CHRONIC COMPLICATIONS IN 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 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.
CHRONIC COMPLICATIONS IN DIABETES Animal Models and Chronic Complications Edited by
Anders A.F.Sima Departments of Pathology and Neurology Wayne State University Detroit, USA
harwood academic publishers Australia • Canada • France • Germany • India • Japan • Luxembourg Australia • Canada • France • Germany 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 © 2000 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 A catalogue record for this book is available from the British Library. ISBN 0-203-30380-6 Master e-book ISBN
ISBN 0-203-34320-4 (Adobe eReader Format) ISBN: 90-5702-433-0 (Print Edition) ISSN: 1029-841X Cover Illustration: Trypsin digested retinal preparation from an alloxan diabetic dog showing saccular microaneurysm (Courtesy Dr T.S.Kern, Department of Ophthalmology, University of Wisconsin, Madison).
CONTENTS
Preface to the Series
vii
Preface
viii
Contributors
ix
1
Diabetic Animals for Research into the Complications: A General Overview Eleazar Shafrir and Anders A.F.Sima
1
2
Studies in Animal Models on the Role of Glycation and Advanced Glycation End-Products (AGEs) in the Pathogenesis of Diabetic Complications: Pitfalls and Limitations Jesús R.Requena and John W.Baynes
45
3
Utility of the Transgenic Mouse in Diabetes Research Soroku Yagihashi, Shin-Ichiro Yamagishi and Chihiro Nishimura
73
4
Oxidative Stress and Abnormal Lipid Metabolism in Diabetic Complications Norman E.Cameron and Mary A.Cotter
99
5
Diabetic Neuropathy in Various Animal Models Ashutosh K.Sharma and Penelope A.Ricbards
133
6
Neurotrophism in Diabetic Neuropathy David R.Tomlinson and Paul Fernyhough
169
7
Diabetes Mellitus: Evidence for Altered Calcium Signaling in Excitable Tissues Karen E.Hall and john W.Wiley
185
8
Experimental Diabetic Nephropathy Mark E.Cooper, Richard E.Gilbert, Darren J.Kelly and Terri J.Allen
203
9
Diabetic Retinopathy in Experimental Animal Models and their Feasibility for Understanding the Human Disease Subrata Chakrabarti
229
Fetal Malformations in Diabetes Ulf J.Eriksson
251
Index
271
10
vi
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-todate 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
The material contained in this first volume of Frontiers in Animal Diabetes Research consists of a series of review chapters by internationally renowned investigators. The chapter by Yagihashi and co-workers deals with the utility of transgenic mouse strains modelling specific potential defects underlying the genesis of diabetic syndromes as well as the chronic complications occurring in both type I and type II diabetes. Induced and spontaneous animal models which have contributed in a major way to our present understanding of the pathogenesis and the natural history of the chronic complications are reviewed by Sharma and Richards. Specific metabolic perturbations contributing to the development of the dreaded complications such as abnormal lipid metabolism and oxidative stress as well as the role of abnormal Ca+-metabolism are extensively reviewed. Requena and Baynes have contributed a critical review of the pathophysiological role of glycation end products in diabetic complications. The role of perturbed neurotrophism in diabetic neuropathy and potential therapeutic implementations are extensively reviewed by Tomlinson and his group. Specific complication such as neuropathy, retinopathy and nephropathy are expertly dealt with in detail. A seldom considered complication, namely fetal malformations in the offspring of diabetic mothers is reviewed in detail by Eriksson. In this volume a determined effort was made to provide detailed reviews of the diverse but often interrelated pathogenic mechanisms involved in the chronic complications of diabetes as well as their functional and structural consequences in the target organs. These are accompanied by extensive lists of up-to-date references. Reflected in these reviews is the enormous contribution animal diabetes research has made toward a better understanding of the pathogenesis of diabetic complications. Only by a more complete understanding of the underlying mechanisms will we be able to design targeted and biologically meaningful therapies to curtail and/ or prevent the feared complications of diabetes. I am therefore bopeful that students, trainees and fellows, as well as clinicians and investigators interested in the complexities of diabetes and it’s complications will find this volume informative and useful. Anders A.F.Sima Detroit, October 1998
CONTRIBUTORS
Terri J.Allen Department of Medicine University of Melbourne Austin & Repatriation Medical Centre Repatriation Campus Heidelberg West 3081 Australia John W.Baynes Department of Chemistry and Biochemistry University of South Carolina Columbia SC 29208 USA Norman E.Cameron Department of Biomedical Sciences University of Aberdeen Marischal College Aberdeen AB9 1AS Scotland Subrata Chakrabarti Department of Pathology Dental Sciences Building University of Western Ontario London, Ontario Canada N6A 5C1 Mark E.Cooper Department of Medicine University of Melbourne Austin & Repatriation Medical Centre Repatriation Campus Heidelberg West 3081 Australia Mary A.Cotter Department of Biomedical Sciences University of Aberdeen Marischal College Aberdeen AB9 1AS Scotland Ulf J.Eriksson Department of Medical Cell Biology Uppsala University Biomedical Center PO Box 571 S-751 23 Uppsala Sweden Paul Fernyhough Department of Pharmacology Queen Mary and Westfield College Mile End Road London El 4NS UK Richard E.Gilbert Department of Medicine University of Melbourne Austin & Repatriation Medical Centre Repatriation Campus Heidelberg West 3081 Australia Karen E.Hall Department of Internal Medicine VA Medical Center GRECC 11G, D318 Ann Arbor MI 48105 USA Darren Kelly Department of Medicine University of Melbourne Austin & Repatriation Medical Centre Repatriation Campus Heidelberg West 3081 Australia Chihiro Nishimura Department of Pharmacology Kyoto Prefectural Medical University Kyoto Japan Jesús R.Requena Department of Chemistry and Biochemistry University of South Carolina Columbia SC 29208 USA Penelope A.Richards Department of Anatomy Faculty of Medicine University of Pretoria Pretoria 0001 South Africa Eleazar Shafrir Department of Biochemistry Hadassah University Hospital Kiryat Hadassah PO Box 1200 IL-91120 Jerusalem Israel
x
Ashutosh K.Sharma Department of Human Anatomy Faculty of Medicine and Health Sciences United Arab Emirates University 33521 Jameah EM PO Box 17666 Al Ain United Arab Emirates Anders A.F.Sima Departments of Pathology and Neurology Wayne State University 540 E. Canfield Avenue Detroit MI 48201 USA David R.Tomlinson Department of Pharmacology Queen Mary and Westfield College Mile End Road London El 4NS UK John W.Wiley Department of Internal Medicine VA Medical Center GRECC 11G, D318 Ann Arbor MI 48105 USA Soroku Yagihashi Department of Pathology Hirosaki University School of Medicine 5 Zaifu-Cho Hirosaki Japan Shin-Ichiro Yamagishi Department of Pathology Hirosaki University School of Medicine 5 Zaifu-Cho Hirosaki Japan
1. DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATIONS: A GENERAL OVERVIEW ELEAZAR SHAFRIR and ANDERS A.F.SIMA Department of Biochemistry, Hadassah University Hospital, Jerusalem, Israel Departments of Pathology and Neurology, Wayne State University, Detroit, MI, USA
The renal, ocular, neural, and other complications in animal diabetes, whether caused by cytotoxic agents, spontaneous autoimmunity, or other etiopathologies, show similarities among the species in their development and histological appearance. Hyperglycemia has now been established as the major culprit of these complications and renal, ocular and nervous tissues are especially prone to diabetic lesions, both in humans and animals, since they are freely glucose penetrable. There are differences in susceptibility to complications in various species, predisposition of cellular sites and differences in the time-course of development of changes. Lesions in animals, though not identical, resemble those of humans, since they share the hyperglycemia-linked causation particularly in the initial stages of their development. The background of a similar pathogenic milieu provides excellent opportunities for a study of the mechanisms, which are most probably common to animals and humans. Extensive information on diabetes induced lesions in various animals has been surveyed in the past (Salans and Graham, 1982; Shafrir and Renold, 1984; Shafrir and Renold, 1988; Velasquez et al., 1990; Winegrad, 1987; Robison and Laver, 1993; Sima et al., 1992). We shall present here an updated review on the lesions in animals with cytotoxic and spontaneous diabetes. NEPHROPATHY Early abnormalities observable in streptozotocin (STZ)- or alloxan-diabetic animals are kidney hypertrophy, increase in glomerular filtration rate (GFR) and filtration surface (Carney et al., 1979; Seyer-Hansen et al., 1980; Gotzsche et al., 1981; Zatz et al., 1985). Proteinuria may be present at this stage, even before the increase in glomerular basement membrane (GBM) thickness. STZ-diabetic rats demonstrate an increase in GFR, single nephron GFR, total renal plasma flow and single nephron plasma flow, consistent with findings in human nephropathy. Micropuncture studies in such animals show that the increases in single nephron GFR are due to the combination of augmented plasma flow with a rise in the glomerular transcapillary pressure. Apart from increased GFR, it is glomerular hypertension which is most injurious to the glomerular microcirculation (Zatz et al., 1985). Corresponding author: Eleazar Shafrir, Ph.D., Department of Biochemistry, Hadassah University Hospital, Kiryat Hadassah, POB 1200, IL-91120 Jerusalem, Israel.
2 E.SHAFRIR AND A.A.F.SIMA
Various mechanisms have been proposed to account for hyperfiltration in diabetic rats: increased expression of angiotensinase which stimulates arterioral and mesangial smooth muscle contraction increasing the intraglomerular pressure (Mathis and Banks, 1996; Thaiss et al., 1996), growth factor excess (IGF and TGFȕ) (Nakamura et al., 1993; New et al., 1996) the mitogenic activity of which promotes the growth of mesangium and smooth muscle, cellular hypertrophy and mesangial collagen synthesis; increased renal glucose metabolism (Scholey and Meyer, 1989) resulting in enhanced oxygen consumption, which per se contributes to hyperperfusion (Korner et al., 1994) and directs a substantial proportion of glucose to the aldose reductase (AR) initiated polyol pathway (Goldfarb et al., 1988; Ghahary et al., 1989; Cohen, 1986), enhanced renal kallikrein activity (Jaffa et al., 1995), augmented production of vasodilatory prostaglandins (Schambelan et al., 1985), reduced expression of nitric oxide synthase in macula densa (Yagihashi et al., 1996), and increased presence of atrial natriuretic peptide (ANP) in the circulation (Ortola et al., 1987; Kaneko, et al., 1987). Although the contribution of each of these factors to hyperfiltration and glomerulopathy has been demonstrated, it is uncertain from the available animal studies which of them is the main culprit of the failure in kidney function (Allen et al., 1990). In early studies of STZ-diabetic rats, the polyol pathway has been linked to the decrease in renal ATPase activity (Goldfarb et al., 1988). It has been postulated that hyperglycemia interferes with the maintanance of the intracellular pool of myoinositol, the precursor of diacylglycerol (DAG), a cofactor for the activation of ATPase by protein kinase C (PKC). Myoinositol depletion is related to competition with glucose for entry into the cell (Guzman and Crews, 1992; Haneda et al., 1990). In several tissues of diabetic rats, including glomerular preparations, a decreased ATPase activity has been reported, associated with low tissue myoinositol levels (Goldfarb et al., 1988; Cohen et al., 1985), which can be prevented by the administration of AR inhibitors or by strict control of hyperglycemia. Low ATPase activity is supposed to affect the glomerular function by altering the intracellular calcium homeostasis, impairing the Na+ gradient and changing the membrane potential. AR inhibition or myoinositol supplementation lower hyperfiltration, prevent glomerular polyol accumulation, diminish the proteinuria and inhibit glomerular changes (Goldfarb et al., 1988; Mauer et al., 1989; Tilton et al., 1989; Cole et al., 1995; Daniels and Hostetter, 1989; Beyer-Mears et al., 1984). However, other experiments with myoinositol supplementation produced different results. Hyperfiltration was not corrected and was not correlated with ATPase activity changes, creatinine clearance and glomerular presssure were not improved (Chen, 1993; Cohen et al., 1990; Pugliese et al., 1990; Wald et al, 1993). Similar findings were also reported in the sucrose-induced, insulin resistant Cohen diabetic rat (Cohen et al., 1995). It is also pertinent that transgenic mice, overexpressing human AR, develop histopathological renal changes, mainly fibrinous deposits in the Bowman capsule, without overt effect on hyperfiltration (Yamaoka et al., 1995). It may be concluded, therefore, that the nephropathy resulting from hyperglycemia is mediated by mechanisms additonal to the lack of myoinositol and
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 3
low ATPase activity. It is most plausible that the hyperglycemia-related lesion is distal to the polyol pathway and caused by glycation of the glomerular and tubular components. This may include oxidative stress related to glycooxidation products and reactive oxygen radicals produced during the persistent hyperglycemia (Baynes, 1991; Ha et al., 1994) as well as detrimental interactions of glomerular components with end products of advanced glycation, as reviewed by Brownlee (Brownlee, 1994). Comparison of Human and Animal Morphological Kidney Changes in Diabetes GBM thickening and mesangial expansion, diffuse and nodular Kimmelstiel-Wilson glomerusclerosis, fibrin cap and capsular drop lesions (or better termed exudative lesions) are the hallmarks of human diabetic glomerulopathy. Similar lesions are seen in several diabetic animal species. Diabetic animals do not develop all of these lesions, and those present are not histologically identical to human lesions. However, research into the causative factors underlying their appearance, structure and prevention by therapeutic modalities are likely to produce useful information for the elucidation of mechanisms of their development. In glomeruli, the deposition of basement material in the mesangial matrix starts focally, but unlike human Kimmelstiel-Wilson nodules, it continues in various diabetic rodents in a diffuse fashion, gradually filling the capillary lumen and diminishing the GFR (Osterby and Gundersen, 1980; Osterby, 1988; Bendayan, 1985). Nodular deposits are seen in alloxan-diabetic dogs (Engerman and Kramer, 1982) and in type 2 diabetic monkeys (Kopp et al., 1990), probably because their longer life span provides sufficient time for the formation of hyperglycemia induced changes. Analogous to human nephropathy, additional lesions are noted in prolonged diabetes: deposition of immunoglobulins and complement in the enlarged, hyperplastic mesangium, glomerular aneurysms and adhesions, multiple tubular alterations with glycogen accumulation (Rasch and Holck, 1988). The deposition of IgA and IgG complexes in the kidney of STZ-diabetic rats occurs in correlation with the increase in plasma immunoglobulin levels and may contribute to the pathogenesis of focal segmental glomerulosclerosis (Sabatino and Val Liew, 1997). Hyaline deposits, seen in human renal lesions are infrequent in diabetic rodents. Mesangial expansion and GBM thickening occur both in diabetic humans and animals. Synthesis of various mesangial and glomerular components is considerably enhanced (Fukui et al., 1993; Yoshida et al., 1995). Several metabolic alterations may be contributory. High glucose levels alone inhibit the degradation of certain mesangial components in cell cultures, attributed to PKC activity (McLennan et al., 1994), and to the reduced content of heparan sulfate proteoglycan (Olgemoller et al., 1992). Decreased sulfated glomerular glycosaminoglycans are found in STZdiabetic rats and are linked to increased synthesis of hyaluronan acid derivatives (Mahadevan et al., 1995). On the other hand, high glucose concentration in glomerular cell cultures increases the synthesis of fibronectin, laminin and other collagen types (Pugliese et al., 1994; Wakisaka et al., 1994; Nahman et al., 1992; Cagliero et al., 1991). Earlier, Brownlee and Spiro (Brownlee and Spiro, 1979) reported that the synthesis of collagen is accelerated through GBM polypeptide
4 E.SHAFRIR AND A.A.F.SIMA
synthesis and proline hydroxylation. Alterations in glomerular proteoglycan metabolism show a different pattern in the hyperinsulinemic Zucker rat at the initial stage of nephropathy. Radiosulfate incorporation into proteoglycan is increased but the proportion of releasable sulfated proteoglycan is unchanged, suggesting an increased electronegativity (Fioretto et al., 1993). The activity of several glycosidase and glucosyltransferase enzymes related to GBM proteoglycan breakdown as well as proteases (Reckelhoff et al., 1993; Shankland et al., 1996) is decreased in diabetic mice, rats and hamsters (Fushimi and Tarui, 1976; Fushimi and Tarui, 1976; Fushimi et al., 1980; Draeger et al., 1984; Haft and Reddi, 1979; Reddi et al., 1979; Chang, 1979). Abnormal enzymuria of glucoaminidase, alanine aminopeptidase and Ȗ-glutamyl transpeptidase is induced by infusion of glucose to nondiabetic Wistar rats (Ishii et al., 1995). It was also observed that autophagic degradation of cytoplasmic components in proximal tubular cells was inhibited independently of the growth stimuli induced by unilateral nephrectomy or STZ-diabetes (de Almeida Barbosa et al., 1992). All these “anticatabolic” changes may contribute to the expansion of the mesangial components by retarding their metabolic breakdown. Proteinuria is an additional hallmark of human nephropathy and is evident in most diabetic animals, although not as abundant as in humans. The major sieving defect may be due to loss of selective macromolecular size permeability by the glomerular capillary (Michel et al., 1982), possibly related to diminished heparan sulfate synthesis (Rohrbach, 1986), resulting in reduced GBM barrier density and ionic charge (Van den Born et al., 1995). High salt concentration was also shown to be deleterious to selective permeability in hypertensive rats (Hertzan-Levy et al., 1997). Ultrastructural changes in the lamina densa are consistent with enlarged porosity (Inoue and Bendayan, 1995). One may speculate that the stimulus for growth and thickening of the GBM is restoration of normal permeability. In humans, glycated albumin is preferentially transported across the glomerular capillary (Ghiggeri et al., 1985). Likewise, protein glycation has been shown to confer an increased permeability across the rat glomerulus (Williams and Siegal, 1985; Hauser et al., 1990). These parallel findings result from enhanced glycation of plasma proteins in diabetic humans and animals alike. Perhaps the most impressive similarity between effects on renal function in human and experimental diabetes is the reponse to theraputic treatments. This should not be surprising since many therapeutic approaches have been first tested in animal models. Treatment of diabetes by islet transplantation, may not be fully corrective (Bretzel et al., 1984; Steffes et al., 1980; Bretzel et al., 1979). Strict control of glycemia by exogenous insulin normalizes renal hemodynamic deficits (Stackhouse et al., 1990; Diabetes Control and Complications Research Group, 1993), minimizes proteinuria and often restrains the GBM thickening as demonstrated in the extensive studies of Rasch (Rasch, 1979; Rasch, 1979; Rasch, 1979; Rasch and Dorup, 1997). Insulin also prevents the changes in heparan sulfate proteoglycan species occurring during incubation of glomerular cells in high glucose mediium (Kasinath, 1995). Further, it is of note that maintenance of STZ-diabetic rats on a diet low in carbohydrate and high in protein for a period of 1 year resulted in amelioration of proteinuria and prevented kidney glycogen deposition (Schmidt et al., 1984). Another successful treatment in diabetic humans and animals is inhibition of the angiotensin converting enzyme (Whitty and Jackson, 1988; Nakamura et al., 1995;
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 5
Cooper et al., 1989), retarding both GBM thickening and proteinuria and decreasing the synthesis of extracellular matrix components. Aminoguanidine or similar molecules may attenuate the advanced glycation in human patients, since they are effective in diabetic rats (Oxlund and Andreassen, 1992; Soulis et al., 1997). Nephropathy in Different Animal Species with Diabetes Models of type 1 diabetes BB/W rats develop spontaneous diabetes due to insulitis and destruction of pancreatic ȕ-cells due to autoimmune assault culminating at 2 to 3 months of age. Renal abnormalities, mainly increased GFR and renal blood flow appear early, 1 to 3 months after the onset of diabetes corresponding to the extent of hyperglycemia (Brown et al., 1983; Cohen et al., 1987). The particular feature of BB rats is that they do develop only mild proteinuria and moderate immunoglobulin and complement deposits without mesangial expansion. Thickening of GBM is associated with minimal mesangial or other glomerular structural abnormalities. NOD mice lose their insulin secretion capacity as a result of autoimmune ȕ-cell destruction between 3 and 6 months of age (Tochino, 1984). Insulitis and diabetes is much more severe in females than males and accompanied by early proteinuria. However, surprisingly, males show more extensive proteinuria than females at 4 to 5 months of age (Tochino et al., 1983). Apart from GBM thickening, deposition of GBM-like components in glomeruli, and immunoglobulin accumulation in the mesangium, renal abnormalities are mild. NON mice are genetically similar to NOD mice but without autoimmune etiology (Tochino, 1984). They are not really insulin deficient, but are mentioned here because of their propensity to develop glomerular pathology and proteinuria. This occurs particularly in males despite absence of hyperglycemia except for abnormal glucose tolerance and low pancreatic insulin reserve. Protein excretion is massive and may reach 1 g/day. Glomerulopathy is of nodular type with deposits of PASpositive and GBM-like materials under endothelial cells of glomerular capillaries. Lipid deposition in the glomerular lumina has also been reported (Watanabe et al., 1991). Chinese hamsters (Cricetulus griseus) lose almost completely their insulin secretion capacity and ȕ-cells early in life probably because of inability to cope with the nutritional overload in captivity. They have been inbred and intensively studied in the past as recently reviewed by Frankel (Frankel, 1996). Renal abnormalities involve moderate mesangial enlargement and GBM thickening, more diffuse than nodular, and proteinuria that is not well correlated with the severity or duration of diabetes (Shirai et al., 1967; Soret et al., 1974). Only ketonuric hamsters show glycogen and PAS positive material deposited at various sites including Bowman’s capsule and tubules. No arteriolar hyalinization or exudative lesions are seen. High levels of sorbitol, fructose and glucose are noted in the kidney, and are not diminished by treatment with an AR inhibitor (Sekiguchi et al., 1991).
6 E.SHAFRIR AND A.A.F.SIMA
Hyperinsulinemic and insulin resistant animal species db/db mice on the Ks background are derived from the Jackson Laboratory in Bar Harbor, ME. They are initially spontaneously hyperinsulinemic and obese, but after a few months their pancreatic function declines, associated with pronounced hyperglycemia, weight loss, severe nephropathy (Gartner, 1978; Like et al., 1972; Bower et al., 1980) and early death. The ob/ob mice on the BL6J background, renown for their extreme obesity, show longstanding hyperinsulinemia and moderate hyperglycemia, but do not exhibit significant kidney lesions, except for occasional hyaline deposits within capillary walls (Bailey et al., 1985). In db/db mice, in contrast, the GFR is elevated at onset of diabetes and is followed by renomegaly, progressive glomerulosclerosis and proteinuria (Meade et al., 1981; Lee, 1984; Wehner et al., 1972). Kidney lesions include both diffuse and nodular enlargements of the mesangium and GBM, nodular deposits in the basal lamina, mesangial proliferation, hyaline exudative lesions, accumulation of collagen fibrils and immunoglobulins and vacuoli in the mesangium and tubuli. It is remarkable that these changes occur despite the scarcity of AR and virtual absence of the polyol pathway in this species (Bianchi et al., 1990). This model may therefore be useful for studying kidney lesions as consequences of hyperglycemia distal to the polyol pathway. Various treatments have ben used to prevent or slow down the progression of diabetes in db/db mice. Diet restriction to match the body weight with nondiabetic siblings, feeding a diet with 20% nonabsorbable fiber, administration of a FFA oxidation inhibitor 2tetraglycidate, maintenance on the intestinal glucosidase inhibitor acarbose, all resulted in amelioration of db/db nephropathy (Lee, 1984). The common denominator of these treatments was reduction in hyperglycemia. A carbohydratefree high protein diet in diabetic db/db mice reduced the glycemia, prolonged ȕ-cell survival and prevented some of the glucose toxicity effects (Leiter et al., 1981; Leiter et al., 1983; Shafrir, 1988) without pronouncing the proteinuria. Zucker fa/fa rats are extremely obese, hyperlipidemic and insulin resistant but maintain their capacity of insulin secretion and hyperinsulinema during most of their life, with mild glucose intolerance only. The rat fa gene appears to be a homologue of the mouse db gene, however, the nephropathy in fa/fa rats differs considerably from that in db/db mice. Fa/fa rats develop spontanous focal segmental glomerulosclerosis, mesangial enlargement, loss of podocytes and endothelial obliteration of capillary lumina as well as accumulation of intraglomerular fibronectin (Kasiske et al., 1985; Lash et al., 1989; Paczek et al., 1991). Proteinuria and mesangial enlargement appear at 3 to 5 months of age and precede the development of focal segmental glomerulosclerosis. Interestingly, the GFR and filtration fraction remains similar to that in nonobese siblings. With age, the GFR and creatinine clearance decrease, the proteinuria and glomerulosclerosis worsen with evident uremia in fa/fa rats surviving for 12 months. Renal failure is the primary cause of death of fa/fa rats. Food restriction slows down the progression to glomerulosclerosis and renal insufficiency (Shimamura, 1982), whereas the angiotensin II receptor antagonist losartan reduces the hypertension but not the renal lesions (Crary et al., 1995). Estrogen treatment accelerated the renal disease in female fa/fa rats, when administered to ovariectomized animals. The renal injury appeared to be promoted not by a direct effect of the hormone but by the increase in triglyceride-rich proteins in the circulation (Gades et al., 1998).
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 7
KK mice develop obesity slowly, mild hyperinsulinemia and hyperglycemia peaking at 4–9 months and subsiding after 1 year. They are a series of substrains with polygenic inheritance raised in Japan (Ikeda, 1995) and were also investigated in the USA (Wyse and Dulin, 1974). Obesity and hyperglycemia are promoted by high energy feeding (Matsuo et al., 1971). Nephropathy in KK mice bears similarity to that in humans (Duhault et al., 1973; Reddi et al, 1978; Emoto et al., 1982). Glomeruli show both diffuse and nodal enlargement with proliferation of mesangial cells and peripheral GBM thickening, although the nodules are not identical in structure and distribution with the characteristic human Kimmelstiel Wilson nodules. Proteinuria increases with age. There is intense fluorescence of IgA and IgG in the mesangium, nodules and along the capillary wall. Exudative fibrinoid caps are also seen in glomeruli of > 1 year old KK mice but not in very old animals. Splitting of the GBM due to the deposition of newly formed basal membrane components is occasionally observed. Renal amyloidosis has been seen in KK mice and may be involved in the pathogenesis of glomerulosclerosis (Soret et al., 1977). The extent of the mesangial enlargement correlates with hyperglycemia, and becomes prominent on high energy diet and diminishes under food restriction. KKAy mice were reared in Japan by inserting the yellow agouti Ay obesity gene into KK mice. They show an early onset of severe and prolonged hyperglycemia and hyperinsulinemia with marked insulin resistance (Diani et al., 1987). Renal abnormalities appear already after 3 months with GBM thickening and proteinuria progressing with age. The contour of the GBM is smooth rather than nodular in contrast to KK mice and other diabetic animals. Than et al. (Than et al., 1992) reported that KKAy mice receiving an allogeneic bone marrow transplantation from normal BALBc mice exhibited morphological and functional recovery of both glomeruli and ȕ-cells. NZO mice are a polygenic model developed by selective inbreeding of a mixed colony, which included agouti mice, in New Zealand (Melez et al., 1980; Proietto and Larkins, 1993). NZO mice are obese, moderately hyperglycemic, hyperinsulinemic and insulin resistant with diabetes and obesity progressing with age. Nephropathy is seen at 6 months and consists of increased cellularity of glomerular tufts and mesangium, mild GBM thickening with some eosinophilic noduli, hyalinization of glomerular arterioles and arteriolar inflammation. There is also deposition of IgG in glomeruli. These changes occur in > 60% of NZO mice and are more pronounced in females. SHR obese rats were developed from SHR females and Kyoto Wistar males by Koletzky (Koletsky, 1973). These rats are hypertensive and hyperlipidemic. A proportion becomes hyperglycemic with pancreatic islet hyperplasia. Hypertension is exhibited early, prior to renal lesions. Proteinuria appears first, followed by glomerular, vascular and tubular damage and terminal uremia (Michaelis et al., 1986). Mesangial proliferation is associated with focal necrosis, collagen deposition and hyalinization. Capillary basement membrane thickening and arteriolar ncpbrosclerosis are prominent. In terminal stages, glomerular and vascular damage lead to glomerulo-sclerosis, interstitial fibrosis and tubular atrophy. WDF/Ta-fa rats are also referred to as Wistar fatty rats. They are genetically obese, hyperphagic and hyperglycemic, obtained by transfer of the fa gene to the Wistar Kyoto rat in Japan (Ikeda et al., 1981). The characteristics of males are similar to Zucker fa/fa rats, but they are less obese and more glucose intolerant and insulin
8 E.SHAFRIR AND A.A.F.SIMA
resistant. In females the hyperglycemia may be induced by feeding a sucrose-rich diet (Kava et al., 1989; Matsuo et al., 1984). Nephropathy is evident at 20 weeks of age with depressed GFR but marked proteinuria which increases dramatically with age (25x that of lean siblings) (Diani et al., 1988). Significant GBM thickening is seen at 12 weeks of age (Yagihashi et al., 1978). Renomegaly, glomerular hypertrophy, expansion of GBM area and thickness are marked at 5 months and peak at 10 months. SHR/N-cp rats are a congenic strain developed together with other corpulent cp rat strains at the NIH (Michaelis et al., 1986) by mating Koletzky males, heterozygous for the cp gene, with female SHR rats. followed by backcrossing. The cp/cp males exhibit obesity, mild hypertension, hyper-insulinemia and glucosuria (Michaelis et al., 1991). The predominant diabetic complication in these rats is early proteinuria and glomerulopathy, probably abetted by hypertension. Already at 5 months of age they show significant proteinuria and large kidneys with hypertrophied glomeruli and decreased GFR (Velasquez et al., 1989; Kimmel et al, 1992). Diffuse and segmental mesangial expansion and proliferation with nodular lesions and focal segmental glomerulosclerosis in some glomeruli are the prominent features. Thickening of GBM is seen in older rats. Males and females show similar morphological changes which are accentuated by, sucrose rich diet, particularly in males. SHHF/Mcc-cp rats may serve as a model of the human insulin resitance syndrome (syndrome X) (Reaven, 1991), since they exhibit a cluster of obesity, hyperlipidemia, glucose intolerance, and hypertension, predisposing to human cardiovascular disease. The SHHF/Mcc-cp rats, maintined by McCune and colleagues (McCune et al., 1995), have a genomic background for cardiomyopathy, presenting as congestive heart failure. Renal lesions also appear in these animals and are more pronounced in males than in females. They consist of diffuse intercapillary sclerosis and are similar to those of SHR/N-cp rats. They become severe after the onset of congestive heart failure. Interestingly, another strain derived from the cp group, the insulin resistant hyperlipidemic but not hypertensive LA-cp rat, maintained by Russel et al. (Russel, 1992), exhibits an ischemic, atherosclerotic rather than congestive cardiopathy without significant kidney changes. Cohen diabetic rats were developed from an albino rat strain of the Hebrew University in Jerusalem by genetic selection and inbreeding of individuals exhibiting low glucose tolerance on a 72% sucrose, copper-deficient diet. They are hyperglycemic, glucosuric, hyperinsulinemic and insulin resistant without weight gain. Nephropathy and proteinuria occur in up to 60% of Cohen rats already at 4 months of age. Nephropathy consists of renomegaly, diffuse glomerulosclerosis with acellular thickening of the mesangium and of peripheral GBM and segmental lipohyaline exudative changes resembling the human hyaline cap lesion, tubular atrophy and cystic dilatation (Rosenmann and Cohen, 1984). Arteriolar sclerosis is seen in some cases. Detailed description of their nephropathy and hormonal influences on its development have been provided in a monograph (Cohen and Rosenmann, 1990). GK rats, (Goto-Kakizaki rats), were inbred in Japan by selective repetetive mating of a normal rat strain exhibiting marginal glucose intolerance. A diabetic, inheritable pattern was obtained after 35 generations kept on a regular laboratory chow (Suzuki et al., 1992). GK rats may be defined as a polygenic overexpression of defective
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 9
factors related to metabolic-endocrine pathways, manifested particularly in pancreatic islets and kidneys. GK rats are nonobese and their impaired glucose tolerance does not proceed to marked hyperglycemia or ketosis. Their pancreatic islets are deformed and reduced in mass (Ostenson et al., 1996; Movassat et al., 1997). Glomerulopathy, evident by thickening of GBM, is first discernible at 3 months of age and can be reduced by an AR inhibitor (Goto et al., 1988). Impaired renal Vitamin D metabolism has been reported (Ishimura et al., 1995). Psammomys obesus (“sand rat”) is a desert gerbil, nondiabetic in its native habitat, but showing a genetic propensity to progression to diabetes when transferred to relatively affluent laboratory diet (Ziv and Shafrir, 1995). The progess to fully fledged diabetes involves stages of hyperglycemia and insulin resistance associated with moderate weight gain, terminating in loss of insulin secretion and insulin dependence. The kidneys of Psammomys, living in arid region, are sturdy and resilient being able to excrete urine salty like sea water. However, the diabetic state is accompanied by significant differences in sodium pump activity in the renal cortex and medulla. Differences in GFR are paralelled by changes in ATPase activity, independently of changes in plasma glucose and insulin levels Ziv, and Shafrir, 1995). In severely diabetic Psammomys, kidney tubules fill with glycogen-rich cytoplasmic vacuoles, but no glomerular or GBM abnormalities have been recorded (Ziv et al, 1998). Kidney changes have been observed in several other diabetic animals, which have either not been sufficiently investigated recently or the access to these models is rather difficult. They are mentioned here in brief. Spiny mice (Acomys cahirinus) are a desert rodent investigated in Geneva and Jerusalem, becoming obese and lapsing into ketotic diabetes at about 1 year of age (Gutzeit, 1979). GBM thickening and mesangial changes occur at this time along with glycogen infiltration of the tubular epithelial cells consisting of lysosome-like inclusions (Orci et al., 1970). GBM thickening and PAS-positive mesangial proliferation was found in the hyperglycemic white tailed rat Mystromys albicaudatus (Riley et al., 1975), in association with increased activity of glucosyl transferases, but decreased activity of sialyzation enzymes. Mystromys also shows pronounced thickening of muscle basement membrane (Schmidt et al., 1980). In the eSS rat with spontaneous late-onset diabetes, bred in Argentina, glucose intolerance is conspicuous in males at about 1 year of age in association with fibrotic islet lesions causing partial loss of ȕ-cells (Martinez et al., 1992). Kidneys exhibit focal, interstitial and pyelic inflammatory infiltrates. With age, the glomeruli show diffuse hypertrophy of the mesangium, thickening of GBM, reduction in capillary lumina proceeding to necrosis. Proteinuria starts at about 6 months and is pronounced at 1 year. In the Wbn/Kob rat, that shows spontaneous fibrotic lesion in both the endocrine and exocrine pancreas (Mori et al., 1990), the hyperglycemia is associated with weight loss and is not due to insulin resistance. These rats excrete substantial amounts of protein, show thickened GBM and enlarged mesangium. The BHE/cdb rat, developed at the USDA Bureau of Home Economics in the USA manifests mild diabetes at maturity (Berdanier, 1995). Renal disease is characterized by kidney enlargement and a variety of histological abnormalities such
10 E.SHAFRIR AND A.A.F.SIMA
as various grades of hyaline changes in glomeruli, Bowman’s capsule and proximal tubules accompanied by tubular hyperplasia and dilatation. The tuco tuco (Ctenomis talarus) is a roaming rodent in Argentinian arid regions (Wise et al., 1972) which exhibits mild hyperglycemia, hyperphagia and obesity when restrained in captivity. ȕ-cells tend to degranulate and accumulate “amyloid”, presumably islet amyloid polypeptide. Glomerular lesions and diffuse mesangial argyrophylia have been reported in tuco tuco along with GBM thickening and hyaline deposits in afferent arterioles. Tubular lesions or nodular glomerulosclerosis are not seen. A recently discovered NIDDM model in Japan, the OLETF rat, exhibits glomerular lesions which can be prevented by islet transplantation (Katsuragi et al., 1996). Spontaneously diabetic guinea pigs reveal diffuse GBM thickening, as well as focal expansion of the mesangial core of glomerular tufts with frequent scarring and fibrosis in the Bowman’s capsule (Munger et al., 1973). Among nonrodent mammals, mesangial enlargement and immunoglobulin deposits are present in the basement membrane of glomeruli and tubuli of several species of spontaneously diabetic dogs, reminiscent of human nephropathy (Jeraj et al., 1984). Maintenance of normoglycemia for 2.5 years, even after a period of 2.5 years of poor control arrested the progression of nephropathy, but did not reverse it (Kern and Engerman, 1990). The nonhuman primate Macaca fuscata, after 25 months of STZ-diabetes, shows thickening of glomerular and muscle basement membranes (Yasuda et al., 1984). Diffuse mesangial expansion is also observed in pancreatectomized or STZdiabetic rhesus monkeys and baboons after several years of diabetes duration without insulin treatment (Stout et al, 1986) and in spontaneously diabetic Macaca nigra (Howard, 1982). OCULAR COMPLICATIONS Retinopathy and cataracts are the major causes of human ocular pathology both in IDDM and NIDDM patients. Chakrabarti and Sima (1988) reviewed the lesions in animals and observed that they are mainly the consequence of hyperglycemia but occur with less frequency and different morphology. Most diabetic rodents do not display retinal microaneurysms, capillary occlusions, ‘cotton wool’ spots from nerve fiber infarcts and proliferation of blood vessels, which lead to vision loss by hemorrhage into the vitreous, or retinal detachment. This may be due to the relative brevity of their diabetic life span, rather than to pathophysiological differences. Alloxandiabetic dogs and spontaneously diabetic monkeys better reproduce human retinopathy (save for neovascularization), but these changes take several years of diabetes to develop. They exhibit microaneurysms, capillary basal membrane thickening, alternating acellular and hypercellular capillaries with pericyte-devoid ‘ghosts’. Cataract Lesions Cataracts occur in many diabetic animals, mostly in association with hyperglycemia. There is substantial evidence that the polyol pathway bears more resposibility for
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 11
the cataractous than the renal lesions both in humans and animals (Lerner et al., 1984; Kinoshita and Nishimura, 1988). Cataract was extensively investigated in diabetic dogs and rats (Schofleld and Gould, 1979; Kuwabara et al., 1969; Fukushi et al., 1980; Kinoshita et al., 1981). The common initiating mechanism appears to be the activation of AR and shunting of a substantial proportion of glucose to the polyol pathway, with intracellular accumulation of sorbitol, which cannot dififuse through the cell membrane. Its disposal is dependent on the conversion to fructose by the intracellular polyol dehydrogenase. In rats and dogs galactose was administered to elicit an early appearance of cataracts, since the affinity of AR for galactose is higher than for glucose (Sato et al., 1991) and galactitol accumulates more readily than sorbitol in the lens because of its slower metabolism. The first deleterious event is cell edema and swelling, due to the accumulation of polyol, regardless whether sorbitol or galactitol. An osmotic damage is caused, affecting the membrane function, especially its selective permeability, ensuing in the loss of potassium, amino acids and myoinositol and in the rise of cellular sodium and chloride. These changes may be prevented or reduced by inhibiting AR (Hayman and Kinoshita, 1965; Ao et al., 1991; Datile et al., 1982; Yeh et al., 1986; Unakar et al., 1989). Cell edema is the forerunner of the damage to the structural integrity of the lens (Kuwabara et al., 1969; von Sallman et al., 1958; Robison et al., 1990), starting at the anterior central region of the epithelial cell layer. This is inferred from the observation that initiation of caractogenesis occurs in the epithelial layer where AR is abundant (Hayman et al., 1966; Ludvigson and Sorenson, 1980). It is also consistent with the fact that epithelial cells are involved in the uptake and exchange of ions and metabolites of the lens. Next, hydropic lens fibers and vacuole-like opacities appear in the cortical region of the equatorial zone of the lens (Sakuragawa et al., 1975) and lenticular proliferation is observed (Grimes and von Sallmann, 1968). The fact that the first affected site is the lens epithelium rather than the equatorial cortex was recognized by the histological studies of Kinoshita and associates (Kuwabara et al., 1969; Robison et al., 1990) and confirmed later (Nagata et al., 1989). In addition to the polyol pathway, oxygen free radicals have been recently implicated in lens opacification (Delacruz et al., 1994; Ahmad et al., 1992; Kilic et al., 1994). Furthermore, strong evidence has been presented implicating the contribution of direct glycation and interaction of end products of advanced glycation with lens components in diabetic rats and dogs (Harding, 1994; Nagaraj et al., 1996; Perry et al., 1987; Yarat et al., 1995; Nakayama et al., 1993; Turk et al., 1997). In this respect, an accumulation of unusual metabolites has been discovered in lenses of aging and diabetic rats, including fructose-3-phosphate, sorbitol-3-phosphate and galactitol 2and 3-phosphates (Lal et al., 1995a; Lal et al., 1995b; Kappler et al., 1995). These metabolites are potent glycating agents and a probable source of 3deoxyglucosozone, which may importantly contribute to lens protein crosslinking and opacity. Moreover, an involvement of hyperlipidemia, usually associated with diabetic hyperglycemia, should not be overlooked. It has to be considered as a potential risk factor for diabetic cataracts, as their onset could be suppressed in rats by agents decreasing the plasma lipid concentration (Tsutsumi et al., 1996). Observations strongly supporting the role of the polyol pathway in the initiation of caractogenesis come from studies with transgenic mice overexpressing AR, which developesd cataracts and occlusion of retinocorneal vessels already after 7 days of
12 E.SHAFRIR AND A.A.F.SIMA
galactose-rich diet (Yamaoka et al., 1995). On the other hand, db/dbmice do not develop cataracts despite being consistently hyperglycemic, even when fed galactose (Kinoshita et al., 1979; Varma and Kinoshita, 1974). Mice, in general, are genetically characterized by scant AR activity in their lenses and in other tissues (see the nephropathy section). At the opposite end is degu (Octodon degus), a porcupine-like rodent of South American origin, which develops cataracts even on a regular laboratory chow (Varma et al., 1977; Varma, 1980) at blood glucose levels not exceeding 150 mg/dl. The degu exhibits a very high lenticular AR activity and sorbitol accumulation. The desert rodent Psammomys obesus (sand rat) also suffers from cataracts when maintaned on laboratory diet (Gutman et al., 1975) and even more if placed on high energy diet, displaying lenticular elevation of sorbitol. The cataracts in Psammomys develop already after 1 month on the high energy diet and are preceded by swelling and degeneration of cortical cells and formation of fibrous tissue around proliferating lens cells, events that mimic human caractogenesis (Kuwabara and Okisakaa, 1976; Zahnd and Adler, 1984). Cataracts have been also described in Mongolian gerbils (Aguizy et al., 1980) and in rhesus and Macaca nigra monkeys (Howard, 1982), generally in relation to the severity of hyperglycemia. Additional survey of animal models illusttrating different aspects of cataract pathogenesis have been recently published (Hockwin and Sasaki, 1994). Retinopathy Diabetic retinopathy poses a risk for blindness, greater than any other ocular complication. Retinopathy is mainly a vascular disease affecting the retinal capillary plexus (Cogan et al., 1961; de Venecia et al., 1976; Robison and Nagata, 1988). Because of the long latency, diabetic rats which manifest cataracts and keratopathy, show only initial lesions of retinopathy such as basement membrane thickening, intramural pericyte degeneration, endothelial cell proliferation and acellularity (Papachristodoulou et al., 1976; Little, 1983). One of the reasons for vascular proliferation appears to be the retinal hypoxia due to diminished oxygen supply by aggregated red cells and other rheological abnormalities (Boot-Handford and Heath, 1980). This may stimulate the compensatory production of angiogenic growth factors stimulating new vessel formation (Lowe et al., 1995). These include VEGF, ȕPGF and IGF-1. Their role in the pathogenesis of diabetic retinopathy has been reviewed (King et al., 1993; Miller et al., 1997; Pfeiffer et al., 1997). The basement membrane thickening was demostrated to be associated with enhanced collagen and fibronectin synthesis (Roy and Lorenzi, 1996). Also, early after the onset of rat diabetes the permeability of blood-retinal barrier to fluorescein is increased, with leakage into the vitreous, probably due to disruption of the retinal pigment epithelium (RPE) (MacGregor and Matshinsky, 1986; Tso et al., 1980; Kirber et al., 1980). A reduced ability of the retinal microvasculature to retain the permeation selectivity results in the leakage of small molecules such as fluorescein which is probaly one of the earliest derangements in the retinal circulation. These phenomena, analogous to incipient human retinopathy, are demonstrable in diabetic guinea pigs (Klein et al., 1980), BB rats (Sima et al., 1985; Blair et al., 1984) and rhesus macaques (Farnsworth et al., 1980; Jones et al., 1986). The BB rat develops pericyte and endothelial cell degeneration and, in addition, a specific loss of anionic
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 13
sites in the basement membrane which is at least in part responsible for the increased permeability abnormalities (Chakrabarti et al., 1991). Areas of capillary proliferation and depletion, occluded acellular capillaries and pericyte loss are seen, which seem to precede the thickening of basement membrane. Changes in the RPE and visual cells, as well as platelet thrombosis, have also been identified in rats (Leuenberger et al., 1981; Grimes and Laties, 1980; Ishibashi et al., 1981). Spontaneously diabetic KK mice develop microaneurysms and acellular capillaries (Duhault et al., 1976) and STZ-diabetic mice exhibited pericyte loss, basement membrane thickening and endothelial cell proliferation (Agren et al., 1979). Retinal changes in spontaneously diabetic Chinese hamsters are confined to increased intramural/endothelial cell pericyte ratio, glycogen deposits in the inner nuclear layer (Soret et al., 1974) and thickening of basement membrane but without capillary pathology or leakage. However, microaneurysm-like lesions have been observed in STZ-treated Chinese hamsters (Sibay et al., 1971). Generally, paucity of capillary microaneurysms is characteristic of most rodents (Cogan et al., 1961; Papachristodoulou et al., 1976) including Cohen sucrose-induced diabetic rats (Cohen and Rosenmann, 1990), in contrast to dogs and monkeys. In this regard, the retinal anatomical diversity should be taken into account: e.g. vascularization in guinea pigs and rabbits is poor, whereas dogs, monkeys, rats and hamsters possess an elaborate vascular network (Frank et al., 1983). Studies with galactose-fed rats (Wise et al., 1971; Robison et al., 1986; Robison et al., 1989; Robison et al., 1990) and dogs (Engerman and Kern, 1984; Kador et al., 1988; Kador et al., 1990) became an important advance. Sorbitol and glucose were found to be elevated in the diabetic rat retina early in hyperglycemia (MacGregor et al., 1986). However, galactose-fed rats and dogs develop a complication pattern similar to that of humans, despite normoglycemia. The earliest histopathological lesions are the degenaration of intramural pericytes and thickening of the capillary basement membrane (Roy and Lorenzi, 1996), occurring before any changes in the fundus. The function of pericytes is not well established. It has been proposed that they may be involved in blood flow regulation because of their contractile properties (Tilton et al., 1979; Das et al., 1987), barrier/transport function (DeOliveira, 1966), and the proliferation of endothelial cells (Orlidge and D’Amore, 1987; Carlson, 1988). After the pericyte loss empty pockets (“ghosts”) are left and the capillary loses its endothelial cells, becomes occluded and acellular. Other capillaries may proliferate and considerably dilate (Robison et al., 1989; Robison et al., 1990). The loss of pericytes, attributed in diabetic animals to hyperglycemia (King et al., 1986), appears to represent a link between this initial triggering event in galactose-induced polyol pathway and the various types of microangiopathies seen in this disease process. The capiliary dilatations are in fact microaneurysms of various types e.g. fusiform, saclike and cylindrical, similar to the human intraretinal microvascular abnormaiities. New vessel formation, if it occurs, is seen in the retina rather than in the vitreous. A word of caution is in order here, since in other tissues (e.g. nerves) AR activity does not result in sorbitol concentrations high enough to produce the damaging osmolarity (Sima, 1983; Stewart et al., 1967). As discussed in the nephropathy section, the decreased myoinositol levels, associated with polyol accumulation and resulting in reduced DAG concentration with consequently deficient PKC activa tion might lead to another mechanism, based on decreased ATPase activity. The
14 E.SHAFRIR AND A.A.F.SIMA
altered sodium equilibrium might affect the RPE, which actively transports metabolites out of the retinal extracellular fluid. Reduced transport of fluorescein out of the retina may be the early functional derangement (Kaufman and Lacoste, 1986). Increased RPE sorbitol, decreased myoinositol levels, ATPase activity and sodium gradient across the RPE have been demonstrated in diabetic rabbits (MacGregor et al., 1986a; MacGregor et al., 1986b). Support for the polyol triggering hypothesis comes from the successful preventive experience with AR inhibitors sorbinil and tolrestat, in diabetic or galactose fed rats (Robison et al., 1989; Robison et al., 1990; Lowe et al., 1995; McCaleb et al., 1991; Robison et al., 1989; Chandler et al., 1984). Similar findings, though not as striking, were obtained in galactose fed dogs (Kador et al., 1988; Kador et al., 1990). In these animals significant delays in aneurysm appearance, though not total prevention was observed. However, negative results with sorbinil were also reported in diabetic and galactosemic dogs treated with sorbinil (Engerman and Kern, 1993). Thus, other effects of hyperglycemia have to be considered as well. The importance of nonenzymatic glycation and the interaction of retinal components with end products of advanced glycation is now being intensively evaluated (Brownlee, 1990; Beisswenger et al., 1995), and the effect of free oxygen radicals is also at an early stage of investigation (Kowluru et al., 1996). Advanced glycation alters signal transduction pathways and may alter gene expressions of basoactive substances such as thrombomodulin and endothelins (Esposito et al., 1992; Chakrabarti et al., 1997). Studies in nonhyperglycemic galactose-fed animals convincigly indicate that the polyol pathway is the initiating reaction. It is, however, probable that these two processes have a cumulative impact on diabetic retinopathy in its advanced stages. Inhibition of advanced glycation with aminoguanidine seems to protect against capillary acellularity and microaneurysms in diabetic rats but does not prevent the loss of pericytes (Hammes et al., 1995), the primary lesion in retinopathy (King et al., 1986). Hyperglycemia may exert other effects as well, seemingly contradictory to the above mentioned decrease in the cellular myoinositol level, PKC and ATPase activities. PKC and its isoforms are involved in many intracellular functions, including transmembrane signaling and insulin receptor phosphorylation (Kibbawa and Nishizuka, 1986; Hashiya et al., 1987). Insulin receptors in the retina of diabetic rats are sensitive to regulation by the insulin/glucose ratio (Zetterstrom et al., 1992), whereas functional deterioration of the G protein dependent signaling system, which regulates PKC activity, has been reported (Kowluru et al., 1992). Evidence is available that high glucose concentrations may increase rather than decrease PKC activity in several tissues including microvascular cells (Lee et al., 1989) and retinal capillary cells (Wolf et al., 1991). In a skin chamber granulation model (Lee et al., 1989) the flow of blood and vascular permeation of albumin were found to be increased along with a severalfold elevation in the inositol-derived DAG concentration, consistent with PKC activation. Further, PKC activity was increased, while ATPase was reduced in bovine retinal capillary cells. AR inhibition by sorbinil prevented the reduction in ATPase activity while PKC activity remained increased. These results do not vitiate the polyol pathway hypothesis of retinal and other diabetic complications but suggest that multiple biochemical mechanisms are affected by increased glucose availability and flow through the polyol pathway. Especially, the role of changes in myoinositol concentrations, their occurrence in
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 15
specific cellular sites and the changes in the turnover of this effector vs. its concentration, require an exhaustive investigation. Likewise the biochemical exploration of retinopathy should be targeted on the defects in the signaling pathway which may be primarily responsible for the functional and morphological abnormalities. NEUROPATHY The elucidation of the pathogenesis of diabetic neuropathy, like nephropathy and retinopathy, heavily depends on information gained from a variety of diabetic animals. Most of the investigations have been done in STZ- or alloxan-diabetic rats, but animals with spontaneous diabetes like BB rats and db/db mice have importantly contributed to the understanding of the neuropathic complications. Not all morphologic characteristics of sensory or autonomic neuropathy are demonstrable in diabetic animals, however they display metabolic and electrophysiological disorders common with humans. As in other complications the assessment of nerve lesions in animals is limited by their short lifespan, but the initial derangements are very similar and constructive for the understanding of human neuropathy, since they may be detected at a reversible stage. There is now a concensus that the primary metabolic anomaly is diabetic hyperglycemia. The secondary cellular alterations induced by hyperglycemia are a group of functional abnormalities, still reversible in part, such as dysequilibrium in cellular metabolites and signaling effectors consequent, to the increased flow through the polyol pathway, e.g. the reduced sensory and motor nerve conduction velocity (NCV). Other, possibly interrelated metabolic derangements are enhanced protein glycation, oxidation by free oxygen radicals, hypoxia-ischemia, and impaired neurotrophic support, giving origin to more advanced functional disturbances. These alterations are followed by the deterioration of the structural integrity of nerve cells and supporting tissue elements with irreversible consequences, for example axonal shrinkage and atrophy, demyelination, and nodal changes resulting in irreversible nerve impulse defects. The degeneration of peripheral nerve myelinated axons and segmental demyelination occur both in type 1 or type 2. Some animals like the BB rat and rodents with cytotoxin induced diabetes are models for type 1 diabetes, while the db/db mice resemble type 2 diabetes, but the ensuing neuropathy depends to a large extent on the chronicity of diabetes rather than on its causation. NCV Slowing and the Polyol Pathway; Role of Aldose Reductase and its Inhibitors Of paramount importance in the recognition of the initial aberrations in diabetic neuropathy was the experimental demonstration of the retarded NCV in alloxandiabetic rats by Eliasson (Eliasson, 1964), which could be improved or reversed by insulin-effected reduction in hyperglycemia (Sima and Brismar, 1985; Jakobsen, 1979). It occurred in the absence of demyelination or axonal degeneration and was reversible by correction of the metabolic dysfunction (Greene et al., 1975; Greene et al., 1987). The most plausible connection between hyperglycemia, NCV and other disturbed nerve functions is the enhanced shunting of glucose through the polyol
16 E.SHAFRIR AND A.A.F.SIMA
pathway (Greene et al., 1987), by a route discussed earlier in nephropathy and ocular lesions. The reduced energy production in the nervous tissue of diabetic animals is attributed to lowered Na+/K+ ATPase activity (Greene and Lattimer, 1983; Clements and Stockard, 1984; Greene et al, 1988; Greene et al., 1985). This has been demonstrated particularly in BB rats by Sima and associates (Sima and Brismar, 1985; Greene et al., 1987; Brismar et al., 1987; Brismar and Sima, 1981) by studying the ionic currents in the node of Ranvier in peripheral nerves responsible for the polarization state of the axonal membrane. The polarization state of this membrane is regulated by the intra-axonal sodium equilibrium and sodium permeability, which is markedly increased as a result of the reduced sodium pump activity. These changes in nodal function in the acutely diabetic BB rats are reversible by upregulating the ATPase activity and reducing the intra-axonal sodium concentration. They are consistent with a conduction block of large myelinated fibers leading to NCV slowing. However, chronically diabetic BB/W rats show irreversible nodal changes which are associated with a breakdown of the paranodal ion channel barrier (axo-glial dysjunction, Sima et al., 1986), a change that has also been identified in neuropathy of type 1 patients (Sima et al., 1988). ATPase activity is tightly associated with excessive glucose flow throught he polyol pathway, intracellular sorbitol elevation and myoinositol depletion (Simmons et al., 1982), as mentioned already in renal and ocular lesions. The high glucose levels competetively diminish the uptake of myo-inositol (Guzman and Crews, 1992; Haneda et al., 1990) and the cellular elements of peripheral nerve are unable to maintain their high tissue/plasma myoinositol gradient (Greene et al., 1984; Palmano et al., 1977) and the consequent elaboration of DAG, the ATPase activity. Lack of activation of ATPase under these circumstances has a prompt effect on ion fluxes, across cellular membranes, leading to intracellular sodium retention and defective transmission of electrical impulses with decreased NCV. Additionally, the accumulation of sorbitol may also induce osmotic stress within the nerve cell or the Schwann cell, abetted by the elevation of other small molecules e.g. amino acids (Burg, 1988). Other metabolites, e.g. taurine may be depleted, as the concentrations of organic osmolytes are interdependent (Stevens et al., 1993). However, the osmolar edema due to polyol accumulation is not likely to be responsible for fiber damage since water accumulation occurs in endoneurial spaces rather than in Schwann cells (Jakobsen, 1978). The increased flow through the polyol pathway is dependent on AR activity, the high Km gate for glucose entry. The AR activity is thought to be enhanced by the increased intracellular osmolarity in nerve tissue (Ghahary et al., 1991) as it is by glomerular hyperosmolarirty (Hohman et al., 1990). The key function of AR is further emphasized by beneficial effects on inhibition of its activity, which prevents the elevation of sorbitol and depletion of myoinositol in nerves of STZ-diabetic rats (Price et al., 1988; Yoshida et al., 1987; Greene and Lattimer, 1984; Yagihashi et al., 1990) and diabetic BB rats (Greene et al., 1987), as well as restores the NCV, vagal nerve dysfunction (Zhang et al., 1990) in rats and endothelial cell function in rabbits (Tesfamariam et al., 1993) even in the face of continuing hyperglycemia. Structural changes such as axo-glial dysjunction in the long term diabetic BB rat (Greene et al., 1987) and in human patients (Sima et al., 1988) have been partially normalized by AR inhibitors. A prolonged treatment has been demonstrated to be particularly effective in STZ-diabetic and BB rats in promoting nerve fiber regeneration
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 17
(Yagihashi et al., 1990; Sima et al., 1990). These findings suggest that the impact of polyol pathway is carried beyond the initial functional defects and may underlie the structural nerve damage of experimental animal diabetes. Furthermore, galactose feeeding of transgenic, AR overexpressing rats induced neuropathy in spite of normoglycemia (Yagihashi et al., 1996) demonstrating that polyol accumulation and its consequences constitute the pathogenic background. Dogs are also an interesting model of AR inhibitor action. The AR inhibitor was effective in preventing NCV retardation and sorbitol accumulation in alloxan-diabetic dogs, but in those fed galactose NCV remained normal, despite manifold greater cellular concentration of galactitol (Engerman et al., 1994). The authors concluded that the NCV defect does not occur when the accumulated polyol does not proceed through further metabolism. Dietary replenishment of inositol in diabetic rats also produced an improvement in NCV and normalization of ATPase activity (Greene and Lattimer, 1983; Greene et al., 1982; Kim et al., 1991). Administration of diets containing up to 1% inositol restored the nerve myoinositol concentration and resulted in normalized ATPase activity (Greene et al., 1987; Greene et al., 1982; Gillon et al., 1983). Myoinositol supplement was also effective in correcting NCV and ATPase activity in rats fed fucose, a potent competetive inhibitor of myoinositol transport (Yorek et al., 1993). In BB rats, prolonged maintenance on an inositol-rich diet, resulted not only in amelioration of the NCV defect but also prevented structural nodal alterations (Schmidt et al., 1991). It appears that ATPase activity can also be improved nonadditively by PKC agonists in vitro and myoinositol in vivo (Kim et al., 1991). There is some indication, however, that AR inhibition may be a preferable treatment since inositol supplementation did not elicit nerve fiber regeneration in diabetic rats (Sima et al., 1990; Schmidt et al., 1991), did not prevent the progression of neuroaxonal dystrophy (Kim et al., 1991) and was not yet shown to be decisively effective in diabetic patients. Nerve Hypoxia-Ischemia, Oxidative Stress and Prostacyclin Effects in Diabetic Animals Apart from derangements linked to the polyol pathway there is ischemia, abnormal microcirculation and deficient oxygen supply in peripheral nerves of STZ-diabetic rats (Low et al., 1987; Low et al., 1985; Low et al., 1989; Smith et al., 1991). The nerves in diabetic animals show hypoxia, due to the reduced blood flow (Tuck et al., 1984) with lowered creatine phosphate and increased lactate levels, indicating a switch to nonoxidative glycolytic pathway, as well as to an increase in superoxide radicals (McCord, 1985; Low and Nicklander, 1991). Exposure of diabetic rats to hyperbaric oxygen ameliorates some of these abnormalities (Low et al., 1985). The microvascular circulation has been proposed to be influenced by the equilibrium between the vasodilating action of the endothelial prostacyclin and nitric oxide and vasoconstricting tromboxane A2, from platelets (Moncada, 1979). An oxidative stress appears to result from reduced prostacyclin and nitric oxide release with a negative influence on the vasorelaxant capacity of the vascular endothelium (Oberley, 1988; Pieper et al., 1993; Tesfamariam and Cohen, 1992a; Tesfamarian and Cohen, 1992b).
