity is believed to mediate most of the biological actions of the three ßAR through the activation of the cAMP-dependent protein kinase (PKA). However, it has also been proposed that Ga s, upon ßAR stimulation, directly activates L-type calcium channels (Yatani and Brown, 1989). It was also suggested that the Na+/H+ exchanger type-1 is under the regulatory influence of the ß2AR via its interaction with Ga13 (Barber et al., 1989, 1992; Barber, 1991; Voyno-Yasenetskaya et al., 1994). More recently, it was shown that a direct interaction of the ß2AR with the Na+/H+-exchanger regulatory factor (NHERF) has a modulatory influence on the activity of the NA+/H+ exchanger type-3 (Hall et al., 1998). Coupling of the ß2AR (Crespo et al., 1995; Bogoyevitch et al., 1996; Yamamoto, J. et al., 1997b) ß1AR (Williams et al., 1990) and ß3AR (Gerhardt et al., 1999; Soeder et al., 1999) to the ERKl/ERK2/p38 MAP kinase signalling pathways through the ßg subunit has also been demonstrated. This activation of the MAP kinase systems has been shown to be pertussis toxin-sensitive and to involve Gai (Daaka et al., 1997). Whether the three subtypes will show selective efficacies toward these effectors, remains to be investigated. This point may have important physiological implications since the distinct physiological end-points of the three ßAR isoforms could result from subtypeselective coupling to various pathways. Even with regard to the relative signalling efficacy of the three ßAR via the more classical adenylyl cyclase stimulatory pathway, relatively little data is available. The only systematic studies comparing the capacity of equivalent receptor numbers to stimulate cAMP production in the same cellular background were carried out for the ß2- and ß1AR. In three independent studies using different expression systems, the human ß2AR was found to stimulate the adenylyl cyclase activity more efficiently than the ß1AR, suggesting a better coupling of the former to G
s (Green et al., 1992; Levy et al., 1993; Rousseau et al., 1996). Unfortunately, no study systematically compared the efficacy of the ß3AR to that of the two other subtypes. However, results obtained in several studies would indicate that the coupling efficacy of the ß3AR to the adenylyl cyclase system is very similar to that of the ß2AR and thus greater than that of the ß1AR (Nantel et al., 1993, 1994). The preceding discussion thus raises the possibility that the three ßAR may be linked to various signalling pathways with subtype-selective efficacy. Until now, however, studies on the ß3AR mostly considered the Gs/adenylyl

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cyclase signalling cascade. It follows that all that we know concerning the regulation of the signalling efficacy of this receptor relates to its coupling with the production of cAMP. This review will therefore focus on the mechanisms that are known to modulate cAMP-mediated signalling.

2.2 General concepts of G protein-coupled signalling regulation Constraints on signalling networks undoubtedly exist, and provide a high degree of plasticity to transduction pathways. In fact, the concerted action of many regulatory processes assures the plasticity required to adapt to various stimulatory conditions. As the initial point of interaction with the transmitters, receptors are uniquely positioned to regulate the responsiveness of a given signalling pathway. As mentioned in the general introduction, these regulatory processes play an important role in the development of altered ßAR responsiveness related to cardiovascular and metabolic diseases. Therefore they have been the object of intense research both under normal and pathological conditions. Most efforts have concentrated on the best-characterized ß2AR, and the molecular processes regulating the signalling efficacy of this receptor have begun to be unravelled. Because much is known about the regulation of ß2AR signalling efficacy, it will be used in this section as a model system to which the ß3AR will then be compared. An important regulatory mechanism of receptor function is known as agonistinduced desensitization. This is characterized by the fact that the intensity of a response mediated by the receptor wanes over time, despite the continuous presence of the stimulus. The appearance of agonist-induced desensitization of ß 2 ARstimulated adenylyl cyclase was at first thought to be related solely to the decrease in ß2AR number (down-regulation) which follows prolonged stimulation (Lefkowitz, 1979). However, it was quickly realized that the regulatory processes are far more complex, and it is now generally accepted that at least three distinct processes are involved in the desensitization phenomenon. These are known as functional uncoupling, sequestration and down-regulation (Benovic et al., 1988; Hausdorff et al., 1990). Uncoupling corresponds to a decreased receptor-mediated activation of the adenylyl cyclase with no change in receptor number or distribution. Sequestration consists of a cellular redistribution of the ß2AR from the cell surface to an intracellular vesicular fraction (Lefkowitz et al., 1980; Staehelin and Simons, 1982; Harden, 1983). Sequestration is therefore observed as a decreased receptor density at the cell surface with no change in total cellular receptor number, whereas down-regulation refers to a loss of receptor sites and results in a decrease in total ß2AR number expressed in a given cell.

2.2.1 Agonist-promoted receptor phosphorylation and receptor uncoupling Functional receptor uncoupling represents the fastest regulatory process regulating signalling efficacy. Reduction in the ß-adrenergic-stimulated adenylyl cyclase activity can be observed as rapidly as one minute following the beginning of the stimulation, and is believed to reflect a decreased capacity of the receptor to interact productively with Gs. Phosphorylation of the ß2AR has been shown to play an important role in the expression

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of such agonist-induced desensitization. At least two distinct protein kinases, PKA and the ß-adrenoreceptor kinase (ßARK) have been implicated. Whereas PKA has broad substrate specificity, ßARK demonstrates a much more restricted specificity and phosphorylates only agonist-occupied receptor (Benovic et al., 1986). Using sitedirected mutagenesis, we and others have identified several phosphorylation sites involved in desensitization (see Figure 2.1) (Bouvier et al., 1988; Clark et al., 1989; Hausdorff et al., 1989). ßARK-mediated phosphorylation of serine and threonine residues in the distal portion of the carboxyl terminus (Ct) of the receptor has been shown to promote the association of the protein ß-arrestin with the ß2AR thus inhibiting functional coupling of the receptor to Gs (Lohse et al., 1990b; Pitcher et al., 1992b). In contrast, phosphorylation of ß2AR by PKA does not favour the interaction of the receptor with ß-arrestin (Pitcher et al., 1992b), and the mechanisms by which it promotes functional uncoupling remain largely unknown. Two phosphorylation sites for PKA have been identified within the ß2AR structure (Bouvier et al., 1989). One is located in the third intracellular loop of the receptor (Ser261,262), while the other is in the proximal portion of the carboxyl tail (Ser345,346). Upon direct activation of PKA, and in the absence of agonist occupancy of the receptor, only the third loop site is phosphorylated and contributes to the desensitization (Bouvier et al., 1989; Clark et al., 1989). However, following agonist stimulation of the receptor, the site in the carboxyl tail also becomes available to the kinase and contributes to the desensitization (Moffett et al., 1996). As discussed further below, agonist-dependent regulation of the palmitoylation state of a cysteine located four amino acids upstream of this second phosphorylation site is believed to control its accessibility to the kinase (Loisel et al., 1996; Morello and Bouvier, 1996). Because of the important role of the third cytoplasmic loop in the functional interaction of the receptor with Gs, it has been proposed that the change in charge distribution imposed to this domain by the PKA-mediated phosphorylation could be responsible for the functional uncoupling. Arguing against this hypothesis, however, is the observation that substituting acidic residues for Ser261,262, to mimic the addition of the phosphate negative charges, failed to promote receptor uncoupling and desensitization (Yuan et al., 1994). More recently, however, it was proposed that phosphorylation of the third cytoplasmic loop by PKA promotes the interaction of the receptor with G
i to the expense of G
s, thus favouring the activation of the MAP kinase and reducing the activation level of the adenylyl cyclase (Daaka et al., 1997). This would suggest that phosphorylation of the third loop sites results in a change in G protein coupling specificity rather than a loss of overall signalling efficacy. For the PKA site located in the carboxyl tail, it has recently been proposed that its phosphorylation increases the rate of phosphorylation of the more distal ßARK sites in the carboxyl tail. Although the precise mechanisms by which PKA contributes to the rapid desensitization of the ß2AR remain to be clarified, it is clear that both PKA and ßARK play important roles in receptor-specific or homologous desensitization (Hausdorff et al., 1989; Lohse et al., 1990a). In addition to its role in homologous desensitization, PKA—and in some instances PKC, but not ßARK—is involved in the heterologous desensitization of the ß2AR that occurs upon activation of other receptor systems (Bouvier et al., 1991; Yuan et al., 1994). Unlike PKA and PKC, ßARK is not activated by a second messenger and its mode of activation makes it an enzyme of choice for a role in homologous desensitization. Indeed, upon agonist binding of the receptor and G protein activation, ßARK is translocated from the cytosol to the membrane and phosphorylates only the agonist-

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occupied receptors (Benovic et al., 1986; Chen et al., 1993). Interaction between dissociated Gßg complexes and ßARK is responsible for the translocation of the enzyme to the membrane and has been shown directly to increase its activity (Pitcher et al., 1992a). Prenylation of the g subunit is believed to play a crucial role in this translocation and activation mechanism (Inglese et al., 1992). ßARK is only one member of a growing family of G protein-coupled receptor kinases. To date, six kinases named GRK1 to GRK6 (ßARK1 corresponding to GRK2) have been cloned and characterized. Although absolute specificity of a given kinase for a subset of receptors has not been demonstrated, every kinase is believed to have distinct selectivity patterns towards the various G protein-coupled receptors. Selectivity towards activating Gß g has also been recently reported (Daaka et al., 1997). Interestingly, two members of the family, GRK4 and GRK6, do not require any interaction with Gßg subunits to be translocated to the plasma membrane and to phosphorylate receptors. Indeed, these two kinases are palmitoylated at their carboxyl terminus, and the lipid modification was found to be responsible for membrane association and activity (Stoffel et al., 1994; Premont et al., 1996). From the previous discussion, it is clear that phosphorylation is a post-translational modification that plays a central role in modulating ß2AR signalling efficacy by regulating its functional interaction with Gs. However, during the past few years, several studies suggested that another post-translational modification of the receptor, palmitoylation of its Cys341, may also play an important role in dictating the ability of the ß2AR to couple to Gs and to stimulate adenylyl cyclase. Like phosphorylation, palmitoylation was found to be a reversible modification that is regulated by agonist stimulation. Indeed, ß2AR stimulation leads to an increased turnover rate of the receptor-bound palmitate that ultimately favours the unpalmitoylated form of the receptor (Mouillac et al., 1992; Loisel et al., 1996). This agonist-promoted depalmitoylation of the receptor is believed to increase the accessibility of its carboxyl tail to regulatory kinases, and thus to regulate the extent of its phosphorylation and desensitization. This idea is supported by the observation that mutation of the palmitoylation site greatly increases the basal phosphorylation level of the receptor that promotes its desensitization (Mouillac et al., 1992; Moffett et al., 1993, 1996). In contrast to the wealth of information concerning the enzymes catalysing the phsophorylation of the ß2AR, nothing is known about the enzymes that control its palmitoylation state.

2.2.2

Receptor sequestration; a process involved in desensitization and resensitization

Although less rapid than functional uncoupling, sequestration of receptors away from the cell surface occurs within a few minutes after the beginning of receptor stimulation. It is intuitively sensible to propose that such a mechanism which leads to the removal of receptor sites from the cell surface would greatly contribute to the reduction of responsiveness. However, over the past few years, it has become increasingly evident that the contribution of sequestration to the overall rapid desensitization is relatively modest (Lohse et al., 1990a). This is probably due to the fact that the receptors that are removed from the cell surface have already been functionally uncoupled from Gs via phosphorylation. More recently, it was in fact found that sequestration plays a more important role in the resensitization than in the

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desensitization of the system. Indeed, sequestered receptors have been shown to be dephosphorylated and returned to the cell surface in a functional conformation (Sibley et al., 1986; Yu et al., 1993; Krueger et al., 1997). It follows that upon arrest of the stimulation, sequestration plays a crucial role in rapidly restoring a control level of responsiveness to the system. Although sequestration clearly involves internalization of the receptor in an intracellular vesicular compartment, both the cellular compartments and the molecular mechanisms involved remain poorly characterized. Sequestration of the ß2AR can readily be measured using hydrophobic and hydrophilic ligands that allow a distinction to be made between cell surface (accessible to both hydrophilic and hydrophobic ligands) and the sequestered (accessible only to the hydrophobic ligands) receptors. The sequestered receptors can also be measured following subcellular fractionation, as they are associated with a light membrane vesicular fraction. However, the nature of the vesicles involved remains a matter of controversy. Using immunofluorescence, it was shown that at least in some cells, internalization of the ß2AR occurs through classical endocytosis involving clathrin-coated vesicles (Von Zastrow and Kobilka, 1992, 1994). In contrast, electron microscopic studies demonstrated that the ß 2AR is internalized through smooth vesicles that were identified as caveolae (Raposo et al., 1989; Dupree et al., 1993). Whether the differences between the studies reflect the existence of cell type-specific pathways, or else suggest that distinct internalization pathways (for example, one being involved in recycling the other in lysosomal degradation) can co-exist in the same cell remains to be clarified. With regard to the clathrin-coated vesicle pathway, recent studies (Ferguson et al., 1996) have shown that binding of ß-arrestin to the receptor plays a central role in internalization. It was demonstrated that, by binding to both the ß2AR and clathrin, ß-arrestin serves as a clathrin-adapter protein (Goodman et al., 1996). More recently, it was also shown that ß-arrestin binds to the AP1 clathrin adapter protein that can in turn attract the ß-arrestin-bound receptor to the clathrin-coated pits (Laporte et al., 1999). However, the identity of the receptor residues involved in the interaction with ß-arrestin and in the promotion of agonist-induced sequestration remains elusive. Indeed, several studies have shown that mutation of the ßARK and PKA phosphorylation sites do not abolish sequestration, demonstrating that phosphorylation is not the main determinant of sequestration (Strader et al., 1987; Bouvier et al., 1988; Hausdorff et al., 1989; Pippig et al., 1995). Since phosphorylation of the receptor is believed to be required for ß-arrestin binding, some unanswered questions concerning the role of ß-arrestin in sequestration are left open. Interestingly, some G protein-coupled receptors were found to undergo agonistpromoted sequestration in an arrestin-independent manner (Lee et al., 1998), thus suggesting that both arrestin-dependent and independent pathways may exist. The fact that the clathrin-coated vesicle pathway has been associated with the binding of ßarrestin raises the interesting possibility that the ß-arrestin-independent pathway may involve the caveolae-like vesicular system. The NPXXY motif located at the bottom of the seventh transmembrane domain of the ß2AR has been proposed as a potential determinant for sequestration. However, mutation of this motif in the gastrin-releasing peptide (Slice et al., 1994) and the type 1 angiotensin II (Hunyady et al., 1995) receptors did not affect their agonist-promoted sequestration, arguing against a general role for this sequence. More recent studies have shown that mutation of the NPXXY motif of the ß2AR have detrimental effects on many receptor functions, suggesting that the effect of this mutation on sequestration may be

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rather non-specific (Gabilondo et al., 1996). Thus, the specific receptor sequence(s) or structural motif(s) involved in ß2AR sequestration remain to be determined.

2.2.3 Agonist-promoted down-regulation Following longer-term (hours) stimulation of the receptor, another phenomenon known as down-regulation also contributes to the development of agonist-promoted desensitization. This process, which is defined as a reduction of the total number of ß2AR present in the cells, involves mechanisms acting both at the level of gene expression and of intracellular receptor processing following internalization. Many lines of evidence (reviewed in Nantel and Bouvier, 1993b) support the notion that an agonist-promoted increase in the rate of receptor degradation is involved in down-regulation. Classical models suggest that this increased rate of degradation results from the targeting of the internalized receptors toward the lysosomal degradation pathway. In a recent study, Gagnon et al. (1998a) suggested a role for the clathrinmediated endocytotic pathway in such agonist-induced down-regulation. However, this dependence of the down-regulation on the clathrin-mediated endocytosis process appears to be cell type-dependent, and different degradation pathways—including a plasma membrane restricted receptor degradation—could be involved (Jockers et al., 1999). Similarly to the case of sequestration, very little is known about the molecular motifs directing the receptors towards the degradative pathways. Only one potential molecular determinant of receptor processing has been proposed. It consists of two tyrosine residues located within a predicted ß-turn in the carboxyl tail of the receptor. Mutation of these two tyrosines (Tyr 350 and Tyr354) was found to greatly impair the agonist-induced down-regulation of the ß2AR (Valiquette et al., 1990, 1993). Similar motifs involving tyrosine residues have also been shown to play important roles in agonist-promoted down-regulation of other receptors. These include the G protein-coupled M2-muscarinic receptor (Goldman and Nathanson, 1994), as well as receptors belonging to different families such as those for low density lipoprotein, poly-immunoglobulin and mannose-6-phosphate (Vega and Strominger, 1989), epidermal growth factor (EGF; Helin and Beguinot, 1991) and transferrin (Girones et al., 1991). The internalization of these receptors involves endocytosis by means of clathrin-coated vesicles, and it is believed that the tyrosine motifs favour the interaction of receptors with proteins of the clathrin-coated protein complex, termed adaptins (Collawn et al., 1990). In addition to increasing its rate of degradation, sustained stimulation of the ß2AR has been found to regulate its synthetic pathway. First, a cAMP regulatory element located in the promoter region of its gene (Collins et al., 1990), is responsible for a rapid but transient increase in the rate of transcription of the gene. However, in the cell types studied, this transiently increased transcriptional activity was not accompanied by an elevation in receptor number (Collins et al., 1989). This may be due to the fact that, shortly following this transcriptional activation, the mRNA steady-state level begins to decline and reaches levels that are significantly lower than those observed under basal conditions. This reduction in mRNA level has since been shown to result from a cAMP-dependent destabilization of ß2AR mRNA that contributes to the agonist-induced down-regulation of the receptor (Hadcock and Malbon 1988; Bouvier et al., 1989; Hadcock et al., 1989). Although the precise

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mechanisms still need to be investigated, the cAMP-dependent binding of a ‘destabilizing’ protein to the 3' untranslated domain of the ß2AR transcript has been shown to be involved.

2.3 The ß3-adrenoreceptor; a prototypic system to study subtypespecific regulation Over the years, a number of studies carried out on many G protein-coupled receptors have confirmed the importance of the mechanism described above for the regulation of their signalling efficacy. At the molecular level it was shown in several instances that, as for the ß 2 AR, phosphorylation by GRKs and second messenger-activated kinases is responsible for the functional uncoupling that leads to rapid agonist-promoted desensitization. For down-regulation, the role of tyrosine residues in the carboxyl tail of the receptors for the degradation path, and of the mRNA level regulation for the synthetic arm, have also been confirmed for several receptors. From these studies, it was often assumed that the mechanisms described for the ß 2 AR would apply to all G protein-coupled receptors. However, primary sequence analysis of receptors that are otherwise closely related to the ß 2 AR (for example, ß 1 - and ß 3 AR) reveals that many of the sites and motifs known to play important roles in ß 2 AR regulation are not strictly conserved. This could indicate either that different sites are involved, or that some of the processes do not apply universally to all G protein-coupled receptors. As discussed below, the ß 3AR represent an excellent model to study this question, as many of the regulatory sites identified for the ß 2 AR are lacking from its primary structure.

2.3.1 Comparative analysis of the structural determinants of receptor regulation As shown in Figure 2.1, in contrast with the strong sequence identity observed between the transmembrane domains of the two receptors, the sequences of the cytosolic domains are greatly divergent between the ß 3- and the ß 2AR (see also Chapter 1). This lack of conservation within the intracellular regions of the receptors also applies to the motifs that were shown to play regulatory functions for the ß2AR. Indeed, a comparison of the ß3- and ß2AR sequences reveals that neither of the two PKA phosphorylation sites found in the ß 2AR is conserved within the ß 3 AR structure. Similarly, whereas the ß2AR has six serine and five threonine residues within the distal portion of its carboxyl tail that have been proposed as potential GRK phosphorylation sites, only five serines are found within the corresponding domain of the ß3AR. Moreover, only one of these serines is located in a context that is believed to be favourable for GRK phosphorylation. Indeed, the presence of acidic residues near serines or threonines has been proposed as a requirement for GRK phosphorylation (Onorato et al., 1991) and only one serine is found within three amino acids from an aspartate or a glutamate residue in the ß3AR carboxyl tail. This contrasts with the three threonines and five serines that are found in such an acidic context within the ß 2AR tail. Also, the two consensus sequences for phosphorylation by PKC that were proposed to play a role in the heterologous desensitization of the

Figure 2.1 Primary sequences of the ß2AR and ß3AR and schematic representation of their proposed membrane topology. The one-letter amino acid code is used. The confirmed phosphorylation sites for PKA and PKC are indicated by the closed diamonds, whereas the putative ßARK phosphorylation sites (defined as S or T located close to an acidic residue within the distal portion of the carboxyl tail) are indicated by open diamonds. Arrows identify a putative down-regulation signal. The boxes numbered 1 to 4 delimit important regulatory domains for the ß2AR and their corresponding regions within the ß3AR.

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ß2AR, upon stimulation of the phospholipase C signalling pathway (Bouvier et al., 1991), are absent from the ft sequence. Finally, the two tyrosine residues of the carboxyl tail that were found to be important in the down-regulation of the ß2AR (Valiquette et al., 1990, 1993) are absent from the sequence of the ß3AR. This lack of conservation of many of the regulatory motifs that were identified for the ß2AR has led us and others to propose that these sequence differences may underlie subtype-specific regulation profiles.

2.4 The ß3-adrenoreceptor is resistant to rapid agonist-promoted uncoupling and sequestration Based on the importance that was attributed to the phosphorylation of the ß2AR for the occurrence of functional uncoupling and rapid desensitization that follows agonist stimulation, and also given the lack of putative phosphorylation site for PKA and ßARK in the ß3AR sequence, it was reasonable to propose that the latter may be more resistant to rapid agonist-promoted desensitization than the ß2AR. This hypothesis was assessed directly using heterologous expression systems that allow comparison of the regulatory profiles of the two receptors in the same cellular background. When expressed in Chinese hamster (CHW) or murine (Ltk-) fibroblasts, the human ß3AR was found to be completely resistant to rapid desensitization. Indeed, stimulation of the ß 3AR for up to 1 h did not affect receptor responsiveness, whereas similar treatment of the ß2AR led to a reduction of 25–50% of the ß-adrenergic-stimulated adenylyl cyclase activity, depending on the cell type considered (Liggett et al., 1993; Nantel et al., 1993a). A similar resistance to rapid desensitization was also observed in cells naturally expressing the ß3AR. Indeed, exposure of isolated rat adipocytes to the non-selective ß-adrenergic agonist isoproterenol did not promote any desensitization of the ß3-adrenergic-stimulated adenylyl cyclase activity, whereas the ß1-stimulated response was significantly blunted by pretreatment with the agonist (Granneman et al., 1992). A comparable refractoriness to desensitization was also observed in hamster fat cells (Carpéné et al., 1993b). In the non-adipocytic human SKN-MC neurotumour cells, sustained treatment with isoproterenol also failed to cause desensitization of the endogenously expressed ß 3 AR, while promoting a rapid desensitization of the ß1AR also expressed in these cells (Curran and Fishman, 1996). In contrast to this general observation that ß3AR-stimulated adenylyl cyclase activity does not undergo rapid desensitization, Chaudry and Granneman observed agonistpromoted desensitization of this response both in HEK-293 cells heterologously expressing the human ß3AR and in SK-N-MC cells (Chaudhry and Granneman, 1994). However, an agonist stimulation of 1 h was the shortest assessed in this study. It could therefore be proposed that the desensitization observed did not result from receptor uncoupling, but rather from processes occurring downstream of the receptor. Consistent with this hypothesis, longer-term treatment with agonists has been shown to lead to desensitization in several cell types. It has been proposed that such desensitization results from a down-regulation of G
s (Chambers et al., 1994) or from an increased activity of phosphodiesterase activity (Unelius et al., 1993). An additional technical point needs to be raised when addressing the desensitization of the ß3AR in tissues or cells that expressed other ßAR subtypes. Indeed, in contrast to the studies using heterologous cell systems that lack endogenous ßAR (for example, CHW, CHO, Ltk-), investigations carried out using

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tissues or cells such as SK-N-MC or HEK-293 that endogenously express ß1 or ß2AR cannot use the non-selective agonist isoproterenol to assess ß 3AR responsiveness. Therefore, CGP-12177A was used, in many instances, as a selective partial ß 3 agonist (being a ß 2AR antagonist) to evaluate the ß3AR response. However, caution should be used when employing CGP-12177A as a selective ß3AR agonist, as it was shown also to have partial agonistic properties towards the ß1AR (Pak and Fishman, 1996). Overall, the studies carried out during the past 5 years show clearly that, in contrast to the ß2 and ß 1AR, the ß3AR does not undergo rapid functional uncoupling as a result of short-term agonist stimulation. This of course is entirely consistent with the fact that the ß3AR lacks most of the phosphorylation sites that are believed to promote the rapid functional uncoupling and that it does not become phosphorylated upon agonist stimulation (Liggett et al., 1993). The blunted ß3AR response observed in some cell types following longer-term stimulation therefore most likely results either from a down-regulation of the ß3AR number (see below) or else from processes affecting the signalling efficacy of elements located beyond the receptor itself. As discussed in Section 2.2, shortly following functional uncoupling, the ß2AR becomes sequestered in intracellular vesicles where it is no longer accessible to the natural hydrophilic ligands, but can be dephosphorylated and recycled back to the cell surface (Sibley et al., 1986; Pitcher et al., 1995). This process is believed to contribute largely to the rapid resensitization of the system that follows interruption of the stimuli (Yu et al., 1991). Given that the ß 3AR is neither phosphorylated nor rapidly desensitized, one could predict that there is no need for sequestration of this receptor. As predicted, no agonist-promoted sequestration of the ß3AR could be observed in any of the cell systems examined (Liggett et al., 1993; Nantel et al., 1993a; Curran and Fishman, 1996; Jockers et al., 1996). Indeed, sustained stimulation of cells expressing the ß3AR does not promote translocation of the receptor from the cell surface to intracellular compartments, as assessed by either the accessibility to hydrophilic ligands or subcellular fractionation. Although one can establish a teleological link between the lack of phosphorylation and the absence of agonist-promoted sequestration (there is no need to dephosphorylate a receptor that does not become phosphorylated upon activation), the causal relationship between the lack of phosphorylation and the absence of sequestration has not yet been fully established (see Section 2.6). It should be noted that the ß3AR harbours within its transmembrane domain VII the NPXXY sequence that has been proposed as a sequestration signal for the ß2AR. The lack of agonist-promoted sequestration of the ß3AR therefore suggests that this signal is either not sufficient to permit sequestration or that, as argued in Section 2.2, it does not represent a bona fide internalization signal.

2.5

Cell type-specific down-regulation of the ß3-adrenoreceptor

In contrast to the generally observed resistance of the ß 3AR to rapid agonistpromoted desensitization and sequestration, no general consensus can be easily drawn from the existing literature on the effect of longer-term stimulation on ß3AR responsiveness. In hamster, long-term stimulation did not lead to a reduction of the ß3-adrenergic-

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mediated lipolytic response even after 6 days of continuous infusion with noradrenaline (Carpéné et al., 1993b; Langin et al., 1995), suggesting that the ß3AR was not downregulated. Similarly, no desensitization nor down-regulation of the ß3AR number could be observed in 3T3-F442A adipocytes upon exposure to the ß-adrenergic agonist isoproterenol for up to 30 h (Thomas et al., 1992). In fact, a significant increase in both ß3AR mRNA and receptor number was observed in that study, while ß1AR expression declined by approximately 70% in response to the same stimuli. This increase in ß3AR number and message was attributed to a positive-feedback mediated by transcriptional activation through three cAMP responsive elements located in the receptor gene promoter region. This resistance of the ß 3AR to long-term down-regulation is, however, not universally observed. Using the same 3T3-F442A adipocyte cell line, Granneman and Lahners (1995) observed an important reduction in ß3AR mRNA levels upon sustained stimulation with isoproterenol. A reduction in ß3AR transcript was also observed in cultured hamster brown adipocytes following treatment with ß3-adrenergic agonists and dibutyryl cAMP (Klaus et al., 1995a,b) and in white adipose tissue (WAT) of mice treated in vivo with the ß3-adrenergic agonist BRL-26830. Treatment of rats with the same agonist or with isoproterenol for 8 h also led to a significant reduction of the ß3AR mRNA levels in both WAT and brown adipose tissue (BAT) (Granneman and Lahners, 1992). In that study, the agonist-promoted down-regulation of the ß3AR mRNA was shown to be accompanied by a reduction of the ß3-adrenergic-stimulated adenylyl cyclase activity in WAT, suggesting the occurrence of a functional downregulation. However, in none of the studies mentioned above was the actual number of ß3AR shown to be down-regulated as a result of the decrease in ß3AR mRNA content. In one study, Revelli and colleagues showed, using Zucker rats, that such a decrease in ß3AR mRNA levels indeed preceded the down-regulation of ß3AR number observed in interscapular BAT following a 72-h treatment with the ß-adrenergic agonist RO 16– 8714 (Revelli et al., 1992). The possibility that agonist-mediated down-regulation of the ß 3AR mRNA may play a physiologically relevant regulatory role is further supported by the fact that sympathectomy promotes an increase in the ß3AR mRNA level, whereas exposure of rats to 4°C (which increases sympathetic activity) reduces this level (Granneman and Lahners, 1992; Onai et al., 1995). This suggests that expression of the ß 3 AR is physiologically under the dynamic control of the sympathetic nervous system activity. The reasons for the difference in the phenotype of resistance to down-regulation observed in different studies, and with the different models used, are not completely understood. However, studies carried out in heterologous expression systems may have shed some light on the apparent inconsistencies and have helped better appreciate the relative resistance of the ß3AR to agonist-promoted down-regulation. Indeed, the expression of both the ß 2- and ß 3AR subtypes in the same cellular background showed clearly that although the ß3AR can undergo agonist-promoted down-regulation, it does so less efficiently and following a pattern that is distinct from that of the ß2AR. When expressed in either hamster CHW or murine Ltk- fibroblasts, the human ß2AR was found to be down-regulated more rapidly and to a greater extent than the human ß3AR expressed at comparable density. In fact, almost no down-regulation of ß3AR number could be observed in CHW cells, even following a 24-h exposure to an agonist (Liggett et al., 1993; Nantel et al., 1994), whereas a significant loss of sites could be observed only after 6 h of agonist treatment in Ltk- cells (Nantel et al., 1994). This

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contrasts with the down-regulation of 20–40% of the ß2AR sites observed as early as 1 h after the beginning of the stimulation in the two cell lines. These data indicate two things: (i) that the ß3AR is less prone to down-regulation than the ß2AR when expressed in the same cellular background; and (ii) that the extent of down-regulation of the ß3AR varies significantly from one cell type to the next. Analysis of the mechanisms leading to the agonist-promoted loss of ß3AR sites in Ltk- and CHW cells helped in our understanding the distinct down-regulation patterns of the two receptor subtypes. As indicated in Section 2.2, at least two processes contribute to the down-regulation of the ß2AR number: first, an increased rate of receptor degradation; and second, a decreased rate of receptor synthesis due to a reduction in the receptor mRNA steady-state level. The increase in receptor degradation rate can be easily appreciated for the ß2AR by the agonist-promoted reduction of the receptor half-life observed in the presence of the protein synthesis inhibitor cycloheximide (Nantel et al., 1994). In contrast, agonist treatment did not affect the half-life of the ß3AR in either CHW or Ltk- cells, thus excluding the contribution of accelerated receptor degradation to the ß 3AR down-regulation. Interestingly, the two tyrosine residues that were shown to play an important role in the down-regulation of the ß 2AR are not conserved in the ß 3 AR (see Figure 2.1), suggesting that this structural difference may account at least in part for the distinct down-regulation patterns. In contrast, sustained agonist treatment of CHW or Ltk- cells expressing the ß3AR led to a reduction of the receptor mRNA concentrations that reached statistical significance after 6 h of a maximal level of stimulation (Nantel et al., 1994). This reduction which was modest (25% at 6h) and transient (mRNA levels returning to control values after 24 h of continuous stimulation) in CHW cells, was sustained and reached 60% in Ltk-cells. The good correlation between the ß3AR mRNA reduction and the loss of binding sites observed in each cell type indicates that the ß3AR downregulation results primarily from regulatory control at the mRNA level. This regulatory mechanism was found to be cAMP-dependent, as the decline in both mRNA and ß3AR receptor number could be mimicked by membrane-permeant cAMP analogues and direct stimulation of adenylyl cyclase by forskolin. The difference in the extent of ß3AR down-regulation observed between CHW and Ltk-cells was thus attributed to the fact that the receptor stimulated cAMP production less efficiently in CHW than in Ltkcells. The reasons for this cell type-specific difference remain unknown, but could include different contingents of G protein subunits, adenylyl cyclase and phosphodiesterases isoforms. Taken together, these studies suggest that ß 3 AR down-regulation results primarily, if not exclusively, from the regulation of the mRNA steady-state levels, and that the agonist-promoted degradation that contributes to accelerate and amplify the down-regulation of the ß 2 AR is not a factor involved in ß 3 AR regulation. Differences in the extent of ß 3AR down-regulation observed in various cell types or tissues could then reflect different efficacy with which ß-adrenergic stimulation can activate the cAMP-dependent mRNA regulatory pathway. It should be noted, however, that even in cells in which significant ß 3AR down-regulation was observed, the extent of ß-adrenergic-stimulated adenylyl cyclase desensitization seen after 24 h of continued stimulation was considerably less than that for the ß 2AR (Nantel et al., 1998). This most likely reflects the absence, for the ß3AR, of the other mechanisms (uncoupling, sequestration) that contribute to the ß 2AR desensitization.

