Animal Models in Diabetes: A Primer (Frontiers in Animal Diabetes Research)

ANIMAL MODELS OF DIABETES

Frontiers in Animal Diabetes Research Each volume of this series will be topic oriented with timely and liberally referenced reviews and provide in depth coverage of basic experimental diabetes research. Edited by Professor Anders A.F.Sima, Wayne State University, Detroit, USA and Professor Eleazar Shafrir, Hadassah University Hospital, Jerusalem, Israel. Volume 1 Chronic Complications in Diabetes: Animal Models and Chronic Complications edited by Anders A.F.Sima Volume 2 Animal Models of Diabetes: A Primer edited by Anders A.F.Sima and Eleazar Shafrir Volumes in preparation Insulin Signaling: From Cultured Cells to Animal Models edited by George Grunberger and Yehiel Zick Muscle Metabolism in Animal Models in Diabetes edited by Harriet Wallberg-Henriksson and Juleen R.Zierath This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.

ANIMAL MODELS OF DIABETES A PRIMER

Edited by

Anders A.F.Sima Departments of Pathology and Neurology Wayne State University Detroit, USA and

Eleazar Shafrir Department of Biochemistry Hadassah University Hospital Jerusalem, Israel

harwood academic publishers Australia • Canada • France • Germany • India • Japan • Luxembourg Malaysia • The Netherlands • Russia • Singapore • Switzerland

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © 2001 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands British Library Cataloguing in Publication Data ISBN 0-203-30473-X Master e-book ISBN

ISBN 0-203-34329-8 (Adobe eReader Format) ISBN: 90-5823-096-1 (Print Edition) ISSN: 1029-841X

CONTENTS

Preface to the Series

vii

Preface

viii

Contributors

ix

1

Autoimmune Diabetes Mellitus in the BB Rat John P.Mordes, Rita Bortell, Herman Groen, Dennis Guberski, Aldo A.Rossini and Dale L.Greiner

1

2

The NOD Mouse and its Related Strains Hiroshi Ikegami and Susumu Makino

38

3

Obesity/Diabetes in Mice with Mutations in the Leptin or Leptin Receptor Genes Lieselotte Herberg and Edward H.Letter

56

4

The Zucker Diabetic Fatty (ZDF) Rat Richard G.Peterson

95

5

KK and KKAy Mice Shigehisa Taketomi and Hitoshi Ikeda

113

6

The Obese Spontaneously Hypertensive Rat (SHROB, Koletsky Rat): A Model of Metabolic Syndrome X Richard J.Koletsky, Jacob E.Friedman and Paul Ernsberger

126

7

Characteristics of Wistar Fatty Rat Hiroyuki Odaka, Yasuo Sugiyama and Hitoshi Ikeda

141

8

The New Zealand Obese Mouse: A Polygenic Model of Type 2 Diabetes Sofianos Andrikopoulos, Anne W.Thorburn and Joseph Proietto

152

9

The NSY Mouse: An Animal Model of Human Type 2 Diabetes Mellitus with Polygenic Inheritance Hironori Ueda, Hiroshi Ikegami, Masao Shibata and Toshio Ogihara

164

10

The Goto-Kakizaki Rat Claes-Göran Östenson

174

11

The OLETF Rat Kazuya Kawano, Tsukasa Hirashima, Shigehito Mori, Zhi-wei Man and Takashi Natori

188

vi

12

The JCR:LA-cp Rat: An Animal Model of Obesity and Insulin Resistance with Spontaneous Cardiovascular Disease J.C.Russell and S.E.Graham

199

13

The Neonatally Streptozotocin-Induced (n-STZ) Diabetic Rats, a Family of NIDDM Models Bernard Portha, M.H.Giroix, P.Serradas, J.Movassat, D.Bailbe and M.Kergoat

216

14

Galactosemic Animal Models W.Gerald Robison Jr.

237

15

The Rhesus Monkey (Macaca mulatto): A Unique and Valuable Model for the Study of Spontaneous Diabetes Mellitus and Associated Conditions Noni L.Bodkin

269

16

Psammomys obesus: Primary Insulin Resistance Leading to Nutritionally Induced Type 2 Diabetes Ehud Ziv and Rony Kalman

286

17

The C57BL/6J Mouse as a Model of Diet-Induced Type 2 Diabetes and Obesity Ann E.Petro and Richard S.Surwit

301

Index

313

PREFACE TO THE SERIES

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

PREFACE

Since Oscar Minkowski discovered, about 110 years ago, that the removal of the pancreas causes diabetes in dogs, many other animal models with spontaneous diabetes or nutritionally induced diabetes have been used to obtain a better understanding of this disease. This holds true for both type 1 and type 2 of the disease. Because of the continuing increase of diabetes to epidemic proportions, mainly of type 2, its pathophysiology in many respects is still largely unknown and animal models of type 2 diabetes are of great importance for future research in diabetology. Much that we know about the pathological processes, immune derangements, insulin secretion and insulin signaling abnormalities, as well as nutritional influences in diabetes, has been derived from studying diabetes in animals. An effective diabetes model demonstrates tissue functions which have been compromised by genetic mutation or environmental effects often associated with longstanding complications. Studies on models of diabetes became fundamental, since laboratory investigation in human subjects is limited by the availability of tissues and the long duration of observations required for the basic approach to the study of cellular changes. In addition, there is an urgent need for preventive and curative diabetes research. This book is intended to provide a review of the characteristics of the more commonly used animal species with various diabetic syndromes which were developed and extensively investigated during the last few decades. Animal models have been included which are readily available, reasonably well described and proven to be of value in the research of both types of diabetes. It is hoped that this extensively referenced book will be helpful to established investigators, as well as to graduate students, young investigators and pharmaceutical scientists working on the development of antidiabetic and preventive modalities in various areas of diabetes and its complications.