18 E.SHAFRIR AND A.A.F.SIMA
The role of oxidative stress in neuropathy and other diabetic complications has been extensively reviewed by Van Dam et al. (1995), with the conclusion that hyperglycemia leads to increased production of reactive oxygen species by glucose autooxidation and/or reduced antioxidant activity. The superoxide radicals may generate lipid peroxides due to the rise in cyclooxygenase activity and an increase in tromboxane/prostacyclin ratio. This produces in turn endoneurial constriction and platelet aggregation. Superoxide radicals may damage the blood/nerve barrier by lesioning the endothelial cells (Rechthand et al., 1987; Sima et al., 1991) and produce other detrimental changes through lipid peroxidation. Among the sources for increased activity of superoxide radicals are the decrease in superoxide dimutase activity in the nerve of diabetic animals (Low and Nicklander, 1991) due to an oversupply of glucose-derived substrates in diabetes. Dietary probucol, butylated hydroxytoluene and n-acetylcysteine are effective antioxidants, reducing plasma peroxidation products in STZ-diabetic rats (Chisolm and Morel, 1993; Cameron et al., 1994; Cameron et al., 1993; Sagara et al., 1996). These antioxidants and Vitamin E (Thompson and Lee, 1993; Karasu et al., 1995) may assist the oxidatively stressed cell and stimulate the production of glutathione, which by itself was shown to be an antioxidant in diabetic rats and mice (Bravenboer et al., 1992; Hermenegildo et al., 1993). The importance of ischemia in the deterioration of nerve function is emphasized by the improvement of hypoxia-delayed NCV by treatment of STZ-diabetic rats with endoneurial blood flow promoting compounds (Cameron et al., 1991), angiotensin converting enzyme inhibitors (Cameron et al., 1992), prostaglandin analogs (Cotter et al., 1993; Hotta et al., 1995) or prostaglandin precursors, such as arachidonate (Cotter and Cameron, 1998) and other long chain fatty acids (Lockett and Tomlinson, 1992; Cameron et al., 1993). The enhanced endogenous prostacyclin production prevents the acute NCV defect and ATPase pump activity in diabetic rats (Tomlinson et al., 1989; Stevens et al., 1993). Another correction of the disturbed nerve function in STZ-diabetic rats and BB rats as well as the structural changes in BB rats was demonstrated by treatment with acetyl- or propionyl-Lcarnitine (Morabito et al., 1993; Hotta et al., 1996; Cotter et al., 1995; Lowitt et al., 1995; Sima et al., 1996), which act by promoting fatty acid oxidation and augment the intracellular energy supply. Thus, the defective NCV and reduced ATPase activity may have another etiology in addition to the polyol pathway (Carrington et al., 1991; Calcutt et al., 1990). Whether these changes are metabolically separated or integrated with the pathogenesis elicited by the polyol pathway, requires further investigation. Evidence that the polyol pathway, nitric oxide generation, essential fatty acid, proctacylin and cyclooxygenase systems may synergistically interact in diabetic rats was recently presented (Cameron et al., 1996; Yasuda et al., 1992). Glycation of Nerve Components and its Prevention The role of nonenzymatic glycation of nerve proteins requires particularly intensive study, since glycation has been demonstrated to induce numerous detrimental changes in many tissues (Brownlee, 1994). Presumed candidates might be the peripheral nerve tubulin, and/or neurofilaments resulting in alterations in
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 19
selfassembly and solubility (Williams et al., 1982). The first stage of glycation by attachment of fructosyl residues to protein lysine constituents is followed by advanced end-product formation, inducing covalent protein crosslinking and cytokine production (Brownlee, 1994). Evidence for such reactions in peripheral nerve myelin and of uptake of myelin by macrophages is available (Vlassara et al., 1985). In addition, glycation of plasma proteins facilitates their penetration into nerve cells with deleterious consequences on nerve function (Patel et al., 1991). Prevention of nerve protein interaction with advanced glycation end products with aminoguanidine is a promising treatment possibility, with initial beneficial results in STZ-diabetic rats, and may restore nerve blood flow, vascular permeability and prevent the excessive rise of oxidants (Yagihashi et al., 1992; Kihara et al., 1991). Neurotropic Factors Abnormalities in the transport and synthesis of structural proteins have been reported in the nerve cells of STZ-and alloxan-diabetic rats, BB rats (Medori et al., 1988; McLean and Meiri, 1980; Marini et al., 1986), db/db mice (Vitadello et al., 1985) and galactose-fed nonhyperglycemic rats (Sidenius and Jakobsen, 1980). These proteins may be endogenous enzymes, e.g. acetylcholine tranferase and esterase, enolase, actin and calmodulin, the transport of which is slowed down and may contribute to improper alignment of neurofilaments (Sima, 1980). Retrograde transport, which moves tropic factors and hormones from the periphery into the cell, notably of exogenous nerve growth factor to the dorsal root or the mesenteric ganglia is decreased in STZ-diabetic rats (Schmidt et al., 1983). Several neurotrophins and neurotrophic factors as well as their receptors have been reported to be decreased in diabetic nerve. Most notably NGF and its high affinity receptor TrkA are reduced in STZ-diabetic rats (Thomas, 1994). Similarly both IGF-1 and its receptor show decreased gene expression in peripheral nerve of STZ- and BB rats (Ishii, 1995; Sima, 1996; Sima et al., 1997) Lately the potential neurotropic effect of insulin itself and simultaneously secreted C-peptide have been implemented in the pathogenesis of diabetic neuropathy (Ishii, 1995; Sima et al., 1997). The latter changes may account for the differences seen between neuropathy in type 1 and type 2 human diabetes (Sima et al., 1988) and similar differences reported between the neuropathies occurring in type 1 and type 2 animal models (Sima et al., 1997). These neurotropic factors enhance phosphoinositide turnover and phosphorylation of structural proteins which might hence be decreased. PKC agonists, involved in the phosphoinositide chain, also have a neurotropic activity and their action may be impaired in diabetic animals (Ishii et al., 1987). The neurotropic support and its alterations have been extensively described (Tomlinson et al., 1995). An overall conclusion can be inferred that neurotropic factors, essential both for regeneration and maintenace of normal structure of the nervous system, may be either deficient or not accessible to their target sites in diabetic animals. Neuropathy in Other Animal Models The polyol pathway does have an impact on the neuropathy in the Chinese hamster, causing demyelination in peripheral and pelvic visceral (autonomic) nerves
20 E.SHAFRIR AND A.A.F.SIMA
of this insulin deficient animal. (Schlaeper et al., 1974; Dail et al., 1977). These nerves show glycogen deposition, fibrillar degeneration, lysosome accumulation and axon swelling. This autonomic neuropathy may be responsible for neurogenic bladder, delayed gastric emptying and intestinal atony (Diani et al., 1979). Segmental demyelination and acute axonal (Wallerian) degeneration also takes place in peripheral tibial nerves in severe diabetes, however, there was no reduction in nerve fiber diameters in moderate diabetes (Kennedy et al., 1982). A surprising characteristic of Chinese hamster is that the NCV and polyol accumulation are not relieved by AR inhibition, neither in the nerve nor in the retina or kidney, indicating a species specificity in response to inhibition of AR (Sekiguchi et al., 1991). In insulin-dependent NOD mice, no morphological abnormalities were evident early after the onset of hyperglycemia. Only after several months of diabetes did the myelinated fiber size became reduced and their density increased. Myelin wrinkling and early paranodal demyelinization was found in a proportion of mice by teased fiber studies, suggesting the presence of axonal atrophy and nodal changes (Kamijo et al., 1990). WKY fatty rats exhibit peripheral nerve abnormalities, decreased NCV, paranodal swelling and some segmental demyelination even in the face of moderate hyperglycemia. These changes, including phosphoinositide metabolism are similar to the findings in STZ-diabetic rats (Berti-Mattera et al., 1989). The selectively inbred GK rats simulate human neuropathy of type 2 diabetes by exhibiting first low NCV, followed by reduction in the size of nonmyelinated sural nerve fibers and distorted sheaths of myelinated flbers at 6 months after the onset of moderate hyperglycemia, but no reduction in myelin thickness or axon size (Yagihashi et al., 1982; Suzuki et al., 1988). Psammomys obesus (sand rat), rendered hyperglycemic by a high energy diet, exhibits sensory dysfunction demonstrable by hyperalgesia, as shown by a low pain threshold, presumably due to impaired function of unmyelinated fibers (Wuarin-Bierman et al., 1987). These findings resemble those of STZ-diabetic rats in which hyperalgesia is related to the increased flow through the polyol pathway (Calcutt et al., 1995). Functional and structural expressions of neuropathy in WBN/Kob rats feature demyelinating motor neuropathy, which differs from that observed in other diabetic animals (Hotta et al., 1996). OLETF rats exhibit delayed NCV, decreased R-R interval variability in the electrocardiogram, reduced nerve blood flow, and platelet clumping (Hotta et al., 1996). These rats are non-insulin dependent and sucrose feeding was applied to elicit these changes, which were reversed by treatment with cilostazol, an antithrombotic agent preventing platelet aggregation. Peripheral nerve damage with decreased NCV is also documented in db/db mice (Carson et al., 1980; Hanker et al., 1980; Moore et al., 1980; Robertson and Sima, 1980). The db/db mice are markedly hyperglycemic displaying an abnormal axonal epinephrine, acetylcholine and phosphofructokinase transport (Calcutt et al., 1988; Giachetti, 1979; Sima and Robertson, 1979) but no segmental demyelination. Their impaired nervous function was ascribed to a maturation deficit (Sharma et al., 1983). However, axonal atrophy of both myelinated and nonmyelinated fibers was seen after protracted hyperglycemia (Giachetti, 1979; Sima and Robertson, 1979). The incorporation ratio of fiicose/leucine into myelin was increased, similarly to the observations in diabetic rats (Chez and Peterson, 1983).
DIABETIC ANIMALS FOR RESEARCH INTO THE COMPLICATION 21
It is remarkable that the neuropathy in db/db mice was accompanied by only small decreases in ATPase activity in several nerves without accumulation of sorbitol or depletion in myoinositol (Bianchi et al., 1990; Calcutt et al., 1988). As mentioned earlier (Bianchi et al., 1990), db/db mice are virtually devoid of AR activity. Thus, the axonal malfunction has to be explained on a basis other than enhanced polyol pathway. The neuropathy of db/db mice was a model for investigation of the beneficial effects of gangliosides (Calcutt et al., 1988; Norido et al., 1984; Schiavinato et al., 1984). Gangliosides are essential components of the nerve membranes, but the effect of hyperglycemia and other diabetic alterations on their structure and function is not well known. Treatment with exogenous gangliosides ameliorated the conduction defect. These results contrast the experience with STZ-diabetic rats in which ganglioside treatment corrected the ATPase defect and prevented the decrease in axonal transport despite reduction in DAG content (Bianchi et al., 1990; Bianchi et al., 1993). In BB rats, treatment with gangliosides reversed the neuropathy changes in urinary bladder and had a prolonged positive effect on structural changes in the parasympathetic limb of the micturition reflex (Paro et al., 1991). The recently described BBZ/WORDR rat shows spontaneous onset of type 2 diabetes at 3 months of age. It develops obesity preceding onset of diabetes and shows normal or elevated insulin levels and peripheral insulin resistance (Guberski et al., 1993). it develops hypertension and hyperlipidemia (Murray et al., 1996). The BBZ/WORDR rat shows background retinopathy with basement membrane thickening and pericyte loss and progressive increases in urine albumin and protein excretion, mesangial expansion and basement membrane thickening in the kidney (Murray et al., 1996). The neuropathy develops slower in this model than in type 1 BB rats with a milder NCV defect and axonal atrophy. The nodal changes are mild or absent (Sima et al., 1997), similar to type 2 human diabetic neuropathy. Despite this the abnormalities of the polyol-pathway and Na+ 1K+-ATPase are more severe and persist for a longer duration of diabetic neuropathy compared with type 1 BB rats (Sima et al., 1997). The reviewed results in diabetic animals repeatedly point out that neuropathy is widespread with the common denominator of hyperglycemia, whether due to diabetogens or genetic diabetes, insulin-dependent or insulin-resistant. Polyneuropathy, from the initial decline in ATPase activity through decreases in NCV, sensory perception and autonomic reflex and transmitter levels, to the final structural-morphologic deficits, shows a wide, heterogenous spectrum of effects, related to the affected species and probably type of diabetes. Each of the different models may provide a window on a part of the whole spectrum, which requires careful integration with respect to the specific causes and consecutive interrelations. REFERENCES Agren, A., Rehn. G. and Naeser, P. (1979) Morphology and enzyme activities of retinal capillaries of streptozotocin diabetic mice. Acta Ophtalmol., 57, 1065–1069. Aguizy, H.K., Richards, R.D., Varma, S.D. (1980) Sugar cataracts in Mongolian gerbils. Invest. Ophthalmol. Vis. Sci., 19(Suppl.).
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Ahmad, H., Sharma, R., Mansour, A. and Awasthi, Y.C. (1992) tert-Butylated hydroxytoluene enhances intracellular levels of glutathione and related enzymes of rat lens in vitro organ culture. Exp. Eye Res., 54, 41–48. Allen, T.J., Cooper, M.E., O’Brien, R.C., et al. (1990) Glomerular filtration rate in streptozocininduced diabetic rats. Diabetes, 39, 1182–90. Ao, A., Kikuchi, C., Ono, T. and Notsu, U. (1991) Effect of instillation of aldose reductase inhibitor FR74366 on diabetic cataract. Invest. Ophthalmol. Vis. Sci., 32, 3078–3083. Bailey, C.V., Flatt, P.R. and Radley, N.S. (1985) Effect of high fat and high carbohydrate cafeteria diets on the development of the obese hyperglycemic (ob/ob) syndrome in mice. Nutr. Res., 5, 1003–1010. Baynes, J.W. (1991) Role of oxidative stress in development of complications in diabetes. Diabetes, 40, 405–412. Beisswenger, P.J., Makita, Z., Curphey, T.J., et al. (1995) Formation of immunochemical advanced glycosylation end products precedes and correlates with early manifestations of renal and retinal disease in diabetes. Diabetes, 44, 824–829. Bendayan, M. (1985) Alteration in the distribution of type IV collagen in glomerular basal laminae in diabetic rats as revealed by immunocytochemistry and morphometrical approach. Diabetologia, 28, 373–378. Berdanier, C.D. (1995) Non-insulin-dependent diabetes in the nonobese BHE/cdb rat. In Lessons from Animal Diabetes, edited by E.Shafrir, 5, 231–246. London: Smith-Gordon. Berti-Mattera, L.N., Lowery, J., Day, S.-F., et al. (1989) Alteration of phosphoinositide metabolism, protein phosphorylation, and carbohydrate levels in sciatic nerve from Wistar fatty diabetic rats. Diabetes, 38, 373–378. Beyer-Mears, A., Ku, L. and Cohen, M. (1884) Glomerular polyol accumulation in diabetes and its prevention by oral sorbinil. Diabetes, 33, 604–607. Bianchi, R., Berti-Mattera, L.N., Fiori, M.G. and Eichberg, J. (1990a) Correction of altered metabolic activities in sciatic nerves of streptozocin-induced diabetic rats. Diabetes, 39, 782–788. Bianchi, R., Marelli, C. and Marini, P. (1990b) Diabetic neuropathy in db/db mice develops independently of changes in ATPase and aldose reductase. A biochemical and immunohistochemical study. Diabetologia, 33, 131–136. Bianchi, R., Zhu, X., Fiori, M.G. and Eichberg, J. (1993) Effect of gangliosides on diacylglycerol content and molecular species in nerve from diabetic rats. Eur. J. Pharmacol., 239, 55– 61. Blair, N.P., Zeimer, R., Ruain, M. and Cunha-Vaz, J. (1983) Outward transport of fluorescein from the vitreous in normal subjects. Arch. Ophthalmol., 101, 1117–11121. Blair, N.P., Tso, M.O.M. and Dodge, J.T. (1984) Pathological studies on the blood retinal barrier in the spontaneously diabetic BB-rat. Invest. Ophtalmol. Vis. Sci., 25, 302–3011. Boot-Handford, R. and Heath, H. (1980) Identification of fructose as the retinopathic agent associated with the ingestion of sucrose-rich diets in the rat. Metabolism, 29, 1247– 1252. Bower, G., Grown, D.M., Steffes, M.W., et al. (1980) Studies of the glomerular mesangium and the juxtaglomerular apparatus in the genetically diabetic mouse. Lab, Invest., 43, 333– 341. Bravenboer, B., Kappelle, A.C., Hamers, F.P.T., et al. (1992) Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozotin-induced diabetic rat. Diabetologia, 35, 813–817.
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Bretzel, R.G., Breidenbach, G., Hofmann, J. and Federlin, K. (1979) Islet transplantation in experimental diabetes in the rat VI. Rate of regression in diabetic kidney lesion after isogeneic islet cell transplantation: quantitative measurement. Horm. Metab. Res., 11, 200– 207. Bretzel, R.G., Brocks, D.G. and Federlin, K.F. (1984) Reversal and prevention of nephropathy by islet transplantation in diabetic rats. In Lessons from Animal Diabetes, edited by E.Shafrir and A.E. Renold, 1, 425–435. London: J. Libbey. Brismar, T., Sima, A.A.F. and Greene, D.A. (1987) Reversible and irreversible nodal dysfunction in diabetic neuropathy. Ann. Neurol., 21, 504–507. Brismar, T. and Sima, A.A.F. (1981) Changes in nodal function in nerve fibres of the spontaneously diabetic BB-Wistar rat. Potential clam analysis. Acta Physiol. Scand., 113, 499– 506. Brown, D.M., Steffes, M.W., Thibert, R, et al. (1983) Glomerular manifestations of diabetes in the BB rat. Metabolism, 32, 131–135. Brownlee, M. and Spiro, R.G. (1970) Glomerular basement membrane thickness in the diabetic rat. Diabetes, 28, 121–125. Brownlee, M. (1990) Advanced glycosylation products and the biochemical basis of late diabetic complications. In Current status of prevention and treatment of diabetic complications, edited by N.Sakamoto, K.G.M.M.Alberti and N.Hotta, pp. 92–98. New York: Elsevier. Brownlee, M. (1994) Glycation and diabetic complications. Diabetes, 43, 836–841. Burg, M. (1988) Role of aldose reductase and sorbitol in maintaining the medullary intracellular milieu. Kidney Int., 33, 635. Cagliero, E., Roth, T., Roy, S. and Lorenzi, M. (1991) Characteristics and mechanisms of highglucose-induced overexpression of basement membrane components in cultured human endothelial cells. Diabetes, 40, 102–110. Calcutt, N.A., Li, L., Yaksh, T.L. and Malmberg, A.B. (1995) Different effects of two aldose reductase inhibitors on nociception and prostaglandin E. Eur. J. Pharmacol., 285, 189– 197. Calcutt, N.A., Tomlinson, D.R. and Biswas, S. (1990) Coexistence of nerve conduction deficit with increased Na+-K+-ATPase activity in galactose-fed mice. Implications for polyol pathway and diabetic neuropathy. Diabetes, 39, 663–666. Calcutt, N.A., Tomlinson, D.R. and Willars, G.B. (1988) Ganglioside treatment of streptozotocin-diabetic rats prevents defective axonal transport of 6phosphofructokinase activity. J.Neurochem. 50, 1478–1483. Calcutt, N.A., Willars, G.B. and Tomlinson, D.R. (1988) Axonal transport of choline acetyltransferase and 6-phosphofructokinase activities in genetically diabetic mice. Muscle Nerve, 11, 1206–1210. Cameron, N.E., Cotter, M.A., Archibald, V., et al. (1994) Anti-oxidant and pro-oxidant effects on nerve conduction velocity, endoneurial blod flow and oxygen tension in non-diabetic and streptozotocin-diabetic rats. Diabetologia, 37, 449–459 Cameron, N.E., Cotter, M.A., Dines, K.C., et al. (1993) The effects of evening primrose oil on nerve function and capillarization in streptozotocin-diabetic rats: modulation by the cyclo-oxygenase inhibitor flurbiprofen. Br. J. Pharmacol., 109, 972–979 Cameron, N.E., Cotter, M.A., Ferguson, K., et al. (1991) Effects of chronic a-adrenoceptor blockade on peripheral nerve conduction, hypoxic resistance, polyols, Na-K-ATPase activity and vascular supply in streptozotocin-diabetic rats. Diabetes, 40, 1652–1658.
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Yeh, L.A., Rafford, C.E., Beyer, T.A. and Hutson, N.J. (1986) Effects of the aldose reductase inhibitor sorbinil on the isolated cultured rat lens. Metabolism, 35, 4–9 Yorek, M.A., Wiese, T.J., Davidson, E.P., Dunlap, J.A., et al. (1993) Reduced motor nerve conduction velocity and Na+-K+-ATPase activity in rats maintained on L-fucose diet. Diabetes, 42, 1401–1406. Yoshida, E, Isobe, K.-I. and Matsuo, S. (1995) ln vivo effects of hyperglycemia on the outcome of acute mesangial injury in rats. J. Lab. Clin. Med., 125, 46–55. Yoshida, T, Nishioka, H., Yoshioka, K., et al. (1987) Effect of aldose reductase inhibitor ONO 2235 on reduced sympathetic nervous sysem activity and peripheral nerve disorders in STZ-induced diabetic rats. Diabetes, 36, 6–13. Zahnd, G.R. and Adler, J.H. (1984) Sand rat as a model of diabetic cataract—a major blinding condition. In Lessons from Animal Diabetes, edited by E.Shafrir and A.E.Renold, 1, 500– 502. London: J.Libbey. Zatz, R., Meyer, T.W., Rennke, H.G. and Brenner, B.M. (1985) Predominance of hemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc. Natl. Acad. Sci. USA, 82, 5963–5907. Zetterstrom, C., Benjamin, A. and Rosenzweig, S.A. (1992) Differntial expression of retinal insulin receptors in STZ-induced diabetic rats. Diabetes, 41, 818–825. Zhang, W.X., Chakrabarti, S., Greene, D.A. and Sima, A.A.F. (1990) Diabetic autonomic neuropathy in BB-rats: The effect of ARI-treatment on heart-rate variability and vagus nerve structure. Diabetes, 39, 613–618. Ziv, E. and Shafrir, E. (1995) Psammomys obesus: nutritionally induced NIDDM-like syndrome on a ‘thrifty gene’ background. In Lessons from Animal Diabetes, edited by E.Shafrir, 5, 285–300. London: Smith-Gordon.
2. STUDIES IN ANIMAL MODELS ON THE ROLE OF GLYCATION AND ADVANCED GLYCATION ENDPRODUCTS (AGEs) IN THE PATHOGENESIS OF DIABETIC COMLICATIONS: PITFALLS AND LIMITATIONS JESÚS R.REQUENA and JOHN W.BAYNES Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208 INTRODUCTION Animal models provide unique opportunities for researchers studying the etiology and treatment of human disease. The most significant strides in the treatment of diabetes stem from research on diabetic dogs, which led to the discovery of insulin. Research on diabetes continues today in a wide variety of animals, from rodents to primates, representing chemical and genetic models of both insulin-dependent and non-insulin-dependent diabetes mellitus (IDDM, NIDDM). In this review we will discuss research in animal models on the role of the Maillard or browning reaction and the formation of advanced glycation end-products (AGEs) in the development of diabetic complications. The hypothesis under consideration, known as the Maillard or AGE hypothesis, proposes that the accumulation of AGEs in tissues contributes to the chemical modification and crosslinking of tissue proteins, lipids and DNA. These alterations in biomolecules affect their structure, function and turnover, contributing to a gradual decline in tissue function and the pathogenesis of diabetic complications (Bucala and Cerami, 1992; Vlassara, 1994; Schmidt et al., 1994b). In addition to considering the direct role of AGEs in diabetic complications, we will also evaluate recent evidence on the use of aminoguanidine as an AGE inhibitor, the role of AGE receptors in the removal of AGEs from blood and tissues, and the activity of AGEs as a source of oxidative stress and cytotoxicity in tissues. The majority of the chapters in this volume may emphasize the usefulness of animal models in understanding human diabetes, but the theme that we would like to develop in this article is one of caution, acknowledging the merit, but also illustrating the limitations and pitfalls in the interpretation of experiments in animal models. Most of the discussion will focus on studies in rodents, primarily the
Corresponding Author: John W.Baynes, Ph.D., Carolina Distinguished Professor, Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Coiumbia, SC 29208, Phone: 803– 777–7272, FAX: 803–777–9521, Email:
[email protected]
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streptozotocin (STZ)-induced diabetic mouse and rat, because these animals have been used most extensively in studies on the sequelae of non-enzymatic glycation of protein in diabetes. However, evidence on the role of AGE-receptors in the recognition and metabolism of AGE-proteins in non-diabetic animals will also be reviewed since the metabolism, toxicity and pathology induced by AGE-proteins is widely studied in control, non-diabetic animals. Thus, the animal models under consideration include both the STZ-diabetic rat and the non-diabetic rodent exposed to AGE-proteins. To provide some background, this chapter begins with an introduction to the chemistry of the Maillard reaction, the role of oxidative stress in the formation of AGEs, and the proposed role of AGEs in the development of diabetic complications. A more detailed discussion of the AGE hypothesis and the role of oxidative stress in development of diabetic complications can be found in previous reviews (Baynes, 1991, 1995, 1996). The chapter is organized around a series of questions addressing critical elements of the AGE hypothesis. The discussion of these questions considers a range of studies in animal models which have been interpreted in support of the AGE hypothesis, but which, in our judgment, have yielded, at best, ambiguous insights into the significance of the Maillard reaction and AGEs in the pathogenesis of diabetic complications. PRODUCTS AND PATHWAYS OF THE MAILLARD REACTION Nonenzymatic glycation, leading to formation of a ketoamine or Amadori adduct, is the first step in the Hodge pathway (Hodge, 1953) of the Maillard or browning reaction between reducing sugars and amines (Figure 1, top). Following glycation of amino groups in proteins, lipids and nucleic acids, a complex cascade of reactions ensues, leading to further chemical modification, crosslinking, fragmentation and insolubilization of biomolecules, accompanied by the formation of brown and fluorescent products. Maillard himself had noted in his earliest studies that increased rates of reactions between blood sugar and amino acids during hyperglycemia might explain the wasting of muscle protein associated with diabetes (Maillard, 1912). While this hypothesis proved incorrect, the characterization of the Amadori adduct on glycated hemoglobin (HbA1c) in the mid-1970s confirmed that the first stage of the Hodge pathway of the Maillard reaction actually occurred in vivo. Measurements of glycated hemoglobin and plasma proteins are now widely used for the clinical assessment of glycemic control in diabetes. The recent Diabetes Control and Complications Trial (1993) showed that there was a strong correlation between glycated hemoglobin concentration and the rate of development of renal, retinal and vascular complication of diabetes. In addition to the Amadori adduct on glycated protein, blood and tissue proteins also contain a number of AGEs (Figure 2, reviewed by Wells-Knecht et al., 1996). AGEs were measured first in long-lived proteins, such as lens crystallins and tissue collagens, because these proteins constitute a stable substrate for the age-dependent accumulation of AGEs (Baynes, 1991). Although the retina, kidney and vasculature
are the major sites of pathology in diabetes, skin collagen has been studied in greatest detail because of its ready accessibility. Implicit in these studies is the assumption that systemic alterations in metabolism in diabetes would affect
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Figure 1 Pathways for formation of AGEs and glycoxidation products and their effects on tissues. AGEs and glycoxidation products are formed via the Wolff pathway by autoxidative glycosylation (Wolff and Dean, 1987; Wolff et al., 1991), the Namiki pathway by autoxidation of Schiff base adducts (Hayashi and Namiki, 1986; Glomb and Monnier, 1995), or the Hodge pathway by reactions of the Amadori adduct on protein (Hodge, 1953). Some of the intermediates in these pathways have been identified. AGEs and glycoxidation products induce structural and functional changes in tissue proteins, including formation of crosslinks and fluorescent compounds, and are thought to affect the metabolism of tissue proteins and to induce oxidative stress and cytotoxicity in tissues.
Figure 2 Structure of AGEs and glycoxidation products which have been identified in tissue proteins in vivo (Wells-Knecht et al., 1995). The glycoxidation products CML and pentosidine are the only AGEs which have been quantified in human tissue proteins by chemical methods and which are known to increase in tissue proteins with age and to accumulate at an accelerated rate in diabetes. Pentosidine and pyrraline were described by Monnier and colleagues (Sell and Monnier, 1989; Hayase et al., 1989; Miyata and Monnier, 1992). Crosslines were described by lenaga et al. (1996).
extracellular matrix proteins in all tissues similarly. In fact, levels of AGEs in skin collagen correlate with the severity of renal, retinal and vascular complications of
diabetes (Sell et al., 992; Beisswenger et al., 993; McCance et al., 993). These observations support the hypothesis that AGE-induced chemical and physical damage to long-lived proteins impairs the function of the extracellular
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matrix and leads to the development of diabetic complications. However, it is important to recognize that correlations between age-adjusted levels of AGEs and risk for complications do not exclude alternative mechanisms, e.g. Maillard reactionindependent effects of glucose, since the severity of long-term hyperglycemia also correlates with risk for development of complications. For over 50 years, formation of Amadori adducts, such as the ketoamine fruc toseLys-ine selysine (FL), was considered the rate-limiting step in the Maillard reaction (Figure 1, Hodge pathway). Reactive dicarbonyl sugars, either proteinbound or free in solution, formed by rearrangement or decomposition of Amadori compounds, were identified as critical intermediates in the formation of AGEs. Indeed, 3deoxyglucosone (3DG), a product of decomposition of FL, has been detected in human and rat plasma, and plasma concentrations of both 3DG and its metabolite 3-deoxyfructose are increased in diabetes (Knecht et al., 1992; Niwa et al., 1993; Wells-Knecht et al., 1994; Yamada et al., 1994). Formation of 3DG by the Hodge pathway does not require oxygen, but other reactive dicarbonyl intermediates, such as glyoxal (GO) and methylglyoxal (MGO) are formed by oxidative fragmentation of Amadori compounds (Figure 1, Hodge pathway) or of Schiff base intermediates (Hayashi and Namiki, 1986; Glomb and Monnier, 1995) (Figure 1, Namiki pathway), or even by direct oxidation of sugars prior to their reaction with protein, a process known as autoxidative glycosylation (Wolff and Dean, 1987; Wolff al., 1991; Wells-Knecht et al., 1995) (Figure 1, Wolff pathway). Although there are many possible routes to their formation in vitro and in vivo, Baynes (1991) noted that autoxidative conditions were required for formation of both Ne-(carboxymethyl) lysine (CML) and pentosidine during reaction of protein with glucose or ascorbate in vitro. Among the AGEs, these two compounds have received the most attention because of their stability to acid hydrolysis of proteins and the specificity with which they can be assayed, CML by gas chromatography—mass spectrometry (GC/MS) and pentosidine by reversed phase high performance liquid chromatography (HPLC) with fluorescence detection. They are the only AGEs which are known to accumulate in proteins with age and at an accelerated rate in diabetes, leading to the conclusion that oxygen is a “fixative” of irreversible Maillard reaction damage to protein and that diabetes accelerates the natural, chemical aging of tissues proteins, as measured by the increased rate of accumulation of AGEs. Thus, at least at the chemical level, diabetes may be described as a disease of accelerated aging. As products of both glycation and oxidation reactions, CML and pentosidine have been described as glycoxidation products (Baynes, 1991). These AGEs are considered specific biomarkers not only for quantifying the extent of nonenzymatic damage to tissue proteins via the Maillard reaction, but also for assessing the role of both glycative and oxidative stress in the development of diabetic complications (Baynes 1995, 1996). In the discussion below, we will use the term AGE to refer in general to products of advanced glycation reactions, and the term glycoxidation product to refer specifically to CML and pentosidine. At this point the glycoxidation products are also the only AGEs which have been rigorously quantified in tissue proteins by analytical chemical techniques. Despite their importance as indicators of the Maillard reaction, the known AGEs and glycoxidation products are only trace components of tissue proteins (Table 1). Among these, CML is quantitatively the most significant AGE in lens crystallins and skin collagen and has been identified as the major AGE antigen in AGE-proteins (Reddy et al., 1995). However, CML
STUDIES ON THE ROLE OF GLYCATION AND AGES 49
Table 1 Concentrations of AGEs in skin collagen of control and diabetic humans and ratsa.
a
Data are estimated for 20 year old humans (Dyer et al., 1993) and for 8 month old rats with 6 months’ duration of STZ-induced diabetes (Trachtman et al., 1995). Analyses for CML were performed by GC/ MS and pentosidine by reversed phase HPLC with fluorescence detection.
accounts for only 0.2% of lysine residues in proteins, even in long-lived, insoluble skin collagen from older diabetic patients, and is a relatively inert product. Although pentosidine is fluorescent and crosslinks proteins, its concentration in proteins is typically less than 1 % that of CML. At this time, glycoxidation products, such as CML, the related compound Ne-(carboxyethyl)lysine (Wells-Knecht et al., 1996; Ahmed et al., 1997), and pentosidine (Figure 2), should probably be considered biomarkers of the Maillard reaction, rather than pathogenically relevant chemical modifications of tissue proteins. Other lysine modifications or products of modification of arginine, cysteine, histidine and tryptophan are also formed during the Maillard reaction in vitro, but remain to be detected and measured in tissue proteins. Among these uncharacterized AGEs may be redox-active compounds which produce reactive oxygen and initiate local inflammatory processes, contributing to oxidative stress and pathology in diabetes and other diseases (Kirstein et al., 1990; Miyata et al., 1994; Schmidt et al., 1994b; Smith et al., 1994; Vitek et al., 1994; Wautier et al., 1994; Yan et al., 1994). THE STREPTOZOTOCIN-INDUCED DIABETIC RAT AND MOUSE MODELS The STZ-induced diabetic rodent is, by far, the most commonly used animal model for diabetes research. Diabetes is induced in mice by several low-dose treatments with STZ, which lead to destruction of ȕ-cells by immune mechanisms, or in mice or rats by a single high dose of STZ sufficient to destroy essentially all ȕ-cells, apparently by induction of oxidative stress (reviewed in Baynes, 1995). A serious limitation of this model is that the aninsulinemic diabetic animals are commonly maintained in extremely poor glycemic control, with chronic ketosis and probably ketoacidosis. These animals typically experience substantial weight loss and high mortality during the course of experimental studies. In contrast, the patient with diabetes is rarely, if ever, aninsulinemic, and ketoacidosis, when present, is transient; nor is severe weight loss and early mortality as a result of chronically poor glycemic control a common problem among diabetic patients.
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Because of the severe diabetes in the STZ-diabetic rat, there is valid concern about the relevance of observations in this model to the understanding and management of human diabetes. In an effort to address this problem, Pugliese et al. (1989) introduced a mildly diabetic rat model in which a limited dose of STZ was used to produce only partial destruction of ȕ-cells. These animals retained partial ȕ-cell function, displayed abnormal glucose tolerance, had elevated blood glucose and HbAlc, and gained weight normally. They exhibited less severe, vascular and renal abnormalities, compared to severely diabetic rats. Brennan (1989a,b) selected mildly diabetic animals from a cohort of STZ-treated animals in order to improve survival during a 45-week experiment, while others (Soulis-Liparota et al., 1995; Reaven et al., 1997) have treated STZ-diabetic animals with a maintenance dose of ultralente insulin. We believe that similar regimens should be more widely adopted to limit starvation and weight loss in diabetic animals and that, in any case, the biochemical characterization of these animals should include information on blood pH, pCO2 and levels of ketone bodies. Even with these precautions, however, it should be noted that diabetic rodents are unusually resistant to development of vascular disease and atherosclerosis, common complications of human diabetes. Sugar cataracts are also much more common in rats than humans. The efficacy of aldose reductase inhibitors in diabetic rats, compared to their modest effects on human disease (Williamson et al., 1993), suggests that differences in the severity of diabetes in STZ-rats and in the genetic complement of rodents and humans will limit the broad applicability of studies in rodent models of human diabetes. On the other hand, there are several genetic models of diabetes now available (discussed below) that promise to provide better insights into the pathogenesis of diabetes and its complications. EVALUATION OF THE AGE HYPOTHESIS IN THE STZRAT MODEL Are levels of AGEs and glycoxidation products in tissue proteins consistent with their proposed role in the development of diabetic complications in humans and rodents? In general, CML, pentosidine and other AGEs have been detected first in chemical model systems in vitro, then detected and measured in human tissues, and finally in tissues of shorterlived animal models. The reason for this sequence is technical—AGEs are present at higher concentration and therefore are easier to measure in human than in rodent tissues, regardless of the presence of diabetes (Table 1). In fact, diabetic rats develop microalbuminuria within three months of induction of diabetes, at a time when levels of glycoxidation products in rat skin are much lower than those in skin of young non-diabetic humans (Table 1). One might ask then, how can AGEs be central to the pathogenesis of diabetic complications (or aging) if levels of AGEs are so much lower in shorter-lived animals with diabetic complications than in nondiabetic humans. Are rodents more sensitive or reactive to AGEs? It is also paradoxical that older humans have levels of AGEs or glycoxidation products comparable to those found in younger diabetic patients with complications, yet older humans do not typically have a characteristic spectrum of diabetes-like complications. These observations suggest that, in both rats and humans, the absolute concentrations of
STUDIES ON THE ROLE OF GLYCATION AND AGES 51
AGEs are not a determining factor in the development of diabetic complications or the process of aging. According to the free radical theory of aging (Harman, 1981), oxidative stress and damage are a fundamental mechanism of biological aging. This theory provides an explanation for the inverse relationship between the maximum lifespan of species and their rates of oxidative metabolism and production of reactive oxygen species. Sell et al. (1996) reported recently that the rate of pentosidine accumulation in skin collagen of mammals is inversely related to their maximal lifespan. Since glycemia is comparable in rodents and humans, these observations suggest that oxidative stress is increased in shorter-lived animals, consistent with the free radical theory. However, these authors also noted that the absolute levels of pentosidine were lower in skin collagen of old, but shorter-lived mammals, suggesting that AGEs are an epiphenomenon, rather than an etiological factor, in aging. Lyons (1995) suggested that it is the rate of accumulation of glycoxidation products which is important in the development of diabetic complications. The rate of accumulation of CML and pentosidine is accelerated in diabetic patients, compared to controls, in association with accelerated development of complications in diabetes. The AGEs themselves, which do not cause widespread diabetic complications in the elderly, appeared to be side-products or biomarkers of acceleration of a more fundamental pathological process in diabetes, rather than pathogenically relevant species. Studies on x-ray or radiation-induced cataracts in animals may provide insight into the relationship between AGEs and pathology. Irradiation of the lens of young rabbits leads to rapid development of cataracts, while older rabbits are more resistant to radiation-induced cataracts (Cogan and Donaldson, 1951; Hockwin, 1962). This difference in sensitivity appears to result from more active cell division in young lenses, amplifying radiation-induced damage to DNA, leading to proliferation of abnormal lens epithelia and development of cataracts. In contrast, lower rates of cell division in the mature lens delay the appearance of radiation-induced cataracts in older animals. Similar differences in rates of cell growth and division between young and old humans might explain why tissue levels of AGEs and glycoxidation products appear to be more damaging in younger diabetic than in older non-diabetic individuals. Higher levels of glycoxidation products in young diabetic persons are an indication that these individuals are exposed to greater oxidative damage at an earlier age. Thus oxidative damage, as measured by the formation or accumulation of glycoxidation products, may be the underlying source of injury to endothelial cells, explaining, for example, the loss of pericytes from the retina. These considerations do not explain a priori why rodents develop diabetic complications at much lower levels of tissue AGEs, than found in humans. However, it is possible that rodents are in fact more sensitive to glycoxidation reactions. Consider, for example, that rodents are more susceptible to radiation damage than humans because of their lower capacity for DNA repair. To continue the cataract analogy, if accumulation of AGEs is comparable to radiation-induced damage via oxidative stress, then, by analogy to radiation sensitivity, younger animals (diabetic humans) and shorter-lived animals (rodents) may be more sensitive to AGEs—not necessarily to the glycoxidation products themselves, but to a process that contributes to their formation, i.e. glycative and oxidative stress. In summary, the significant differences in levels of AGEs between humans and rodents with diabetic complications and the fact that accumulation of AGEs does
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not cause diabetic complications in older non-diabetic individuals argues that AGEs are an index of Maillard reaction damage to tissue, but not pathogenically relevant species. However, as with measurement of glycated hemoglobin, which is increased in diabetes without major hematological sequelae, the measurement of AGEs in tissue proteins, blood or urine, whether they are pathogenic or not, may be useful for quantifying the overall rate of tissue damage and for assessing risk for development of complications. Therapies which are designed to minimize the accumulation of AGEs and glycoxidation products may be indirectly beneficial to the diabetic patient by reducing the underlying mechanisms of damage. In all studies on the Maillard reaction and AGEs in diabetes, it is essential to consider that AGEs cannot explain all diabetic complications in all tissues and that there are alternative, non-AGE sources of complications. The following sections discuss other weaknesses of the AGE hypothesis, emphasizing that metabolic imbalances secondary to hyperglycemia, hyperlipidemia and ketoacidosis may be relatively more important in the pathogenesis of complications. CHEMICAL AND IMMUNOLOGICAL ASSAYS FOR AGEs Do current assays for AGEs have adequate sensitivity and specificity for reliable assessment of the role of AGEs in diabetic complications? The requirement for increased assay sensitivity, coupled with the complexity of the GC/MS and RP-HPLC assay procedures, has limited the widespread measurement of specific AGEs in rodent tissues. In some cases, researchers have relied on measures of collagen crosslinking as an index of AGE formation. The extent of crosslinking of collagen has been assessed by measuring its resistance to solubilization in acetic acid or to chemical or enzymatic digestion, or changes in tail tendon elasticity or breaking time. However, interpretation of changes in crosslinking as an index of AGE formation is complicated by the fact that collagen crosslinking may also be altered either nonenzymatically or enzymatically, independent of AGE formation. Alternative nonenzymatic mechanisms include oxidative crosslinking of collagen, either directly by reactive oxygen species or indirectly by products of lipid peroxidation. Changes in enzymatic crosslinking of collagen, altered metabolic turnover of collagen, or changes in the distribution of types of collagen or in the collagen: elastin ratio in tissues may also be inappropriately attributed to AGE-induced crosslinking. For example, oxidative stress induced by ascorbate stimulates the synthesis of collagen by fibroblasts in vitro (Chojkier et al., 1989), as well as the degradation of collagen in vivo, e.g. during the respiratory burst accompanying phagocytosis. Thus, alterations in intracellular oxidative stress, induced by hyperglycemia (Kunisaki et al., 1995; Giugliano and Ceriello, 1996), hormonal imbalances, e.g. hyperinsulinemia (Niskanen et al., 1995) or compromised antioxidant defenses (Oberley, 1988; Godin and Wohaieb, 1988) may also affect collagen synthesis, crosslinking and turnover in tissues in diabetes, independent of effects on AGE formation. In many studies, AGE formation in diabetic animals has been assessed indirectly by measurement of fluorescence at Maillard-type (Ex§370 nm, Em§440 nm) or pentosidine (Ex§325, Em§375 nm) wavelengths. Conclusions based on these measurements are compromised because similar types of fluorescence are produced by direct oxidative modification of proteins by oxygen radicals (Dyer et al., 1990;
STUDIES ON THE ROLE OF GLYCATION AND AGES 53
Huggins et al., 1993), by reactions of lipid peroxidation products with protein (Cominacini et al., 1991; Esterbauer et al., 1992), and even by natural, enzymatically formed crosslinks in collagen, such as pyridinoline. Measurements of collagen crosslinking as an index of AGE formation must also be interpreted with caution. For example, Brennan (1989a,b) observed a significant decrease in acid-soluble collagen in tendons of STZ-diabetic rats, but concluded that the decrease in solubility resulted from maturation of lysine-derived enzymatic crosslinks, rather than from increased crosslinking by AGEs. Thus, increases in crosslinking and fluorescence in collagen of diabetic rats are consistent with the AGE hypothesis, but are not conclusive. ELISA assays are now available for the measurement of AGEs in human crystallins and collagens, plasma proteins, red cells and urine (Araki et al., 1992; Makita et al., 1992; Papanastasiou et al., 1994; Reddy et al., 1995; Ikeda et al., 1996), as well as in rat tissues (Mitsuhashi et al., 1993; Nakayama et al., 1993). Several of these antibodies have also been used for immunohistochemical detection of AGEs in tissues in diabetes and atherosclerosis (Kume et al., 1995; Rumble et al., 1997; Schleicher et al., 1997; Soulis et al., 1997). Most of the anti-AGE-protein monoclonal and polyclonal antibodies are now known to be specific for CML (Reddy et al., 1995; Ikeda et al., 1996), although antibodies specific for pentosidine (Taneda and Monnier, 1994), pyrraline (Hayase et al., 1989; Miyata and Monnier, 1992), imidazolones (Niwa et al., 1997; Uchida et al., 1997) and crosslines (lenaga et al., 1996) have also been described. Because of their simplicity and sensitivity, ELISA assays provide a powerful tool for assessing changes in AGEs in tissue proteins in diabetes and other diseases. As is the case with measurements of crosslinking and fluorescence, however, measurement of unidentified AGEs, i.e. using antisera of unknown antigenic specificity, may yield uninterpretable results. In one recent study with an anti-AGE antibody of unknown specificity, the authors reported that AGEs increased in skin collagen from diabetic patients, but did not increase with chronological age (Beisswenger et al., 1995). This observation indicates that, in contrast to CML and pentosidine, some AGEs may not accumulate in long-lived proteins with chronological age, and that pathology may develop as a result of a steady-state increase in AGEs in tissues. This might occur, for example, with pyrraline, an AGE that is not known to accumulate in proteins with age, but may be a precursor to crosslinks formed in AGE-proteins (Nagaraj et al., 1996). While the observations are intriguing, without knowledge of the specificity of the antibody, it is difficult to interpret the experiments in a mechanistic fashion. Indeed, the specificity of the antibody for AGEs may be in question. During the preparation of AGE-proteins in vitro, many products are formed on the protein, in addition to AGEs. H2O2 is generated, for example, during autoxidation of glucose under conditions used to make the AGEproteins in vitro (Jiang et al., 1990; Elgawish et al., 1996). Oxidative modifications of the protein resulting from exposure to H2O2 may include methionine sulfoxide, 0-tyrosine, dityrosine, hydroxyamino amino acids, dihydroxyphenylalanine or protein carbonyls (Simpson et al., 1992). Any one of these products may be the antigenic determinant recognized by an uncharacterized “anti-AGE" antibody. In summary, because of the complexity and limitations in sensitivity of HPLC and GC/MS assays for specific AGEs, it is more convenient to use indirect measures of AGE formation, such as protein crosslinking, fluorescence and ELISA assays.