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The regulation of the ß3AR expression through modulation of its mRNA level is not unique to the process of homologous agonist-promoted down-regulation. Indeed, various stimuli including treatment with dexamethasone (Fève et al., 1992), insulin (El Hadri et al., 1998), phorbol ester (El Hadri et al., 1998) and butyrate (Krief et al., 1993) have been shown to promote heterologous down-regulation of both ß3AR transcript and binding sites in various cells.

2.6 Use of chimeric ß3/ß2-adrenoreceptors to delineate regulatory motifs The above sections have conveyed the notion that structural differences between the ß3AR and the ß2AR may underlie their subtype-specific down-regulation profiles. In order to test this hypothesis formally, chimeric ß 2/ß3 receptors in which specific domains of one subtype were transferred into the equivalent position within the other receptor were generated. The construction of such chimeric receptors offers a powerful tool to study the molecular determinants of subtype-specific regulation. In particular, it has the advantage of allowing a search to be made for a gain of regulatory phenotypes rather than for a loss, as is often the case in classical site-directed mutagenesis studies. Substituting the third intracellular loop (i3) and carboxyl tail (Ct) of the ß2AR for their counterparts within the ß3AR allowed confirmation to be made that the resistance of the ß3AR to rapid agonist-promoted desensitization resulted at least in part from the absence of PKA and GRK phosphorylation sites. Indeed, the substitution of the ß2ARderived i3 and Ct (that harbour the PKA and ßARK phosphorylation sites) within the ß3AR restored the ability of this receptor to become rapidly desensitized upon agonist stimulation (Liggett et al., 1993; Nantel et al., 1993a). Interestingly, however, the level of desensitization attained by the chimeric receptor was significantly less (by ~50%) than that observed for the ß2AR, suggesting that additional motifs would be required to fully restore a ß2-like desensitization profile. The construction of additional chimeric receptors including substitution of i1, i2, i3 and Ct individually or in various combinations allowed identification of the second cytoplasmic loop as being an important contributor to the desensitization phenotype (Jockers et al., 1996). The presence of the ß2AR i2 alone within the ß3AR restored approximately 20% of the desensitization normally observed with the ß 2 AR, whereas the simultaneous substitution of i2, i3 and Ct restored a rapid desensitization phenotype that was quantitatively indistinguishable from that of the ß2AR. Therefore, in addition to confirming the regulatory importance of the i3 and C t phosphorylation sites, the use of chimeric receptors allowed the proposal to be made that residues within the second cytoplasmic loop of the ß 2 AR also play a role in rapid agonist-promoted desensitization. However, the identity of the specific residues involved, and the mechanism by which they contribute to the desensitization process, remain to be determined. The chimeric receptor constructs also provided information concerning the determinants of agonist-promoted sequestration. Indeed, as reviewed above, the ß3 AR does not become sequestered upon agonist stimulation, but substitution of the ß2AR Ct within the ß3AR allowed some sequestration of the receptor (Liggett et al., 1993; Jockers et al., 1996). This result is consistent with the hypothesis that phosphorylation of the receptor by ßARK and the subsequent binding of ß-arrestin could play an important role

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in agonist-promoted sequestration (see Section 2.2.2). This ß2AR domain was, however, not the only one that could confer a sequestration phenotype to the ß3AR. Indeed, substitution of i2 alone had similar effects to that of the substitution of the Ct on the sequestration profile. Furthermore, their simultaneous substitution had additive effects, and led to a sequestration phenotype very similar to that of the ß2AR. The fact that the i2 alone conferred agonist-promoted sequestration to the chimeric receptor is difficult to reconcile with the hypothesis that phosphorylation of the receptor by ßARK is a prerequisite for sequestration, but may suggest that this domain represent a binding site for ß-arrestin, even in the absence of phosphorylation. Although questions remain unanswered concerning the precise determinants involved in some aspects of desensitization and sequestration, there is no doubt that the use of chimeric receptors has confirmed the hypothesis that the resistance of the ß3AR to rapid desensitization is an intrinsic property of the receptor itself which results from the absence of specific regulatory motifs in its primary sequence.

2.7 Potential physiological consequences of the relative resistance of the ß3-adrenoreceptor to desensitization Teleologically, it has been proposed that the refractoriness of the ß3AR to desensitization could be the trademark of an emergency receptor that would become activated only in extreme situations upon a high level of stimulation, and thus should not undergo rapiddesensitization. This notion is supported by the fact that the affinity of the ß3AR for its known endogenous ligands, adrenaline and noradrenaline, is significantly lower that that of the other adrenoreceptors. One could then argue that the physiological contribution of the ß3AR to energetic metabolism would come into play only upon very high level of sympathoadrenal activation at a time when both ß1- and ß2AR have already been desensitized. The ß3AR-stimulated lipolysis would then proceed in adipose tissue for an extended period of time as an ultimate source of energy. In order for such a receptor to play a valuable role, resistance to the mechanism that normally leads to rapid desensitization would be an imperative feature. The relative resistance of the ß 3AR to desensitization may also have clinical implications. Indeed, the lipolytic action of the ß 3AR and its almost exclusive expression into adipose tissues, make it a prime target for the development of antiobesity agents. The observations that both the number and the signalling efficacy of the ß3AR are decreased in the ob/ob mice model of obesity (Collins et al., 1994; Begin-Heik, 1996) have reinforced this notion. It follows that selective ß3-adrenergic agonists have been developed with the idea of generating drugs that could promote weight loss with limited side effects. Treatment for 5–7 weeks with the selective ß3 agonist CL-316,243 has been shown to induce a significant weight loss associated with a reduction of abdominal fat in dogs. The fact that the ß3AR cannot undergo rapid desensitization may have important consequences in that context. It may suggest that agonists targeted at the ß3AR will not lead to the development of rapid tolerance that often limits the effectiveness of agonist drug therapy. However, as discussed above, long-term agonist-promoted down-regulation has been observed to some extent in cultured cell lines and animal tissues. Moreover, desensitization occurring down-stream of the receptor itself in the signal transduction pathway could lead to some tolerance. Because some of these processes have been shown to be cell type-specific, the tachyphilactic properties of ß 3-adrenergic agonists that

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could be clinically useful in humans, will need to be assessed directly in human adipocytes. If some form of desensitization is observed in these cells, a detailed understanding of the molecular processes that are involved will be required in order to develop therapeutic strategies that would minimize tolerance.

2.8 Conclusions Regulation of the G protein-coupled receptor’s signalling efficacy is a well-recognized phenomenon that has important pathophysiological implications. It may also have a direct impact on the therapeutic efficacy and undesirable effects of drugs which act through this class of receptor. Among the various regulatory mechanisms identified, agonist-promoted desensitization, that leads to a loss in tissue responsiveness to endogenous hormones in some pathological conditions and to the development of tolerance in the course of specific drug therapy, has been particularly well characterized. However, studies carried out during the past ten years have shown clearly that, despite the generality of this phenomenon, different receptors have distinct desensitization patterns. In particular, the ß3AR was found to be relatively resistant to agonist-promoted desensitization when compared with the other ßAR subtypes. Indeed, due to the lack of regulatory phosphorylation sites within its sequence, no rapid receptor uncoupling nor receptor internalization could be observed in any of the tissues or cell types studied, indicating that the desensitization refractoriness is an intrinsic property of the receptor that finds its source in its primary structure. In contrast, the extent of longer-term desensitization resulting in part from the regulation of the ß3AR gene expression via mRNA destabilization was found to be cell type-dependent. The role that this type of regulation might play in pathophysiological conditions or in limiting the efficacy of anti-obesity therapies based on the use of ß3AR agonists remains uncertain. Further studies carried out on physiologically relevant tissues and cells such as human adipose tissues and human adipocytes obtained from normal and obese subjects should help to clarify this important question.

3

Using Transgenic and Gene Knockout Techniques to Assess ß3-Adrenoreceptor Function BRADFORD B.LOWELL, VEDRANA S.SUSULIC1, DANICA GRUJIC2 AND MORIKO ITO 3 Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA 1 Present address: Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543, USA 2 Present address: Transkaryotic Therapeutics, 195 Albany St., Cambridge, MA 02139, USA 3 Present address: Novartis, Basel, Switzerland

3.1 Introduction Given that ß3-adrenoreceptors (ß3AR) are abundantly expressed on white and brown adipocytes of rodents, and that ß3AR-selective agonists have potent effects on the function of white and brown adipocytes, it has been presumed that ß3AR play a critical role in mediating effects of the sympathetic nervous system on white and brown adipocytes. However, it has been difficult to test this hypothesis critically, given the general unavailability of ß3ARselective blockers. As a means of assessing the functional importance of ß3AR, our laboratory (Susulic et al., 1995) and another group (Revelli et al., 1997) have used gene targeting to create mice which lack ß3AR. Like any method, the gene knockout approach has advantages and disadvantages. Since gene knockout mice have a complete and selective absence of ß3AR, there are no concerns regarding the incompleteness or non-specificity of ‘blockade’. However, because ß3AR are absent from the first day of embryonic life, questions regarding long-term compensation for loss of ß3AR arise. Despite this and other potential limitations, the gene knockout approach is one of many important tools for assessing adrenoreceptor function in vivo. By generating gene knockout mice lacking a given receptor, it is possible to establish unequivocally the relationship between a cloned receptor and pharmacologically defined activities—an issue which is sometimes a source of controversy.

3.2

3.2.1

Mice lacking ß3AR

Phenotype of mice lacking ß3AR

As shown in Figure 3.1, homologous recombination was used to create mice which lack ß3AR (Susulic et al., 1995). As expected, mice with targeted disruption of the ß3AR gene 36

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Figure 3.1 The ß3AR gene, targeting vectors and recombinant allele. Shown here is a partial restriction enzyme map of the ß3AR gene, the ß3-knockout (KO) targeting vectors and the predicted structure of the recombinant allele. The empirically determined map is consistent with a previously reported genomic map (Nahmias et al., 1991). The targeting vectors contain 12 kb of homologous ß3AR genomic DNA, with 5 kb located 5' and 7 kb located 3' of the PGKNEO-PolyA cassette. The PGK-NEOPolyA vector replaces 306 bp of ß3AR coding sequence between Nhel and Xhol, corresponding to ß3AR residue 120, in the middle of the third transmembrane domain, to residue 222, at the C-terminal end of the fifth transmembrane domain. Boxes refer to exons, the locations of which have been described previously (Granneman et al., 1992; van Spronsen et al., 1993). The translated segments are shown in black. Arrow refers to orientation of transcription. B, BamHl; H, Hind III; N, Nhel; P, Pstl; S, Sall; X, Xhol. (Figure reproduced from Susulic et al., 1995.)

lack intact ß3AR mRNA, and fail to respond to the ß3AR-selective agonist, CL-316,243 (to be discussed later in greater detail). From this it can be concluded that ß3AR gene knockout mice lack functional ß 3AR. Surprisingly, the phenotype of ß3AR gene knockout mice is relatively mild. Brown adipose tissue weight, protein content, DNA content and UCP1 protein content in mice maintained at 23°C or at 4°C is unaffected by the absence of ß3AR (Susulic et al., 1995). However, more recently (in unpublished observations), we have noted that the body temperature and UCP1 mRNA response to cold exposure is sometimes impaired in ß3AR gene knockout mice. This issue is presently being explored in greater detail. Total body fat content is slightly increased in ß3AR gene knockout mice (Susulic et al., 1995; Revelli et al., 1997). This increase in body fat supports the view that ß3AR play a role in maintaining energy homeostasis; however this effect—as observed in gene knockout mice—is not large. The lack of a major phenotype in ß3AR gene knockout mice suggests the following two possibilities: (i) that ß3AR play only a small role in regulating energy homeostasis; and (ii) that other gene products compensate for the loss of ß3AR. Given that ß1, ß2 and ß3AR all recognize the same extracellular ligands (noradrenaline and adrenaline) and signal via adenylyl cyclase to increase cAMP levels, and that ß1 and ß2ARs are expressed in white and brown adipocytes, it is reasonable to speculate that ß1 and/or ß2AR compensate for the absence of ß3AR. Indeed, we noted that ß1AR mRNA levels were up-regulated by 76% in brown fat and by 42% in white fat of ß3AR gene knockout mice (Susulic et al., 1995). However, these findings are in contrast to that of

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Revelli et al. (1997), where ß1AR mRNA levels were actually found to be 66% lower in brown fat of ß3AR gene knockout mice. The reason for this difference is unknown, but could be due to differences in background strain between the two studies. It is also possible that ß1 and ß2AR activity may have increased, independent of effects on gene expression. For example, it is known that agonist exposure results in the desensitization of ß1- and ß2AR. This raises the possibility that an absence of ß3AR, with its predicted decrease in adrenergic tone, might result in compensatory increases in the activity of ß1- and ß2AR.

3.2.2 Effects of ß3AR deficiency on catecholamine-mediated stimulation of adenylyl cyclase and lipolysis in adipocytes In normal mice, CL-316,243—a ß3AR-selective agonist—increases adenylyl cyclase activity in white and brown adipocyte membranes by 4- to 8-fold, and stimulates lipolysis in isolated white adipocytes by approximately 5-fold. These effects are completely absent in membranes and adipocytes isolated from ß3AR gene knockout mice (Susulic et al., 1995). Such results demonstrate that ß3AR gene knockout mice lack functional ß3AR. Isoproterenol is a ß1, ß2 and ß3AR agonist, and its effects on adenylyl cyclase activity and lipolysis in ß3AR gene knockout mice are of interest (Figure 3.2) (Susulic et al., 1995). The ability of isoproterenol to stimulate adenylyl cyclase activity maximally, was markedly impaired in membranes derived from ß3AR gene knockout mice. Specifically, maximally stimulated adenylyl cyclase activity was decreased by 70–80% in membranes derived from white and brown adipocytes, an observation which suggests that much of ßAR signalling in adipocytes is mediated, in large part, by ß3AR. With regards to a functional response such as lipolysis, however, the effect of ß3AR deficiency on adrenergic signalling is more complex, being heavily influenced by the presence or absence of negative effects on adenylyl cyclase activity (Susulic et al., 1995). Isolated adipocytes incubated in vitro normally produce adenosine, which activates adipocyte A1-adenosine receptors. These A1-adenosine receptors couple negatively with adenylyl cyclase, thus decreasing lipolysis. In order to avoid the confounding effects of adenosine during incubation of adipocytes, it is common to add adenosine deaminase, which degrades endogenously produced adenosine. When adipocytes were incubated in the presence of adenosine deaminase, isoproterenol-stimulated lipolysis was decreased by only 33% in adipocytes from ß3 AR gene knockout mice when compared with adipocytes from control mice. However, when adipocytes were incubated with adenosine deaminase and PIA (N 6[R-(–)-1-methyl-2-phenyl]adenosine), an A1-adenosine receptor agonist, isoproterenol-stimulated lipolysis was completely absent in ß 3 AR-deficient adipocytes. In other words, in white adipocytes isolated from control mice, PIA inhibited isoproterenol-induced lipolysis minimally, whereas in adipocytes isolated from ß3AR-deficient mice, PIA completely blocked isoproterenol-induced lipolysis. Thus, the ability of A1-adenosine receptors to inhibit catecholamine-induced lipolysis is critically dependent upon the abundance of ßAR (all three ßAR in control mice versus only ß1- and ß2AR in knockout mice). These findings demonstrate that the ratio of G i -coupled receptor activity to total ßAR activity has marked consequences for functional responses such as lipolysis.

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Figure 3.2 Adenylyl cyclase activity and lipolysis in response to CL-316,243 and isoproterenol. (A) Adenylyl cyclase activity. Membranes were obtained from isolated white adipocytes and brown adipose tissue of 8 to 12-week-old male wild-type (+/+) and ß3AR-deficient (-/-) littermates, and then assayed for adenylyl cyclase activity. Adenylyl cyclase response to maximally effective doses of CL-316,243 (CL) and isoproterenol (ISO) are shown. Results are expressed as the mean (±SEM) of 10 experiments. (B) Lipolysis in isolated white adipocytes. White adipocytes were isolated from epididymal fat pads of 8- to 12-week-old male wild-type (+/+) and ß3AR-deficient (-/-) littermates and then assayed for glycerol release as an indicator of lipolysis. Previous studies using wild-type adipocytes demonstrated that 10µM CL and 100µM isoproterenol produced maximal increases in lipolysis. Lipolysis. assays were performed in the presence of adenosine deaminase (ADA) and N6-phenyliso-propyladenosine (PIA) (left panel, +ADA, +PIA) or with adenosine deaminase only (right panel, +ADA, -PIA). Results are expressed as the mean (±SEM) of three experiments. (Figure reproduced from Susulic et al., 1995.)

These findings most likely relate to the observation that in mouse white adipocytes, ß1- and ß2AR mRNA transcripts are only 1/50 to 1/150 as abundant as ß3AR mRNA transcripts (Collins et al., 1994), implying that white adipocytes possess many more ß3AR than ß1- and ß2ARs (Figure 3.3). Consequently, maximal activation of ß1- and/or ß2AR produces a small stimulatory effect on adenylyl cyclase activity, and this smaller effect is more readily inhibited by negative influences on adenylyl cyclase activity, such as that induced by PIA treatment. This observation could have important implications for signalling in human white adipocytes, which possess few ß3AR and abundant a2AR (Lafontan and Berlan, 1995). a2AR, like A1-adenosine receptors, are negatively coupled to adenylate cyclase.

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Figure 3.3 Regulation of lipolysis in mouse adipocytes (a working model). ß3AR are significantly more abundant than ß1- and ß2AR in rodent adipocytes. In adipocytes from a wildtype mouse, combined stimulation of ß1, ß2 and ß2AR by isoproterenol easily overcomes the inhibitory influence of the A1-adenosine receptor on stimulation of lipolysis. In contrast, in gene knockout mice which lack ß3AR, inhibition via the A1-adenosine receptor easily overrides the stimulatory effect of the less abundant ß1- and ß2AR.

3.2.3

Effects of ß3AR deficiency on in vivo effects of CL-316,243

In normal mice, CL-316,243 increases whole body energy expenditure, increases insulin levels acutely, and decreases food intake acutely. CL-316,243 treatment also markedly reduces leptin gene expression in adipose tissue (Mantzoros et al., 1996). All of these actions are completely absent in ß3AR gene knockout mice (Susulic et al., 1995; Mantzoros et al., 1996). In fact, we have been unable to identify any response to CL316,243 which is preserved in ß3AR gene knockout mice. Thus, it can be concluded definitively that the actions of CL-316,243 are mediated exclusively by ß3AR. CL316,243 does not appear to interact with any additional receptors in causing the effects listed above; thus, there can be little doubt that CL-316,243 can be used as a reference standard for ß3AR in mice.

3.3 Role of ß3AR on white versus brown adipocytes in mediating effects of ß3-selective agonists on energy expenditure, insulin secretion and food intake As mentioned above, acute treatment of normal mice stimulates whole body energy expenditure 2-fold, increases insulin secretion 50- to 140-fold (Yoshida, 1992; Susulic et al., 1995) and decreases food intake by 45% (Tsujii and Bray, 1992; Himms-Hagen et al., 1994; Susulic et al., 1995). The relative role of ß3AR in white versus brown adipocytes, as well as ß3AR in other sites in mediating each of these effects has been unknown. In general, it has been difficult to determine the relative role of various target tissues in mediating complex physiological responses. Genetic engineering in mice, however, provides a means by which these issues can be addressed. In this respect, we

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Figure 3.4 Transgenic tissue-specific re-expression of murine ß3AR in knockout mice. ß3AR gene knockout mice were bred to produce embryos which are homozygous for the ß3AR gene knockout allele. These embryos were injected with either the aP2-ß3AR transgene or the UCPß3AR transgene to generate mice which express ß3AR in white and brown fat only (WAT+BATmice), or in brown fat only (BAT-mice), respectively.

have combined gene knockout and transgenic techniques to create mice in which functional ß3AR are completely absent (knockout mice; Susulic et al., 1995), or are expressed exclusively in selected tissues, namely white and brown adipose tissue (WAT+BAT-mice), or brown adipose tissue only (BAT-mice) (Grujic et al., 1997). To create WAT+BAT-mice and BAT-mice (Figure 3.4), transgenic constructs were generated in which murine ß3AR gene expression is driven by the tissue-specific promoter/ enhancers, aP2 for white and brown adipose tissue expression (Ross et al., 1990) and UCP1 for brown adipose tissue expression (Boyer and Kozak, 1991; Cassard-Doulcier et al., 1993). These transgenic constructs were then injected into fertilized mouse zygotes homozygous for the ß3AR gene knockout allele (Susulic et al., 1995), thus creating mice in which functional ß 3 AR are restricted to white and brown fat (WAT+BAT-mice), or brown fat only (BAT-mice). Control, knockout, WAT+BAT and BAT-mice were then used to investigate the relative role of ß3AR in white versus brown adipose tissue, as well as to rule out involvement of ß3AR in other sites, in mediating a number of responses to ß3-selective agonists. As will be summarized below, the effects of CL-316,243 on energy expenditure, insulin secretion and food intake were found to be mediated exclusively by ß3AR on white and brown adipocytes.

3.3.1

Role of white versus brown adipocyte ß3A R in mediating effects of CL-316,243 on energy expenditure

In general, it is assumed that the stimulatory effect of CL-316,243 on energy expenditure is mediated by ß3AR on brown adipocytes. In order to test this hypothesis, control, knockout, WAT+BAT and BAT-mice were treated with CL-316,243 and effects on energy expenditure (oxygen consumption) were assessed. As before, CL produced

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a large increase in oxygen consumption in control mice, and no response in knockout mice. In WAT+BAT mice, CL produced a full stimulation of oxygen consumption. In BAT-mice, however, CL produced only a partial stimulation of oxygen consumption (20% of control response). These findings indicate that the full thermogenic response to acute ß3-agonist treatment requires the presence of ß3AR on both white and brown adipocytes. The mechanism by which stimulation of white adipocytes greatly augments the thermogenic response is unknown, but could be related to mobilization of free fatty acids which are then used as substrates by brown fat. Alternatively, ß3agonist-stimulated white adipocytes, which express high levels of uncoupling protein2 (Fleury et al., 1997; Gimeno et al., 1997), might contribute directly to energy expenditure.

3.3.2

Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on Insulin secretion

The mechanism by which ß 3-selective agonists acutely cause large increases in insulin secretion has been unknown. This is especially true since pancreatic beta cells do not express ß3AR. In order to test the role of adipocytes in mediating this response, control, knockout, WAT+BAT and BAT-mice were treated with CL316,243 and effects on insulin secretion were assessed. CL produced a large increase in insulin secretion in control and WAT+BAT mice, but not in knockout or BAT-mice. This demonstrates that ß 3AR on white adipocytes are required for this response. Thus, a signal emanating from white adipocytes can, directly or indirectly, profoundly alter pancreatic beta-cell function. This signal may be free fatty acids, which are known to be secretagogues for insulin secretion (Prentki et al., 1992; Warnotte et al., 1994).

3.3.3

Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on food intake

Although acute treatment with ß3AR-selective agonists acutely decreases food intake (Tsujii and Bray, 1992; Himms-Hagen et al., 1994; Susulic et al., 1995; Mantzoros et al., 1996), the mechanism for this effect has been unknown. In order to test the role of adipocytes in mediating this response, control, knockout, WAT+BAT and BAT-mice were treated with CL-316,243 and effects on food intake were assessed. CL reduced food intake in control and WAT+BAT mice, but not in knockout or BAT-mice. Like the insulin secretion response, ß3AR on white adipocytes are required for inhibition of food intake. Since expression of ß3AR in adipose tissue (WAT+BAT-mice) completely restored the inhibitory food intake response, it can be concluded that ß3AR in other sites such as the brain are not involved. The nature of the signal emanating from adipose tissue which mediates the inhibitory effect of ß3-selective agonists on food intake is unknown. Leptin is a fat-derived protein that regulates appetite (Zhang et al., 1994); however, it is unlikely to meditate this response since leptin levels decrease substantially following ß3-agonist treatment (Giacobino, 1996; Mantzoros et al., 1996), and this would be predicted to have a stimulatory effect on food intake. Insulin is another factor that has been shown to suppress food intake (Woods and Gibbs, 1989; Woods et al., 1990). Since insulin levels rise acutely following ß3-agonist treatment, it

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could mediate decreased food intake. Finally, heat has long been known to have inhibitory effects on appetite (Strominger and Brobeck, 1953; Brobeck, 1960), and it has been postulated that heat generation by brown fat regulates food intake (HimmsHagen, 1995a,b). Increased heat production could be responsible for ß3-agonistinduced inhibition of food intake.

3.4

Creation of mice which express human, but not murine, ß3AR

Important similarities and differences exist between human and rodent ß3AR, and these differences have significant implications for the development of anti-obesity drugs. The receptors are similar in that they are both expressed predominantly in adipose tissue, their amino acid sequences are about 80% identical, and their pharmacological profiles are similar in that both receptors are relatively resistant to blockade by conventional ßAR antagonists and both are stimulated by ß3AR-selective agonists (Emorine et al., 1989; Granneman et al., 1991; Muzzin et al., 1991; Nahmias et al., 1991). Human and rodent ß 3AR differ, however, in two important ways. First, their relative expression in white versus brown adipocytes, and second, the degree to which they can be stimulated by specifie ß3AR-selective agonists. In rodents, ß3AR mRNA is abundant in WAT and BAT (Granneman et al., 1991; Muzzin et al., 1991; Nahmias et al., 1991), while in humans, ß 3AR mRNA is abundant in BAT only (Granneman et al., 1992; Granneman and Lahners, 1994; Granneman, 1995), with much less (Krief et al., 1993; Revelli et al., 1993; Berkowitz et al., 1995) or no (Thomas and Liggett, 1993) ß 3AR mRNA being found in WAT. In contrast with analyses of mRNA expression, some pharmacological studies support the existence of ß3AR in human white adipocytes (Lönnqvist et al., 1993; Enocksson et al., 1995; Hoffstedt et al., 1996a; Tavernier et al., 1996), though another study did not provide such support (Rosenbaum et al., 1993). For the most part, pharmacological evidence supporting the existence of ß3AR in human white adipocytes depends upon the demonstration that CGP-12,177, a ß1- and ß2AR blocker with partial ß3AR agonist activity, stimulates lipolysis in human white adipocytes. However, the significance of this finding in the absence of abundant ß3AR mRNA expression is unknown since CGP-12177 might possibly interact with another, as yet unidentified, receptor (Galitzky et al., 1997) (see below). Human ß3AR also differ from rodent receptors with respect to their ability to be activated by ß3AR-selective agonists. Many agonists which are extremely potent against the murine ß3AR, such as CL-316,243 and BRL-37,344, are only weakly effective against the human ß3AR (Blin et al., 1994; Dolan et al., 1994). As might be expected, these agents have not performed well as anti-obesity compounds in human clinical trials. Presently, cell lines expressing recombinant human ß3AR are being used to screen for compounds capable of potently activating the human ß3AR. In order to address differences in sites of expression and pharmacology between human and rodent ß 3AR, we have transgenically introduced a P1 genomic clone containing a large fragment of the human ß3AR genomic locus into ß3AR gene knockout mice (Ito et al., 1998) (Figure 3.5). Using a sensitive RNase protection assay, we observed that human ß3AR mRNA was expressed almost exclusively in BAT of mice transgenic for the human ßAR P1 genomic clone (Ito et al., 1998) (Figure 3.6), with little or no expression being

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Figure 3.5 Transgenic expression of human ß3AR in knockout mice. Embryos homozygous for the ß3AR gene knockout allele were injected with a P1-genomic clone bearing the human ß3AR genomic region.

Figure 3.6 RNase protection analysis of human (HU) ß3AR gene expression in multiple tissues. An RNase protection assay was used to detect human ß3AR mRNA in RNA samples isolated from brown adipose tissue (B), perigonadal white adipose tissue (W), liver (L), stomach (ST), small intestine (SI) and muscle (M). The RNA samples were isolated from tissues obtained from four different transgenic lines (H2, H3, H4 and H10). All RNase protection analyses were performed using 40 µg of total. K, ß3AR gene knockout mice; T, human ß3AR transgenic mice on the ß3AR gene knockout background. (Figure reproduced from Ito et al., 1998.)

detected in three WAT depots (perigonadal, inguinal and perirenal). Given the large size of the genomic clone employed in this study (~33 kb of 5' flanking sequence and ~44 kb of 3' flanking sequence), and that four out of four transgenic lines generated the same result, it is extremely likely that human versus rodent cis-regulatory elements within the ß3AR gene differ with respect to their ability to direct gene expression to white versus

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brown adipocytes. The human ß3AR cis-regulatory elements direct expression to brown, but not white, adipocytes while the rodent cis-regulatory elements direct expression to both brown and white adipocytes. Our finding that human ß3AR cis-regulatory elements are active in brown but not white adipocytes is in agreement with negative studies of mRNA expression in human WAT samples (Thomas and Liggett, 1993; Granneman and Lahners, 1994), suggesting that ß3AR do not exist or are rare in human white adipocytes. This conclusion then raises the possibility that CGP-12,177-stimulated lipolysis in human white adipocytes is mediated by another ‘atypical’ receptor. Functional evidence for an additional ‘atypical’ receptor, i.e. a ‘ß4AR’, has recently emerged, and will be discussed below in greater detail. Given that these transgenic animals express human but not murine ß3AR in a ‘humanlike’ pattern, i.e. in brown but not white fat, they should be useful in evaluating the potential efficacy of newly identified ß3AR-selective agonists which are potent for the human ß 3 AR. In order to assess this possibility, we have observed that acute administration of CGP-12,177 causes oxygen consumption in ‘humanized’ mice to increase by 91%, compared with an increase of only 49% in ß3AR gene knockout controls (Ito et al., 1998) (Figure 3.7). The stimulatory effect observed in the control animals (ß3AR gene knockout mice) is presumably mediated by an additional receptor (possibly the ‘ß4AR’ referred to earlier). Since the only difference between ‘humanized’ mice and gene knockout mice is the presence of the human ß3AR transgene, the larger effect of CGP-12,177 on oxygen consumption in ‘humanized’ mice strongly indicates that human ß3AR in these transgenic mice can effectively couple with increased energy

Figure 3.7 In vivo effects of CGP-12,177 on O2 consumption. ß3AR gene knockout mice (KO) and human transgenic mice (lines H3 and H4) on the ß3AR gene knockout background were treated with CGP-12,177 (s.c. injection of 1 mg/kg body weight) and effects on O2 consumption were assessed. Mice were awake and unrestrained for analysis. The results are expressed as % change in Vo2 over basal (i.e. pre-injection resting oxygen consumption), and are the mean (±SEM) for each group. Basal and post-injection Vo2 are the averages of seven consecutive measurements. Numbers of animals for each group were as follows: KO=11; H4=6. *=P<0.05, compared with KO. (Figure reproduced from Ito et al., 1998.)

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expenditure. These animals should assist in the analysis and development of drugs which might possibly become effective anti-obesity agents in humans.

3.5

3.5.1

Using ß3AR gene knockout mice to study ‘ß4AR’ activity

Evidence for the existence of ‘ß4AR’

Substantial evidence suggests that additional, not yet cloned ‘atypical’ ßAR exist (Arch and Kaumann, 1993; Kaumann, 1997). One or more of these novel ßAR may play key roles in regulating processes important in normal biology and pathophysiology. Data supporting the existence of additional ßAR are summarized below. Using isolated atria preparations, it has been demonstrated that CGP-12,177, a partial ß3-agonist with ß1- and ß2AR antagonist activity, increases heart rate (Arch and Kaumann, 1993; Kaumann and Molenaar, 1996; Kaumann, 1997). This finding has been made with CGP-12,177 as well as with other related compounds, using a number of independent approaches and in multiple species, including rats and humans (Kaumann and Molenaar, 1996; Malinowska and Schlicker, 1996; Kaumann, 1997). Since CGP-12,177 antagonizes ß1 and ß 2AR, these receptors cannot mediate this chronotropic effect. Furthermore, ß1 and ß2-selective blockers have no effect on this activity. Importantly, ß3AR mRNA is undetectable in rat atria, even when sensitive polymerase chain reaction (PCR) assays are used (Evans et al., 1996); thus, it is unlikely that the ß3AR mediates this effect. In support of this, CL-316,243—a much more potent and selective agonist against the rat ß3AR—has no effect on heart rate in isolated rat atrial assay (Kaumann and Molenaar, 1996). In addition, SR-59,230, a new ß3-selective blocker, does not inhibit the stimulatory effect of CGP-12,177 on heart rate (Kaumann and Molenaar, 1996). Thus, evidence is overwhelming that the heart ‘atypical’ receptor is not the ß1, ß2 or ß3AR. Nevertheless, the ‘atypical’ receptor is pharmacologically more similar to the ß3AR than to any other receptor. Both are stimulated by CGP-12,177, both are relatively resistant to antagonism by propranolol, both are effectively antagonized by bupranolol (a ß1, ß2 and ß3 blocker), and both are coupled positively with adenylyl cyclase (Kaumann and Lynham, 1997). As was discussed above at length, data suggest that human adipocytes express little or no ß3AR mRNA. Therefore, stimulation of lipolysis in human adipocytes by CGP12,177 is likely to be mediated by another ‘atypical’ ßAR. In addition, it has been demonstrated recently that CGP-12,177-mediated stimulation of lipolysis in human white adipocytes is resistant to blockade by the ß3-selective blocker, SR-59,230 (Galitzky et al., 1997). These data suggest that human adipocytes also possess an additional ‘atypical’ ßAR. Evidence for additional ‘atypical’ ßAR in skeletal muscle is based upon observations of effects with ß3-selective agonists not prevented by ß1 and ß2 blockade, and the absence of detectable levels of ß 3AR mRNA. Specifically, studies have demonstrated that a number of ß3-agonists such as BRL-37,344, ICI-D7114 and CGP12,177 increase oxygen consumption (Thurlby and Ellis, 1986; Gainer et al., 1993; Ye et al., 1995) and glucose utilization (Abe et al., 1993; Liu et al., 1996a) in isolated skeletal muscle preparations. These effects have been observed despite the fact that skeletal muscle does not express ß3AR mRNA (Granneman et al., 1991 ; Evans et al., 1996). Therefore, it is likely that a presently unidentified ‘atypical’ ßAR mediates these actions.