CONTRIBUTORS

Sofianos Andrikopoulos Department of Medicine University of Melbourne Royal Melbourne Hospital Parkville, Victoria 3050 Australia D.Bailbe Lab. Physiopathologie Nutrition CNRS ESA 7059 Université Paris 7/D. Diderot 2 place Jussieu 75251 Paris Cedex 05 France Noni L.Bodkin Obesity and Diabetes Research Center Department of Physiology School of Medicine University of Maryland 10 South Pine Street MSTF 6–00

x

Baltimore, Maryland 21201 USA Rita Bortell Department of Medicine University of Massachusetts Medical Center Worcester, Massachusetts 01605 USA Paul Ernsberger Departments of Nutrition and Medicine Case Western Reserve University School of Medicine Cleveland, Ohio 44106–4935 USA Jacob E.Friedman Departments of Nutrition and Medicine Case Western Reserve University School of Medicine Cleveland, Ohio 44106–4935 USA M.H.Giroix Lab. Physiopathologie Nutrition CNRS ESA 7059 Université Paris 7/D. Diderot 2 place Jussieu 75251 Paris Cedex 05 France S.E.Graham Department of Surgery University of Alberta Edmonton, Alberta Canada Dale L.Greiner Department of Medicine University of Massachusetts Medical Center Worcester, Massachusetts 01605 USA Herman Groen Department of Medicine University of Massachusetts Medical Center Worcester, Massachusetts 01605 USA Dennis Guberski

xi

Department of Medicine University of Massachusetts Medical Center Worcester, Massachusetts 01605 USA lieselotte Herberg Diabetes Research Institute Heinrich-Heine University of Düsseldorf Düsseldorf Germany Tsukasa Hirashima Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd. Otsuka Japan Hitoshi Ikeda Pharmaceutical Research Laboratories Takeda Chemical Industries Ltd. Yodogawa-ku, Osaka 532–8686 Japan Hiroshi Ikegami Department of Geriatric Medicine Osaka University Medical School 2–2 Yamadaoka Suita, Osaka 565 Japan Rony Kalman Diabetes Research Unit Hadassah-Hebrew University Medical Center Jerusalem Israel Kazuya Kawano Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd. Otsuka Japan M.Kergoat Merck-Lipha Centre de Recherché 91380 Chilly-Mazarin

xii

France Richard J.Koletsky Departments of Nutrition and Medicine Case Western Reserve University School of Medicine Cleveland, Ohio 44106–4935 USA Edward H.Leiter The Jackson Laboratory Bar Harbor, Maine 04609 USA Susumu Makino AC Center Shionogi Laboratories Osaka Japan Zhi-wei Man Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd. Otsuka Japan John P.Mordes Department of Medicine University of Massachusetts Medical Center Worcester, Massachusetts 01605 USA Shigehito Mori Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd. Otsuka Japan J.Movassat Lab. Physiopathologie Nutrition CNRS ESA 7059 Université Paris 7/D. Diderot 2 Place Jussieu 75251 Paris Cedex 05 France Takashi Natori Tokushima Research Institute Otsuka Pharmaceutical Co., Ltd.

xiii

Otsuka Japan Hiroyuki Odaka Pharmaceutical Research Division Takeda Chemical Industries Ltd. Yodogawa-ku, Osaka 532–8686 Japan Toshio Ogihara Department of Geriatic Medicine Osaka University Medical School 2–2 Yamadaoka Suita, Osaka 565 Japan Claes-Göran Östenson Department of Molecular Medicine The Endocrine and Diabetes Unit Karolinska Institute and Hospital SE-171 76, Stockholm Sweden Richard G.Peterson Indiana University School of Medicine, Anatomy MS 5035, 635 N.Barnhill Drive Indianapolis, Indiana, 46202–5120 USA Ann E.Petro Duke University Medical Center Durham, North Carolina 27710 USA Bernard Portha Lab. Physiopathologie Nutrition CNRS ESA 7059 Université Paris 7/D. Diderot 2 place Jussieu 75251 Paris Cedex 05 France Joseph Proietto Department of Medicine University of Melbourne Royal Melbourne Hospital Parkville, Victoria 3050

xiv

Australia W.Gerald Robison, Jr. National Eye Institute National Institutes of Health Bethesda, Maryland 20892–2735 USA Aldo A.Rossini Department of Medicine University of Massachusetts Massachusetts Medical Center Worcester, Massachusetts 01605 USA J.C.Russell Department of Surgery University of Alberta Edmonton, Alberta Canada P.Serradas Lab. Physiopathologie Nutrition CNRS ESA 7059 Université Paris 7/D. Diderot 2 place Jussieu 75251 Paris Cedex 05 France Masao Shibata Department of Health Aichi-Gakuin University College of General Education Iwasaki, Nishincho Aichi-gun, Aichi 470–01 Japan Yasuo Sugiyama Pharmaceutical Research Division Takeda Chemical Industries Ltd. Yodogawa-ku, Osaka 532–8686 Japan Richard S.Surwit Duke University Medical Center Durham, North Carolina 27710 USA

xv

Shigehisa Taketomi Discovery Research Laboratories Takeda Chemical Industries Ltd. Yodogawa-ku, Osaka 532–8686 Japan Anne W.Thorburn Department of Medicine University of Melbourne Royal Melbourne Hospital Parkville, Victoria 3050 Australia Hironori Ueda Department of Geriatric Medicine Osaka University Medical School 2–2 Yamadaoka Suita, Osaka 565 Japan Ehud Ziv Diabetes Research Unit Hadassah—Hebrew University Medical Center Jerusalem Israel

1. AUTOIMMUNE DIABETES MELLTTUS IN THE BB RAT JOHN P.MORDES, RITA BORTELL, HERMAN GROEN, DENNIS GUBERSKI, ALDO A.ROSSINI and DALE L.GREINER Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01605, USA