54 J.R. REQUENA AND J.W. BAYNES
These assays are especially convenient in animal models where levels of AGEs are much lower than in humans, however lack of specificity may compromise conclusions regarding the validity of the AGE hypothesis. Fortunately, antibodies are now available for measurement of specific AGEs, and ELISA assays using these antibodies should provide more reliable information for assessing the AGE hypothesis in future studies. INHIBITION OF AGE FORMATION Do aminoguanidine (AG) and related carbonyl traps inhibit the formation of AGEs in vivo? Brownlee et al. (1986) observed that AG inhibited development of AGE-like fluorescence during incubation of albumin with glucose in vitro, and that administration of AG also inhibited the increase in fluorescence and crosslinking of aortic collagen of diabetic rats. Kochakian et al. (1996) also demonstrated recently that AG and related compounds inhibited the crosslinking of tail tendon collagen in diabetic rats, although the effects were modest and dependent on the strain and supplier of the rats. Brownlee et al. (1986) proposed that AG acted as a nucleophilic trap for early glycosylation products (reactive carbonyl and dicarbonyl intermediates) and that studies on the mechanism of action of AG should allow “precise experimental definition of the pathogenic significance” of AGE formation in diabetic complications. Numerous studies have confirmed the beneficial effects of AG on the development of nephropathy, retinopathy and neuropathy in STZdiabetic rats (reviewed in Baynes, 1995), and this compound is now in clinical trials. Several AG-related compounds have also been described, which inhibit crosslinking of collagen (Kochakian et al., 1996) and development of nephropathy (Nakamura et al., 1997) in diabetic rats. It is surprising, however, that there are still no published chemical measurements of the effect of AG on formation of specific AGEs in collagen. Thus, the hypothesis that AG inhibits arterial wall protein crosslinking by blocking advanced glycation reactions has not been rigorously tested. Degenhardt (1995) recently reported that AG, administered either by intraperitoneal injection or in drinking water, had no effect on the diabetes-induced increase in CML and pentosidine in rat skin or tail collagen, despite demonstrable inhibition of nephropathy, as measured by reduction in microalbuminuria. A possible limitation of this study is its focus on measurement of AGEs in skin and tail collagen, rather than in renal, retinal or aortic basement membrane proteins. Although it is possible that AG may inhibit AGE formation in a tissue specific manner, e.g. in the kidney or retina, but not the skin, there are strong correlations between ageadjusted levels of glycoxidation products in skin collagen and the severity of complications in other tissues (see above). Both AGEs and intermediates in their formation, such as 3DG, are also present at higher concentration in plasma in diabetes (Makita et al., 1992;Niwa et al., 1993; Papanastasiou et al., 1994; Taneda and Monnier, 1994; Yamada et al., 1994), so that AG inhibition of modification of extracellular proteins should be observed if entrapment of reactive carbonyl compounds were its primary mechanism of action. Degenhardt’s observations raise questions about the mechanism of action of AG in vivo. AG is known to inhibit formation of AGEs by autoxidation of free glucose (Figure 1, Wolff pathway) (Fu et al., 1994; Wells-Knecht et al., 1995) or Schiffbase
STUDIES ON THE ROLE OF GLYCATION AND AGES 55
adducts (Figure 1, Namiki pathway) (Glomb and Monnier, 1995) in vitro. However, the Hodge pathway, involving the Amadori adduct as an intermediate (Figure 1), appears to be the most likely route to formation of AGEs in vivo (Wells-Knecht et al., 1995; Elgawish et al., 1996). Other studies (Requena et al., 1993; Glomb and Monnier, 1995; Khalifah et al., 1996; Booth et al., 1996) indicate that AG is not an effective inhibitor of formation of AGEs from Amadori compounds, providing a possible explanation for the failure of AG to inhibit AGE formation in vivo. Under these circumstances, alternate hypotheses regarding the mechanism(s) of action of AG deserve more serious consideration. AG is a potent inhibitor of amine oxidases, including semicarbazide sensitive amine oxidase (Yu and Zuo, 1997) and nitric oxide synthase (Corbett et al., 1992; Tilton et al. 1993), and also inhibits metal-catalyzed oxidation of lipoproteins (Picard et al., 1992; Requena et al., 1992; Philis-Tsimikas et al., 1995). Indeed, at plasma concentrations of AG attained in experimental animals (~50 µM; Degenhardt, 1995), the drug is much more likely to act as an inhibitor of enzymatic pathways and nonenzymatic lipid peroxidation reactions; much higher concentrations, typically25 mM (Requena et al., 1993; Brownlee et al., 1986; Fu et al, 1992, 1994), are commonly required to inhibit glycoxidation reactions in vitro. Soulis et al. (1997) have also shown that AG and inhibitors of nitric oxide synthase, methylguanidine and nitro-L-arginine methyl ester, are equally effective in inhibiting the increase in CML, measured by radioimmunoassay, in both glomerular and tubular collagen in diabetic rats. These observation suggest that AG inhibits AGE formation, but not through its carbonyl trapping activity. The role of AG as an antioxidant deserves serious consideration. Consider that AG (Kihara et al., 1991; Cameron et al., 1992; Yagihashi et al., 1992) and numerous antioxidants (Cameron et al., 1994; Cotter et al., 1995; Sagara et al., 1996) inhibit the development of neuropathy in STZ-diabetic rats. AG (Hammes et al., 1991; Kihara et al., 1991; Corbett et al., 1992) and vitamin E (Keegan et al., 1995; Kunisaki et al., 1995) also inhibit the development of retinal and vascular complications in diabetic rats. AG has both pro-oxidant and anti-oxidant activity in vitro (Picard et al., 1992; Ou and Wolff, 1993; Philis-Tsimikas et al., 1995) suggesting that, like ascorbate and tocopherol, it may also have antioxidant activity under appropriate conditions in vivo. Rumble et al. (1997) demonstrated increased TGF-ȕl and collagen gene expression in STZ-diabetic rats, characteristic responses to oxidative stress, and reversal of these changes by AG therapy, although the effects of antioxidant supplementation were not explored. Overall, while its proposed mechanism of action as an AGE inhibitor is still unproven, AG is effective in retarding diabetic complications in animal models, albeit severely diabetic animals, suggesting that further studies into its mechanism of action may lead to important insights in the pathogenesis of, and therapy for, diabetic complications. CYTOTOXICITY OF AGE-PROTEINS IN NON-DIABETIC RATS Are AGE-proteins cytotoxic? Do they induce pathophysiology? Injection of AGE-proteins into non-diabetic mice, rats and rabbits induces renal and vascular pathology (Kirstein et al., 1990; Poduslo and Curran, 1991; Vlassara et al., 1992, 1994, 1995a; Zimmerman et al., 1995), including increased vascular permeability and deposition
56 J.R. REQUENA AND J.W. BAYNES
of AGEproteins in the extracellular matrix. These observations have provided a basis for research into the cytotoxicity of AGE-proteins. However, the AGE-proteins used in these experiments were prepared by incubation of proteins for long periods of time, typically several months, with high concentrations of glucose (or more reactive sugars or sugar phosphates) in concentrated phosphate buffers. These brown and fluorescent, highly modified AGE-proteins are questionable models for natural AGE-proteins. Reddy et al., (1995) have shown, for example, that 50–80% of the lysine residues are modified in AGE-keyhole limpet hemocyanin or AGEalbumin, prepared by standard protocols. More than 10% of lysine residues were glycated, i.e. modified with the Amadori adduct, in one preparation of AGEalbumin, while only about 0.5% of lysines are glycated in natural albumin (Baynes et al., 1989). More importantly, 20–3 5 % of the original lysine residues in these proteins were converted to CML, while less than 0.1% of lysine residues in natural albumin is modified as CML. Even skin collagen from older, poorly controlled diabetic patients contains only about 0.2% CML (2 mmol CML/mol lysine) (Dyer et al., 1993). Thus, typical preparations of AGE-proteins are highly modified by known (as well as unknown) products, are brown in appearance, and are hydrophobic and denatured. These proteins may yield a range of biological responses which are not necessarily characteristic of natural AGE-proteins. Although extensive glycation of a protein alters its biological properties, glycation, and probably carboxymethylation of proteins to extents observed in diabetes is unlikely to affect overall protein function in vivo (reviewed in Baynes et al., 1989). In addition to concerns about the excessive modification of artificial AGEproteins, these proteins are prepared in phosphate buffer containing traces of metal O2-, OH•, ions and are exposed to reactive oxygen species, such as H2O2, metaloxo complexes and organic radicals, generated by sugar autoxidation in vitro (Wolff and Dean, 1987;Jiang et al., Wolff et 1991; Elgawish et al., 199 6). Indeed, over 50% of the methionine residues in collagen are oxidized to methionine sulfoxide under conditions traditionally employed for glycoxidation of proteins with glucose (Fu et al., 1992, 1994; Wells-Knecht, 1995). Glycoxidation of proteins in vitro is likely to produce AGE-proteins which are not only both crosslinked and fragmented by oxygen radicals, but are also carriers of reactive and toxic intermediates formed during metal-catalyzed oxidation reactions, includipg amino acid hydroperoxides, protein carbonyls and redox-active compounds such as dihydroxyphenylalanine (Simpson et al., 1992; Gebicki and Gebicki, 1993; Fu et al., 1995). The list of possible modifications on AGE-lipoproteins can be expanded to include lipid derived organic peroxides and aldehydes (Hunt et al., 1990; Lyons and Jenkins, 1996) formed during AGEing of lipoproteins. All of these reactive, potentially toxic products may be present in AGE-proteins, in addition to AGEs, and any of these, rather than true AGEs, may act as the toxic component in AGE-proteins. Some of these products, not necessarily AGEs, may affect vascular tone and permeability, may initiate an oxidative stress response in tissues, and may initiate the irreversible binding of AGEproteins to other proteins in the vascular wall. More anionic and polymeric components may even fix Complement and initiate other metabolic cascades, not necessarily characteristic of natural AGE-proteins isolated from plasma. Another factor to consider in the practical use of AGE-proteins is that they are often injected into animals in large boluses (Zimmerman et al., 1995), or injected chronically at high doses (Vlassara et al., 1992; Yang et al., 1994). The half-life of
STUDIES ON THE ROLE OF GLYCATION AND AGES 57
these artificial AGE-proteins is short, the major fraction being cleared with a halflife of less than 10 minutes (Schmidt et al., 1994a) or returning to baseline values within 60 minutes (Zimmerman et al., 1995). Under these circumstances, large quantities of AGE-proteins are delivered instantaneously to tissues, probably by non-specific phagocytosis of denatured protein, rather than AGE-specific receptormediated processes. Following injection of 125I-AGE-albumin into rats, over 50% of the protein was recovered in liver within 10 minutes (Yang et al., 1991). This observation is at odds with evidence that the kidney has a major role in the normal clearance of AGEproteins from the human circulation (Makita et al, 1991; Hricik et al., 1993; Papanastasiou et al., 1994; Gugliucci and Bendayan, 1996). It also conflicts with a recent report that AGE-LDL, modified to extents observed in vivo, actually has a longer circulating half-life than the native protein in mice (Bucala et al., 1994). A major limitation in the execution and interpretation of many studies on AGEproteins is the failure to consider dose-response and threshold effects in the experimental design. While it is clear that chronic injection of large quantities of highly modified AGE-proteins is pathogenic, it has not been shown that natural AGEproteins, present at plasma concentrations observed in diabetes, have similar properties or induce similar pathology. In summary, it is debatable whether studies on the properties and effects of extensively modified, poorly characterized, heterogeneous AGE-proteins injected into non-diabetic rodents have contributed to an understanding of the metabolism or toxicity of AGEs or the role of AGE-proteins in the development of complications in human diabetes. The high extent of modification and dosage of AGE-proteins used in many experiments makes it difficult to assess the significance of experimental observations regarding the toxicity of AGE-proteins. The resolution of this issue will depend on the isolation of endogenous AGE-proteins by affinity chromatography, using either AGE-receptors or specific antibodies, followed by a careful study of the properties of these proteins in cell culture and in vivo. RECEPTORS FOR AGE-PROTEINS Do receptors for AGE have a role in turnover of AGE-proteins? Nearly a dozen AGEbinding proteins have been identified in various laboratories during the last ten years (Horiuchi et al., 1988; Yang et al., 1991; Skolnik et al., 1991; Schmidt et al., 1992; Wu and Cohen, 1993; Vlassara et al., 1995b). One of these proteins, known as RAGE (Receptor for AGE), has been characterized in greatest detail (Schmidt et al., 1992). RAGE was isolated from bovine lung, along with two other AGE-binding proteins, a lactoferrin-like protein and a non-histone, high mobility group, basic nuclear protein. The assay used for the identification and isolation of RAGE was a microplate assay measuring the binding of putative receptor proteins to wells coated with AGE-proteins. The fact that a nuclear protein with high affmity for AGEprotein was also isolated during the purification of RAGE underscores the potential role of nonspecific, ionic interactions in AGE-receptor interactions. AGE-proteins are anionic because of glycation and carboxymethylation of lysine residues and other modifications of arginine residues (Fu et al., 1994). The microplate assay for RAGE binding to AGE (Schmidt et al., 1992) has an inherent limitation because it may select for proteins which bind adventitiously to AGE-proteins because of charge-charge
58 J.R. REQUENA AND J.W. BAYNES
interactions, rather than by specific recognition of an AGE ligand. For example, the binding of AGE-proteins to the basic enzyme lysozyme (pI§12) (Li et al., 1995) is most likely the result of non-specific interactions. Because of heterogeneous nature of chemical modifications on AGE-proteins and the lack of information on the ligand recognized by AGE-receptors, as well as the lack of specificity of assays used for identification of these receptors, it is likely that some proteins have been wrongly identified as AGE-receptors. In future studies, proteins modified to a similar extent with physiologically irrelevant agents, such as glutaraldehyde, acetic anhydride or fluorescein isothiocyanate, should be used to assess non-specific recognition of modified proteins by candidate AGE-receptor proteins. Some effort should also be made to establish the threshold of modification required for recognition of AGEproteins by these receptors. Receptors that recognize only highly modified AGEproteins, rather than proteins incubated for shorter times or at lower glucose concentrations, may not have a role in the recognition of natural AGE-proteins in blood and tissues. Among the unusual features of RAGE and other AGE-receptors is their distribution in a wide variety of cell types and their multiple binding specifcities. AGE receptors have been described in monocytes, macrophages, lymphocytes, endothelial cells, neuronal cells, smooth muscle cells and mesangial cells of various animals (Takata et al., 1989; Skolnik et al., 1991; Yang et al., 1991; Imani et al., 1993; Schmidt et al., 1994b; Vlassara et al., 1995b). In contrast, other scavenger-type receptors are localized primarily in macrophages, monocytes and endothelial cells, consistent with their function in turnover of modified proteins. RAGE itself also recognizes ligands other than AGE-proteins, such as amphoterin and ȕ-amyloid protein, with 10 to 100 fold higher affinity (Hori et al., 1995), while Leishmania promastigotes compete for the binding of AGE-albumin to a mouse macrophage AGE-receptor (Mosser et al., 1987). Horiuchi and colleagues (Takata et al., 1988; Araki et al., 1995) have also demonstrated that the macrophage scavenger receptor, which has a role in clearance of oxidized LDL from the circulation, mediates the clearance of AGE-proteins from the circulation of rats. Takata et al. (1989) have proposed that the scavenger receptor recognized structural features of aldehydemodified protein, perhaps common structures formed during oxidative modification of proteins by lipids and carbohydrates. Recent work by Fu et al., (1996) supports this hypothesis since the glycoxidation product CML was formed from products of lipid peroxidation during metal-catalyzed oxidation of LDL. The rapid uptake of injected AGE proteins (and oxidized LDL) by liver (Zimmerman et al., 1995) is consistent with a role for the reticuloendothelial system and the scavenger receptor in the clearance of AGE proteins from the circulation. Overall, despite their description in many studies in animal models, the biological relevance of AGE-specific binding proteins is still uncertain. The role of the liver in clearance of AGE-proteins from the rodent circulation, the importance of the kidney in the clearance of AGE-proteins from the human circulation (Makita et al., 1991; Hricik et al., 1993; Papanastasiou et al., 1994) and the long half-life of AGE-LDL in the mouse circulation (Bucala et al., 1994) suggest that AGEreceptors in other tissues may not have a major role in the clearance of AGE-proteins from the circulation. At the same time, it is difficult to explain the detection of AGE-proteins in the circulation, considering the presence of numerous AGE-specific receptor systems and the capacity of the scavenger receptors in the reticuloendothelial system. There
STUDIES ON THE ROLE OF GLYCATION AND AGES 59
is, in fact, no evidence that any of the AGE or scavenger receptors bind natural AGEproteins in plasma. Although putative AGE-proteins have been isolated from plasma using an affinity column with immobilized RAGE and these proteins shown to induce oxidative injury to endothelial cells in vitro (Yan et al., 1994), these AGEproteins were not structurally characterized with respect to their AGE content by chemical or ELISA assays for specific AGEs. Anti-RAGE antibodies also inhibit the clearance of AGE-albumin from the circulation of mice (Schmidt et al., 1994a), but antibody to RAGE may also cause endothelial and reticuloendothelial cell dysfiinction, interfering with endocytic processes. In a related study, a fraction of erythrocytes from diabetic rats were shown to have a shortened lifespan in nondiabetic rats, and their rapid clearance was inhibited, in part, by antibody to RAGE (Wautier et al., 1994). However, it is difficult to interpret these experiments in the absence of evidence that erythrocyte survival is shortened in diabetic rats or that tissue sites of sequestration of erythrocytes in diabetes are consistent with the distribution of RAGE in tissues. The red cell is an attractive model, however, because this cell has a long residence time in the circulation, compared to plasma proteins. Accumulation of arrays of AGEs on its surface may enhance its recognition by AGE receptors. The resolution of issues regarding the specificity and physiological function of RAGE and other receptors for AGE-proteins will require definition of the ligand or motif recognized on AGE-proteins and careful delineation of the differences in recognition and function among AGE receptors, scavenger receptors, and other receptors for denatured and oxidized proteins (Schnitzer and Bravo, 1993). AGES AND OXIDATIVE STRESS Do AGE proteins induce oxidative stress? Stern and colleagues have shown that AGEproteins reduce cytochrome c, i.e. generate superoxide, in vitro and induce oxidative stress in endothelial cells (Wautier et al., 1992, 1994; Schmidt et al., 1994b; Yan et al., 1994) and macrophages (Miyata et al., 1996) bearing the RAGE receptor in vitro. In some cases the cell binding experiments are conducted in serum free medium, so that the cells are already compromised with respect to oxidative defenses and binding is measured in an abnormal, protein deficient environment. Many cellular responses observed under these conditions are characteristic of oxidative stress, including an increase in thiobarbituric acid reactive substances and expression of heme oxygenase, tumor necrosis factor-Į and nuclear factor NF-KB, responses similar to those observed in lung endothelial cells of rats injected with high doses of AGE-proteins (Yan et al., 1994). AGE-proteins also induce increased vascular permeability in endothelial cell monolayers in vitro (Esposito et al., 1989) and hyperpermeability in vivo (Wautier et al., 1996), in both cases probably in response to AGE-protein-induced oxidative stress. However, as noted above, AGE-proteins may carry a complement of reactive oxygen intermediates, such as amino acid hydroperoxides and dihydroxyphenylalanine, so it is uncertain whether the AGEs themselves are the source of oxidative stress. As in other studies with AGE-proteins in animal models, the observations are not in question, but their relevance is uncertain in the absence of evidence that natural AGE-proteins have similar reactivity, and that this activity can be ascribed to specific AGE ligands.
60 J.R. REQUENA AND J.W. BAYNES
Another issue that must be considered in the interpretation of studies on cellular effects of AGE-proteins is that AGE-proteins are glycated proteins, i.e. they may contain substantial amounts of the Amadori adduct FL which is a good reducing agent for iron, and a source of superoxide and dicarbonyl sugars. Thus, in some experiments the Amadori product, rather than AGEs produced at later stages in the Maillard reaction, may be a source of the oxidative stress induced by AGE-proteins. AGEs may also bind or activate metal ions on the protein surface, serving as a source of reactive oxygen. In one study, for example, generation of reactive oxygen by AGEproteins was measured in the presence of lactoferrin-like protein (Yan et al., 1994). This protein could release iron into the medium and contribute to artifactual oxidative stress. These issues can be addressed by assessing the activity of proteins glycated under antioxidative conditions, which permits the formation of Amadori adducts, but inhibits the formation of glycoxidation products and AGEs (Fu et al., 1992, 1994), and by careful exclusion of metal ion contamination from reagents and proteins. SUMMARY AND CONCLUSIONS The roles of glycoxidation, lipoxidation and oxidative stress in diabetes and other diseases. There is increasing evidence for the involvement of oxidation chemistry in the irreversible or cumulative chemical modification of proteins in diabetes and other diseases (reviewed in Baynes, 1991, 1995, 1996; Baynes and Thorpe, 1996; Thorpe and Baynes, 1996). Based largely on studies in animal models, chemical modification of proteins in disease and aging may derive from a number of sources, including hyperglycemia per se (Giugliano and Ceriello, 1996), alterations in metabolite flux through the sorbitol pathway, glycolysis and intermediary metabolism (Williamson et al., 1993), as well as the generalized increases in basal metabolic rate, and oxygen and food consumption in diabetic animals. AGEs and glycoxidation products may participate in a cycle of glycative and oxidative damage, followed by cytotoxicity and cell injury, weakened antioxidant defenses, leading to cell death and a continuing cycle of oxidative damage. This non-enzymatic cycle includes not only AGEs, but also Maillard reaction products derived from lipid peroxidation. Recent studies by Fu et al., (1996) illustrate that at least one AGE/glycoxidation product, CML, is formed during both carbohydrate autoxidation and lipid peroxidation reactions. Immunohistochemical studies show that CML is enriched in plaques formed in dialysis-related amyloidosis (Miyata et al., 1994, 1995), atherosclerosis (Kume et al., 1995) and age-related neuropathies, including Alzheimer’s disease (Smith et al., 1994; Vitek et al., 1994; Yan et al., 1994; reviewed by Colaco and Harrington (1994) and by Thorpe and Baynes (1996)). Other AGEs in these plaque deposits may act as macrophage chemoattractants, leading to inflammation, oxidative stress and propagation of tissue damage. It is not yet certain whether the accumulation of CML in these diseases or in diabetes is derived from glycoxidation or lipoxidation reactions, but oxidation chemistry is implicated in both processes and is likely to be a common feature of degenerative diseases. Strengths and limitations of animal models. Although the STZ-diabetic rodent is the most widely used animal model for studying the role of the Maillard reaction in the development of diabetic complications, the usefulness of rodents is limited by the
STUDIES ON THE ROLE OF GLYCATION AND AGES 61
fact that the Maillard reaction is a slow process under physiological conditions and that rodents are short-lived species, compared to humans. Animal models are also typically acute models of untreated diabetes, leading to complications within a few months, while humans develop complications more gradually over a period of years. Levels of AGEs are significantly lower in animals than in human diabetic plasma and tissues, so that experiments using bolus and chronic injection of highly modified AGE-proteins into non-diabetic animals must be viewed cautiously. The attribution of pathological effects to AGEs must also be balanced against evidence for the presence of other non-AGE, oxidative modifications in AGE-proteins, and the role of AGE receptors in the recognition and catabolism of AGE-proteins must be reconciled with evidence of the stability of AGE-proteins in the circulation and on the role of liver and kidney in catabolism of AGEs in the circulation. Overall, although we have raised questions about the interpretation of a number of experimental approaches used in animal models to evaluate the AGE hypothesis, there is growing evidence, from both animal and human studies, that non-enzymatic chemistry and oxidative damage are involved in the development pathology in diabetes and other age-related diseases (reviewed in Thorpe and Baynes, 1996). Future experiments in animal models will undoubtedly continue to yield insights into the pathogenesis of diabetic complications in humans. A number of genetic models of IDDM and NIDDM are now available to expand the range of animal models for diabetes research, including the db/db and ob/ob mouse and the BB, fa/ fa, OLEFT and NOD rat (Carnaud, 1995; Mathe, 1995; Nakamura et al., 1997). STZ-mouse models of accelerated atherosclerosis in diabetes have also been described recently (Kunjathoor et al., 1996; Reaven et al., 1997), providing unique opportunities for evaluating the role of AGEs, oxidative stress and metabolic derangements in both lipid and carbohydrate metabolism in the development of diabetic complications. ACKNOWLEDGMENT JRR is supported by a postdoctoral fellowship from the Juvenile Diabetes Foundation International. Research in the authors laboratories was supported by National Institutes of Health Research Grants DK-19971 and AG-11472. The authors thank Dr. Suzanne R.Thorpe, Department of Chemistry and Biochemistry, University of South Carolina, for helpful discussion and editorial suggestions.
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Takata, K., Horiuchi, S., Araki, N., Shiga, M., Saitoh, M. and Morino, Y (1988) Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified protein. J. Biol. Chem., 263, 14819–14825. Takata, K., Horiuchi, S., Araki, S., Shiga, M., Saitoh, M. and Morino, Y. (1989) Scavenger receptor of human monocytic leukemia cell line (THP-1) and murine macrophages for nonenzymatically glycosylated proteins. Biochim, Biophys. Acta, 986, 18–26. Taneda, S. and Monnier, V.M. (1994) ELISA of pentosidine, an advanced glycation end product, in biological specimens. Clin. Chem., 40, 1766–1773. Thorpe, S.R. and Baynes, J.W. (1996) Role of the Maillard reaction in diabetes and diseases of aging. Drugs & Aging, 9, 69–77. Tilton, R.G., Chang, K., Hasan, K.S., Smith, S.R., Petrash, J.M., Misko, T.P., Moore, W.M., Currie, M.G., Corbett, J.A., McDaniel, M.L. and Williamson, J.R. (1993) Prevention of diabetic vascular dysfunction by guanidines. Inhibition of nitric oxide synthase versus advanced glycation end-product formation. Diabetes, 42, 221–232. Trachtman, H., Futterweit, S., Maesaka, J., Ma, C., Valderrama, E., Fuchs, A., Taretecan, A.A., Rao, P.S., Sturman, J.A., Boles, T.H., Fu, M.X. and Baynes, J.W. (1995) Taurine ameliorates chronic streptozocin-induced diabetic nephropathy in rats. Am. J. Physiol., 269, F429–438. Uchida, K., Khor, T.O., Oya, T., Osawa, T., Yasuda, Y. and Miyata, T. (1997) Protein modification by a Maillard reaction intermediate methylglyoxal: immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo. FEBS Letters, 410, 313– 318. Vitek, M.P., Bhattacharya, K., Glendening, J.M., Stopa, E., Vlassara, H., Bucala, R., Manogue, K. and Cerami, A. (1994) Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc. Natl. Acad. Sci. (USA), 91, 4766–4770. Vlassara, H. (1994) Recent progress on the biologic and clinical significance of advanced glycosylation end products. J. Lab. Clin. Med., 124, 19–30. Vlassara, H., Brownlee, M. and Cerami, A. (1985) High-affinity-receptor-mediated uptake and degradation of glucose-modified proteins: a potential mechanism for the removal of senescent molecules. Proc. Natl. Acad. Sci. (USA), 82, 5588–5592. Vlassara, H., Fuh, H., Makita, Z., Krungkrai, S., Cerami, A. and Bucala, R. (1992) Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals: a model for diabetic and aging complications. Proc. Natl. Acad. Sci. (USA), 89, 12043–12047. Vlassara, H., Striker, L.J., Teichberg, S., Fuh, H., Li, Y.M. and Steffes, M. (1994) Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc. Natl. Acad. Sci. (USA), 91, 11704–11708. Vlassara, H., Fuh, H., Donnelly, T. and Cybulsky, M. (1995a) Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits. Molecular Medicine, 1, 447–456. Vlassara, H., Li, Y.M., Imani, F., Wojciechowicz, D., Yang, Z., Liu, F.T. and Cerami, A. (1995b) Identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (AGE): a new member of the AGE-receptor family. Molec. Med., 1, 634–636. Wautier, J.L., Wautier, M.P., Schmidt, A.M., Anderson, G.M., Hori, O., Zoukourian, C., Capron, L., Chappey, O., Yan, S.D., Brett, J., Guillausseau, P.J. and Stern, D. (1994) Advanced glycation end products (AGEs) on the surface of diabetic
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Yang, Z., Makita, Z., Horii, Y, Brunelle, S., Cerami, A., Sehajpal, P, Suthanthiran, M. and Vlassara, H. (1991) Two novel rat liver membrane proteins that bind advanced glycosylation endproducts: relationship to macrophage receptor for glucose-modified proteins. J. Exp. Med., 174, 15–524. Yang, C.H., Vlassara, H., Peten, E.P., He, C.J., Striker, G.E. and Striker, L.J. (1994) Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease. Proc. Natl. Acad. Sci. (USA), 91, 9436–9440. Yu, P.H. and Zuo, D.M. (1997) Aminoguanidine inhibits semicarbazide-sensitive amine oxidase activity: implications for advanced glycation and diabetic complications. Diabetologia, 40, 1243–1250. Zimmerman, G.A., Meistrell, M. III, Bloom, A., Cockroft, K.M., Bianchi, M., Risucci, D., Broome, J., Farmer, P, Cerami, A., Vlassara, H. and Tracey, K.J. (1995) Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine. Proc. Natl. Acad. Sci. (USA), 92, 3744–3748.
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3. UTILITY OF THE TRANSGENIC MOUSE IN DIABETES RESEARCH SOROKU YAGIHASHI, SHIN-ICHIRO YAMAGISHI and CHIHIRO NISHIMURA Department of Pathology, Hirosaki University School of Medicine, Hirosaki and Department of Pharmacology, Kyoto Prefectural Medical University, Kyoto
INTRODUCTION Recent drastic increases in the population of diabetic patients urges the medical community to resolve the questions of the pathogenesis of diabetes mellitus and its complications. Clinical epidemiology has given the direction to which basic research should be oriented. The DCCT trial has established the role of metabolic abnormalities and related factors in the development of diabetic complications (The Diabetes and Complications Clinical Trial Group, 1993; Edelman and Henry, 1995). In contrast to clinical studies, animal models are extremely valuable for the longitudinal and systematic studies, which are not feasible in human diabetic patients (Shafrir, 1990; Karasik and Hattori, 1994). It has been difficult, however, to specifically address single factors responsible for the pathophysiology in animal models. Alternatively, studies in in vitro culture systems have demonstrated considerable difficulties in correlating such data with in vivo conditions. The transgenic technology has now been widely used to elucidate the role of regulating or oncogenic genes in complicated life phenomena, human development, organogenesis, immunoregulation, and oncogenesis (Goldwin et al., 1980; Palmiter et al., 1982; Jaenisch, 1988; Hanahan, 1989). It has also made it possible to recapitulate and analyze human genetic disorders and their basis for progression of pathological lesions. In the field of diabetes research, a variety of transgenic animal models have been produced. In relation to the etiology of insulin-dependent diabetes mellitus (IDDM), several immune factors have been demonstrated to be responsible for the autoimmune processes (Eisenbarth, 1986; Todd and Bain, 1992). By this reasoning, the pathogenesis of IDDM is now being examined using transgenic mice incorporating specific genes for cytokines or immunomodulatory or immunogenetic factors or targeting of specific immune-related genes has been attempted (Adams et al., 1987; Sarvetnik et al., 1988; Stewart et al., 1993; Higuchi et al., 1991). On the other hand, genetic loci are now in the process of being clarified in non-insulin dependent diabetes mellitus (NIDDM) (Garvey et al., 1992; Mueckler, 1993). Transgenes for glucokinase or glucose transporters are now employed for studies in NIDDM (Liu et al., 1993; Shepherd et al., 1993). Manipulations to produce mutant Correspondence to: Dr. Soroku Yagihashi, Department of Pathology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036–8562 Japan
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genes or knockouts of these genes have been attempted to recapitulate human NIDDM (Bali et al., 1995; Grupe et al., 1995; Terauchi et al., 1995). Furthermore, mechanisms of diabetic complications are being studied using tratisgenic animals with excessive expression of cytokines, growth hormones, and other peptides (Doi et al., 1991; Pesce et al., 1991). Recently, transgenic animals for a specific enzyme, aldose reductase, were produced for studies on diabetic complications (Yamaoka et al., 1995; Lee et al., 1995; Yagihashi et al., 1996). By the list of these newly developed animal models, our knowledge regarding the mechanisms and pathogenesis of diabetes is expanding. In this review, utilization of transgenic animal models in the field of diabetes research will be introduced and future development of the field will be proposed. TRANSGENIC ANIMALS FOR STUDIES ON THE PATHOGENESIS OF INSULIN DEPENDENT DIABETES (IDDM) IDDM is a disorder with progressive destruction of ȕ-cells largely due to autoimmune processes (Rossini et al., 1993; Skowronski et al., 1990). It is not yet known what kind of factors trigger this autoimmune process. Conceivably, viral infections or toxic substances may initiate the antigen presentation of ȕ-cells. Once, an injury of ȕ-cells occurs, activation of immunological systems which have been tolerant under normal conditions may be perpetuated leading to the progressive destruction of target cells. Immunogenetic factors contribute to the initiation and progression of this immune process (Rossini et al., 1993; Skowronski et al., 1990). With this background, various transgenic mice have been produced by using vectors in which specific candidate genes for the autoimmune process are bound on the lower stream of the insulin promoter (Table 1) (Skowronski et al. 1990; Lipes and Eisenbarth, 1990). The insulin promoter shows high tissue specificity and thereby transgenes can be expressed only in ȕ-cells. These animals if they develop diabetes may serve to clarify the specific genes which may be involved in the pathogenesis of islet ȕ-cell destruction. On the other hand, spontaneously occurring diabetic NOD (nonobese diabetic) mice are used to explore the role of specific genes by incorporating modified immune-related genes to examine as to whether they affect the onset of diabetes (Lund et al., 1990). Viral Antigens Viral infection is considered to be important for the initiation of autoimmune processes in the pancreatic islets. As an exogenous viral antigen, Adams et al. (1987) first introduced SV40 T-antigen into islet ȕ-cells. This model is known to develop insulinoma (Palmiter et al., 1985; Murphy et al, 1987). When T-antigen appears after 10 weeks of age in the ȕ-cells, autoimmune reaction to islet T-antigen develops due to the lack of immune tolerance, thus inducing lymphocytic infiltration in the
*Tumor necrosis factor
Table 1 Transgenic mouse models for insulin dependent diabetes mellitus
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pancreatic islets (insulitis) and antibody production directed against the T-antigen. In this model, however, a diabetic syndrome with hyperglycemia and polyuria was not encountered. Thus the autoimmune reaction to the ȕ-cells occurs by integration of exogenous SV40 T-antigen in this model. It was suggested, however, that the insulitis was not sufficient to cause ȕ-cell destruction (Adams et al., 1987; Skowronski et al., 1990). On the other hand, transgenic mice with incorporated hemagglutinin influenza virus in their ȕ-cells were produced which develop insulitis with antibody production (Roman et al., 1990). It has been shown that a small percentage of these mice do develop hyperglycemia. Ohashi et al. (1991) produced transgenic mice which expressed LCM-virus encoded glycoprotein specifically on the cell surface membranes of ȕ-cells (GP transgenic mice). They also produced transgenic mice which expressed TCR (T cell receptor) in cytotoxic T-cells and crossed TCRtransgenic mice with GP transgenic mice (double transgenic mice). When the hybrid transgenic mice were infected with LCM virus, they developed severe diabetes shortly (3–4 days) after inoculation. In these diabetic transgenic mice, cytotoxic Tcells infiltrated the pancreatic islets and were accompanied by CD4+ T cells. In this model, pretreatment with antibodies to CD4 or CD8 prior to LCM virus inoculation prevented the induction of diabetes. It is therefore likely that both CD4 and CD8 of T-cells may be relevant to the onset of diabetes. In this series of experiments, it has been suggested that cytotoxic T-cells cannot induce immune reaction to ȕ-cells if helper T-cells are not fully activated (Oldstone et al., 1991). These models thus confirm the important role of viral antigens in the induction of autoimmune processes in the pancreatic islets. It still remains unclear, whether human viruses activate immune processes in pancreatic islets in a similar fashion. Apparently, further molecular analysis of viral genomes and their role in initiating the onset of immune reaction in the pancreatic islets are needed. Cytokine Expression in ȕ-cells of Transgenic Mice In pancreatic islets undergoing insulitis, the major infiltrating cells have been demonstrated to be T-lymphocytes, which are involved in the ȕ-cell destruction in IDDM patients (Bottazzo et al., 1985). It is not known, however, whether the lymphocytes directly attack ȕ-cells or if cytokines like interferon (IFN), tumor necrosis factor (TNF), interleukin (IL)-l and IL-2 secreted from lymphocytes are responsible for the ȕ-cell destruction. To answer this question, transgenic mice with an integrated gene of IFN-Ȗ on the insulin promoter have been established (Sarvetnick et al., 1990). In this model, pancreatic islets were infiltrated with lymphocytes and macrophages soon after birth, followed by the destruction of ȕcells. The results indicated that IFN-Ȗ induces stimulatory activity leading to T-cell activation with recruitment of other immunoreactive cells, resulting in an immunesensitizing process. It is not known, however, whether IFN-Ȗ directly injures ȕ-cells or if other factors secondary to the overexpression of IFN-Ȗ may be responsible. Transgenic mice in which ȕ-cells express IFN-Į also exhibit a diabetic syndrome and insulitis (Stewart et al., 1993). On the other hand, transgenic mice which express IL-2, TNFĮ, TNF-ȕ in ȕ-cells of pancreatic islets do not develop diabetes, although insulitis occurs (Picarella et al., 1992, 1993). While TNF-ȕ transgenic mice show marked peri-insular lymphocytic infiltration with perivascular predominance
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(Picarella et al., 1992), TNF-Į transgenic mice show marked intraislet lymphocytic infiltration around the ȕ-cells (Picarella et al., 1993). It has thus been concluded that during the islet autoimmune processes the basis for lymphocytic infiltration is different from that of islet ȕ-cell destruction. This difference may in part be accounted for by the different roles of various cytokines in the islet pathology underlying the development of IDDM. In contrast to the cytokines which activate immune reactions to ȕ-cells, overexpression of IL-10, known to have immunosuppresive function, also elicits conspicuous inflammatory reactions in the exocrine pancreatic ducts but not in the pancreatic islets (Wogensen et al., 1993). ȕ-cell Specific Major Histocompatibility (MHC) Antigen (Constitutive) Expression in Transgenic Mice Overexpression of major histocompatibility (MHC) antigen in ȕ-cells may trigger autoimmunity in IDDM models. In pancreatic islets undergoing insulitis, MHCI antigen expression is enhanced, accompanied by ectopic appearance of Class II antigen in ȕ-cells (Bottazzo et al., 1985, 1983). This is probably caused by influences of excessive cytokines like IFN and TNF. As a consequence, ȕ-cells are themselves committed to antigen-presenting cells and activate T-lymphocytes, resulting in the induction of autoimmune processes predisposing to the development of diabetes. To explore this mechanism, Class I-H2Kb, Class II-I-EbĮ, ȕ or Class II- I-AkĮ, ȕ genes were connected with insulin promoters in transgenic mice so that MHC antigens could be specifically overexpressed in ȕ-cells (Allison et al., 1988; Böhme et al., 1989; Lo et al., 1989). In these mice, severe diabetic conditions developed rapidly, possibly irrelevant to the immune mechanisms, because it was demonstrated that these mice acquired immunological tolerance to MHC antigens. The diabetes is probably accounted for by the overexpression of MHC antigen which itself damages islet ȕ-cells. When I-AkĮ, ȕ were expressed in small amounts, the mice did not acquire immune tolerance and did not develop diabetes (Lo et al, 1989). Thus the expression of MHC I and II did not necessarily cause the autoimmune reaction. MHC-I antigen, a heterodimer molecule located on the cell membrane surface, consists of ȕ2-rnicroglobulin initslightchain. Allison et al. (1991) produced transgenic mice, which expressed ȕ2-microglobulin withtheinsulin promoter (RIPȕ2Mice), in which the insulin content in ȕ-cells was greatly reduced. In addition, when homotypic genes were integrated, severe hyperglycemia developed. When these mice were crossed with mice which expressed MHC I Kb in ȕ-cells, the incidence of diabetes was greatly reduced, in spite of increased expression of H-2Kb on the cell membranes of ȕ cells. In RIP-ȕ2M mice, the ȕ2-microglobulin is present in insulincontaining secretory granules, thereby inhibiting insulin secretion due to interference of ȕ2-microglobulin within the secretory granules. NOD Transgenic Mice In patients with IDDM, a particular type of HLA has been found to correlate with the onset of disease (Todd et al., 1987). MHC Class II antigen molecules are heterodimers which consist of Į- and ȕ-chains. They are expressed in Blymphocytes, macrophages and dendritic cells. Immune reaction of class II antigen
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is mediated by T-cells. In humans, class II antigen consists of loci of DP, DQ, DR and each locus shows polymorphism. The 57th aminoacid in the normal class IIDQȕ gene is aspartic acid, which is replaced by serine or alanine in IDDM patients. Therefore, DQȕ Asp57 is considered to inhibit the onset of IDDM (Todd et al, 1987, 1991). It is interesting to note that the alterations of the MHC II haplotype are compatible with the onset of diabetes in NOD mice (Hattori et al., 1986; Ghosh et al., 1993). Microsatellite analysis of the chromosome demonstrated at least 10 genetic loci (Idd1~10) for the induction of diabetes (Hattori et al., 1986; Prochazka et al., 1987). Idd-1 is linked with MHC region (H-2) on the chromosome 17 (Hattori et al., 1986). The I-A and I-E regions of class II antigen have been shown to be important for autoimmune processes. Insulitis in NOD mice occurs around 4 weeks of age and its incidence reaches 90% at 20 weeks of age and the incidence of diabetes is also high (female 80%, male 20%). In NOD mice, the I-E antigen is not expressed due to the defect in the 5’area of the EĮ gene (Prochazka et al., 1987). This deficit is not specific to NOD mice, but is found in other mice which have H-2 of b, s-haplotype (Hattori et al., 1986; Prochazka et al., 1987). Another characteristic of NOD mice is the specific pleomorphism of the ȕ! domain of the Aȕ chain, which shows replacement of the 57th aminobase of aspartic acid by serine (Ach-Orbea and McDevitt, 1987). This finding is compatible with human IDDM DQȕ. Based on the findings of MHC analysis, transgenic technology has been applied to the NOD mice by introducing specific immune-responsive genes and subsequent evaluations of pathological findings of pancreatic islets and incidence of diabetes. The incorporation of EdĮ gene to express I-E in transgenic NOD mice completely inhibited the onset of insulitis at 20 weeks of age and the subsequent onset of diabetes (Uehira et al., 1989). In addition to I-E gene, the involvement of I-A gene in the onset of insulitis and diabetes was confirmed in the transgenic NOD mice. Incorporation of the AkĮ and Akȕ genes and expression of the I-Ak antigen (the 57th aminoacid is aspartic acid) demonstrated significant inhibition of the incidence of insulitis in these mice (Lund et al., 1990; Uno et al., 1991; Hurtenbach et al., 1993). Recently NOD transgenic mice expressing the TCRĮ gene specifically oriented to islet ȕ-cells derived from NOD mice were produced (Katz et al., 1995; Katz and Benoist, 1995). Rapid onset of insulitis and diabetes were detected in the H-2 hapltotype transgenic NOD mice. On the other hand, the transgenic NOD mice expressing TCRȕ did not develop diabetes, indicating the important role of TCRĮ gene in the development of diabetes in NOD mice. In contrast, NOD mice which incorporated the TCR gene, not directed to NOD islet cells, did not develop diabetes (Lipes et al., 1993). In addition to these transgenic mice, gene targeting technology has also been applied to NOD mice to clarify each factor involved in the pathogenesis of islet cell destruction. NON-INSULIN DEPENDENT DIABETES MELLITUS (NIDDM) MODEL Insulin secretion in response to glucose uptake, and action of insulin in the target organs are the key factors that regulate blood glucose levels. Abnormalities during this process are implicated in the pathogenesis of NIDDM, which can be classified into two major categories; insulin resistance and ȕ-cell dysfiinction with low insulin
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secretion (Moller and Flier, 1991; DeFonzo et al., 1992; Kahn, 1994). Glucose sensing is mediated through an increase in the rate of intracellular catabolism of glucose rather than a ligand-receptor interaction. Specific glucose transporters at the cell membrane facilitate the uptake of glucose into the cytoplasm. Potassiumsensitive ATP channels are then activated to open calcium ion entry, which in turn results in insulin secretion (White and Kahn, 1994). It is likely that there is still a need for a rate-limiting step in glucose catabolism to serve as a glucose sensor. Glucokinase (GK), which catalyzes a rate-limiting step in glucose metabolism, the phosphorylation of glucose to glucose 6-phosphate, has been shown to fulfill this role (Matschinsky and Sweet, 1996). For the insulin secretion, energy production by mitochondria and its utilization for the transport of insulin-containing granules are responsible for the exocytotic secretion of insulin. It is therefore extremely important how mitochondria, microtubules and actin filaments are organized. In the target tissues, insulin first binds with the insulin receptor, followed by phosphorylation at the site of the tyrosine kinase domain, to which the insulin receptor substrate is attached (Efrat et al., 1994). After binding with receptor, the signaling system is activated to nuclear levels. Although mutation of insulin receptor genes at several points are reported, this does not constitute a major factor in the pathogenesis of NIDDM. For the elucidation of the pathogenesis of NIDDM, transgenic technology has been applied by targeting the glucokinase gene, glucose transporters, or genes related to insulin receptors (Table 2) (Epstein et al., 1992; Mueckler, 1993; Treaday et al., 1994). Transgenic mice which overexpress islet amyloid polypeptide (IAPP) has also been produced to explore the amyloidogenesis in the pancreatic islets (D’Alessio et al., 1994; Yagui et al., 1995). Glucokinase (GK) Gene GK is one mediator of the hexokinase family. It has a high substrate specificity for glucose and has a relatively high Km of about 10 mM (versus 0.1–0.001 mM for other hexokinases). Pancreatic ȕ cells and hepatocytes are two major active sites for this particular enzyme (White and Kahn, 1994; Matschinsky and Sweet, 1996). The enzyme has a sigmoidal glucose dependance with an inflection point around the physiological glucose concentration (5 mM glucose). Furthermore, the rate of glucose phosphorylation appears to be significantly less than the rate of glucose entry into the ȕ-cells via GLUT 2, the ȕ-cell-liver glucose transporter. These characteristics are consistent with the possibility that GK is the main glucose sensor in the ȕ-cell (Matschinsky and Sweet, 1996). Recent studies have established that heterozygous point mutations in the GK gene are associated with the development of diabetes in patients with maturity onset of diabetes of the young (MODY), a form of NIDDM (Katagiri et al., 1992; Froguel et al., 1993; Randle, 1993). Similar mutations of the GK gene have been detected in some populations of diabetics in other races (Chiu et al., 1992, 1993). The mutations result in reduced enzymatic activity which causes abnormal glucose sensing and decreased insulin secretion. With this background, Bali et al., (1995) recently established an animal model with gene tartgeting of glucokinase, considered to be a suitable model for MODY. In this model, the expression of GK in the pancreatic ȕ-
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cells and liver is very low due to the incorporation of the mutated GK gene. Reduced islet glucokinase activity is associated with mild hyperglycemia. On the other hand, transgenic knockout of the glucokinase gene elicited marked hyperglycemia and early death in mice, who were completely lacking the GK gene in both hepatocytes and islet ȕ-cells (Grupe et al., 1995; Terauchi et al., 1995). In this model, incorporation of the GK gene with the insulin promoter in ȕ-cells has resulted in reduced hyperglycemia to modest levels and prolonged survival. It has thus been suggested that GK of ȕ-cells has a greater impact on glucose homeostasis than liver GK and provides strong support for the concept that GK is the primary ȕ-cell glucose sensor. Glucose Transporter Gene Glucose entry into the cell is mediated by specific transporters located in the cell membranes. Different kinds of transporters are elaborated in various cells for intracellular uptake of glucose. Different tissues have their specific glucose transporters named GLUTl for erythrocytes, GLUT2 for liver, GLUT3 for brain, GLUT4 for muscle and fat and GLUT5 for small intestine (Bell et al., 1990; Malaisse, 1996). Pancreatic islet ȕ-cells have GLUT2 proteins. Skeletal muslce cells and hepatocytes are the major sites for glucose disposal, mediated by GLUT4 and GLUT2, respectively. For the improvement of glucose uptake to facilitate the clearance of glucose, localization of transporter proteins (GLUT4) in small vesicles are translocated to the plasma membrane in response to insulin and several other stimuli (Kraegen et al., 1993). It has been shown that abnormalities in glucose transporters occur primarily due to mutated transporter genes in a diabetic patient (Mueckler et al., 1994) or as a consequence of insulin deficiency in diabetic BB/W rat (Orci et al., 1990) or glucotoxicity in long-standing hyperglycemia (Ogawa et al., 1995). To explore the pathogenesis of abnormal glucose metabolism and to understand the diabetic condition, molecular manipulations, mainly of GLUT4, have been attempted by using transgenic mice (Liu et al., 1993; Leturque et al., 1996; Tsao et al., 1996). The overexpression of GLUT4 targeted to adipose tissue, by a tissue-specific promoter, induces obesity with extra glucose being taken up and stored as fat (Shepherd et al., 1993). Transgenic mice overexpressing GLUT4 under the regulation of its own promoter in adipose tissue and in skeletal muscle exhibit increased adiposity and improved oral glucose tolerance (Liu et al., 1993). The improvement of hyperglycemia has been demonstrated to be due to more effective insulin action on the overexpressed GLUT4, specifically in muscle cells of streptozotocin-induced diabetic mice (Gibbs et al., 1995; Leturque et al., 1996). The increase in skeletal muscle glucose uptake by GLUT4 has led to an increase in whole body glucose turnover and alterations in lipid metabolism mediated by the improved insulin action on the muscle cells. A similar improvement in blood glucose levels was obtained by selectively overexpressing GLUT4 in skeletal muscle in db/db diabetic transgenic mice with incorporated human GLUT4 (Gibbs et al., 1995). From these experiments, diabetic and insulin-resistant patients should benefit from physiological or pharmacological approaches aimed at specifically increasing GLUT4 protein in skeletal muscle.