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47

Assessment of ‘ß4AR’ activity in ß3AR gene knockout mice

Taken as a whole, the evidence presented above makes a strong case for the existence of one or more additional ‘atypical’ ßARs. Of course, pharmacological studies alone cannot completely rule out the possibility that one of these activities is mediated by one of the known ßAR. Given the nature of the pharmacological evidence cited above, the most likely receptor to cause confusion is the ß3AR. ß3AR gene knockout mice make it possible to exclude any role of the ß3AR. CGP-12,177, which is a partial ß3AR agonist with ß1 and ß2-blocker activity, becomes a useful reference compound for ‘ß4AR’ activity when used in ß3AR gene knockout mice. Along these lines, we have begun to assess CGP-12,177-stimulated activities in ß 3AR gene knock-out mice. An example of such studies was shown in Figure 3.6. As mentioned earlier, CGP-12,177 caused a 49% increase in oxygen consumption in ß 3AR gene knockout mice (Ito et al., 1998). Of note, no such effect is observed in ß3AR gene knockout mice when either saline or CL-316,243, a pure rodent ß3AR agonist, was injected. This finding provides strong evidence that CGP-12,177 can stimulate energy expenditure by interacting with an additional ‘atypical’ ßAR. Thus, this novel receptor might be relevant to the regulation of energy balance, and efforts are underway to further identify its nature.

3.6

Conclusions

Transgenic and gene knockout approaches can be useful tools in pharmacology research. Such studies make it possible to establish definitively the receptor and tissue site where agonists have their effects in vivo. We have used such methods to study the ß3AR. These studies have established that all effects of the agonist, CL316,243, are mediated exclusively by ß3AR on white and brown adipocytes. We have also used these approaches to create mice which express human, but not murine, ß3AR under the control of human ß3AR gene regulatory elements. These animals should be useful in evaluating the potential in vivo potency of various agonists capable of stimulating the human ß3AR. Finally, it is anticipated that ß3AR gene knockout mice, because they lack ß 3ARs, will assist in the characterization of additional ‘atypical’ ßAR. Note added in proof Results published in a recent paper by Kankar et al. (2000) suggest that the stimulatory effects of CGP-12177 on brown adipocytes may be due to an atypical interaction with the ß1AR.

Acknowledgements We wish to thank the following collaborators who were extremely helpful in completing the investigations discussed above: E.Dale Abel, Barbara E.Corkey, Barbara A.Cunningham, Jeffrey S.Flier, Robert C.Frederich, Jean Himms-Hagen, Mary-Ellen Harper, Barbara B.Kahn, Joel Lawitts, A.Donny Strosberg, Effie Tozzo and Antonio Vidal-Puig.

4

ß3-Adrenoreceptor Ligands and the Pharmacology of the ß3-Adrenoreceptor JONATHAN R.S.ARCH Department of Vascular Biology, SmithKline Beecham Pharmaceutical, Harlow CM19 5AD, United Kingdom

4.1 Introduction It was apparent that a third ß-adrenoreceptor (ßAR) existed some years before the cloning of the human ß3AR removed any remaining doubts (Emorine et al., 1989). Just as adrenoreceptors (ARs) had been classified into aARs and ßARs by Ahlquist, and ßARs into ß1- and ß2AR by Lands et al. (1967a,b), it was the availability of ligands which displayed a unique affinity or efficacy for the ß3AR that pointed to their existence. It was the availability of these ligands that also enabled the cloned ß3AR to be rapidly characterized, and its role in mammalian tissues to be understood. Early evidence for the ß3AR stemmed not from the existence of ligands with higher potency at the ß3- than ß1- or ß2ARs, but, on the contrary, from the discovery that ß1and ß2AR antagonists lacked potency in certain tissues. Furchgott (1972) summarized early data of this type for various gut preparations, while Zaagsma et al. (1985) summarized the early work of their group on adipocytes. However, evidence for the ß3AR that was based on the ‘negative selectivity’ of ßAR antagonists was unconvincing to many workers, especially since the ß3AR could not, at that time, be detected by binding studies. Binding studies were difficult because the available radiolabelled ligands were antagonists with low affinity for the ß3AR, and the ß3AR is always expressed in tissues alongside ß1- or ß2ARs. The ß3AR has subsequently been detected using the ‘negatively selective’ antagonists [125I]-iodocyanopindolol and [3H]-CGP-12177, but it has only been by using higher concentrations than would be used for ß1- or ß2AR binding that this has been possible. In the absence of ß3AR-selective antagonists, it was the discovery by Beecham Pharmaceuticals of selective ß3AR agonists that gave the impetus to the field. These novel agonists were more potent as stimulants of rat brown or white adipocyte lipolysis than as stimulants of atrial contraction, or tracheal or uterine relaxation (Arch et al., 1984a; Wilson et al., 1984). The Beecham compounds and ßAR agonists from HoffmanLa Roche and Eli Lilly were found to stimulate metabolic rate and to have anti-obesity and anti-diabetic activity in rats and mice, giving therapeutic relevance to the novel receptor (Arch et al., 1984b; Cawthorne et al., 1984; Meier et al., 1984; Yen et al., 1984). 48

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When the human ß3AR was cloned and expressed in 1989 (see Chapter 1), one of the Beecham compounds, BRL-37344, was found to be a potent stimulant of cyclic AMP synthesis. However, as studies continued with the human cloned ß3AR and human adipocytes, it was realized that the first-generation agonists, which had been selected for their thermogenic and anti-obesity activity in rats and mice, were far from ideal agonists of the human ß 3AR. Their efficacy at the human ß 3AR is especially poor (Wilson et al., 1996; see also comment in introduction of Fisher et al., 1998); it may be sufficient for them to be potent stimulants of adenylyl cyclase in cells transfected with high numbers of the ß3AR, but in human tissues the firstgeneration agonists elicited either little or no response, or a response mediated via ß1- or ß2ARs (Hoffstedt et al., 1996a). During the 1990s therefore, a number of pharmaceutical companies attempted to identify agonists with high efficacy and selectivity for the human cloned ß3AR. Difficulties persist in studies of the role of the ß3AR, even in rodents, in part because there are still no highly selective antagonists. Sanofi have described a series of aryloxypropanolaminotetralins that show some selectivity (about 30-fold) between rodent tissues (Manara et al., 1996), but their utility is questionable (Strosberg and PietriRouxel, 1997). Merck have described two compounds with moderate selectivity for the human cloned receptor, but neither binds to the rodent ß3AR (Candelore et al., 1998). An even greater problem is that certain aryloxypropanolamine ß3AR agonists also stimulate another non-ß1/2AR. (Hoey et al., 1996a; Galitzky et al., 1997; Kaumann et al., 1998; Preitner et al., 1998). This receptor has been called the putative ‘ß4’AR, a term now recognized by the IUPHAR nomenclature committee (Bylund et al., 1998), although it seems premature to assume that it is an AR, let alone a ßAR. This chapter therefore describes ‘ß4’AR (for want of a better term) as well as ß 3AR ligands and their pharmacology.

4.2

4.2.1

Antagonists

ß1/2AR-selective antagonists

Most standard ß1AR and ß2AR antagonists (Figure 4.1), whether ß1AR- or ß2ARselective, or non-selective between these subtypes, have 100- to 1000-fold lower affinity for the ß3AR than for ß1- and/or ß2ARs. Some of these compounds are agonists at the ß 3AR and are described in Section 4.3. The present section focuses on compounds that are antagonists of all three receptors, except for the inclusion of the standard antagonist propranolol, which is a partial agonist of the human cloned ß3AR (Blin et al., 1993). The low affinity of standard antagonists for the rodent ß3AR has, for the most part, been deduced from studies of ßAR pharmacology in tissues. pA2 or pKB values for antagonism of white and brown adipocyte lipolysis, and gut relaxation have been collated by Arch and Kaumann (1993) and compared with similar values for antagonism of atrial rat stimulation (ß1AR-mediated) or uterine or tracheal relaxation (ß2ARmediated). These data are exemplified in Table 4.1. Interpretation of tissue pharmacology studies is complicated by the presence of ßARs other than the ß3AR in these tissues. For example, receptor binding studies show that both ß1- and ß2ARs are present in rat white adipocytes (Bojanic and Nahorski, 1983), and in one study it appeared that isoprenaline stimulated lipolysis via both ß1- and ß3ARs

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Figure 4.1 Chemical structures of selective antagonists of ß1- and ß2ARs.

(Hollenga and Zaagsma, 1989). Moreover, the ‘ß4’AR also has a low affinity for at least some of the standard antagonists, but it does show some differences from the ß3AR in this respect (see Chapter 8). If any doubts remained in the interpretation of rodent tissue pharmacology experiments, they have, in any event, been allayed by the demonstration

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Table 4.1 Affinities (pA2 or pKB) of ß1/2AR-selective antagonists for ßARs determined from functional studies using rodent tissues*

* Isoprenaline was used as the agonist in each case. Similar or slightly lower pA2 or pKB values were obtained for the antagonists when a selective ß3AR agonist was used † Rat right atrial rate [ref. 1]; guinea-pig right atrial rate [refs. 2, 3, 4, 6] ‡ Rat diaphragm contraction [ref. 1]; guinea-pig uterine relaxation [ref. 2]; rat uterine relaxation [ref. 4]; guinea-pig tracheal relaxation [ref. 6] § Rat white adipocyte lipolysis ** Difference in pA2 or pKB values for (-)- and (+)-enantiomers [1] Harms et al., 1977; [2] Molenaar and Summers, 1987; [3] Hollenga and Zaagsma, 1989; [4] Bilski et al., 1983; [5] Wilson et al., 1984; [6] Lemoine and Kaumann, 1983; [7] Langin et al., 1991

Table 4.2 Affinities of ß1/2AR-selective antagonists for human and rodent cloned ßARs*

* Ki values were determined from displacement of [125I]-iodocyanopindolol binding. ‘pKB’ values are -log10 of the concentration of antagonist that caused 50% inhibition of the stimulation of cyclic AMP accumulation by isoprenaline at its Kact concentration (5 nM) in Chinese hamster ovary cells transfected with the ß3AR [1] Blin et al., 1993; [2] Muzzin et al., 1991 for rat; [3] Arch and Wilson, 1996b; [4] Blin et al., 1994 for mouse; [5] Tate et al., 1991; [6] Emorine et al., 1989

that propranolol and ß1AR- and ß2AR-selective antagonists have low affinity in binding studies for the rat cloned ß3AR (Table 4.2). Few studies on the pharmacology of antagonists at the human ß3AR have been conducted using human tissues. There is some evidence from studies of isoprenaline-

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stimulated white adipocyte lipolysis that CGP-20712A (ß1AR-selective) and ICI-118,551 (ß2AR-selective) have low affinity for the human ß3AR (Zaagsma and Hollenga, 1991), but the preponderance of ß1- and especially ß2ARs in human white adipocytes makes such studies far more difficult to interpret than similar studies on rodent adipocytes. Stimulation of ß1- and ß2ARs can be avoided by using the ß3AR agonist CGP-12177, but CGP-12177 also stimulates the ‘ß4’AR (see Section 4.3). However, Sennitt et al. (1998) used novel agonists that stimulate only ß3- and not ‘ß4’ARs and showed that nadolol has a low affinity for the ß3AR in human white adipocytes (see Section 4.4.2). By working with the human cloned ß3AR it has been clearly demonstrated that standard antagonists have low affinities for these receptors (Table 4.2). For the most part, affinity has been determined from binding affinity, but low functional potencies for antagonists have also been described. There is some evidence that ICI-118,551 has a higher affinity for the human than the rat ß3AR, but this difference has not been seen in all studies (Table 4.2). Early tissue pharmacology studies showed that the rat white adipocyte ß3AR is less able than the rat cardiac ß1AR and the rat hemidiaphragm ß2AR to distinguish between the more potent (—)- and the less potent (+)- enantiomers of ß1/2AR antagonists (Table 4.1). Unfortunately, it seems that there has been no attempt to confirm this finding using the rodent cloned ß3AR. The human cloned ß3AR distinguishes well between the (—)and (+)- enantiomers of propranolol in both binding (Ki ratio= 154) and functional (Ki ratio=74) studies (Table 4.2). In the absence of readily available and well-validated selective ß3AR antagonists, potent but ‘negatively selective’ ß3AR antagonists have been employed to characterize ß3AR-mediated responses. (—)-Bupranolol is now often used for this purpose. Its pKB value for antagonism of human or rat white adipocyte lipolysis, or the ß3AR-mediated negative inotropic effect of BRL-37344 in human ventricle ranges from 6.3 to 7.6 (Gauthier et al., 1996; Galitzky et al., 1997; see also Arch and Kaumann, 1993). However, (—)-bupranolol antagonizes the ‘ß4’AR with similar affinity (Galitzky et al., 1997; Kaumann, 1997), which limits its utility.

4.2.2

Non-selective and ß3AR-selective antagonists

Selective ß3AR antagonists do not have the obvious therapeutic potential of ß3AR agonists, and so there has been less incentive to identify them. Nevertheless, it is surprising that until recently no ß3AR antagonists with a selectivity better than 30-fold were known. Two tolamolol derivatives (Compounds 3 and 11 in Figure 4.2) have been described that are almost as potent as antagonists of rat white adipocyte lipolysis as of rat atrial rate or diaphragm contraction (Table 4.3), but these have not been employed in other studies. There has, by contrast, been considerable interest in a series of Sanofi aryloxypropanolaminotetralins (Manara et al., 1996), of which the most potent, SR59230A (see Figure 4.2), has been studied by a number of workers. SR-59230A was about 30-fold more potent as a relaxant of rat isolated proximal colon (ß3) than as a stimulant of guinea-pig right atrial rate (ß1) or as a relaxant of guinea-pig trachea (ß2) (Table 4.3). In studies in vivo, SR-59230A was 20 and 12 times more potent against the colonic response than against cardiac or bronchial responses respectively. SR59230A has the S configuration at both points of asymmetry in the molecule, and it is interesting that, in contrast to the ß1/2AR-selective antagonists described above (Table

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Figure 4.2 Chemical structures of non-selective and ß3AR-selective antagonists.

4.1), the RR enantiomer was at least 75-fold less potent. In other words, the relatively poor ability of the rat ß3AR to distinguish between stereoisomers of ßAR antagonists does not extend to SR-59230A, or indeed to the related molecule SR-58894A (Manara et al., 1996). It should be noted that the potent antagonism of rat colon relaxation originally reported by Manara et al. (1996) has not been confirmed by others (Kaumann and Molenaar, 1996) (Table 4.3).

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Table 4.3 Affinities of non-selective and ß3AR-selective antagonists for human and rodent ßARs

* Isoprenaline was used as the agonist in functional studies † Schild slope (0.54) less than 1 ‡ Partial agonist; intrinsic activity=0.45 [1] de Vente et al., 1980: ß1 rat atria, ß2 rat diaphragm; [2] Manara et al., 1996: ß1 guinea-pig atria, ß2 guinea-pig trachea; [3] Kaumann and Molenaar, 1996; [4] Kubo et al., 1997: ß2 rat white adipose tissue; [5] Nisoli et al., 1996a; [6] Bardou et al., 1998; [7] De Ponti et al., 1996; [8] Strosberg and Piétri-Rouxel, 1997; [9] Candelore et al., 1998: ß1 and ß2 human cloned receptors

SR-59230A showed very high affinity binding (pKi=9.47) for a ß3AR site on rat white adipocytes and low affinity (pKi=5.14) for a ß2AR site (Kubo et al., 1997). The original report on the aryloxypropanolaminotetralins (Manara et al., 1996) commented that they were only weak antagonists of in vitro lipolysis, and suggested that they bound to albumin present in the medium, but SR-59230A has been shown to be a potent (pA2=8.9) antagonist of lipolysis in rat cultured brown adipocytes incubated in the absence of albumin (Nisoli et al., 1996a). It also antagonized rat white adipocyte lipolysis in the presence of albumin, but with much lower potency (pA2 values 6.4, 6.9; Galitzky et al., 1997). The utility of SR-59230A as a selective antagonist of the human ß3AR is unclear. It was a potent antagonist (pA2=8.31) of the relaxant effect of isoprenaline in human isolated colonic circular smooth muscle, and since the ß1AR antagonist CGP-20712A and the ß2AR antagonist ICI-118,551 were poor antagonists, this effect of SR-59230A appears to be mediated via the ß3AR (De Ponti et al., 1996). However, the affinity of SR59230A for the human ß3AR was 2.8-fold lower than for the rat ß3AR (Table 4.3). This may seem insignificant, but if SR-59230A has similar affinity for rat and human ß1- and ß2ARs, its selectivity for the human ß3AR is only 10-fold. Moreover, in a recent study a pKB of only 7.26 for antagonism of isoprenaline in human colonic smooth muscle was reported, but higher potencies (pKB 8.4–8.6) were reported for antagonism of selective ß3AR agonists (Bardou et al., 1998). SR-59230A does, however, appear from most studies to be a far more potent ß3AR antagonist than ‘ß4’AR antagonist. There are no reports on the pharmacology of SR-59230A using the rodent cloned ß3AR, and work conducted with the human cloned ß3AR shows it to be a partial agonist

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(intrinsic activity relative to isoprenaline=0.46) with a Kact value of 18nM (Strosberg and Piétri-Rouxel, 1997). It is possible that it has low efficacy, so that it behaves as an antagonist in tissues, but as a partial agonist in cells that express high numbers of cloned receptors. Recently, two selective antagonists of the human cloned ß3AR have been described (Candelore et al., 1998). L-748,328 and L-748,337 are more than 80-fold selective over the human ß1AR, and 24- and 46-fold respectively selective over the human ß2AR. Unfortunately, they do not bind to the rodent ß3AR.

4.3

4.3.1

Agonists

ß1/2AR-selective agonists

Isoprenaline, noradrenaline, adrenaline and most standard synthetic ß 1- and ß2AR agonists are less potent agonists of ß3AR-mediated tissue responses or of the human cloned ß3AR than they are of ß1- and/or ß2AR-mediated responses (Tables 4.4 and 4.5). Standard agonists also have very low affinities (high Ki values) when displacing labelled antagonists from the native or cloned ß3AR (Galitzky et al., 1993a, 1997; Emorine et al., 1994; Germack et al., 1997; Kubo et al., 1997). However, agonists bind selectively to G protein-coupled receptors when they are indeed G proteincoupled, whereas antagonists bind with similar affinities to G protein-coupled and uncoupled receptors. Therefore, affinities determined for agonists when displacing radiolabelled antagonists may be lower than their affinities for the receptor when it is G protein-coupled (Kenakin, 1997) and differ markedly from their true functional affinity (Section 4.4.1). Moreover, antagonists such as [125I]-iodocyanopindolol and [3H]-CGP-12177 may bind to the ‘ß4’- as well as the ß3AR in tissues. This type of study should not therefore be used to assess the ß3AR selectivity of agonists. Studies using radiolabelled agonists (Deng et al., 1996) may be less open to misinterpretation, but even here there is the possibility that a labelled low efficacy agonist may bind with similar affinity to G protein-coupled and uncoupled receptors, while higher efficacy unlabelled agonists may be selective for the G protein-coupled receptors. Since noradrenaline and adrenaline have low potency as stimulants of the ß3AR, it is generally assumed that the ß3AR is normally only activated by the high concentrations of noradrenaline that can be achieved at sympathetic nerve endings. ß3AR-mediated responses may be favoured when release is prolonged, because ß1-and ß2-, but not ß3ARs, are subject to rapid down-regulation (Arch and Wilson, 1996a). It has also been suggested that octopamine (Figure 4.3), which is stored within sympathetic nerve endings and co-released with noradrenaline, might be the natural ß3AR agonist (Galitzky et al., 1993b).

4.3.2

Arylethanolamine ß3AR-selective agonists

Early agonists: Beecham, Lilly, Roche, Sanofi With the exception of the structurally related ICI/Zeneca compounds ICI-198157, ICI201651, ICI (or ZM)-215001 and ICI D (or ZD)-7114, the first-generation ß3AR-

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Table 4.4 Potencies (pD2, i.e. pEC50 values) of ßAR agonists in rodent tissues*

* Superscripts: r=rat; g=guinea-pig; u=uterus; s=soleus; t=trachea; a=white adipocytes; ba=brown adipocytes; c=colon; i=ileum; o=oesophagus † Partial agonist ‡ No effect at l0ìM [1] Arch et al., 1984a; [2] Wilson et al., 1984; [3] Wilson and Lincoln, 1984; [4] Piercy, 1988; [5] Hollenga et al., 1990; [6] O’Donnell and Wanstall, 1981; [7] Kirkham and Kelly, 1992; [8] Dolan et al., 1994; [9] Yen et al., 1998; [10] Arch and Kaumann, 1993; [11] Badone and Guzzi, 1994; [12] Landi et al., 1995; [13] Hioki et al., 1995; [14] Dow, 1997; [15] Sugasawa et al., 1997; [16] Grant et al., 1994; [17] Grant et al., 1995; [18] Barrionuevo et al., 1996; [19] Langin et al., 1991; [20] Zhao et al., 1998; [21] Mayers et al., 1996; [22] Konkar et al., 1996

selective agonists were phenylethanolamines (Figure 4.3). Many of these compounds have acidic N-substituents which often, but not always, increase their selectivity (Table 4.6). Others are esters that are converted to acids in vivo. Following oral administration, the SmithKline Beecham (BRL) esters are rapidly and totally de-esterified to their parent

Table 4.5 Binding affinities and functional potencies of ßAR agonists in human cloned ßARs*

Table 4.5 Continued

* Compounds are listed mainly in the order that they are described in the text. Values may vary according to level of expression of the receptor and, for pEC50, according to whether whole-cell cyclic AMP accumulation or membrane adenylyl cyclase was measured References 1, 3, 4, 6, 7, 15 are from Strosberg and co-workers, who like most other workers measured whole-cell cyclic AMP Reference 17 employs Strosberg’s ß3AR cell line. These data are also collated in Strosberg and Piétri-Rouxel (1996) References 2, 5, 17, 18 are from SmithKline Beecham who measured membrane adenylyl cyclase † Intrinsic activity relative to isoprenaline or noradrenaline which gave very similar maximum effects ‡ Data for the rat ß3AR § R-enantiomer of ICI-201651 ** pIC50 [1] Emorine et al., 1989; [2] Sennitt et al., 1998; [3] Méjean-Galzi et al., 1995; [4] Tate et al., 1991; [5] S.M.Henson and J.R.S.Arch, unpublished means of at least three determinations; [6] Blin et al.. 1993; [7] Pietri-Rouxel and Strosberg, 1995; [8] Dolan et al., 1994; [9] Dow, 1997; [10] Hargrove et al., 1999; [11] Harada et al., 1997; [12] Sher et al., 1997; [13] Gavai et al,. 1998; [14] Weber et al., 1998a; [15] Parmee et al., 1999; [16] Naylor et al., 1998; [18] Green et al., 1996; [19] Nahmias et al., 1991; [20] Pak and Fishman, 1996; [21] Molenaar et al., 1997b; [22] Arch et al., 1999; [23] Fisher et al., 1996; [24] Fishers et al., 1998; [25] Weber et al., 1998b; [26] Jesudason et al., 1998; [27] Zheng et al., 1999; [28] Konkar et al., 1996

Figure 4.3 Chemical structures of phenylethanolamine agonists.

Figure 4.3 Continued

Figure 4.3 Continued

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Figure 4.3 Continued

acids in all species that have been studied, including man. BRL-26830, BRL-33725 and BRL-35135 are as potent as their parent acids as ß3AR agonists, but they are also potent ß1- and ß2AR agonists (Table 4.6). SR-58611 is rapidly and totally biotransformed to SR58878, which is ß3AR-selective (Landi et al., 1995). Most evidence that SR-58611 is a ß3AR-selective agonist comes from in vivo studies; there are in vitro data that also demonstrate that SR-58611 is ß3AR-selective, but the possibility that it is de-esterified in vitro has not been excluded (Badone and Guzzi, 1994). There is no evidence that some of the compounds shown in Figure 4.3 are selective ß3AR agonists, either in their own right or via a metabolite, although they do have antiobesity activity in rodents. Thus, the Eli Lilly compound LY-79771 was only marginally more potent as a stimulant of adenylyl cyclase mediated by the human ß3AR compared with ß1- (5-fold) or ß2ARs (17-fold) (Blin et al., 1993), and there are no reports that LY-104119, or the Hoffman-La Roche compounds Ro-16–8714 or Ro40–2148 selectively stimulate ß3AR-mediated responses in vitro. It is interesting that Eli Lilly chose to progress the less potent RS molecules as anti-obesity agents and the more potent RR enantiomers as intropic agents, but whether these compounds differ in ß3/ß1AR selectivity is unclear. The Roche compounds are tertiary amines and, although Ro-16–8714 stimulated rat brown adipocyte thermogenesis in vitro, it is possible that it is metabolized to a more potent secondary or primary amine. Thus, Ro-16–8714 had

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Table 4.6 ß3AR selectivities of esters or amides and related acids in animal tissue and human cloned receptors

* Tissues are ratr or guinea-pigg right atrial rate † Tissues are rat uterineu relaxation or insulin-stimulated glucose incorporation into glycogen in soleuss muscle ‡ Tissues are rat white adipocyte lipolysisa or colon relaxationc § CONH2 replaces SO3H of BMS-187413 [1] Arch et al., 1984b; Wilson et al., 1984 and unpublished data of the same authors; [2] Dolan et al., 1994; [3] Badone and Guzzi, 1994; [4] Sher et al., 1997

no effect on rat right atrial rate in vitro, but it was a potent chronotropic agent in vivo (Table 4.7). Later compounds and salmeterol The arylethanolamines tend to stimulate responses mediated by all three receptors, the ß3AR-selective compounds being more potent as stimulants of the ß3AR (see Tables 4.4 and 4.5). Some arylethanolamines have little or no agonist activity at ß1-or ß2ARs, however. CL-316,243 (American Home Products) had little or no activity at human cloned ß1- and ß2ARs (Table 4.5), but it has a little activity in the rat soleus muscle (Table 4.4). AZ-002 (Banyu/Merck) also has no ß1- or ß2AR-mediated agonist activity in rat tissues, but its potency as a ß3AR agonist is low (Hioki et al., 1995). ZD-9989 (10mM) increased basal atrial rate by only 12% and elicited a 4-fold rightward shift of the concentration-chronotropic response curve of guinea-pig right atria to isoprenaline, but had no significant agonist or antagonist effects in guinea-pig tracheal chains (Grant et al., 1995). ZD-2059 had little or no agonist or antagonist activity in guinea-pig right atria or tracheal chains (Grant et al., 1994).

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Table 4.7 Comparison of the effects of BRL-26830/28410 and Ro-16–8714 on rat right atrial rate and in vivo heart rate

* 90% of orally administered BRL-26830 is absorbed in the rat, but it is totally de-esterified to BRL-28410. Therefore BRL-28410 was used for the in vitro study † Data from Arch et al., 1984 ‡ Unpublished results of C.Wilson. Heart rate was measured 30, 60 and 90 min after dosing, using a tail cuff (n=6)

Many of the arylethanolamines shown in Figure 4.3 have low efficacy as agonists of the human ß3AR. Some of the more recent compounds are said to have high intrinsic activities as agonists of the human cloned ß3AR (Table 4.5), but since intrinsic activity depends on the level of receptor expression, it is important that there is a comparison with known low and high efficacy compounds (e.g. BRL-37344, CGP-12177) in the same system. The Pfizer compound CP-114,271 has been described as having a similar in vivo efficacy and selectivity profile to its close structural congener BRL-37344. Like BRL37344, its potency and intrinsic activity at the human cloned ß3AR are poor (Dow, 1997). CP-209,129 represented an advance on CP-114,271 in having a better intrinsic activity at the human ß3AR. At a concentration of 30 µM it had no activity at ß1- or ß2ARs, but since its EC50 at the ß3AR was only 20 µM in the system used, its selectivity is unclear. CP-331,679 is a more potent, higher efficacy agonist at the human ß3AR that is more than 100-fold selective relative to ß1- and ß2AR stimulation (Dow, 1997). The structurally related compound CP-331,684 has a similar pharmacological profile (Hargrove et al., 1999). FR-149175 (Fugisawa) is a potent (IC50=0.8 nM) and selective (>900-fold versus ß1and ß2AR-mediated effects) inhibitor of rat distal colon that stimulates the human cloned ß3AR (Dow, 1997; Yamamoto et al., 1997a). FR-165914 is a less potent relaxant of rat intestinal preparations (Hattori et al., 1995). The Dainippon compound AD (or AJ)-9677 was a potent full agonist of the human cloned ß3AR in the system studied, and more than 100-fold less potent and a partial agonist at human cloned ß1- and ß2ARs (Harada et al., 1997). The Bristol-Myers Squibb compound BMS-187413 is approximately 10-fold more potent as a stimulant of the human cloned ß3AR than BRL-37344 (Sher et al., 1997), but in view of its similar structure it seems unlikely that it has significantly better efficacy at the human receptor. The structures of BMS-194449 (‘clinical candidate’), BMS196085 (‘selected for evaluation in clinical trials’) and BMS-210285 (‘pre-clinical backup to BMS-196085’) (Gavai et al., 1998; Sher et al., 1998; Washburn et al., 1998) seem more likely to give good efficacy at the human receptor.

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The Merck compound L-757,793 has weak partial agonist activity at the human ß1AR, but only at concentrations 1000-fold higher than those that activate the ß3AR (EC50=0.43 nM). It displays 500-fold selectivity over binding to the ß2AR (Naylor et al., 1998; Weber et al., 1998b). L-770,644 is a partial agonist at both ß1- and ß2ARs and is less selective than L-757,793. L-771,047 is a potent (0.9 nM) full ß3AR agonist with more than 1000-fold selectivity over binding to ß1- and ß2ARs (Weber et al., 1998b; Mathvink et al., 1999). L-760,087 and L-766,892 (Parmee et al., 1999) and L-764,646 (Naylor et al., 1998) are less potent ß3AR agonists than L-771,047, and only L-764,646 approaches L-771,047 in selectivity over binding to ß1- and ß2ARs. The functional activity of these compounds at ß1- and ß2ARs has not been described. SM-11044 (Sumitomo) showed moderate selectivity as a relaxant of guinea-pig ileum via the ß3AR relative to its potencies in guinea-pig atrial and tracheal preparations. It was a full agonist of the human cloned ß3AR, but not selective relative to ß2AR stimulation (Sugasawa et al., 1992). Tecradine® induces rat oesophageal muscularis mucosae relaxation via the ß3AR, but the evidence for it being ß3AR-selective was based on lack of activity in atria and tracheal preparations from the guinea-pig (Barrionuevo et al., 1996). Guinea-pig atria are less sensitive than rat atria to ß1AR-agonists, and so it is possible that the ‘selectivity’ of Tecradine in these experiments was due to it having low efficacy. Salmeterol is a potent agonist of the human cloned ß3AR (Piétri-Rouxel and Strosberg, 1995), but it is an even more potent ß2AR agonist, characterized by a long duration of action (Coleman et al., 1996).

4.3.3

Aryloxypropanolamine ß3AR agonists

CGP-12177 and other ‘non-conventional partial agonists’ CGP-12177 (Figure 4.4) has become the standard aryloxypropanolamine ß3AR agonist. CGP-12177 is a highly potent ß1- and ß2AR antagonist, but the discoveries that it stimulates respiration in hamster brown adipocytes (Mohell and Dicker, 1989) and

Figure 4.4 Chemical structures of aryloxypropanolamines.