INTRODUCTION Since last reviewed in detail by us in 1992, more than 250 studies of the BB rat have appeared. This literature testifies to the continuing scientific interest in this animal as a model of human Type 1 diabetes mellitus, even among proponents of the NOD mouse (Atkinson and Leiter, 1999). Many lines of evidence document that Type 1 or insulin-dependent diabetes (IDDM) in humans is a disease of autoimmunity. These include observations of pancreatic insulitis, islet autoantibodies, reappearance of disease after syngeneic pancreas allografts, induction of disease in bone marrow allograft recipients, successful immunoprophylaxis with cyclosporin, and major histocompatibility complex (MHC) associations. Analogous evidence suggests that the hyperglycemic syndrome of the BB rat is a similar disorder. SPONTANEOUSLY DIABETIC BB RATS The Original BioBreeding Colony The spontaneously diabetic BB rat was discovered in 1974 in a colony of outbred Wistar rats at BioBreeding Laboratories in Ottawa, Canada (Nakhooda et al., 1977). Many breeding colonies have subsequently been established (Table 1). The cumulative frequency of spontaneous diabetes among the original BioBreeding animals was about 10%. Selective breeding has increased the cumulative frequency to

2

AUTOIMMUNE DIABETES MELLITUS IN THE BB RAT

>90% in some colonies. These animals are all described as “diabetes prone” BB rats to distinguish them from “diabetes resistant” or “control” sublines developed later. Genetic Heterogeneity Among BB Bats from Different Sources As detailed below, all spontaneously diabetic BB rats share the class II RT1U rat MHC, develop pancreatic insulitis with selective beta cell destruction, and are Table 1 Pedigree of BB rat originating from the bio-breeding laboratory Ottawa Ontario CN, spontaneous mutation 1974. U.S. Colony Founded 1977 in Worcester, MA

MRC Colony Founded in Ottawa Ontario Canada 1978

BB/Wor Rat Diabetes Prone Lines

BB/Wor-Derived Colonies: Location

Year of Derivation and designation

Diabetes Prone BB Lines

dpBB-Derived Colonies: Location

Designation

BBBA/Wor// Brm F70

Gentofe, Denmark (Hagedorn) Toronto Ontario (Hospital for Sick Children) NIH Reference Colony

1983 BB/H

dpBB

Edinburgh, UK

dpBB/E

1984

Edinburgh, UK

dpBB/Ed

1983–1998

Univ. Greifscoald, Karlsburg, Germany Philadelphia, USA

BBDP/Wor//Brm F70

BBNB//Wor/ Brm F71

BBPA/Wor//Brm F71

M&B (Formerly Møllegaard) Brussells Belgium Memphis, TN U.S.A. Denver CO, USA, Bucharest Romania Westmead Australia Pittsburgh, PA USA None

1987 BBDP/ Wor//Mol 1988 BBDP/Wor

dpBB/OK dpBB/Ph

1989 BBDP/ Wor//Utm 1986 BBDP/Wor 1996 BBDP/Wor 1986 1988

This work was supported in part by grants DK25306 (AAR), DK36024 (DLG), and DK41235 (JPM) from the National Institutes of Health, and by grant DFN98.501 (HG) from the Diabetes Foundation of the Netherlands. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the officiai views of the National Institutes of Health.

JOHN P.MORDES ET AL.

U.S. Colony Founded 1977 in Worcester, MA BB/Wor Rat Diabetes Prone Lines BBIWor Diabetes Resistant Lines BBDR/Wor// Brm F71

BBVB/Wor// Brm F71

BB/Wor-Derived Colonies: Location

3

MRC Colony Founded in Ottawa Ontario Canada 1978 Year of Derivation and designation

Diabetes Prone BB Lines

dpBB-Derived Colonies: Location

Designation

MRC Resistant BB Rats Gentofe, Denmark Hagedorn 1983 Westmead Australia 1986 Pittsburgh, PA USA 1988 Denver CO, USA, 1986 Brussells Belgium, 1988 M&B (Formerly Møllegaard) 1987 None

BBDR/Wor

drBB (BBc, BBn) BBDR/Ed

BBDR/Wor BBDR/Wor BBDR/Wor//Mol

Partial listing of BB rat sublines and their origins. All BB rats were derived from the outbred Wistar rats discovered at the BioBreeding Laboratory in Canada in 1974. A subset of these animals formed the nucleus of two colonies that later became the source of all tertiary colonies. BB/Wor rats have been inbred for 70+ generations. The tertiary colonies of Worcester-origin were derived from one of three sublines, as indicated. Diabetes prone dpBB rats have remained outbred, and the tertiary colonies from these Ottawa animals have undergone various degrees of inbreeding. BBDR/Wor animals were derived from diabetes prone BBBA/Wor forebears in the fifth generation of inbreeding. The BBVB/Wor rat was derived directly from outbred forebears from the original BioBreeding colony. Additional information is available from the website of the Institute for Laboratory Animal Research (ILAR) at http://www2.nas.edu/ilarhome/ and from Festing (Festing, 1993).

severely lymphopenic. Despite these uniform features, however, “BB rats” are not all identical. Table 1 summarizes some of the history of the BB rat. The original outbred BioBreeding animals gave rise to two broad classes of contemporary BB rats. The first class is derived from the colony established in 1977 in Worcester, MA, USA and sponsored for two decades by the National Institutes of Health (NIH). The second class is derived from a colony established in 1979 in Ottawa, Canada from BioBreeding progenitors under the sponsorship of the Canadian Medical Research Council (MRC). The Worcester facility developed inbred sublines of diabetes prone “BB/Wor” rats. In the 1980s, it supplied nuclear breeders for several tertiary colonies around the world. The Ottawa colony has remained outbred. It has been the source of several other tertiary colonies that in turn have been partially or competely inbred (See Table 1). All available BB rats are thought to belong to one of these two progenitor classes. For BB/Wor derived animals, Table 1 indicates both when and from which subline tertiary colonies were derived. Available data for the Ottawa-derived colonies are less detailed. All secondary and tertiary colonies