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On the other hand, disruption of the GLUT4 gene elicited growth retardation with reduced adipose tissue, and decreased longivity due to cardiac dysfiinction, while maintaining near normoglycemia but with attenuated sensitivity to insulin action (Katz et al., 1995). Consequently GLUT4-null mice can compensate for the lack of the insulin-sensitive glucose transporter by retaining normal levels of blood glucose, yet GLUT4 seems essential for sustaining growth, normal cellular glucose and fat metabolism. Islet Amyloid Polypeptide (IAPP, amylin) Gene The most significant pathologic feature in NIDDM is amyloid deposition in the pancreatic islets (Opie, 1901; Clark et al., 1990). The extent of amyloid deposition increases in parallel with the extent of glucose intolerance. A major component of islet amyloid is islet amyloid polypeptide (lAPP/amylin), a novel 37 aminoacid polypeptide. IAPP is synthesized as an 89 aminoacid precursor that has a typical signal peptide followed by a propeptide containing mature IAPP (Cooper et al., 1987). The amino acid sequence of the central part of IAPP (residues 20–29) varies among the species, and therefore the amyloid deposition is considered to be related to this region, because islet amyloid is formed only in humans and the cat, but not in the rat or the mouse. Thus the IAPP molecule itself may be responsible for aggregation to amyloid fibrils. However, the relationship between increased production of IAPP and islet amyloid deposits in the pathogenesis of NIDDM is unknown. To explore the amyloidogenesis, and its implication in the pathogenesis of NIDDM, transgenic mice which express human IAPP have been produced (D’Alessio et al., 1994; Yagui et al., 1995). D’Alessio et al (1994) developed transgenic mice using a human IAPP cDNA connected to an insulin promoter and confirmed the expression of human IAPP in pancreatic ȕ cells. In these mice, a twofold increase in immunoreactive insulin of the pancreas was detected, without accumulating amyloid in the islet even after 19 months of age. On the other hand, Yagui et al. (1995) could detect fibril formation within the secretory granules. Thus the IAPP transgene elicited increased insulin production which may be related to insulin resistance in NIDDM. Genes of Calmodulin, Insulin Receptor Substrate-1 and Insulin Receptor Transgenic mice carrying a calmodulin minigene regulated by the rat insulin promoter develop severe diabetes right after birth (Epstein et al., 1989). Calmodulin, the primary transducer of the calcium signal, has been implicated as a regulator of insulin secretion in the ȕ-cell (Niki et al., 1981; Watkins and Cooperstein, 1983). A five-fold increase in ȕ-cell calmodulin content induced progressive destruction of ȕcells, consistent with insulin depletion of pancreas in transgenic mice. Abnormal calcium homeostasis thus altered both the function and viability of the ȕ-cell. Various elements in insulin signaling pathways are targets for gene disruption. The cytoplasmic protein insulin-receptor substrate-1 (IRS-1) is the principal substrate for insulin and insulin-like growth factor-1 (IGF-1) receptors that are ligand activated tyrosine kinases (Kasuga et al., 1990). Phosphorylated IRS-1 binds and activates
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several protein kinases thus coupling to a group of signaling pathways (White et al., 1985; Kasuga et al., 1990; Sun et al., 1991). To elucidate how critical IRS-1 is for insulin and IGF-1 action, mice deficient in IRSl were generated (Tamemoto et al., 1994; Araki et al., 1994). The phenotype of these mice was, however, unexpectedly normal, although their growth was substantially retarded with impaired glucose tolerance and a decrease in insulin/IGF-1 -stimulated glucose uptake. These results provided evidence for IRS-1 dependent and IRS-1-independent pathways for signal transduction of insulin and IGF-1. Lately, mice lacking in the insulin receptor gene were generated to investigate whether metabolic and growth-promoting actions of insulin are both mediated by the insulin receptor (Accili et al., 1996). These mice were born at term with normal growth and development, but within hours of birth they developed severe hyperglycemia and died. These data endorse the assumption that the insulin receptor functions primarily as a mediator of metabolic actions. The different results demonstrated between mice with defects in the insulin receptor gene or the IRS-1 gene suggest that multiple components are involved in the insulin receptor signaling pathway. TRANSGENIC MICE FOR STUDIES ON DIABETIC COMPLICATIONS Prevention and treatment of diabetic complications are the most important issue for the clinical management of diabetic patients. The pathogenesis of diabetic complications, represented by the involvement of eye, kidney and peripheral nervous systems, is not fully understood. This is partly due to the lack of suitable animal models which recapitulate the pathology of human diabetic complications. There are several transgenic mice for the study on diabetic complications particularly of kidney complications, using vectors whose constructs consist of genes of growth factors or insulin-like growth factor-1 (IGF-1) (Table 3) (Doi et al., 1991; Pesce et al., 1991; Yang et al., 1993). For the purpose of the pathogenesis of diabetic complications, transgenic mice which express human aldose reductase (hAR), a key enzyme of the polyol pathway, has been produced for the study of diabetic neuropathy and cataract (Yamaoka et al., 1995; Lee et al., 1995; Yagihashi et al., 1996). By using such models, new treatment modalities can now be explored. Growth Factors like Growth Hormone (GH) and Insulin-like Growth Factor (IGF) The kidney disease of diabetes mellitus is a major cause of end-stage renal disease. Its cause appears to be multifactorial. Diabetic glomerulopathy is characterized by early glomerular hypertrophy, followed by mesangial expansion (proliferation and increase in matrix production), the pathological hallmark of glomerulosclerosis. Based on the hypothesis that the humoral growth factors may play a role in the genesis of glomerulosclerosis, transgenic mice for growth factors and oncogenes have been developed (Doi et al., 1988, 1990, 1991; Pesce et al., 1991; Yang et al., 1993). In SV40 transgenic mice, significant glomerular hypertrophy and glomerulosclerosis were detected (MacKay et al., 1988, 1990). However, kidney size
Table 3 Transgenic mouse models for studies of diabetic complications.
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did not parallel glomerular size in this model and it was therefore considered that they were independently regulated. Doi et al. (Doi et al., 1990, 1991) found that in transgenic mice for bovine growth hormone (bGH), human insulin-like growth factor-1 (IGF 1) or human growth hormone releasing factor (hGHRF) all developed progressive glomerular hypertrophy, whereas mesangial proliferation followed by progressive glomerulosclerosis was only depicted in the bGH and hGHRF transgenic mice, as well as glomerulosclerosis, which finally led to death from uremia (Figure 1) (Doi et al., 1988). The microscopic appearance of the glomeruli in bGH mice showed a strong resemblance to that found in the kidney of diabetic patients. In this model, complete glomerulosclerosis characterized by marked mesangial sclerosis occurred at 30 to 37 weeks of age, with albuminuria, leading to death from uremia (Doi et al., 1988, 1991). During the development of glomerulosclerosis, there was a progressive increase in the cell number of glomeruli in the early stage, whereas in the later stages an increase in mesangial matrix and loss of glomerular cells became conspicuous, suggesting increased cell turnover as a significant component of the sclerotic process (Striker et al., 1993). For the expansion of the mesangial matrix, increased mRNA levels of type IV collagen, laminin B2, and basement membrane heparan sulfate (HSPG) proteins appeared to be responsible rather than a reduction of protein turnover (Figure 2) (Striker et al., 1993). In contrast, transgenic mice for insulinlike growth factor-1 (IGF-1) showed only glomerular hypertrophy but not glomerulosclerosis (Doi et al., 1990). Since the IGF-1 mice failed to develop glomerulosclerosis, even though they had higher circulating levels of IGF-1 than bGH mice, the circulating level of IGF-1 did not appear to be important in mediating glomerular proliferation and sclerosis in bGH mice. Thus the development of glomerulosclerosis in bGH mice is associated with an upregulation of type IV collagen mRNA, the appearance of type I collagen mRNA, and an increase in cell turnover. Mutant GH Gene The association between GH and glomerulosclerosis in GH transgenic mice has been supported by the finding that GH deficiency may prevent glomerulosclerosis. Hypophysectomy significantly reduced spontaneous glomerulosclerosis in rats and dwarf rats had only mild sclerotic lesions after subtotal nephrectomy (Yang et al., 1993). It is therefore assumed that the transgene of mutant GH may alter the development of glomerulosclerosis in mice. Individual structural domains of the bGH molecule have been shown to mediate specific physiologic effects (Chen et al., 1990). There is an amphiphilic Į-helical structure in helix III between aminoacid residues 109 and 126. Mice transgenic for mutations in this region in which Į-helix III was destabilized were of normal body size (m-11), whereas, if the Į-helix III was altered to a perfect amphiphilic structure, the mice were dwarfs (m-8) (Chen et al., 1991). The bGH-mll mice developed marked glomerulosclerosis and glomerular enlargement, 1.5 times larger than that of littermate control mice, despite having a comparable body size (Chen et al., 1991). There was a significant correlation between mean glomerular volume and sclerosis index in bGH-mll mice. In contrast, the glomerular histology of dwarf bGH-m8 mice did not differ from littermate control mice. The Į exponent in the allometric equation
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Figure 1 Glomerular hypertrophy found in the growth hormone transgenic mouse. In this figure A, serial changes of mean glomerular volume in growth hormone transgenic mice (GH mice) and their control mice (wild type mice; WT mice) are shown. The glomerular volumes in GH mice were significantly different from those in WT mice (4– 37 weeks, all p < 0.01). The glomeruli in insulin-like growth factor (IGF)-l mice were also significantly larger than in WT mice (7 weeks and 19 weeks, p < 0.01). In figure B, mean body weights in each group are shown. GH mice were significantly heavier than WT mice (4, 30 and 37 weeks, p < 0.05; 7 and 19 weeks, p < 0.01), as were IGF-1 mice (7 and 19 weeks, p < 0.01). This figure is adapted from Doi et al. Am. J. Pathol., 137(3) 541– 552, 1990.
Figure 2 Increased matrix protein production in the glomeruli of growth hormone (GH) transgenic mice. In this figure, results from densitometric analysis of mRNA for extracellular matrix proteins are shown. Data are expressed as the mean ± SE. Open bars are control (wild type) mice and crossed bars are transgenic mice. a, p < 0.05. This figure is adapted from Doi et al.J. Exp. Med., 173(May); 1287–1290, 1991. a, p < 0.05.
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of littermate control and bGH-m8 mice were parallel, suggesting that glomerular development was normally regulated in the bGH-m8 strain. On the other hand, Stewart et al. (1992) have shown that mice transgenic for a mutated human GH gene, not affecting the Į-helix III, demonstrated increased body size and glomerulosclerosis. Furthermore, mice transgenic for a mutated human GH gene which lacked expression of the peptide containing Į-helix III (5kD), showed normal body weight and no renal lesions. These data confirm that (Į-helix III region of bGH is responsible for both body growth and the development of glomerulosclerosis in mice transgenic for both bGH and human GH. However, the IGF-1 binding protein profile, IGF-1 receptor, and the possibility of autocrine/paracrine effects of locally produced IGF-1 on glomerulosclerosis have yet to be examined. Effects of Hyperglycemia on GH Transgenic Mice GH transgenic mice were made diabetic by streptozotocin and compared with nondiabetic transgenic mice and littermate mice (Chen et al., 1995). In these studies GH antagonist (mutated bGH) transgenic mice were made diabetic to test the hypothesis that GH antagonist could protect animals from end organ damage caused by STZ-treatment. Retardation in body growth was characteristic for both GH, GH antagonist (dwarf) transgenic and nontransgenic mice compared with respective nondiabetic animals. Diabetic GH transgenic animals possessed kidney lesions similar to those found in humans with diabetic end-stage organ damage. This may be indicative of a synergistic effect between GH and hyperglycemia in generating a more advanced stage of nephropathy as compared to non-diabetic GH transgenic animals. In contrast, glomerulosclerosis and mesangial lesions were not found in diabetic GH antagonist mice, suggesting the ability of the GH antagonist to protect the transgenic mice from diabetic end-stage organ damage. Thus this system may serve as an animal model for studying human diabetic end-stage kidney damage. Aldose Reductase (AR) Gene Along with the degree of glycemic control, the progress of diabetic complications is known to be affected by various factors including activation of the polyol pathway and enhanced nonenzymatic glycation (Yagihashi et al., 1995). Among these factors activation of the polyol pathway in diabetes was suggested to play a key role in the initial damage leading to derangement of the functional integrity of peripheral nerve, retina and lens (Kinoshita and Nishimura, 1988). It was not fully explained, however, how and to what extent the polyol pathway accounted for the development of diabetic complications. To this end, we (Yamaoka et al., 1995; Yagihashi et al., 1996) and others (Lee et al., 1995) have generated transgenic mice expressing high levels of human aldose reductase (hAR) in various tissues. Since AR is known to be species specific and that the effects of aldose reductase inhibitors are different in different species (Stribling et al., 1985; Nishimura et al., 1991), the transgenic mice for hAR would be ideal to offer valuable information about the dynamic changes of the polyol pathway mediated by hAR. They may also serve as a basis for the clinical application of aldose reductase inhibitors (ARI) in human diabetic patients. In our experiment, the promoter used for the expression of hAR cDNA was derived
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from murine MHC class I antigen, H-2Kd gene (Nishi et al., 1988; Yagihashi et al., 1996). We obtained several founder mice integrating high copy numbers of the transgene (Yagihashi et al., 1996). One of these lines designated as Kd-ARl was crossed to B6D2F1 female mice to generate heterozygous hAR transgenic mice. Unfortunately homozygous transgenic mice could not be obtained since all of the female heterozygous mice were infertile. Northern blot analysis of tissues isolated from the heterozygous mice indicated the presence of hAR mRNA in all of the tissues examined. Simultaneous expression of hAR protein was clearly detected in these tissues by immunoassay as well as by Western blot analysis using specific anti-hAR antibody (Nishimura et al., 1993). The highest level of hAR was demonstrated in the liver, followed by the sciatic nerve and kidney. Very low level of hAR was detected in the lens. In tissues of littermate mice negative for the transgene, only the presence of mouse aldose reductase was detected by Western blot analysis using anti-rat aldose reductase. When the transgenic mice for hAR were fed with 30% galactose for 16 weeks, progressive accumulation of galactitol was detected in sciatic nerve (Figure 3) (Yagihashi et al., 1996). Littermate control mice showed only modest accumulation of galactitol. These biochemical changes were associated with signifiant slowing of motor nerve conduction velocity in galactose-fed hAR transgenic mice. Littermate control mice both with and without galactose feeding did not exhibit significant slowing of nerve conduction, suggesting the activation of polyol pathway was the cause of impaired nerve function. These changes were accompanied by structural changes of myelinated fibers showing severe fiber atrophy in galactose-fed hAR transgenic mice. With progression of the galactose feeding, neuropathic changes became more severe in hAR transgenic mice, indicating sustained activation of the integrated hAR transgene by galactose feeding (Yagihashi et al., 1995). However, in this condition, galactose-fed littermate control mice also showed significant accumulation of galactitol and delay in nerve conduction velocity, although to a lesser extent compared to galactose-fed hAR transgenic mice. Hence long-term galactose feeding led to activation of the polyol pathway and neuropathic changes in mice, which are considered to contain little AR. Myo-inositol levels were equally depressed in both transgenic and littermate control mice, suggesting that myoinositol does not play a principal role in the development of the neuropathy. Under diabetic conditions, hAR transgenic mice showed the functional and structural derangement of peripheral nerve, similar to those encountered in other diabetic animal models and humans (Yagihashi et al., 1994). The transgenic mice made diabetic by streptozotocin injection showed marked delay in motor nerve conduction velocity, and a more severe atrophy of myelinated fibers compared to diabetic nontransgenic mice. Not only the peripheral nerve, but renal glomeruli were also severely affected in diabetic transgenic mice as indicated by glomerular hypertrophy and mesangial expansion, when compared with diabetic nontransgenic mice (Yamagishi et al., 1996). The transgenic mice also showed cataract formation (unpublished observation) in the presence of galactosemia or hyperglycemia. The hAR transgenic mouse therefore appears to be a suitable animal model in which to explore the pathogenesis of diabetic complications with special reference to polyol pathway and the development of specific inhibitors for hAR. The presence of AR and its activation are found to be crucial for the cataractogenesis (Kinoshita and Nishimura, 1988). As the level of intrinsic AR in
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Figure 3 Neuropathic changes detected in galactose-fed transgenic mice which express human aldose reductase. (A) Significant acumulation of dulcitol occurs in the peripheral nerve in a time-dependent manner, when transgenic mice were fed a 30% galactose diet. Littermate control mice also show small accumulation. (B) Motor nerve conduction velocity was significantly reduced in galactose-fed transgenic mice compared with galactos-firee mice. Galactosefed littermate mice showed a small delay which was not significant. (C) Myelinated fiber underwent significant fiber atrophy in galactose-fed transgenic mice. Other groups did not show differences. This figure is adapted from Yagihashi et al., Diabetes Metab. Rev., 11, 193– 225, 1995.
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the mouse lens is low, sugar cataract formation is rarely seen in this species. The possibility that transgenic induction of AR may alter the cataractogensis in mice have been raised by several laboratories, which have demonstrated significant levels of AR mRNA induction in mice lens epithelial cells induced by the transgene of SV40 coupled with the ĮA crystalline promoter (Russel et al., 1990; Limjoco et al., 1991). Lee et al. (Lee et al., 1995) reported generation of transgenic mice that overexpressed hAR exclusively in the lens. They used hAR cDNA fused to the mouse ĮAcrystalline promoter that directs the expression of the hybrid gene to the lens epithelial cells. When the offsprings of these transgenic lines were made galactosemic, they rapidly developed cataract at a rate proportional to the level of AR expressed in the lens, thus confirming the critical role of AR in cataract formation. CONCLUSION Embryonic stem (ES) cell technology has been used to create numerous mouse strains with targeted gene alterations. This approach has promoted our understanding of the functional significance of individual constituents in whole animals. Modifications of diabetes onset by augumentation of specific integrated genes related to autoimmune processes has elucidated the mechanisms of pancreatic islet ȕ cell damage in IDDM animal models. Targeted mutations in genes that participate in insulin action, like glucokinase and glucose transporters have provided important evidence for interpreting their roles in vivo. These transgenic mice can be utilized in the development of animal models of autoimmune-based IDDM as well as polygenically inherited NIDDM. The new gene technology that allows us to add, alter, or eliminate the genes of interest offers a new experimental system to aid in our understanding of the pathogenesis of diabetes and to investigate approaches in its prevention. Up till the present, a large number of transgenic animals and mutant mouse lines with specific structural changes in the genome have been generated worldwide. A computerized database TBASE was developed to organize the information on transgenic animals and targeted mutations (Woychik et al., 1993). Each record in the database contains most of the relevant information on the mouse line, including reference to a corresponding person who can give details about acquiring the line. Unpublished information may also be available through the database if those data are voluntarily deposited and open to the public. As more lines are submitted to this database, it should become a valuable source for those investigators who wish to obtain animal lines useful for diabetic research. ACKNOWLEDGMENT This study was supported in part by a grant from the Japanese Ministry of Science and Education and the Juvenile Diabetes Foundation International.
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Yamaoka, T., Nishimura, C., Yamashita, K., Itakura, M., Yamada, T., Fujimoto, J. and Kokai, Y. (1995) Acute onset of diabteic pathological changes in transgenic mice with human aldose reductase. Diabetologia, 38, 255–261. Yang, C.-W., Striker, L.J., Pesce, C, Chen, W.Y., Peten, E.P., Elliot, S., Doi, T., Kopchick, J J. and Striker, G.E. (1993) Glomerulosclerosis and body growth are mediated by different portions of bovine growth hormone. Lab. Invest., 68, 62–70. Watkins, D.T. and Cooperstein, S.J. (1983) Role of calcium and calmodulin in the interaction between islet cell secretion granules and plasma membranes. Endocrinology’, 112, 766–768. White, M.F. and Kahn, C.R. (1994) The insulin signalling system. J. Biol. Chem., 269, 1–4. White, M.F., Maron, R. and Kahn, C.R. (1985) Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature (Lond), 318, 183–186. Wogensen, L., Huang, X. and Sarvernick, N. (1993) Leukocyte extravasation into the pancreatic tissue in transgenic mice expressing interleukin 10 in the islets of Langerhans. J. Exp. Med, 178, 175–185. Woychik, R.P., Wassom, J.S. and Kingsbury, D. TBASE (1993) A computerized database for transgenic animals and targeted mutations. Nature, 363, 375–376.
4. OXIDATIVE STRESS AND ABNORMAL LIPID METABOLISM IN DIABETIC COMPLICATIONS NORMAN E.CAMERON and MARY A.COTTER Department of Biomedical Sciences, University of Aberdeen, Mariscbal College, Aberdeen AB9 1AS, Scotland U.K.
INTRODUCTION Diabetes causes a state of hyperlipidemia coupled with impaired essential fatty acid metabolism, which has adverse consequences, contributing to the development of diabetic complications. This occurs in the context of increased oxidative stress, defined as an elevated production of reactive oxygen species (ROS) combined with weakened endogenous free radical scavenging capacity. These factors alone, and particularly together, have the capability to damage tissues. A notable target is the vascular system and diabetes is a risk factor for cardiovascular disease and atherogenesis. The purpose of this review is to bring together experimental evidence highlighting the contribution of ROS and abnormalities of lipid metabolism to diabetic complications, with a particular focus on changes in vascular function and their role in neuropathy. OXIDATIVE STRESS AND ANTIOXIDANT THERAPY Reactive Oxygen Species and Cell Damage The role of free radicals in biochemistry and medicine has been the subject of several reviews (Halliwell and Gutteridge, 1989; Sies, 1991; Cheeseman and Slater, 1993). While molecular oxygen is reactive, it becomes damaging when reduced products are formed. The major species are superoxide, O2í•, hydrogen peroxide, and the hydroxyl radical, •OH. The superoxide anion is formed by the addition of an electron to molecular oxygen and is produced by many metabolic reactions. The autoxidation of several small molecules including glucose, ascorbic acid, hydroquinones, catecholamines and thiols is another source greatly enhanced by transition metal ions. Increased ROS production is linked to vascular disease and ischaemia-reperfusion effects resulting from the conversion of xanthine to uric acid by xanthine oxidase (McCord, 1985). Another major source of ROS is leakage from electron transport chains in mitochondria. Phagocytes produce high levels of
Correspondence to: Dr. Norman E. Cameron and Dr. Mary A. Cotter, Department of Biomedical Sciences, University of Aberdeen, Marischal College, Aberdeen AB9 1AS, Scotland U.K. Tel: +44 1224 273013 (Cameron) or 273014 (lab) or 273015 (Cotter), Fax: +44 1224 273019.
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superoxide and peroxide when activated. Superoxide is normally rather short-lived and is converted to hydrogen peroxide by superoxide dismutase (SOD). Hydrogen peroxide is also produced by a variety of metabolic reactions and is directly formed from superoxide via the unstable hydroperoxyl radical.
Hydrogen peroxide is not particularly reactive but is highly diffusible and can move across cell membranes. It is normally rapidly dealt with (see below) but if these mechanisms are impaired hydrogen peroxide will cause damage via formation of hydroxyl radicals. The hydroxyl radical, while very short-lived, is the most toxic of the ROS. Many of the damaging effects of superoxide and hydrogen peroxide result from the formation of hydroxyl radicals by the Haber-Weiss and Fenton reactions. The Fenton reaction is catalyzed by free transition metal ions, particularly iron and copper.
Free radicals damage lipids, proteins and DNA. Polyunsaturated fatty acids are particularly susceptible because the result of activation, for example by a hydoxy1 radical, is a destructive self propagating chain reaction, iilustrated in Figure 1. Thus, the fatty acid radical adds oxygen to generate a peroxyl radical, which is responsible for perpetuating the process by oxidizing further fatty acids. The lipid hydroperoxides so formed can undergo further reactions, catalyzed by transition metals to give more peroxyl radicals. Damage to proteins and DNA is more restricted as chain reactions are not involved. For DNA, oxidation of bases could lead to point mutations if not detected before reduplication. For proteins, oxidation effects could be important if they occurred at an active site; the formation of protein carbonyls and oxidation of sulphydryl groups to form S-S bonds have been noted. As proteins may bind transition metals at specific sites, these would be particularly susceptible to hydrogen peroxide mediated hydroxyl radical attack. One ROS-sensitive target is the glycolytic and mitochondrial phosphorylation of ADP, which could have profound effects on cell energy balance and viability (Hyslop et al., 1988). Free Radical Defense Mechanisms Enzymatic mechanisms comprise SOD in the cytosol (Cu-Zn dependent) and mitochondria (Mn dependent) which scavenges superoxide, normally keeping the intracellular concentration very low. The hydrogen peroxide so formed is converted to water and molecular oxygen by catalase, located mainly in peroxisomes. Glutathione peroxidase, a selenium requiring enzyme, is found in cytosol and
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Figure 1 Lipid peroxidation chain reaction due to activation of lipid (LH) by a hydroxyl ion resulting in the formation of lipid hydroperoxides.
mitochondria and deals with both lipid hydroperoxides and hydrogen peroxide, using glutathione (GSH) as a reductant. Most free radical scavengers are not enzymes, rather they are compounds that react with free radicals but form only poorly reactive radicals themselves. The major constituent of vitamin E, Į-tocopherol, reacts with lipid peroxyl and hydroxyl radicals to form the relatively stable tocopheroxyl radical. Thus, it is termed a chainbreaking antioxidant because it interrupts the self-perpetuating chain reaction of lipid peroxidation outlined in Figure 1. Ubiquinone is another lipid soluble chain breaking antioxidant found in the mitochondrial respiratory chain and in circulating lipoproteins. Carotenoids from plant sources, such as ȕ-carotene and lycopene (in tomatoes), are also effective lipophilic free radical scavengers. In the aqueous phase, aside from GSH, other antioxidants include ascorbic acid, uric acid and cysteine. Ascorbic acid has been shown to regenerate Į-tocopherol from the tocopheroxyl radical in vitro (Packer et al., 1979), which has been suggested to be a potentially important mechanism in vivo although this has been disputed (Burton et al, 1990). In more modern times, addition of antioxidants to food to increase shelf life may add to protection from dietary sources. Commonly used artificial agents are butylated hydroxytoluene and butylated hydroxyanisole. Another way in which the body is protected from oxidative stress is the regulation of free transition metal ion concentrations. Iron is tightly bound by ferritin in tissues and by transferrin and lactoferrin in plasma; similarly copper is bound by ceruloplasmin. Vascular Actions of Reactive Oxygen Species in Diabetes and Effects of Antioxidant Treatment The increased ROS in diabetes has a variety of effects on the vascular system. Oxidation of LDL provides a source of circulating cytotoxic material (Evensen et al., 1983; Morel and Chisolm, 1989) that is implicated in the increased susceptibility to atherosclerosis of diabetes (Lyons, 1991). Vascular endothelium is very vulnerable (Arbogast et al., 1982), and several studies report early morphological
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signs of damage in experimental diabetes (Arbogast et al., 1984; Moore et al., 1985; Lin et al., 1993), presumably because of oxidant injury. Vessels from diabetic rats are more susceptible than normal to injury by ROS generating systems in vitro and in vivo (Pieper and Gross, 1988; Chang et al., 1993; Cameron et al., 1994a). Hyperglycemia compromises the glutathione redox cycle, rendering endothelium and red blood cells more susceptible to peroxide and xenobiotic induced damage (Tagami et al., 1992; Kashiwagi et al., 1994; Yoshida et al., 1995). In addition to physical damage, exposure to oxidative stress causes changes in gene expression and endothelial function. The local renin-angiotensin system is upregulated in several tissues including mesenteric vessels and vasa nervorum (Maxfield et al., 1993, 1995; Cooper et al., 1994). Circulating plasma angiotensin converting enzyme (ACE) levels are increased in diabetic patients (Hallab et al., 1992) and rats (Cameron et al., 1994a), in the latter case this was prevented by treatment with the antioxidant, probucol. Oxidative stress also stimulates endothelin-1 production, probably via nuclear factor-kB (NF-kB) (Collins, 1993; Rubanyi and Polokoff, 1994). Elevated circulating levels of endothelin-1 are seen in diabetic subjects with microvascular disease and in diabetic rats where increased production by mesenteric vessels has also been demonstrated (Takahashi et al., 1990; Takeda et al., 1991). Both angiotensin II and endothelin are potent vasoconstrictors, therefore increased synthesis would promote reduced tissue blood flow. Activation of NF-kB also increases leucocyte adhesion to endothelium, and may contribute to the enhanced thrombosis and atherosclerosis in diabetes, which would further exacerbate vascular complications (Ceriello, 1993; Collins, 1993). Antioxidants such as a-tocopherol, probucol and acetyl cysteine inhibit monocyte adhesion to endothelial cells (Faruqi et al., 1994). For peripheral nerve, reduced nutritive perfusion leading to endoneurial hypoxia has been strongly implicated in the etiology of neuropathy in both animal models and diabetic patients (reviewed in Low et al., 1989; Cameron and Cotter, 1994a; Tesfaye et al, 1994). Pharmacological blockade of the renin-angiotensin system, using ACE inhibitors or angiotensin II AT1 receptor antagonists largely corrects deficits in nerve blood flow, endoneurial oxygenation and NCV in diabetic rats as well as preventing the increased resistance to hypoxic conduction failure and a blunting of regenerative capacity following nerve injury (Cameron et al., 1992a; Maxfield et al., 1993, 1995). Single center short term open label trials in diabetic patients have shown improvements in NCV and sensory thresholds with ACE inhibitor treatment (Reja et al., 1995; Al-Memar et al., 1996) and encouraging preliminary results of a larger placebo controlled double-blind study have recently been reported (Malik et al., 1997). Furthermore, ACE inhibitors are commonly used antihypertensive agents in diabetes and slow the progression of nephropathy (Elving et al., 1994). In diabetic rats, ACE or AT1 inhibition partially protected against myopathic changes in cardiac and skeletal muscles (Cameron et al., 1992a; Boy et al., 1994) and vessel wall hypertrophy and deficits in nitric oxide (NO) mediated relaxation of mesenteric vessels (Cooper et al., 1994; Olbrich et al., 1996). The effects of endothelin-1 antagonists have been examined in less detail, however, for peripheral nerve, the ETA receptor antagonists BQ 123 and BMS 182874 corrected impaired blood flow and NCV in diabetic rats (Cameron et al., 1994c; Cameron and Cotter, 1996a), whereas the nonspecific ETA/ETB antagonist, bosentan, had more modest effects (Stevens and Tomlinson, 1995). The lesser action
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of blocking both ETA and ETB receptors compared to ETA probably depends on altered endothelial vasodilator action. In many vascular beds, stimulation of endothelial ETB receptors causes NO and prostacyclin release, the vasodilator effects of which offset ETA-mediated contraction of vascular smooth muscle (Rubanyi and Polokoff, 1994). Thus, ETA blockade would eliminate the vasoconstrictor action of endogenous endothelin-1, while still allowing the ETB-stimulated component of NO/ prostacyclin release, both of which favor increased blood flow. In contrast, joint ETA —ETB blockade while reducing vasoconstriction also inhibits the vasodilator components; the result being a diminished effect on flow, hence NCV. Oxidative stress diminishes vessel endothelium-dependent relaxation, which is apparent in some experimental preparations even after acute exposure to hyperglycemia. Superoxide neutralizes NO (Gryglewski et al., 1986) and the peroxynitrite so formed is a source of hydroxyl radicals that can cause endothelial damage (Beckman et al., 1990). Incubation of rabbit aorta or rat mesenteric vessels in high-glucose containing Ringers solution for several hours induces a reduction in endothelium-dependent relaxation to acetylcholine that may be partially prevented by antioxidant (probucol) pretreatment or addition of SOD to the tissue bath (Tesfamariam and Cohen, 1992; Taylor and Poston, 1994). This deficit depends both on the NO mechanism and an increase in the production of vasoconstrictor prostanoids, the cyclooxygenase pathway being a further source of ROS. In contrast, rat aorta is fairly resistant to acute high glucose exposure (Archibald et al., 1996). However, many studies have shown deficits in endothelium-dependent vasorelaxation in chronic experimental diabetes (reviewed in Cameron and Cotter, 1992, 1994a; Cohen, 1993; Poston and Taylor, 1995). This has also been observed in type I and type II diabetic subjects (McVeigh et al., 1992; Elliott et al, 1993; Johnstone et al., 1993; Nitenberg et al., 1993; Morris et al., 1995). In some in vitro studies endotheliumdependent relaxation of diabetic tissues may be partially improved by acute addition of SOD or antioxidants to the bathing fluid (Hattori et al., 1991; Diederich et al., 1994), although others have reported negative findings (Otter and Chess-Williams, 1994). Impaired endothelium-dependent relaxation of aorta, corpus cavernosum and coronary vessels of diabetic rats can be prevented by chronic treatment with free radical scavengers, such as butylated hydroxytoluene, dimethylthiourea and Įtocopherol, or the sulfhydryl donor acetyl cysteine, as shown in Figures 2A and B for the last two treatments respectively (Keegan et al., 1995, 1997; Rösen et al., 1995; Archibald et al., 1996; Pieper et al., 1996). Some of these antioxidants prevent or correct impaired nerve blood flow and NCV (Cameron et al., 1993a; Cotter et al., 1995a; Love et al., 1996a; Sagara et al., 1996), as do other scavengers including probucol and probucol analogues, lipoic acid, ȕ-carotene, GSH and ascorbic acid (Bravenboer et al., 1992; Cameron et al., 1994a; Cameron and Cotter, 1995a; Cotter et al, 1995a; Karasu et al., 1995; Nagamatsu et al., 1995). Recently, Į-lipoic acid treatment has been shown to prevent dysfiinction of small nitrergic vasodilator fibers that innervate corpus cavernosum in diabetic rats, which could be potentially important for the treatment of diabetic impotence (Keegan et al., 1997). The effects of probucol on nutritive blood flow, endoneurial oxygen tension and NCV are shown in Figure 3. Acetyl cysteine and Į-tocopherol treatments also prevented axon dwindling and the blunting of regenerative capacity following nerve damage in diabetic rats (Love et al., 1996a, 1997; Sagara et al., 1996). As with large vessels such
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Figure 2 Endothelium-dependent relaxation to acetylcholine in vitro for phenylephrineprecontracted aortas from diabetic ( ) and nondiabetic ( ) rats. Diabetes duration was 2 months, and the effects of chronic treatment with (A) (Į-tocopherol (1g kgí1 dayí1) and (B) acetyl-L-cysteine (250 mg kgí1 dayí1) fromdiabetes induction are shown ( and dashed lines) In both experiments, there was a deficit in maximum relation to acetylcholine and antioxidant treatment provided a high degree of protection. Data are means ± SE (group n=13í44). See Keegan et al. (1995) and Archibald et al. (1996) for further details.
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Figure 3A–C Effects of the diabetes and probucol treatment on A, sciatic nutritive endoneurial blood flow, B, mean endoneurial oxygen tension and C, motor nerve conduction velocity. Experimental groups are identified as C, nondiabetic controls; D, 2 month diabetic controls; DP, 2 month diabetic rats treated with the free radical scavenger probucol (1g kgí1 dayí1) from induction. For all measures, the diabetic deficit was markedly attenuated by probucol treatment. Data are means+SE (group n=9í16) See Cameron et al. (1994a) for further details.
as aorta, it is likely that free radical scavenger effects on nerve blood flow were mediated by an improvement of endothelium-dependent relaxation in the epi and perineurial resistance vessels of vasa nervorum, where a diabetic NO deficit has been located (Kihara and Low, 1995; Maxfield et al, 1997). Different antioxidant treatments have varying efficacy, although both lipid and water soluble drugs such as probucol and acetyl cysteine can be highly effective. However, ascorbic acid on its own was not very effective and it exhibited a biphasic dose response curve such that high doses were less effective than low doses (Figure 4A) in correcting diabetic NCV deficits (Cotter et al., 1995a). This may be due to an action as a prooxidant due to autoxidation (Hunt et al., 1992). A very high level of treatment with antioxidants such as Į-tocopherol is needed to protect against nerve dysfunction in diabetic rats, more than an order of magnitude above the normal dietary intake; the dose-response curve for correcting NCV deficits is shown in Figure 4B. This suggests that a better therapeutic approach might be to prevent ROS production rather than scavenging them once formed. The autoxidation of glucose and some of the reactions of the advanced glycation process produce ROS, catalyzed by trace amounts of transition metal ions, which are also necessary for the production of damaging hydroxyl ions by the Fenton reaction (Baynes, 1991; Wolff, 1993; Fu et al, 1994). Low-dose transition metal chelator treatment with deferoxamine or trientine rapidly corrected nerve blood flow and NCV deficits in diabetic rats (Cameron and Cotter, 1995b). Defective vasa nervorum NO action in diabetes, presumably due to increased ROS production, causes increased reactivity of epi and perineurial vessels to vasoconstrictors such as norepinephrine (Maxfield et al., 1997). An example is shown in Figure 5 for 8 week diabetic rats, deferoxamine treatment over the last 2 weeks completely returning sensitivity to normal. Chronic trientine treatment also prevented the development of impaired endothelium-dependent relaxation of aortas from diabetic rats (Keegan et al., 1996). Thus, metal catalyzed autoxidation and related reactions are an
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Figure 4A, B Dose-response curves for the effects of ascorbate and Į-tocopherol on nerve conduction velocity. A, effects of ascorbate treatment for 2 weeks after 6 weeks of untreated diabetes on sciatic motor ( ) and saphenous sensory ( ) nerves. Note the biphasic shape of the curves. B, effects of Į-tocopherol treatment in protecting against the development of sciatic motor conduction deficits over 1 month of diabetes. Data are means ± SE (group n=6í10). See Cotter et al, (1995a) for further details.
extremely important source of ROS in diabetes, making a large contribution to neurovascular deficits in experimental models. In man, the acute cardiovascular effects of elevated ROS production in diabetes (Wieruszwysocka et al., 1995) can be seen by monitoring blood pressure changes during vascular infusion of antioxidants such as acetyl cysteine (Ceriello et al., 1991). These cause a reduction in blood pressure in nondiabetic subjects and an even greater fall in diabetic subjects without microvascular complications. This suggests a widespread involvement of ROS-related mechanisms in the control of tone in the major vascular beds, and could potentially be explained by greater ROS neutralization of NO in diabetes. Markers of endothelial damage which result, at least in part, from long term ROS exposure, such as elevated von Willibrand factor, have been linked with the development of nerve and renal complications in diabetic subjects (Yaqoob et al., 1993; Plater et al., 1996). In the retina of diabetic rats there is an early reduction in blood flow paralleling that for nerve (Bursell et al., 1992; Cameron et al., 1991a). This is accompanied by increased de-novo synthesis of diacylglycerol (DAG), which exerts an effect on retinal vessels by stimulation of protein kinase C (PKC). The reduction in blood flow with diabetes can be acutely mimicked in nondiabetic rats by vitreal injection of phorbol esters to stimulate PKC (Shiba et al., 1993). Conversely, treatment of diabetic rats with a ȕ isoform specific PKC inhibitor corrects the retinal perfusion
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Figure 5 Dose-response curves for superfusion of sciatic vasa nervorum with norepinephrine containing physiological solutions. Alterations in blood flow were monitored by laser-Doppler flowmetry and the data are expressed as changes in vascular conductance to eliminate the effects of blood pressure fluctuations. Compared to the curve for nondiabetic rats (O), there was a marked leftward shift towards greater sensitivity after 8 weeks of diabetes ( ) This was corrected by deferoxamine treatment (8 mg kgí1dayí1) during the last 2 weeks of diabetes (Cameron and Cotter, unpublished observations). Data are means ± SE (group n=8í12). See Maxfield et al. (1997) for methodological details.
deficit. For the kidney, PKC inhibition also prevented the early increase in glomerular filtration rate and urinary albumin excretion (Ishii et al., 1996). Treatment with Į-tocopherol reduces DAG levels in aorta and cultured vascular smooth muscle (Kunisaki et al., 1994), probably via a mechanism involving increased DAG kinase activity (Lee et al., 1996). It is not known whether other antioxidants have this action. If so, the mechanism depends on a reduction in oxidative stress, otherwise it reflects a nonantioxidant effect of Į-tocopherol. The DAG—PKC mechanism is unlikely to have direct relevance for neurons or Schwann cells because nerve DAG levels are reduced by diabetes and PKC activity is either unchanged or diminished (Zhu and Eichberg, 1990; Kim et al., 1991; Borghini et al., 1994; Mathew et al., 1997). However, it is plausible that elevated PKC activity in vasa nervorum contributes to reduced nerve blood flow and therefore function. Recent experiments have shown that PKC inhibition increased nerve blood flow in diabetic rats. At low doses, NCV was improved whereas at high doses it remained at the diabetic control level despite improved nerve perfusion. Thus, high levels of PKC inhibition have a
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direct deleterious effect on nerve fibers in diabetic rats, although it is not known which PKC isoforms are involved since the inhibitors used in that investigation were relatively non-specific (Cameron et al., 1997a). Tissue Markers of Oxidative Stress and Antioxidant Treatment Effects in Non-vascular Tissues As well as establishing that ROS in diabetes have deleterious neurovascular effects, recent research has revealed evidence of oxidative modification of nerve components and alterations in endogenous free radical scavenging systems. Thus, within one month of diabetes induction in rats, there was an increase in sciatic nerve lipid peroxidation and decreases in SOD and GSH levels with no changes in glutathione peroxidase and reductase (Low and Nickander, 1991; Nickander et al., 1994; Nagamatsu et al., 1995). Changes in SOD were prevented by insulin treatment that moderated but did not abolish hyperglycemia (Low and Nickander, 1991). In nondiabetic rats fed a diet deficient in the natural antioxidant, Į-tocopherol, NCV deficits were noted. The diabetic NCV reduction was also markedly exacerbated by this diet, as were the increase in conjugated dienes, a marker of lipid peroxidation, and the decrease in GSH (Nickander et al., 1994). In nondiabetic rats, prooxidant treatment to increase ROS caused NCV and blood flow abnormalities (Cameron et al., 1994a). Increased lipid peroxidation is also found in plasma, red cells, aorta, kidneys and most other tissues in diabetic rats and patients (Jain et al., 1991; Lyons, 1991;Ceriello, 1993; Chang et al., 1993; Yaqoob et al., 1993). ROS effects, amenable to antioxidant treatment, have also been implicated in diabetic cardiomyopathy (Dai and McNeill, 1995), embryopathy (Eriksson and Borg, 1991) and the formation of lens cataracts (Ross et al., 1983). Roles of Advanced Glycation and the Polyol Pathway in Oxidative Stress Glucose and its metabolic products, including fructose formed by the polyol pathway, are highly reactive and nonenzymatically glycate free amino groups on proteins and other molecules. From this reversible step, a series of progressively less reversible reactions follow: the formation of Schiff’s base, Amadori products (for example, HbAlc) and then a diverse set of advanced glycation end products (AGEs) (Baynes, 1991; Brownlee, 1992; Bucala et al, 1995). There is an accumulation of AGEs in virtually all tissues; for example, in peripheral nerve they have been localised to endoneurial and perineurial vessels, axons and Schwann cells in diabetic rats (Sugimoto and Yagihashi, 1995). Crosslinking of structural proteins is increased by AGE reactions (Fu et al., 1994), which could alter their function. Many cells have receptors for AGEs and their activation causes changes in gene expression via the free-radical sensitive transcription factor, NF-KB (Schmidt et al., 1994) which may be suppressed by antioxidant treatment (Bierhaus et al., 1997). AGEs also have direct vascular effects via their ability to quench NO (Bucala et al., 1991). The advanced glycation process is a source of ROS (Baynes, 1991) and contributes to the oxidation of LDL, which further promotes vessel dysfunction (Bucala et al., 1994). There is positive feedback in this pathogenetic mechanism; ROS increase the formation of
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AGEs, the process being termed glycoxidation or autoxidative glycation (Baynes, 1991; Wolff, 1993). Aminoguanidine, although not an antioxidant or metal chelator, prevents AGE formation by irreversibly binding with reactive carbonyl intermediates (Fu et al., 1994). Chronic treatment of diabetic rats with aminoguanidine attenuates the development of impaired depressor responses to intra-arterial acetylcholine in vivo and protects against reduced endotheliumdependent relaxation in vessels studied in vitro (Bucala et al., 1991; Archibald et al., 1996). For peripheral nerve, aminoguanidine treatment prevents impaired nutritive blood flow and NCV in diabetic rats (Kihara et al., 1991). The effects of aminoguanidine on NCV are relatively rapid, reversal of an established deficit occurring within 12 days of the start of treatment. Similarly, nerve blood flow is easily corrected following a period of untreated diabetes (Cameron and Cotter, 1996b). This suggests that the short-term effects of the advanced glycation process are not caused by a buildup of AGEs, which have a low turnover rate so that nerve dysfiinction would not be rapidly reversible. Rather, along with the effects of antioxidant treatment in suppressing AGE formation and protein crosslinking (Fu et al., 1994), and improving neurovascular function (Cameron et al., 1994a; Nagamatsu et al., 1995), it may be argued that the inhibition of ROS production by glycoxidation is the most relevant action of aminoguanidine. This emphasizes the importance of flux through the advanced glycation pathway rather than steady state AGE levels for diabetic neurovascular pathogenesis. As with antioxidants, the effects of aminoguanidine on nerve blood flow and NCV are abolished by cotreatment with a NO synthase inhibitor (Cameron and Cotter, 1996b), which further sustains the view that the action is predominantly vascular. This is supported by the lack of effect of aminoguanidine treatment on nerve lipid peroxidation (Kihara et al., 1991), which suggests that there is little direct neuronal effect. Aminoguanidine treatment also has beneficial actions on other tissues in experimental diabetes. These include attenuation of the increased pericyte loss, endothelial proliferation and incidence of acellular capillaries in the retina (Hammes et al., 1994, 1995), prevention of the blunted autoregulatory response of muscle arterioles (Hill and Ege, 1994), partial normalization of large vessel distensibility (Huijberts et al., 1993), and attenuation of mesangial expansion and albuminuria in the kidney (Soulis-Liparota et al., 1991). The extent to which these benefits depend simply on prevention of AGE accumulation and protein crosslinking as opposed to the formation of ROS is not known. However, free radical scavengers have beneficial effects on kidney (SoulisLiparota et al., 1995) and aorta (Kunisaki et al., 1994; Keegan et al., 1995; Archibald et al., 1996). The polyol pathway is also involved in oxidative stress, and ARI treatment prevented the accumulation of malondialdehyde, a marker of lipid peroxidation, in nerves of diabetic rats (Lowitt et al., 1995). As with antioxidants and aminoguanidine, ARI treatment prevents deficits in NO-mediated endothelium-dependent relaxation in aortas from animal models of diabetes (Cameron and Cotter, 1992, 1993; Tesfamariam et al., 1993; Otter and Chess-Williams, 1994). Furthermore, ARIs prevent or correct deficits in nerve blood flow, endoneurial oxygen tension, conduction and regenerative capacity in diabetic rats (Calcutt et al., 1994; Cameron et al., 1994b, 1996a,b; Hotta et al., 1995; Love et al., 1995). ARIs promote changes in the pattern of endoneurial perfusion, favoring flow through the capillary bed at
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Figure 6 Relations between polyol pathway activity, advanced glycation and glucose autoxidation in oxidative stress. ROS are generated by glucose by autoxidation and glycation and polyol pathway flux leads to impaired endogenous scavenging. The oxidative stress reduces endoneurial perfusion, which leads to nerve dysfunction. Advanced glycation end products (AGEs) also contribute. Note the dashed lines indicating positive feedback in the scheme. Free radicals increase advanced glycation, and impaired perfusion generates more free radicals. The targets for drug intervention are shown by the curly arrows.
the expense of arteriovenous shunt flow (Cameron et al., 1994b, 1996a, b). This is very similar to vascular effects observed with acetyl cysteine treatment of diabetic rats (Love et al., 1996a). Acetyl cysteine is a GSH precursor, which may give a clue to the mechanism of ARI action. Aldose reductase requires NADPH as a cofactor, as does glutathione reductase in the glutathione redox cycle. Thus, when polyol pathway flux is high, a competitive NADPH deficit will impair the recycling of GSH from GSSG. This reduces the neutralization of peroxide by the glutathione redox cycle, hence contributing to an ARI-preventable component of oxidative stress. In lens and sciatic nerve of diabetic and galactose-fed rats, GSH is markedly diminished and this is prevented by ARI treatment (Lou et al., 1988; Hohman et al., 1997). It appears that the first half of the polyol pathway is crucial for the short term effects on nerve in diabetes. In the galactosemic rat model, galactose is converted to galactitol by aldose reductase, however galactitol is a poor substrate for sorbitol dehydrogenase and it is not further metabolized by the second step of the polyol pathway. Galactosemia causes similar ARI-preventable effects on nerve conduction, regenerative capacity and vascular function to those found in diabetes (Cameron et al., 1992b; Cameron and Cotter, 1993; Mizisin and Powell, 1993; Kamijo et al., 1994). These defects are also attenuated by free radical scavenger and transition metal chelator treatments (Love et al., 1996b), emphasizing the importance of the glycoxidation process in this model of polyol pathway hyperactivity. Furthermore, inhibition of the second polyol pathway step in diabetic rats, sorbitol to fructose conversion by sorbitol dehydrogenase, had little effect on nerve blood flow and
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NCV (Cameron et al., 1997b). This stresses the importance of the aldose reductase catalyzed step and shows that the contribution of polyol pathway fructose production to the glycoxidation process is relatively minor. Thus, the polyol and advanced glycation pathways make a marked contribution to oxidative stress mechanisms as part of their pathophysiological action, and the interrelationship is schematized in Figure 6. This scheme is applicable not only to diabetes but also to the galactosaemic model because galactose participates in autoxidation reactions, as well as being a good glycating agent and a substrate for aldose reductase. It may also be relevant for other models where nerve complications result from ingestion of high levels of sugars or other substances prone to autoxidation, such as the fiicose-fed rat, however in that case increased polyol pathway activity is not involved (Yorek et al., 1993). While the hypothesis is applicable to nerve and perhaps some other vascular beds, it may not cover all tissues. Thus, comparison of the effects of aminoguanidine, butylated hydroxytoluene and the ARI, ponalrestat, on renal collagen fluorescence and urinary albumin excretion in diabetic rats showed that while aminoguanidine markedly attenuated these measures, butylated hydroxytoluene had somewhat lesser effects on both whereas ponalrestat was ineffective (Soulis-Liparota et al., 1995). However, this was a single dose study, and other investigations have shown that very high levels of polyol pathway blockade or free radical scavenger treatment are required for optimal effects in nerve (Cameron et al., 1994b; Cotter et al, 1995a). Thus, the dose of ponalrestat in particular, but also butylated hydroxytoluene, used in that renal study may have been too modest. ALTERED FATTY ACID METABOLISM AND DIABETIC COMPLICATIONS The abnormalities in lipid and lipoprotein levels in diabetes contribute to the increased risk of micro and macrovascular complications (Ginsberg, 1991; Lyons, 1991). Glycation and oxidation of LDL are atherogenic and changes in VLDL composition involving an enrichment of free and esterified cholesterol also constitutes a major risk factor. Fatty acid handling by tissues is compromised by reduced levels of L-carnitine in diabetes (Rodrigues et al., 1988). Furthermore, the metabolism of n-6 and n-3 essential fatty acids is inhibited by diabetes-induced impairments of delta-6 and delta-5 desaturation (Horrobin, 1988). This is particularly unfortunate as unsaturated fatty acids are a major target for ROS damage, thus, enhanced rather than reduced availability of these essential components is likely to be required in diabetes (Horrobin, 1991). Essential Fatty Acids and Neurovascular Dysfunction The n-6 essential fatty acid deficit has proved to be particularly important for neurovascular dysfiinction. Figure 7 outlines the production of vasoactive prostanoids from the main dietary source, linoleic acid. There is an alternating series of desaturation and elongation steps, the former being relatively slow, particularly for delta-6 desaturation. Diabetes further depresses hepatic desaturation, probably by a combination of effects due to hyperglycemia, hypoinsulinemia and oxidative
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Figure 7 Scheme for the production of vasoactive prostanoids from n-6 series essential fatty acids. The normal dietary source is linoleic acid (18:2ní6) In diabetes, defects in desaturation, particularly at the delta-6 step, result in reduced production of prostanoids. PG, prostaglandin; TX, thromboxane.
stress (Horrobin and Carmichael, 1992), the result being reduced plasma and tissue levels of Ȗ-linolenic acid (GLA), dihomo-GLA and arachidonic acid (ARA). Thus, the desaturation defect limits the synthesis of cyclooxygenase products; vasodilator and anti-aggregation prostaglandins (PG) I2, and E2 from ARA and PGE1 from dihomoGLA, as well as the platelet aggregator and vasoconstrictor, thromboxane (TX) A2, from ARA. Synthesis of PGI2 by sciatic nerve epi and perineurial vessels is reduced by diabetes, mainly because of diminished ARA availability (Ward et al., 1989). Treatment of diabetic rats with evening primrose oil (which contains approximately 9% GLA) bypasses the delta-6 desaturation deficit, and improves vasa nervorum PGI2 production (Stevens et al., 1993). This suggests that while delta-5 desaturation is also impaired, the delta-6 defect predominates and is rate limiting. Chronic evening primrose oil treatment prevents the development of impaired nerve blood flow, endoneurial hypoxia, reduced NCV and increased resistance to ischemic conduction failure in diabetic rats (Julu, 1988; Cameron and Cotter, 1994b; Cameron et al., 1991b, 1996a; Karasu et al., 1995). Interestingly, evening primrose oil also improves
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nerve function in the galactosemic rat model of polyol pathway hyperactivity (Dines et al., 1995a). The effects of evening primrose oil are mimicked by pure GLA and the triglycerides tri-GLA and di-linolein mono-GLA; the latter is the predominant form of GLA in evening primrose oil and is, therefore, the active component (Dines et al., 1993, 1995b; Cameron and Cotter, 1996c). Chronic GLA and evening primrose oil treatments caused an increase in endoneurial capillary density in diabetic and galactosemic rats (Cameron et al., 1991b, 1993b; Dines et al., 1993, 1995a), further supporting the view that their main action is on vasa nervorum rather than directly on nerve fibers themselves. The vascular and NCV effects of GLAcontaining oils are completely blocked by cyclooxygenase inhibition, emphasizing the importance of prostanoid synthesis for their action (Cameron et al., 1993b, 1996a; Dines et al., 1995b). There also appears to be an interaction with the NO pathway since evening primrose oil effects on NCV and blood flow are attenuated by NO synthase inhibition (Cameron et al., 1996a). This may be a result of the increase in blood flow promoted by GLA-stimulated prostanoid synthesis. Flowinduced shear stress enhances NO production by vascular endothelium (Pohl et al., 1991), which would cause further vasodilation to magnify the GLA effect. Consequently, blockade of NO synthesis would reduce the apparent neurovascular action of GLA. While evening primrose oil’s efficacy is accurately predicted from the GLA content (Dines et al., 1995b), this is not the case for some other GLA-rich oils. Thus, a study on correction of motor and sensory NCV in diabetic rats ranked the effectiveness of evening primrose > fungal > borage > blackcurrant oil when equated for GLA content (Dines et al., 1996). This ranking agrees with that obtained for the ratio of 6-keto PGF1Į/TXB2 (stable metabolites of PGI2 and TXA2 respectively) outflow from the mesenteric vascular bed of nondiabetic rats in response to these oils (Jenkins et al., 1988). The differences in prostanoid production are presumably because natural oils have complex compositions and there may be metabolic interactions between components. For example, blackcurrant and borage oils also contain n-3 essential fatty acids whereas fungal and evening primrose oils do not. The n-3 series use the same enzymes that metabolize n-6 fatty acids .There is mutual competition, with the n-3 fatty acids exerting a larger inhibitory effect than the n-6 series (Lands, 1992). Treatment of diabetic rats with fish oil, which contains n-3 eicosapentaenoic acid, only had very modest effects on NCV. However, fish oil cotreatment markedly attenuated the effects of evening primrose oil and GLA on NCV, resistance to ischemic conduction failure and endoneurial capillary density (Cameron et al., 1991b; Dines et al., 1993). While fish oil has some beneficial effects on the general vasculature in diabetic subjects, involving improvements in endothelium-dependent relaxation (McVeigh et al, 1993), it has also been reported to increase plasma endothelin-1 levels in microalbuminuric patients (Selvais et al., 1995), which could be considered potentially deleterious. A further possible vascular benefit of fish oil is that competition with arachidonic acid metabolism results in preferential synthesis of TXA3 which is much less potent for platelet aggregation and vasoconstriction than TXA2 (Lands, 1992). However, at least in diabetic rats, increased platelet activation and TXA2 synthesis do not appear to make a major contribution to nerve dysfunction as treatment with a TX receptor and synthase antagonist only slightly improved NCV (Dines et al., 1996). n-3 Essential fatty acid treatment exacerbated retinopathy in diabetic rats (Hammes et al., 1996), the mechanism is unknown but it could relate to inhibition of n-6 prostanoid metabolism.