Figure 4.4 Continued

Figure 4.4 Continued

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Figure 4.4 Continued

lipolysis in white adipocytes of various species (Langin et al., 1991) led to the demonstration that it is an agonist of murine and human cloned ß3ARs (Granneman et al., 1991; Nahmias et al., 1991). CGP-12177 is, however, 1000-fold less potent as a ß3AR agonist than as a ß1- or ß2AR antagonist. In cell lines that express high numbers of the human cloned receptors it can also act as a potent full ß1AR agonist and as a potent weak partial ß2AR agonist (Pak and Fishman, 1996). It is therefore important to exclude the possibility that any effect of CGP-12177 is mediated by ß1- or ß2ARs by using an appropriate antagonist. CGP-12177 is one of a number of ß 1/2AR antagonists that Kaumann and his colleagues had found to stimulate cardiac function. The other ß1/2 antagonists were oxprenolol, alprenolol, carazolol, pindolol and derivatives of carazolol and pindolol (Figure 4.4). All these compounds have pD2 values for their cardiac effects that are lower than their pKB values for ß1- or ß2ARs, leading to their description as ‘non-conventional partial agonists’ (Kaumann, 1989; see also Arch and Kaumann, 1993). Their agonist activities reside exclusively in their (-)-enantiomers. (-)-RO-363 appears to have a similar pharmacological profile, except that it is a partial ß1AR agonist in cardiac tissues as well as acting at higher concentrations via another receptor (Molenaar et al., 1997b). As well as CGP-12177, all the other ‘non-conventional partial agonists’ and (-)-RO363 stimulate the cloned ß3AR (see Emorine et al., 1994). However, the cardiac effects of CGP-12177 in both rat and man, and its lipolytic activity in human adipocytes are antagonized with a pharmacology that differs from that expected for ß3AR-mediated responses (Galitzky et al., 1997; Kaumann, 1997; see Section 4.4.1). The pharmacology of the cardiac effects of the other non-conventional partial agonists has not been differentiated from ß3AR pharmacology, but it is probable that they too stimulate this ‘ß4AR’. Except for SB-226552, SB-236923 and SB-251023, which have no stimulant effect in human right atrial appendage (see below), it is possible that all the other compounds shown in Figure 4.4 also stimulate this putative receptor.

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The disadvantages of CGP-12177 as a ß3AR agonist are therefore that it is a far more potent ß1- and ß2AR antagonist (and potentially ß1AR agonist), and that it stimulates the ‘ß 4 AR’. On the other hand, it has advantages over most phenylethanolamine ß3AR agonists of having much higher efficacy at the human ß3AR (Hoffstedt et al., 1996a; Wilson et al., 1996; see also comment in introduction to Fisher et al., 1998) and of stimulating human white adipocyte lipolysis via ß3- rather than ß1- or ß2ARs (Hoffstedt et al., 1996a; see Section 4.4.2). These features should also be borne in mind when judging the utility as pharmacological tools of other aryloxypropanolamines.

Other ß1/2AR antagonist/ß3AR agonists with small N-substituents The ß1/2AR antagonist carteolol has anti-obesity activity in mice (Takahashi et al., 1994). This effect is probably mediated by the ß3AR because propranolol is a weak antagonist of the stimulation of hamster brown adipocyte oxygen consumption by carteolol. However, carteolol appears to relax guinea-pig cecum via the ß2AR (Zhao et al., 1998). The a/ßAR antagonist arylthiopropanolamine arotinolol activates brown adipose tissue thermogenesis in vivo (Yoshida et al., 1994a). Although not demonstrated by detailed in vitro studies, it is probable that this effect is also mediated by the ß3AR. Propranolol, nadolol, carazolol and (—)-teratolol are ß1/2AR antagonists which have been shown directly to activate the human cloned ß3AR, but agonism has yet to be demonstrated in isolated tissues or in vivo (Méjean-Galzi et al., 1995; PiétriRouxel and Strosberg, 1995).

ZM-215001 and related molecules Prior to CGP-12177 being recognized as a ß3AR agonist, the aryloxypropanolamine ICI198157 was reported to have anti-obesity and anti-diabetic activity in obese (fa/fa) Zucker rats. Metabolic rate and brown adipose GDP binding in vivo (an index of brown adipose tissue thermogenesis) were stimulated at dose levels well below those that affected heart rate, and there were no ß2AR-mediated effects on muscle tremor or blood potassium (Holloway, 1989). However, ICI-198157, like some phenylethanolamines described in Section 4.3.2, is a methyl ester, and it is its acid metabolite ICI-201651, specifically the S enantiomer of this metabolite, ZM-215001, that is a ß3AR-selective agonist (Holloway, 1989; Holloway et al., 1991). Although ICI-201651 and ZM-215001 have slight ß1- and ß2AR agonist activity in some tissue preparations (Holloway, 1989), they are predominantly ß1- and ß2AR antagonists. ZM-215001 has pA2 values of 6.7 and 7.3 for antagonism of the effects of isoprenaline in guinea-pig atria and trachea (Tesfamarian and Allen, 1994). These values are similar to the pD2 value of ZM-215001 (7.4) for stimulation of rat white adipocyte lipolysis (Mayers et al., 1996). Thus, in contrast to CGP-12177, ZM-215001 can stimulate the ß3AR without totally blocking ß1- and ß2ARs, but some ß1/2AR antagonism will occur. Blin et al. (1993) found that ICI-201651 was a full agonist of the human cloned ß3AR, and Fisher et al. (1996) reported an intrinsic activity of 0.71 for ZM-215001 relative to isoprenaline. However, other workers (Fisher et al., 1998) commented that all the first-generation ß3AR agonists that they have tested in man are only weak partial agonists of the human ß 3 AR. It seems unlikely that a compound which lacks

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substituents in the phenyloxypropanol moiety can have good efficacy at the human receptor, and it is possible that the systems used by Blin et al. (1993) and Fisher et al. (1996) flatter these compounds. ZM-215001 must have poor efficacy at the rat ß3AR since it had an intrinsic activity of only 0.30 relative to isoprenaline or BRL-37344 as a stimulant of rat white adipocyte lipolysis (Mayers et al., 1996). Its pA2 value for antagonism of isoprenaline-stimulated lipolysis was 7.3. This value is similar to its pA2 values for antagonism of atrial and tracheal responses, showing that it has similar affinities for the three cloned ßARs. ICI/Zeneca chose to develop the amide ZD-7114 rather than the ester ICI-198157, presumably to achieve a better pharmacokinetic profile. The amide is also metabolized to the acid (Mayers et al., 1996). BMS-187257 (Bristol-Myers Squibb) has a similar profile at the human cloned ß3AR to BRL-37344 (Fisher et al., 1996). Like the ICI/Zeneca compounds, the substitution pattern in its aryloxypropanol moieties seems unlikely to provide good efficacy at the human ß3AR. The high intrinsic activity (0.72) reported for BMS-187257 may reflect the use of Strosberg’s cell line.

Recent SmithKline Beecham, Merck and Eli Lilly compounds When the first-generation ß3AR agonists and their prodrugs were evaluated in man, the results were largely disappointing (Table 4.8). These compounds either lacked adequate efficacy or their thermogenic, anti-obesity or anti-diabetic activity was associated with ß1- or ß2AR-mediated side effects (tachycardia or tremor). Poor oral bioavailability and rapid clearance was one reason for these failures (Section 4.3.5), but it was also recognized that the first-generation compounds lacked good selectivity and especially efficacy at the human ß3AR (Wilson et al., 1996; Fisher et al., 1998). Therefore, in recent years pharmaceutical companies have tried to identify ß 3AR agonists that are not only high selective but also have good efficacy at the human ß3AR. Table 4.8 Clinical experience with first-generation ß3AR agonists

[1] Arch and Wilson, 1996a; [2] Smith et al., 1990; [3] Toubro and Astrup, 1995; [4] Weyer et al., 1998

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Sennitt et al. (1988) have described six aryloxypropanolamines that vary in their pharmacology at the human cloned ßAR. Four of these and a further compound, SB251023, are shown in Figure 4.4. SB-226552, SB-229432 and SB-251023 had intrinsic activities that were at least as high as that of CGP-12177 at the human ß3AR. Like CGP12177, they lacked agonist activity at ß1- and ß2ARs at the level of expression used. However, in contrast to CGP-12177, their binding affinities as antagonists (pKi values) at ß1- and ß2ARs were much lower than their pD2 values at the ß3AR. SB-236923 and SB246982 had higher intrinsic activities than isoprenaline at the ß3AR, but they were partial agonists at ß1ARs, though at higher concentrations than their ß3AR agonism. A key feature of these SmithKline Beecham aryloxypropanolamines is that Kaumann, the originator of the ‘ß4AR’ hypothesis, found that they either do not stimulate the force of contraction of human right atrial appendage (SB-226552, SB-229432, SB-251023), or any slight effect that they do have is blocked by 200 nM (-)-propranolol (SB-236923). Thus, they appear to have the advantage over CGP-12177 and other non-conventional partial agonists of not stimulating the ‘ß4AR’. The Merck compound L-755,507 is a potent agonist of human and rhesus monkey cloned ß3ARs (Fisher et al., 1998; Parmee et al., 1998). Its intrinsic activity at the human ß3AR is significantly higher than the intrinsic activities of ß3AR agonists that have been evaluated in man. It is a highly selective ß3AR agonist, but it does have partial agonist activity at both human and rhesus monkey ß1ARs. High intravenous doses of L-755,507 stimulate heart rate in rhesus monkeys, but it was suggested that this was a reflex response to ß3AR-mediated peripheral vasodilatation rather than a direct ß1AR- or ‘ß4AR’-mediated effect. Pyridines related to L-755,507, a phenol, have been described recently. L-749,372 and L-750,355 are somewhat less potent ß3AR agonists that have partial agonist activity at the ß1AR. Their ß3/ß1AR selectivity is more than 100-fold. They bind weakly to the ß2AR, presumably as antagonists (Weber et al., 1998a). LY-362884 is another potent agonist of the human cloned ß3AR that had a high intrinsic activity (0.98) relative to isoprenaline. Like SB-226552, SB-229432 and SB251023, it had only antagonist activity at the human ß1- and ß2ARs, but in contrast to the SB compounds, its ß3AR EC50 (30 nM) was similar to its IC50 values for antagonism of isoprenaline’s actions at ß1- and ß2ARs (Jesudason et al., 1998). A closely related compound, LY- 377604 is reported to be in Phase I clinical development (Cohen et al., 1999). There is no information on whether LY-362884 or LY-377604 have ‘ß4AR’ agonist-like activity.

4.3.4

Trimetoquinol and analogues

Trimetoquinol and its analogues constitute a third class of ß3AR agonists (Figure 4.5). These compounds lack the hydroxy group of arylethanolamines or aryloxypropanolamines, and the amino nitrogen is contained within a semi-rigid tetrahydroisoquinoline ring. Their large 3',4',5'-trimethoxybenzyl substituent resembles the large N-substituents of selective ß3AR agonists. (-)-Trimetoquinol and 3'-iodotrimetoquinol have similar potencies for stimulation of ß1AR- and ß3AR-mediated responses in rat tissues, with a slightly lower potency for ß2AR-mediated tracheal relaxation (Fraundorfer et al., 1994; Konkar et al., 1996) (see Table 4.4). 3',5'-Diiodotrimetoquinol shows, however, about 10-fold selectivity for stimulation of the ß3AR-mediated responses. All three compounds are also potent

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Figure 4.5 Chemical structures of trimetoquinols.

stimulants of cyclic AMP accumulation in Chinese hamster ovary cells expressing the rat ß3AR (see Table 4.5). The activities at the human ß3AR of trimetoquinol and some novel ß3AR-selective analogues have recently been reported (Zheng et al., 1999).

4.3.5

Metabolism and pharmacokinetics

Problems with the oral bioavailability or pharmacokinetics of ß3AR agonists have often limited their value, especially as therapeutic agents for humans. De-esterification of some of the first-generation compounds is beneficial to the extent that it produces the ß3AR-selective acid metabolites. In rats, the acids BRL-28410 and BRL-373444 are excreted in the bile and reabsorbed, resulting in sustained blood levels, but in man they are not excreted in the bile, and rapid renal excretion results in their having very short half-lives. The diacid CL-316,243, by contrast, has a very long plasma half-life in man (16 h), but only 10% of an oral dose is absorbed (Weyer et al., 1998). In rats, 12% of an oral dose is absorbed and in monkeys 3%. The 2,2-dimethylpropyl and 3-methylbutyl ester-type prodrugs of CL-316,243 had 2- and 3-fold enhanced oral bioavailabilities in monkeys. CP-114,271 had both poor oral bioavailability in the monkey (7%) and rat (11%), and a plasma half-life following intravenous administration of less than 30 min in these species (Wilson et al., 1995). Slightly better results were achieved in dogs (Wilson et al., 1995; Yee et al., 1995). The SmithKline Beecham aryloxypropanolamines shown in Figure 4.4 also encountered problems of oral bioavailability and short half-lives in the rat. L-755,507, a phenoxypropanolamine that is subject to rapid presystemic glucuronidation (Weber et al., 1998a), is one of a class of compounds that has poor oral bioavailability and relatively short half-lives in various animal species (Fisher et al., 1998). L-755,507 itself has an oral bioavailability of about 1 %, but the aminopyridinebased analogue L-750,355 has bioavailabilities of 4% in the rat and 47% in the dog, and a half-life of 13 h in the dog (Dow, 1997). The pyridine L-749,372, also has a better pharmacokinetic profile than L-755,507 (Weber et al., 1998a). Attempts to achieve oral bioavailability with 3-pyridylethanolamines have had limited success (Naylor et al., 1998, 1999; Parmee et al., 1999), except perhaps for L-770,644, which had 27% oral bioavailability in the dog. BMS-194449 has an oral systemic bioavailability in monkeys of less than 2% (Washburn et al., 1998). It is well absorbed, but is excreted in the bile as a monoglucuronide. BMS-194449 is one of three compounds (the others are BMS-196085

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and BMS-201620) that have been described as transdermal agents that increase lipolysis, but whose development has been discontinued. BMS-196085 similarly has less than 5% oral systemic bioavailability (Gavai et al., 1998). Oral systemic bioavailability is not markedly improved in BMS-210285 (Sher et al., 1998). The Lilly compound reported to be in Phase I clinical trials, LY-377604, is described as having more than 20% oral bioavailability (Cohen et al., 1999).

4.4

4.4.1

Idiosyncrasies of ß3AR pharmacology

Relative agonist potencies vary with the nature of the assay

Studies conducted using the human cloned ß3AR have shown that agonists are more potent stimulants of whole-cell cyclic AMP accumulation than of adenylyl cyclase in membranes. Moreover, the relative potencies of the agonists differ in these two systems (Emorine et al., 1994; Arch, 1995; Wilson et al., 1996). In particular, the catecholamines and BRL-37344 are much more potent than aryloxypropanolamines, such as CGP-12177 or cyanopindolol, in the whole-cell assay, but not in the membrane assay. Wilson et al. (1996) showed that these findings extend beyond potencies to the functional affinities of the agonists (Table 4.9). They suggested that the aryloxypropanolamines interact with a signal tranduction pathway in the whole cell that impairs cyclic AMP production, but they were unable to identify what this other signalling pathway might be. For example, treatment of cells with pertussis toxin to prevent signalling via Gi did not affect the relative potencies of isoprenaline or CGP12177. One can speculate that interactions of aryloxypropanolamines with the ‘ß4’AR affects their ß3AR-mediated responses, or that the newly discovered RAMP proteins (McLatchie et al., 1998) differentially modulate responses to the agonists, but these influences would have to differ between whole cells and membranes to account for the findings. Whatever the reason, there must be at least two forms of the ß3AR with different absolute and relative affinities for agonists. Interestingly, Clarke and Bond (1998) have recently reviewed evidence that the relative efficacies of agonists can also vary according to the environment of the receptor. Table 4.9 Relative potencies of agonists at the human cloned ß3AR vary with assay*

*From Wilson et al., 1996

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Wilson et al. (1996) also showed that while isoprenaline was 229-fold more potent than cyanopindolol as a stimulant of whole-cell cyclic AMP accumulation, cyanopindolol was 365-fold more potent than isoprenaline in displacing [125I]-iodocyanopindolol from membranes that express the human cloned ß3AR (Table 4.9). The difficulties of interpreting such data were discussed in Section 4.3.1. It seems that cyanopindolol must bind with high affinity to the same population of ß3ARs as its close homologue [125I]-iodocyanopindolol, whereas isoprenaline binds with high affinity to only a fraction of the receptors that bind [125I]-iodocyanopindolol. The to membranes from untransfected cells was negligible. Discrepancies between binding possibility that the labelled ligand bound to the ‘ß4’AR can be excluded, since binding affinities and potencies for cyclic AMP accumulation have also been described for the rat ß3AR: the (–)-(S) isomer of trimetoquinol was 120-fold more potent than the (+)-(R) isomer in binding experiments, but 4700-fold more potent in functional experiments (Fraundorfer et al., 1994).

4.4.2

Prediction of human tissue pharmacology from cloned receptor pharmacology

Isoprenaline, noradrenaline and BRL-37344 each stimulate the human cloned ß3AR (Emorine et al., 1994). However, responses to BRL-37344 (if they occur at all), and to isoprenaline and noradrenaline in human gut and adipose tissue are mediated primarily by the ß1- or ß2- rather than the ß3AR (Langin et al., 1991; MacLaughlin and MacDonald, 1991; Lönnqvist et al., 1993; Rosenbaum et al., 1993; Sennitt et al., 1995), although responses of the gut preparations to isoprenaline and noradrenaline do involve a significant ß3AR-mediated component (MacLaughlin and MacDonald, 1991; De Ponti et al., 1996; Kelly et al., 1998). Indeed, of the arylethanolamines, only CL-316243, which has very low or zero efficacy at ß1- and ß2ARs, has been shown to elicit a lipolytic response in human white adipocytes that is totally resistant to antagonism by 10-7 M propranolol (Hoffstedt et al., 1996a). This compound has also been shown to stimulate insulin-mediated glucose storage in man by what must very probably be a ß3ARmediated mechanism, since no changes in heart rate, blood pressure or tremor could be detected (Weyer et al., 1998). This background led Sennitt et al. (1998) to investigate the lipolytic activities of two phenylethanolamines that had higher intrinsic activities than BRL-37344 in their human cloned ß 3AR assay. One of these compounds, SB-220646, appeared to elicit no stimulation of adenylyl cyclase in membranes that expressed human cloned ß1- or ß2ARs, and yet its lipolytic effect was antagonized by both nadolol and propranolol with KB values that indicated the involvement of one or both of these classical receptors. Moreover, the compound was almost a full agonist of human right atrial appendage contractility, an effect that was shown to be mediated by both ß1- and ß2ARs. Further studies using a cell line that expressed high numbers of ß2ARs revealed weak ß2AR agonist activity in SB-220646. This (and presumably weak ß1AR agonist activity) was apparently sufficient to subvert any ß3AR-mediated effect on lipolysis and elicit the inotropic activity. The second compound, SB-215691, had higher ß1- and ß2AR agonist activity than BRL-37344, but also higher intrinsic activity at ß3ARs. Low concentrations of SB215691 did have a nadolol-resistant component in its lipolytic effect in some experiments, but in every experiment high concentrations of SB-215691 stimulated lipolysis by a nadolol-sensitive mechanism.

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The tendency of these phenylethanolamines to stimulate human white adipocyte lipolysis via ß1- or ß2ARs contrasts with data for CGP-12177 and the SmithKline Beecham aryloxypropanolamines described in Section 4.3.3 (Figure 4.4). The lipolytic effects of all these compounds were insensitive to nadolol, even in the case of the weak partial ß 1 - and ß 2 AR agonist SB-236923. The lipolytic effect of another aryloxypropanolamine (SB-248320; Sennitt et al., 1998) was sensitive to nadolol, but this compound was a strong partial agonist of the ß2AR. Since any atrial inotropic effects of the SmithKline Beecham compounds were sensitive to propranolol, and SB-251023 does not antagonize the inotropic effect of CGP-12177 (Arch et al., 1999), these compounds must stimulate lipolysis via the ß3- rather than the ‘ß4’AR. It seems that cross-talk between ß1/2ARs and ß3ARs tends to override signalling via the ß3AR conformation to which phenylethanolamines bind, but not signalling via the conformation to which aryloxypropanolamines bind. Consequently, for a phenylethanolamine to signal via ß3ARs in human white adipocytes, it must, like CL316243, be virtually devoid of efficacy at ß1- or ß2ARs.

4.5

Conclusions

The ß3AR is characterized by a low affinity for standard ß1- and ß2AR antagonists. In rat tissues, it distinguishes poorly between the more potent (-)- and less potent (+)enantiomers of these antagonists, but this feature has not been demonstrated using the rodent cloned ß3AR and is not found with the human cloned ß3AR. There are no potent and highly selective antagonists of the ß3AR, but SR-59230 displays moderate selectivity in the rat and isolated rat tissues, and L-748,328 and L-748,337 are moderately selective antagonists of the human cloned ß3AR. The ß3AR is selectively stimulated by certain phenylethanolamines, but many of these have low selectivity for and efficacy at the human ß3AR. Unless, like CL-316,243, they lack any efficacy at ß1- and ß2ARs, they tend to stimulate human white adipocyte lipolysis via ß 1- or ß 2 ARs. The ß 3AR is also selectively stimulated by certain aryloxypropanolamines, some but not all of which are potent ß1/2AR antagonists. Some of these compounds have good efficacy at human ß3ARs, and this class of compound generally seems more likely to elicit ß3AR than ß1AR or ß2AR-mediated responses in tissues that express all three receptors. Some aryloxypropanolamines, notably CGP-12177, appear to stimulate another receptor, now known as the ‘ß4’AR. This receptor shares with the ß3AR the property of insensitivity to ß1- and ß2ARs, but there are differences between ß3- and ‘ß4’AR pharmacology, notably that phenylethanolamines are not agonists for the ‘ß4’AR, at least in cardiac tissues (see Chapter 8). One must be cautious in assuming that a novel pharmacology implies a novel receptor, since the pharmacology of even cloned ß3ARs can vary, but ‘ß4’AR pharmacology has been demonstrated in atria and brown adipose tissue from ß3AR knockout mice. Recent work suggests that the ‘ß4’AR is a form of the ß1AR. ß 3AR agonists have potential for the treatment of human obesity and type 2 diabetes, but the first-generation compounds lacked selectivity and efficacy for the human ß3AR. Subsequent compounds have overcome this deficiency, but a compound that combines appropriate pharmacology with good oral bioavailability and sustained blood levels has not been described—at least not in any detail. This remains a major goal of this research area.

5

The Native Human ß3-Adrenoreceptor PETER ARNER AND FREDRIK LÖNNQVIST Department of Medicine at Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden

5.1

Introduction

Long before the ß3-adrenoreceptor (ß3AR) was characterized, it was implicated that certain catecholamine responses in man were mediated through an ‘atypical’ non-ß1AR, ß2AR. Using pharmacological experiments with selective and non-selective ß1AR and ß2AR agonists and antagonists, Zaagsma, Kaumann and their co-workers were the first to demonstrate the existence of such ‘atypical’ ßARs mediating lipolysis in human fat cells and metabolism and contractility in human heart (for reviews see Kaumann, 1989; Zaagsma and Nahorski, 1990). However, the role of this atypical receptor was largely unknown until the first cloning and molecular characterization of the human ß3AR (Emorine et al., 1989). This chapter focuses on the native human ß3AR and the possible physiological and pathophysiological role of the receptor. In the interest of space, review articles have been cited instead of original articles whenever p ossible.

5.2

The human ß3AR gene

While the structural aspects of the human ß3AR are discussed in detail in other chapters of this book, the clinically important factors are species differences in structure-function relationships. Most of our knowledge on ß3AR functions is derived from studies in nonhuman cells, and in particular cells or tissues from rodents. Some structural features predict that the human ß3AR gene may have unique functional properties in comparison with that of rodents. The mouse and rat ß3AR genes contain two introns and three exons, whereas the human gene contains only one intron and two exons (Granneman and Lahners, 1992; Van Spronsen et al., 1993). Furthermore, the ß3AR gene transcription differs markedly between human and rat with regard to the use of polyadenylation sites, promoter start sites and hormonal regulation (Granneman and Lahners, 1994). There are however, also species differences which cannot be predicted by a comparison of the structure of different ß3AR genes. The predicted primary structures of the rat and the human ß3AR are more than 90% similar, yet the receptor properties differ substantially in their pharmacological response to ßARagonists (Granneman et al., 1991). In contrast, the mouse ß3AR gene shows only 80% 77

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homology with the human ß3AR gene, although the pharmacological properties of the mouse and human receptors, when expressed in Chinese hamster ovary cells, are very similar (Granneman et al., 1991; Blin et al., 1994). As yet, our knowledge of the regulation of expression of the native human ß3AR gene is limited. However, it is likely that this knowledge will increase in the near future through results from experiments on transgenic animals, where the native gene product has been replaced by the human counterpart (Ito et al., 1998). Until then, caution should be exercised when data derived from experiments on laboratory animals or cell lines are extrapolated to the human ß3AR gene.

5.3

Tissue expression of mRNA for ß3AR

It is now evident that specific mRNA is present in a variety of human tissues (Table 5.1). The expression of mRNA has been documented with various techniques such as RTPCR, RNase protection and Northern blotting (Rodriguez et al., 1995; Deng et al., 1996). The measurement of ß3AR mRNA in man is mainly of a semi-quantitative nature, and therefore the true order of magnitude of gene expression in human tissues is unknown. It appears, however that brown adipose tissue and gallbladder have the highest levels of expression. In most tissues, mRNA for ß3AR co-exists with mRNA for ß1AR and ß2AR. The relative abundance of the three gene products in these various tissues is also unclear, but by using semi-quantitative techniques, it has been shown that quantities of ß3AR mRNA are less than those of ß1AR and ß2AR mRNA in both brain and brown adipose tissue (Krief et al., 1993; Berkowitz et al., 1995; Deng et al., 1996). For obvious reasons, it is likely that a direct ß3AR function is expected solely in tissues that express the corresponding gene, and the role of the receptor in these tissues will be discussed in the following text.

5.3.1

White adipose tissue

The major metabolic function of white adipose tissue (WAT) is to store and release energy-rich fatty acids that are mobilized through lipolysis in white fat cells. The role of ß3AR in human lipolysis has been examined in some detail (see Arner, 1996 for an overview of lipolysis regulation in man). The gene has been shown to be expressed in all human white adipose depots examined to date (Rodriguez et al., 1995; Deng et al., 1996). However, early studies of lipolysis in vitro in easy-to-obtain subcutaneous adipose tissue failed to show a significant lipolytic response following ß3AR stimulation, despite the same adipocytes responding to lipolytic stimulation of ß1AR and ß2AR (for details see Galitzky et al., Table 5.1 Human tissues expressing ß3AR

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1995). It is, however, likely that methodological factors contribute to the difficulties in demonstrating a functional ß3AR in these earlier in vitro studies on lipolysis. The lipolytic effect of ß3AR stimulation is dependent on a number of factors such as adipose tissue region (visceral fat is much more responsive than subcutaneous fat), the type of tissue preparation (isolated cells are more responsive than tissue pieces), and overall lipolytic activity in the in vitro preparation (Hoffstedt et al., 1995). Using a sensitive lipolysis assay it has been possible to demonstrate and characterize in detail the pharmacological properties of a ß3AR in omental (visceral) human fat cells with regard to lipolysis in vitro. ß3AR functionally co-exists with ß1AR and ß2AR (Lönnqvist et al., 1993), and its pharmacological properties clearly differ from those reported for the ß3AR in rat fat cells. A number of ‘rat-selective’ ß3AR agonists are either ineffective or non-selective on lipolysis in human fat cells (Hoffstedt et al., 1996a). However, the lipolytic properties of the receptor in omental fat cells is almost identical as in the mouse 3T3-L1 adipocyte cell line (Shimizu et al., 1996). So far, only two adrenergic agonists have been identified as selective ß3AR lipolytic agonists in human fat cells: CGP-12177 which is also a ß1AR/ß2AR non-selective antagonist (Lönnqvist et al., 1993), and CL-316,243 (Hoffstedt et al., 1996a), although some data suggest that the latter agent is non-selective (Umekawa et al., 1996). The strongest proof for a functional ß3AR in human WAT has been obtained from in vivo studies using microdialysis. This technique has proven very useful in pharmacological investigations of in situ lipolysis in humans (Lafontan and Arner, 1996), as it permits the monitoring and manipulation of local lipolysis and blood flow in vivo in human subcutaneous adipose tissue. In this tissue it was shown that all three ßAR subtypes regulate lipolysis in vivo, but ß2AR was shown to be the most important for lipid mobilization as only this receptor type stimulated lipolysis as well as local blood flow (Enocksson et al., 1995). The existence of a functional ß3AR in vivo in human subcutaneous adipose tissue was later confirmed by other microdialysis experiments (Barbe et al., 1996). The question remains as to whether the ß 3AR in white fat cells is subject to regulation. Extensive evidence exists to show that the adipocyte ß3AR is influenced by physiological and pathophysiological factors (Table 5.2), the most important factor probably being obesity. Subjects with upper-body obesity have a higher rate of catecholamine-induced lipolysis in visceral fat cells than non-obese subjects, this variation being ascribed mainly to enhanced lipolytic function of ß3AR in visceral adipocytes (Lönnqvist et al., 1995). Many metabolic and cardiovascular abnormalities found in upper-body obese subjects, which together form a so-called metabolic

Table 5.2 Modulation of ß3AR lipolytic function in white human fat cells

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(insulin resistance) syndrome, are related to the lipolytic function of ß3AR in visceral fat cells (Hoffstedt et al., 1996b). As discussed in detail (Arner, 1996), increased mobilization of fatty acids from the visceral fat depot may be a key factor behind many metabolic abnormalities observed in upper-body obesity. Only visceral fat is drained by the portal vein, so this fat depot has a unique direct link to the liver. Liver function is altered by increased ‘portal’ fatty acids, resulting in hyperinsulinaemia, dyslipidaemia, hyperglycaemia and liver insulin resistance, all of which form part of the metabolic syndrome in upper-body obese subjects (for details see Arner, 1996). Obese male subjects have higher lipolytic ß3AR function in visceral fat cells than obese women, which at least in part can explain the more marked metabolic aberrations in the obese males (Lönnqvist et al., 1997). The lipolytic ß3AR function is dependent on the adipose tissue region. In the two visceral depots, omental and mesenteric adipose tissue, the function is similar, but within these two regions ß 3 AR is more sensitive to stimulation than in the subcutaneous region (Van Harmelen et al., 1997). The regional differences in ß3AR function are much more attenuated in obese than in lean men (Hoffstedt et al., 1997). When all available data on lipolysis are considered together, it appears that the ß3AR is functional in all WAT regions, though ß3AR does not seem to play a leading role in lipolysis in the quantitatively dominating subcutaneous depot. In contrast, ß3AR appears to be of greater importance for lipolysis in visceral fat cells, and in particular in obese men. These subjects have very high rates of catecholamine-induced fatty acid mobilization from visceral fat, mainly owing to a high lipolytic function of ß3AR in their visceral fat cells. Thus, ß3AR may play a significant role in the pathogenesis of the metabolic syndrome, which often accompanies male obesity and is also seen in obese women who have an upper-body fat distribution (for detailed discussion see Arner, 1996). In these conditions, elevated ‘portal’ fatty acid levels could be the result of high ß3AR activity in visceral adipose tissue. In addition, ß3AR is responsible, at least in part, for the well-known regional variations in lipolytic activity between visceral and subcutaneous fat depots (Arner, 1995). The increased lipolytic activity in visceral as compared with subcutaneous fat cells is largely explained by the higher lipolytic function of ß3AR in visceral fat cells.

5.3.2

Brown adipose tissue

It is difficult to study brown adipose tissue (BAT) in humans because in general, only small amounts of tissue are present in adults. However, available data suggest that ß3AR is functional in human BAT. As in other species, the probable role of ß3AR is to stimulate thermogenesis through activation of uncoupling protein 1. ß3AR agonists increase uncoupling protein 1 mRNA levels in primary cultures of perirenal adipose tissue, which contains more brown fat cells than most other human fat depots (Champigny and Ricquier, 1996). Some of the thermogenic response in vivo to isoprenaline (a non-selective ßAR-agonist) infusion and to intravenous adrenaline administration is mediated by ß3AR (Wheeldon et al., 1993; Liu et al., 1995). Longterm oral treatment with CL-316,243, which is a selective ß3AR agonist in human fat cells (Hoffstedt et al., 1996a), increases thermogenesis in healthy volunteers (Weyer et al., 1998). Thus, it appears that the ß3AR is involved in the regulation of thermogenesis in BAT, although its role in relation to other adrenoreceptor sub-types in human brown fat cells is yet unknown.