4

AUTOIMMUNE DIABETES MELLITUS IN THE BB RAT

obviously vary with respect to source of the original breeders and duration of genetic isolation; not surprisingly they also vary with respect to frequency and severity of disease and immunological characteristics (Crisá et al., 1992; Guberski, 1994). The genetic heterogeneity of the various outbred and inbred “BB rats” implicit in this history is well documented (Prins et al., 1991). These authors investigated the extent of the heterogeneity among 26 distinct lines (24 inbred, 2 outbred) of the BB rat by analyzing 19 protein markers. They observed polymorphisms in 9 markers and used them to define 7 distinct haplotypes. In the decade since the appearance of that report, additional divergence may have occurred. In addition to genetic drift, environmental factors may also affect BB rat colonies (Crisá et al., 1992). As will be discussed in detail below, the incidence of diabetes in BB rats can be modified by environmental factors including diet and infectious agents. These factors can affect experimental outcomes. In addition, breeding programs based on the identification of diabetic rats may select for environmentally activated (or suppressed) genes if they contribute locally to the appearance (or suppression) of diabetes. In summary, there is significant genotypic and phenotypic variation among lines of BB rats. Interpretations and comparisons of results obtained using “the BB rat” should take into account the origin and status of the colonies from which the experimental animals were obtained. Nomenclature An additional complication in interpreting BB rat data is inconsistent nomenclature. Some Institute for Laboratory Animal Research (ILAR) designations are listed in Table 1, but for the most part these have not been used in the literature. Few reports use the newest designations. For the sake of consistency with the published literature, this review generally uses designations employed by the authors of original reports. DIABETES RESISTANT BB RATS Important assets in the study of autoimmunity in the BB rat are lines of diabetes resistant animals, the origins of some of which are listed in Table 1. In the Ottawa colony, resistant animals (usually designated BBdr but sometimes BBc or BBn) are outbred and non-lymphopenic. A diabetes-resistant (DR) subline of the BB rat based on animals obtained from the Ottawa colony has also been established in Edinburgh, U.K (Joseph et al., 1993). These BB-DR/Ed rats are unusual in that, like diabetes-prone BB-DP/Ed rats, they are lymphopenic. In the Worcester colony, there are two sublines of diabetes resistant BB/Wor rats: BBDR/Wor and BBVB/ Wor (Crisá et al., 1992; Guberski, 1994). BBDR/Wor animals were derived from diabetes prone BBBA/Wor forebears in the fifth generation of inbreeding. The BBVB/Wor rat was derived directly from outbred forebears from the original BioBreeding colony. Both the BBDR/Wor and BBVB/Wor sublines have been continuously selected for resistance to diabetes (Guberski, 1994). What is now officially designated as the BBDR/Wor rat was frequently referred to as the DR-BB/Wor rat in the literature. Unlike diabetes prone BB/ Wor rats, diabetes resistant BB/Wor rats are not lymphopenic (Crisá et al., 1992). They have been used in the majority of studies of disease resistance in the BB rat. The BBDR/Wor rat has been inbred for >70 generations. When housed in a viral antibody free (VAF) vivarium, none (0%) become spontaneously diabetic (Guberski, 1994). Important features that distinguish these animals from DP-BB/Wor rats are summarized in Table 2. One of the most useful characteristics of the DR-BB/Wor rat is its propensity to become diabetic in response to environmental perturbation. As will be discussed, it is clear that diabetes resistant BB/Wor rats harbor populations of diabetogenic effector cells that are normally held in check by

JOHN P.MORDES ET AL.

5

populations of regulatory cells, among them cells that express the RT6+ phenotype (Crisá et al., 1992). BBDR/Wor rats are susceptible to collagen-induced arthritis (Watson et al., 1990) and inducible lymphocytic thyroiditis (Crisá et al., 1992). OBTAINING BB RATS Information on the status of BB rat lines and colonies is available from the International Index of Laboratory Animals (Festing, 1993) and the ILAR web site (http://www2.nas.edu/ilarhome/). The web site provides contact information for many BB rat colonies. The major American supplier of BB rats is Biomedical Research Models, Inc., Worcester, MA (http://www.brm.com); the major European vendor is M&B, A/S, Ry, Denmark (http://www.m-b.dk/). At this writing, the approximate costs, exclusive of shipping, for weanling diabetes prone and diabetes resistant rats from BRM, Inc. are USD26 and USD21, respectively. From M&B, the costs are roughly comparable. The production facility for BB rats at BRM, Inc merits comment because of the well defined status of its animals. This company was formed when the NIH decided to privatize the colony that had been maintained under their sponsorship at the University of Massachusetts Medical Center in Worcester (Guberski, 1994). The “BB/Wor//Brm” animals in this colony are inbred, have clearly defined Table 2 Selected clinical and immunological characteristics of diabetes prone and diabetes resistant

Cumulative frequency of diabetes Age at onset Insulitis Diabetic rats Nondiabetic rats Thyroiditis Peripheral lymphocytes Lymphocyte Subsets CD4+, CD5+ OX22, OX32 (CD45R+) CD8+, RT6+ RT6+ intraepithelial Lymphocytes NK Cell Activity Environmental Perturbants KRV infection LCMV infection Hydrolysed casein diet

Diabetes Prone

Diabetes Resistant

>90% >85% of cases between 55 and 120 days of age

0% in VAF vivaria Inducible in animals if treatment begun by 30 days

100% 50% 5% in BBVB/Wor 100% in BBDR/Wor Lymphopenic

100% 0% in VAF vivaria ~20% in animals given poly I:C + anti-RT6 mAb Nonlymphopenic

Low Low Very low

Normal Normal Normal

Present at reduced levels Increased

Present Low

No effect Reduces IDDM incidence Reduces IDDM

Induces diabetes Not known No effect

Partial listing of characteristics that distinguish diabetes prone and diabetes resistant BB/Wor rats. Susceptible and resistant animals that originated from the Ottawa colony (see Table 1) may differ. Citations are provided in the text.