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Figure 8 Comparison of the effects of evening primrose oil, rich in Ȗ-linolenic acid, and arachidonic acid on (A) sciatic nutritive endoneurial blood flow and (B) motor conduction velocity. C, nondiabetic group; D, 8 week diabetic control group; DEPO, diabetic rats treated with evening primrose oil for 8 weeks (dose of Ȗ-linolenic acid ~ 800 mg kgí1 day í1); DARA, 8 week diabetic rats treated for the last two weeks with arachidonic acid rich oil purified from a fungal oil source (dose of arachidonic acid ~ 400 mg kgí1 dayí1). Bothtreatment markedly attenuated the diabetic flow and conduction deficits. Data are means ± SE (group n=8í12). Data obtained from Cameron and Cotter (unpublished observations and 1994b) and Cotter and Cameron (1997).
Certainly, PGI2 analog treatment prevented the development of electroretinographic abnormalities in diabetic rats (Hotta et al., 1996a). Thus, the vascular actions of n-6 essential fatty acids are likely to explain their beneficial effects on neuropathy in experimental models. Evening primrose oil/GLA effects on NCV and nerve blood flow may be mimicked by treatment with ARArich oils in diabetic rats (Cotter and Cameron, 1997) as illustrated in Figure 8. This reinforces the view that delta-6 rather than delta-5 desaturation deficits are rate limiting for prostanoid synthesis and suggests that PGI2 or PGE2 are the active vasodilator metabolites. In support of this proposition, PGI2 analogues have very similar effects to GLA on NCV, blood flow, resistance to ischemic conduction failure and endoneurial capillary density in diabetic rats (Ohno et al., 1992; Cotter et al., 1993; Hotta et al., 1996a). Multicenter clinical trials of evening primrose oil revealed improvements in NCV, evoked potential amplitudes, hot and cold perception thresholds, muscle strength, tendon reflexes and sensation in treated patients compared with a deterioration in a placebo group (Keen et al., 1993). Non-vascular Effects of Essential Fatty Acids Essential fatty acids are components of cell membranes and influence their structure and fluidity, the function of membrane proteins, and electrical characteristics. Sciatic
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nerve glycerophospholipid composition is altered in diabetic rats, with a decline in arachidonyl-containing molecular species (Zhu and Eichberg, 1993) as predicted from the defective essential fatty acid desaturation. This may also account for the reduced DAG content of sciatic nerve (Zhu and Eichberg, 1990). DAG activates PKC as does the inositol triphosphate derived from phosphoinositide breakdown. In turn, PKC can phosphorylate a subunit of Na+-K+-ATPase, increasing its activity. Therefore, depression of this mechanism, perhaps via reduced turnover of phosphoinositides due to myo-inositol depletion, was postulated to be responsible for reductions in Na+-K+-ATPase activity, hence NCV (Greene et al., 1992). Although phosphoinositides are a major source of DAG in nondiabetic nerves, compensatory changes occur in diabetes, with phosphatidylcholine becoming an important precursor (Zhu and Eichberg, 1993). NCV improvements by prostanoid analogues in diabetic rats are accompanied by an increase in nerve Na+-K+-ATPase activity, stimulated via an elevation in neuronal cAMP (Sonobe et al., 1991). In contrast, evening primrose oil treatment did not increase Na+-K+ pump activity in diabetic rats (Lockett and Tomlinson, 1992). This suggests that n-6 essential fatty acid effects are not mediated by a direct neuronal prostanoid action to alter Na+-K +-ATPase activity. Carnitine Metabolism, Vascular Function and Diabetic Complications Another metabolic change in diabetes, which has a potential effect on other aspects of fatty acid related metabolism, is a reduction in plasma and tissue L-carnitine content (Vary and Neily, 1982; Ido et al., 1994). Treatment with L-carnitine or the acetyl and proprionyl derivatives has been used to prevent cardiomyopathy (Rodrigues et al, 1988), neuropathy (Cotter et al, 1995b; Lowitt et al., 1995; Hotta et al., 1996b,c; Sima et al, 1996; Cameron and Cotter, 1997) and electroretinographic changes related to retinopathy (Lowitt et al., 1993; Hotta et al., 1996b) in experimental models. In nondiabetic rats, acetyl-L-carnitine improves nerve regeneration following crush injury and reduces senescence-related deterioration in neuromuscular function (De Angelis et al, 1992; Scarfo et al, 1992). Correction of nerve morphological abnormalities and an increase in regenerating fibers was noted with long term acetyl-L-carnitine treatment in the bio-breeding Worcester genetically diabetic rat model (Sima et al., 1996). The mechanisms of L-carnitine action have been the subject of some debate. Cellular energy metabolism could be improved as L-carnitine increases the transport of long chain fatty acids into mitochondria for ȕoxidation. This would have the further advantage of reducing the accumulation of long chain acyl derivatives including DAGs in diabetes, which can alter membrane processes, for example sarcoplasmic reticulum Ca2+-ATPase activity (Lopaschuk et al, 1983). Acetyl-L-carnitine has also been suggested to have neurotrophic properties; it stimulates nerve growth factor receptor synthesis in cultured rat phaechromocytoma cells, which increases neurite outgrowth in response to nerve growth factor (Taglialatela et al., 1991). Acetyl-L-carnitine may be an antioxidant; treatment prevented an increase in malondialdehyde content in an ischaemic mouse brain model (Fariello and Calabrese, 1988) and in sciatic nerves of diabetic rats (Lowitt et al., 1995), and
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protected cultured fibroblasts from free radical damage (Tesco et al., 1992). In keeping with a putative antioxidant action, L-carnitine and its derivatives increase nerve blood flow in diabetic rats, proportionate to the increase in NCV (Cotter et al., 1995b; Hotta et al. 1996b,c). Furthermore, the effects of L-carnitine on NCV and blood flow are abolished by cotreatment with a NO synthase inhibitor (Cameron and Cotter, 1997), which provides further indirect support for the notion that the primary action is vascular. L-Carnitine derivatives also have direct neurochemical effects. A 30% deficit in sciatic nerve carnitine content was seen in diabetic rats, associated with a halving of DAG content and Na+-K+-ATPase activity. Treatment with acetyl-L-carnitine restored nerve L-carnitine levels and prevented the Na+-K+-ATPase deficit (Ido et al., 1994; Sima et al., 1996). However, nerve DAG content was not restored; instead acetyl-L-carnitine treatment caused a further reduction. Thus, the mechanism of improved Na+-K+-ATPase activity in this case does not depend on an elevation of DAG causing stimulation of PKC as proposed by Greene et al (1992) and outlined above. It is also possible that the Na+-K+-ATPase deficit in diabetes is not sufficiently severe to be the main factor causing impaired NCV. Thus, vasodilator and evening primrose oil treatment studies have shown that normal NCV can be found in the presence of a diabetic level of nerve Na+-K+-ATPase activity (Cameron et al., 1991c; Lockett and Tomlinson, 1992). While there does not appear to be a direct link between nerve function and neuronal DAG/PKC mechanisms in the action of L-carnitine derivatives, these elements could be involved in the beneficial effects on nerve blood flow, as shown in Figure 9. Thus, long chain fatty acid entry into mitochondria is enhanced by the availability of L-carnitine for the transporter. Subsequent disposal by ȕ-oxidation would reduce the accumulation of DAG caused by the esterification of acyl-CoA. DAG stimulation of PKC has vascular effects involving a decrease in NO production by endothelium (Cohen, 1993), and a sustained contraction of vascular smooth muscle due to phosphorylation of contractile elements (Shimamoto et al., 1993). Thus, by indirectly reducing the stimulation of PKC, L-carnitine would have a vasodilator action in diabetes. This hypothesis could account for L-carnitine’s effects on nerve blood flow. It is also consistent with the effects of PKC inhibitors on the deficit in nerve and retinal blood flow in diabetes rats (Ischii et al., 1996; Cameron et al., 1997a) and the actions of acetyl-L-carnitine and vasodilator treatment on electroretinographic changes (Lowitt et al., 1993; Hotta et al., 1996b). Clinical trials of acetyl-L-carnitine treatment on neuropathy produced sufficiently disappointing effects to cause early termination. This may have been predictable from the very high doses found necessary to produce benefits in rat models, which far exceeded those used in the trials (Ido et al., 1994; Cotter et al, 1995b; Hotta et al., 1996b,c). INTERACTIONS BETWEEN OXIDATIVE STRESS AND FATTY ACID MECHANISMS A major effect of oxidative stress in diabetes is to reduce the effectiveness of NOmediated vasodilation whereas essential fatty acid dysmetabolism attenuates prostanoid mediated vessel relaxation. Together, such pathophysiological changes
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Figure 9 Relation between fatty acid ȕ-oxidation. the carnitine mitochondrial transporter, activation of protein kinase C (PKC) and neurovascular dysfunction. The L-carnitine deficit in diabetic tissues reduces transport of acyl-coA into the mitochondria for ȕoxidation. The resultant accumulation leads to an elevation of diacylglycerol and stimulation of PKC. In turn this has deleterious effects on vascular endothelium nitric oxide (NO) release and also stimulates a sustained contraction of vascular smooth muscle. Together these contribute to reduced nerve perfusion, which may be partially corrected by L-carnitine dietary supplementation.
promote increased vascular reactivity to endogenous vasoconstrictors. However, these individual mechanisms do not act in isolation but interact (De Nucci et al., 1988) to form a large part of the local blood flow control system in most tissues, including peripheral nerve. Therefore, it is not surprising that changes in one mechanism can affect the others. High levels of oxidative stress inhibit cyclooxygenase (Moncada et al., 1976), vitamin E restores reduced prostacyclin synthesis in aortic endothelial cells cultured under a high glucose concentration and also normalizes the PGI2/ TXA2 ratio in diabetic rats (Karpen et al., 1982; Kunisaki et al., 1992). As a polyunsaturated fatty acid, GLA is susceptible to free radical destruction and a high level of antioxidant treatment would have a protective effect, thus indirectly promoting PGI2 synthesis. Antioxidant treatment would also protect neurons and vascular cells from cumulative damage caused by ROS, and essential fatty treatment would complement this by supplying components necessary for the repair of their membranes (Horrobin, 1988, 1991; Zhu and Eichberg, 1993; Nickander et al., 1994). In experiments on nondiabetic rats designed to mimic some of the vascular changes in diabetes and assess the consequences for nerve function, chronic treatment with a low dose of a cyclooxygenase or a NO synthase inhibitor caused modest NCV reductions. However, with combined treatment, there was a 5-fold amplification of drug effects on NCV compared to that expected for simple summation (Cameron et al., 1993c). This demonstrates a marked synergism between
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Figure 10 Effects of diabetes and treatment with low doses of Ȗ-linolenic acid or the free radical scavenger, BM150,369, alone or in combination, on (A) sciatic motor conduction velocity and (B) endoneurial nutritive blood flow. C, nondiabetic group; D, 8 week diabetic control group; DG, diabetic group treated for the last 2 weeks with Ȗ-linolenic acid (20 mg kgí1 dayí1); DB, diabetic group treated for the last 2 weeks with BM150,369 (6 mg kgí1 dayí1); DGB, diabetic group given 2 weeks joint Ȗ-linolenic acid—BM150,369 treatment. The horizontal dashed lines indicate expected flow and velocity values for a simple additive interaction between these 2 drugs. This level was greatly exceeded in the joint treatment group, indicating a synergistic interaction. Data are means ± SE (group n=10í14). See Cameron and Cotter (1996c) for further details.
blockade of the prostanoid and the NO systems, suggesting that they normally act in a mutually compensatory manner to limit nerve perfusion changes. The converse effect, joint treatment of diabetes with drugs that improve the NO and prostanoid mechanisms, could potentially have therapeutic value. Thus, a combination of low doses of an ARI and evening primrose oil resulted in NCV and blood flow improvements matching those obtained with an approximately 8-fold increase in the dose of either drug alone (Cameron et al., 1996a). A similar interaction, illustrated in Figure 10, was seen for joint treatment with GLA and the free radical scavenger, BM150,639, a probucol analogue (Cameron and Cotter, 1996c). In that case there was an approximately 7.5-fold amplification of individual drug effects. Recent experiments have examined hybrid drugs combining antioxidant and n-6 essential fatty acid moieties, for example, ascorbyl-6-GLA. While ascorbate is not particularly effective against NCV deficits in diabetic rats (Figure 4A), combination with GLA increases lipid solubility and therefore the ability to enter cell membranes. Compared to GLA alone, ascorbyl -6-GLA was 4.4-fold more efficacious in correcting NCV (Cameron and Cotter, 1996d). More recently, a GLA-lipoic acid combination proved to be extremely potent (Cameron et al., 1998). These synergistic effects, seen when different vascular mechanisms are targeted, contrast with results when both
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drugs affect the same mechanism. Thus, joint treatment of diabetic rats with ascorbate and Į-tocopherol gave NCV improvements that were in good agreement with predictions based on a simple summation of individual drug effects (Cotter et al., 1995a). SUMMARY AND CONCLUSIONS From the evidence considered in this review, it is clear that defective nerve perfusion is a very important cause of functional abnormalities in experimental models of diabetes. Although the evidence is less abundant, there are also strong indications that neurovascular insufficiency contributes to human diabetic neuropathy (Tesfaye et al., 1994). Animal experiments suggest that oxidative stress and impaired essential fatty acid metabolism are two fundamental pillars in the pathophysiological process as far as vasa nervorum is concerned. The main result of defective n-6 essential fatty acid desaturation is a reduction in prostanoid-mediated vasodilation, reduced PGI2 synthesis from ARA being the likely mechanism. Increased oxidative stress in diabetes depends on autoxidation of glucose and its metabolites, but also includes the advanced glycation process and the polyol pathway. A major target of ROS appears to be NO action. Together, essential fatty acid and oxidative stress mechanisms give rise to reduced vasodilation of vasa nervorum, increased reactivity to endogenous vasoconstrictors, and prothrombotic state. Oxidant damage to the vascular system, perhaps coupled to reduced repair capacity due to diminished availability of essential fatty acids, causes further changes in local vasoconstrictor production (angiotensin II and endothelin-1) which contribute to impaired nerve perfusion and which are also involved in atherogenesis. While these mechanisms primarily apply to peripheral nerve, it is plausible that they are relevant for some other tissues, for example the retina. This will depend on the special characteristics of the vascular bed involved; the capacity for autoregulation, the degree of neural control of the vasculature and hence susceptibility to autonomic neuropathy, the sensitivity to circulating vasoactive substances, and nonvascular alterations in the tissue being supplied. Vascular changes in the kidney appear to be rather unique; there is an early hyperperfusion phase following diabetes induction which is absent in nerve and retina (Cameron et al., 1991a; Wright and Nukuda, 1994; Ishii et al., 1996) and increases in NO and prostanoid synthesis have been implicated (Larkins and Dunlop, 1992; Bank and Aynedjian, 1993). Nonetheless, some renal changes appear to respond to treatments targeting oxidative stress, including antioxidants, ARIs and aminoguanidine (Bank et al., 1989; Soulis-Liparota et al., 1995). While the vasculature may be considered the primary or at least the early target for oxidative stress and essential fatty acid dysmetabolism, the longer term contribution of cumulative ROS-mediated tissue damage and problems with repair must not be overlooked in the etiology of diabetic complications. Similarly, although the contributions of the polyol pathway and advanced glycation have been considered in terms of oxidative stress in this review, these mechanisms also have other unrelated actions, for example, sorbitol accumulation in the pathogenesis of lens cataracts. Thus, recent research has revealed several mechanisms that are important for diabetic complications, including some that are amenable to drug treatment. The synergism noted between treatments targeting oxidative stress and n-6 essential fatty
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Keen, H., Payan, J., Allawi, J., Walker, J., Jamal, G.A., Weir, A.I., Henderson, L.M., Bissessar, E.A., Watkins, P.J., Sampson, M., Gale, E.A.M., Scarpello, J., Boddie, H.G., Hardy, K.J., Thomas, P.K., Misra, P. and Halonen, J.-P. (1993) Treatment of diabetic neuropathy with Ȗ-linolenic acid. Diabetes Care, 16, 8–15. Kihara, M. and Low, P.A. (1995) Impaired vasoreactivity to nitric oxide in experimental diabetic neuropathy. Exp. Neurol., 132, 180–185. Kihara, J., Schmelzer, J.D., Poduslo, J.F., Curran, G.L., Nickander, K.K. and Low, P.A. (1991) Aminoguanidine effects on nerve blood flow, vascular permeability, electrophysiology and oxygen free radicals. Proc. Natl. Acad. Sci. USA, 88, 6107–6111. Kim, J., Rushovich, E.H., Thomas, T.P., Ueda, T., Agranoff, B.W. and Greene, D.A. (1991) Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocindiabetic rats and its correction by dietary myo-inositol. Diabetes, 40, 1545– 1554. Kunisaki, M., Bursell, S., Umeda, R, Nawata, H. and King, G.L. (1994) Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes, 43, 1372– 1377. Kunisaki, M., Umeda, F., Inoguchi, T. and Nawata, H. (1992) Vitamin E restores reduced prostacyclin synthesis in aortic endothelial cells cultured with a high concentration of glucose. Metabolism, 41, 613–621. Lands, W.E.M. (1992) Biochemistry and physiology of n-3 fatty acids. FASEB J., 6, 2530– 2536. Larkins, R.G. and Dunlop, M.E. (1992) The link between hyperglycaemia and diabetic nephropathy. Diabetologia, 35, 499–504. Lee, I.-K., Koya, D., Ishii, H. and King, G.L. (1996) Vitamin E (vit E) prevents hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle by an increase of DAG kinase activity. Diabetes, 45(Suppl. 2), 64A. Lin, S.-J., Hong, C.-Y, Chang, M.-S., Chiang, B.N. and Chien, S. (1993) Increased aortic endothelial cell death and enhanced transendothelial macromolecular transport in streptozotocin-diabetic rats. Diabetologia, 36, 926–930. Lockett, M.J. and Tomlinson, D.R. (1992) The effects of dietary treatment with essential fatty acids on sciatic nerve conduction and activity of the Na+/K+ pump in streptozotocindiabetic rats. Br. J. Pharmacol., 105, 355–360. Lopaschuk, G.D., Tahiliani, A.G., Vadlamudi, R.V.S.V., Katz, S. and McNeill, J.H. (1983) Cardiac sarcoplasmic reticulum function in insulin or carnitine treated diabetic rats. Am. J. Physiol., 245, H969-H976. Lou, M.F., Dickerson, J.E., Garadi, R. and York, B.M. (1988) Glutathione depletion in the lens of galactosemic and diabetic rats. Exp. Eye Res., 46, 517–530. Love, A., Cotter, M.A. and Cameron, N.E. (1995) Impaired myelinated fiber regeneration following freeze-injury in rats with streptozotocin-induced diabetes: involvement of the polyol pathway. Brain Res., 703, 105–110. Love, A., Cotter, M.A. and Cameron, N.E. (1996a) Effects of the sulphydryl donor, NacetylL-cysteine, on nerve conduction, perfusion, maturation, and regeneration following freeze-damage in diabetic rats. Eur. J. Clin. Invest,, 26, 698–706. Love, A., Cotter, M.A. and Cameron, N.E. (1996b) Nerve function and regeneration in diabetic and galactosaemic rats: antioxidant and metal chelator effects. Eur.J. Pharmacol., 314, 33– 39.
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Love, A., Cotter, M.A. and Cameron, N.E. (1997) Effects of (Į-tocopherol on nerve conduction velocity and regeneration following a freeze lesion in immature diabetic rats. NaunynSchmeideberg’s Arch. Pharmacol., 355, 126–130. Low, P.A. and Nickander, K.K. (1991) Oxygen free radical effects in sciatic nerve in experimental diabetes. Diabetes, 40, 873–877. Low, P.A., Lagerlund, T.D. and McManis, P.G. (1989) Nerve blood flow and oxygen delivery in normal, diabetic and ischemic neuropathy. Int. Rev. Neurobiol, 31, 355–438. Lowitt, S., Malone, J.I., Salem, A., Kozak, W.M. and Orfalian, Z. (1993) Acetyl-Lcarnitine corrects electroretinographic deficits in experimental diabetes. Diabetes, 42, 1115–1118. Lowitt, S., Malone, J.I., Salem, A.F., Korthals, J. and Benford, S. (1995) Acetyl-L-carnitine corrects altered peripheral nerve function in experimental diabetes. Metabolism, 44, 677– 680. Lyons, T.J. (1991) Oxidised low density lipoproteins—a role in the pathogenesis of atherosclerosis in diabetes. Diabetic Med., 8, 411–419. Malik, R.A., Abbot, C.A., Williamson, S., Abu-Aisha, B. and Boulton, A.J.M. (1997) A double blind placebo trial of the effect of an ACE inhibitor, trandolapril on diabetic polyneuropathy. Diabetologia, 40(Suppl. 1), A31. Mathew, J., Bianchi, R., McLean, W.G., Peterson, R.G., Roberts, R.E., Savaresi, S. and Eichberg, J. (1997) Phosphoinositide metabolism, Na, K-ATPase and protein kinase C are altered in peripheral nerve from Zucker diabetic fatty rats (ZDF/Gmi-f alpha) Neurosci. Res. Commun., 20, 21–30. Maxfield, E.K., Cameron, N.E., Cotter, M.A. and Dines, K.C. (1993) Angiotensin II receptor blockade improves nerve function, modulates nerve blood flow and stimulates endoneurial angiogenesis in streptozotocin-diabetic rats. Diabetologia, 36, 1230–1237. Maxfield, E.K., Love, A., Cotter, M.A. and Cameron, N.E. (1995) Nerve function and regeneration in diabetic rats: effects of ZD-7155, an AT1 receptor antagonist. Am. J. Physiol., 269, E530-E537. Maxfield, E.K., Cameron, N.E. and Cotter, M.A. (1997) Effects of diabetes on reactivity of sciatic vasa nervorum in rats. J. Diabet. Complications, 11, 47–55. McCord, J.M. (1985) Oxygen-derived free radicals in postischemic tissue injury. New Engl. J, Med., 312, 159–163. McVeigh, G.E., Brennan, G.M., Johnston, G.D., McDermott, B.J., McGrath, L.T., Henry, W.R., Andrews, J.W. and Hayes, J.R. (1992) Impaired endotheliumdependent and independent vasodilation in patients with type 2 (non-insulindependent) diabetes mellitus. Diabetologia, 35, 771–776. McVeigh, G.E., Brennan, G.M., Johnston, G.D., McDermott, B.J., McGrath, L.T., Henry, W.R., Andrews, J.W. and Hayes, J.R. (1993) Dietary fish oil augments nitric oxide production or release in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia, 36, 33–38. Mizisin, A.P. and Powell, H.C. (1993) Schwann cell injury is attenuated by aldose reductase inhibition in galactose intoxication. J. Neuropatbol. Exp. Neurol,, 52, 78–86. Moncada, S., Gryglewski, R.J., Bunting, S. and Vane, J.R. (1976) A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins, 12, 715–737.
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Rösen, R, Ballhausen, T., Bloch, W. and Addicks, K. (1995) Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia, 38, 1157–1168. Ross, W.M., Creighton, M.O., Trevithick, J.R., Stewart-DeHaan, J.R and Sanwal, M. (1983) Modelling cortical cataractogenesis IV. Induction by glucose in vitro or in diabetic rats: prevention and reversal by glutathione. Exp. Eye Res., 37, 559–573. Rubanyi, G.M. and Polokoff, M.A. (1994) Endothelins: molecular biology, biochemistry, pharmacology, physiology and pathophysiology. Pharmacol. Rev., 46, 325–415. Sagara, M., Satoh, J., Wada, R., Yagihashi, S., Takahashi, K., Fukuzawa, M., Muto, G., Muto, Y. and Toyota, T. (1996) Inhibition of development of peripheral neuropathy in streptozotocin-induced diabetic rats with N-acetylcysteine. Diabetologia, 39, 263–269. Scarfo, C, Falcinelli, M., Pacifici, L., Bellucia, A., Reda, A., De Angelis, C., Ramacci, M.T. and Angelucci, L. (1992) Morphological and electrophysiological changes of peripheral nerve-muscle unit in the aged rat prevented by levocarnitine acetyl. Int. J. Clin. Pharm. Res., 12, 253–262. Schmidt, A.M., Hasu, M., Popov, D., Zhang, J.H., Chen, J., Yan, S.D., Brett, J., Cao, R., Kuwabara, K., Costache, G., Simonescu, N., Simonescu, M. and Stern, D. (1994) Receptor for advanced glycation end products (AGEs) has a central role in vessel wall interactions and gene activation in response to circulatory AGE proteins. Proc. Natl. Acad. Sci, USA, 91, 8807–8811. Selvais, P.L., Ketelslegers, J.M., Buysschaert, M, and Hermans, M.P. (1995) Plasma endothelin1 immunoreactivity is increased following long-term dietary supplementation with ˰-3 fatty acids in microalbuminuric IDDM patients. Diabetologia, 38, 253. Shiba, T., Inoguchi, T., Sportsman, J.R., Heath, W.F., Bursell, S. and King, G.L. (1993) Correlation of diacylglycerol level and protein kinase C activity in the rat retina to retinal circulation. Am. J. Physiol., 265, E783-E793. Sima, A.A.F., Ristic, H., Merry, A., Kamijo, M., Lattimer, S.A., Stevens, M.J. and Greene, D.A. (1996) Primary preventive and secondary interventionary effects of acetyl-Lcarnitine on diabetic neuropathy in the bio-breeding Worcester rat. J. Clin. Invest., 97, 1900–1907. Shimamoto, Y, Shimamoto, H., Kwan, C. and Daniel, E.E. (1993) Differential effects of putative protein kinase C inhibitors on contraction of rat aortic smooth muscle. Am. J. Physiol., 264, H1300-H1306. Sies, H. (1991) Oxidative Stress: Oxidants and Antioxidants., London: Academic Press. Sonobe, M., Yasuda, H., Hisanaga, T., Maeda, K., Yamashita, M., Kawabata, T., Kikkawa, R., Taniguchi, Y. and Shigeta, Y. (1991) Amelioration of nerve Na+-K+ATPase activity independently of myo-’inositol level by PGE1 analogue OP-1206-Į-CD in streptozotocininduced diabetic rats. Diabetes, 40,726–730. Soulis-Liparota, T., Cooper, M., Papazoglou, D., Clarke, B. and Jerums, G. (1991) Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes, 40, 1328–1334. Soulis-Liparota, T., Cooper, M.E., Dunlop, M. and Jerums, G. (1995) The relative roles of advanced glycation, oxidation and aldose reductase inhibition in the development of experimental diabetic nephropathy in the Sprague-Dawley rat. Diabetologia, 38, 387– 294.
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5. DIABETIC NEUROPATHY IN VARIOUS ANIMAL MODELS ASHUTOSH K.SHARMA and PENELOPE A.RICHARDS
INDUCTION OF DIABETES Alloxan and Streptozotocin Alloxan and streptozotocin have been widely employed to induce diabetes in a variety of experimental animals. Alloxan, a pyrimidine with structural similarities to uric acid and glucose, has been investigated in depth. Jacobs (1937) observed hypoglycaemia and convulsions in rabbits following the administration of alloxan. In 1943 the diabetogenic property of alloxan was discovered, following which, diabetes was successfully induced in rabbits (Bailey and Bailey, 1943), rats (Dunn and McLetchi, 1943; Gomori and Goldner, 1943) and dogs (Lukens, 1948). Diabetes has also been induced, by alloxan, in a number of other species including cats, sheep, monkeys, pigeons and mice (Lukens, 1948; Lazarow, 1947). Guinea pigs, however, were found to be resistant to the diabetogenic action of alloxan (Johnson, 1950a, 1950b; West and Highet, 1948). During the last five decades the literature on alloxan, as a diabetogenic agent, has expanded greatly and was reviewed, by the present author, in 1987 (Sharma and Thomas, 1987). Alloxan has a pronounced cytotoxic effect, particularly on pancreatic islet beta cells (Heikkila et al., 1976; Rerup, 1970; Webb, 1966; Wellmann et al., 1967) although other cell types are also sensitive, albeit to a varying and usually much lesser degree (Harman and Fischer, 1982; Ishibashi et al., 1981; Sagström et al, 1987; Watala et al, 1989; Zhang et al., 1991). Although the exact mechanism of alloxan cytotoxicity is not fully understood; it is considered to be mediated by the formation and effects of a number of reactive oxygen species, such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals (Grankvist et al, 1979; Grankvist and Marklund, 1986; Takasu et al., 1991a). The uniquely weak antioxidative defense system of beta cells has been suggested to underlie their sensitivity to oxidative stress (Malaisse et al., 1982). This hypothesis is however not universally accepted (Grankvist et al., 1981). Cytotoxic mechanisms that do not necessitate the cellular uptake of alloxan have also been proposed. These include; (a) hydroxyl radical
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attack at the plasma membrane level (Zhange and Brunk, 1993) and (b) diffusion of hydrogen peroxide through the cytoplasm into lysosomes, where intralysosomal Fenton reactions may occur, in the presence of reactive lysosomal iron (Zhange et al., 1991, 1992a, 1992b). As a consequence of these two mechanisms, peroxidation may damage, not only the plasma membrane, but also the lysosomal limiting membranes, resulting in leakage of potent hydrolytic enzymes into the cytosol. During the 1980’s a group of researchers (Okamoto, 1985; Uchigata etal., 1982; Yamamoto et al., 1981) proposed a model which has enjoyed increasing acceptance and is now termed ‘Okamoto’s model’ (Renold, 1988). Central to this model is fragmentation of the nuclear DNA of the pancreatic beta cell. DNA fragmentation appears to be important in the development of diabetes and supposedly results from the accumulation of superoxide or hydroxyl radicals, especially in the case of alloxan (Okamoto, 1985). Direct evidence for such an accumulation is however lacking. Takasu and colleagues (Takasu et al., 1991a, 1991b) have also demonstrated that alloxan stimulates both hydrogen peroxide generation and DNA fragmentation both in vitro and in vivo. Thus it is possible that alloxan-induced hydrogen peroxide generation may play a role in DNA fragmentation and consequently in the development of diabetes mellitus. Streptozotocin, another remarkable substance with regard to its specificity for the beta cell, has also been extensively used in the induction of insulin-dependent diabetes mellitus in experimental animals. Streptozotocin, isolated from Streptomyces achromogenes (Herr et al., 1959), is also effective against gram-positive and gramnegative organisms (Lewis and Barbiers, 1959). Rakieten and associates (1963) were the first to report that the intravenous injection of a streptozotocin solution produced diabetes in both rats and dogs. Since then streptozotocin has been used in the induction of diabetes in rats (Evans et al., 1965; Junod et al., 1969), mice (Evans et al., 1965; Brosky and Logothetopoulos, 1969), guinea pigs (Brosky and Logothetopoulos, 1969) and monkeys (Pitkin and Reynolds, 1969). Rabbits, unlike the other species tested, have been found to be resistant to the diabetogenic action of streptozotocin (Kushner et al., 1969)- As with alloxan, the mechanism of action of streptozotocin has not been fully elucidated. It is probable, according to Okamoto’s model, that streptozotocin initiates beta cell destruction in much the same manner as alloxan (Renold, 1985). Histopathology The histopathologic changes that occur in pancreatic beta cells, in response to treatment with alloxan and streptozotocin, suggest that similarities exist between the two substances; despite differences in the time scales for the occurrence of these changes (Rakieten et al., 1963). The characteristic degenerative changes include shrinkage of the beta cell, a decrease or complete loss of cytoplasmic granules and nuclear pyknosis (Arison et al., 1967; Rakieten et al., 1963). With time these changes become more marked; the nucleus undergoes karyolysis; vacuolization and disintegration of cytoplasm occurs and finally the cell boundaries disappear, leading to the formation of a mass of cellular debris. In contrast, the cytoplasm of the alpha cells appears to remain unscathed (Junod et al., 1969). When compared to alloxan, streptozotocin is more effective in its induction of experimental diabetes (Rakieten
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et al., 1963). Alloxan is also not entirely specific for the beta cell and has been noted to induce reversible changes in other organs, such as the kidney and adrenal medulla, when a dose greater than that required to produce diabetes is administered (Harman and Fischer, 1982; Malaisse, 1982). Metabolic Effects The most notable metabolic change that follows the administration of streptozotocin is a state of pronounced hyperglycaemia, while the serum levels of ketones and plasma free fatty acids remain within normal limits (Mansford and Opie, 1968). Likewise, the levels of glycolytic intermediaries, glycogen and citrate, in the perfused heart, also remain normal. Alloxan, however, when compared to normal controls produces elevations in all of the metabolic parameters examined. Junod and colleagues (1969) partly confirmed these observations and also demonstrated that ketonuria was a feature only seen after large dosages of streptozotocin. In contrast, Veleminsky and associates (1970) were unable to demonstrate any qualitative differences, in respect of plasma glucose levels, free fatty acids and triglyceride responses, following the administration of alloxan and streptozotocin. They suggested that the metabolic events, induced by these agents, are primarily the result of beta cell destruction and not due to extrapancreatic toxicity. The insulinopenia that is induced by both alloxan and streptozotocin is characterised by increased gluconeogenesis, glycogenolysis and protein catabolism, as well as hyperlipidaemia and subnormal levels of hepatic glycogen (Grodsky et al., 1982). Thompson and Mikhailidis (1993) evaluated, in streptozotocin-induced diabetic rats, several of the biochemical parameters that form an integral part of the diabetologist’s armory in the assessment and management of human diabetes mellitus. They noted numerous similarities, between the human diabetic state and that in streptozotocin-induced long-term nonketonuric and diabetic ketoacidosis animals, which could prove useful in the investigation of drugs intended for use in the treatment of diabetes mellitus. Dosages Alloxan and streptozotocin are of particular interest in that they duplicate the pancreatic lesions that occur following the destruction of the beta cells in human insulin-dependent diabetes mellitus. Furthermore, they provide a relatively permanent diabetic state which is suitable for longitudinal electrophysiological and morphological studies of the peripheral nerves in experimental diabetes. The effective diabetogenic dosage of either alloxan or streptozotocin is usually four to five times less than that which is lethal. Both the effective and lethal dosages for alloxan and streptozotocin vary considerably from species to species and are highly sensitive to the age, gender and nutritional state of the animal (Dulin et al., 1983; Gold et al., 1981). A single intraperitoneal or intravenous injection of a solution of either alloxan or streptozotocin, when administered during the fasting state to rats, is usually sufficient to induce a diabetic state that is maintainable, without insulin treatment, for up to two years (Powell et al., 1977; Sharma and Thomas, 1974; Zemp et al, 1981). Unfortunately the mortality rate in rabbits may be as high as 75%, due to convulsions and coma that accompany the severe hypoglycaemia that develops
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within the initial 24 hours following diabetic induction (Wellmann et al., 1969). However, careful monitoring of the blood glucose level and the appropriate administration of 50% dextrose, in order to reduce the risk of convulsions, has been shown to reduce this mortality rate to approximately 23% during the first 24 hours (Bajada et al., 1980). Large Animal Models Even though diabetes mellitus has been successfully induced in small animals, the basic scientific investigation of the disease and its complications has been limited due to the dearth of appropriate large animal models. The disease has been successfully induced in pregnant ewes by the intravenous injection of a streptozotocin solution, on two separate occasions, 4 days apart (Dickinson et al., 1991). These ewes demonstrated some of the characteristic features of human diabetes mellitus including hyperphagia, polyuria and polydipsia. The alterations in both maternal glucose levels and insulin response, that result from streptozotocin-induced pancreatic beta cell destruction, combined with the elevations in fetal glucose, insulin and weight, suggest that the ewe may provide a suitable model for the investigation of gestational diabetes (Dickinson et al., 1991). The pancreatic beta cells of fetal lambs are significantly damaged when subjected to the intrauterine injection of a streptozotocin solution (Brinsmead and Thorburn, 1982). A concomitant rapid and prolonged appearance of hyperglycaemia occurs as well as a deficit in glucose and tolbutamidestimulated fetal insulin release and an absolute deficiency of pancreatic insulin (Philipps et al., 1986, 1991). These effects are also associated with significant retardation of somatic and skeletal growth, as well as protein deposition in lategestation lambs (Philipps et al., 1991). In monkeys, the induction of diabetes has been successfully achieved with both alloxan and streptozotocin. Although alloxan was initially utilised (Bloodworth et al., 1973; Gibbs et al., 1966) streptozotocin has become the more widely used agent (Chopra et al, 1977; Pitkin and Reynolds, 1969; Salazar et al., 1973) as it produces a mild to moderate diabetes which undergoes progression to overt diabetes (Howard, 1982). Adolescent baboons have been utilised as a model for the study of graded beta cell dysfunction (McCulloch et al., 1988), where lower doses of streptozotocin induce a diabetic state, similar to that seen in the preclinical phase of IDDM and in non-diabetic first degree relatives of IDDM patients (Johnston et al., 1987; McCulloch et al., 1990). Within the same species, higher doses of streptozotocin produce a clinical diabetic state which may or may not require insulin intervention, where the degree of beta cell dysfunction correlates closely with the quantitative estimates for beta cell mass and pancreatic insulin content (McCulloch et al., 1990, 1991). The induction of experimental diabetes in dogs is possible with both alloxan and streptozotocin. Engerman and Kramer (1982) described an induction method using alloxan in which approximately 85%, of the 42 dogs used, developed permanent insulin dependent diabetes. As canine death, within the first two days following induction, is usually due to severe hypoglycaemia, a number of investigators use alloxan and streptozotocin simultaneously or in multiple subdiabetogenic doses (Black et al., 1980; Issekutz et al., 1974; Rakieten et al., 1963).