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Heart

There is clear evidence that ß3AR co-exists with ß1AR and ß2AR and is functional in the human heart. For example, RO-363 (a ß1AR/ß3AR agonist) can increase atrial force in vitro through ß3AR (Molenaar et al., 1997b), while CGP-12177 mediates positive inotropic effects in human atrial myocardium in vitro (Kaumann, 1996). In addition, BRL-35135 and isoprenaline also mediate some of their cardiotropic effects in vivo through ß3AR (Wheeldon et al., 1993, 1994). Interestingly, cardiodepressive ß3AR effects which have been demonstrated in vitro in human heart and seem to be mediated by inhibition of cyclic AMP formation through a Gi-coupled receptor pathway (Gauthier et al., 1996). This must be a unique feature of human ß 3AR, since in man (and in other species) all other effects by this receptor subtype (as by ß 1AR and ß 2AR) are mediated by G s proteins. The negative cardiac inotropic effects of ß3AR vary markedly depending on the species, these being observed more readily in humans than in either rats or ferrets (Gauthier et al., 1999). Although a functional ß3AR is present in human heart, the physiological role of the receptor in relation to ß1AR and ß2AR remains unclear. Neither has the influence of ß3AR on pathophysiological conditions in the heart yet been determined.

5.3.4

Colon

The role of ß3AR in the regulation of colonie function has been demonstrated in humans in vitro (De Ponti et al., 1996; Roberts et al., 1997). The receptor is present in the colonie smooth muscle, but not in the colonie mucosa. ß 3AR mediates catecholamine-induced muscle relaxation, the role of ß3AR (and ß1AR) being to enhance mucosal blood flow in colon as a result of relaxation of vascular and nonvascular smooth muscle.

5.3.5

Brain

There is evidence for the presence of ß3AR mRNA in the human brain, although mRNA expression may be subject to regulation as its levels are much higher in the brain of children than in adults (Rodriguez et al., 1995). Although it remains to be established whether ß3AR is functional in human brain, a functional ß3AR has been reported in human neuroblastoma cells (Esbenshade et al., 1992).

5.3.6

Urinary bladder

mRNA for all three ßAR subtypes is present in the detrusor muscle of human urinary bladder (Takeda et al., 1999), and it is possible that ß3AR is of unique importance for the adrenergic regulation of urinary bladder motility. Only this ßAR subtype has been shown to have any marked functional effect on human detrusor muscle in pharmacological experiments (Igawa et al., 1999).

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5.3.7

Other tissues

mRNA for ß3AR is found in the gallbladder, small instestine and prostate gland of humans (Krief et al., 1993; Berkowitz et al., 1995), though as yet no evidence has been presented that ß3AR is functional in any of these tissues. However, a ß3AR protein has been shown to be present in gallbladder, as evidenced by the use of antibodies (Guillaume et al., 1994). Immunohistochemical studies have also suggested that a ß3AR protein is present in vascular smooth muscle in several regions of the gastrointestinal tract (Anthony et al., 1998).

5.4

Is there a fourth ßAR?

Some pharmacological data support the view that there is a fourth functional ßAR in human heart and white fat cells, and which is proposed to co-exist with the other three ßAR subtypes (Kaumann and Molenaar, 1997; Molenaar et al., 1997a; Galitzky et al., 1997, 1998; Kaumann et al., 1998). However, these data should be interpreted with some caution, since they are based on the use of the agonist CGP-12177 and the antagonist SR59,230A. The CGP compound is a partial ß3AR agonist, and its pharmacological behaviour is dependent on the amount and coupling of ß3AR. It has been shown in Chinese hamster ovary cells transfected with the cloned ß3AR that the sensitivity and potency of CGP-12177 is strongly dependent on receptor expression level and the nature of the pharmacological assay (Wilson et al., 1996). In the case of SR-59,230A, this compound is classified by the manufacturer as a ß3-selective antagonist. In our hands, however, the results of lipolysis experiments with human omental fat cells suggest that it is non-selective for all three known ßAR subtypes (P.Arner, unpublished results). Furthermore, unpredicted effects have been shown on human fat cells with regard to ßAR selectivity of novel agonists that are selective against ß3AR in cloned cells (Sennitt et al., 1998). Studies on the differentiation between native ß3AR and ß4AR in man have been hampered by lack of specific tools, and no full ß3AR agonist or truly selective ß3AR antagonist is available. However, data received suggest that it may be possible to develop selective antagonists for the human ß 3 AR (M.R.Candelore et al., unpublished results). There is no reliable radioligand available, although tritiumlabelled CGP-12177 and SB-206606 have each been used to monitor binding to ß3AR in human fat cells (Revelli et al., 1993; Deng et al., 1996). The CGP ligand binds to ß3AR with low affinity, but this is very difficult to differentiate from nonspecific binding. The SB ligand is a stereoisomer of BRL-37344, and the racemic form of the BRL compound is a non-selective ßAR ligand (Lönnqvist et al., 1993; Hoffstedt et al., 1996a). Difficulty also occurs in the correct pharmacological characterization of ß3AR in tissues that have a high number of ß1- and ß2ARs, but a low number of ß3AR (Sennitt et al., 1998). These unfavourable proportions of the three known ßAR subtypes exist in human adipose tissue. So far there are no genetic data in support of the existence of a ß4AR.

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Structural variations in the native human ß3AR

In 1995, three independent investigations demonstrated the existence of a coding polymorphism in the ß3AR gene that results in the replacement of tryptophan by arginine at position 64 (Trp64Arg) of the receptor (Walston et al., 1995; Clément et al., 1995; Widen et al., 1995). The less common Arg allele was associated with weight gain, early onset of non-insulin-dependent diabetes, and certain features of the metabolic (insulin resistance) syndrome. The polymorphism has attracted enormous scientific interest, and to date more than 100 studies have been published (MEDLINE) dealing with the genetic variance (see Walston et al., 1995; Widen et al., 1995 for detailed reviews). Some additional clinical features of the Arg allele have been presented (Table 5.3) such as decreased metabolic rate, increased fertility and increased risk of developing coronary heart disease. Furthermore, the association with obesity and increased weight gain seems gender-specific, as it is observed only in women. The phenotypic effect of the polymorphism is usually more marked in homozygotes than among heterozygotes. It should be stressed that the association between the Trp64Arg polymorphism and pathophysiology is controversial, as many studies have failed to demonstrate an influence of the polymorphism on obesity, weight gain, metabolic syndrome or noninsulin-dependent diabetes (Strosberg, 1997; Arner and Hoffstedt, 1999). One recent meta-analysis of 23 studies did not find any association between body mass index and the Trp64Arg polymorphism (Allison et al., 1998), though another meta-analysis did show such an association (Fujisawa et al., 1998). The reason for this divergence in results is not known, but it is not due to race, gender or population size. With regard to any association with obesity, evidence both for and against has been found in Finnish, Japanese, adult female and large populations. In contrast, most of the genetic studies have been performed on cross-sectional materials. In a paired sibling analysis, the Arg64 allele was associated with significant higher values in body mass index (Mitchell et al., 1998). An important aspect of a structural variance in a protein is whether it is functional or not. Again, conflicting results have been published regarding the Trp 64 Arg polymorphism. In Chinese hamster ovary cells transfected with the mutant ß3AR variant, receptor function has been either normal (Candelore et al., 1996) or reduced (Piétri-Rouxel et al., 1997) in comparison with cells carrying the ‘wild’ variant. In a small number of subjects the lipolytic function of ß3AR in omental fat cells was not significantly different between carriers and non-carriers of the Arg 64 allele (Li et al., 1996). However, in a larger study group the same investigators recently demonstrated that the lipolytic sensitivity of the ß3AR agonist CGP-12,177A was

Table 5.3 Phenotypes influenced by the Trp64Arg polymorphism in human ß3AR

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significantly and 10-fold reduced in omental fat cells of the carriers of the Arg 64 allele (Hoffstedt et al., 1999). Other investigators have also demonstrated that the Trp64Arg variant influences ß3AR function in visceral human fat cells (Umekawa et al., 1999). It is possible that ß3AR is a so-called ‘thrifty’ gene (Groop and Tuomi, 1997) which is important for survival among ancient tribes whose living conditions were dependent on the availability of food. As mentioned, the Trp64Arg variant of ß3AR is associated with rapid weight gain, low metabolic rate and improved fertility. All these features would be advantageous in a primitive society where food is sparse. Recent data suggest that the Trp64Arg polymorphism is an old genetic variant, as it is associated almost completely with several other (non-coding) structural variations in the ß3AR gene (Hoffstedt et al., 1999). When all available data are considered together, it is our opinion that the Trp64Arg polymorphism of ß3AR is functional and has an influence on body weight and on several metabolic parameters associated with obesity. However, the Arg64 variant cannot be considered as a major obesity gene abnormality.

5.6

ß3AR as a therapeutic target

Because of the central role of ß3AR in the regulation of thermogenesis in many animal models, selective ß3AR agonists have been considered suitable candidates for anti-obesity drugs (Arch and Wilson, 1996a; Lipworth, 1996; Weyer et al., 1999). A low metabolic rate is believed to be of pathophysiological significance for the development of obesity (Ravussin, 1995). Thermogenesis is mainly activated by ß3 AR agonists through stimulation of BAT (Lowell and Flier, 1997). As mentioned earlier, there is evidence that thermogenesis in humans can be stimulated by ß3AR and that this might occur in brown fat cells. However, a number of anti-obesity drug trials using selective ß3AR agonists have failed to induce weight loss and/or have been associated with ß 1AR or ß2AR-mediated side effects (Arch and Wilson, 1996a; Groop and Tuomi, 1997); these were most likely due to the agonists having been developed using ß 3 AR in rats as a pharmacological model. With reference to previous discussion in this chapter, the rat ß3AR differs greatly from its human counterpart, and thus most of the ‘rat-derived’ ß3AR agonists used in trials were either non-effective or non-selective on the native human ß3AR (Hoffstedt et al., 1996a). Several pharmaceutical companies have developed ß3AR agonists using the human ß3AR as a pharmacological tool in drug testing. For example, a highly potent human ß 3AR agonist, (L-755,507) developed by Merck (Parmee et al., 1998) increased metabolic rate in rhesus monkeys (Fisher et al., 1998). The compound appeared to be a selective ß3AR agonist on lipolysis in human fat cells (Zilberfarb et al., 1997). Several such second-generation ß3AR agonists are presently under test as anti-obesity drugs in clinical trials, but the outcome of these investigations is not yet known (Weyer et al., 1999). ß3AR agonists may also be beneficial in non-insulin-dependent diabetes (Arch and Wilson, 1996a; Lipworth, 1996; Weyer et al., 1998). The anti-diabetic effect can in part be secondary to weight reduction, but may also occur directly due to stimulation of the combustion of fatty acids, as the latter have several adverse effects on glucose metabolism. In addition, ß3AR agonists have been found to stimulate insulin secretion in some animal models (Arch and Wilson, 1996b; Lipworth, 1996), though

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Table 5.4 Possible clinical use of ß3AR agents

whether the same effect can be obtained in man remains to be established. The early ß3AR agonists have been used as anti-diabetic drugs, but results were disappointing (Arch and Wilson, 1996a; Lipworth, 1996; Weyer et al., 1999), probably for the same reasons that the agonists failed as anti-obesity drugs. Another possible indication for ß3AR agonists is that of anti-inflammatory agents for the gastrointestinal tract (Anthony, 1996). As mentioned above, the intestinal receptor is functional in man and may stimulate blood flow in the intestinal mucosa through the relaxation of smooth muscles. In addition to their spasmolytic properties, animal experiments have suggested that ß3AR agonists might heal gastric and small intestinal ulcers, though their clinical use in ulcero-inflammatory disorders of the gastrointestinal tract remains to be established. In theory, ß3AR antagonists might also be used as therapeutic agents, bearing in mind two points: first, the increased liploytic action of ß3AR in visceral adipose tissue of obese subjects; and second, the association between increased ß3AR function and the metabolic syndrome (see Section 5.3.1). In upper-body obese subjects with signs of the metabolic syndrome, ß3AR antagonists might preferentially inhibit fatty acid release from visceral adipose tissue and improve some of the metabolic abnormalities associated with the high ‘portal’ fatty acid flux (Lönnqvist et al., 1993; Arch and Wilson, 1996b; Arner, 1996). The putative clinical indications for ß3AR-directed agents are summarized in Table 5.4. Because the receptor is expressed mainly in adipose tissue it is an important target in anti-obesity and anti-diabetic drug programmes for the pharmaceutical industry. However, side effects from the intestine and heart—and perhaps also from the urinary bladder, gallbladder, brain and prostate gland, where the ß3AR is also functionally expressed—should be considered in both drug design and drug trials.

5.7

Conclusions

There is accumulating evidence that ß3AR is of functional importance in man, as the receptor has a clear role in promoting lipid mobilization from white adipose tissue— and in particular from visceral adipose tissue—because of its stimulatory effect on lipolysis in fat cells. The ß3AR appears to have a physiological role in the development of the metabolic (insulin resistance) syndrome because of its increased function in visceral adipose tissue of upper-body obese subjects, which is observed primarily in obese men. The receptor is also involved in the regulation of thermogenesis and contractility of the heart, urinary bladder and gallbladder, although its physiological

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importance and pathophysiological role in these processes is less well defined. A polymorphism, Trp64Arg, in the ß3AR has been described which has been shown to be associated with obesity and obesity complications in some, but not all, studies. This polymorphism may also alter the native receptor function; however, although it may influence body weight and metabolism, the polymorphism is probably not a major obesity gene abnormality. Although in the past ß3AR agonists and antagonists have been proposed as antiobesity and anti-diabetes drugs, the first-generation agonist drugs were either ineffective or had undesirable side effects. Consequently, second-generation agonists are currently being developed using the human ß3AR as a pharmaceutical model. Indeed, these agents might be useful in the future treatment of both obesity and non-insulin-dependent diabetes mellitus.

6

ß3-Adrenoreceptor-Mediated Responses in Heart and Vessels MAX LAFONTAN, DOMINIQUE LANGIN, JEAN GALITZKY, MICHEL BERLAN, CHANTAL GAUTHIER 1 AND GENEVIÈVE TAVERNIER Unité INSERM 317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangueil, 31403 Toulouse Cédex 4, France 1 Laboratoire de Physiopathologie et Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96–01, Hôpital Hôtel-Dieu and Faculté des Sciences et Techniques, Université de Nantes, 44093 Nantes Cedex, France

6.1

Introduction

The cloning of the gene encoding the ß3-adrenoreceptor (ß3AR), rapidly followed by the delineation of its structural determinants in transfected cell lines, has opened a new avenue for our undertanding of the adrenergic regulation of a number of cells (Emorine et al., 1989, 1991; Piétri-Rouxel et al., 1995). The role and the regulation of the ß3AR have mainly been studied in white and brown fat cells, and in the gastrointestinal tract in various species, including humans. Other studies, based on ß3AR mRNA detection and/or functional studies with selective ß3AR agonists and antagonists have also revealed that its expression is probably not limited to these tissues (Arch and Kaumann, 1993). The majority of studies on the beta-adrenergic control of the cardiovascular system have focused on ß1- and ß3AR, and until now relatively little was known regarding ß3AR cardiovascular control. Here we present an overview of recent reports addressing the existence and role of the ß3AR in the cardiovascular system. Moreover, the existence of another putative ‘atypical’ ßAR—named the ß4AR—in heart will be discussed.

6.2

ßAR subtypes in the heart

A large number of studies have shown the co-existence of ß1- and ß2AR in the human heart. Their stimulation produces positive inotropic effects in in vitro human atrial and ventricular preparations (Brodde, 1991) as well as in single myocytes (Del Monte et al., 1993). However, a series of non-selective adrenergic antagonists with high affinity for myocardial ß1- and ß2AR have been shown to promote stimulant effects on atrium and ventricle. These actions were resistant to blockade by the highly selective ß1- and ß2antagonists. In 1989, Kaumann suggested that these partial agonists such as pindolol and related indoleamines may act through a third cardiac ßAR ressembling the ß3AR. These 87

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agents were causing stimulant effects at concentrations greatly in excess of those that usually caused blockade of the myocardial effects of catecholamines (Kaumann, 1989). This putative ßAR appeared to be coupled to adenylyl cyclase via a G protein of the Gs type. These preliminary studies have been followed by two major observations.

6.2.1

Functional ß3AR in the human heart

In isolated preparations of human ventricle, obtained from endomyocardial biopsies in patients having open-heart surgery or cardiac transplants, the ß3AR agonists promoted negative inotropic effects and reduction in the amplitude and duration of the action potentials (Gauthier et al., 1996). When considering the mechanical responses, the non-selective beta-adrenergic agonist, isoproterenol, at micromolar concentrations and under blockade of ß1- and ß2AR by nadolol, exerted negative inotropic effects. Nadolol is a ß1-/ß2-antagonist which has low affinity for the native and recombinant ß3AR (Bond and Clarke, 1987; Emorine et al., 1989; Galitzky et al., 1993a). The ß3adrenergic selective agonists (BRL-37,344, CL-316,243, SR-58,611A and CGP12,177), which are known to exert lipolytic and thermogenic effects in white and brown rodent fat cells respectively (Lafontan and Berlan, 1993), exerted concentration-dependent negative inotropic effects in human heart ventricle biopsies (Figure 6.1). They also induced a reduction in the amplitude and an acceleration in the repolarization phase of the action potential. The relative rank order of potency of the various ß 3 AR agonists (BRL-37,344>SR-58,611~CL-316,243>CGP-12,177) was similar to that observed in Chinese hamster ovary cells expressing human ß3AR (PiétriRouxel and Strosberg, 1995). CGP-12,177, the non-conventional ß 3-adrenergic agonist, having ß 1 - and ß 2 -adrenergic antagonist properties, also exerted a cardiodepressant action which was weaker than that elicited by the other ß3-agonists. The cardiodepressant effects of the various ß3AR agonists were unaffected by nadolol and metoprolol blockade, but were antagonized by bupranolol, which combines ß1-, ß2and ß 3AR antagonist properties. This pharmacological profile demonstrates the expression of a functional ß3AR in human heart. Treatment of endomyocardial biopsies with pertussis toxin blunted the cardiodepressant effect of BRL-37,344. This result is consistent with the coupling of the heart ß3-adrenoreceptor with a pertussis toxin-sensitive protein of the Go and Gi family. A more complete study is required to establish the Gi protein isoform activated by the ß3AR as well as the nature of the different steps of the receptor-effector pathway, downstream to Gi activation, involved in the control of electrical and mechanical responses. A recent report has shown that the decrease in human cardiac contractility promoted by activation of ß3AR operates through the activation of a nitric oxide synthase (NOS) pathway. Increases in nitric oxide (NO) production and intracellular cGMP levels were observed after treatment of endomyocardial biopsies with a ß3AR agonist. Pertussis toxin abolishes the effect of ß3AR stimulation both on cardiac contraction and on cGMP generation, suggesting the involvement of a Gi/o protein. Finally, immunohistochemical analysis have revealed abundant endothelial NOS (eNOS) but not intracellular NOS (iNOS) proteins in sections of human endomyocardial biopsies (Gauthier et al., 1998). Coupling of the ß3AR to Gi in heart is noticeable. At a basic research level, it is the first report implicating a ßAR whose primary biochemical function appears to be inhibitory and Gi-mediated. Most reports in native cell systems have shown that ßAR are coupled to Gs protein. However, it has

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Figure 6.1 Effects of BRL-37,344 and SR-58,611 on the twitch tension of human endomyocardial biopsies. (A) Time-course of the effects of cumulative concentrations of BRL37,344 (left) and SR-58,611 (right) on twitch tension. After control value settlement (C), the ß3AR agonists were perfused for 10 min with each concentration to obtain a steady state of the effect. (B) Superimposed twitches obtained from the experiments illustrated in (A). (C) Concentration-response curves for the negative inotropic effect of BRL-37,344 and SR-58,611. Values are the means ±SEM of six to seven experiments. *Significant statistical difference (P<0.05) from basal peak tension. (Reproduced from Gauthier, C. et al., J.Clin. Invest. 1996, 98:556–62.)

been shown previously that fat cell ß3AR can also, in addition to a primary coupling to Gs, be coupled to Gi (Chaudhry et al., 1992; Begin-Heick, 1995). Furthermore, the stimulation of ß2AR has been shown to activate both Gs and Gi in mouse cardiomyocytes (Xiao et al., 1995). Since Gi protein can be up-regulated in certain types of heart failure (Feldman et al., 1988), an increase of this G protein may produce an increase in ß3ARmediated effects in patients with heart failure. This is a point which merits further attention. In addition to the pharmacological and functional studies, the expression of a myocardial ß3AR was further shown by the detection of ß3AR transcripts in the human ventricle. The authors verified that the presence of ß3AR transcripts was not linked to the presence of contaminating adipose tissue in the heart biopsy (Gauthier et al., 1996). Previous studies reporting the presence of ß3AR transcripts in human atria either took into account (Krief et al., 1993), or did not (Berkowitz et al., 1995), the possible presence of fat.

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The results also focus attention on the risks of exclusive screening on a single second messenger pathway in transfected cells and the interest to perform functional assays in native receptors-expressing organs. Most of the protocols for the screening of ß3-adrenergic agonists have been based on measurements of adenylyl cyclase activation and increase of cAMP levels in cells transfected with the ß 3AR gene. It is likely that, depending on the type of assay and the host cell used, some ß 3ARdependent coupling and effects have been missed. In addition, it is not possible from studies on transfected cells to determine which ßAR will mediate responses to agonists in tissues that have a high number of ß1- and ß2AR and a low number of ß3AR. The existence of a functional ß3AR in human myocardium, whose activation inhibits contractility, brings another dimension to our understanding of the adrenergic regulation of heart function and its role in heart pathology. ß3AR may become important in pathological situations linked to alterations of the ß1/ß2AR-dependent positive inotropic effect that have been described when marked increases in sympathetic tone and cardiac noradrenaline level occur. Under such conditions, changes in the expression of the ß3AR and/or efficiency of ß3AR-dependent pathways may alter the balance between positive and negative inotropic effects of catecholamines on the heart and lead to possible myocardial dysfunction.

6.2.2

The putative ß4AR in cardiac tissue

Based on the use of non-conventional partial agonists such as CGP-12,177, cyanopindolol and pindolol, the existence of a putative ß4AR in the heart has been proposed. These agonists can cause tachycardia in vivo in the rat, and exert positive inotropic effects in vitro in the atrium of human and all mammalian species studied (Kaumann, 1996; Malinovska and Schlicker, 1996, 1997; Kaumann and Molenaar, 1997). However, selective ß3-adrenergic agonists such as BRL-37,344, CL-316,243 and SR-58,611 failed to elicit cardiostimulation. Atrial and ventricular contractile force are increased by non-conventional partial agonists. The effects are probably mediated by the activation of Gs protein and cAMP-dependent protein kinase. The relative efficacy of various antagonists in the suppression of ß4AR-mediated effects has been established. A similar pharmacological profile has been found in the heart of various species (Molenaar et al., 1997a) and in human adipose tissue (Galitzky et al., 1998). The arguments for the existence of another ßAR called the ‘ß4AR’ have been discussed extensively in a recent review (Kaumann and Molenaar, 1997). The results obtained in human fat cells by us are in accordance with the arguments developed by these authors (Galitzky et al., 1998). Recently published binding data have shown that [3H]-CGP-12,177 binding sites, different from the ß3AR, exist in rat and mice atrium membranes (Kaumann et al., 1998; Sarsero et al., 1998). Although the pharmacological demonstration has been questioned (Strosberg et al., 1998), the recent results obtained in heart and brown fat of mice bearing an invalidation of the ß3AR gene support the views of Kaumann. All the ß4AR-mediated effects described in mice heart are preserved in mice lacking ß3AR (Kaumann et al., 1998). Furthermore, CGP-12,177, the only compound used as a reference compound to assess the presence of a functional ß3AR in human fat cells, exerts potent thermogenic effects in vivo (Ito et al., 1998) and stimulates oxygen consumption in vitro in brown adipose tissue of mice devoid of ß3AR (Preitner et al., 1998). These results demonstrate that some of the

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effects on non-conventional partial agonists, in particular CGP-12,177, cannot be attributed to ß3AR. Nevertheless, the molecular proof for the existence of a ß4AR is still missing; controversial views remain valid and the debate merits being kept open (Galitzky et al., 1998; Strosberg et al., 1998).

6.3

ß3AR-mediated vasodilatation

ßAR-agonists have been shown to exert profound effects on peripheral vasodilatation, primarily in skin and adipose tissue, in unanaesthetized dogs (Berlan et al., 1994; Shen et al., 1994). Studies that used radioactive microspheres demonstrated a different pattern of distribution of regional blood flow increase with ß3AR stimulation compared with isoproterenol-induced ß1- and ß2AR-mediated peripheral vasodilatation. BRL-37344, associated with ß1- and ß2AR blockade, selectively increased blood flow to the skin and adipose tissue (Shen et al., 1994). Similar vasodilating effects were obtained in dog skin, after BRL-37,344 and CL-316,243 infusion, when measuring directly cutaneous blood flow with a laser Doppler flowmeter (Berlan et al., 1994). Bupranolol, the non-selective ß-adrenergic antagonist, known to display antagonistic effects against the dog ß3AR (Galitzky et al., 1993a,b) antagonized the blood flow changes promoted by the ß3adrenergic agonists (Figure 6.2). Vasodilatation has also been observed in brown adipose tissue after administration of a ß3-adrenergic agonist (BRL-26830A) associated with a ß1-/ß2-adrenergic antagonist having a-adrenergic blocking properties (arotinolol) in anaesthetized rats (Takahashi et al., 1992). A comparative study of the effects of two ß3AR agonists (BRL-37,344 and CL316,243) administration has been performed in conscious dogs, rats and non-human primates. CL-316,243 appeared to be a more specific ß3AR agonist than BRL-37,344 as previously described in rodent fat cells. ß3AR stimulation was most profound in dogs, where even a direct effect on left ventricular contractility was identified, but was diminished although significant in rats and essentially absent in non-human primates (Shen et al., 1996). The experimental conditions used by the authors have not permitted the demonstration of ß3AR-mediated negative inotropism. The effect of ß3-adrenergic agonists on human subcutaneous adipose tissue blood flow was studied using in situ microdialysis and the ethanol escape method (determination of the ethanol outflow/inflow ratio) in order to evaluate semiquantitatively local blood flow changes in adipose tissue (Lafontan and Arner, 1996). CGP-12,177 promotes a vasodilatation in human subcutaneous adipose tissue, which is weaker than that initiated by dobutamine ß 1AR agonist) or terbutaline (ß2AR agonist) (Barbe et al., 1996). In the absence of selective ß3AR antagonist it is difficult to ascertain whether the effect is mediated by ß 3- or ß 4 AR, or by some other mechanism. Although the ß 3AR-mediated vasodilating effects are not questionable, the mechanisms initiating the vasodilating effects appear to be independent of generally recognized mediators of peripheral vasodilatation [agonists of a-adrenergic, cholinergic, histaminergic or purinergic receptors; prostaglandins and endotheliumderived relaxing factor (EDRF)] (Shen et al., 1994). Other mechanisms that mediate vasodilatation in skin and fat may be proposed. Vasodilating effects are related to a potent increase in metabolism rather than mediated by autonomic or hormonal mechanisms, ß3AR agonists elicit a strong increase in plasma non-esterified fatty acid concentrations and an increase in insulin release (Galitzky et al., 1993a),

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Figure 6.2 (A) Effect of the intravenous (i.v.) infusion (from 0 to 10min) of CL-316,243 (0.4 nmol/kg/min) on cutaneous blood flow in normal dogs. (B) Effect of the i.v. infusion of BRL37,344 (0.4 nmol/kg/min) on cutaneous blood flow in normal dogs before and after bupranolol administration. The probe holder was positioned on the internal part of the left ear. CL-316,243 and BRL-37,344 promoted striking vasodilating effects in dog skin. Bupranolol, the nonselective ßAR antagonist known to exert antagonist effects at the ß3AR, antagonized the blood flow changes initiated by the ß3AR agonist BRL-37,344. Values are means ±SEM.

suggesting that blood flow increases could be secondary to a strong increment of metabolic processes (Bülow and Madsen, 1981). To date, the presence of ß3AR mRNAs has not been investigated in skin and adipose tissue vessels. However, it cannot be excluded that the systemic administration of ß3AR agonists promotes the release of a circulating factor, in an unidentified location, which exerts vasodilating effects in vessels. Until recently, the beta-adrenergic vasodilating effects were known to occur preferentially through ß2AR and mainly at the microcirculation level, the action on vessels of larger resistance and on capacitance vessels being weak. Together, these data, obtained in various species, indicate that ß3AR could exist in the micro vasculature of some specific vascular territories and confirm that the distribution of ßAR subtypes varies, as that of a1- and a2-subtypes, according to the localization of the vessels and the animal species (McGrath et al., 1989). From a physiological point of view, the role of vessel ß3AR is currently poorly known. They are probably under the

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control of noradrenergic innervation, and appear to exert redundant functions in vessels which also express ß2AR. Catecholamines stimulate ß3AR (in rat and dog fat cells) at higher concentrations than those required to recruit ß1- or ß2AR (Granneman, 1992; Galitzky et al., 1993b). The search for a specific involvement of ß3AR in conditions characterized by hyperactivity of the sympathetic nervous system merits consideration.

6.4

6.4.7

ß3AR regulation of blood flow Regulation of pancreatic islet blood flow

ß3AR agonists have been reported to have an anti-diabetic effect in rats and mice (Cawthorne et al., 1984; Caroll et al., 1985), though the exact mechanism of action is not well understood. It has been shown that these agents increase both insulin secretion and action (Smith et al., 1985). Increase of insulin release has been demonstrated in vivo in rats and mice and in the perfused mouse pancreas (Bloom et al., 1992; Yoshida, 1992; Yoshida et al., 1994b). The stimulatory effect of ß3AR agonists on insulin secretion is no longer present in pancreatic islets in vitro (Yoshida, 1992). These results suggest that the ß3AR agonist-induced insulin release is not due to a direct effect of the drug on the pancreatic beta cell. Utilization of mice having a targeted disruption of the ß3AR gene has confirmed the contribution of ß3AR in the insulin-secreting effects. Acute treatment with CL-316,243 produced a 140-fold increase in plasma insulin levels in control mice, but had no effect in the mice lacking ß3AR. The effects of acute CL-316,243 treatment on insulin secretion seems to be mediated exclusively by the presence of ß3AR (Susulic et al., 1995). Regulation of pancreatic islet blood flow, which is known to be under the influence of both parasympathetic and sympathetic nervous systems (Atef et al., 1992), is an important component of the control of insulin secretion (Jansson, 1994). Noradrenaline has been reported to inhibit islet microcirculation via an aAR-mediated mechanism (Meyer et al., 1982; Rooth et al., 1985). Administration of the nonselective ßAR agonist, isoproterenol leads to stimulation of islet blood flow (Meyer et al., 1982), whereas the ß2AR agonist, terbutaline promotes its reduction (Jansson et al., 1989). There is evidence that ß 3 AR are under the influence of noradrenergic innervation (Taneja and Clarke, 1992). Thus, islet blood flow could be under the influence of the sympathetic nervous system via both a- and ß3AR. The two types of receptors would have opposite effects on the regulation of blood flow, as already reported for insulin secretion. The exact role of both receptor types remains to be clearly established. Experiments have been performed to determine if a ß3AR agonist could modify islet blood flow in rats, using the highly selective ß3AR agonist, CL-316,243 (Atef et al., 1996). Islet blood flow measurements were performed with a method utilizing nonradioactive microspheres (Jansson and Hellerström, 1981; Atef et al., 1992). CL316,243 was shown to promote a marked increase in both islet blood flow and plasma insulin concentration without changes in whole pancreatic blood flow. The increase was totally prevented when the rats were pretreated with bupranolol, but not with nadolol. These results suggested the presence of ß3AR in microvessels of Langerhans islets (Figure 6.3). Due to the differences existing in the effects of ß3AR agonists on insulin secretion according to whether it was studied in vivo or in vitro, it seems

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Figure 6.3 Plasma insulin concentrations and islet blood flow, a percentage of pancreatic blood flow, in rats under basal conditions, after perfusion of CL-316,243 alone or in combination with an injection of bupranolol or nadolol. Values are means ±SEM of four to seven determinations. Statistically significant difference between control and treated rats: **P<0.01, ***P<0.001.

reasonable to propose that the increased islet blood flow induced by the ß3AR agonist stimulation could be one of the mechanisms involved in the increased insulin secretion described in the rats treated with ß3AR agonists. These data provide insights into the mechanism of the anti-diabetic and insulinotropic effect of ß3AR agonists, though some points need to be clarified. The presence of ß3AR mRNAs has not yet been shown in rat Langerhans islets. Mouse pancreatic islets appear not to express detectable levels of ß3AR mRNA (Grujic et al., 1997). Recent studies performed on mice with targeted disruption of the ß3AR gene and genetically engineered to express exclusively the receptor in brown adipose tissue alone (BAT-mice) or in both brown and white adipose tissue (BAT+WAT-mice) have raised further questions (Grujic et al., 1997). The insulin-secreting effect of CL316,243, which is completely suppressed in mice lacking ß3AR, was not recovered in BAT-mice, but appeared to be largely recovered in BAT+WAT-mice. This report suggests that the stimulatory effect of CL-316,243 on insulin secretion is not mediated via a direct stimulation of ß3AR within islets [since ß3AR are only driven in BAT and WAT by tissue-specific uncoupling protein-1 (UCP-1) and aP2 gene promoters respectively]. Islet blood flow measurements were not performed in this study, though species-specific differences might exist in the regulation of islet blood flow. These puzzling results suggest that the effects of CL-316,243 on insulin secretion depend largely upon the presence of ß3AR in white fat cells. It may be proposed that a signal or signals emanating from adipocytes and tightly controlled by ß3AR stimulation can directly or indirectly control pancreas beta-cell function. The identity of such signals is unknown; but free fatty acids which are known to promote insulin secretion are putative candidates (Boden, 1997; Grujic et al., 1997; Bollheimer et al., 1998). This observation does not completely exclude a possible modulation of islet blood flow by ß3AR in Langerhans islets of normal mice and rats.