6

AUTOIMMUNE DIABETES MELLITUS IN THE BB RAT

pedigrees, and are the progenitors of many other colonies worldwide (Table 1). They are housed in viral antibody free (VAF) conditions, and available for shipment worldwide. Diabetes prone BB rats in the BRM, Inc. colony are available from three of the six sublines listed in Table 1: BBDP/Wor, BBNB/Wor, and BBDR/ Wor (Guberski, 1994). Two lines of diabetes resistant BB rats, BBDR/Wor and BBVB/Wor, are also available from BRM. All BB/Wor animals have been inbred for >70 generations. The diabetes prone BB/Wor rat has also been crossed with the Zucker fatty rat to yield two coisogenic lines, the BBZDP/Wor rat, an obese animal with autoimmune features, and the BBZDR/Wor, a model of NIDDM available from BRM. MAINTAINING AND USING BB RATS BB rats should be obtained from a VAF production facility and maintained in microisolators in VAF or specific-pathogen free quarters. A laminar flow hood should be used as a workstation for animal husbandry and for experimental procedures. These precautions are necesssary because, as detailed below, the frequency of both spontaneous and induced diabetes in BB rats is modified by viral infection. For example, lymphocytic choriomeningitis virus (LCMV) prevents dia betes in diabetes prone rats, and Kilham rat virus (KRV) can induce IDDM in resistant animals (Crisá et al., 1992). We recommend that all materials taken into rooms housing BB rats be autoclaved. Non-autoclavable materials and the exposed surfaces of tubes, equipment, and other objects should be disinfected with a solution of Clidox™ or its equivalent. Personnel handling BB rats should wear sterile outer garments, including bonnets, masks, gloves, and shoe covers. Frequent disinfection of handling areas, racks, and other objects in the facility with fresh Clidox™ is recommended to the facility free of contaminating viruses. The pathogen status of BB rat vivaria should be monitored by serological testing of sentinel animals housed without microisolation in the same rooms. We routinely add some used bedding from randomly selected cages of BB rats to the sentinel cages at regular intervals. Because DP-BB rats are lymphopenic, it is preferable to use DR-BB rats or immunocompetent non-BB rats as sentinels. Procedures for detecting diabetes and basic experimental protocols for use with BB rats are available (Whalen et al., 1996). Diabetic BB rats require daily treatment with insulin and may require additional treatments for ketonemia; detailed therapeutic protocols have been published (National Research Council, 1996). Breeding diabetes prone BB rats requires special procedures. Mating diabetic male BBDP rats with female BBDP rats that have been prophylactically transfused to prevent diabetes (see page 11) enhances both fertility and nursing (Guberski et al., 1992). CLINICAL FEATURES AND PATHOLOGY Clinical autoimmunity and organ pathology are similar in spontaneously diabetic BB rats and in DR-BB/ Wor rats that are induced to become hyperglycemic. Clinical Diabetes in BB Rats Like human IDDM, spontaneous diabetes in BB rats appears during adolescence, typically between 60 and 100 days of age (Crisá et al., 1992). Incidence in males and females is similar. Insulin response to glucose challenge may be normal up to 10 days before disease onset; it then declines as beta cell mass declines (Teruya et al., 1993). Clinical onset is abrupt, and hyperglycemia is accompained by weight loss, hypoinsulinemia, and ketonuria. Fatal ketoacidosis ensues unless exogenous insulin is given. The

JOHN P.MORDES ET AL.

7

cumulative frequency of diabetes through 120 days of age generally ranges between 60 and 90% depending on the colony; it is >85% in the Worcester colony (Guberski, 1994). In a study of old BB/Mol rats that had not developed spontaneous diabetes, it was observed that they nonetheless harbored populations of autoreactive cells capable of inducing disease in adoptive recipients (MacKay, 1995). Hyperglucagonemia and other endocrine abnormalities characteristic of IDDM (e.g. decreased growth hormone secretion) are present (Crisá et al., 1992). The clinical diabetic syndrome observed after induction in the DR-BB/Wor rat has not been reported in as much detail. It is, however, similarly characterized by susceptibility to ketoacidosis in the absence of exogenous insulin. Pancreatic Insulitis Intense mononuclear infiltration within and around the islets of Langerhans (“insulitis”) is the characteristic histopathological lesion of spontaneous diabetes in BB rats at the time of onset (Nakhooda et al., 1977; Crisá et al., 1992). Serial pancreatic biopsies have shown that the lesions start as early as 2–3 weeks before overt diabetes and rapidly progress to complete, selective destruction of islet beta cells. In chronically diabetic animals, “end stage” islets with few or no inflammatory cells are observed. In contrast, islet alpha, delta, and pancreatic polypeptide (PP) cell numbers and morphology appear to be preserved. Sub-populations of lymphoid cells present at different stages of insulitis have been extensively characterized (Crisá et al, 1992). Macrophages are among the earliest, possibly the first, of the cellular elements observed (Hanenberg et al, 1989). Alternatively, it has been suggested that the earliest infiltrating elements may include dendritic cells, which may have enhanced antigen presenting capability in the BB rat (Tafuri et al., 1993). Both CD4+ and CD8+ T cells, natural killer (NK) cells and, to a lesser extent, B cells subsequently infiltrate the islets. Insulitis has also been studied immunocytochemically in resistant BB rats in which diabetes was induced by immunological perturbation (Jiang et al, 1990). This approach permitted kinetic analyses of islet pathology in relationship to induction by administration of a cytotoxic anti-RT6 antibody (See page 24). The study revealed a prodromal period of 10 days during which no morphologic abnormalities of the pancreas were detected. This was followed by a second phase of early insulitis in which a few islets were infiltrated by both macrophages and T cells. The lesions rapidly progressed, and by day 18 insulitis was generalized and intense. Pancreatic ductular lymphocytic infiltration (clusters of lymphocytes around small ductules and venules of the pancreas) can also antedate the appearance of insulitis (Bone et al, 1990). Both the early periductular lesion and infiltrative insulitis may be found in animals that do not become diabetic. Periductular lesions can also be found in F1 crosses of BB rats with other non-diabetes prone inbred strains, although these animals never become diabetic (Colle, 1990). The relationship of this lesion to insulitis remains unclear. Hyper-expression of class I antigens occurs on islet and exocrine cells in diabetic BB rats (Bone et al, 1990). Class II antigen expression is not seen on BB rat pancreatic beta cells (Timsit et al, 1989). In situ hybridization studies have demonstrated the presence of tumor necrosis factor (TNF), IL-1 and IL-6 mRNA in cells at the site of lymphocytic infiltration in newly diabetic BB rats (Jiang and Woda, 1991). RT-PCR has been used to measure mRNA encoding T helper (Th) type 1 and 2 cytokines present in inflamed islets (Zipris et al, 1996; Kolb et al, 1996). In both DP and RT6-depleted DR-BB/Wor rats, interferon-gamma (IFN-) mRNA was present in islets before and during disease onset. IL-2 and IL-4 mRNAs were minimal or undetectable in infiltrated islets but present in activated peripheral T cells (Zipris et al., 1996). IL-10 mRNA was present at low abundance. The data suggest that insulitis in the BB rat is a Th1 type inflammatory response. Consistent with this interpretation, it was found that mRNA encoding the p40 chain of IL-12 was