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Chemically Induced Diabetic Models for IDDM Recently, attempts have been made to gain a greater understanding of what changes occur in insulin secretion and sensitivity during the preclinical phase of insulin dependent diabetes mellitus. Such investigations have included the use of genetically matched siblings of IDDM patients (Raghu et al., 1985; McCulloch et al., 1988, 1990) as well as the assessment of insulin autoantibodies, in order to predict the occurrence of the disease (Dotta and Eisenbarth, 1989). These studies suggest that beta cell dysfunction and insulin resistance may exist in nondiabetic siblings of IDDM patients. The development of an animal model, for insulin deficiency, which closely resembles beta cell dysfunction progression, would be advantageous in order to investigate the effect of this gradual impairment on the other parameters of glucose tolerance. The current conventional animal models of diabetes, such as those using diabetogenic doses of streptozotocin or alloxan, or spontaneously diabetic rats such as the BB/W rat, unfortunately do not simulate the course of beta cell dysfunction that is hypothesized to precede the onset of human IDDM. In the six theoretical stages of IDDM (Eisenbarth, 1986), the stage immediately prior to overt diabetes is represented by a progressive decrease in insulin release, while glucose tolerance generally remains normal (Eisenbarth, 1986; Eisenbarth et al., 1987). In order to investigate the longitudinal relationship between progressive beta cell dysfunction and other parameters of glucose tolerance, repeated low-dose streptozotocin models in both dogs (Tobin and Finegood, 1993) and baboons (McCulloch et al., 1991) have been used. The hypothesis that insulin resistance is not necessarily a direct consequence of beta cell dysfunction is supported by inducing diabetes with streptozotocin in rats during the neonatal period (Kergoat et al., 1991; Blondel et al., 1989). Neonatal rats treated with streptozotocin (Weir et al, 1981; Levy et al., 1984; Hiramatsu et al., 1994) and alloxan (Kodama et al., 1993) demonstrate similarities of insulin secretion and action to that in human NIDDM and may thus be useful for studying chronic diabetic complications. Low-dose streptozotocin-induced diabetes in mB7–1 transgenic mice (Harlan et al., 1995) and C57BL/KsJ male mice (Ellias et al., 1994) is considered to be an immune mediated process with distinct potential advantages over existing insulin-dependent diabetes models. CHANGES IN NERVE CONDUCTION Alloxan-induced diabetes in rats is associated with a reduction in the motor nerve conduction velocity (Eliason, 1964). Within 10 days of treatment, the conduction velocity in the sciatic nerve of treated rats may be reduced to as little as 30–50% of the initial velocity (Eliason, 1969). Both motor and sensory nerves of the myelinated variety are affected while those nerves with unmyelinated axons are not. Within six weeks of the administration of alloxan the sciatic conduction values return to near normal velocities; this suggests that the initial decrease in velocity occurs as a consequence of the toxic effects of alloxan. However, in those animals that fail to develop diabetes the conduction values show little to no decrease, suggesting that the change in conduction velocity is dependent on the development of diabetes as opposed to direct toxicity (Eliasson 1964, 1969). Reduced nerve conduction velocity was subsequently confirmed in alloxan (Preston, 1967; Hildebrand et al., 1968;
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Miyoshi and Goto, 1973) and streptozotocin-induced diabetic rats (Sharma and Thomas, 1974; Greene et al., 1975; Jakobsen, 1979), as well as in alloxan-induced diabetic rabbits (Bajada et al., 1980). In general, reductions in conduction velocity are dependent upon the severity of the diabetes. Rats with severe uncontrolled diabetes, induced with either alloxan or streptozotocin, demonstrate an acute fall in nerve conduction velocity and do not usually survive (Sharma and Thomas, 1974). The reduction in velocity tends to be less marked in animals with a milder form of the disease. The prolonged survival of animals with mild diabetes, in the absence of insulin treatment, enables the assessement of morphological change that accompanies the disease, as well as the evaluation of the influence of the various therapies proposed for the management of human diabetic neuropathy. To whit it has been shown that conduction velocity can be improved, in streptozotocin-diabetic rats, at least in the early stages, by employing insulin therapy (Greene et al., 1975; Jakobsen, 1979; Julu and Mutamba, 1991). The degree of reduction of conduction velocity is dependent upon multiple factors, including the duration and severity of the diabetes. In severely diabetic animals the acute reduction in conduction velocity may be partly related to the dehydrational shrinkage of affected axons, with a concomitant reduction in the axonal contents, in response to high tissue osmolality (Sugimura et al., 1980). Dyck and colleagues (1981) observed that the intravenous administration of a hypertonic solution of dextrose, to cats, led to a progressive fall in nerve conduction velocity. This was accompanied by a diminution in the cross sectional area of the axons and crenation of the axolemma, confirming the hyperosmolality theory. A complicating factor in the long-term observation of conduction velocity in rats, is that growth occurs for a significant period in the life span of this species. Both nerve fibre diameter and conduction velocity increase consistently, in normal rats, until at least nine months of age (Birren and Wall, 1956; Sharma and Thomas, 1974; Sharma et al., 1980). Growth retardation occurs, in rats, in the presence of untreated diabetes (Sharma et al., 1981, 1985a). Hence, at least part of the difference observed between diabetic and control animals, may be attributable to a maturational deficit. Even though conduction velocity continues to increase for a period of eight months, following induction in one month old rats, it remains consistently less than that of age-matched controls (Moore et al., 1981). Similarly the conduction velocity, at the end of a two-month observation period, is noted to be intermediate between the onset-control and end-control values (Sugimra et al., 1980; Cameron et al., 1986). Should rats be made diabetic when they have ceased to grow, i.e. at the age of nine months, they demonstrate only a slight reduction in their conduction velocities (Thomas et al., 1981). Recently Wright and Nukada (1994) reported that sciatic and caudal nerve conduction velocities decreased significantly at 16 weeks following the induction of diabetes in mature nine month old rats, although the trends were apparent after 4 weeks following induction. The Role of Myoinositol In short-term experiments, conduction velocity has been restored to normal by the use of either the aldose reductase inhibitor, sorbinil, (Yue et al., 1982; Gillon and Hawthorne, 1983; Mayer and Tomlinson, 1983) or by dietary supplementation with
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myoinositol (Greene et al., 1975; Mayer and Tomlinson, 1983). The complex interactions of metabolic factors such as hyperglycaemia, increased peripheral nerve glucose, sorbitol, fructose, and decreased nerve myoinositol levels, in experimental diabetic animals, and their possible roles in the reduction of conduction velocity, have been extensively reviewed (Clements, 1979; Greene, 1986; Greene and Lattimer, 1987; Greene et al., 1988a, 1988b, 1988c, 1992; Dvornik and Porte, 1987). According to the sorbitol or polyol hypothesis, activation of the polyol pathway by glucose, via the high Km aldose reductase, promotes sorbitol and fructose accumulation, myoinositol depletion, and nerve conduction slowing (Greene et al., 1975). Extensive biochemical and electrophysiological studies in experimental diabetic rats attribute nerve conduction slowing, in acute diabetes, to a polyol pathway induced, myoinositol-related alteration in neural Na+-K+-ATPase activity (Greene and Lattimer, 1983). The changes in peripheral nerve myoinositol levels in experimental diabetes are more controversial. Decreased nerve myoinositol levels in diabetic animals have been reported to occur after periods of two (Greene et al., 1975) and thirteen weeks of consistent hyperglycaemia respectively (Palmano et al., 1977). Cameron et al. (1986) reported that the sciatic myoinositol levels, in diabetic animals, decreased significantly after 3 months of hyperglycaemia, while after 6 months the levels were not significantly different to the control values. Ward et al. (1972), however, reported no significant differences in myoinositol levels when the hyperglycaemia duration ranged from 3 days to 3 months. Similarly, Jefferys et al. (1978) reported no alteration in myoinositol levels following 4 weeks of hyperglycaemia, when diabetes was induced in rats of nine months of age. The effects of long periods of hyperglycaemia, on nerve myoinositol levels, were studied by Poulsom and associates (1983); they reported that given 8 to 12 months of continual hyperglycaemia, no significant difference existed between the diabetic and control group levels. The decreased levels of nerve myoinositol reported after a short duration of diabetes, and the lack of reported differences after extended periods of hyperglycaemia, led Cameron and associates (1986) to postulate that nerve myoinositol level changes may be a transient phenomenon, lasting only until the metabolic derangement reaches a new steady state, and that in long-term diabetes alterations in nerve myoinositol may not be a prerequisite for the observed abnormalities that occur in peripheral nerve function. THE EFFECTS OF ALDOSE REDUCTASE INHIBITION The prevention and reversal of nerve conduction abnormalities in experimental diabetic animals, with aldose reductase inhibitors has been firmly established. These inhibitors include sorbinil (Yue et al., 1982; Gillon et al, 1983; Tomlinson et al., 1984), ponalrestat (Stribling et al., 1980; Cameron and Cotter, 1992), imirestat (Carrington et al., 1991), 5-thienyltetrazol-1-y1-acetic acid (TAT) (Inukai et al, 1993; Hotta et al., 1995), M16209 and M12687 (Kato et al., 1991), FR-62765 (Nishikawatf al., 1991), SNK-860 (Kato et al., al., 1994) ONO-2235 and isoliquiritigenin (Shindo et al., 1992). Although the efficacy of the aldose reductase inhibitors in the reduction of conduction abnormalities is established, the specific mechanisms of action of all of these compounds remains open to theoretical interpretation and debate (Green
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et al., 1988b, 1988c, 1992; Tomlinson, 1992; Tomlinson et al., 1992, 1994; Dvornik and Porte, 1987; Cameron and Cotter, 1993). As aldose reductase inhibitors, in general, correct myoinositol deficits in experimental nerves, their effects are speculatively linked to alterations in phosphoinositide metabolism and Na+K+ATPase activity. Unfortunately the evaluation of Na+K+ATPase function, in the peripheral nerves of various experimental diabetic animal models, has produced conflicting reports. The majority of studies utilizing enzymatic methods (Das et al., 1976; Greene and Lattimer, 1983, 1984; Greene et al., 1984; Llewelyn and Thomas, 1987; Bianchi et al., 1988; Lambourne et al., 1988; Hirata and Okada, 1990), although not all (Carrington et al., 1991), have demonstrated significantly decreased Na+K+ATPase activity in experimental diabetes. Enzymatic methodology is not without its disadvantages; these disadvantages have been reviewed by Nøgaard (1986). Using a previously described [3H] ouabain binding method (Clausen and Hansen, 1974; Nøgaard, 1986), Kjeldsen and associates (1987) reported a 16% decrease in the Na+K+ATPase concentration in diabetic animals, when compared to a control group. Sonobe and colleagues (1991), however, using Kjeldsen’s method were unable to demonstrate any significant difference between diabetic and control groups after 1 month of hyperglycaemia. Similarly, when employing Kjeldsen’s ouabain method to the sciatic nerve of rats with induced diabetes, of 24 weeks duration (induction at 9 months of age), Wright and Nukada (1994) found no discernible differences in the total Na+K +ATPase concentrations between the experimental and control groups. Rubidium ion uptake is a useful method for measuring the functional activity of the Na+K+ATPase pump. No difference occurs in the functionality of this pump after 4 weeks of hyperglycaemia (Simpson and Hawthorne, 1986), when compared to appropriate endoneurial controls. A 54% (Llewelyn and Thomas, 1987) and 63% (Simpson and Hawthorne, 1986) reduction is however noted at 8 and 6 weeks respectively following diabetic induction. The actual effects of aldose reductase inhibitors on Na+K+ATPase activity are disputed by some researchers (Tomlinson et al., 1992), and may be an artifact resulting from the feeding of a high sucrose diet to rats (Sredy et al., 1991). EFFECTS OF ISCHAEMIA The ischaemic-hypoxic hypothesis stresses the early occurrence of reduced endoneurial blood flow and oxygen tension, and an increased endoneurial vascular resistance, in order for nerve dysfiinction to develop in experimental diabetes (Low, 1987; Low et al., 1987). Endoneurial blood flow is reduced and neural vascular resistance is increased, in rats, within 1 week of the induction of experimental diabetes, and remains low for several months (Cameron et al., 1991a; Wright and Nukada, 1994). The resultant endoneurial hypoxia (Tuck et al., 1984) impairs neuronal energy production resulting in a greater reliance on anaerobic metabolic mechanisms (Low et al., 1985). This state produces oxidative stresses which may damage neurons directly; for example, by the peroxidation of lipids within cell membranes (Baynes, 1991). The majority of animal studies, employing a variety of methodologies, including hydrogen clearance (Cameron et al., 1991a; Tuck et al., 1984; Kihara et al., 1991; Hotta et al., 1992), butanol accumulation (Monafo et al.,
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1988) and laserdoppler flowtometry (Yasuda et al., 1989), agree that a reduction in endoneurial blood flow occurs in experimental diabetes, preceding a significant change in nerve conduction velocity. In contrast, Tilton and associates (1989), using a microsphere entrapment technique have shown increased ‘trapping’ in diabetic nerve vasculature; this was postulated to be due to elevated nerve blood flow. This phenomenon could however be due to either vessel alterations, that do not directly produce flow changes, or to an increase in the ‘stickiness’ of the microvessel walls (Bohlen and Niggl, 1980; Simpson, 1988). Studies of non-diabetic rats exposed to chronically hypoxic environments reproduce the electrophysiological and endoneurial vascular flow abnormalities observed in experimental diabetic rats (Low et al., 1986; Smith et al., 1991) and thus lend support to the ischaemic-hypoxic hypothesis. The efficacy of various vasodilators in the prevention and treatment of the early conduction abnormalities that occur in experimental diabetic rats has been clearly demonstrated. These vasodilators include noradrenergic antagonists (Cameron et al., 1991a, 1991b), calcium channel blockers (Kappelle et al., 1992; Robertson et al., 1992a), agents inhibiting the renin-angiotensin system (Cameron et al., 1992a, 1993; Maxfield et al., 1992) and vasomodulator prostanoid analogues (Cotter et al., 1993; Ohno et al., 1992; Yassuda et al., 1989). Various other treatment regimes, all of which have vascular related effects, have also been demonstrated to improve nerve conduction in experimental diabetic rats. These include dietary supplementation with essential fatty acids (Julu, 1988; Tomlinson et al., 1989; Cameron et al., 1991c; Robertson et al., 1992b), aldose reductase inhibitors (Cameron et al, 1992a; Tesfamariam et al., 1992; Yasuda et al., 1989; Hotta et al., 1992), aminoguanidine, which prevents the formation of advanced glycation end-products (Kihara et al., 1991; Yagihashi et al, 1990; Cameron et al., 1992b), and anti-oxidants (Bravenboer et al., 1992; Cameron et al., 1993b). The early and sustained alterations in vascular indices, as well as the efficacy of treatments which potentially improve endoneurial blood flow and nerve conduction, lend support to the theory that ischaemic-hypoxic factors may play a role in the causation of diabetic neuropathy. Even so, the ischaemic-hypoxic hypothesis does not diminish the importance of the multitude of metabolic alterations that occur rapidly, subsequent to the onset of hyperglycaemia. In this respect a series of studies have focused on axonal growth and regeneration, impaired neuronal synthesis, the transport of growth-related chemicals, as well as on neurotrophic abnormalities. MORPHOLOGICAL CHANGE Anterior Horn and Dorsal Root Ganglion Cells Characteristic of streptozotocin-induced diabetic rats, that have survived the condition for a period of 16 weeks, is a reduction in the total number of anterior horn cells, as well as alterations in the dendritic field patterns (Felten, 1979). When compared with age-matched controls the perikaryal volume of both the lower motor and primary sensory neurons, of the experimental rats, is reduced (Sidenius and Jakobsen, 1980). Similarly, after only 4 weeks of streptozotocininduced diabetes the volume of dorsal root ganglion cells decreases by up to 18%; this is due to a shift in
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the relative proportions between the larger A-cells and the smaller B-cells (Sidenius and Jakobsen, 1980). Although the specific functional differences between these two cell types have not been fully elucidated it appears as if neuro-transmitter substanceP is confined to the B-cells (Hökfelt et al.,1975), while the uptake of nerve growth factor is restricted to the A-cells (Stoeckel et al., 1975). Nerve Trunks The widespread, severe degenerative changes that occur in established cases of human diabetic neuropathy are not usually replicated in experimental diabetic rats (Sharma and Thomas, 1974). Initially, qualitative examination of single fibres, isolated from alloxan-induced diabetic rats, and fixed in either formal saline or osmium tetroxide, demonstrated the occurrence of paranodal and segmental demyelination (Hildebrand et al., 1968; Preston, 1967; Seneviratne and Peiris, 1969). These features were however not observed in single fibres obtained from the sural, peroneal or tibial nerves of experimental rats and fixed, either in situ or by perfusion, in an aldehyde solution (Sharma and Thomas, 1974; Jakobsen, 1976b; Brown et al., 1980; Sharma et al., 1981, 1985a; Sugimura et al., 1980). Thus it appears unlikely that either segmental demyelination or axonal degeneration occur to any significant degree in the peripheral nerve trunks of chemically-induced diabetic rats. Furthermore, morphological analysis of the sural (Sharma and Thomas, 1974; Sharma et al, 1985a; Sugimura et al., 1980), peroneal (Jakobsen, 1976a), and tibial nerves (Brown et al, 1980; Sharma and Thomas, 1974; Sharma et al., 1981), of streptozotocin diabetic rats, has clearly established that no loss of myelinated fibres occurs. Even so, some subtle morphological alterations, such as decreased myelinated fibre diameter, are detectable in these nerves when compared to agematched controls (Jakobsen and Lundbaek, 1976; Sharma et al., 1976, 1977). When using light and electron microscopical morphometric techniques, it is apparent that axon diameter is affected to a greater degree than myelin thickness (Jakobsen, 1976a), regardless of whether the peroneal (Jakobsen, 1979), sural (Sugimura et al., 1980) or tibial nerve (Britland et al., 1986; Bhoyrul et al., 1988) of diabetic rats is examined. The mechanisms that lead to changes in fibre and axon size, in diabetic rats, are probably multiple and remain ill defined. On the basis of serial morphometric observations, utilising the same rats, prior to and five weeks following diabetic induction, no significant change in external fibre diameter was noted; the control group however demonstrated a significant increase in fibre diameter (Sharma et al., 1977), suggesting that the diabetic state impairs fibre maturation. In normal rats, myelinated fibre size continues to increase until at least nine months of age (Sharma et al., 1980). Morphometric analysis, at both the light and electron microscope level, has revealed that both fibre (Sharma et al., 1985a; Sugimura et al., 1980; Dockery and Sharma, 1990) and axon size (Britland et al., 1986; Bhoyrul et al., 1988) are significantly less in diabetic animals than in their age-matched controls, but not less than that of the onset controls. Other variables such as body weight, long bone length (Sharma et al., 1981, 1985a) and myelinated fibre internodal length (Jakobsen, 1976b), are also affected by the diabetic state, probably as a consequence of a maturational deficit secondary to the metabolic effects of the disease.
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The interactions between the metabolic derangement’s of diabetes and maturational deficits are far from understood. Suggested mechanisms include: a) reduced protein synthesis in nerve cell bodies with defective delivery of structural proteins to the axons, possibly secondary to decreased retrograde axonal transport (Sidenius and Jakobsen, 1981), b) impaired amino acid uptake by dorsal root ganglion cells in response to reduced Na+K+ATPase activity (Thomas et al., 1984; Green et al., 1985) and c) impaired anterograde axonal transport of structural proteins (Sidenius and Jakobsen, 1987). Whatever the final explanation, the abnormalities already identified in experimental diabetic rats are of considerable interest and importance. It is hoped that long term chemically-induced diabetic rats, in reproducing some of the structural alterations characteristic of early human diabetic neuropathy, may ultimately aid in the elucidation of the underlying pathogenic mechanisms of the disease. EXPERIMENTAL DIABETIC THERAPY A variety of therapies have been tested in an attempt to prevent or reverse structural abnormalities in the peripheral nerves of experimental diabetic animals. To whit the influence of conventional insulin treatment (CIT), continuous subcutaneous insulin infusion therapy (CSII), aldose reductase inhibition as well as pancreatic islet transplantation have been examined. Conventional Insulin Treatment (CIT) Four weeks of conventional insulin treatment, administered by daily subcutaneous injection, produces a light microscopic improvement in axonal and myelinated fibre size (Jakobsen, 1979). Such treatment, when administered for two consecutive months, to streptozotocin diabetic rats, results in normalisation of their body weight, improved skeletal growth and the correction of glucose, sorbitol, fructose and myoinositol concentrations in the sciatic nerve; however, deficits in myelinated fibre size in the tibial and sural nerves are not corrected (Sharma et al., 1985a) (Figure 1). Ultrastructural studies of the tibial nerves of diabetic rats reveal that after 3 to 4 months of conventional insulin therapy, both axonal diameter and myelinated fibre size are only partially corrected, whereas the metabolic and biochemical parameters are fully restored to normality (Britland et al, 1986; Bhoyrul et al, 1988). Unfortunately each of these animal studies differs from the real situation that diabetic patients face, in that insulin treatment was initiated promptly following the induction of diabetes, rather than in the midst of an established condition. In response to this experimental shortfall, Britland and Sharma (1990) designed a study in which conventional insulin therapy was only initiated 2 months after diabetic induction. After 8 weeks of therapy the metabolic concentrations of glucose, sorbitol and fructose were completely normalised, while the myoinositol levels were only partially corrected; myelinated fibre size and axonal diameter however, were not completely normalised. Thus it may be concluded that conventional insulin treatment does not completely reverse or prevent the structural abnormalities that occur in the peripheral nerves of experimental diabetic rats.
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Figure 1 Mean myelinated fibre diameter in the tibial nerve of onset control (OC), diabetic (D), conventional insulin-treated (CIT) diabetic and end control (EC) rats. * End control values significantly greater than onset control values. ** Significantly less than end controls. (Number of animals in brackets).
Conventional insulin therapy not only fails to prevent or reverse peripheral neuropathy, in alloxan and streptozotocin diabetic rats, but is also associated with a paradoxical increase in axonal degeneration (Sharma et al., 1985a; Westfall et al., 1983). As axonal degeneration occurs in healthy rats, rendered hypoglycaemic by means of insulin administration (Sidenius and Jakobsen, 1983), it appears likely that fluctuations in blood glucose levels, especially in the hypoglycaemic range in insulin treated diabteic rats, may be the inducing factor in this pathology. Even though the satisfactory control of blood glucose levels is often achieved in insulin-treated diabetic animals, diurnal fluctuations still occur and produce hypoglycaemic episodes (Duguid, 1984). Both severe and mild-recurrent hypoglycaemia produce a detrimental effect on the peripheral nerve structure and function of streptozotocin diabetic rats (Potter et al., 1988). More specifically a significant correlation exists between observed episodes of hypoglycaemia (<2.0 m.mol/L) and the incidence of axonal degeneration in insulin-treated diabetic animals (Potter et al., 1988). Acute hypoglycaemia is also associated with a decrease in the fast component of axonal transport in both diabetic and non-diabetic rats (Sidenius and Jakobsen, 1987). The
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occurrence of either severe or asymptomatic mild hypoglycaemia is thus best avoided in the management of diabetes. Continuous Subcutaneous Insulin Infusion Therapy (CSII) Another effective form of diabetic therapy is the administration of insulin via continuous subcutaneous infusion thus providing for the optimum control of blood glucose levels on a 24 hour, day to day basis. When CSII is commenced, in diabetic rats, two months following induction, the paradoxical axonal degeneration characteristic of CIT does not occur (McCallum et al., 1986; Mandelbaum et al., 1983; Britland and Sharma, 1990). Furthermore, myelinated fibre size and skeletal growth are normalised within 2 to 4 months of the commencement of therapy (McCallum et al., 1986) (Figure 2). Although both CIT and CSII were thought to be equally effective in the correction of metabolic abnormalities, in streptozotocin diabetic rats, the concentrations of tissue glucose, fructose, sorbitol and myoinositol were assessed at the time of biopsy and therefore the effects of blood glucose fluctuations on these levels were uncertain (Sharma et al, 1985b). When compared to age-matched controls the concentrations of glucose, sorbitol and fructose are indeed normalised by both forms of therapy. The levels of myoinositol however, in the CIT group, are intermediate between untreated diabetic and age-matched controls, whereas in the CSII group the myoinositol concentrations are normalized to those of age-matched controls (Britland and Sharma, 1990). Assuming that the results of animal studies can be extrapolated to man, CSII therapy appears superior to CIT for the achievement of normoglycaemia, the restoration of skeletal growth and the prevention or reversal of both morphological and biochemical abnormalities. However, when managing diabetic patients a number of hazards related to this form of treatment must be recognised. These include the risk of instrument failure; nosocomial infection related to the subcutaneous catheter delivery system; the possibility of intercurrent stress and its impact on metabolic control; systemic amyloidosis and an increased incidence of hypoglycaemia (Dupre, 1985). Such risks aside, animal studies have revealed that the maintenance of normoglycaemia on a 24 hour, day to day basis is essential to prevent or reverse nerve abnormalities in diabetes. Aldose Reductase Inhibition Cytosolic glucose levels increase in induced-diabetic rats, activating the aldose reductase pathway and resulting in the conversion of glucose to sorbitol and fructose; the levels of which then increase in the peripheral nerves (Gabbay et al., 1966; Stewart et al., 1966). These changes are accompanied by, and in some way linked to, a concomitant reduction in the myoinositol levels (Greene et al., 1975; Stewart et al., 1967; Greene and Lattimer, 1987). The hypothesis that sorbitol accumulation may directly lead to Schwann cell oedema and hence dysfiinction, has not been borne out by morphological studies in streptozotocin-diabetic rats (Sharma and Thomas, 1974; Jakobsen, 1978). Although aldose reductase inhibition has beneficial effects on the electrophysiological abnormalities associated with induced diabetes in rats, it has
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Figure 2 Mean myelinated fibre diameter in the tibial nerve of onset control (OC), diabetic (D), continuous subcutaneous insulin infusion-treated (CSII) diabetics and end control (EC) rats. * Diabetic values significantly less than age-matched end controls. ** Significantly greater than untreated diabetic rats but not different from age-matched controls. (Number of animals in brackets).
not been conclusively demonstrated to reduce or reverse the structural abnormalities that occur in the peripheral nerves of these animals. Using light microscopy techniques, Cameron et al. (1986) reported that the treatment of streptozotocindiabetic rats with sorbinil, corrected both the reduced axonal area and the abnormal axon-myelin ratio in the fibres of the tibial nerve. Light microscopy however, is inadequate for the measurement of these parameters, leaving some doubt as to the accuracy of these results. On the other hand, ponalrestat administration fully corrects sorbitol concentration, partially corrects fructose and myoinositol levels, and leaves glucose levels unaffected in the sciatic nerves of experimental diabetic rats (Bhoyrul et al., 1988). Electron microscopic examination of the sciatic nerve of ponalrestat treated rats shows, in contrast to light microscopy, that fibre and axon size are not fully corrected, even when ponalrestat is used in conjunction with insulin therapy. Such an experimental design serves as a good comparison to the clinical trials that involve human insulin-dependent diabetic patients, where a) metabolic control of diabetes is achieved with insulin therapy and b) an aldose reductase inhibitor is administered in an attempt to prevent or reverse diabetic neuropathy. Rats receiving ponalrestat, in conjunction with CIT, experience wide fluctuations in their blood glucose levels which probably initiates the resultant excess in axonal degeneration. Aldose reductase inhibition does therefore not fully correct all of the defects in axonal transport noted in experimental diabetes (Tomlinson et al., 1985), nor does it sustain the normal growth related increase in conduction velocity that occurs in young diabetic rats (Stribling et al., 1980), suggesting a heterogeneous aetiology for these defects.
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Figure 3 Mean myelinated fibre area in onset control (OC), diabetic (D), transplanted diabetic (TD) and end control (EC) rats. Transplantation was either early (one week following induction and examined six months later) or delayed (six months after induction and examined twelve months later).
However, these observations do not preclude the possible advantageous effects of aldose reductase inhibition on long term diabetes. A recent report by Kato et al. (1994) indicates that long term therapy (26 weeks) with SNK-860, a new aldose reductase inhibitor, prevents an increase in the number of abnormal fibres in teased preparations, from streptozotocin-diabetic rats. Morphometric analysis of myelinated fibres from the same group of animals also reveals the preservation of large size fibres as well as the prevention of axonal atrophy and distorted axonal circularity (Kato et al, 1994). Pancreatic Islet Transplantation Ultimately, the transplantation of pancreatic islets may prove more successful than other therapies, in producing normal or near normal 24 hour plasma glucose levels, for extended periods, in experimental diabetes. In this respect, good control of blood glucose levels, for between 4 and 14 months, has been achieved in streptozotocin diabetic rats, following the transplantation of allogenic islet cells, across a major histocompatibility barrier and in the absence of immunosuppression (Bretzel et al, 1981; Schmidt et al., 1983; Tze et al., 1985). When streptozotocin diabetic rats undergo transplantation, immediately or up to 6 months following diabetic induction, excellent metabolic control of the disease, as judged by body weight, plasma glucose and glycosylated haemoglobin levels, is achieved (Britland et al.,
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1991). Successful early allotransplantation of the pancreatic islets, in streptozotocin diabetic rats, also prevents the characteristic reductions in fibre and axon diameter and axon to myelin ratio (Britland et al., 1991; Sima et al., 1988) (Figure 3); should transplantation be delayed then myelinated fibre size and axon diameter are not completely normalised (Britland et al., 1991). Excessive axonal degeneration, usually seen with CIT, is not observed in either early or late transplanted diabetic animals, indicating that normoglycaemia is achieved by this therapy for periods of up to 14 months (Britland et al, 1991; Sima et al., 1988). Thus it appears that the development of structural abnormalities in the peripheral nerves of diabetic rats are preventable by early transplantation; however, once established, abnormal fibre morphology is not ameliorated merely by achieving and sustaining euglycaemia through delayed transplantation. Transplantation therapy could therefore play an important role in preventing the development of human diabetic neuropathy, it may take a long time for nerve structure and function to normalise in established cases of diabetic neuropathy. SPONTANEOUS DIABETIC ANIMAL MODELS The study of neuropathy, associated with the diabetic state, is not purely limited to experimental animal models, but can also be effectively examined in animals where the occurrence of diabetes is spontaneous. Alloxan and streptozotocin diabetes in animals suffer from the objection that some of the changes described may well be the direct result of chemical toxicity (Jefferys and Brismar, 1980). An increasing number of such models are being described and used, with the best known being the BB-rat and the db/db mouse. The db/db Mouse The autosomal recessive genetic mutation, associated with the db/db mouse, was first described in 1966 (Hummel et al., 1966) when it arose in an inbred C57BL/Ks mouse. The db/db mutation, which demonstrates full penetrance, results in grossly obese mice due to abnormal fat deposition commencing at about 5 weeks after birth (Sharma et al., 1983). The heterozygous state (db/m) is readily recognizable from the homozygote (db/db) due to distinct black coloration of the animals coat. Those mice that do not carry the db mutation (m/m) are usually grey in color. Affected diabetic mice rapidly become hyperglycaemic with concomitant glycosuria and polyuria. During the initial stages of diabetes the plasma levels of insulin increase in these animals and are associated with increased gluconeogenesis, lipogenesis and glucose utilisation. With time the plasma insulin levels fall, returning close to normal base line levels; gluconeogenesis, on the other hand, continues unabated while peripheral glucose utilisation becomes reduced (Coleman and Hummel, 1967). Due to the degree of obesity that db/db mice experience, they often display minor disturbances in motor behaviour, including an inability to maintain posture on a rotator wheel (Carson et al., 1980; Hanker et al., 1980). Nerve conduction velocity decreases significantly, in affected mice, with the onset of clinical diabetes and remains depressed in the absence of therapy (Sima and Robertson, 1978). Insulin therapy however, when commenced soon after the recognition of the diabetic state,
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only partially restores conduction velocity (Robertson and Sima, 1980). Conduction velocity is not affected in the same manner in the different functional nerve types, when compared to age-matched controls, where: motor nerve fibres usually demonstrate decreased velocities while only a few sensory fibres are affected (Moore et al., 1980). The neuropathy associated with the db/db mouse does not replicate that, that occurs in either experimental diabetes or man. Such differences may reflect metabolic variation between the groups. Where, for instance, mice do not appear to possess a sorbitol pathway and thus the accumulation of this metabolite, in nerves, does not occur. Similar to experimental diabetic rats, the segmental demyelination and axonal degeneration, that is characteristic of established human diabetic neuropathy, is not replicated in isolated peripheral nerve fibres of db/db mice (Hanker et al., 1980; Sharma et al., 1983; Sima and Robertson, 1978). Even though marked neuronal degeneration is not a feature of the db/db mouse, a decrease in fibre size is (Hanker et al., 1980; Robertson and Sima, 1980). Two main schools of thought predominate as to the pathogenesis of reduced fibre size: Robertson and Sima (1980) demonstrated that axon size is reduced in relation to myelin thickness; suggestive of primary axonal pathology. Sharma et al. (1983), on the other hand, described an equal reduction in axonal area and myelin thickness, suggesting that both structures were equally affected by the diabetic state. Alternatively they suggested that the equal reduction, when viewed in the light of decreased skeletal growth, may reflect a maturational defect (Sharma et al., 1983). Morphological studies reveal that a minor degree of myelinated fibre degeneration and regeneration is common in the peripheral nerves of mice, throughout their life span, regardless of whether they are diabetic or not (Stanmore et al., 1978). Unmyelinated nerves are not usually affected (Sharma et al., 1983). Detailed electron microscopic analysis of the myelinated fibres of db/db mice illuminates a myriad of axonal and Schwann cell inclusions. These include axonal glycogenosomes, polyglucosan bodies, Schwann cell and axonal networks, as well as Schwann cell Reich granules (Sima and Robertson, 1979). While all these inclusions increase with age in both db/db and age-matched control mice, glycogenosome quantity always remains significantly greater in the diabetic mice (Sharma et al., 1983). The endoneurial capillaries of diabetic mice are usually normal although occasional thickening and duplication of the basal lamina, surrounding the endothelial cells, is noted (Sima and Robertson, 1979). The Spontaneously Diabetic BB Wistar Rat The BB Wistar rat strain (Nakhooda et al., 1977, 1978; Like et al., 1982) develops spontaneous diabetes. Such diabetes is genetically determined and mediated via immune mechanisms, where diabetes-prone animals may be identified by the presence of lymphopenia. In order for overt diabetes to occur, the diabetic gene, various background genetic combinations and specific environmental factors (Butler et al., 1991) are required. Full blown diabetes, in the BB Wistar rat, has been achieved by whole body radiation, depletion of the RT6.1+T cell subset and by infection with Kilhams’s rat virus (Butler et al., 1991). The onset of diabetes, which is rapid, occurs between 60 and 120 days of age and may be attributed to marked
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hypoinsulinaemia. Typically, the affected rats develop hyperglycaemia with concomitant polyuria, glycosuria, ketonuria, weight loss and dehydration (Marliss et al., 1982; Nakhooda et al., 1977) and subsequently require insulin therapy to prolong their survival. Three varieties of neuropathy occur in these animals in addition to the metabolic abnormalities; these are mononeuropathy, autonomic neuropathy and distal symmetric polyneuropathy (Sima et al., 1987; Sima, 1993, 1995). Single motor nerve pathology, or mononeuropathy, as a result of endoneurial vascular occlusion by platelets, in response to abnormal prostacyclin metabolism, may, on occasion, lead to acute weakness (Sima and Thibert, 1982). A similar proximal motor neuropathy also occurs in human diabetes mellitus. The autonomic nerves that innervate the cardiovascular, urogenital and gastrointestinal systems of the diabetic BB Wistar rat may develop morphological and functional abnormalities. Axonal sequestration and atrophy of both myelinated and unmyelinated fibres of the vagus nerve (Yagihashi and Sima, 1986), accompany a progressive decline in the R-R interval of BB rats with diabetes of 8 weeks duration (McEwen and Sima, 1987). Similar changes occur in the penile and distal myenteric parasympathetic nerves (Yagihashi and Sima, 1986), as well as in postganglionic sympathetic unmyelinated fibre axons from the grey ramus. Even though axonal atrophy is absent, dystrophy of terminal axons and denervation of paravertebral ganglion cells is characteristic of preganglionic myelinated sympathetic fibres from BB rats with diabetes of 11 months duration (Yagihashi and Sima, 1985). Autonomic bladder dysfunction in BB diabetic rats is characterised by decreased contraction frequencies and increased amplitudes, which occur due to progressive axonopathy of the afferent sensory pelvic nerve fibres and axonal atrophy of the myelinated efferent preganglionic fibres of the hypogastric nerve (Paro et al., 1991). The structural and functional changes that accompany the third form of BB rat diabetic neuropathy, termed distal symmetric polyneuropathy, may be divided into acute and chronic categories. In the acute phase and as early as 3 weeks following diabetic onset, both nerve conduction velocity and evoked muscle potential amplitude decrease significantly (Sima and Hay, 1981; Sima et al., 1984). The conduction velocity deficit remains stable for about 4 months, after which it steadily worsens to about 60% of normal values at the end of a one year period. Single nerve fibre analysis, by the voltage clamp technique, reveals specific Na+-current abnormalities. These include intracellular Na+ accumulation, probably due to decreased Na+K+-ATPase activity, and a decrease in maximum available Na+permeability (Brismar, 1993). The increase in intra-axonal [Na+], that occurs in acutely diabetic BB Wistar rats, results in nodal and paranodal swelling; both of which are reversed following insulin treatment and/or myoinositol supplementation (Brismar and Sima, 1981; Greene et al., 1987). In the absence of treatment progressive axonal atrophy occurs, with a proximo-distal gradient; resulting in a dying-back process of the distal axons, degeneration and ultimately fibre loss. Up to 50% of sural nerve fibres may be lost in this way following one year of clinical diabetes. The central axons of sensory ganglia exhibit a similar but milder form of axonopathy (Sima and Yagihashi, 1986). Pathology of the node of Ranvier, which occurs independently to the dying-back axonopathy, is characterised by axo-glial dysjunction and progresses with the duration of the diabetic state. Ultimately myelin detaches completely from the paranode, producing paranodal demyelination, which
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is subsequently repaired by remyelinating Schwann cells (Sima et al., 1990) forming intercalated internodes. In the chronically diabetic BB Wistar rat, incremental axonal atrophy and axoglial dysjunction occur, contributing to progressive nerve conduction velocity deficits; where the magnitude of the conduction deficit correlates to the degree of dysjunction (Sima et al., 1990). In the chronic diabetic state, aldose reductase activity decreases, while sorbitol dehydrogenase activity increases (Sima et al., 1995), as does NAD consumption; these factors in combination contribute to a state of ‘pseudohypoxia’ (Williamson et al., 1993) with increased concentrations in the reducing sugars (e.g. glucose and fructose). Thus, non-enzymatic glycation and fructation of various structural proteins (e.g. tubulin and neurofilaments) occurs contributing to the progressive axonopathy. Abnormalities, in the form of increased Ca2+ currents, occur in the dorsal root ganglion cells of chronically diabetic BB rats but not in the acutely diabetic animal and are preventable with aldose reductase therapy (Hall et al., 1995). The increase in cytoplasmic Ca2+ seen in these rats appears to be arbitrated by a defect in the G-protein mediated receptorgated Ca2+ channels, which ultimately contributes to dorsal root ganglion cell death in chronic diabetic neuropathy (Hall et al, 1995). In addition to the above abnormalities, recent studies have shown that the decrease in the levels of vasodilatory Prostaglandin E1, in chronically diabetic BB Wistar rats, may be prevented and/or corrected by aceyl-L-carnitine intervention (Sima et al., 1995). In light of the above discussion both the functional and structural changes that occur in chronically diabetic BB Wistar rats are most likely a reflection of a myriad of pathogenic mechanisms and differ from those that occur in the acute stage of the disease. The diabetic BB Wistar rat, with its three distinct types of neuropathology, serves as an excellent model for the investigation of the molecular and metabolic factors responsible for diabetic neuropathy. As such it also proves useful for the testing various therapies aimed at the management of human diabetic neuropathy. Other Spontaneously Diabetic Animal Models In order to elucidate the causation of human diabetic neuropathy, other spontaneously diabetic animal models have also been studied with respect to the biochemical, functional and structural changes that arise in their peripheral nerves. The fatty Wistar diabetic rat (WKY/N-cp) has proved to be a useful animal model for the study of non-insulin-dependent diabetes (NIDD) (Peterson et al., 1988a, 1990). The motor nerve conduction velocity, in this model, is found to be reduced while morphological studies of the peripheral nerves reveal paranodal and segmental demyelination, paranodal swelling and axonal degeneration, occuring at the age of 42–49 weeks (Peterson et al., 1988b). The GK rat (Goto-Kakizaki SSDR) a selectivelyinbred spontaneously diabetic animal, is also studied with specific reference to the aetiology of human diabetic neuropathy (Yagihashi et al., 1982). More recently the WBN/Kob rat has been described as a model for chronic pancreatitis and lateonset persistent diabetes that occurs spontaneously and does not require insulin therapy in order to ensure the survival of the animal (Mori et al., 1988, 1990; Nakama et al., 1985; Tsuchitani et al., 1985). Slowing of motor nerve conduction velocities and demyelination of the tibial nerve has been demonstrated in WBN/ Kob rats with
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diabetes of long duration (Narama and Kino, 1985). However, ultrastructural findings in WBN/Kob rats of 28 months of age are not significantly different to those seen in age-matched controls, suggesting that the neuropathy is related to metabolic aberrations, and that it is essentially indistinguishable from age-related neuropathy in the rat (Narama and Kino, 1985). More recently the development of demyelination in the sciatic and tibial nerve, of this animal model, has been confirmed, with less severe changes occuring in the sural nerve; such changes are more conspicuous around the endoneurial vessels and tend to be accompanied by axonal alteration (Yagihashi et al., 1993). Of the large animal models, the spontaneously diabetic dog provides a unique opportunity to study the effects of long term diabetes by eliminating complicating factors such as continued growth and the changes that accompany old age, all of which tend to beset rodent models where stable control groups are not available. In the superficial peroneal nerve, of spontaneously diabetic dogs, motor nerve conduction velocity is reduced while morphological analysis demonstrates a large number of fibres undergoing paranodal and segmental demyelination (Sharma et al., 1995). The nerve abnormalities observed in spontaneously diabetic rats and dogs tend to be similar to those observed in newly diagnosed asymptomatic diabetic patients, in whom a higher frequency of segmental demyelination has been observed (Dyck et al., 1980; Sharma et al., 1990). It is possible that with a longer duration of diabetes, the moderate electrophysiological and morphological changes in these animal models may lead to the more prominent changes that are observed in established cases of human diabetic neuropathy. CONCLUSION Assuming that the lessons learnt from experimental diabetes in animals can be related to the human situation, it is hoped that the changes observed in these animal models may reproduce some of the alterations characteristic of early human diabetic neuropathy and thus provide clues to the specific pathogenic mechanisms. Such animal studies have already revealed how important it is to maintain normoglycaemia on a 24 hour, day to day basis in order to prevent or reverse diabetic neuropathy. REFERENCES Arison, R.N., Ciaccio, E.I., Glitzer, M.S., Cassaro, J.A. and Pruss, M.P. (1967) Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin. Diabetes, 16, 51–56. Bailey, C.C and Bailey, O.T. (1943) The production of diabetes mellitus in rabbits with alloxan. JAMA, 122, 1165–1166. Bajada, S., Sharma, A.K. and Thomas, P.K. (1980) Axoplasmic transport in vagal afferent fibres in normal and alloxan-diabetic rabbits. J. Neurological Sci, 47, 365–378. Baynes, J.W. (1991) Role of oxidative stress in the development of complications in diabetes. Diabetes, 40, 405–412.
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Bhoyrul, S., Sharma, A.K., Stribling, D., Mirlees, D.D., Peterson, R.G., Farber, M.O., et al. (1988) Ultrastructural observations on myelinated fibres in experimental diabetes: effect of the aldose reductase inhibitor ponalrestat given alone or in conjunction with insulin therapy. J Neurol Sci, 85, 131–147. Bianchi, R., Marini, P., Merlini, S., Fabris, M., Triban, C., Mussini, E., et al. (1988) ATPase activity defects in alloxan-induced diabetic sciatic nerve recovered by ganglioside treatment. Diabetes, 37, 1340–1345. Birren, J.E. and Wall, P.D. (1956) Age changes in conduction velocity, refractory period, number of fibres, connective tissue space and blood vessels in sciatic nerve of rats. J Comp Neurol, 104, 1–16. Black, H.E., Rosenblum, I.Y. and Capen, C.C. (1980) Chemically induced (streptozotocinalloxan) diabetes mellitus in the dog. Am J Pathol, 98, 295. Blondel, O., Bailbe, D. and Portha, B. (1989) Relation of insulin deficiency to impaired insulin action in NIDDM adult rats given streptozotocin as neonates. Diabetes, 38, 610– 617. Bloodworth, J.M., Anderson, P.J. and Engerman, R.L. (1973) Microangiopathy in the experimentally diabetic animal. Adv Metab Res, 2, 245–250. Bohlen, H.G. and Niggl, B.A. (1980) Early arteriolar disturbances following streptozotocininduced diabetes mellitus in adult mice. Microvasc Res, 20, 19–29. Bravenboer, B., Kapelle, A.C., Hamers, F.P.T., van Buren, T., Erkelens, D.W. and Gispen, W.H. (1992) Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. Diabetologia, 35, 813– 817. Bretzel, R.G., Beule, B. and Federlin, K. (1981) Function and morphology of adult rat islets after culture and transplantation. In Islet Isolation, Culture and Cryopreservation, edited by K.Federlin and R.G.Bretzel. New York: Georg Thieme Verlag. Brinsmead, M.W. and Thorburn, G.D. (1982) Effect of streptozotocin on foetal lambs in midpregnancy. Aust J Biol Sci, 35, 517–525. Brismar, T. (1993) Abnormal membrane currents in diabetic rat nerve nodal membrane. In Diabetes mellitus and its complications, edited by A.K.Sharma, I. Galadari, M.Behara, S.K. Manchanda, Y.Abdulrazzaq and N.K.Mehra. New Delhi: MacMillan. Brismar, T. and Sima, A.A.F. (1981) Changes in nodal function in nerve fibres of the spontaneously diabetic BB-Wistar rat. Potential clamp analysis. Acta Physiol Scan, 113, 499– 506. Britland, S.T. and Sharma, A.K. (1990) A comparison of conventional insulin treatment and continuous subcutaneous insulin infusion therapy in the reversal of ultrastructural abnormalities of myelinated fibres in experimental diabetes. Diab Res, 15, 143–150. Britland, S.T., Sharma, A.K., Duguid, I.G.M. and Thomas, P.K. (1986) Ultrastructural observations on myelinated fibre size in the tibial nerve of streptozotocin-diabetic rats: effect of insulin treatment. In ESAO Proceedings, edited by S. Raptis. London: W.B.Saunders. Britland, S.T., Von Zimmerman, O., Sharma, A.K., Bretzel, R.G. and Federlin, K. (1991) The effect of pancreatic islet transplantation on experimental diabetic neuropathy.J Neurol Sci, 105, 168–174. Brown, M.J., Sumner, A.J., Greene, D.A., Diamond, S.M. and Asbury, A.K. (1980) Distal neuropathy in experimental diabetes. Ann Neurol, 8, 168–178. Brosky, G. and Logothetopoulos, J. (1969) Streptozotocin diabetes in the mouse and guinea pig. Diabetes, 18, 606–611.
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6. NEUROTROPHISM IN DIABETIC NEUROPATHY DAVID R.TOMLINSON and PAUL FERNYHOUGH Department of Pharmacology, Queen Mary and Westfield College, Mile End Road, London E1 4NS, U.K.
BACKGROUND Peripheral nerves exhibit extreme polarity of structure to subserve their role as longdistance information conduits. All of the biosynthetic capacity of the neurone is retained close to the source of messenger RNA (mRNA) in the nucleus and this requires the translocation of macromolecules and organelles from their site of synthesis to the axonal extremity. In primary afferents and motoneurones of the lower limb in a tall individual, this represents a cytological marathon of over 1 metre. Maintenance of this process of intra-axonal transport is an absolute requirement for the survival of the distal axon and the degeneration consequent on experimental interruption of transport resembles the degeneration seen in diabetic neuropathy (Cuénod et al., 1972). Thus, substantial early work concentrated on the potential of failures in the process of axonal transport of organelles and proteins to the periphery in the search for the aetiology of axonal transport. In summary, this work has revealed that many components of axoplasm are delivered to the periphery in reduced amounts in rats with experimental diabetes (see Tomlinson and Mayer, (1984) for review). However, it appears that the processes of translocation itself functions more or less normally in experimental diabetes (Robinson et al., 1987; Tomlinson et al., 1988), so that attention is now more properly focused on deficits in synthesis of material in the nerve cell body; effective transport with deficient production is equally detrimental to export. In neurones regulation of the synthesis of the various classes of protein is adjusted via production of neurotrophic molecules by the neuronal target cells—these are called neurotrophic factors and they are captured by the axon and delivered to the cell body by retrograde axonal transport. In this way the nature and activity of the target cell maintains appropriate expression of the phenotype of its innervating neurones. Nerve growth factor (NGF) is a member of a family of neurotrophic factors that includes brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5) (see Ebendal (1992) for review). NGF is retrogradely transported in sensory and sympathetic neurones in adult rats (Hendry et al., 1974; Schmidt and Yip, 1985) and 50% of the adult rat lumbar sensory neurones can bind
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NGF with high affinity (Richardson et al., 1986). BDNF and NT-3 are also retrogradely transported from an injection site in the sciatic nerve to the dorsal root ganglia (DRG) and motoneurons of adult rats (DiStefano et al., 1992). Not surprisingly, most of the work has been done on the hindlimb of the rat, with skeletal muscle and skin as the major sources of neurotrophins and genes expressed in the cell bodies of primary afferents in the lumbar dorsal root ganglia as the major targets for neurotrophin response. There is mounting evidence to suggest that NGF and other neurotrophins are involved in regulating the mature phenotype in adult neurones. Sciatic nerve transection results in a decrease in mRNA for the medium neurofilament protein (NFM) in large DRG neurons as measured by in situ hybridisation (Verge et al., 1990). Administration of NGF by intrathecal infusion was shown in the same study to prevent this injury-induced decrease in NF-M mRNA in neurons which express high affinity NGF receptors. The major role of the neurofilaments is maintenance of axonal calibre, which decreases proximal to the injury site after axotomy (Gold et al., 1991). Delivery of NGF to the proximal stump of an injured nerve partially prevents this decrease in axonal calibre and the associated decrease in neurofilament content (Gold et al., 1991). In addition, daily injection of NGF antiserum to normal adult rats causes a decrease in axonal calibre in the proximal axon of intact DRG neurons (Gold et al., 1991). Thus, it is possible that one role of retrogradely transported NGF in the adult rat is the maintenance of axonal calibre via regulation of neurofilament synthesis. Retrogradely transported NGF may also regulate the levels of its own receptors. Levels of mRNA for both the high and the low affinity NGF receptors in the L5 DRG are also down-regulated after peripheral nerve transection and restored by exogenous NGF (Verge et al., 1992). Interestingly, exogenous NGF has no effect on mRNA for the high affinity NGF receptor in uninjured sensory neurons, but upregulates mRNA for the low affinity receptor (Verge et al., 1992). The substance P content of cervical sensory ganglia was significantly increased 72 hours after NGF was injected into the forepaw of adult rats (Goedert et al., 1981). It has since been shown that, although regenerating adult sensory neurones in culture do not require NGF or BDNF for survival, exogenous NGF or BDNF enhance neurite extension and NGF mediates increases in both the peptide content (Lindsay et al., 1989) and mRNA (Lindsay and Harmar, 1989) for substance P and calcitonin gene-related peptide (CGRP). The rat and bovine promoter sequences of the preprotachykinin-A (PPT-A) gene contain regions which confer NGF responsiveness and are putative transcription binding sites (Gilchrist et al., 1992). This suggests that NGF can influence the binding of regulatory protein(s) to the promoter region of the PPT-A gene, indicating that NGF can directly stimulate and regulate transcription of substance P. No such regulatory mechanisms have yet been described for CGRP. NGF is also thought to play a role in nociception, subcutaneous injections of exogenous NGF causing mechanical and heat hyperalgesia (Lewin and Mendell, 1993); this could cause problems in the event of therapeutic use of NGF. It has also been demonstrated that intradermal injection of NGF into the foot pad of adult rats leads to an increase in the rate of nociceptive fibre sprouting and evokes de novo sprouting (Diamond et al., 1992). In denervated skin there is also collateral sprouting from neighbouring undamaged nerve fibres. This is associated with an increase in
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NGF mRNA in the skin (Mearow et al., 1993). Treatment of rats with anti-NGF antibodies prevented denervation-induced sprouting (Diamond et al., 1992). These data suggest that altered nociception and collateral sprouting in disease states could be related to alterations in trophic support. This presents an overwhelming case for a physiological role for NGF in regulating expression of endoskeletal and transmitterrelated genes in primary afferent neurones of the adult nervous system. By association the other neurotrophins probably complement this role for other neurone types and may be selective for other genes. It follows, therefore, that the expression and action of the neurotrophins could present a primary focus for failure in peripheral neuropathies. REGULATION OF NEUROTROPHIN EXPRESSION A range of molecules and second messengers stimulate NGF gene expression in a variety of cell types, including Schwann cells, glioma cell lines and fibroblasts (Carswell, 1993), but in the intact adult system, the relevance of these mechanisms remains to be elucidated. However, failing expression of neurotrophins might be counteracted pharmacologically as an alternative to replacement therapy. Of major interest is the ability of agonists stimulating adrenoceptors, coupled to adenylate cyclase, to up-regulate NGF synthesis and secretion (Mocchetti et al., 1989)Recently, cultured smooth muscle cells have been shown to increase NGF synthesis in response to contractile stimuli (Tuttle et al., 1993). It has also been demonstrated that NGF levels in the heart correlate with the density of sympathetic innervation (Shelton and Reichardt, 1984), and that depolarising stimuli increase NGF mRNA levels in cultured rat hippocampal neurons (Lu et al., 1991). These observations suggest that impulse activity of neurons may influence NGF secretion by the target organ. This functional regulation of NGF synthesis in intact nerves may be supplanted or invigorated on damage to the nerve. Sciatic nerve section increases NGF mRNA in fibroblasts and Schwann cells of both proximal and distal sections of the nerve (Heumann et al., 1987; Lindholm et al., 1988). This initial increase in NGF mRNA is believed to be due to the induction of immediate early genes such as c-fos (Heumann et al, 1991). Following this early boost in NGF mRNA, interleukin 1, released from macrophages, is thought to produce a more lasting increase in NGF synthesis (Heumann et al., 1991). This mechanism is not uniform for all trophic factors, in that BDNF expression is also up-regulated in response to sciatic nerve transection, but the time course of induction is much slower than for NGF, starting 3–4 days post-lesion and reaching maximal levels after 3–4 weeks (Meyer et al., 1992). In addition, interleukin-1 is without effect on BDNF mRNA levels in cultured nerve explants (Meyer et al., 1992). PRODUCTION AND ACTION OF NEUROTROPHINS IN DIABETES Criteria for demonstration of the involvement of deficiency of a trophic factor in the aetiology of diabetic neuropathy are outlined below.
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• Deficient expression or retrograde transport of the neurotrophic factor must be demonstrated in tissues from diabetic models and diabetic patients. • Deficient expression of genes, which respond tonically to the neurotrophic factor, must be demonstrated in neurones in diabetes • Deficient expression of these genes in diabetes should be corrected by administration of the appropriate neurotrophic factor or selective stimulation of its endogenous production. • Administration of the neurotrophic factor should attenuate functional neuronal deficits characteristic of diabetic neuropathy. Decreased retrograde transport of NGF may reflect decreased expression in target organs in experimental diabetes. Thus, in diabetic rats there is reduced axonal transport of exogenous NGF in the sciatic and mesenteric nerves (Jakobsen et al., 1981; Schmidt et al., 1986) and reduced levels of NGF in the submandibular gland, superior cervical ganglion and sciatic nerve (Hellweg and Hartung, 1990; Hellweg et al., 1991). These reductions were attenuated or prevented by either insulin treatment or allogenic pancreatic islet transplantation (Hellweg et al., 1991). NGF levels have also been shown to be decreased in the serum of diabetic patients with peripheral neuropathy (Faradji and Sotelo, 1990). The latter study demonstrated a significant correlation between serum NGF levels and motor nerve conduction velocity (MNCV). The importance of this finding is unclear and the correlation may be coincidental as NGF is not thought to act on motoneurons, nor is the relevance of serum levels established. Recent work in our laboratory has shown that, with increasing durations of diabetes, progressive reductions in NGF mRNA appear in different tissues. We have concentrated on production of neurotrophins by neuronal target tissues of the lower (hind) limb, so as to correlate findings with data from the sciatic nerves and lumbar dorsal root ganglia. Thus, in this system, the major producers of neurotrophin are skeletal muscle and skin. Leg muscles show reductions in NGF expression, in terms of both mRNA and immunoreactivity (Figure 1) and these changes are evident after just four weeks of diabetes (Fernyhough et al, 1993a; Fernyhough et al., 1993b). In foot skin the reduction in NGF expression takes longer to develop (Figure 2), because at 4 weeks diabetes NGF mRNA levels are normal, though a clear reduction is present in skin from rats with diabetes of 12 weeks duration (Fernyhough et al., 1992). In both tissues these reductions can be prevented or reversed by maintenance of tight glycaemic control with intensive insulin treatment (Figures 1 and 2). More recently we have demonstrated a clear reduction in the amount of NGF-like immunoreactivity (NGF-LI) accumulating distal to a crush applied to the sciatic nerves of diabetic rats (Figure 3). This indicates a deficit in retrograde axonal transport of NGF, an assertion borne out by the finding of a reduction of a similar proportion in the NGF-LI content of the contralateral dorsal root ganglia at L4 and L5, which house the cell bodies of the sciatic primary afferents (Figure 3). These data also show prevention of these deficits via maintenance of tight glycaemic control with insulin. Part of the same experiment involved treatment of further diabetic groups with human recombinant NGF together with measurement of NGF-LI distal to a sciatic nerve constriction and in the contralateral dorsal root ganglia; this was done with the object of demonstrating that exogenous NGF could gain access to
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Figure 1 Production of nerve growth factor (NGF) by soleus muscle. The main figure shows NGF mRNA levels—measured from northern blot hybridisations and expressed relative to controls (open columns)—and NGF protein levels in untreated diabetic rats (black columns), diabetic rats treated with intensive insulin for the final 4 weeks of the protocol (diagonal hatching) and diabetic rats treated with human recombinant (hr) NGF (stippled; at 0.5 mg/ kg 3 times per week for the last 4 weeks). Duration of diabetes was 8 weeks. Note the reductions in NGF mRNA and protein in diabetic rats, with normalisation of both by insulin treatment. hrNGF treatment was associated with increased NGF-like immunoreactivity in the soleus, but this was not derived from endogenous production, as shown by the reduced mRNA levels. The inset figure (top right) shows reduced levels of NGF mRNA (normalised to each of 4 different control groups) in soleus at different durations of diabetes.