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Regulation of gastric mucosal blood flow

Several lines of evidence suggest that the vasodilative action of isoproterenol, in addition to gastric secretory inhibition due to ß-adrenergic stimulation, protects the gastric mucosa from experimental ulcerations. It is considered that some gastric ulcers are promoted by an imbalance between aggressive factors (such as gastric acid) and defensive factors (such as blood flow). Vasodilators with a-adrenergic blocking activity have been shown to attenuate indomethacin-induced antral ulcers in refed rats (Kuratani et al., 1992). Administration of ß3AR agonists, which enhances gastric mucosal blood flow, also attenuates the indomethacin-induced antral ulcers in refed rats. Conversely, activation of ß1- and/or ß2AR attenuates indomethacin-induced corpus erosions through an inhibition of gastric secretion (Kuratani et al., 1994). Various ß3AR agonists (BRL351,35, CL-316,243 and SR-58,611 A) have been shown to enhance gastric mucosal blood flow, suggesting the presence of ß3AR in the microvasculature of the antrum and their involvement in the relaxation of vascular smooth muscle (Kuratani et al., 1994). Thus, ß3AR-induced vasodilatation attenuates the appearance of indomethacin-induced antral ulcers in rats, and there is evidence to suggest that ß3AR-dependent enhancement of mucosal blood flow is a mechanism of protection. If the same phenomenon exists in humans, ß 3 AR agonists might offer some therapeutic applications in ulceroinflammatory disorders (Anthony, 1996).

6.5

ß3AR-mediated cardiovascular effects

ß3AR agonists promote potent cardiovascular changes in dogs, including positive chronotropic effects induced by the administration of various ß3-adrenergic agonists (BRL-37,344, CGP-12,177 and CL-3 16,243). This effect was not related to a direct stimulation of cardiac ß3AR, but was due to a baroreflex mechanism, since it was suppressed after sinoaortic denervation in conscious animals (Tavernier et al., 1992). Furthermore, the ß3-adrenergic agonist, BRL-37,344 has no direct ß3AR-mediated effects in isolated blood-perfused dog atria (Takayama et al., 1993) or in the pithed or conscious rat (Cohen et al., 1995).

6.6

Conclusions and future trends

Although characterization of the ß3AR first triggered interest in this novel receptor subtype among research groups working in the fields of brown and white adipose tissue, there is now no doubt that the receptor is expressed also in other tissues. Both pharmacological studies and detection of the ß3AR mRNA in the target cells enabled ß3AR to be localized in rodent brown and white adipose tissues, human heart and gut. A number of heterogeneous pharmacological studies in rodents and other species also detected atypical ßAR, which might be ß3AR if convincingly demonstrated, in other tissues (e.g. oesophagus, stomach, skeletal muscle, hypothalamus, liver, bowel and prostate gland). In a number of cases the pharmacology was not complete, and to date mRNAs have not been investigated (Arch and Kaumann, 1993). Most of these studies must be followed by more convincing demonstrations, as discussed for the ß4AR concept. With regard to cardiac ß3AR, it is now necessary to investigate the level of expression

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of ß3AR in various cardiac locations and to delineate their functional role in the intact heart. Moreover, a detailed investigation of the transduction pathways involved in the ß3AR-mediated effects in heart is also necessary. In addition, it is a major goal to establish if the desensitization-resistant ß3AR are becoming more operative as the heart fails (when the ß1-/ß2AR-dependent effects have been rendered relatively unresponsive by an increased sympathetic tone). If such a mechanism were to operate, one might speculate that drugs which block ß3AR might be of therapeutic importance in such pathological conditions. The use of animal models will be helpful in solving some fundamental questions. Preliminary studies in mice have not revealed ß3AR mRNA in heart (G.Tavernier et al., unpublished results), although this animal could represent a valuable model for the production of transgenic animals expressing various levels of human ß3AR in different regions of the heart (depending upon the nature of the promoter used to target the expression of the ß3AR gene in the organ). With regard to the putative ß4AR, the final verdict will be pronounced when the receptor gene is cloned and the pharmacological properties of this receptor are fully elucidated. The involvement of ß3AR in the control of local blood flow in tissues and organs represents a wide open field. Until now, ßAR-dependent vasodilating effects have been shown to occur preferentially through ß2AR, and mainly at the microcirculation level. It is essential to establish whether ß3AR could exist in the microvasculature of some specific vascular territories and explain the vasodilating effects of ß3AR agonists. To date, the presence of ß3AR mRNAs and ß3AR has not been investigated in vessels from skin, adipose tissue, gastric mucosa and Langerhans islets. This question needs to be clarified first. In addition to the adrenergic-dependent effects, vasodilatation is the result of complex and balanced tissue-specific actions of cholinergic, peptidergic and purinergic nerves. It cannot be excluded that the systemic administratation of ß3AR agonists, which initiates vasodilatation, promotes in fact the release of a circulating factor that is thought at present to originate from an unidentified location, and which is able to exert vasodilating effects in vessels. Finally, research on the regulation of ß 3AR gene expression will permit the identification of the nervous, endocrine or metabolic factors that govern the tissuespecific expression of this gene in human adults.

7

ß3-Adrenoreceptors in Brown and White Adipocytes: Roles in Thermogenesis and Energy Balance JEAN HIMMS-HAGEN Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa ON, Canada K1H 8M5

7.1

Introdu ction

Brown adipocytes represent a unique type of cell by virtue of their content of a specific uncoupling protein (UCP-1). Indeed, the presence of UCP-1 defines an adipocyte as brown (Himms-Hagen and Ricquier, 1998). This protein, which is not found in any other mammalian cell, has a key role in the production of heat (thermogenesis) in the body. In experimental animals, this role has been shown to be important in the maintenance of body temperature in a cold environment, and in the maintenance of energy balance. Thermogenesis in brown adipocytes is brought into play by impulses from the central nervous system reaching the adipocytes via sympathetic nerves. The nervous stimuli arriving at the brown adipocytes release noradrenaline which, by reacting with adrenergic receptors on the cell surface, initiates the adipocytes’ thermogenic response. Prolonged sympathetic stimulation can also induce hyperplasia of the brown adipocytes, with proliferation of their mitochondria, and thereby lead to hypertrophy of the brown adipose tissue (BAT). In most species, the principal adrenergic receptor of brown adipocytes is the ß3-adrenoreceptor (ß3AR). This receptor is not, however, unique to brown adipocytes but is also present in white adipocytes. A number of drugs that can serve as ß3AR agonists are now available. They are proving valuable in experiments designed to unravel the physiological functioning of brown adipocytes in the body. ß3AR agonists are also candidates for anti-obesity drugs to raise energy expenditure in the treatment of obesity (Himms-Hagen and Danforth, 1996; Danforth and Himms-Hagen, 1997). One impediment to the use of ß3AR agonists in treatment of obese humans has been the very low number of readily identifiable brown adipocytes in adult human adipose tissues. However, BAT is abundant in newborn babies. Immortalized cell lines derived from BAT of newborn babies express ß3ARs as their major ßAR subtype (Zilberfarb et al., 1997; Jockers et al., 1998). They also express the characteristic uncoupling protein-1 (UCP-1) (Zilberfarb et al., 1997). Moreover, precursor cells isolated from adult human adipose tissues can differentiate in culture and 97

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develop into brown adipocytes when stimulated by ß3AR agonists (Champigny and Ricquier, 1996) or by a thiazolidine dione (Digby et al., 1998). Brown adipocytes have been reported in adult human white adipose tissue (WAT) under the influence of excessive sympathetic stimulation due to phaeochromocytoma (Ricquier et al., 1982; Lean et al., 1986; Garruti and Ricquier, 1992; Zilberfarb et al., 1997) and these brown adipocytes do express ß3ARs (Jockers et al., 1998). In rodents, ß3AR agonists induce not only hypertrophy and thermogenic activation of brown adipocytes in BAT, but also the appearance of ‘ectopic’ brown adipocytes in certain WAT depots where they are otherwise not usually apparent and where they presumably contribute to the increase in energy expenditure induced by the drug. The origin of these ‘ectopic’ brown adipocytes is not understood and remains the subject of much investigation. Thus, continued elucidation of factors involved in the proliferation and differentiation of brown adipocytes at different locations in both BAT and WAT depots and in the expression of UCP-1 and of the role of ß3ARs in these processes—particularly in humans—is anticipated to lead to novel approaches to raising human energy expenditure that exploit the thermogenic function of brown adipocytes. BAT has been the subject of two books (Lindberg, 1970; Trayhurn and Nicholls, 1986) and many recent reviews (Himms-Hagen, 1989, 1990, 1991, 1996; Ricquier et al., 1991; Ricquier and Cassard-Doulcier, 1993; Himms-Hagen and Ricquier, 1998), and the reader is referred to these for a detailed analysis of the previous literature. This chapter will concentrate on concepts of brown adipocyte function and of its control by ß3ARs that are changing as a consequence of the recent discovery of other uncoupling proteins (besides the long-known UCP-1), of the often perplexing results of studies of transgenic mice with altered BAT function, and of new information about the ‘ectopic’ appearance of brown adipocytes in traditional WAT depots.

7.2

7.2.1

Brown and white adipocytes and tissues

Functions of brown and white adipocytes

The longest known physiological function of brown adipocytes is their increased heat production when they are stimulated by noradrenaline released from the sympathetic nerves which innervate them in BAT depots. Heat can be regarded as a secretory product of brown adipocytes, exported via the extensive blood flow through the BAT, and contributing to thermoregulation in a cold environment. However, brown adipocytes also secrete other products (see Section 7.5 below). For example, the socalled satiety hormone, leptin, which is derived mainly from white adipocytes, can also be expressed in and secreted by brown adipocytes. Its synthesis in BAT is rapidly suppressed by sympathetic nervous system activity (Moinat et al., 1995; Dessolin et al., 1997), and is generally inversely related to UCP-1 expression (Cinti et al., 1997). The existence of another specific brown adipocyte satiety hormone, in addition to leptin, has also been suggested on other grounds (Melnyk and Himms-Hagen, 1998; Himms-Hagen, 1999a,b) (see Section 7.4.2 below). In contrast, the longest known physiological function of white adipocytes is their storage of triacylglycerol and the secretion by them of free fatty acids (FFA), when lipolysis is stimulated in response to needs of other tissues for an energy source, as during fasting or exposure to a cold environment. White adipocytes also secrete products other than FFA. Best known is the secretion in proportion to adipose mass, of leptin, one component of the

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neuroendocrine systems involved in energy balance of the body (Flier, 1998; HimmsHagen, 1999a) (see Section 7.5.1 below).

7.2.2

Definition and tissue distribution of brown and white adipocytes

There are usually considered to be two basic types of adipocyte, white and brown. The former are characteristically unilocular, with a thin film of cytoplasm containing a few small mitochondria with sparse cristae (Figure 7.1). The latter are characteristically multilocular and contain abundant large mitochondria with densely packed cristae (Figure 7.1). Recent evidence supports the existence of a third type of adipocyte, variously termed convertible white adipocyte (Loncar, 1991a,b) or masked brown adipocyte (Casteilla et al., 1999), that usually assumes the appearance of a typical unilocular white adipocyte but can take on the morphological and biochemical characteristics of a brown adipocyte when stimulated via ß3ARs. These convertible white adipocytes are the probable precursors of most of the ‘ectopic’ brown adipocytes which appear in certain WAT depots in response to intense stimulation of ß3ARs (see Section 7.3.4). Brown adipocytes express UCP-1; indeed they are defined as brown because of the expression of this protein, whereas white adipocytes do not (Himms-Hagen and Ricquier, 1998). Precursors to white adipocytes predominate in WAT depots, whereas precursors to brown adipocytes predominate in BAT depots. BAT depots contain some white adipocytes that do not express UCP-1 (Morroni et al., 1995; Cinti et al., 1997).

Figure 7.1 Diagrammatic representation of the principal features that distinguish a brown adipocyte from a white adipocyte. Both are controlled primarily by their sympathetic innervation, mediated by noradrenaline, but central control is selective (increased in both in cold; increased in WAT but decreased in BAT during fasting) (see Section 7.2.6). Despite the remarkable morphological difference between the two cells types, very few proteins are known which are expressed in brown adipocytes but not in white adipocytes (see Section 7.2.2 for discussion). No protein is known that is expressed in white adipocytes but not brown adipocytes. Not shown in this figure is the convertible white adipocyte, which can appear brown or white according to its environment (see Sections 7.3.1 and 7.3.4).

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Moreover, characteristic brown adipocytes, expressing UCP-1, have appeared in cultures of precursor cells derived from certain WAT depots (Klaus et al., 1995a). Precursors to brown adipocytes are even present in tissues not traditionally regarded as adipose tissues, such as bone marrow (Marko et al., 1995). Bone marrow does contain white adipocytes, but mature brown adipocytes are not seen there. Unlike brown adipocytes—which can be distinguished by their expression of UCP1—no genetic marker distinguishes white adipocytes (Klaus, 1997). The only distinction between brown and white adipocytes has been considered to be expression of UCP-1 in brown adipocytes (Klaus, 1997; Himms-Hagen and Ricquier, 1998). There are, however, some other differences between proteins expressed in BAT and those expressed in WAT, but these differences do not provide a specific marker for brown or white adipocytes because the proteins in question are also expressed in other, non-adipose, tissues. Also, there is no marker for convertible white adipocytes. Thus, the recent discovery of a PPAR gamma coactivator-1 (PGC-1) has revealed a protein expressed in brown but not white adipocytes (Puigserver et al., 1998; Wu et al., 1999) (see Section 7.3.1 below). However, because the expression of PGC-1 is not limited to BAT but also occurs in other mitochondria-rich tissues such as skeletal muscle, heart, kidney and brain, it cannot serve as a marker for brown adipocytes. A recent report claims expression in rat BAT, but not WAT, of a muscle type of carnitine palmitoyltransferase I (CPT I), and expression in WAT, but not BAT, of a liver type of CPT I (Yamazaki et al., 1996). However, the liver-type CPT I in WAT is expressed in pre-adipocytes and not in mature white adipocytes, which express muscle type CPT I, the same isoform that is present in BAT (Esser et al., 1996; Brown et al., 1997). Thus, it can be concluded that muscle CPT I is not a feature that distinguishes brown adipocytes and white adipocytes in rats. However, a remarkable species difference shows that this conclusion does not apply to mice. In contrast to white adipocytes isolated from rat WAT, white adipocytes isolated from mouse WAT express liver CPT I almost exclusively, whereas mouse BAT, like rat BAT, expresses muscle CPT I (Brown et al., 1997). In this respect human white adipocytes resemble those of rats rather than those of mice (Brown et al., 1997). The authors of these studies (Brown et al., 1997) caution that although transgenic mice can be very useful in studies of rodent WAT and BAT, results may not always apply to human adipose tissues. Another protein important in fatty acid metabolism, fatty acid binding protein of the adipose type (A-FABP, also known as aP2), is usually regarded as a marker for adipocytes in both WAT and BAT, and the promoter of its gene has been used to drive adipose-specific expression of a variety of genes in transgenic mice. A-FABP is indeed expressed in brown pre-adipocytes in culture, where its abundance is not altered by noradrenaline. However, mRNA for another isoform of FABP abundant in heart (HFABP) is the major FABP expressed during differentiation of brown adipocytes in culture, where its expression is up-regulated by noradrenaline. This isoform is also upregulated in BAT of rats by exposure of the animals to cold, to an even greater extent (100×) than the up-regulation of UCP-1 (5×) (Daikoku et al., 1997). In contrast, expression of A-FABP in BAT is not altered by exposure of rats to cold (Daikoku et al., 1997). Again, while H-FABP is not a marker specific for brown adipocytes, since it is also present in heart, it can serve to distinguish them from white adipocytes. Another cold-inducible protein expressed in BAT but not in WAT is metallothionein (Beattie et al., 1996). This is a cysteine-rich, metal-binding protein which is believed to play an antioxidant role. This protein is also not a marker for BAT since it is expressed also in liver. Another cold-inducible protein in BAT is guanosine monophosphate

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reductase; it is not expressed in WAT, but is present in other tissues (Salvatore et al., 1998).

7.2.3

Definition of BAT and WAT

Much recent work has entailed experiments in which stimulation of the development of BAT has occurred at sites which have hitherto been believed to be essentially devoid of brown adipocytes. Reports of the appearance of apparently new brown adipocytes at unusual locations have raised skepticism concerning their precise identity. Are such cells genuine brown adipocytes or some other type of cell masquerading as such? Such ‘ectopic’ brown adipocytes appear in depots of WAT following intense sympathetic stimulation, enhanced stimulation of a sympathetic transduction component, or the mimicking of sympathetic stimulation by administration of a drug acting on ß3ARs (see Section 7.3.4 below). The existence of precursors to these ‘ectopic’ brown adipocytes in WAT depots creates a problem in the definition of BAT and WAT. Because, under certain circumstances, brown adipocytes, expressing UCP-1 do appear in both BAT and WAT depots, the term brown adipocyte will be used, rather than BAT, to describe these cells, whether they are present in BAT or WAT. The terms BAT and WAT are here used to denote the anatomical location of the depot, e.g. interscapular BAT, inguinal WAT, epididymal WAT, rather than the type of adipocyte contained therein. Brown or white, as applied to the tissue, denotes that is usually the major adipocyte type present, but does not exclude the presence of the other type or of convertible white adipocytes. BAT contains a mixture of cell types. These include large mature, multilocular brown adipocytes that contain UCP-1, small endothelial cells associated with the vasculature, pre-adipocytes, interstitial cells, unilocular white adipocytes that do not contain UCP-1, and others. WAT also usually contains a mixture of cell types, including precursors to ‘ectopic’ brown adipocytes that can be induced to appear by treatment with ß 3AR agonists or by acclimatization to cold. In studies of gene expression or of protein levels in WAT or BAT a problem arises from the potential presence of thermogenically active brown adipocytes in WAT depots and characteristic unilocular white adipocytes in BAT depots. When the experimental procedure adopted involves isolation of material from the intact tissue, such as mRNA or mitochondria, there is the possibility that any protein or mRNA identified may not have come from white adipocytes in WAT or from brown adipocytes in BAT, but from brown adipocytes in WAT or from white adipocytes in BAT. For this reason, an effort is made in this review to specify when the location of protein or mRNA in brown adipocytes has been confirmed by immunohistochemistry, by in situ hybridization, or by studies with cultured adipocytes. When identification of mRNA or protein in the tissue (BAT or WAT) is specified, this implies that the cells or origin were not identified conclusively.

7.2.4

Multiple UCPs in BAT

In 1997, the uniqueness of a single mitochondrial uncoupling protein in brown adipocytes was reversed with the cloning of two other mammalian uncoupling

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proteins. The formerly well-recognized UCP is now referred to as UCP-1, which is still believed to be unique to brown adipocytes. UCP-2 is expressed in both BAT and WAT as well as in muscle and in tissues rich in macrophages, including Kupffer cells in liver (Boss et al., 1997b, 1998; Fleury et al., 1997; Gimeno et al., 1997; Gong et al., 1997; Larrouy et al., 1997; Masaki et al., 1997; Camirand et al., 1998; Hodny et al., 1998). UCP-3 is expressed predominantly in BAT and skeletal and cardiac muscles (Boss et al., 1997a; Gong et al., 1997; Larkin et al., 1997; Matsuda et al., 1997; Vidal-Puig et al., 1997). Thus, all three UCPs are expressed in BAT but the function(s) of each in this tissue are as yet not understood. Most publications report only mRNA levels for specific UCPs (Boss et al., 1998). The level of mitochondrial protein, when this has been reported, has not followed closely changes in mRNA level (Sivitz et al., 1999). Thus, since the amounts of UCP-2 or UCP-3 protein in mitochondria in any tissue are as yet mostly unknown, and although they have been shown to be present in mitochondria (Jezek et al., 1999) and the functional operation of these proteins has as yet not been assessed, caution is necessary in ascribing them any role in energy balance. Many believe that novel UCPs expressed in tissues other than BAT may mediate the long-known mitochondrial proton leak present in most tissues. This leak represents a large part of resting metabolic rate (Rolfe and Brand, 1996; Rolfe and Brown, 1997). Moreover, since hepatocytes possess a thyroid hormone-sensitive proton leak (Harper and Brand, 1993,1994), but no hitherto identified UCP, either the existence of even more novel UCPs remains to be discovered or the proton leak may not be mediated exclusively by UCPs. A complete discussion of the role of UCPs in BAT and other tissues is beyond the scope of this chapter, which will concentrate on their presence in brown and white adipocytes and on their potential regulation by ß3AR agonists.

7.2.5 Adrenoreceptors in brown adipocytes In most species, the principal adrenoreceptors (ARs) mediating most effects of noradrenaline on mature brown adipocytes are usually considered to be the ß3AR, responsible for the thermogenic effect, and the
1 AR, which enhances the thermogenic effect of noradrenaline (see Zhao et al., 1997). The subtype a 1aAR is present in the mature brown adipocyte, and its level is increased by ß3AR agonists (Granneman et al., 1997). ß 1ARs predominate in the precursor cells to brown adipocytes, where they mediate a proliferative response to noradrenaline. Targeted disruption of the ß3AR completely abolishes the increase in metabolic rate induced by a selective ß3AR agonist (Susulic et al., 1995; Revelli et al., 1997), but does not abolish the response to the non-selective agonists, noradrenaline and isoproterenol (Susulic et al., 1995). The retention of a normal amount of UCP in BAT and a normal increase in UCP content of BAT in response to acclimation to cold in ß3AR knockout mice suggests replacement of ß3AR function by ß1AR function. In fact, in this study, ß1AR expression was found to be up-regulated in BAT of the ß3AR knockout mice (Susulic et al., 1995). In another study of ß3AR knockout mice, ß1ARs were to some extent down-regulated in BAT, and the level of mRNA for UCP-1 was very low in the mice with the greatest extent of down-regulation of ß1ARs (Revelli et al., 1997). The ectopic emergence of brown adipocytes in WAT depots of mice overexpressing ß1ARs (Soloveva et al., 1997) and the fairly mild predisposition to obesity of mice

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with targeted disruption of ß3ARs but up-regulation of ß1ARs in their BAT (Susulic et al., 1995; Revelli et al., 1997) indicates considerable plasticity in the use of the subtypes of ßARs in mice. In rat brown adipocytes in culture, low concentrations of noradrenaline stimulate lipolysis via a ß3AR, but stimulate UCP-1 expression via a ß1AR (Chaudhry and Granneman, 1999). Both responses involve increased cAMP production but different internal signalling pathways (Chaudhry and Granneman, 1999). At high levels of noradrenaline, such as are present in the synaptic cleft, it is likely that ß3ARs mediate most effects of noradrenaline (D’Allaire et al., 1995; Atgié et al., 1997). The thermogenic and lipolytic responses are closely linked (Atgié et al., 1997) and persist despite marked desensitization of ß3ARs in brown adipocytes of cold-acclimatized rats (Zhao et al., 1998b). Even two days of cold-exposure can desensitize ß3ARs in brown adipocytes (Scarpace et al., 1999). Desensitization and down-regulation of ß3ARs in human brown adipocytes also occurs readily in the presence of noradrenaline (Jockers et al., 1998). ß3ARs in white adipocytes are likewise desensitized by long-term stimulation by a ß3AR agonist (Atgié et al., 1998; Vicario et al., 1998). Guinea-pigs have a ß3AR that differs from that of rats and is expressed only at very low levels in both BAT and WAT (Carpéné et al., 1994, 1998; Himms-Hagen et al., 1995; Atgié et al., 1996), yet the guinea-pig is able to adapt to cold and grow more thermogenically active BAT with brown adipocytes that have a markedly enhanced capacity for a thermogenic response to noradrenaline (Rafael et al., 1986; Himms-Hagen et al., 1995), presumably mediated by ß1ARs.

7.2.6

Control of brown adipocytes by the sympathetic nervous system and the hypothalamus

The neurotransmitter of the sympathetic nervous system, noradrenaline, plays a major role in the control of thermogenesis in brown adipocytes and of growth of BAT (HimmsHagen, 1991). In rats and mice, noradrenaline also acts directly on precursor cells to promote their proliferation and differentiation, including mitochondrial proliferation and a selective increase in synthesis of UCP-1. BAT receives a sympathetic innervation of diffuse central origin (Bamshad et al., 1999). Sensory or afferent autonomic nerves containing substance P and calcitonin gene-related peptide (CGRP) are also present in BAT, where their exact role is not understood. The loss of some BAT cells and concomitant loss of UCP in capsaicin-desensitized rats, in which these afferent autonomic nerves are destroyed, suggests a role for the afferent autonomic nervous system in the maintenance of a subpopulation of brown adipocytes. The central neural pathways that selectively influence sympathetic nervous system activity in BAT have been extensively studied. Experimental approaches have included central injections of neurotransmitters, central lesions and central electrical stimulation, all in specific brain locations, with assessment of subsequent BAT thermogenic function or of firing activity of the nerves to BAT. A review of over 100 such studies made in 1991 indicated important roles of the ventromedial nucleus of the hypothalamus (VMH) and lateral hypothalamus (LH) in central neural control of BAT thermogenesis (HimmsHagen, 1991). Interest in the central neural mechanisms involved in control of BAT thermogenesis has been revived by the discovery of leptin and the leptin receptor, and the realization that leptin activates BAT thermogenesis at least in part by an action on the brain to increase sympathetic nervous system activity (Collins et al., 1996; Haynes et al.,

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1997a,b, 1999). Leptin receptors are located at multiple sites in the brain, particularly in the hypothalamus (Fei et al., 1997; Elmquist et al., 1998). Thus, elucidation of the neuropeptidergic networks involved in the control of feeding by leptin will undoubtedly yield more information about central control of BAT thermogenesis and energy expenditure (Levine and Billington, 1997). These integrated neural networks involve primarily, but not exclusively, the hypothalamus and the hindbrain (Campfield et al., 1996; Woods et al., 1998). WAT also receives a sympathetic innervation of diffuse central origin, including hypothalamic areas known to be involved in energy balance (Bamshad et al., 1998; Bartness and Bamshad, 1998). WAT likewise has afferent sensory nerves which may also play a role in leptin’s effect to increase the activity of the sympathetic nervous system (Niijima, 1998, 1999). Little attention has been paid to the central neural networks that allow selective activation of sympathetic nervous system activity in WAT. However, the brain is able selectively to influence BAT and WAT function. While cold exposure increases sympathetic nervous system activity in both WAT and BAT (Himms-Hagen, 1991; Garofalo et al., 1996), fasting increases sympathetic nervous system activity in WAT (Migliorini et al., 1997) but decreases it in BAT (Young et al., 1982; Young and Landsberg, 1997). Thus, white adipocytes, as well as brown adipocytes, appear to be subject to regulation by noradrenaline as well as by circulating hormones.

7.2.7

Central and peripheral effects of insulin in maintenance of thermogenic capacity of BAT

In intact animals there is a complex interaction between insulin and the sympathetic nervous system in maintenance of the themogenic capacity of BAT. Lack of insulin action (in streptozotocin diabetic rats or mice) induces rapid atrophy of BAT, with loss of UCP and mitochondria (Shibata et al., 1987; Géloën and Trayhurn, 1990a,b; Burcelin et al., 1993). However, prevention of the BAT atrophy by administration of insulin requires an intact innervation to the BAT (Géloën and Trayhurn, 1990b). Moreover, lack of insulin in itself suppresses sympathetic nervous system activity (Yoshida et al., 1985). Thus, both central and peripheral consequences of the insulin deficiency presumably contribute to the atrophy of BAT. Insulin stimulates glucose transport into brown adipocytes. This effect is associated with translocation of glucose transporter 4 (GLUT 4) to the plasma membrane (Slot et al., 1991), as in other insulin-sensitive tissues. Noradrenaline also increases glucose transport into brown adipocytes. This increase is not associated with translocation of GLUT 4 to the plasma membrane (Omatsu-Kanbe and Kitasato, 1992; Shimizu et al., 1998), but rather with activation of glucose transport by GLUT 1 already present in the plasma membrane via a cAMP-dependent mechanism (Shimizu et al., 1998). Fatty acids can also activate glucose transport, possibly by a similar mechanism (Marette and Bukowiecki, 1991). Cold-exposure, like noradrenaline, does not induce translocation of GLUT 4 to the plasma membrane (Slot et al., 1991), but does induce a large increase in glucose utilization in vivo, presumably mediated by noradrenaline (Vallerand et al., 1990). BAT in coldacclimatized animals is a major site of non-insuln-mediated glucose uptake (Bukowiecki, 1989; Agosto et al., 1997). Under the influence of exaggerated

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sympathetic nervous system activity, therefore, BAT becomes highly active in glucose uptake. The extra glucose is not used as a fuel for thermogenesis, but as a supply of substrate for ATP formation by substrate level phosphorylations in glycolysis, at a time when mitochondrial ATP synthesis is uncoupled from the electron transport oxidative system by activation of the uncoupling protein. Under these conditions the glucose carbon is exported as lactate (see Himms-Hagen, 1990 for discussion of the BAT-liver glucose-lactate cycle).

7.2.8

Control of blood flow in BAT by the sympathetic nervous system and the hypothalamus

A series of studies of blood flow through interscapular BAT by Foster and his colleagues between 1978 and 1982 established for the first time the unexpectedly large quantitative contribution of BAT to noradrenaline-induced thermogenesis in rats (see Foster, 1986). Blood flow through BAT is a useful index of BAT thermogenic activity in vivo. Studies of the vasculature and of the innervation of BAT have extended our understanding of control of its blood flow and demonstrated the importance of arteriovenous anastomoses (Nnodim and Lever, 1988). Studies of hypothalamic control of thermogenesis in BAT have shown that neural mechanisms resident in the VMH and the LH can act separately via nerves to the brown adipocytes and to the arteriovenous anastomoses to bring about either suppressed thermogenesis and diversion of blood away from BAT so that it cools rapidly, or stimulated thermogenesis and diversion of blood flow through the capillary bed so that the BAT warms up and heat is exported (Woods and Stock, 1994, 1996). An understanding of the physiological functioning of interscapular BAT and its control clearly requires more information than can be gained from studies of brown adipocytes in culture or of gene expression in the tissue.

7.3

7.3.1

Origin of brown adipocytes

Differentiation of brown adipocytes and mitochondriogenesis

There is a relative lack of distinguishing features between the brown adipocyte and the white adipocyte, apart from their distinct morphological phenotype and the presence or absence of UCP-1 (see Section 7.2.2 and Figure 7.1). The need to understand the origin of brown adipocytes and how this differs from the origin of white adipocytes has led to an extensive search for the factor (or factors) involved in differentiation of precursors that commit them to becoming a brown adipocyte rather than a white adipocyte. Despite the pronounced differences in phenotype between brown versus white adipocytes, remarkably few differences in the nuclear receptors and transcription factors that govern their differentiation have been identified. The similarities between brown and white adipocytes (enzymes of de novo lipogenesis, lipoprotein lipase, enzymes of lipolysis, ß 3 ARs and components of their signal transduction and regulatory systems; insulin receptors and components of their signal transduction and regulatory systems) appear to extend to the nuclear receptors and transcription factors that govern their differentiation.