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AUTOIMMUNE DIABETES MELLITUS IN THE BB RAT

also present before and during disease onset. Th1 lymphocytes appear to predominate over Th2 lymphocytes in these inflammatory lesions. Nitric oxide is a candidate beta cell cytotoxic agent. The inducible NO synthase (iNOS) has been found in pancreatic lesions of adult diabetes-prone BB rats using both RT-PCR and immunohistochemistry (Kleemann et al., 1993; Kolb et al., 1996). Expression was absent in normal Wistar rats, young DP-BB rats without insulitis, and in DR-BB rats. Parallel staining for ED1+ macrophages showed restriction of iNOS expression to areas of islet infiltration by macrophages, suggesting the possibility of iNOS involvement in islet destruction (Kleemann et al., 1993). Pancreatic Endothelium Activation and selective increase in the leakiness of islet capillaries and postcapillary venules at the onset of spontaneous diabetes have been observed (De Paepe et al., 1992). Other studies have described an inducible venular defect specific to the pancreas (Desemone et al., 1990) that can be prevented by anti-inflammatory agents (Kitagawa et al., 1993). The leakiness defect is also seen in other, non-diabetic rat strains, and its relationship to diabetes pathogenesis remains uncertain. Endothelium could participate in the BB rat autoimmune process in other ways. Hyper-expression of class I and induction of class II MHC molecules on pancreatic endothelium occurs early in the disease (Bone et al., 1990). Anti-endothelial cell autoantibodies also develop in spontaneously diabetic BB/Wor rats and in DR-BB/ Wor rats during the course of diabetes induction (Doukas et al., 1996). Thyroiditis Autoimmune thyroiditis is more common among humans with IDDM than in the general population. Lymphocytic infiltration of the thyroid has also been described in both diabetic and non-diabetic DP-BB/ Wor animals, but the lesion does not progress to frank hypothyroidism in the absence of dietary or other manipulation (Reimers et al., 1996; Mooij et al., 1993; Mori et al., 1998). Pathologically, thyroiditis is associated with infiltration of both dendritic cells and lymphocytes (Simons et al., 1998). RT-PCR analyses of cytokine mRNA in the thyroid glands of RT6-depleted DR-BB/Wor rats revealed a Th1-type cytokine profile similar to that observed in inflamed islets (Zipris, 1996). Thyroiditis can also be adoptively transferred from DP-BB rats to MHC compatible naïve recipients using splenocytes (McKeever et al, 1990), and it can be prevented by the transfusion of normal MHCcompatible lymphocytes (Burstein et al., 1989). Lines of thyroid-reactive cells have also been derived from diabetes prone animals (Allen and Thupari, 1996). Like IDDM, thyroiditis can also be induced in diabetes resistant BB/Wor rats (Thomas et al., 1991), and DR-BB/Wor thymocytes can adoptively transfer thyroiditis (Kimura and Davies, 1996; Whalen et al., 1994). In early reports, thyroiditis was observed in about 59% of diabetic and 11% of non-diabetic DP-BB/Wor rats. It is MHC-associated (Awata et al., 1995). The prevalence of lymphocytic thyroiditis varies among different diabetes prone sublines in the Worcester colony (Rajatanavin et al., 1991). At ~110 days of age, for example, the prevalence was 100% in BBNB/Wor rats but only 4.9% in BBBE/Wor animals. This observation, together with the low rate of concordance of diabetes and thyroiditis (Pettersson et al., 1995), suggests that these two hereditary autoimmune diseases are not tightly linked genetically. Understanding the relationship between IDDM and lymphocytic thyroiditis in the BB rat remains a major unsolved problem.