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Figure 2 Production of nerve growth factor (NGF) mRNA by foot skin. NGF mRNA (measured as for Figure 1) was unaffected by diabetes of 4 weeks duration (black columns) relative to controls (open columns), but was reduced significantly at 12 weeks diabetes. At this duration, intensive insulin for the final 4 weeks of the protocol (diagonal hatching) normalised NGF mRNA levels.
neurones whose gene expression it might influence. As is clearly shown (Figure 3), such treatment achieved dose-related increases in NGF-LI in both nerve and ganglia; indeed the amounts registered were greater than those seen in tissues from nondiabetic untreated rats. It is clear, therefore, that expression and retrograde transport of NGF is deficient in diabetic rats, satisfying the first criterion listed above. Furthermore, appropriate pharmacological intervention can surmount this deficit. More recently still Anand and his colleagues, working in our hospital, have demonstrated reduced expression of NGF-like immunoreactivity in a small cohort of diabetic neuropathic patients (Anand et al., 1996), correlating this change with impaired cutaneous responses to capsaicin. This correlation extends the observed reduction in NGF expression to a possible reduction in release of neuropeptides from C-fibres, a major gene target for NGF. The cause of the reductions in NGF mRNA and protein in tissues of diabetic animals is unknown. The vitamin D metabolite, 1,25-dihydroxyvitamin D3 is known to induce NGF mRNA in vitro (Wion et al., 1991). The serum concentration of 1,25-dihydroxyvitamin D3 is decreased in diabetic rats, whereas corticosterone concentration is increased. When fibroblast-like L929 cells were exposed to concentrations of corticosterone and 1,25-dihydroxyvitamin D3 similar to those found in diabetic rats, there was a decrease in NGF mRNA (Neveu et al., 1992). Besides changes in the synthesis and transport of NGF itself, alterations in NGF receptors may mediate a reduced response. The extracelluar cleavage product of the
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Figure 3 Retrograde transport of NGF-like immunoreactivity (NGF-LI) in sciatic nerves. Rats of 5 different groups were subject to unilateral ligation of the sciatic nerve for 6 hours to collect retrogradely transported material accumulating distal to the ligature (the linearity of this accumulation was determined as described elsewhere (Fernyhough et al., 1995)). NGFLI was measured in the 1 cm segment distal to the ligature (Dl, shaded grey) and in the contralateral lumbar dorsal root ganglia (L4 and L5, cross hatched). Reductions in retrograde transport of NGF-LI in diabetic rats (8 weeks duration) were prevented by treatment with insulin (as Figure 1). Treatment of diabetic rats with hrNGF (0.5 or 1.0 mg/kg, timing as Figure 1) increased retrograde transport above control levels.
low affinity NGF receptor (truncated p75NTR) is found in plasma and urine and the urine concentration was increased in diabetic patients with neuropathy (Hruska et al., 1993). The same study demonstrated an increase in immunoreactivity for this low-affinity receptor in Schwann cells of neuropathic diabetic patients. Thus, undisclosed mechanisms may provoke an increased turnover of the low-affinity NGF receptor. This may have functional consequences and additionally may offer useful diagnostic information via the change in urine. We have also seen clear reductions in the levels of the mRNA coding for the highaffinity NGF receptor, trkA in dorsal root ganglia from diabetic rats (Maeda et al., 1996). If this change translates to reduced trkA receptor protein levels, then there would be an additional flaw in NGF-derived neurotrophic support, since these receptors are involved in the capture of target cell-derived NGF by its respondent neurones and in the transduction of their response to the neurotrophin (see Chao and Hempstead, 1995; Kaplan and Stephens, 1994; Maness et al., 1994, for reviews). The above criteria also demand that there should also be deficient expression of NGF-responsive genes in neurons in diabetes. The neuropeptides substance P and calcitonin gene-related peptide (CGRP), are known to be up-regulated by NGF in cultures of adult DRG (Lindsay and Harmar, 1989). The levels of substance P and
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CGRP are decreased in the sciatic nerve and DRG of diabetic rats (Willars et al., 1989; Diemel et al., 1992). Recent work has demonstrated that there is also a reduction in the levels of mRNA for both CGRP and the substance P precursor, preprotachykinin-A (PPT-A) in the DRG of these animals (Diemel et al., 1994). This data is illustrated in Figure 4, where clear reductions in both substance P and CGRP can be seen in sciatic nerve segments from diabetic rats. Furthermore these reductions in neuropeptides are closely associated with reductions in NGF-like immunoreactivity in adjacent sciatic segments from the same animals. Administration of neurotrophic factor, or stimulation of its production, should correct the diabetes-associated deficits in expression of those neuronal gene which form targets for NGF. The decrease in PPT-A and CGRP mRNA in the DRG and the decrease in substance P and CGRP in the sciatic nerve of diabetic rats can be corrected by treatment with NGF (Diemel et al., 1994; Apfel et al., 1994). Again, Figure 4 illustrates this capacity for reversal of both substance P and CGRP deficits by treatment of diabetic rats with exogenous NGF. The association between NGF deficits and reduced expression of neuropeptides is strengthened further by the effect of insulin treatment as shown in Figure 4, whereby tight glycaemic control normalised the levels of both peptides and of NGF in insulin-treated diabetic rats. Regeneration is defective in both clinical and experimental diabetes. We have examined the capacity of targeted delivery of NGF to normalise parameters of regeneration following a nerve crush in diabetic rats (Whitworth et al., 1995). Specifically, this study examined the influence of fibronectin conduits, joining the two halves of a sectioned sciatic nerve, with and without pre-impregnation of the fibronectin with nerve growth factor (NGF), on regeneration in rats with streptozotocin induced diabetes. Regeneration, measured morphometrically in fibres containing immunoreactivity to calcitonin gene-related peptide (CGRP) and growthassociated protein 43 (GAP-43), was significantly impaired (p < 0.0001 for all comparisons) in diabetic rats with fibronectin grafts without NGF, compared to similarly-treated controls. Regeneration distances in diabetic rats were reduced to 43% (CGRP reactive fibres) and 44% (GAP-43 reactive fibres) of controls and the total amounts of immunoreactivity in the conduits were also reduced, though by lesser amounts (55% and 61% of controls respectively for CGRP and GAP-43). Impregnation of the conduits with NGF before implantation increased the distance and amounts of regenerating immunoreactivity in both control and diabetic rats for both CGRP and GAP-43, such that these regeneration parameters were similar in diabetic rats with NGF-fibronectin conduits to those in control rats implanted with untreated fibronectin conduits. These findings implicate impaired neurotrophic support in the defective regeneration characteristic of diabetic neuropathy and show that such defects can be corrected in NGF-responsive fibres by targeted administration of the human recombinant neurotrophin. Many molecules have been shown to stimulate NGF expression (see Riaz and Tomlinson (1996a) for review). Treatment of diabetic rats with 4-methylcatechol has been reported to prevent the decreased MNCV and increase, but not normalise, sciatic nerve NGF levels (Hanaoka et al., 1992). This suggests that 4-methylcatechol can increase endogenous NGF in vivo. The effect on MNCV is unexpected as NGF is not thought to act on adult motoneurons. Retrograde axonal transport labelling studies have shown that following injection of labelled neurotrophins at the site of
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Figure 4 Correction of deficient neuropeptide expression by administration of insulin or hrNGF. In sciatic nerves from untreated diabetic rats (black columns; 8 weeks duration) there were reduced levels of NGF-like immunoreactivity (NGF-LI), substance P and calcitonin generelated peptide (CGRP). These deficits were reversed by intensive insulin treatment (D-I, cross hatched; treatment as Figure 1) and increased above controls, in a dose-related manner by treatment with three doses (mg/kg, stipple; timing as Figure 1) of human recombinant NGF (D-hrNGF).
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a crush in the sciatic nerve, BDNF and NT-3 are found in motoneurons, whereas NGF is not (DiStefano et al, 1992). In addition, trkA (the high affinity NGF receptor) mRNA is not detected in postnatal rat lumbar motoneurones, whereas trkB and trkC mRNA (which encode the high affinity receptors for BDNF and NT-3 respectively) are detected in these cells (Chao and Hempstead, 1995). It is possible, however, that 4-methylcatechol is not specific for NGF and also stimulates BDNF and/or NT-3. Thus, although BDNF has no effect on neuropeptide (substance P and CGRP) levels, when administered to control or diabetic rats (Diemel et al., 1994), it may still be of importance in the treatment of some aspects of diabetic neuropathy. We have examined the capacity of the atypical ȕ-adrenoceptor, clenbuterol, to stimulate NGF expression in control and diabetic rats (Riaz and Tomlinson, 1996b). Figure 5 shows the effect of daily treatment of rats with clenbuterol on NGF and neuropeptide levels in sciatic nerves, with well-defined correlation between the effect on NGF and on the peptides. This suggests strongly that treatment with drugs that can stimulate expression of NGF in diabetes may boost neurotrophic support with beneficial functional consequences. OTHER NEUROTROPHINS NGF could participate in some of the signs and symptoms of diabetic neuropathy, but it could not be responsible for all or even the major neurotrophic deficit. One of the most dangerous clinical manifestations of this condition is loss of protective sensation in the feet. This leads to insensible trauma and foot ulceration, with a very poor prognosis. The neurones responsible for this sensation are probably unresponsive to NGF and may be dependent upon support from other neurotrophic factors. NT-3 mRNA levels are reduced in leg muscle from diabetic rats (Figure 6), but as stated above, it is difficult to make a systematic assessment of the tonic response to NT-3 because its gene targets are unidentified. We have, however, made a pragmatic assessment of the possible influence of NT-3 in experimental diabetes. Diabetic rats develop reduced velocity of conduction in both motor and sensory fibres of the sciatic nerve; these can be measured as differences in motor and sensory latencies from the M wave and the H reflex, respectively. The data shown in Figure 7 illustrate the reductions in both motor and sensory velocity in rats with streptozotocin-induced diabetes of 12 weeks duration. The third group of rats were diabetic treated with human recombinant NT-3, given subcutaneously at the back of the neck at a dose of 1 mg/kg three times per week for the last four weeks of the period of diabetes. It is clear that the NT-3 treatment, which did not attenuate the hyperglycaemia of diabetes, had a powerful selective effect on sensory nerve conduction velocity. The groups of sensory fibres which elicit the H reflex might be expected to include those responsible for protective sensation in the foot, implying that NT-3 may be more instrumental than NGF in the development of important functional deficits in diabetic neuropathy and that normalisation of its expression or pharmacological replacement may be an important therapeutic target.
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Figure 5 Stimulation of NGF expression, with corrective effects on its neuronal target genes, by treatment with clenbuterol. Four groups are shown in the scatterplots— untreated controls (open circles), clenbuterol-treated (0.25 mg/kg/day s.c.) controls (filled circles). Untreated diabetics (open squares) and clenbuterol-treated (as above) diabetics (filled squares). The regressions show close association between the effects of diabetes and of treatment on expression of NGF and of the neuropeptide products of its target genes.
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Figure 6 Neurotrophin 3 (NT-3) mRNA levels in soleus muscle from diabetic rats. At both 6 and 12 weeks diabetes (black columns) there is reduced expression of NT-3 mRNA in soleus muscle relative to controls (open columns). In the 12 week study, tight glycaemic control (cross hatched) for the final 4 weeks normalised NT-3 mRNA levels.
other neurotrophins and of other target neuronal processes in the aetiology of diabetic neuropathy has barely begun, but there is reason to suspect that the clear deficiencies in NGF and NT-3 expression and action in diabetic rats are but the tip of the iceberg. The therapeutic prospects offered by these findings are particularly exciting. CONCLUSIONS In summary, therefore, alterations in nerve growth factor and neurotrophin 3 synthesis in the target organs of sensory neurons is associated with reduced delivery of NGF—and possibly also of NT-3—to the nerve cell body in experimental diabetic neuropathy. This could have far reaching effects on the capacity of the affected neurones to maintain their phenotype. The ability of exogenous NGF, upregulators of endogenous NGF synthesis and of exogenous NT-3 to prevent at least some of the diabetes-associated neuronal dysfunctions, is an important step in understanding the pathogenesis of diabetic neuropathy. Examination of potential involvement of
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Figure 7 Neurotrophin 3 (NT-3) treatment selectively increases sensory nerve conduction velocity in diabetic rats. The effects of untreated diabetes and of treatment of diabetic rats with NT-3 (1.0 mg/kg s.c. 3 times/week for the last 4 weeks of 8 weeks diabetes) on motor (open columns) and sensory (filled columns) nerve conduction velocities. Both were decreased in diabetic rats, but NT-3 selectively improved sensory conduction velocity.
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Diemel, L.T., Brewster, W.J., Fernyhough, P. and Tomlinson, D.R. (1994) Expression of neuropeptides in experimental diabetes; effects of treatment with nerve growth factor or brain-derived neurotrophic factor. Mol. Brain Res., 21, 171–175. DiStefano, P.S., Friedman, B., Radziejewski, C., Alexander, C., et al. (1992) The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron., 8, 983–993. Ebendal, T. (1992) Function and evolution in the NGF family and its receptors. J. Neurosci. Res., 32, 461–470. Faradji, V. and Sotelo, J. (1990) Low serum levels of nerve growth factor in diabetic neuropathy. Acta Neurol Scand., 81, 402–406. Fernyhough, R, Carrington, A.L. and Tomlinson, D.R. (1992) Reduced nerve growth factor mRNA in skin of diabetic rats: effects of insulin. Br, J. Pharmacol., 107, 462P. Fernyhough, R, Brewster, W.J., Diemel, L.T. and Tomlinson, D.R. (1993a) Nerve growth factor mRNA in diabetic rat sciatic nerve; effects of neurotrophic factor treatment. Br. J. Pharmacol., 110, 173P. Fernyhough, R, Diemel, L.T., Smith, W.J. and Tomlinson, D.R. (1993b) Reduced mRNA for neurotrophic factors and GAP-43 in peripheral nerve and target tissue of diabetic rats: effects of brain-derived neurotrophic factor. Br. J. Pharmacol., 108, 37P Fernyhough, R, Diemel, L.T., Hardy, J., Brewster, W.J., et al. (1995) Human recombinant nerve growth factor replaces deficient neurotrophic support in the diabetic rat. Eur. J. Neurosci., 7, 1107–1110. Gilchrist, C.A., Morrison, C.F. and Harmar, AJ. (1992) A single-stranded DNA binding protein which interacts with sequences within the bovine preprotachykinin promoter: regulation by nerve growth factor. Biochem. Biophys. Res. Commun., 187, 1395–1400. Goedert, M., Stoeckel, K. and Otten, U. (1981) Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons. Proc. Natl. Acad. Sci. USA, 78, 5895– 5898. Gold, B.G., Mobley, W.C. and Matheson, S.F. (1991) Regulation of axonal caliber, neurofilament content, and nuclear localization in mature sensory neurons by nerve growth factor. J. Neurosci., 11, 943–955. Hanaoka, Y., Ohi, T., Furukawa, S., Furukawa, Y, et al. (1992) Effect of 4-methylcatechol on sciatic nerve growth factor level and motor nerve conduction velocity in experimental diabetic neuropathic process in rats. Exp. Neurol., 115, 292–296. Hellweg, R., Wöhrle, M., Hartung, H.-D., Stracke, H., et al. (1991) Diabetes mellitusassociated decrease in nerve growth factor levels is reversed by allogeneic pancreatic islet transplantation. Neurosci. Lett., 125, 1–4. Hellweg, R. and Hartung, H.-D. (1990) Endogenous levels of nerve growth factor (NGF) are altered in experimental diabetes mellitus: A possible role for NGF in the pathogenesis of diabetic neuropathy. J. Neurosci. Res., 26, 258–267. Hendry, I.A., Stoeckel, K., Thoenen, H. and Iversen, L.L. (1974) The retrograde axonal transport of nerve growth factor. Brain Res., 68, 103–121. Heumann, R., Korsching, S., Bandtlow, C.E. and Thoenen, H. (1987) Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection.J. Cell Biol., 104, 1623–1631. Heumann, R., Hengerer, B., Brown, M. and Perry, H. (1991) Molecular mechanisms leading to lesion-induced increases in nerve growth factor synthesis. Ann. N.Y. Acad. Sci., 633, 581–582.
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Hruska, R.E., Chertack, M.M. and Kravis, D. (1993) Elevation of nerve growth factor receptortruncated in the urine of patients with diabetic neuropathy. Ann. N.Y. Acad. Sci., 679, 349–351. Jakobsen, J., Brimijoin, S., Skau, K., Sidenius, P., et al. (1981) Retrograde axonal transport of transmitter enzymes, fiicose-labeled protein, and nerve growth factor in streptozotocindiabetic rats. Diabetes, 30, 797–803. Kaplan, D.R. and Stephens, R.M. (1994) Neurotrophin signal transduction by the Trk receptor. J. Neurobiol, 25, 1404–1417. Lewin, G.R. and Mendell, L.M. (1993) Nerve growth factor and nociception. Trends Neurosci., 16, 353–359. Lindholm, D., Heumann, R., Hengerer, B. and Thoenen, H. (1988) Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J. Biol. Chem., 263, 16348–16351. Lindsay, R.M., Lockett, C., Sternberg, J. and Winter, J. (1989) Neuropeptide expression in cultures of adult sensory neurons: Modulation of substance P and calcitonin generelated peptide levels by nerve growth factor. Neuroscience, 33, 53–65. Lindsay, R.M. and Harmar, A.J. (1989) Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature, 337, 362–364. Lu, B., Yokoyama, M., Dreyfus, C.F. and Black, I.B. (1991) Depolarizing stimuli regulate nerve growth factor gene expression in cultured hippocampal neurons. Proc. Natl. Acad. Sci. USA, 88, 6289–6292. Maeda, K., Fernyhough, P. and Tomlinson, D.R. (1996) Regenerating sensory neurones of diabetic rats express reduced levels of mRNA for GAP-43, gamma-preprotachykinin and the nerve growth factor receptors, trkA and p75 NGFR. Mol. Brain Res., 37, 166–174. Maness, L.M., Kastin, A.J., Weber, J.T., Banks, W.A., et al. (1994) The neurotrophins and their receptors: Structure, function, and neuropathology. Neurosci. Biobehav. Rev., 18, 143– 159. Mearow, K.M., Kril, Y. and Diamond, J. (1993) Increased NGF mRNA expression in denervated rat skin. Neuroreport, 4, 351–354. Meyer, M., Matsuoka, I., Wetmore, C, Olson, L., et al. (1992) Enhanced synthesis of brainderived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J. Cell Biol., 119, 45–54. Mocchetti, I., De Bernardi, M.A., Szekely, A.M., Alho, H., et al. (1989) Regulation of nerve growth factor biosynthesis by ȕ-adrenergic receptor activation in astrocytoma cells: A potential role of c-Fos protein. Proc. Natl. Acad. Sci USA, 86, 3891–3895. Neveu, L, Jehan, F. and Wion, D. (1992) Alteration in the levels of 1,25-(OH)2D3 and corticosterone found in experimental diabetes reduces nerve growth factor (NGF) gene expression in vitro. Life Sci., 50, 1769–1772. Riaz, S.S. and Tomlinson, D.R. (1996a) Neurotrophic factors in peripheral neuropathies: pharmacological strategies. Prog. Neurobiol., 49, 125–143. Riaz, S.S. and Tomlinson, D.R. (1996b) Clenbuterol stimulates nerve growth factor expression in control and diabetic rats: effects on neuropeptides. Br. J. Pharmacol., 117, 56P. Richardson, P.M., Verge Issa, V.M.K. and Riopelle, R.J. (1986) Distribution of neuronal receptors for nerve growth factor in the rat. J. Neurosci., 6, 2312–2321. Robinson, J.P., Willars, G.B., Tomlinson, D.R. and Keen, P. (1987) Axonal transport and tissue contents of substance P in rats with long-term streptozotocin-diabetes. Effects of the aldose reductase inhibitor ‘statil’. Brain Res., 426, 339–348.
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Schmidt, R.E., Grabau, G.G. and Yip, H.K. (1986) Retrograde axonal transport of [125I]nerve growth factor in ileal mesenteric nerves in vitro: effect of streptozotocin diabetes. Brain Res., 378, 325–336. Schmidt, R.E. and Yip, H.K. (1985) Retrograde axonal transport in rat ileal mesenteric nerves. Characterization using intravenously administered {125I}-nerve growthfactor and effect of chemical sympathectomy. Diabetes, 34, 1222–1229. Shelton, D.L. and Reichardt, L.F. (1984) Expression of the beta nerve growth factor gene correlates with the density of sympathetic innervation in effector organs. Proc. Natl. Acad. Sci. USA, 81, 7951–7955 Tomlinson, D.R., Robinson, J.P., Willars, G.B. and Keen, P. (1988) Deficient axonal transport of substance P in streptozocin-induced diabetic rats. Effects of sorbinil and insulin. Diabetes, 37, 488–493. Tomlinson, D.R. and Mayer, J.H. (1984) Defects of axonal transport in diabetes mellitus — a possible contribution to the aetiology of diabetic neuropathy. J. Auton Pharmacol., 4, 59–72. Tuttle, J.B., Etheridge, R. and Creedon, D.J. (1993) Receptor-mediated stimulation and inhibition of nerve growth factor secretion by vascular smooth muscle. Exp. Cell Res., 208, 350–361. Verge, V.M.K., Tetzlaff, W., Bisby, M.A. and Richardson, P.M. (1990) Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons. J. Neurosci., 10, 2018–2025. Verge, V.M.K., Merlio, J.-R, Grondin, J., Ernfors, R, et al. (1992) Colocalization of NGF binding sites, trk mRNA, and low-affinity NGF receptor mRNA in primary sensory neurons: responses to injury and infusion of NGF. J. Neurosci., 12, 4011–4022. Whitworth, I.H., Terenghi, G., Green, C.J., Brown, R.A., et al. (1995) Targeted delivery of nerve growth factor via fibronectin conduits assists nerve regeneration in control and diabetic rats. Eur. J. Neurosci., 7, 2220–2225. Willars, G.B., Calcutt, N.A., Compton, A.M., Tomlinson, D.R., et al. (1989) Substance P levels in peripheral nerve, skin, atrial myocardium and gastrointestinal tract of rats with longterm diabetes mellitus. Effects of aldose reductase inhibition.J. Neurol. Sci., 91, 153–164. Wion, D., MacGrogan, D., Neveu, I., Jehan, E, et al. (1991) 1,25-Dihydroxyvitamin D3 is a potent inducer of nerve growth factor synthesis. J. Neurosci. Res., 28, 110–114.
7. DIABETES MELLITUS: EVIDENCE FOR ALTERED CALCIUM SIGNALING IN EXCITABLE TISSUES KAREN E.HALL and JOHN W.WILEY Department of Internal Medicine, University of Michigan and Ann Arbor Veterans Affairs Medical Centers
INTRODUCTION The mechanisms underlying the pathophysiological consequences of diabetes mellitus are multifactorial. Accumulating evidence suggests that diabetes mellitus is associated with altered calcium homeostasis in a variety of tissues. The potential relationship of altered calcium homeostasis and the pathophysiology of diabetes mellitus has been the subject of a recent review (Levy et al., 1994). In this chapter we will focus on recent evidence that diabetes mellitus is associated with altered calcium homeostasis in excitable tissues. Therefore, we will emphasize evidence for altered calcium signaling in diabetic myopathy and neuropathy. Some studies employing non-excitable tissues will be included where they illustrate potentially relevant hypotheses examining the relationship of altered calcium signaling and the pathophysiologic consequences of diabetes. Cytosolic calcium levels [Ca2+]i in excitable tissues reflect the contribution of extra- and intracellular sources of calcium. Calcium influx via voltage-activated calcium channels appears to be the dominant contributor to [Ca2+]i when neurons undergo depolarization (Thayer and Miller, 1990). Changes in the magnitude of calcium influx in diabetes could reflect either altered expression or altered regulation of calcium channels. Alterations in basal cytosolic calcium levels may reflect changes in the magnitude of sequestered intracellular calcium stores or the ATP-dependent membrane extrusion pumps that maintain low physiological concentrations of intracellular calcium. We will review the literature that addresses each of these potential mechanisms. BACKGROUND Diabetes mellitus is characterized by hyperglycemia associated with either absolute insulin deficiency (Type 1) or relative insulin deficiency and insulin resistance (Type 2). Hypotheses that have been proposed to explain the pathophysiologic changes associated with diabetes include: 1. production of toxic metabolic products such as sorbitol, fructose and glycosylation end products as well as a reduction in myoinositol-dependent signal transduction events (Greene et al., 1988), and 2. impaired
Correspondence: John W Wiley, MD, VA Medical Center, Gastroenterology Division (lllD), Room B501a, 2215 Fuller Road, Ann Arbor, MI48105. Tel: (734)761–7981, Fax: (734)761–7549, E-mail:
[email protected]
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tissue vascular perfusion possibly as a result of decreased production of nitric oxide and overproduction of vasoconstricting eicosanoids (Craven et al., 1994). The role of altered regulation of calcium homeostasis in these events and its potential contribution to the complications of diabetes mellitus is the subject of on-going studies. Abnormal regulation of cytosolic calcium levels appears to be associated with decreased insulin secretion (Draznin, 1988) and increased insulin resistance (Draznin et al., 1989), that may contribute to the pathophysiology of diabetes and its complications. Increased intracellular calcium is a common finding in both Type 1 and Type 2 diabetes (Mazzanti et al., 1990; Tschope et al., 1991; Segal et al., 1990), although normal (Ischii et al., 1990) and decreased levels (Studer and Ganas, 1989; Chan and Junger, 1984; Levy et al, 1989a) have also been found in some tissues. Influx of calcium into coronary, aortic, and mesenteric smooth muscle cells is increased in insulin-deficient rats (Agrawal and NcNeill, 1987; Pieper and Gross, 1989). The density of calcium channels is increased in the leg skeletal muscle of diabetic rats (Lee and Dhalla, 1992). In contrast, no difference in channel expression was observed in ȕ-cells from diabetic GK rats that demonstrated an increase in calcium channel activity (Kato et al., 1996). Thus, the altered calcium signaling associated with diabetes demonstrates considerable variability and tissue specificity.
EVIDENCE THAT DIABETES-ASSOCIATED MYOPATHY INVOLVES ALTERED REGULATION OF BASAL CYTOSOLIC CALCIUM LEVELS
The changes in intracellular calcium homeostasis in Type 1 and Type 2 diabetes may overlap, as intracellular calcium levels tend to be increased in both conditions (Levy et al., 1994). However, the specific abnormalities in regulatory mechanisms may be different in the two syndromes. For example, membrane phospholipid content plays an important role in maintaining physiologic intracellular calcium levels (Levy et al., 1988) and may affect calcium influx and efflux mechanisms (Ganguly et al., 1986; Ishii et al., 1990; Mazzanti et al., 1990; Levy et al., 1988). Diabetes is associated with changes in membrane phospholipid composition that are different for insulindependent and non-insulin-dependent diabetes (Baldini et al., 1989). Therefore, the differences in intracellular calcium regulation between the two conditions may reflect different membrane phospholipid compositions. In addition, tissue-specific variation in membrane phospholipid composition may help explain the differences observed in intracellular calcium regulation between various tissues from the same model of diabetes (Levy et al., 1988). Variability has also been reported with regard to the plasma membrane calcium adenosine triphosphatase (ATPase) and sodium:potassium adenosine triphosphatase (Na+-K+-ATPase) activities. These two ATPase-associated cation pumps help maintain the low physiologic levels of intracellular calcium. The plasma membrane calcium-ATPase pumps calcium out of the cell. Calcium is also extruded
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in exchange for sodium via the sodium:calcium exchange pump, and sodium levels in the cell are, in turn, regulated by the Na+-K+-ATPase, which exchanges sodium for potassium. Thus, alterations in the activity of Na+-K+-ATPase that lead to changes in intracellular sodium concentration will modulate the activity of calciumATPase and the sodium:calcium exchange pump (Greene and Mackway, 1987). Decreased calcium-ATPase activity has been observed in heart sarcoplasma and sarcolemma from insulin-deficient diabetic rats (Pierce and Dhalla, 1981; Heyliger et al., 1987). In addition, mRNA expression of this pump is decreased in cardiomyocytes from insulin-deficient diabetic rats (Makino et al., 1987). However increased calciumATPase has been described in kidney cortex, erythrocytes and platelets from human and animal models of type 2 diabetes mellitus (Levy et al, 1986; Ishii et al., 1990; Mazzanti et al., 1990), suggesting that the discrepancy may be related to differences in either insulin availability, or relative resistance to insulin action. The effect of diabetes on Na+-K+-ATPase is also variable. Increased activity was observed in skeletal muscle membranes obtained from rat models for diabetes (Taira et al., 1991; Ganguly et al., 1986), and decreased activity was reported in nerve (Greene and Lattimer, 1983) and aortic smooth muscle cell membranes (Ohara et al., 1991) from insulin-deficient rats. Cardiovascular Muscle High levels of intracellular calcium in the myocardium can affect myocardial cell function and contribute to the development of diabetic cardiomyopathy (Allo et al., 1991; Russ et al., 1991). This cardiomyopathy is characterized by reduced cardiac reserve, delayed relaxation, and decreased shortening velocity, and can be prevented by calcium antagonist therapy (Afzal et al., 1988). Diabetes is associated with reduced myosin calcium-ATPase activity, decreased sarcolemmal and sarcoplasmic calciumATPase activities, decreased sarcolemmal Na/Ca exchange activities, and reduced mitochondrial ability to accumulate calcium (Allo et al., 1991; Makino et al., 1987; Afzal et al., 1988). Both sarcoplasmic reticulum (SR) calcium content and SR ryanodine binding sites were reduced in diabetes, suggesting that that SR calcium sequestration and release were impaired in diabetes mellitus (Yu et al, 1994). This could explain the diminished contraction observed in diabetic hearts. Collectively, these changes in cellular calcium regulation predispose the myocardium to cytosolic calcium overload, with adverse hemodynamic consequences (Afzal et al., 1988). Tam et al. (1997) examined the effect of norepinephrine on intracellular cytosolic calcium levels in vascular smooth muscle obtained from streptozotocininduced diabetic and non-diabetic control rats. The authors observed increased sensitivity to norepinephrine-induced changes in intracellular free calcium levels. These alterations may contribute to the peripheral vascular abnormalities observed in diabetes mellitus. Gastrointestinal Smooth Muscle Diabetic gastroparesis is frequently attributed to neuropathic changes in extrinsic or intrinsic innervation of the stomach. There is evidence that, in addition to neuropathy, myopathy may also play a role. Takahashi et al. (1996) observed
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impaired gastric circular muscle function in response to directly acting stimulants in the spontaneously-diabetic BB/W rat. Carbachol-induced production of IP3 and translocation of PKC were decreased in gastric smooth muscle obtained from rats 6 months after onset of diabetes. However, release of acetylcholine was not impaired in the diabetic rats. These observations suggest that a myopathic process may contribute to diabetic gastroparesis in the BB/W rat. Sakai et al. (1994) observed that the contractile response of smooth muscle from the gastric fundus, and protein kinase C activity was greater in diabetic rats compared to controls. Of interest, binding studies with the calcium channel antagonist [3H]PN200–110 revealed that the affmity and density of binding sites on smooth muscle plasma membrane-enriched fractions were similar in control and diabetic preparations. This suggests that the principal abnormality in calcium signaling in this model of diabetes mellitus involves altered intracellular signal transduction pathways, rather than increased numbers of membrane calcium channels. It is relevant to note that in the gastrointestinal tract, the relative contributions of extraand intracellular calcium pools is both region- and agonistdependent (Makhlouf, 1995). EVIDENCE THAT DIABETES MELLITUS-ASSOCIATED MYOPATHY INVOLVES ALTERED CALCIUM INFLUX Cardiovascular Muscle Chronic diabetes mellitus was associated with prolongation of the action potential duration in ventricular muscle in the streptozocin-induced diabetic rat which was possibly related to enhanced calcium influx (Nobe et al., 1990). In contrast, Wang et al. (1995) observed reductions in potassium currents and the L-type calcium current in ventricular myocytes from streptozotocin-induced diabetic rats. The authors suggest that the reduced K+- and Ca2+-currents can account for the action potential prolongation and depressed contraction, respectively, that is observed in ventricular myocytes from diabetic rats. Gotzsche et al. (1996) examined the number of myocardial calcium channels and beta-receptors in streptozotocin-diabetic rats. After 90 and 200 days of untreated diabetes the calcium channel number was significantly increased. The increase in number of calcium channels was normalized after 20 days of strict blood glucose control with insulin. Total myocardial beta receptor number did not differ in controls and diabetic rats. The authors postulate that the increase in sarcolemmal calcium channels may compensate for the impaired coupling of the beta-receptor to adenylate cyclase. Shimabukuro et al. (1995) observed that treatment with the L-type calcium channel antagonist nifedipine was associated with a partial recovery in impaired cardiac mechanical response in streptozotocin-induced diabetic rats. Hattori et al. (1995a, 1995b) observed that phorbol ester and serotonin-mediated activation of protein kinase C caused a delayed phase of contraction in diabetic rat aorta, possibly related to a greater influx of calcium as a consequence of increased PKC activity (Hall et al., 1995a). Augmented calcium influx correlated with enhanced proliferation of cultured vascular smooth muscle cells obtained from spontaneously diabetic Goto-Kakizaki rats (Yoo et al., 1997).
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Gastrointestinal Smooth Muscle Uchida et al. (1994) performed muscle tension studies on isolated ileum from streptozotocin-induced diabetic rats and controls. The authors observed a hyperreactivity of contractile response to elevated KCl in preparations from the diabetic rats and suggest that this may reflect enhanced calcium influx through voltageactivated calcium channels. Skeletal Muscle Nojima et al. (1995) observed that diaphragm muscle from streptozotocin-diabetic mice demonstrated desensitization of nicotinic acetylcholine receptor channels. This process was accelerated by activating protein kinase C, but not protein kinase A. The authors suggest that accelerated activation of PKC is caused by an increase in the amount of available intracellular calcium. The number of calcium channels expressed in skeletal muscle may also be affected in diabetes. Ogawa et al. (1995) observed a significant increase in the maximal binding (Bmax) of an L-type calcium channel ligand in 10 week streptozotocin-induced diabetic rats. Of interest, there was no change in binding affinity (Kd), and treatment with insulin for 8 weeks normalized the Bmax to control levels. DIABETIC NEUROPATHY The beneficial effects of achieving improved glycemic control on the development and progression of neuropathy in insulin-dependent humans are now clear (DCCT, 1993). It remains unclear, however, whether the mechanism(s) underlying the pathogenesis of diabetic neuropathy involve hyperglycemia per se, insulin deficiency, or associated defects in hormone, growth factor, lipid or amino acid metabolism. As we observed with changes in muscle function associated with diabetes, it is also not clear which tissue compartment(s) are principally involved in the derangements associated with diabetic neuropathy. Reduction of motor nerve conduction velocity and pathological alteration of peripheral nerve structure and function have been described in the diabetic human and rat models of experimental diabetes (Behse et al., 1977; Brismar and Sima, 1981). Prominent alterations in nerve carbohydrate metabolism in diabetic animals include increased levels of glucose, sorbitol and fructose, and reductions in nerve myo-inositol (MI) content and resting energy utilization. Considerable evidence implicates involvement of the polyol pathway by which glucose is metabolized to sorbitol and fructose by aldose reductase and sorbitol dehydrogenase, respectively. Hyperglycemia-associated alterations in the metabolism of sorbitol and MI and their associated effects on phosphoinositide metabolism, protein kinase C (PKC), Na/ KATPase and redox perturbations secondary to mitochondrial dysfunction may be particularly important in the pathogenesis of neuropathy (Greene and Mackway, 1987; Greene et al., 1988). Production of sorbitol by the aldose reductase pathway in response to hyperglycemia is thought to contribute to the pathogenesis of diabetic neuropathy because treatment with oral aldose reductase inhibitors and dietary supplementation with myo-inositol improves nerve conduction delay and reverses some metabolic alterations (Sima et al., 1990; Greene et al., 1993). Hyperglycemia
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and abnormal polyol and myo-inositol metabolism may represent a common link for the pathology observed in diabetes. The relationship may be tissue-specific, however, as it is observed mainly in tissues in which glucose entry into the cell is insulinindependent (Greene et al., 1993). According to this concept, cellular hyperglycemia increases the polyol pathway, decreases myoinositol pools, and decreases Na+-K+ATPase activity (Greene et al., 1987). The resulting increase in intracellular sodium inhibits the sodium-calcium exchanger, thus increasing intracellular calcium, which is associated with impaired axoplasmic transport and Schwann cell function (Winegrad, 1986; Lowery et al., 1990). The altered cellular calcium homeostasis subsequently contributes to changes in glucose-polyol-activity. Diabetic Neuropathy is Associated with Altered Calcium Signaling in Primary Sensory Neurons Kostyuk et al. (1995) observed that depolarization-induced calcium transients were prolonged in dorsal root ganglia (DRG) from diabetic mice. Of interest, this difference was noted in the sub-population of smaller (18–25 µm diameter) neurons, not larger (30–45 µm) DRGs. The smaller neurons are thought to primarily subserve nociception, raising the possibility that this abnormality may contribute to the increased pain sensitivity observed in some patients with diabetic neuropathy. Hall et al. (1995b) reported an increase in calcium current density in DRGs from longterm diabetic BB/W rats (Figure 1). The enhancement in calcium influx involved multiple calcium currents and was prevented by long-term treatment with an aldose reductase inhibitor. Therefore, alterations in neuronal function associated with the aldose reductase pathway may contribute to altered calcium signaling in diabetic neuropathy (Greene et al., 1993). In addition, the observation that diabetic neuropathy is associated with increased calcium influx involving multiple calcium channels provides a rationale for the limited improvement in nerve conduction velocities observed in diabetic rats treated with selective L-type calcium channel antagonists (Kappelle et al., 1992; Ristic et al., 1996). In separate studies, Hall et al. (1996) demonstrated that opioid-mediated inhibition of calcium currents in DRGs from diabetic BB/W rats was impaired compared to the magnitude of current reduction observed in age-matched controls. The impairment in opioid-mediated inhibition of calcium influx may contribute to the increased pain sensitivity experienced by some patients with diabetic neuropathy. Activation of opioid receptors decreases calcium influx in neurons via activation of inhibitory G proteins that couple the receptors to calcium channels (Moises et al., 1994). Evidence suggests that inhibitory G protein (Gi2Į) expression and/or function is decreased in hepatocytes in streptozotocin-induced diabetic rats (Bushfield et al., 1990). Of interest, hepatocytes in this model demonstrate enhanced phosphorylation of Gi2Į at a protein kinase C site that is associated with impaired function (Morris et al., 1996). Thus, decreased expression and/or impaired regulation of the inhibitory G protein-calcium channel complex may contribute to the observed increase in calcium influx in diabetes.
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Figure 1 Enhancement of calcium currents in diabetes was prevented by an aldose reductase inhibitor (ARI). Means+S.E.M. of currents elicited by depolarization to =10 mV from a holding potential of-80 mV in neurons from control ( ), diabetic ( ) and ARI- treated ( ) diabetic animals (diabetic duration 4 months: 8 control, 8 diabetic neurons; diabetic 6 months: 18 control, 9 diabetic, 2 ARI-treated diabetic neurons; diabetic 8 months: 15 control, 11 diabetic, 9 ARI-treated diabetic neurons; *** P < 0.005, **P < 0. 01 by ANOVA). (Reprinted from: Hall et al., 1995).
DiabeticNeuropathy: Role of Vas oactive Age nts Diminished nitric oxide (NO) production has been implicated in the pathogenesis of the ischemic injury observed in diabetes (Williams et al., 1996). Evidence in support of metabolic interdependence of the polyol pathway and NO synthesis is provided by the observations that in the diabetic rat model, aldose reductase inhibitor treatment restores endothelium-dependent relaxation to within normal limits, and that a stereospecific nitric oxide synthase (NOS) inhibitor can block the beneficial effects of an aldose reductase inhibitor on diabetic nerve conduction slowing (Stevens et al, 1995; Cameron et al., 1996). Activation of aldose reductase by glucose could blunt NO synthesis by either a PKC-mediated mechanism or possible metabolic competition between aldose reductase and NO synthase (NOS) for the cofactor NADPH. Recently, evidence has been provided that PKC can regulate NOS by direct phosphorylation (Bredt et al., 1992). The constitutive form of nitric oxide synthase is calcium and calmodulin-dependent, thus altered calcium homeostasis in diabetes may also contribute to the above abnormalities in NO production. The relationship between altered calcium signaling in diabetes mellitus and the activity of constitutive NOS remains to be elucidated.
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Diabetic Neuropathy: Role of Serum Factors Recent evidence suggests that serum factor(s) may also play a role in the pathophysiology of diabetes mellitus, possibly by facilitating calcium influx. Exposure of insulin-producing cells to serum from humans with Type 1 diabetes resulted in increased L-type calcium channel activity (Figure 2) (Juntti-Berggren et al., 1993). Initial characterization of the serum factor suggested that it was an immunoglobulin belonging to the IgM family, and that it induced apoptosis. Ristic et al. (1998) observed that 24 hour exposure of acutely dissociated non-diabetic rat primary sensory neurons to serum from diabetic BB/W rats caused an enhancement in multiple calcium currents. The authors present data suggesting that impaired inhibitory G-protein regulation of calcium channels may be involved. Other studies have implicated a complement-fixing autoantibody (IgG class) as the factor in serum from Type 1 diabetics that induces calcium-associated neuronal injury (Pittenger et al., 1993, 1995, 1997). However, proof of the cause and effect relationship between elevated cytosolic calcium and neuronal injury has not been established. The potential relationship to altered calcium signaling was not examined in these latter studies. Diabetic Neuropathy: Role of Neurotrophic Growth Factors Peripheral sensory and autonomic neuropathy of diabetes appears to also involve impaired neurotrophic “tone”, which could in turn reflect diminished synthesis or secretion of, or decreased responsiveness to, neurotrophic factors such as nerve growth factor (NGF) (Thomas, 1994). Neurotrophic factors such as NGF are required not only for the development but also for the maintenance of sensory and autonomic neurons and their axonal processes (Ruit et al., 1990). During neural development and regeneration, Schwann cells also synthesize and express growth factors such as NGF, ciliary neurotrophic factor (CNTF), a small polypeptide originally purified from chick and rabbit sciatic nerve, and insulin-like growth factor I (IGF 1). NGF is required for normal function of sympathetic and sensory neurons and loss of NGF leads to neuronal dysfiinction and/or death (Rich et al., 1987). NGF levels are reduced in sympathetically innervated target organs of streptozocindiabetic rats (Hellweg and Hartung, 1990). Retrograde axonal transport of NGF is reduced in somatic sensory neurons in streptozocin-diabetic rats. Some of these transport defects are corrected in the diabetic rat by treatment with aldose reductase inhibitors or myo-inositol, suggesting involvement of the polyol pathway in axonal transport defects (Greene et al., 1993). Patients with diabetic neuropathy also have significantly lower levels of serum NGF than non-diabetic control subjects. These data suggest that NGF production may be altered in diabetes, and are consistent with the contention that disruption of normal neurotrophism may contribute to the pathogenesis of diabetic neuropathy. IGF-1 immunoreactivity is also present in Schwann cell cytoplasm, is increased by sciatic nerve transection, and enhances regeneration in a dose-dependent fashion in lesioned sciatic nerve (Ishii, 1995). In streptozocin-diabetic rats, serum IGF-1 levels are diminished, as is IGF-1 mRNA in liver, kidney, lung and heart. Since successful axonal maintenance, regeneration and reformation of axon-Schwann cell contacts
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Figure 2 Increased activity of single Ca2+channel currents in cells treated with IDDM serum. Examples of cell-attached, single channel recordings from individual cells of an insulinproducing pancreatic cell line (RINm5F) treated with normal (A) and IDDM (B) serum. Cells exposed to diabetic serum demonstrated significantly increased single channel open time and decreased closed time. This resulted in a ten-fold increase in the associated mean current (bottom traces) in cells exposed to IDDM serum (B) compared to cells exposed to control serum (A). The bottom traces represent the mean currents obtained by averaging 125 (A) and 90 (B) traces. (Reprinted from: Juntti-Berggren et al., 1993).
is likely dependent on local growth factor synthesis, blunted local neurotrophic effects may contribute to the pathogenesis of diabetic neuropathy. Impaired neurotropic “tone” in diabetes appears to have significant effects on calcium signaling and susceptibility to injury. The mechanism(s) of excitotoxic/ ischemic injury have been studied extensively in the CNS and appear to involve elevation of intracellular calcium and generation of free radicals (Nicotera et al., 1992; Mattson et al., 1993; Morley et al., 1994). Several growth factors including neurotrophins and IGF-1 can protect neurons from excitotoxic/ischemic injury, possibly by preventing the excessive elevation of calcium (Johnson et al., 1992) and attenuating the effects of free radical-induced injury such as metal-catalyzed oxidation. Treatment of diabetic rats with NGF was associated with improvement in structural and functional correlates of neuropathy (Apfel et al., 1994). Neurons failing to obtain sufficient trophic factor are thought to undergo apoptosis (programmed cell death) (Bredesen, 1995). Deprivation of adult sympathetic neurons of NGF results in extensive neuronal death (Rich et al., 1987). In addition, NGF blocks the death caused by axotomy, target removal, and even certain chemical and virological insults to these neurons. Removal of NGF from culture medium
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causes condensation of chromatin, blebbing of neurites, somatic atrophy, and DNA fragmentation into oligonucleosomes, a hallmark of apoptosis (Bredesen, 1995). PATHOPHYSIOLOGIC IMPLICATIONS OF ALTERED CALCIUM SIGNALING IN DIABETES MELLITUS The current view of the calcium hypothesis of cellular injury proposes several interrelated postulates (Johnson et al., 1992; Nicotera et al., 1992). First, it proposes that cellular mechanisms that regulate the homeostasis of cytosolic free calcium ion [Ca2+]i, theso-called calcium “set-point”, play a critical role in a variety of neurodegenerative processes, and that altered (Ca2+)i might account for a number of the changes in neural function observed in diabetes mellitus. Second, it postulates that the plasticity of neuro-architecture is regulated by functional equilibrium between molecular mechanisms promoting growth/regeneration and processes that control regression/degeneration. Third, it proposes a systematic interaction between the magnitude of the change in cytosolic calcium and the duration of the deregulation in calcium homeostasis. Therefore a small change in cytosolic calcium that is sustained over a long period can result in similar cellular damage as a large change in [Ca2+]i overa short period. Fourth, it suggests that altered regulation of [Ca2+]i homeostasis may be part of the final common pathway for the cellular changes leading to cell dysfunction and death. This hypothesis accounts for several alternative mechanisms through which the regulation of cytosolic calcium can be disrupted. These include changes in ion channel functioning or formation of new channels; changes in membrane structure altering the functioning of transmembrane proteins; and alterations in the behavior of calcium binding proteins, extrusion pumps, buffers, and sequestration. Fifth, it proposes that cell injury may not be due to a single event or insult, but is brought about by a series of different antecedent events occurring, in a combination or sequence, over a long period. Most of the events triggered by a rise in cytosolic calcium reflect the action of calcium on enzymes, notably lipases, protein kinases (or phosphatases), proteases, and endonucleases. Potential Sites of Defective Calcium Regulation in Diabetes It is reasonable to speculate that a defect in calcium signaling will involve either increased calcium influx or decreased calcium efflux, or both. Increased calcium entry into the cell or decreased calcium extrusion from the cell could lead to calcium overload and increased intracellular calcium. Studies on adipocytes by Draznin and collegues (Draznin, 1988; Draznin et al., 1988, 1989) suggest that the primary defect involves a site regulated by insulin. Their studies implicate elevated intracellular calcium as a factor contributing to impaired insulin action in diabetes. However, it is not clear which site(s) insulin may act on to regulate intracellular calcium. Insulin stimulates plasma membrane calcium-ATPase activity, an effect that appears to be mediated by phosphorylation (Levy and Rempinski, 1991). This enzyme likely plays an important role in the acute effects of insulin, and as outlined earlier, may contribute to abnormal calcium influx in diabetes. However, other factors have also been shown to affect calcium-ATPase function. There may be a genetic
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predisposition to abnormal calcium-ATPase function in diabetes (Levy et al., 1989b) or affected by diet-such as a high-fat diet-that can change membrane function (Storlien et al., 1987). Hyperglycemia-induced glycosylation of erythrocyte calciumATPase can also decrease its activity (Flecha et al., 1990). In contrast, insulin does not have direct effects on the Na+-K+-ATPase pump in isolated membranes (Resh, 1985). As discussed earlier, the effect of insulin on glucose and potassium metabolism may be dissociated, with both elevated and decreased Na+-K+-ATPase activity observed in diabetes. Interestingly, glucagon has been implicated in the decreased ouabain sensitivity of ileal Na+-K+-ATPase in streptozotocin-induced diabetic rats (Fedorak et al., 1991), as application of glucagon to tissue from age-matched controls mimicked the effect of diabetes on Na+K+ATPase activity. The relationship between insulin, intracellular calcium levels and Na+-K+-ATPase activity is further complicated by the fact that insulin requires an optimal range of intracellular calcium to effectively stimulate glucose transport and other intracellular events (Draznin et al., 1987; Pershadsingh et al., 1987), and therefore, decreased Na+-K+-ATPase activity with concomitant increased intracellular calcium levels could impair insulin action. It is likely that both hyperglycemia and insulin deficiency affect intracellular calcium regulation (Ohara et al., 1991; Sowers, 1992). Changes in intracellular calcium metabolism may lag behind the development of hyperglycemia and hypoinsulinemia in experimentally induced diabetes (Makino et al., 1987; Studer et al., 1989). Differences in the duration of diabetes may help explain the discrepancies in the literature regarding regulation of calcium signaling in diabetes mellitus. Since destruction of pancreatic ȕ-cell mass in insulin-dependent diabetes leads to hyperglycemia and hyperlipidemia (Winegrad, 1986) which, in turn, can affect intracellular calcium handling (Winegrad, 1986; Davis et al., 1987), it is possible that the changes in intracellular calcium homeostasis are secondary to the metabolic derangements. Thus, the acquired increased intracellular calcium is observed in experimental models of insulinopenic diabetic animals (Levy et al., 1989b; Ohara et al., 1991; Studer et al., 1989; Lowery et al., 1990). The situation with noninsulindependent diabetes is less clear. SUMMARY AND CONCLUSIONS Diabetes mellitus is associated with abnormal calcium signaling. This defect appears to be ubiquitous, and is observed in both type 1 and type 2 diabetes. Abnormal calcium signaling may be of significance in the pathogenesis of impaired insulin secretion and action, and in the pathogenesis of microvascular and macrovascular complications. Increased intracellular calcium may contribute to the altered functions of hormones other than insulin in diabetes. Abnormal intracellular calcium homeostasis could contribute to the diversity observed in both diabetic syndromes. Studies involving diabetic humans and experimentally-induced diabetes in animals indicate that altered cytosolic calcium homeostasis is a common defect in both insulindeficient and non-insulin-deficient diabetes resulting in increased cytosolic calcium levels. Metabolic overstimulation by abnormally elevated cytosolic calcium may contribute to cell injury and trigger cell death, possibly by exceeding the intracellular calcium “set-point”. Diabetes is associated with increased calcium influx (via voltage-
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Figure 3 Intracellular mechanisms that may contribute to altered Ca2+ signaling in diabetes mellitus. Elevated cytosolic calcium ([Ca2+]i) may result as a consequence of altered regulation by ligands, including neurotrophins and serum factor(s), as well as impaired signal transduction. Tyrosine kinase-associated receptors (Trk, Tk) located on the plasma membrane may directly modulate calcium channels, or indirectly regulate calcium channels via G protein-dependent pathways and protein kinase C (PKC). Basal [Ca2+]i levels are regulated by activity of membrane-associated ion exchange pumps (Na +-K+ ATPase, Na+-Ca2+ exchange pump, Ca-ATPase) shown in the lower part of the figure. The activity of Na+-K+ ATPase can also be modulated by PKC, leading to intracellular Na+ accumulation, and subsequent elevation of [Ca2+]i.
dependent calcium channels in neurons), as well as decreased inhibition of the calcium channel (Figure 3). This increase may be due to impaired expression and/ or function of signal transduction pathways at the level of the receptor or inhibitory G protein: Ca2+ channel complex. Impaired signal transduction may involve abnormal phosphorylation/dephosphorylation by intracellular intermediates such as protein kinase C and phosphatases. An emerging body of data suggests that serum factors may contribute to the pathophysiology of diabetic complications, perhaps by stimulating calcium influx. Growth factors such as neurotrophins and insulin-like growth factor 1 appear to have cytoprotective actions that may involve modulating intracellular calcium levels. Abnormal regulation of the membrane pumps (calciumATPase, Na+-K+-ATPase and sodium-calcium exchange pump) appear to contribute to the abnormal cytosolic calcium levels in diabetes. This, in turn, can affect insulin sensitivity and responsiveness, leading to more generalized metabolic derangements. It is likely that genetic and biochemical studies of families of diabetic patients will help identify potential initial defects contributing to altered cellular calcium homeostasis in diabetes mellitus.