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Adipocytes are of mesodermal origin. The exact stage at which mesodermal precursor cells become committed to either the white or the brown adipocyte lineage is unknown, as is the nature of the gene or genes involved (Klaus, 1997). Brown adipocytes are present during fetal life in mice and rats (Ricquier et al., 1986; Houstek et al., 1988, 1990; Obregon et al., 1989) as well as in humans (Lean, 1992; Houstek et al., 1993; Kortelainen et al., 1993). In rodents, expression of UCP-1 occurs before birth, but increases very rapidly following the cold exposure and feeding associated with birth and the neonatal state (Houstek et al., 1988, 1990; Obregon et al., 1989; Giralt et al., 1990; Dessolin et al., 1997). UCP-2 expression in BAT occurs earlier in fetal life than UCP1 expression, but does not increase as rapidly at birth (Carmona et al., 1998). However, UCP-3 expression is not detectable before birth, appearing only after birth, and then increasing rapidly (Carmona et al., 1998). It is not known whether differences in timing of appearance of the three UCPs during fetal and neonatal development are due to sequential changes in gene expression in one population of brown adipocytes, or whether these differences are due to sequential appearance of more than one population of brown adipocytes. Because UCP-1 expression is unique to brown adipocytes, and indeed identifies an adipocyte as brown, much effort has gone into analysis of the regulatory mechanisms that control its tissue-specific and differentiation-dependent expression, in the hope of obtaining insight into what features make a brown adipocyte brown and not white (Ricquier and Bouillaud, 1997; Silva and Rabelo, 1997; Spiegelman, 1997; HimmsHagen and Ricquier, 1998). A very complete analysis of the multiple nuclear receptors, their ligands and other transcription factors can be found in recent reviews (Ricquier and Bouillaud, 1997; Silva and Rabelo, 1997; Spiegelman, 1997), and only a summary is presented here. So far, only one transcription factor—PGC-1 (PPAR gamma co-activator-1)—that has been identified in brown adipocytes is not present in white adipocytes. PGC-1 may be responsible for directing the synthesis of UCP-1 in the former; this protein is capable of driving expression of not only UCP-1, but also of other mitochondrial proteins in a white adipocyte cell line (3T3-F442A cells) where overexpression is induced via introduction by a retroviral vector into the pre-adipocytes (Puigserver et al., 1998). PGC-1 is not expressed exclusively in BAT, but also in other mitochondria-rich tissues such as skeletal muscle, heart, kidney and brain. It is not a marker for brown adipocytes, but appears to play an important role in mitochondriogenesis (Wu et al., 1999). Moreover, PGC-1 itself is cold-inducible in both BAT and skeletal muscle in association with increased mitochondriogenesis, whereas UCP-1 is cold-inducible only in BAT and not in muscle (Puigserver et al., 1998). Thus, PGC-1 needs to interact with a committed adipocytic environment to create thermogenic mitochondria containing UCP-1. PGC-1 cannot play this role in skeletal muscle, and the nature of the factors with which it interacts in this environment is unknown. A fundamental part of the long-term thermogenic response of brown and convertible adipocytes to ß3AR agonists is stimulation of mitochondriogenesis. This can occur in both brown adipocytes in BAT and in a subpopulation of white adipocytes in WAT, and results in an increased capacity for such a thermogenic response (see Section 7.3.4). Still unresolved is the nature and regulation of the gene(s) involved in the coordinated synthesis of the multitude of proteins needed for mitochondriogenesis, which necessarily accompanies the proliferation of mitochondria as well as the

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selective increase in synthesis of UCP-1 induced by noradrenaline. There is almost certainly a role for thyroid hormone in the coordinated synthesis of over 100 gene products, most of these being coded by the nuclear DNA with only ~10% encoded by the mitochondrial genome (Klingenspor et al., 1996; Pillar and Seitz, 1997; Villena et al., 1998). The burst of tri-iodothyronine (T3) production within brown adipocytes brought about at an early stage of differentiation by the increase in activity of thyroxine (T4) 5'-deiodinase that occurs in response to noradrenaline (see Encke et al., 1997; Silva and Rabelo, 1997) is probably involved, but the nature of the genes and the way in which T3 interacts with them is unknown. Simply uncoupling mitochondria by the forced expression of UCP-1 induces mitochondriogenesis in HeLa cells (Li et al., 1999) and in white adipocytes (Cinti et al., 1999a). In muscle cells, PGC-1 increases mitochondriogenesis via increased expression of UCP-2 (Wu et al., 1999). Much of the complex control of the UCP-1 gene occurs in a high upstream enhancer region, initially identified as a DNase-sensitive site (Boyer and Kozak, 1991), which contains multiple response elements to various nuclear receptors as well as a brown adipocyte specific enhancer element, for which a nuclear receptor has as yet not been identified (Cassard-Doulcier et al., 1993, 1994; Kozak et al., 1994; Silva and Rabelo, 1997). Transcription factors involved in the very early response of BAT to cold include C/EBPß (CCAAT enhancer binding protein) and C/ EBP (Rehnmark et al., 1993; Yubero et al., 1994). Mice with knockout of either of these factors fail to develop BAT normally in fetal or neonatal life (Wang et al., 1995; Tanaka et al., 1997). In rat BAT, C/EBP is cold-inducible and its level correlates with and precedes the rise in UCP-1 mRNA (Manchado et al., 1994). At least two thyroid response elements (TREs) are also important in the regulation of the UCP-1 gene, with thyroid receptor-RXR (retinoid X receptor) heterodimers implicated in their control (Rabelo et al., 1995, 1996a; Silva and Rabelo, 1997). A retinoic acid response element (RARE) and RAR/RXR heterodimers (RAR=retinoic acid receptor) are also implicated (Alvarez et al., 1995; Puigserver et al., 1996; Rabelo et al., 1996b; Silva and Rabelo, 1997). Two cyclic AMP response elements (CREs) are also present (Rabelo et al., 1997). Peroxisome proliferator activated receptor? (PPAR?) plays a central role in control of the UCP-1 gene (Sears et al., 1996; Tai et al., 1996). Potential endogenous ligands for this nuclear receptor include prostaglandins of the D2 and J2 series (Forman et al., 1995; Kliewer et al., 1995) and polyunsaturated fatty acids (Krey et al., 1997). The production of a higher affinity ligand as a consequence of ADD1/ SREBP1 activation has also been suggested (Kim et al., 1998). PPAR? is of particular interest because it is the binding site for a class of drugs, the thiazolidine diones. Such drugs have long been known to promote the capacity for thermogenesis in BAT in rats and mice (Mercer and Trayhurn, 1986; Rothwell et al., 1987; Thurlby et al., 1987), as they enhance the effect of noradrenaline to increase UCP-1 expression in brown adipocytes in vitro (Foellmi-Adams et al., 1996) and promote differentiation of precursor cells and enlargement of BAT in vivo (Tai et al., 1996). The thiazolidine diones can direct differentiation of a pluripotent stem-cell line of mesodermal origin (C3H10T1/2 cells) into brown adipocytes (Paulik and Lenhard, 1997), and also increase expression of UCP-2 in a brown adipocyte cell line (Camirand et al., 1998). The observation that a thiazolidine dione can induce the appearance of UCP-1 mRNA and UCP-1 protein in human adipose tissue preadipocytes differentiating in culture is particularly exciting (Digby et al., 1998)

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because it indicates the presence of cells of the brown adipocyte lineage in various adult human adipose depots in various locations. When UCP-1 was first cloned in 1985 (Bouillaud et al., 1985; Jacobsson et al., 1985), eight major cold-inducible clones were isolated, of which one (CIN-1) was identified as UCP-1 (Jacobsson et al., 1985). A second clone (CIN-2) has now been identified as a cold-inducible glycoprotein probably present in the plasma membrane and expressed in both BAT and liver (Tvrdik et al., 1997). A third has been identified as GMP reductase, expressed in BAT but not in WAT (Salvatore et al., 1998). Control of expression of coldinducible proteins other than UCP-1 is as yet unknown.

7.3.2

Induction of hyperplasia of brown adipocytes in BAT

Noradrenaline induces proliferation of precursor cells in BAT (Rehnmark and Nedergaard, 1989; Bronnikov et al., 1992). The precursor cells can go on to differentiate fully via pre-adipocytes into mature brown adipocytes characterized by abundant large mitochondria (Bukowiecki et al., 1986; Géloën et al., 1988, 1990, 1992). An intermediate immature brown adipocyte in which T4 5'-deiodinase is transiently increased may occur (Encke et al., 1997). The mature brown adipocytes express the ß3AR whereas the precursor cells do not, but ß3AR expression precedes expression of UCP-1 during differentiation (Klaus et al., 1995b). In cultures of precursor cells from BAT the mitogenic action of noradrenaline is rather weak unless serum is present (Bronnikov et al., 1992). The noradrenaline appears to act by potentiating the mitogenic effect of one or more growth factors present in the serum (Garcia and Obregon, 1997). Basic fibroblast growth factor (bFGF) is a likely candidate since it does interact with noradrenaline in promoting mitogenesis (Garcia and Obregon, 1997). Its expression in BAT is increased when the proliferative response occurs during cold exposure. It is produced by endothelial cells (Yamashita et al., 1994), which also proliferate under these conditions (Bukowiecki et al., 1986). A newly discovered member of the FGF family, FGF-16, is expressed at a high level in fetal BAT, and some expression persists in adult BAT; it too may also be involved in BAT growth (Miyake et al., 1998). Chronic stimulation with a highly selective ß3AR agonist is unable to induce hyperplasia of BAT in vivo (Himms-Hagen et al., 1994; Ghorbani and Himms-Hagen, 1997; Ghorbani et al., 1997). On the other hand, a ß3AR agonist with some activity at ß1ARs can induce both hyperplasia and cellular hypertrophy (Arbeeny et al., 1995). Thus, in cold-exposed animals the initial cell proliferation in BAT is thought to be mediated by ß 1ARs (Rehnmark and Nedergaard, 1989; Bronnikov et al., 1992), whereas the later stimulation of mitochondriogenesis is thought to be mediated by ß3ARs. Once the cold stimulus to hyperplasia is removed, cells disappear again (Desautels et al., 1986; LeBlanc and Diamond, 1988). This disappearance is not simply due to the reduction in sympathetic nervous system activity, since it is not mimicked by denervation. One proposed explanation is the reappearance of a circulating factor responsible for removal of cells from BAT that is suppressed in the cold-acclimatized state (see Himms-Hagen, 1996). A candidate for such a factor would be tumour necrosis factor a (TNF- a ), which induces apoptosis of brown adipocytes in culture—an effect that is opposed by noradrenaline (Nisoli et al., 1997; Porras et al., 1997). Moreover, in an animal model of obesity that is

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characterized by high TNF-a levels and low sympathetic nervous system activity, apoptosis of adipocytes in BAT is exaggerated and can be reduced by acclimatization of these rats to cold (Nisoli et al., 1997). Thus, the cellular constitution of BAT probably depends not only on the degree of stimulation of both proliferation and differentiation by noradrenaline, but also on the level of factors that promote apoptosis of brown adipocytes, such as TNF-a .

7.3.3

Expression of multiple UCPs in brown adipocytes: function and regulation

All three known UCPs are expressed in BAT. Early studies showed that UCP is an abundant protein located in the brown adipocyte mitochondrial inner membrane. Its concentration can change 10-fold and reach almost 10% of membrane proteins in direct relation to temperature of adaptation over a wide range, from thermoneutrality down to 14°C in mice (Ashwell et al., 1983). The level of protein of the individual UCPs has not yet been determined; indeed, it is not yet clear whether all brown adipocytes express all three UCPs or whether there might be more than one type of brown adipocyte (see Section 7.3.4).

7.3.4

Induction of emergence of ‘ectopic’ brown adipocytes in WAT

A puzzling observation made initially in 1984 (Young et al., 1984), and subsequently several times over the past few years (see below), has been the appearance of multilocular mitochondria-rich brown adipocytes, as defined by their expression of UCP, in what had previously been regarded as simply WAT depots. The observation has led to a need to redefine WAT and BAT (discussed in Section 7.2.3.). These ‘ectopic’ brown adipocytes appear in certain WAT depots whenever there is intense sympathetic stimulation or mimicking of intense sympathetic stimulation by the use of a drug acting on ß3ARs. Thus, chronic cold stress has been shown to induce emergence of these cells in periovarian WAT of rats (Cousin et al., 1992, 1993, 1996) and in inguinal WAT of mice (Loncar, 1991a). The new cells acquire a sympathetic innervation (in periovarian WAT of rats) (Giordano et al., 1996) and remain in place as long as the stimulation persists (in inguinal WAT of mice), after which they disappear again (Loncar, 1991a). Likewise, constitutively high activity of protein kinase A in adipose tissues, mimicking intense sympathetic stimulation, also induces appearance of multilocular adipocytes in WAT (Cummings et al., 1996). This high activity of protein kinase A was achieved by disruption of the gene for its usual regulatory subunit (RIIß) and compensation by the mouse by substitution of another subunit (RI
), more sensitive to cAMP, so that the enzyme became more active. Overexpression of ß1ARs under the control of the aP2 promoter in both BAT and WAT also leads to the appearance in WAT of multilocular cells expressing UCP (Soloveva et al., 1997). ß3AR agonists induce emergence of multilocular brown adipocytes in WAT depots in lean rats (Cousin et al., 1992), in genetically obese rats (Ghorbani and Himms-Hagen, 1997; Umekawa et al., 1997), in rats with diet-induced obesity (Ghorbani et al., 1997) and in obese and lean mice (Nagase et al., 1996; Picó et al., 1998; Yoshida et al., 1998). Their appearance is accompanied by marked mitochondriogenesis in the WAT depots (Liu et al., 1998; Melnyk et al., 1999).

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Mitochondria isolated from the WAT depots contain more UCP-1 as well as more UCP-3 (Himms-Hagen, 2000). UCP-3 mRNA levels in the WAT may (Gong et al., 1997; Emilsson et al., 1998; Yoshitomi et al., 1998) or may not (Savontaus et al., 1998) be found to increase. In mice, the ß3AR agonist-induced appearance of brown adipocytes in WAT varies considerably from one strain to another and is under complex genetic control (Guerra et al., 1998). Do these ‘ectopic’ brown adipocytes have any physiological function? One possibility is that they represent a reserve of thermogenically inactive brown adipocytes, with few mitochondria and little or no UCP-1 and with little or no sympathetic stimulation. This reserve is apparently called upon by intense sympathetic stimulation in a cold environment and serves to produce extra heat when needed. This reserve is probably more important in the mouse than in the rat because of the former’s small size. What is the origin of the ‘ectopic’ brown adipocytes? One possible origin for these reserve cells is that their precursors have the appearance of unilocular white adipocytes (‘masked’ brown adipocytes or ‘convertible’ white adipocytes). A second possible origin for the ‘ectopic’ brown adipocytes is that they are small committed brown preadipocytes, already expressing ß 3 ARs, that are stimulated to differentiate by noradrenaline or a ß3AR agonist. In studies of ß3AR agonist-treated rats, we have shown that the precursor cells are already present in the WAT, since the multilocular cells are not derived from cells that have undergone mitosis (Cinti et al., 1997; Himms-Hagen, 2000). Morphological and immunohistochemical assessment of these cells show progressive transformation of a subpopulation of unilocular adipocytes into multilocular, mitochondria-rich adipocytes, only some of which contain UCP-1. However, the protein composition of the mitochondria isolated from the WAT depot of ß3AR agonist-treated rats differs markedly from that of mitochondria isolated from BAT of the same animal, although these mitochondria do contain both UCP-1 and UCP-3. Most of the cells would thus not appear to be typical brown adipocytes (Himms-Hagen, 2000), and most would appear to be derived from convertible white adipocytes rather than from brown adipocyte precursors.

7.3.5

Is there more than one type of brown adipocyte?

Just as WAT depots might contain more than one subtype of adipocyte (white and convertible adipocytes), so may BAT depots contain more than one subtype of brown adipocyte, differing perhaps in their expression of ß 3ARs and hence showing different reactivity to ß3AR agonists. There is some evidence for heterogeneity of brown adipocytes in BAT, first described on the basis of histological appearance of the cells (Vallin, 1970; Pellet and Lheritier, 1975), and now revived by the finding of heterogeneity of immunoreactivity for UCP-1 in animals in which the BAT has been stimulated by cold-acclimatization or by a ß 3AR agonist (Cancello et al., 1999). Thus, neighbouring brown adipocytes, which are otherwise histologically very similar, may stain positively for UCP-1 or show no staining (Cancello et al., 1999). A similar heterogeneity in immunoreactivity has been observed in mouse BAT (Kopecky et al., 1995) and in human BAT (Kortelainen et al., 1993). Heterogeneity in mitochondrial staining of brown adipocytes in BAT of baboons has also been noted (Viguerie-Bascands et al., 1996). The usual definition of a brown adipocyte as a cell that expresses UCP-1 would not include these immunologically non-reactive

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multilocular cells in BAT. It is not clear whether they are convertible white adipocytes or another type of cell. Either the definition of a brown adipocyte needs to be changed, or another name adopted for the multilocular adipocytes in BAT that are immunologically non-reactive for UCP-1. Further suggestive evidence for different adipocyte populations in BAT comes from studies of transgenic mice with partial ablation of BAT. This was achieved by expression of diphtheria toxin A-chain (DTA) under control of the UCP-1 promoter in the UCP-DTA mouse (Lowell et al., 1993). These mice lose brown adipocytes in BAT as these cells express UCP-1 (Lowell et al., 1993). The capacity of the UCP-DTA mouse for a thermogenic response to a ß3AR agonist is reduced to 40% of normal (Lowell et al., 1993). This level of response is similar to the 34% that remains in mice with targeted disruption of the UCP-1 gene (Enerbäck et al., 1997), and indicates substantial loss of UCP-1 expressing brown adipocytes. We have studied the emergence of ‘ectopic’ brown adipocytes in inguinal WAT of UCP-DTA mice living in a cold environment, and have seen many multilocular brown adipocytes in WAT in both control and UCP-DTA mice (A.Melnyk and J.Himms-Hagen, unpublished results). These cells are positive for UCP when stained with a non-selective antiserum. If it is assumed that many brown adipocytes that express UCP-1 are killed by the DTA they express, and areas of necrosis seen in BAT indicate that this does indeed occur, then possibly the cells which emerge in WAT and those that remain in BAT do not express UCP-1 but rather UCP-3. Clearly, further work awaits the development of highly selective antibodies suitable for immunohistochemical recognition of the different UCPs. It has already been pointed out that, besides its brown adipocyte population, BAT also contains white adipocytes and other types of cells. Thus, preparations of BAT mitochondria will comprise mixed populations derived from the different cell types. The heterogeneity of properties of subpopulations of mitochondria isolated from BAT of rats under various conditions has already been noted (Moreno et al., 1994; Gianotti et al., 1998). Because all three known UCPs are present in BAT, caution is required in interpreting reported differences in the expression of each based on mRNA levels since they may be derived from different cell types. Thus, the expression of UCP-2 and UCP3 does not always parallel that of UCP-1. In a situation in which expression of UCP-1 would be expected to increase, namely exposure to cold, there is either no effect of cold on expression of UCP-2 mRNA in BAT (Fleury et al., 1997) or, if any, only a small effect (Boss et al., 1997b; Carmona et al., 1998). UCP-3, on the other hand, is clearly longterm cold-inducible in BAT of rats (Larkin et al., 1997; Boss et al., 1998), but short-term cold exposure does not increase it in BAT of mice, while nonetheless increasing UCP1 mRNA in their BAT (Carmona et al., 1998). Treatment with a ß3AR agonist has been reported to increase (Savontaus et al., 1998; Yoshitomi et al., 1998), to decrease (Emilsson et al., 1998), and to leave unchanged (Gong et al., 1997) mRNA levels for UCP-3 in BAT. In our study (Himms-Hagen, 2000), long-term treatment with CL316,243 increased levels of both UCP-1 and UCP-3 protein in mitochondria isolated from BAT. A high-fat diet generally increases UCP-2 expression in WAT in rats (Matsuda et al., 1997) and in some, but not all, strains of mice (Fleury et al., 1997; Surwit et al., 1998). UCP-3 mRNA in WAT, on the other hand, is generally not detectable or at very low levels in WAT of chow-fed rats and mice (Larkin et al., 1997; Vidal-Puig et al., 1997; Surwit et al., 1998), and there is no effect of a high-fat diet on UCP-3 expression (Larkin et al., 1997; Surwit et al., 1998). In some studies, a small increase in mRNA for UCP-3 in BAT

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in response to a high-fat diet has been reported (Larkin et al., 1997) but this has not been seen in other studies (Matsuda et al., 1997; Surwit et al., 1998). However, fasting reduces both UCP-1 and UCP-3 mRNA levels in BAT of rats (Boss et al., 1997b; Gong et al., 1997). Both increase again on refeeding, whereas UCP-2 mRNA in BAT is not altered by fasting (Boss et al., 1997b). Although results reported so far are preliminary and rather inconsistent, and while only mRNA levels are known, it is possible that what is being measured in both BAT and WAT is due to differential responses of different cell populations to the same stimulus, rather than differential responses of different genes within a single cell type to the same stimulus. That mRNA for UCP-3 appears in mouse BAT only after birth, while UCP-2 and UCP-1 appear sequentially during late fetal life (Carmona et al., 1998) may well indicate that the UCP-3 might be located in a different cell population from that in which UCP-1 and UCP-2 are located.

7.4

Physiological functions of brown adipose tissue

7.4.1 Role in thermoregulation in a cold environment In the small rodents commonly used in metabolic studies of adipose tissues, BAT is a quantitatively major location of heat production for maintenance of body temperature at ambient temperatures below thermoneutrality. Energy expenditure of a rat adapted to living at 6°C is just over twice that of a rat adapted to living at 25°C. Much of the additional oxygen utilized for heat production in the rat at 6°C is used in the rat’s BAT (Foster and Frydman, 1979). Likewise, energy expenditure in a mouse adapted to living at 14°C is about twice that in a mouse adapted to living at 24° C. This in turn is about twice that in a mouse adapted to living at thermoneutrality (33–35°C). Thus, a mouse is able to vary its energy expenditure over a 4-fold range from thermoneutrality down to mild cold (14°C). Again, much of the difference in energy expenditure lies in oxygen utilization for thermogenesis in BAT and probably also in the brown adipocytes which make their appearance in WAT depots of mice adapted to 14°C. Newborn human infants possess abundant BAT (Hull and Hardman, 1970), sufficient to support a doubling of their energy expenditure when they are exposed to mild cold (24°C), just as in rats and mice adapted to living at a lower temperature (Hey, 1974; Hull and Smales, 1978). It would, thus, be expected that a tissue that can be such a major site of energy expenditure would be of importance in determining energy balance in these animals and infants. When BAT thermogenesis cannot be increased to an adequate level in a cold environment, animals frequently succumb to hypothermia; they are described as coldintolerant. Cold-intolerance of some genetic models of obesity, for example, the obese ob/ob mouse and the diabetic db/db mouse, has been known for many years and has been associated with faulty switching on of thermogenesis in their BAT (see Himms-Hagen, 1989). Transgenic mice with altered capacity for BAT thermogenesis may also show cold-intolerance. For example, the mouse with targeted disruption of the UCP-1 gene is unable to use UCP-1-mediated thermogenesis in its BAT when exposed to extreme cold and is cold-intolerant (Enerbäck et al., 1997). These mice are, however, able to adapt to living in a mildly cold environment (12°C), but the way in which they compensate for the lack of UCP-1 in their brown adipocytes is

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unknown (M.Riachi, M.-E.Harper and J.Himms-Hagen, unpublished results). The transgenic mouse that lacks dopamine ß-hydroxylase is unable to synthesize noradrenaline (Thomas and Palmiter, 1997). This mouse is able to maintain only a limited level of mRNA for UCP-1 in its BAT, about 10–20% of the normal level, and is also unable to switch on BAT thermogenesis in a cold environment, being extremely cold-intolerant (Thomas and Palmiter, 1997; Thomas et al., 1998). The transgenic mouse with homozygous ectopic expression of UCP-1 under the control of the aP2 promoter has very marked atrophy of its BAT and is cold-intolerant (Stefl et al., 1998). The transgene is expressed in both BAT and WAT, but cannot substitute for the normal UCP-1 under control of its own promoter in BAT in defence against the cold. The very low abundance of brown adipocytes in BAT of these transgenic mice is suggested to result from the constitutive early expression of UCP-1 under control of the aP2 promoter during differentiation, leading to uncoupling of oxidative phosphorylation and depletion of ATP that prevents further differentiation into mature thermogenically competent brown adipocytes that express UCP-1 under control of its own promoter (Stefl et al., 1998). In contrast to these three transgenic models described above that exhibit coldintolerance—all of which are lean—the obese mouse with partial ablation of BAT is not cold-intolerant. These mice can survive extreme cold (4°C) for at least two days (Lowell et al., 1993), and can adapt to living in a mildly cold environment (14°C) for two weeks (Melnyk and Himms-Hagen, 1998). The way in which the UCP-DTA mouse compensates for loss of UCP-1-expressing brown adipocytes by thermogenesis in other cell types is not yet understood, but may involve ectopic brown adipocytes in WAT (see Section 7.3.5. above). It can be noted in passing that UCP-DTA mice thermoregulate at a slightly lower than normal body temperature, both in a warm environment and in a cold environment (Klaus et al., 1998; Melnyk and Himms-Hagen, 1998), possibly due to their leptin resistance when they are obese (Hamann et al., 1997; Mantzoros et al., 1998). Leptin is known to raise body temperature when this is low during torpor in mice (Döring et al., 1998). Energy conserved by lowering body temperature would also be expected to contribute to the obesity of the UCP-DTA mouse.

7.4.2

Role in food intake

Two distinct characteristics of the physiological function of BAT may be implicated in control of feeding. First, secretion of heat, not only for thermoregulation but also as a satiety signal to the brain. Second, secretion of a putative satiety hormone involved in adjustment of food intake at different environmental temperatures. Discussion of these roles of BAT is beyond the scope of this chapter, and can be found elsewhere (Himms-Hagen, 1995a,b, 1999b; Melnyk et al., 1997; Melnyk and Himms-Hagen, 1998).

7.4.3

Role in defence against obesity

The concept that enhanced thermogenesis in BAT might contribute to the body’s defence against obesity, by accelerating oxidation of excess ingested food, originated in studies of diet-induced thermogenesis in rats fed a varied and palatable ‘cafeteria’

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diet (Rothwell and Stock, 1979). The increase in energy expenditure for thermogenesis in BAT was considered to offset the obesity-inducing effect of the hyperphagia. The concept remains attractive to investigators, with a shift in current emphasis from a role for UCP-1 in BAT to roles for UCP-2 and UCP-3 in WAT (Matsuda et al., 1979; Surwit et al., 1998). The cafeteria diet-fed animals usually overate [in the original publication they ate 80% more than controls fed chow (Rothwell and Stock, 1979)], yet remained fairly lean because their energy expenditure increased to match their increased energy intake. This increased energy expenditure—termed diet-induced thermogenesis—was attributed to the increased sympathetic activity and thermogenesis in BAT induced by the overeating. Thus, dietinduced thermogenesis in BAT was thought to serve as a defence against the obesity that would otherwise develop as a consequence of the overeating. However, if the causal relationship between the overeating of a cafeteria diet and the increased energy expenditure of the rat is viewed from the opposite point of view, a different interpretation can be discerned. The variety and palatability of the food items offered in themselves increased sympathetic nervous system activity (LeBlanc and Labrie, 1997), resulting in stimulation of BAT and increased energy expenditure. This stimulation would be expected to inhibit secretion of the putative BAT-derived satiety signal, hence to increase energy intake. Thus, rats increased their energy intake to match their increased energy expenditure rather than increasing their energy expenditure to match their high energy intake. It is as though the signal that the rats received from the increased BAT stimulation caused by the variety and palatability of the diet resulted in events appropriate for adjustment of food intake upwards in a colder environment. That diet-induced thermogenesis is clearly related to thermal balance is indicated by its virtual disappearance at a thermoneutral temperature (29°C) (Rothwell and Stock, 1986), where potential heat overload is prevented by suppression of sympathetic nervous system activity. According to this view of dietinduced thermogenesis, hyperphagia does not induce diet-induced thermogenesis as a defence against obesity. Rather, diet-induced thermogenesis induces hyperphagia appropriate for a cooler environment, so that energy expenditure and energy intake match and energy balance is maintained. The response protects against wastage of food energy that would otherwise result from the activation of BAT thermogenesis rather than protecting against obesity induced by overeating. There is, nonetheless, support for a role for enhanced thermogenesis in BAT in promoting a lean phenotype. This has come from studies of transgenic animals with modified BAT function. Mice with targeted deletion of the regulatory subunit of protein kinase A (RIIß) were predicted to be resistant to noradrenaline-induced stimulation of thermogenesis in BAT. Unexpectedly, such mice had enhanced BAT function because they compensated by substituting a different subunit of protein kinase A (RIa) that has a higher affinity for cAMP. Thus, protein kinase A became constitutively active in both BAT and WAT (Cummings et al., 1996). BAT is in a continuously stimulated state in these animals and they are hypermetabolic and hyperthermic. Lipolysis in WAT is enhanced (white adipocytes become smaller) and scattered multilocular adipocytes appear in WAT (see Section 7.3.4). Such mutant mice are highly resistant to obesity induced by a high fat diet. The effect of the transgene resembles the effect of chronic treatment with a ß3AR agonist, i.e. chronic enhancement of signal transduction from the ß3AR. A lean phenotype, also resistant to diet-induced obesity, likewise results from overexpression of ß1ARs driven by the aP2 promoter, expressed in both WAT and BAT

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(Soloveva et al., 1997). In this case, enhanced thermogenesis in brown adipocytes occurs in response to endogenous noradrenaline acting on the ß1ARs. In addition, abundant ectopic brown adipocytes appear in WAT and presumably contribute to enhanced thermogenesis (see Section 7.3.4.). A transgenic model expected to resist obesity is the mouse with overexpression of UCP-1 in adipose tissues under control of the aP2 promoter (Kopecky et al., 1995, 1996a,b; Stefl et al., 1998). This mouse was expected to exhibit enhanced thermogenesis in both its WAT and BAT because of the constitutive expression of UCP-1. The initial study of such heterozygous transgenic mice showed a loss of subcutaneous WAT, but enhanced accumulation of abdominal WAT and little abnormality in body weight (Kopecky et al., 1995). Heterozygous transgenic mice resisted obesity induced by a high-fat diet (Kopecky et al., 1996a). Expressed in Avy/+ genetically obese mice, the transgene reduced body weight gain in older mice (Kopecky et al., 1996a). However, expression of the transgene in BAT suppressed endogenous gene expression (Kopecky et al., 1995) and actually reduced the thermogenic capacity of the BAT (Stefl et al., 1998). However, the endogenous gene remained cold-inducible (Kopecky et al., 1995).

7.5

7.5.1

Secretions from brown adipocytes

Satiety factors

Secretions of BAT that can be considered to be potential satiety factors include heat, the putative brown adipocyte satiety hormone involved in adjustment of food intake in accordance with energy needs dictated by ambient temperature (Section 7.4.2) and leptin. Leptin, whether secreted by BAT or WAT, does not appear to be involved in adjustment of food intake in accordance with ambient temperature (Melnyk and Himms-Hagen, 1998; Himms-Hagen, 1999b). Shortly after the discovery of leptin several publications demonstrated weak or absent expression of leptin in BAT (see Himms-Hagen and Ricquier, 1998). Some leptin found to be present in BAT of a variety of obese animals was attributed to the presence of more abundant white adipocytes in the BAT of these animals. This raised the possibility that leptin might be a marker for white adipocytes as UCP-1 was a marker for brown adipocytes. It is now clear that leptin can be expressed by brown adipocytes, but that its expression is very sensitive to suppression by sympathetic nervous system activity. In intact rats, expression of leptin in BAT was shown to be markedly suppressed by a ß3AR agonist and by acute exposure to cold (Moinat et al., 1995). In immunohistochemical studies, leptin is demonstrable in brown adipocytes in BAT of mice and rats (Tsuruo et al., 1996; Cinti et al., 1997) and is co-localized with UCP-1 (Cinti et al., 1997). Moreover, cultured brown adipocytes of mice express mRNA for leptin and secrete leptin into the culture medium (Deng et al., 1997). Expression of leptin in these cells is very sensitive to inhibition by a ß3AR agonist, apparently because the agonist increases degradation of the mRNA, and to a lesser degree by ß1AR and ß 2AR agonists as well (Deng et al., 1997). Leptin is expressed in BAT shortly after birth in rats (Dessolin et al., 1997) and mice (Devaskar et al., 1997), but not in fetal life (Dessolin et al., 1997). The presence of both leptin mRNA and UCP-1 mRNA revealed by in situ hybridization identified the major cell of origin as a brown adipocyte (Dessolin et al., 1997). The relatively high level of leptin in blood during the first few days of life in both mice (Devaskar et al., 1997) and rats (Rayner et al.,

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1997) is suggested to have its origin in the numerous brown adipocytes in the newborn (Dessolin et al., 1997). Not only leptin but also the long form of the leptin receptor (Lep-Rb) is expressed in BAT (Siegrist-Kaiser et al., 1997; Kutoh et al., 1998), raising the possibility of an autocrine influence of leptin on brown adipocytes. Leptin is known to have a central influence on the hypothalamus to increase activity in the sympathetic nerves that supply BAT (Collins et al., 1996; Haynes et al., 1997a,b, 1999). Some effects of administered leptin are probably mediated by these sympathetic nerves, particularly when it is administered into the cerebral ventricles, but some effects appear to be exerted by a more direct action on brown adipocytes. For example, peripheral administration of leptin increases glucose utilization by BAT (Siegrist-Kaiser et al., 1997), as does its central administration (Kamohara et al., 1997). Moreover, leptin can modulate gene expression in cultured brown adipocytes (Siegrist-Kaiser et al., 1997). Chronic treatment with leptin induces the emergence of multilocular adipocytes in inguinal WAT and increases the expression of all three isoforms of UCP (Gong et al., 1997; Sarmiento et al., 1997). Whether this is a direct effect on the precursors, or a result of activation of the sympathetic nervous system, remains to be determined. Marked and prolonged hyperleptinaemia achieved by adenovirus-mediated overexpression of leptin in rats results in loss of triacylglycerol and of white adipocyte markers such as aP2, leptin, acetyl CoA carboxylase, and fatty acid synthase from WAT and acquisition of mitochondrial markers for fatty acid oxidation (such as UCP-1, acyl CoA oxidase and CPTI) (Zhou et al., 1999). Histological changes were not checked in this study (Zhou et al., 1999); however, it seems very likely that the intense sympathetic stimulation brought about by the leptin caused transformation of convertible white adipocytes into mitochondria-rich adipocytes, some of them expressing UCP-1, just as in rats treated with a ß3AR agonist (see Section 7.3.4) and that this transformation, along with the reduction in food intake, was the basis for the very lean phenotype.