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Extrapancreatic Abnormalities and Diabetic Complications Extra-pancreatic diseases other than thyroiditis have also been described in both diabetic and non-diabetic BB rats as reviewed by us previously (Crisá et al., 1992). Some have sialadenitis. The most prominent abnormalities involve the lymphoid system. Lymph nodes of young animals show variable degrees of paracortical and medullary replacement by plasmacytoid lymphocytes. In older rats, B cell lymphomas occur, the frequency being higher in long-term diabetic rats (15%) than in non-diabetic rats (~1%). The observation of similar lymphoproliferative lesions in other autoimmune diseases and in chronic graft vs. host disease suggests that antigen or autoantigen driven clonal expansion of B cells might play a role in the development of these malignancies (Friedman et al., 1991; Shirai et al., 1991). In a necropsy study of BB/E rats, the increased frequency of lymphoma was reportedly associated with a translocation of the c-myc oncogene (Meehan et al., 1993). Diabetes prone BB rats also have an increased susceptibility to pulmonary infections; sterile granulomas in lymph nodes, kidney and pancreas; and prostatic atrophy. As is the case in humans with IDDM, chronically hyperglycemic BB rats gradually develop secondary systemic complications (Crisá et al., 1992). Pathological changes affecting retina (Chakrabarti and Sima, 1997), myocardium (Giles et al., 1998), kidney (Chakrabarti et al., 1989), gut (Young et al., 1995), gonads and sexual function (McVary et al., 1997), bone metabolism (Verhaeghe et al., 1990), vascular endothelium (Lindsay et al., 1997), and autonomic and peripheral nerves (Mohseni and Hildebrand, 1998; Sima and Sugimoto, 1999) have been reported. An advantage of the BB rat in the study of complications is its expression of aldose reductase, which is present at only low levels in the NOD mouse (Yagihashi et al., 1990). BB rats have also been used to model the effects of IDDM on pregnancy (Lea et al., 1996). Thymic Epithelial Defects Given the immunological defects involved in the development of autoimmune diabetes in BB rats, the thymus has been a major focus of study. The expression class II MHC antigen by BB rat thymic epithelial cells is abnormal. In both diabetes prone and diabetes resistant BB/Wor rats, areas of the thymic cortex lack class II MHC expressing epithelial cells (Rozing et al., 1989), and some regions of thymic cortex and medulla lack any thymic epithelium (Doukas et al., 1994). Thymic epithelial defects first appear at 4 weeks of age. The genetic predisposition to thymic epithelial defects in the BB/Wor segregates as an autosomal dominant trait (Doukas et al., 1994), but does not segregate with the diabetes phenotype (DLG, JPM, unpublished observations). HUMORAL IMMUNITY IN THE BB RAT Several different autoantibodies can be detected in the serum of BB rats (Crisá et al., 1992). As is the case with human IDDM, the degree to which abnormal humoral immunity actually contributes to the pathogenic process is unclear. Antibodies directed against the surface of islet cells (ICSA) appear 1–2 months before diabetes onset. The majority are capable of complement mediated cytolytic activity against pancreatic islets in vitro, but no direct pathogenic role of these islet-specific antibodies has been demonstrated in vivo. They may be secondary to the destruction of islet cells. Islet cell cytoplasmic antibodies (ICA) have not been conclusively demonstrated in BB rats (Crisá et al., 1992). DP-BB rats circulate autoantibodies reactive against lymphocytes, gastric parietal cells, smooth * BBBE/Wor//Brm rats are no longer available.

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AUTOIMMUNE DIABETES MELLITUS IN THE BB RAT

muscle and thyroid colloid, but not adrenal tissue. Anti-lymphocyte antibodies can be present before diabetes onset and their presence predicts the development of spontaneous diabetes in Ottawa BBdp rats (Bertrand et al., 1994). Anti-endothelial cell autoantibodies that appear to be capable of inducing endothelial leakiness are detectable in both untreated DP-BB/ Wor rats and RT6-depleted DR-BB/Wor rats before diabetes onset (Doukas et al., 1996). Autoantibodies important in the prediction of human IDDM may or may not have counterparts in BB rats. Anti-glutamic acid decarboxylase (GAD) antibodies are reportedly absent in BB/d (Davenport et al., 1995) and other BB rats (Mackay et al., 1996), but may be present in the BB/OK strain (Ziegler et al., 1994). Another human IDDM autoantibody, IA-2, is either absent (DeSilva et al., 1996) or present only at low titer (Myers et al., 1998) in BB rats. BB rat autoantibodies against heat shock protein 65 (hsp-65) (Mackay et al., 1996) are absent. Insulin autoantibodies (IAA) have been reported in BB/W/D rats (Wilkin et al., 1986), but their presence could not be confirmed in DP or DR-BB/Wor rats (Markholst et al., 1990). The possibility that age or strain specific effects could account for the different results remains open. The pathogenic significance of these autoantibodies and the B lymphocytes secreting them is not clear. An expanded population of CD5+ B cells may be present in DP-BB rats (Shockett and Woodland, 1988). A phenotypically similar CD5+ B cell population appears early in ontogeny in normal humans and secretes antibodies with a pattern of cross-reactive idiotypes directed against some of the autoantigens listed above (Casali and Notkins, 1989). An immunoregulatory role early in ontogeny has been postulated for this subset

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Figure 1 The teeter-totter or equilibrium hypothesis of IDDM expression. Depicted are the diabetes prone and the diabetes resistant BB rats, together with the perturbants known to alter the balance between autoreactive (A) and regulatory (R) cells. Also indicated are the genetic loci associated with the DP and DR strains.

of B-1 lymphocytes (Casali and Notkins, 1989). In the rat, however, the presence of a phenotypically distinct B-1 subpopulation is disputed (Vermeer et al., 1994). An alternative hypothesis is that the pattern of islet specific autoantibodies in the BB rat is due to a defect in antibody response to T cell-dependent but not to T-independent antigens. CELLULAR AUTOIMMUNITY IN THE BB RAT: A MATTER OF IMMUNOLOGICAL BALANCE We have previously conceptualized the etiology of autoimmunity in general, and IDDM in particular, using the analogy of a teeter-totter (Mordes et al., 1996). Our working hypothesis holds that the expression of diabetes depends on the relative balance between autoreactive (TA) cells and “regulatory” (TR) cells that should normally prevent beta cell destruction (Figure 1). The data supporting this concept come principally from studies of the BB/Wor rat and provide a framework for our review of the immunology of these animals. The top half of Figure 1 depicts the absence of self-tolerance, organ destruction, and the development of autoimmune diabetes mellitus. On the left is shown the spontaneously diabetic BB rat. The presence of autoreactive cell populations is inferred from the observation that spleen cells from such animals are capable of the adoptive transfer of the disorder (Koevary et al., 1983; Crisá et al., 1992). The absence of a regulatory cell population that might hold such effector populations in check in these lymphopenic animals

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was first inferred from experiments showing that lymphocyte transfusions prevent disease in lymphopenic, RT6-deficient DP rats if RT6+ donor T cells become engrafted (Rossini et al., 1986; Burstein et al., 1989). This is depicted schematically in the bottom left of the figure as the restoration of a protective equilibrium. The other line of evidence supporting this hypothesis come from diabetes resistant BB rats, depicted on the right of the figure. Although they never develop spontaneous disease, adoptive transfer experiments show that they nonetheless harbor autoreactive cells capable of recognizing and destroying normal beta cells (Greiner et al., 1987; McKeever et al., 1990). The hypothesis, depicted in the lower part of the figure, that they are protected from diabetes by the presence of a regulatory population, has been confirmed by demonstrating that >50% of young non-VAF DR-BB/Wor rats become diabetic if they are depleted in vivo of RT6+ T cells (Greiner et al., 1987). This effect is readily observed in conventionally housed DR rats that have been exposed to common rat viruses, whereas viral antibody free (VAF) DR rats require coadministration of an immune system activator such as polyinosinic-polycytidylic acid (poly I:C) (Thomas et al., 1991; Guberski et al., 1991). Cyclophosphamide and low dose irradiation can also precipitate diabetes in this animal (Crisá et al., 1992). CELLULAR ONTOGENY IN THE BB RAT The Lymphopenia of Spontaneously Diabetic BB Rats It has been recognized for some time that the peripheral lymphoid system in DPBB rats exhibits phenotypic and functional abnormalities (Crisá et al., 1992). The most striking is profound T cell lymphopenia characterized by severe reduction in the number of CD4+ T cells and nearly complete absence of the CD8+ T cell subset. As detailed below, the phenotypic T cell abnormalities in DP rats can be attributed to severely reduced life span of peripheral T cells. The first signs of lymphopenia can be detected in the thymus (Groen et al., 1996; Plamondon et al., 1990). The lymphopenia of BB rats is inherited as an autosomal recessive trait that is discussed in the section on Genetics, page 25. Thymocyte Development in the BB Rat The importance of intrathymic events in BB rat diabetes was recognized in very early thymectomy studies (Like et al., 1982). Subsequent experiments demonstrated that thymocytes from DR-BB/Wor rats can adoptively transfer diabetes to athymic recipients (Whalen et al., 1995). These observations demonstrate that the DR-BB rat thymus harbors abnormal cell populations predisposed to autoreactivity and localize the developmental defect leading to diabetes to an abnormal intrathymic selection process. Studies involving bone marrow and thymus transplantation document that prothymocytes in DP rats have an intrinsic defect leading to the lymphopenia, abnormal phenotypic distribution, and impaired proliferative capacity of T cells (Crisá et al., 1992). Other studies have demonstrated that bone marrow derived thymic antigen presenting cells (APCs) play an additional role in the development of the T lymphopenia (Georgiou et al., 1988; Georgiou and Bellgrau, 1989). Early studies on DP thymi had revealed no major differences between DP-BB, DR-BB, and normal rats with respect to absolute numbers of thymocytes and to the expression and distribution of T cell markers in the thymus. However, DP thymi show a 50% reduction in the numbers of mature thymocytes as compared to control rats in the TcRhiCD4−CD8+ population (Plamondon et al., 1990). This observation has been extended by showing that the TcRhi CD4+ CD8+ precursors of these thymocytes are reduced in absolute number (Groen et al, 1996). The density of cell surface CD8 is slightly decreased on DP rat thymocytes

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(Groen et al, 1996). Speculatively, these observations may relate to altered cytokine expression in the thymi of DP-BB rats (Bieg et al., 1997). These findings could have implications for the efficiency of positive and negative selection and therefore for the generation of autoreactive T cells in DP rats (Groen et al., 1996; Bellgrau and Lagarde, 1990). Another abnormality observed in both DR-BB and DP-BB thymi is a marked reduction in numbers of medullary B lymphocytes (Tullin et al., 1997). Since B lymphocytes play an important role in thymic negative selection, this defect may contribute to the generation of autoreactivity in DP and DR rats (Tullin et al., 1997). A final defect may be a reduction of purine nucleoside phosphorylase (PNP) in the thymus of certain BB rats, but this observation has not been pursued (Wu and Marliss, 1991). BB Rat T Cell Maturation In the rat, T cell maturation can be traced by expression of CD90 (Thy-1), RT6, and CD45RC. CD90 is expressed on stem and progenitor bone marrow cells, thymocytes, and immature peripheral T and B cells (Thiele et al., 1987; Powrie and Mason, 1990; Ritter et al, 1978). CD90 expression is lost during the early stages of post-thymic T cell development (Kampinga et al, 1997; Hosseinzadeh and Goldschneider, 1993). RT6 is a post-thymic T cell alloantigen expressed by the majority of mature peripheral T cells (Bortell et al., 1999). CD45RC is expressed by B cells, ~75% of CD4+ T cells, and most CD8+ T cells (Powrie and Mason, 1990). Recent thymic emigrants (RTE) in the rat are characterized by expression of CD90 and the absence of RT6 and CD45RC (Kampinga et al., 1997; Hosseinzadeh and Goldschneider, 1993). RTEs become CD90−RT6+ CD45RC+ after 7–11 days in the periphery. In the DP-BB rat, the percentage of CD90+ T cells is significantly increased (Groen et al., 1995). In contrast to normal rat strains (Groen et al., 1993),