7.5.2

Other factors

Brown adipocytes secrete numerous other factors involved in development and maintenance of the vasculature and of the innervation of BAT and in uptake by brown adipocytes of an important energy source, triacylglycerol transported in blood lipoproteins. Like white adipocytes, brown adipocytes secrete angiotensinogen (Campbell and Habener 1987; Cassis et al., 1988; Frederich et al., 1992; Crandall et al., 1994). They also secrete the potent angiogenic vascular endothelial growth factor (VEGF) (Asano et al., 1997, 1999; Tonello et al., 1999). Secretion of VEGF is under control of the sympathetic nervous system and is increased in response to ß3AR stimulation (Asano et al., 1997; Tonello et al., 1999). Brown adipocytes also secrete basic fibroblast growth factor-2 (bFGF2), likewise enhanced by sympathetic stimulation (Yamashita et al., 1994, 1995). Nerve growth factor (NGF) is synthesized and secreted by brown adipocytes (Néchad et al., 1994; Nisoli et al., 1996b), its expression being suppressed by noradrenaline (Nisoli et al., 1996b). Expression of NGF in BAT is increased in obese animals in association with low sympathetic nervous system activity in their BAT, and is suppressed in cold-acclimatized rats (Nisoli et al., 1996b). NGF mRNA and protein levels in BAT are rather low during fetal life, but increase dramatically at birth and over the first three days of life, declining somewhat thereafter but remaining at a high level until four weeks

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of age (Nisoli et al., 1998). Rats older than eight months have a very limited amount of NGF in their BAT (Nisoli et al., 1998). Studies of synthesis and secretion of lipoprotein lipase by rat brown adipocytes in culture have shown that it is primarily under control of ß3ARs, with some contribution of ß1ARs when the agonist is noradrenaline (Kuusela et al., 1997a). Curiously, such synthesis is not demonstrable in mouse brown adipocytes in culture, although synthesis in response to noradrenaline is readily seen in BAT of intact mice (Kuusela et al., 1997b). In mice, a role in its synthesis of other cell types, in addition to brown adipocytes is possible (Kuusela et al., 1997b).

7.6

ß3ARs in brown and white adipocytes as a target for anti-obesity and anti-diabetes drugs

A drug that interacts with ß3ARs in both white and brown adipocytes would be expected to exert an anti-obesity effect by promoting mobilization of fat stores from white adipocytes and their oxidation in brown adipocytes with a concomitant production of heat and a reduction in adiposity (Figure 7.2). In rodents, ß3AR agonists bring about hypertrophy of brown adipocytes in BAT depots and, in addition, appearance of brown adipocytes in WAT depots, where they are not normally seen. The substantial increase in resting metabolic rate, of the order of 45–70% reported in various studies with rats (Ghorbani and Himms-Hagen, 1997; Ghorbani et al., 1997), presumably occurs in brown adipocytes at both these locations. ß 3ARs on both brown adipocytes and white adipocytes are essential for a full thermogenic response to a ß3AR agonist (Grujic et al., 1997). Adult humans have no adipose tissue depots described as brown, though whether they would have enough ‘masked’ brown adipocytes or convertible white adipocytes in their adipose tissue depots for a ß3AR agonist to exert a similar effect is unknown. However, it is clear that brown adipocytes do appear in adult human WAT under the influence of excessive stimulation by catecholamines in subjects with phaeochromocytoma (Ricquier et al., 1982; Lean et al., 1986; Garruti and Ricquier, 1992; Zilberfarb et al., 1997; Jockers et al., 1998). Moreover, precursors to brown adipocytes exist in various WAT depots in humans and can be induced by a ß3AR agonist (Champigny and Ricquier, 1996) or by a thiazolidine dione (Digby et al., 1998) to differentiate in culture into brown adipocytes expressing UCP. However, the ß3AR in a human brown adipocyte cell line appears to be poorly coupled to the adenylyl cyclase-lipolysis system and rather sensitive to down-regulation (Jockers et al., 1998). Such sensitivity to down-regulation, if it occurs in brown adipocytes in vivo, may impede the ability of ß3AR agonists to exert a long-term anti-obesity effect in humans. However, in one study of young adult lean healthy human subjects, chronic treatment with a drug that is selective for rodent ß3ARs but has low efficacy at the human ß3AR was able to lower respiratory quotient, increase the level of free fatty acids (FFA) in the blood, and induce negative fat balance (Weyer et al., 1998). Thus, while both lipolysis and fat oxidation were stimulated by the drug (Weyer et al., 1998), no increase in energy expenditure or decrease in fat mass could be detected, probably because changes were too small to be detected by the methods used. It is also possible that unmasking or conversion of adipocytes in WAT depots occurs more slowly in

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Figure 7.2 Use of ß3AR agonists to promote oxidation of excess fat stores. A drug that is completely selective for ß3ARs promotes mobilization of fat from stores in white adipocytes, and oxidation of the fatty acids derived from this fat in brown adipocytes. Thus, the drug stimulates lipolysis in unilocular white adipocytes and release of the triacylglycerol stores as free fatty acids (FFA) that circulate in the blood. The drug also stimulates lipolysis in multilocular brown adipocytes where the FFA released within the cell stimulate the proton leakage function of UCP-1, thus uncoupling respiration from phosphorylation and increasing oxidation of FFA (see Section 7.5). The long-term effect of the drug is to promote hypertrophy of brown adipocytes in BAT and emergence of multilocular mitochondria-rich brown adipocytes in WAT depots, thus enhancing its own effect to promote oxidation of fat stores (see Section 7.3.4).

humans than in rats or mice, and that a much longer duration of treatment would be needed to raise energy expenditure in these cells. In rodents, chronic administration of ß3AR agonists induces a large increase in glucose utilization by BAT and WAT in both lean and obese-diabetic animals (de Souza et al., 1997; Liu et al., 1998). The extent to which the glucose utilization is increased in the abundant brown adipocytes that appear in the WAT depots in the drug-treated animals is unknown (Ghorbani and Himms-Hagen, 1997; Ghorbani et al., 1997). Hyperglycaemia, hyperinsulinaemia and insulin resistance are reversed by the treatment (Ghorbani and Himms-Hagen, 1997; Umekawa et al., 1997; Liu et al., 1998). Little or no effect of such treatment on blood insulin or glucose levels is seen in young lean nondiabetic rats (Himms-Hagen et al., 1994; de Souza et al., 1997; Liu et al., 1998), but some reduction is seen in old, mildly obese rats (Ghorbani and Himms-Hagen, 1997). An increased glucose utilization by skeletal muscle accompanies the improvement in insulin resistance (Liu et al., 1998). This is probably a consequence of the marked decrease in elevated blood fatty acid levels that occurs in the treated obese-diabetic rats (Liu et al., 1998). It is anticipated that a drug that is totally selective and effective at the human ß3AR could be useful in the long-term treatment of obesity in humans. Even a very much

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smaller increase in energy expenditure than the 45–70% seen in rodents would, if associated with a reduction in energy intake, be adequate for humans to reverse slowly and safely even massive obesity over many months of treatment.

7.7

Perspective

ß3ARs are important in the regulation of function of both brown and white adipocytes in most species. The traditional view was of the multilocular, mitochondria-rich brown adipocyte as a site of energy expenditure, and the unilocular, mitochondria-poor white adipocyte as a site of energy storage and mobilization as needed. This view led to the concept that agonists acting on ß3ARs in both cell types could induce mobilization of fat stores and their oxidation in brown adipocytes, a concept underlying the development of ß3AR agonists for the treatment of obesity. Studies with laboratory rodents revealed that an unexpected consequence of chronic treatment with ß3AR agonists was the appearance of ‘ectopic’ multilocular mitochondria-rich brown adipocytes in WAT depots where they are not normally seen. The origin of these cells is still not established. Some may be derived from brown pre-adipocytes present in the WAT, but most appear to be derived from ‘convertible’ unilocular white adipocytes also present in the WAT. The conversion involves a marked stimulation of mitochondriogenesis, as also occurs in brown adipocytes under the influence of ß3AR agonists. Some of the cells may contain UCP1, but the new mitochondria created in WAT are not identical to those in BAT depots in the same animal. Many questions remain to be answered before the remarkable effects of ß3AR agonists to remodel adipose tissues can be understood and exploited in drug development. Which genes determine the commitment of precursors to the brown or white lineage? How many adipocyte lineages are there? At least three can be discerned, brown, white and convertible, but there may well be more. The way in which the function of nuclear and mitochondrial genomes is coordinated during intense mitochondriogenesis in brown and convertible adipocytes is virtually unknown, as is the way in which this differs in these two cell types. Whether adult humans have sufficient precursors to multilocular mitochondria-rich adipocytes in their WAT depots remains to be established. The ongoing development of ß3AR agonists selective for, and efficacious at, the human ß3AR should eventually reveal to what extent the findings with laboratory rodents may be applied to humans.

8

The Putative ‘ß4’-Adrenoreceptor and Other Atypical ß-Adrenoreceptors A.DONNY STROSBERG1 AND JONATHAN R.S.ARCH 2 Institut Cochin de Génétique Moléculaire—Laboratoire d’Immuno-Pharmacologie Moléculaire, CNRS UPR, 0415 and Université de Paris VII, 22, rue Méchain, 75014 Paris, France 2 Department of Vascular Biology, SmithKline Beecham Pharmaceuticals, Harlow CM19 5AD, United Kingdom 1

8.1

Introduction

Throughout this book, the question has arisen whether all the properties ascribed to the atypical ß-adrenoreceptors (ßARs) could be entirely explained by the existence of the ß3AR. Species differences have certainly contributed in a major fashion to confuse the issue. A number of reports argued initially that the human ß3AR was another subtype than the rodent ß3AR, and that in each species the homologue had yet to be discovered. Pharmacological results have now led to the conclusion that the rodent, dog, cow, guinea-pig, monkey and human ß3ARs are all species homologues. This end result has however not laid to rest hypotheses for the existence of other atypical ßARs. A single additional ß4AR, would not be sufficient to reflect all the divergent pharmacological profiles. At the present time, sufficient amounts of pharmacological data justify the search for additional ßAR subtypes, but despite considerable effort, no molecular evidence has yet supported the existence of additional ßAR subtypes. The IUPHAR nomenclature committee (Bylund et al., 1998) thus recognized a ‘ß4AR’ solely on the basis of pharmacological data, which are reviewed in this chapter. A number of authors have also argued for the existence of pharmacological differences between alternative forms of the ß3AR, possibly explaining the variations which others have ascribed to additional subtypes. These forms discussed in various chapters, including polymorphism and splice variants of the ß3AR in Chapter 1, will not be considered here, since they do not provide sufficient explanation for all the atypical ßAR properties.

8.2

Pharmacology of the ß4AR and other atypical ßARs

Reports about the putative ß4AR have been published by the groups of Kaumann (for a review, see Kaumann and Molenaar, 1997) and Galitzky et al. (1997), with contributions by several other laboratories. The properties described by the two main groups do not however define the same ßAR. We will recapitulate here the various observations, and then attempt to draw some tentative conclusions. 120

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8.3 A summary of ‘ß4’AR pharmacology Some aryloxypropanolamines, notably CGP-12177, appear to stimulate—in addition to the ß3AR—another receptor, now known as the ‘ß4’AR. This receptor shares with the ß3AR the property of insensitivity to ß1- and ß2AR antagonists, but there are differences between ß3- and ‘ß4’AR pharmacology, notably that phenylethanolamines are not agonists for the ‘ß4’AR, at least in cardiac tissues. One must be cautious in assuming that a novel pharmacology implies a novel receptor, since the pharmacology of even cloned ß3ARs can vary (see Chapter 4); however, ‘ß4’AR pharmacology has been demonstrated in atria and brown adipose tissue from ß3AR knockout mice (see Chapter 3). Much of the discussion about the atypical ß3AR centres around the inability to explain a number of results involving CGP-12177, a potent ß1/ß2AR antagonist which is a partial agonist of ß3AR. As summarized by Lafontan (Chapter 6), this compound can apparently exert atypical effects even when the ß3AR is blocked (knockout mice) or blocked by a supposed ß3AR antagonist. It is however known that in high concentrations, CGP-12177 can also act as an agonist of the ß1AR (Pak and Fishman, 1996; Konkar et al., 2000). It would be especially useful to know whether propranolol, bupranolol, CGP-20712A and SR-59230 A (Table 8.1) are as potent as antagonists of CGP-12177 at the cloned ß1AR as they are as antagonists of isoprenaline, or whether ß4AR pharmacology merely reflects the pharmacology of CGP-12177 as an agonist at the ß1AR; in fact, Konkar et al. (2000) recently showed that no ß4AR pharmacology can be detected in ß1AR knockout mice, indeed confirming the view that most CGP12177-induced ß 4AR pharmacology can be explained by ‘atypical’ agonistic effects on the ß1AR.

Table 8.1 Comparison of ß3AR and ‘ß4’AR pharmacology

* ß3 data are for rat adipose tissue or colon and ‘ß4’ for cardiac tissues unless stated otherwise in the reference list below † The agonist was isoprenaline or a phenylethanolamine-selective ß3AR agonist for the ß3AR experiments and CGP-12177 for the ‘ß4’AR experiments [1] Arch and Kaumann, 1993; [2] Kaumann, 1997; [3] Gauthier et al., 1996 for human ventricle; [4] ‘ß4’AR result from Gaiitzky et al., 1997 for human white adipocyte

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The pharmacology of the ‘ß4’AR is compared with that of the ß3AR in Table 8.1. Briefly: 1 CGP-12177 has similar potency as an agonist at the two receptors, but phenylethanolamines are ineffective at the ‘ß4’AR. 2 Propranolol has even lower affinity for the ‘ß4’AR than for the rat ß3AR. SR-59230A also has lower affinity for the ‘ß4’AR than the ß3AR, excluding one report that questions its high affinity for the ß3AR (Kaumann and Molenaar, 1996). 3 CGP-20712A has a higher affinity for the ‘ß4’AR than the ß3AR. 4 Bupranolol has a similar affinity for the ‘ß4’AR and the ß3AR. With regard to point (1), the lack of potency of adrenaline and noradrenaline as agonists of the ‘ß4’AR raises the question of whether ‘adrenoreceptor’ is an appropriate term, even if a receptor is eventually cloned with homology to the cloned ßARs. However, there are a number of receptors and ligands with inappropriate names based on the first property discovered for one of their homologues. The pharmacology of the ‘ß 4’AR has been elucidated mainly by Kaumann, Molenaar and colleagues using animal and human cardiac tissue (Kaumann, 1997; Kaumann and Molenaar, 1997; Kaumann et al., 1998). A similar pharmacology has been reported for CGP-12177 in human white adipocytes (Galitsky et al., 1997), but it would be surprising if CGP-12177 elicits all its lipolytic activity via the ‘ß4’AR when it is an agonist of the human cloned ß3AR and when SB-226552, SB-236923 and SB-251023—which are not ß4AR agonists (see Chapter 4)—stimulate lipolysis in a nadolol-resistant manner. If the ß4AR is present in the rat ileum this would explain some surprising results of Hoey et al. (1996a,b). These workers found that a number of cyanopindolol analogues were more potent as antagonists of isoprenaline or BRL-37344-induced relaxation of rat ileum-a non-ß1/2-mediated effect-than they were as agonists (relaxants) in their own right. Moreover, cyanopindolol, at a concentration that had no agonist activity but markedly antagonized the response to isoprenaline or BRL-37344, did not antagonize the response to iodocyanopindolol. The authors concluded that the antagonist effects of cyanopindolol analogues were mediated by the ß3AR and the agonist effects by another receptor. With hindsight, we can now suggest that this other receptor was the ‘ß4’AR. Studies on skeletal muscle from ßAR knockout mice will be of particular interest, not because skeletal muscle is claimed to express the ß4AR, but rather because it has been suggested to express yet another ßAR, one that responds to phenylethanolamines. Very low concentrations of BRL-37344 (10 -11 M) and low doses of its pro-drug ester BRL-35135 stimulate muscle glucose uptake in vitro and in vivo respectively by a mechanism that is resistant to antagonism by ß1 and ß2 antagonists (Abe et al., 1993; Liu and Stock, 1995; Liu et al., 1996a,b). Indeed, the in vivo response is resistant to doses of propranolol that inhibit glucose uptake into brown adipose tissue—a ß3AR mediated effect (Liu et al., 1995)—and the in vitro response has a low sensitivity to the ß 3AR antagonist SR-59230A (Liu et al., 1996a,b). The low sensitivity to propranolol and SR-59230A is reminiscent of ‘ß4’AR pharmacology, except that the ‘ß4’AR is not stimulated by BRL-37344, at least not in cardiac tissue.

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8.4 The search for molecular evidence for atypical ßARs

8.4.1 The discovery of the ß3AR gene and other putative homologues The initial experiments that led to the discovery of the ß 3AR gene utilized DNA hybridization experiments which involved the use of DNA probes derived from the genes of the human ß 2AR and the turkey ß1-like AR (Emorine et al., 1989). In addition to a DNA fragment which was shown to correspond to the ß3AR, several other hybridizing bands were isolated. Each of these was cloned and sequenced: none was shown to correspond to a gene encoding a protein that could belong to the family of G protein-coupled receptors (L.J.Emorine and A.D.Strosberg, unpublished results). Alternative methods, based on the use of degenerate PCR oligomers used as primers were no more successful. Over the past ten years, a number of groups have thus coped with consistently negative results.

8.4.2 The discovery of a receptor which binds iodocyanopindolol and other ß 3AR ligands An alternative, more classical way to search for additional, atypical ß3AR was launched by Sugasawa et al. (1997). This work was based on the initial observation that a nonselective ßAR agonist, SM-11044, stimulates guinea-pig ileum relaxation of KClinduced tonus more efficiently than rat white adipocyte lipolysis (Sugasawa et al., 1992). SM-11044 and BRL-35135, a potent ß3AR agonist, in addition inhibit leukotriene B4induced guinea-pig eosinophil chemotaxis, whereas isoproterenol and BRL-37344 have no such effect. The inhibition is unaffected by the non-selective ß AR antagonist propranolol, but is antagonized by alprenolol, a ß1-ß2AR antagonist and ß3AR partial agonist (see Chapter 4). These observations suggested the existence of a ßAR-like functional site in guineapig ileum that is different from the known ßAR subtypes. This site was characterized in rat ileum cells by ligand binding using a variety of ßAR agonists and antagonists, affinity labelling with iodocyanopindolol diazirine, amino acid sequencing of labelled peptides (Sugasawa et al., 1997) and finally cloning of the corresponding human gene (T.Sugasawa et al., 2000). The SM-11044, iodocyanopindolol binding protein does not display any of the structural features of ßAR, let alone G protein-coupled receptors.

8.4.3 Non-ß3AR-like pharmacological properties of ß3AR proteins Studies of the expression of the ß3AR in a variety of cells and tissues have often led to the conclusion that this protein is much more difficult to characterize than the ß1 AR and ß2AR analysed similarly in the same laboratories, by the same methods. It was thus observed that ß3AR expressed in E.coli or in baculovirus-infected insect displays little or no binding or adenylate-cyclase activity (V.Ravet, J.L.Guillaume and A.D.Strosberg, unpublished observations), even though the mRNA and the protein are synthesized in amounts comparable with those found for the ß1- or ß2AR in the same expression systems.

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A possible explanation for this lack of activity could be that the ß3AR, in contrast to ß1- and ß2AR, is not folded correctly, or that an additional protein is required for appropriate activity. Homo- or heterodimerization of G protein-coupled receptors has recently been shown to occur for a number of receptors, including ß2ARs (homodimers), opioid receptors and dopamine/somatostatin receptors heterodimers (Angers et al., 2000). In these last cases, the pharmacological properties of the heterodimers were strikingly different from those of the monomers or homodimers (Jordan and Devi, 1999). Heterodimers which would include ß3AR and another receptor (e.g. ß1AR or ß2AR) might display the ß4AR or other atypical pharmacologic properties unattributed until now (Rocheville et al., 2000).

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Index ADD1/SREBP1 activation 107 adenosine deaminase 38 adenylyl cyclase 9, 14, 16, 17, 22, 29, 38–40, 75 ß3AR-mediated activation 12–14 adipocyte differentiation 15 adipocyte lipolysis 70 adrenaline 1 adrenoreceptors (ARs) in brown adipocytes 102–3 subclasses 1 subdivision 1 see also ß-AR A-FABP 100 agonist-promoted desensitization 26–7, 33 agonist-promoted down-regulation 26–7 agonist-promoted receptor phosphorylation and receptor uncoupling 22–4 agonist-promoted sequestration 34 agonist-promoted uncoupling and sequestration 29–30, 77 agonists 55–74 relative affinities 74 relative efficacies 74 relative potencies 74 amino acid sequences 8 antagonists 49–55 antibodies, raising and characterization 17–18 anti-diabetic drugs 85, 117–19 anti-obesity drugs 85, 117–19 aP2 94, 100, 115 Arg64 allele 84 arylethanolamine ß3AR-selective agonists 55–66 arylethanolamines 65 aryloxypropanolamine ß3AR agonists 66–72 aryloxypropanolamines 71, 73–6, 76, 121 chemical structures 66–9 aryloxypropanolaminotetralins 54 atypical ß-adrenoreceptors (ßARs) 120–3 molecular evidence 122–3 pharmacology 120–2 ß-adrenoreceptor kinase (ßARK) 23, 29, 33, 34 ßAR agonists 78

binding affinities and functional potencies in human cloned ßARs 57–9 potencies in rodent tissues 56 ßAR antagonists 43 ßAR subtypes 4–7 in the heart 87–91 ßARs, properties 6 ß1AR 4–7 ß1AR, in the heart 87–8 ß1AR antagonists 49, 70 ß1AR-selective agonists 55 ß1AR-selective antagonists 49–52 ß2AR in heart 87–8 primary sequences 28 ß2AR agonist 93 ß2AR antagonists 49, 70 ß2AR-selective agonists 55 ß2AR-selective antagonists 49–52 ß3-adrenergic agonists 91 ß3-adrenergic selective agonists 88 ß3AR 4–7, 20 comparison from various species 7–9 distribution 15–19 function assessment 36–47 functional detection 16–17 human tissues expressing 78–82 in the heart 88 lipolytic function in white human fat cells 79 native human 77–86 overview 1–19 pharmacological properties 6 pharmacology 48–76, 121 idiosyncrasies of 74–6 possible clinical use 85 primary sequences 28 primary structure 5 protein immunodetection 17–18 resistance to desensitization 34–5 structural variations 83–4 structure and function 1–19 structure-function relationships 10–12, 77 subtype-specific regulation 27 therapeutic target 84–5 167

168 ß3AR agonists 88, 91, 94–6 first-generation 71 with small N-substituents 70 ß3AR antagonists 85 ß3AR deficiency effects on catecholamine-mediated stimulation of adnylyl cyclase and lipolysis in adipocytes 38–40 effects on in vivo effects of CL-316,243 40 ß3AR gene 2–4, 122–3 cloning 2–3 coding region 3 expression 96 human 77–8 promoters 3 ß3AR ligands 48–76, 123 ß3AR-mediated activation of adenylyl cyclase 12–14 ß3AR-mediated activation of signalling pathways 14–15 ß3AR-mediated vasodilatation 91–3 ß3AR messenger molecules 3 ß3AR mRNA 26, 31, 32, 43, 92, 94–6 detection 18–19 ß3AR non-selective antagonists 52–5 ß3AR proteins 4 non-ß3AR-like pharmacologicalproperties 123 ß3AR receptor regulation, structural determinants 27–9 ß3AR-selective agonists 43 ß3AR-selective antagonists 52–5 ß3AR selectivities of esters or amides and related acids 64 ß3AR transcripts 89 ß4AR 72, 74–5, 76, 82–3, 96, 120–3 activity assessment 47 existence 46–7 in cardiac tissue 90–1 pharmacology 120–2 ß-arrestin 25, 33–4 baculovirus 17 ßARK 23, 29, 33, 34 basic fibroblast growth factor (bFGF) 108 biological functions 12–15 blood flow control in BAT 105 regulation 93–5 BMS-187257 71 BMS-187413 65 BMS-194449 65, 73 BMS-196085 65, 73 BMS-201620 73 BMS-210285 65, 73 brain 81 BRL-26830 56, 64 BRL-26830A 91 BRL-28410 64

Index

BRL-33725 56 BRL-3513556, 81, 95, 122 BRL-35135A 123 BRL-373444 73 BRL-37344 16, 43, 46, 65, 74–5, 82, 88–92, 122 brown adipocytes 36, 40–3, 45, 97–119 adrenoreceptors in 102–3 control by sympathetic nervous system and hypothalamus 103–4 definition and tissue distribution 99–101 distinguishing features 99, 105–8 ‘ectopic’ 109–10 in WAT 109–10 function 98–9 induction of hyperplasia in BAT 108–9 origin 105–12 secretions from 115–17 subtypes 110–12 UCPs in 101–2, 109, 111, 112 brown adipose tissue (BAT) 15, 18–19, 31, 41, 43, 80–1, 94, 97, 99–101 control of blood flow by sympathetic nervous system and hypothalamus 105 definition 101 induction of hyperplasia of brown adipocytes 108–9 multiple UCPs in 101–2 perspective 119 physiological functions 112–15 thermogenesis 103–4, 112–13 thermogenic capacity 104 UCP-3mRNA in 112 UCPs in 101–2, 109, 111, 112 bupranolol 13, 122 butyrate 33 calcitonin gene-related peptide (CGRP) 103 cAMP 26, 90 cardiovascular effects 95 cardiovascular system 87–96 carnitine palmitoyl transferase I (CPT I) 100 carteolol 70 catecholamine-induced lipolysis 79 catecholamines 74, 77, 93 C/EBPß 107 cell type-specific down-regulation 30–3 cGMP generation 88 CGP-12177 16, 30, 43, 45–7, 65, 66, 69–72, 74–5, 76, 79, 82, 84, 88, 90, 91, 121 CGP-20712A 13, 122 chimeric ß3/ß2-adrenoreceptors 33–4 CIN-1 108 CIN-2 108 CL-316243 34, 38, 40–3, 47, 65, 73, 75, 79, 88, 90–5 clathrin-coated vesicle pathway 25 cloned receptor pharmacology 75–6 colon 81 CP-114,271 65, 73

Index

CP-209,129 65 CP-331,679 65 CP-331,684 65 C-terminal region 18 cyclic AMP response elements (CREs) 4, 107 desensitization 24–6 agonist-promoted 33 ß3AR 34–5 dexamethasone 33 3,5'-diiodotrimetoquinol 73 2,2-dimethylpropyl 73 dobutamine ß1AR agonist 91 dopamine ß-hydroxylase 113 down-regulation agonist-promoted 26–7 cell type-specific 30–3 endomyocardial biopsies 89 endothelial NOS (eNOS) 88 endothelium-derived relaxing factor (EDRF) 91 energy expenditure 41–2 epidermal growth factor (EGF) 26 ERKl/ERK2/p38 MAP kinase signalling pathways 21 extracellular regulated kinase (ERK)1/2 14 fatty acid oxidation 116 fatty acid synthase 116 food intake 42–3, 113 FR-149175 65 free fatty acids (FFA) 117, 118 functional receptor uncoupling 22 G protein-coupled M2-muscarinic receptor 26 G protein-coupled receptor kinases (GRKs) 4, 19, 24, 27, 33 G protein-coupled receptors 4, 6, 11, 18, 20, 27, 123 G protein-coupled signalling regulation, general concepts 22–7 G proteins 1 regions of interaction with 11–12 gastric mucosal blood flow regulation 95 gene knockout techniques 36–47 glucose metabolism 84–5 transporter see GLUT utilization 46 GLUT 1 104 GLUT 4 12, 104–5 GMPreductase 108 heart 81 ß3AR-mediated responses 87–96 HEK-293 cells 29 H-FABP 100 human tissue pharmacology 75–6

169 hyperplasia of brown adipocytes in BAT 108–9 hypothalamus 103–5 ICI compounds 55 ICI-118,551 13, 54 ICI-198157 55, 70, 71 ICI-201651 55, 70 ICI-D711446, 55 immunofluorescence 25 immunofluorescent staining 17 immunohistochemistry 16–18 insulin 33 in maintenance of thermogenic capacity of BAT 104 secretion 42 intracellular NOS (iNOS) 88 iodocyanopindolol 123 isoprenaline 75, 81 isoprenaline-stimulated lipolysis 71 isoproterenol 38 L-749,372 73 L-755,507 72, 73 L-757,793 65 L-760,087 66 L-764,646 66 L-770,644 65 L-771,047 66 L-776,892 66 Langerhans islets 94 lateral hypothalamus (LH) 103 ligand binding 9–11 lipolysis 10, 16, 38–40, 79, 80 low density lipoprotein 26 Ltk-cells 32 LY-79771 63 LY-362884 72 LY-377604 72, 74 mannose-6-phosphate 26 membrane topology 28 messenger RNA 16, 17, 26 tissue expression 78–82 metabolism 73–4 mice expressing human, but nor murine, ß3AR 43–6 lacking ß3AR 36–40 phenotype 36–8 mitochondriogenesis 105–8 myocardial ß1AR and ß2AR 87 myocardial ß3AR 89 Na+/H+-exchanger regulatory factor (NHERF) 21 nadolol 75–6 nerve growth factor (NGF) 116–17 nitric oxide (NO) production 88 nitric oxide synthase (NOS) pathway 88

Index

170 non-conventional partial agonists 66–70 non-insulin-dependent diabetes 84 noradrenaline 1, 75, 108, 113 NPXXY motif 25 NPXXY sequence 30 obesity 79, 85, 117–19 defence against 113–15 oxygen consumption 45–7 p12 antibody 18 pancreatic islet blood flow regulation 93–4 peroxisome proliferator activated receptor (PPAR) 100, 106, 107 pertussis toxin (PTX) 14 pharmacokinetics 73–4 phenoxypropanolamines 73 phenylethanolamine agonists 60–3 phenylethanolamines 55, 70, 75–7 phorbol ester 33 phospholipase C 1, 29 phosphorylation 22–5, 27–9, 33 PI3K 15, 19 PIA 38 plasma insulin concentrations 94 poly-immunoglobulin 26 polymerase chain reaction (PCR) 18 polymorphism 9–10 PPAR gamma coactivator-1 (PGC-1) 100, 106 propanolol 122 propranolol 75 protein kinase A (PKA) 21, 23, 25, 27–9, 33, 109, 114 protein kinase B (PKB) 15 protein kinase C (PKC) 28, 29 pyridines 72, 73 receptor sequestration 24–6 regulatory motifs, delineation 33–4 resensitization 24–6 RIß 109, 114 RIIß 109, 114 Ro-16–8714 63, 64 Ro-363 81 RT-PCR experiments 16–17 salmeterol 65 Sanofi aryloxy-propanolaminotetralins 52 satiety factors 115–16 SB-206606 82 SB-215691 75 SB-220646 75 SB-226552 71, 72, 122 SB-229432 71, 72 SB-236923 72, 76, 122 SB-246982 72 SB-248320 76

SB-251023 71, 72, 122 signal transduction 12–15 signalling efficacy regulation 20–3 signalling pathways 21–2 ß3AR-mediated activation of 14–15 Single Nucleotide Polymorphism (SNIPs) 9 SK-N-MC neurotumour cells 29 SM-11044 66, 123 SR-58611 88–90 SR-58611A 95 SR-59230A 13, 52–3, 82, 122 structure-function relationships 10–12, 77 substance P 103 sympathetic nervous system 103–5, 108 3T3-F442A 19, 31 Tecradine 66 terbutaline ß2AR agonist 91 thermoregulation in cold environment 112–13 transgenic expression of human ß3AR in knockout mice 44 transgenic techniques 41 transmembrane (TM) regions 4 trimetoquinol 72–3 Trp64Arg polymorphism 83–4, 86 tryptophan arginine (W64R) substitution 9 tumour necrosis factor a (TNF-a) 108–9 tyrosines 26 UCP-1 17, 102, 103, 106–15 UCP-2 102, 107, 111, 114 UCP-3 102, 106, 110, 111, 114 UCP-3 mRNA in BAT 112 UCP-3 mRNA in WAT 111 UCPs, expression in BAT 101–2, 109, 111, 112 uncoupling protein-1 (UCP-1) 94, 97–100 urinary bladder 81–2 VEGF 116 ventromedial nucleus of the hypothalamus (VMH) 103, 105 white adipocytes 36, 40–3, 45, 97–119 definition and tissue distribution 99–101 distinguishing features 99, 105–8 function 98–9 white adipose tissue (WAT) 15, 18–19, 31, 41, 43, 44, 78, 79, 80, 94, 97–101, 104 definition 101 ‘ectopic’ brown adipocytes in 109–10 perspective 119 UCP-2 expression in 111 UCP-3 mRNA in 111 ZD-711455, 71 ZD-9989 65 Zeneca compounds 55 ZM-215001 55, 70–1