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Hattori, Y, Kawasaki, H., Kanno, M. and Fukao, M. (1995b) Enhanced 5-HT2 receptormediated contractions in diabetic rat aorta: participation of Ca2+ channels associated with protein kinase C activity. J. Vasc. Res., 32, 220–229. Heliger, C.E., Prakash, A. and McNeill, J.H. (1987) Alterations in cardiac sarcolemmal Ca2+ pump activity during diabetes mellitus. Am. J. Physiol., 252, H540-H544. Hellweg, R. and Hartung, H.-D. (1990) Endogenous levels of nerve growth factor (NGF) are altered in experimental diabetes mellitus: a possible role of NGF in the pathogenesis of diabetic neuropathy. J. Neurosci. Res., 26, 258–267. Ishii, D.N. (1995) Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy. Brain Res., Brain Res. Rev., 20, 47–67. Ishii, H., Umeda, E, Hashimoto, T. and Hawata, H. (1990) Changes in phosphoinositide turnover, Ca2+ mobilization, and protein phosphorylation in platelets from NIDDM patients. Diabetes, 39, 1561–1568. Johnson, E.M., Jr., Kolke, T. and Franklin, J. (1992) A “calcium-set hypothesis” of neuronal dependence on neurotrophic factor. Exp. Neurol., 115, 163–166. Juntti-Berggren, L., Larsson, O., Rorsman, R, Ammala, C., Bokvist, K., Wahlander, K., Nicotera, R, Dypbukt, J., Orrenius, S., Hallberg, A. and Berggren, P.-O. (1993) Increased activity of L-type Ca2+ channels exposed to serum from patients with Type I diabetes. Science, 261, 86–89. Kappelle, A.C., Bravenboer, B., Traber, J., Erkelens, D. and Gispen, W. (1992) The Ca2 + antagonist nimodipine counteracts the onset of an experimental neuropathy in streptozotocininduced diabetic rats. Neurosci. Res. Comm., 10, 95–104. Kato, S., Ishida, H., Tsuura, Y., Tsuji, K., Nishimura, M., Horie, M., Taminato, T., Ikehara, S., Odaka, H., Ikeda, I., Okada, Y. and Seino, Y. (1996) Alterations in basal and glucosestimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of noninsulin-dependent diabetes mellitus GK rats. J. Clin. Invest., 97, 2417–25. Kostyuk, E., Pronchuk, N. and Shmigol, A. (1995) Calcium signal prolongation in sensory neurones of mice with experimental diabetes. Neuroreport, 6, 1010–1012. Lee, S.L. and Dhalla, N.S. (1992) Ca2+-channels andadrenoceptors in diabetic skeletal muscle. Biochem. Biophys. Res. Commun., 184, 353–358. Levy, J. and Rempinski, D. (1991) Insulin phosphorylates the membrane (Ca2+-Mg2+) ATPase in kidney basolateral membranes. Clin. Res., 39, 698. Levy, J., Gavin, J.R., III, Hammerman, M.R. and Avioli, L.V. (1986) Ca2+-Mg2+ATPase activity in kidney basolateral membrane in non-insulin-dependent diabetic rats. Effect of insulin. Diabetes, 35, 899–905. Levy, J., Suzuki, Y, Avioli, L.V., Grunberger, G. and Gavin, J.R., III (1988) Plasma membrane phospholipid content in non-insulin-dependent streptozotocin-diabetic rats: effect of insulin. Diabetologia, 31, 315–321. Levy, J., Reid, I., Halstad, L., Gavin, J.R., III and Avioli, L.V. (1989a) Abnormal cell calcium concentration in cultured bone cells obtained from femurs of obese and noninsulindependent diabetic rats. Calcif. Tissue Int., 44, 131–137. Levy, J., Zemel, M.B. and Sowers, J.R. (1989b) Role of cellular calcium metabolism in abnormal glucose metabolism and diabetic hypertension. Am. J. Med., 87, 7–15. Levy, J., Gavin, EJ.R. and Sowers, J.R. (1994) Diabetes mellitus: a disease of abnormal cellular calcium metabolism. Am. J. Med., 96, 260–273. Lowery, J.M., Eichberg, J., Saubermann, A.J. and Lo Pachin, R.M., Jr. (1990) Distribution of elements and water in peripheral nerve of streptozocin-induced diabetic rats. Diabetes, 39, 1498–1503.
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Makhlouf, G.M. (1995) Smooth muscle of the Gut. In Textbook of Gastroenterology (2nd Edition), edited by T. Yamada, D.H. Alpers, C. Owyang, D.W. Powel and EE. Silverstein. New York: JB Lippincott Co. Makino, N., Dhalla, K.S., Elimban, V. and Dhalla, N.S. (1987) Sarcolemmal Ca2+ transport in streptozotocin-induced diabetic cardiomyopathy in rats. Am. J. Physiol, 253, E202– E207. Mattson, M.P., Cheng, B. and Smith-Swintosky, V.L. (1993) Mechanisms of neurotrophic factor protection against calcium- and free radical-mediated excitotoxic injury: implications for treating neurodegenerative disorders. Exp. Neurol., 124, 89–95. Mazzanti, L., Rabini, R.A., Faloia, E., Fumelli, P, Bertoli, E. and De-Pirro, R. (1990) Altered cellular Ca2+ and Na+ transport in diabetes mellitus. Diabetes, 39, 850–854. Moises, H.C., Rusin, K.L. and Macdonald, R.L. (1994) Mu-opioid receptor-mediated reduction of neuronal calcium currents occurs via a Go-type GTPbinding protein. J. Neurosci., 14, 3842–3851. Morley, R, Hogan, M.J. and Hakim, A.M. (1994) Calcium-mediated mechanism of ischemic injury and protection. Brain Pathol., 4, 37–47. Morris, N.J., Bushfield, M. and Houslay, M.D. (1996) Streptozotocin-induced diabetes elicits the phosphorylation of hepatocyte Gi2 alpha at the protein kinase C site but not at the protein kinase A-controlled site. Biochem. J., 315, 417–420. Nicotera, R, Bellomo, G. and Orrenius, S. (1992) Calcium-mediated mechanisms in chemically induced cell death. Ann. Rev. Pharmacol. Toxicol, 32, 449–470. Nobe, S., Aorrune, M.A., Ito, S. and Takaki, R. (1990) Chronic diabetes mellitus prolongs action-potential duration of rat ventricular muscles: circumstantial evidence for impaired Ca2+ channel. Cardiovascular Res., 24, 381–389. Nojima, H., Tsuneki, H., Kimura, I. and Kimura, M. (1995) Accelerated desensitization of nicotinic receptor channels and its dependence on extracellular calcium in isolated skeletal muscles of streptozotocin-diabetic mice. Br. J. Pharmacol., 116, 1680–1684. Ogawa, T., Kashiwagi, A., Kikkawa, R. and Shigeta, Y. (1995) Increase of voltagesensitive calcium channels and calcium accumulation in skeletal muscles of streptozocin-induced diabetic rats. Metabolism, 44, 1455–1461. Ohara, T., Sussman, K.E. and Draznin, B. (1991) Effect of diabetes on cytosolic free Ca2 + and Na+-K+-ATPase in rat aorta. Diabetes, 40, 1560–1563. Pershadsingh, H.A., Shade, D.L., Delfert, D.M. and MacDonald, J.M. (1987) Chelation of intracellular calcium blocks insulin action in the adipocyte. Proc. Natl. Acad. Sci. USA, 84, 1025–1029. Pieper, G.M. and Gross, G.J. (1989) Diabetes enhances vasoreactivity to calcium entry blockers. Artery, 16, 263–271. Pierce, G.N. and Dhalla, N.S. (1981) Cardiac myofibrillar ATPase activity in diabetic rats. J. Mol. Cell Cardiol., 13, 1063–1069. Pittinger, G.L., Liu, D. and Vinik, A.I. (1993) The toxic effects of serum from patients with Type I diabetes mellitus on mouse neuroblastoma cells: a new mechanism for the development of diabetic autonomic neuropathy. Diabetic Med., 10, 925–932. Pittinger, G.L., Liu, D. and Vinik, A.I. (1995) The neuronal toxic factor in serum of Type 1 diabetic patients is a complement-fixing autoantibody. Diabetic. Med., 12, 380– 386. Pittinger, G.L., Liu, D. and Vinik, A.I. (1997) The apoptotic death of neuroblastoma cells caused by serum from patients with insulin-dependent diabetes and neuropathy may be Fas-mediated.J. Neuroimmunol., 76, 153–160.
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Resh, M.D. (1985) Insulin action on the (Na+-K+)ATPase. In Molecular Basis of lnsulin Action, edited by M.P. Czech. New York: Plenum Press. Rich, K.M., Luszczynski, J.R., Osborne, P.A. and Johnson, J.R. (1987) Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J. Neurocyt., 16, 261–268. Ristic, H., Wiley, J., Hall, K. and Sima, A.A.F. (1996) Failure of nimodipine to prevent or correct the long-term nerve conduction defect and increased neuronal Ca2+ currents in the BB/W rat. Diabetes Res. Clin. Pract., 32, 135–140. Ristic, H., Srinivasan, S., Hall, K.E., Sima, A.A.F. and Wiley, J.W. (1998) Serum from diabetic BB/W rats enhanced calcium currents in primary sensory neurons. J. Neurophysiol, 80, 1236–1244. Ruit, K.G., Osborne, P.A., Schmidt, R.E., Johnson, Jr. E.M. and Snider, W.D. (1990) Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse. J. Neurosci., 10, 2412–2419. Russ, M., Reinauer, H. and Eckel, J. (1991) Diabetes-induced decrease in the mRNA coding for sarcoplasmic reticulum Ca2+-ATPase in adult rat cardiomyocytes. Biochem. Biophys, Res. Commun., 178, 905–912. Sakai, Y, Inazu, M., Shamoto, A., Zhu, B. and Homma, I. (1994) Contractile hyperreactivity and alteration of PKC activity in gastric fundus smooth muscle of diabetic rats. Pharmacol. Biochem. Bebav, 49, 669–674. Segal, S., Lloyd. S., Sherman, N., Sussman, K.E. and Draznin, B. (1990) Postprandial changes in cytosolic free calcium and glucose uptake in adipocytes in obesity and noninsulindependent diabetes mellitus. Horm. Res., 34, 39–44. Shimabukuro, M., Shinzato, T., Higa, S., Nagamine, F., Murakami, K. and Takasu, N. (1995) Impaired mechanical response to calcium of diabetic rat hearts: reversal by nifedipine treatment. J. Cardiovasc. Pharmacol., 26, 495–502. Sima, A.A.F., Prashar, A., Zhang, W.-X., Chakrabarti, S. and Greene, D.A. (1990) Preventative effect of long term aldose reductase inhibition (Ponalrestat) on nerve conduction and sural nerve structure in the spontaneously diabetic BB-rat. J. Clin. Invest., 85, 1410–1420. Sowers, J.R. (1992) Insulin resistance, hyperinsulinemia, dyslipidemia, hypertension and accelerated atherosclerosis. J. Clin. Pharmacol., 32, 529–535. Stevens, M.J., Feldman, E.L. and Greene, D.A. (1995) The aetiology of diabetic neuropathy: the combined roles of metabolic and vascular defects. Diabetic Med, 12, 566–576. Storlien, L.H., Kraegen, E.W., Chisholm, D.J., Ford, G.L., Bruce, D.G. and Pascoe, W.S. (1987) Fish oil prevents insulin resistance induced by high-fat feeding in rats. Science, 237, 885– 888. Studer, R.K. and Ganas, L. (1989) Effect of diabetes on hormone-stimulated and basal hepatocyte calcium metabolism. Endocrinology, 125, 2421–2433. Taira, Y, Hata, T., Ganguly, P.K., Elimban, V. and Dhalla, N.S. (1991) Increased sarcolemmal Ca2+ transport activity in skeletal muscle of diabetic rats. Am. J. Physiol., 260, E626– E632. Takahashi, T., Kojima, Y., Tsunoda, Y, Beyer, L.A., Kamijo, M., Sima, A.A.F. and Owyang, C. (1996) Impaired intracellular signal transduction in gastric smooth muscle of diabetic BB/W rats. Am. J. Physiol., 270, G411–G417.
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Tam, E.S., Ferguson, D.G., Bielefeid, D.R., Lorenz, J.N., Cohen, R.M. and Pun, R.Y. (1997) Norepinephrine-mediated calcium signaling is altered in vascular smooth muscle of diabetic rat. Cell Calcium., 21, 143–150. Thayer, S. and Miller, R. (1990) Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurons in vitro.J. Physiol. (Lond), 425, 85–115. Thomas, P.K. (1994) Growth factor and diabetic neuropathy. Diabetic. Medicine, 11, 732– 739. Tschope, D., Rösen, P. and Gries, F.A. (1991) Increase in the cytosolic concentration of calcium in platelets of diabetics type II. Thromb. Res., 62, 421–428. Uchida, M., Iwata, T., Takagi, S., Sugiyama, Y, Ishitani, K., Honda, H. and Sakai, Y. (1994) Effect of trimebutine maleate on the contractile response of the isolated ileum from diabetic rats. Gen. Pharmacol., 25, 505–508. Wang, D.W., Kiyosue, T., Shigematsu, S. and Arita, M. (1995) Abnormalities of K+ and Ca2+ currents in ventricular myocytes from rats with chronic diabetes. Am. J. Physiol., 269, H1288–1296. Williams, S.B., Cusco, J.A., Roddy, M.A., Johnstone, M.T. and Creager, M.A. (1996) Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J. Am. Coll. Cardiol., 27, 567–574. Winegrad, A.J. (1986) Does a common mechanism induce the diverse complications of diabetes? Diabetes, 36, 396–406. Yoo, H.J., Kozaki, K., Akishita, M., Watanabe, M., Eto, M., Nagano, K., Sudo, N., Hashimoto, M., Kim, S., Yoshizumi, M., Toba, K. and Ouchi, Y. (1997) Augmented Ca2+ influx is involved in the mechanism of enhanced proliferation of cultured vascular smooth muscle cells from spontaneously diabetic Goto-Kakizaki rats. Atherosclerosis, 131, 167–175. Yu, Z., Quamme, G.A. and McNeill, J.H. (1994) Depressed [Ca2+]i responses to isoproterenol and cAMP in isolated cariomyocytes from experimental diabetic rats. Am.J. Physiol., 266, H2334–H2342.
8. EXPERIMENTAL DIABETIC NEPHROPATHY MARK E.COOPER, RICHARD E.GILBERT, DARREN J.KELLY and TERRI J.ALLEN Department of Medicine, University of Melbourne, Austin & Repatriation Medical Centre, Repatriation Campus, Heidelberg West 3081
INTRODUCTION Diabetic nephropathy (DN) affects at least 30% of insulin dependent diabetic patients (Andersen et al., 1983) and a lesser percentage of NIDDM subjects (Mogensen, 1984). This disorder is the commonest cause of end stage renal failure in the Western world (Held et al., 1990) and is a major cause of morbidity and mortality in the diabetic population. The availability of experimental models of DN has allowed investigators to explore not only the morphological abnormalities of the diabetic kidney but also cellular and molecular mechanisms involved in the genesis and progression of DN. Many of the changes observed in the human diabetic kidney have been reported in rodents (Steffes and Mauer, 1984). MODELS OF EXPERIMENTAL DIABETES The original studies of experimental diabetic nephropathy involved diabetic animals in which diabetes was induced by compounds such as streptozocin and alloxan which were toxic to the pancreatic beta cells (Steffes and Mauer, 1984). These compounds are associated with nephrotoxicity. However, most of the changes detected in chemically induced diabetic animals are due to the metabolic milieu rather than to a toxic effect of the agent (Rasch, 1979b). Since alloxan can cause acute renal failure (Pemberton and Manax, 1970), this drug is administered with either clamping of the renal arteries or via a double balloon catheterisation techniques (Wigness et al., 1982). These approaches prevent the kidney from being exposed to high doses of this agent. Streptozocin is also associated with renal morphological changes including tubular abnormalities. In addition, this agent is associated with an increase in renal tumours after 6 or 7 months (Mauer et al., 1974) and may promote the tumorigenic effects of other drugs on the kidney (Oturai et al., 1995). The major model of experimental DN which has been extensively evaluated is the streptozotocin (STZ) model of diabetes in the rat. Over the last 20 years experimental studies predominantly performed in the streptozocin induced diabetic
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rat have assisted in the understanding of diabetic nephropathy and evaluation of various interventions. Streptozocin diabetes in the rat is associated with the universal development of functional and structural features of nephropathy within 6 months (Hirose et al., 1982). The much shorter time course and the predictability of renal changes in experimental diabetes when compared to man are major advantages in initial evaluation before interventions are considered in human subjects. One of the major issues confronting studies of experimental diabetic nephropathy is the appropriateness of the model i.e. whether the changes observed in rats are analogous to those reported in man. Therefore, it is important to consider if the phases that are well described in the evolution of human diabetic nephropathy can also be detected in the rat. Within a week of induction of streptozocin diabetes, the rat develops an increase in GFR (hyperfiltration) and renal plasma flow as well as renal hypertrophy (Seyer-Hansen, 1977; Allen et al., 1990). The link between renal hypertrophy with hyperfiltration is also observed in man (Mogensen and Andersen, 1973). Over the next few months, there is a progressive rise in urinary albumin excretion, with the ultimate development of albustix positive proteinuria after a phase of increased, yet albustix negative, proteinuria (Cooper et al., 1988b) analogous to the phase of microalbuminuria seen in man (Mogensen, 1983). Glomerular ultrastructural changes such as glomerular basement membrane thickening (GBM) and mesangial expansion (Steffes and Mauer, 1984; Cooper et al., 1988b) become apparent. However, a major disadvantage of experimental models of diabetic renal disease is that progressive decline in renal function does not usaully occur. More recently, the renal lesions in other models of diabetes have been evaluated. A summary of these findings has been reported previously (Velasquez et al., 1993). The BB rat, which develops spontaneous diabetes and resembles from a pathogenetic point of view insulin dependent diabetes has less marked changes in the kidney (Brown et al., 1983; Cohen, 1987). Although GBM thickening has been observed in the BB rat, there is no evidence of significant mesangial expansion (Brown et al., 1983). Since DN is commonly observed in patients with NIDDM, various animal models of NIDDM have now been assessed in terms of renal disease. These models have included the obese Zucker rat (Kasiske et al,, 1988) and the corpulent spontaneously hypertensive rat (SHR) (Michaelis et al., 1990) and show evidence of GBM thickening and mesangial expansion, despite modest hyperglycemia. Various models of diabetic mice including chemically induced and genetic models have been evaluated (Velasquez et al., 1990). These have been more difficult to assess in view of their smaller size which make functional studies almost impossible to perform. There are also smaller quantities of tissue available for pathological or molecular biological analysis. The genetic models do not generally have mesangial expansion, which is viewed to be the central lesion involved in the progression of DN (Steffes et al., 1989). These genetic models include models similar to both IDDM such as the NOD mouse (Tochino, 1984) and NIDDM (Bower et al., 1980; Melez et al., 1980; Diani et al., 1987). The alloxan diabetic dog develops GBM thickening, mesangial expansion and ultimately nodular lesions which resemble the Kimmelsteil-Wilson nodules observed in some human diabetic patients with overt renal disease (Kimmelsteil and Wilson, 1936). More recently, studies have been performed in primates (Harano et al., 1992;
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Heffernan et al., 1995). Although technically and logistically more difficult to perform, it is hoped that results obtained from diabetic primates will more readily be able to be extrapolated to man. However, the availability of rodents and their tissues and the ability to detect morphological lesions within a few months of induction of diabetes in these animals has made the rat the most popular choice for exploring experimental diabetic nephropathy. GLYCEMIC CONTROL AND DIABETIC NEPHROPATHY The link between the severity of hyperglycemia and diabetic microvascular complications has been suggested by many investigators (DCCT Research Group, 1993; Gilbert et al., 1993b). To explore this issue, several approaches have been used in experimental models to achieve euglycemia, including insulin therapy and pancreatic transplantation (whole pancreas or islet). Rasch used heat treated ultralente insulin to achieve euglycemia in streptozocin diabetic rats, initiating treatment from the time of induction of diabetes and continuing therapy for 6 months (Rasch, 1979b). Insulin therapy prevented the development of albuminuria (Rasch and Mogensen, 1980), glomerular basement membrane thickening (Rasch, 1979b), renal and glomerular hypertrophy (Rasch, 1979c) and mesangial expansion (Rasch, 1979a). This experiment confirmed that hyperglycemia is necessary for the development of nephropathy. However, these experiments were evaluating prevention rather than intervention in diabetic renal disease. Since nephropathy will only develop in a minority of diabetic patients who cannot be identified at the time of diagnosis (Andersen et al., 1983), the direction of human studies has generally been towards intervention rather than prevention of renal disease. Therefore, studies looking at the effects of improved glycemic control have been designed to intervene in experimental models once functional and structural markers of diabetic renal diseases have become manifest. In such a study, seven weeks of high dose insulin achieving euglycemia after 9 months of poorly controlled streptozocin diabetes in the Munich Wistar rat was associated with reversal of renal hypertrophy and hyperfiltration but did not influence mesangial expansion (Stackhouse et al., 1990). These results are in contrast to another study in which insulin therapy achieving euglycemia was instituted after 6 months of diabetes in the Sprague-Dawley rat (Petersen et al., 1988). In the latter study, institution of normoglycemia resulted in the arrest of mesangial expansion as well as retarding the rise in proteinuria. It is possible that the timing of improved glycemic control may have been a factor in the difference in the results of these 2 studies with respect to mesangial expansion. However, another possible reason for conflicting results is that these 2 studies were performed in different rat strains, a factor which has previously been reported to influence the development of proteinuria and mesangial expansion in rats (Grond et al., 1986; O’Donnell, 1988). For example, induction of diabetes in the DA rat results in less proteinuria and mesangial expansion than in the Lewis rat (Payton and Boulton-Jones, 1989). Pancreatic transplantation has also been used to explore the link between hyperglycemia and diabetic nephropathy. In the original studies by Mauer and Steffes, islet transplantation was performed 7 months after induction of streptozocin diabetes (Steffes et al., 1980). Reversal of mesangial expansion (Mauer, 1978b) and
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reduction in urinary albumin excretion (Steffes et al., 1980) were observed 2 months after islet transplantation without, however, preventing glomerular basement membrane thickening (Steffes et al., 1979). Similar findings with respect to reversal of mesangial expansion have been reported by another group (Orloff et al., 1986) who showed that whole pancreas transplantation after 6 to 21 months of diabetes was associated with reduction in mesangial area. RENAL FUNCTIONAL ABNORMALITIES IN EXPERIMENTAL DIABETES One of the earliest changes detected in STZ diabetes is an increase in GFR (Hostetter et al, 1981; Allen et al, 1990). This is clearly apparent within 1 week of induction of diabetes and does not occur if the animals are rendered euglycemic by intensive insulin therapy. This rise in GFR closely parallels the increase in kidney weight which occurs in the first few days of experimental diabetes (Bach and Jerums, 1990). The cause of the increase in GFR, also known as “hyperfiltration”, is unknown and has been an area of intense investigation over the last two decades. Micropuncture studies of these hyperfiltering diabetic rats have revealed that several of the determinants of GFR are altered in experimental diabetes. These include increases in glomerular flow, primarily due to a decrease in afferent arteriolar resistance and an increase in intraglomerular pressure (Hostetter et al., 1981; Zatz et al, 1985). It has been hypothesised that these changes and, in particular, the increase in intraglomerular pressure is intimately involved in the progression of diabetic renal disease (Hostetter et al., 1982) and in the genesis of many of the ultrastructural abnormalities that are detected in animal models of diabetes. However, several groups have shown that in poorly controlled diabetic rats there is often no increase in GFR, yet the animals develop nephropathy (O’Donnell, 1988). Furthermore, in certain rat strains, it has been more difficult to document an increase in glomerular pressure, yet these animals develop florid diabetic renal lesions (O’Donnell, 1988). Therefore, one cannot exclude a major role for metabolic factors, independent of hemodynamic effects, on the development of DN. Many investigators have explored the possible mechanisms involved in the development of diabetic hyperfiltration (Vora et al., 1994). Much of the original work focussed on the role of glucose per se in increasing GFR. While glucose infusion can modestly increase GFR (Kasiske, 1985), the changes observed in experimental diabetes are much greater than those explained by a direct effect of glucose. Furthermore, as outlined earlier, diabetic rats which are very poorly controlled, do not have elevated and often have a decreased GFR (Hostetter et al., 1981). Volume expansion has been reported in diabetic animals (Ilstrup et al., 1981; Allen et al., 1990) and may be related to elevated sodium and water reabsorption in the proximal tubule as a result of glucose and sodium co-transport. This phenomenon has been confirmed to occur in vivo in micropuncture studies in diabetic animals (Bank and Aynedjian, 1990). It has been suggested that hyperfiltration is a result of an attenuated tubulo-glomerular feedback (TGF) mechanism in diabetes (Blantz et al., 1990). This would occur either as a result of decreased delivery of fluid to the distal tubule or a decrease in TGF responsiveness (Braam et al., 1993). Both mechanisms have been postulated to occur in experimental diabetes.
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The status of the renin-angiotensin system (RAS) in diabetes and in particular in the diabetic kidney is controversial. It has been suggested that a suppressed RAS could result in diabetic hyperfiltration (Bank et al., 1988). However, although plasma renin activity is generally low in experimental diabetes (Christlieb and Boston, 1974; Allen et al., 1990), molecular biological studies have yielded conflicting results with respect to production of renin and angiotensin by the diabetic kidney (Correa-Rotter et al., 1992; Anderson et al., 1993; Kalinyak et al., 1993). Angiotensin II receptors are down-regulated in the diabetic kidney (Ballerman et al, 1984) and could lead to decreased responsiveness to local and circulating AII by the kidney. However, diabetic rats respond to AII infusion normally (Wilkes et al., 1993). Several groups have explored the effects of both acute and chronic ACE inhibition on GFR in diabetic rats. Results have been conflicting with several groups reporting a decrease in GFR (Perico et al., 1994a; Komers and Cooper, 1995) whereas other groups have reported no significant effect on GFR (Zatz et al., 1986; Anderson et al., 1989). These studies cannot be readily interpreted since the ACE inhibitors not only suppress AII formation but also inhibit degradation of kinins (Erdos, 1976). Recent studies by our group using the AII receptor blocker, valsartan, failed to show any effect of this agent on GFR (Komers and Cooper, 1995). In addition, the kinin antagonist, icatibant prevented the reduction in GFR induced by acute ACE inhibition suggesting a role for kinins in mediating some of the renal haemodynamic effects of ACE inhibitors. However, icatibant alone did not affect GFR. A recent preliminary report using icatibant has also failed to detect a significant effect on GFR of this agent in diabetic rats (Vora et al., 1995). However, Jaffa et al. have reported a reduction in GFR using a different kinin antagonist (Jaffa et al., 1995). This issue remains unresolved and requires further studies including more detailed assessment of the renal kallikrein-kinin system in diabetes. Levels of atrial natriuretic peptide, a hormone which is released in response to extracellular volume expansion, are increased in diabetic rats (Ortola et al., 1987; Allen et al., 1990). This peptide will increase GFR in rats (Dunn et al., 1986) and has been postulated to play a central role in mediating diabetic hyperfiltration. Ortola et al reported that infusion of an antibody to ANP normalised the increased GFR of diabetic rats (Ortola et al., 1987). The advent of ANP receptor antagonists such as HS-142–1 has allowed investigators to directly evaluate the role of ANP in diabetic hyperfiltration. Two groups have reported that this antagonist reduced GFR in diabetic rats (Kikkawa et al., 1993; Zhang et al., 1994) consistent with the underlying hypothesis that ANP is involved in the increased GFR in diabetic rats. It has been suggested that the nitric oxide pathway may be involved in diabetic hyperfiltration. Several groups have reported increases in urinary nitrate/nitrite production in experimental diabetes (Bank and Aynedjian, 1993; Tolins et al., 1993; Komers et al, 1994). These findings have been interpreted as evidence of increase renal NO synthase activity in diabetes. Furthermore, NO blockade using competitive antagonists of NO synthase has been reported by several groups to decrease GFR (Mattar et al., 1993; Tolins et al., 1993; Komers et al., 1994). This effect is not seen as clearly in control rats and is consistent with the postulate that NO mediated pathways are involved in diabetic hyperfiltration. There are a large number of other potential mediators of diabetic hyperfiltration that must be considered including dopamine (Barthelmebs et al., 1991) and metabolites of the
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aldose reductase pathway (Goldfarb et al., 1991). Our own group has recently reported that salt restriction will also reduce GFR in diabetic rats (Allen et al., 1997b). Over the last decade, there has been major interest in the possible role of various hormones and their receptors in the progression of renal injury in diabetes (Ballerman et al., 1984; Trinder et al., 1994; Sechi et al., 1995). These studies have focussed on vasoactive hormones which may lead to the renal haemodynamic changes observed in diabetes. It has been shown that many of these vasoactive hormones such as AII and endothelin also have trophic properties (Ruiz Ortega et al., 1994). This is particularly relevant in a state such as diabetes in which there are not only hemodynamic but also trophic changes in the kidney with evidence of both glomerular (Hirose et al., 1982) and tubular hypertrophy (Fioretto et al., 1992) and possibly hyperplasia (Steffes et al, 1996). More recently, studies have involved assessment of tubular and interstitial changes in diabetes (Jerums and Gilbert, 1993). The adverse effects of hyperglycaemia have been attributed to activation of PKC (Inoguchi et al., 1992), a family of serine-threonine kinases that regulates diverse vascular functions, including contractility, blood flow, cellular proliferation (Nishizuka, 1992) and vascular permeability (Lynch et al., 1990). PKC activity, especially the membrane bound form, is increased in the retina, aorta, heart and renal glomeruli of diabetic animals, probably because of an increase in de novo synthesis of diacylglycerol, a major endogenous activator of PKC (Craven et al., 1995). The observation that there is preferential activation of the ȕII isoform of PKC in diabetes (Inoguchi et al, 1992) led to the synthesis of an orally effective PKCȕselective inhibitor, LY333531 (Ishii et al., 1996). LY333531 is a competitive, reversible inhibitor of PKCȕ1 and PKCȕII, with a 50 fold lesser effect on other PKC isoenzymes. In studies over 2 to 8 weeks in streptozotocin diabetic rats, LY333531 ameliorated glomerular hyperfiltration, albuminuria and diabetes related increases in retinal mean circulation time (Ishii et al., 1996). MOLECULAR BIOLOGICAL STUDIES OF THE DIABETIC KIDNEY Much of the recent research has concentrated on the various extracellular matrix proteins and growth factors which have been shown to be synthesised at an increased rate in the diabetic kidney (Gilbert et al., 1995a). These studies have been facilitated by the advent of molecular biological techniques including in situ hybridisation which have allowed investigators not only to assess diabetes associated changes in gene expression but also to evaluate changes in their sites of production. The pathogenesis of the changes in extracellular matrix (ECM) in the diabetic kidney is not well understood but is likely to involve interaction between cells, growth factors, structural proteins and cell receptors for these molecules. In addition to providing tissue structure, ECM is in dynamic interaction with its surrounding cell population, taking part in the regulation of cell morphology, differentiation, migration and macromolecular transport as well as serving as a reservoir for various cytokines and growth factors (Lin and Bissell, 1993). Alterations in tissue morphology, as occurs in diabetes, may reflect complex interactions between various locally acting growth factors, receptors, structural ECM molecules, ECM receptors and ECM degrading enzymes. The ECM expansion found in the diabetic kidney
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may be the result of increased ECM synthesis, reduced degradation or both. Results of animal studies have not been universally concordant showing a decrease (Poulsom et al., 1988), an increase (Roy et al., 1990; Fukui et al., 1992) or no change (Ledbetter et al., 1990; Ihm et al., 1992) in mRNA levels for the various structural proteins. However, the increased ECM found in diabetes may reflect reduced ECM degradation rather than increased ECM synthesis (McLennan et al., 1994). Several types of enzymes capable of degrading ECM have been described. These include the matrix metalloproteinases (MMPs). Gene expression of these MMPs and their naturally occurring inhibitors has been examined in glomeruli of longterm diabetic animals. Nakamura and colleagues found a reduction in MMP-1 and MMP-3 mRNA along with an increase in mRNA for their inhibitors, TIMP-1 and TIMP-2, suggestive of reduced MMP synthesis and activity (Nakamura et al., 1994). These changes could lead to reduced matrix degradation and therefore partly explain ECM accumulation observed in the diabetic kidney. Since diabetes is associated with renal growth and ECM accumulation, there has been intensive investigation into the possible role of growth factors in the diabetic kidney. The main growth factor to have been evaluated in the diabetic kidney is transforming growth Factor-ȕl (TGF-ȕl) (Ziyadeh, 1994). This cytokine has been implicated both in diabetes related renal growth and in the later development of diabetic nephropathy (Ziyadeh and Goldfarb, 1991). Studies in endothelial, mesangial and proximal tubular cells demonstrate increased TGF-ȕ (Cagliero et al., 1988b; Rocco et al., 1992; Sharma and Ziyadeh, 1995), its receptor (Ladson-Wofford et al., 1994) and ECM (Cagliero et al., 1988a; Ayo et al., 1991), when cultured in high glucose. Increased expression of TGF-ȕl in whole kidney preparations has been documented in association with diabetes related kidney growth in the BB rat and in the NOD mouse (Sharma and Ziyadeh, 1994). In long term experimental diabetes increased glomerular TGF-ȕl gene expression has been demonstrated by several groups (Nakamura et al., 1993; Yamamoto et al., 1993; Park et al., 1994). In a study of STZ-diabetic rats, Yamamoto and colleagues described TGF-ȕI gene expression by Northern analysis and protein production by immunohistochemistry (Yamamoto et al., 1993). Comparisons were made between untreated diabetic animals and control animals treated with insulin. This renders the study somewhat difficult to interpret given the recent report that insulin inhibits mesangial cell TGF-ȕ gene expression in culture and inhibits the stimulatory effect of glucose (Badillo et al., 1994). In another study of glomerular TGF-ȕ gene expression, Nakamura compared untreated control and diabetic rats (Nakamura et al, 1993). A significant increase in expression of TGF-ȕ1 as well as several other genes including fibroblast growth factor-2, tumour necrosis factor-a and the B-chain of platelet derived growth factor was reported. These findings illustrate the complex processes underling the pathogenesis of diabetic kidney disease and suggest that a single growth factor is unlikely to be wholly responsible. Recent studies by our group have confirmed the importance of metabolic factors on TGF-ȕ1 gene expression in the diabetic kidney. Diabetic animals were randomised to receive either alternate or daily insulin therapy. This resulted in differences in glycaemic control between the two groups, as assessed by HbA1c levels. The rats receiving alternate daily insulin therapy with the higher HbA1c levels had increased levels of TGF-ȕI mRNA from whole kidney extracts (Figure 1). In
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contrast, in the diabetic rats receiving daily insulin therapy with associated lower HbA1c levels, TGF-ȕ1 mRNA levels were indistinguishable from those in control rats. These results suggest that glucose and/or insulin are important determinants of TGF-ȕ1 gene expression in the kidney. A similar pattern was observed with respect to type IV collagen gene expression (Figure 1). A number of other growth factors/cytokines have been implicated in modulating ECM accumulation and other manifestations of diabetic nephropathy. These include IGF-1 (Bach and Jerums, 1990), platelet-derived growth factor (Fukui et al., 1994), epidermal growth factor (Guh et al., 1991; Gilbert et al., 1997), heparin-binding growth factor (Gilbert et al., 1993a), basic fibroblast growth factor (bFGF) and tumour necrosis factor-Į (TNF-Į) (Fukui et al., 1994). However, data regarding their specific roles in the pathogenesis of experimental diabetic nephropathy are awaited. Cell-matrix interactions can be influenced by proteins with anti-adhesive properties called “anti-adhesins”. Members of the anti-adhesin family include secreted protein acidic and rich in cysteine [SPARC, BM-40, osteonectin] (Sage and Bornstein, 1991). In addition to its antiadhesive properties, SPARC, the prototype anti-adhesin may reduce fibronectin mRNA, inhibit cell proliferation and increase gene expression of MMPs. SPARC is found abundantly in the glomerulus and recent studies by our group have shown that early diabetes related kidney growth is associated with a reduction in renal SPARC mRNA and protein content (Gilbert et al., 1995b). We have proposed that reduced SPARC levels would in turn be associated with increased ECM synthesis, reduced ECM degradation and increased cell proliferation. TREATMENT OF EXPERIMENTAL DIABETIC NEPHROPATHY In the final section of this chapter, we have reviewed the effects of different therapies on the functional and structural parameters of diabetic renal injury. Hypertension has been clearly shown to exacerbate renal injury in diabetic rats. The initial studies were performed in Goldblatt 2 Kidney—1 clip hypertensive rats with chemically induced diabetes (Mauer, 1978a). These studies indicated increased damage in the unclipped kidney exposed to the elevated systemic blood pressure. However, that study could not separate the effect of hypertension from the effect of hyperfiltration on the unclipped kidney. Studies by our group explored the importance of hypertension further by inducing streptozocin diabetes in spontaneously hypertensive rats (SHR) (Cooper et al., 1988b). When comparing diabetic SHR to their normotensive diabetic counterparts, there was evidence of an increase in GBM thickness, fractional mesangial volume and glomerular volume. These changes were associated with a much more rapid development of albuminuria over the 32 week study period. At the time of these studies, there was increasing interest in the role of antihypertensive therapy and in particular ACE inhibitors in retarding the development of overt DN in man (Björck, 1986; Parving, 1989). Using both normotensive and hypertensive models of experimental diabetes, it could be clearly shown that ACE inhibitors were effective in retarding the development of albuminuria (Zatz et al., 1986; Cooper et al., 1990; O’Brien et al., 1993). In these
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Figure 1 Data are shown at week 32 as mean ± SEM for glycated hemoglobin (HbA1c) and mRNA for TGFȕ1 and type IV collagen in control and diabetic Sprague-Dawley rats treated with either daily or alternate daily insulin. *p < 0.01 vs control; fp < 0.01 vs diabetic and insulin daily.
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studies, mesangial expansion was not prevented whereas GBM thickness and glomerular volume were decreased by ACE inhibition (Cooper et al., 1990). These effects were most readily observed in diabetic rats with concomitant hypertension. However, a recent study using a relatively high dose of the ACE inhibitor, trandolapril, reduced mesangial expansion although these effects were rather modest (Sassy Prigent et al., 1995). In contrast, dihydropyridine calcium channel blockers have failed to be renoprotective in streptozocin diabetes (Anderson et al., 1992; Rumble et al, 1995). Several groups have explored the role of ACE inhibitors in animals with early evidence of diabetic renal injury. These agents prevented but did not reverse diabetic nephropathy (Cooper et al., 1988a; Perico et al., 1994b). In uninephrectomised alloxan diabetic dogs, the ACE inhibitor lisinopril reduced glomerular volume and proteinuria (Brown et al., 1993). A diltiazem-like calcium channel blocker had similar effects on these renal parameters. Interestingly, the combination of ACE inhibitor and calcium channel blocker was the most effective regimen in terms of renal protection. These findings suggest that various combinations of antihypertensive agents warrant further investigation to determine the regimen of choice in diabetic patients with renal disease. ACE inhibitors act not only to suppress AII formation but also to inhibit degradation of the vasodilatory kinins (Erdos, 1976). Therefore, it cannot be assumed that their renoprotective effects are related solely to suppression of AII dependent mechanisms. The advent of specific AII receptor antagonists such as losartan (Johnston, 1995) has allowed researchers to explore specifically the role of AII mediated pathways in the genesis of diabetic renal injury. Recent studies by our group have implicated the renin-angiotensin system (RAS) in the genesis of DN. Using a transgenic rat which overexpresses the renin gene (Mullins et al., 1990), it has been shown that induction of streptozocin diabetes in this rat is associated with rapid development of GBM thickening, mesangial expansion and ultimately glomerulosclerosis (Figure 2) (Kelly et al., 1998). These renal changes do not occur in the absence of diabetes in age-matched transgenic Ren-2 hypertensive rats, nor are they observed in aged matched diabetic SHR with similar levels of blood pressure (Cooper et al., 1988b). In a recent study in these diabetic Ren2 rats, the ACE inhibitor perindopril attenuated renal injury (Kelly et al., 1998). These findings provide further evidence for the concept that the RAS plays an important role in the genesis of renal injury in experimental diabetes. Several groups have now documented renoprotective effects of AII antagonists on diabetes associated renal injury in both normotensive and hypertensive rats (Remuzzi et al., 1993; Kohzuki et al., 1995). Recent studies by our group have confirmed that the prevention of albuminuria by ACE inhibitors appears to be via the actions of these agents as inhibitors of AII formation rather than via potentiation of kinin levels (Allen et al., 1997a). The effects of ACE inhibitors were reproduced by the AII antagonist, valsartan, and could not be prevented by concomitant administration of the bradykinin receptor antagonist, Icatibant, with the ACE inhibitor, ramipril. The process of advanced glycation has been implicated in a range of diabetic complications including nephropathy (Bucala and Vlassara, 1995). Advanced glycated end-products are increased in the diabetic kidney and have been detected in glomeruli and tubules (Soulis-Liparota et al., 1991). The inhibitor of advanced
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Figure 2 Micrographs of kidneys counterstained with Periodic Acid Schiff reagent Magnification x330. A: Sham transgenic Ren-2 kidney. B: Diabetic transgenic Ren-2 kidney after 12 weeks. Basement membrane thickening and occluded capillaries within the glomerulus (G) are shown. Some tubules are vacuolated (arrow).
glycation, aminoguanidine, has been shown to be renoprotective (Itakura et al., 1991; SoulisLiparota et al., 1991; Edelstein and Brownlee, 1992). This treatment retards the development of albuminuria and prevents mesangial expansion (Soulis-Liparota et al., 1991). However, GBM thickness is not affected. These findings are similar to those reported with islet transplantation (Steffes et al., 1979; Steffes et al., 1980), consistent with both therapies acting via glucose-dependent pathways. More recent studies by our group have indicated that there was no difference in the renoprotective effects of aminoguanidine if treatment was instituted in the first 16 weeks or the final 16 weeks of the 32 week study period (Soulis et al., 1996). The major determinant of the degree of renoprotection afforded by aminoguanidine appeared to be the duration of drug therapy. Studies by Cohen et al have suggested a pathogenic role for the early glycated products in the development of renal disease in a mouse model of diabetes (Cohen et al., 1994). Treatment with an antibody to the Amadori products was shown to be renoprotective. However, it is possible that
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the antibody led to suppression of advanced glycated product formation since the Amadori product is an important intermediate in that pathway. The role of the aldose reductase inhibitors in preventing DN remains controversial. These agents have been shown to prevent diabetic hyperfiltration (Bank et al., 1989; Goldfarb et al., 1991) and some investigators have reported retardation in the development of albuminuria (McCaleb et al., 1988; Chang et al., 1991; BeyerMears et al, 1992; Kassab et al., 1994). However, despite a large number of studies, there is no consensus as to whether this class of agents is renoprotective, both in terms of effects or albuminuria or glomerular morphology (Daniels and Hostetter, 1989; Mauer et al., 1989; Korner et al., 1992). A 5 year study failed to show a renoprotective role of these agents in alloxan diabetic dogs (Kern and Engerman, 1991; Engerman et al., 1993). IGF-1 has been clearly implicated in the genesis of the early renal hypertrophy of experimental diabetes (Flyvbjerg et al., 1988; Bach and Jerums, 1990). The role of growth hormone and IGF-1 in the progression of renal disease has been exploring using the somatostatin analogue, octreotide. There are conflicting reports of this agent in experimental DN with several groups reported retardation in the development of albuminuria and prevention of renal hypertrophy (Igarashi et al., 1991; Flyvbjerg et al., 1992). However, Muntzel et al., failed to detect a renoprotective role of this agent in diabetic rats (Muntzel et al., 1992). Heparin has been postulated as a therapy to be considered in the prevention and treatment of diabetic renal disease (Gambaro et al., 1992). There are multiple mechanisms by which this agent could potentially ameliorate DN. One important mechanism involves restoration of the anionic charge of the glomerular filtering surface which is reduced in diabetes due to loss of the proteoglycan, heparan sulphate (van den Born et al., 1995). The advent of low molecular weight heparin has allowed investigators to administer a heparinoid without the problems of anticoagulation. Gambarro et al., have reported that heparin treatment retards the development of glomerular hypertrophy and albuminuria without affecting GFR (Gambaro et al., 1992). Another group has also reported a decrease in urinary albumin excretion with low molecular weight heparin. This treatment was associated with restoration of the anionic charge of the GBM (Oshima et al., 1994). However, several other groups have reported a lack of renoprotection with heparin (Yokoyama et al., 1995). Other agents under intense investigation include inhibitors of the potent vasoconstrictor, thromoxane A2 (Craven et al., 1992), either via inhibition of thromboxane synthetase or via specific antagonism of the thromboxane A2 receptor. These agents have been shown to prevent hyperfiltration and reduce albuminuria. In the diabetic mouse, this retardation in the development of albuminuria was associated with a reduction in gene expression of extracellular matrix proteins (Ledbetter et al., 1990). Thromboxane synthetase inhibition has also been reported to decrease albuminuria and prevent mesangial expansion in diabetic SHR (Masumura et al., 1992). Craven et al have reported reduction in albuminuria, glomerular volume, GBM thickness and mesangial expansion with thromboxane synthetase inhibition (Craven et al., 1992). In further experiments by that group, institution of the thromboxane synthetase inhibitor 5 months after the induction of diabetes was not particularly effective, unless there was concomitant administration of a receptor antagonist (Craven et al., 1992). In contrast, Tajiri et al. have reported
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Figure 3 Data are shown at week 24 as mean ± SEM for glomerular volume in control rats and diabetic rats receiving low (LS) or normal salt (NS) diets. *p < 0.01 vs control; tp < 0.05 vs diabetic+NS.
no effect of thromboxane synthetase inhibition on renal morphology or proteinuria despite reduction in urinary thromboxane B2 excretion (Tajiri et al., 1994). Another class of drugs that warrants further investigation is the endothelin receptor antagonists. These agents have been evaluated in detail in non-diabetic models of renal disease (Benigni et al., 1993) and may act via similar mechanisms to drugs which interrupt the renin-angiotensin system. It is intriguing that the ACE inhibitor enalapril which has been shown to be renoprotective in experimental diabetes did not reduce gene expansion of growth factors postulated to be involved in the pathogenesis of DN such as TGF-ȕ, yet reduced the mRNA for preproendothelin I (Fukui et al., 1994). Studies by that group have also described significant abnormalities in gene expression not only of endothelin but of some of its receptors in the diabetic kidney (Fukui et al., 1993). Recently this group reported that an endothelin receptor antagonist reduced proteinuria in diabetic rats and that this was associated with reduction in mRNA for various extracellular matrix proteins (Nakamura et al., 1995). Although most interventions have focussed on pharmacological agents, several dietary interventions have been shown to be beneficial in experimental diabetic nephropathy. Low protein diets have been reported to normalise the raised intraglomerular pressure and retard the development of albuminuria in diabetic Munich Wistar rats (Zatz et al., 1985). Our group has recently observed that salt restriciton not only reduces GFR in diabetic rats but is also associated with
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pre vention of the development of albuminuria in normotensive, diabetic rats (Allen et al, 1997b). Further studies by our group have indicated that these functional effects of salt restriction in diabetic rats were associated with attenuation of glomerular hypertrophy (Figure 3). Although a range of treatments have been shown to be renoprotective, the exact mode of action of these therapies at the molecular level has not been clearly delineated. Recent studies by our group have confirmed that TGFȕ plays a pivotal role in the genesis of diabetes associated microvascular injury. Indeed, the inhibitor of advanced glycation, aminoguanidine, has been shown to prevent overexpression of both TGFȕ and the matrix protein, type IV collagen, in diabetic blood vessels (Rumble et al., 1997). Furthermore, the ACE inhibitor, ramipril, attenuated TGFȕ1 gene expression in the kidney after 24 weeks of experimental diabetes (Gilbert et al., 1998). These effects were associated with a decrease in type IV collagen deposition and reduced glomerular and tubulointerstitial injury (Gilbert et al., 1998). Therefore, it is likely that many of the actions of renoprotective treatments are mediated by cytokine-dependent pathways (Cooper et al., 1997). CONCLUSION It appears likely that as the molecular and cellular mechanisms that link hyperglycemia to diabetic complications are clarified, more rational therapies will be developed which can be investigated in the various animal models of diabetic nephropathy. There is already evidence that various therapies are associated with changes in mRNA levels for matrix proteins and growth factors (Ledbetter et al., 1990; Yang et al., 1994; Nakamura et al., 1995). If these effects represent mechanisms which mediate the renoprotective actions of these agents or are a reflection of attenuation of renal injury remains to be determined. The advent of selective cytokine antagonists will allow this issue to be further investigated. Fortunately, the animal models available exhibit many of the functional and structural characteristics that are observed in human DN. It has already become apparent that many of the treatments that were effective in rodents studies have a role in the prevention, retardation and reversal of human DN. For example, the results of clinical studies have confirmed the role of glycaemic control in the progression of diabetic microvascular disease (Feldt-Rasmussen et al., 1986; DCCT Research Group, 1993). The studies in rodents which explored the role of ACE inhibition in experimental diabetic nephropathy were able to clearly show a renoprotective role for these agents (Zatz et al., 1986; O’Brien et al., 1993). The results of these experiments were a major stimulus for performing trials in both type I and type II diabetic patients which have described beneficial effects of ACE inhibitors on albuminuria and renal function (Lewis et al., 1993; Ravid et al., 1993; Viberti et al., 1994). REFERENCES Allen, T.J., Cao, Z., Youssef, S., Hulthen, U.L. and Cooper, M.E. (1997a) The role of angiotensin II and bradykinin in experimental diabetic nephropathy: functional and structural studies. Diabetes, 46, 1612–1618.
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Allen, T.J., Cooper, M.E., O’Brien, R.C., Bach, L.A., Jackson, B. and Jerums, G. (1990) Glomerular filtration rate in the streptozocin diabetic rat: The role of exchangeable sodium, vasoactive hormones and insulin therapy. Diabetes, 38, 1182–1190. Allen, T.J., Waldron, M.J., Casley, D., Jerums, G. and Cooper, M.E. (1997b) Salt restriction reduces hyperfiltration, renal enlargement and albuminuria in experimental diabetes. Diabetes, 46, 119–124. Andersen, A.R., Christiansen, J.S., Anderson, J.K., Kreiner, S. and Deckert, T. (1983) Diabetic nephropathy in type I (insulin-dependent) diabetes: An epidemiological study. Diabetologia, 25, 496–501. Anderson, S., Jung, F.F. and Ingelfmger, J.R. (1993) Renal renin-angiotensin system in diabetes: functional, immunohistochemical, and molecular biological correlations. Am. J. Pbysiol., 265, F477–86. Anderson, S., Rennke, H.G. and Brenner, B.M. (1992) Nifedipine versus fosinopril in uninephrectomized diabetic rats. Kidney Int., 41, 891–7. Anderson, S., Rennke, H.G., Garcia, D.L. and Brenner, B.M. (1989) Short and long term effects of antihypertensive therapy in the diabetic rat. Kidney Inter., 36, 526–536. Ayo, S.H., Radnik, R.A., Glass, W.F.D., Garoni, J.A., Rampt, E.R., Appling, D.R. and Kreisberg, J.I. (1991) Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. Am. J. Physiol., 260, F185–91 Bach, L.A. and Jerums, G. (1990) Effect of puberty on initial renal growth and rise in kidney insulin-like growth factor 1 in diabetic rats. Diabetes, 39, 557–562. Badillo, L., Sharma, K., Jin, Y. and Ziyadeh, F.N. (1994) Insulin inhibits TGF-ȕl transcriptional activity and mRNA levels in cell culture. J. Am. Soc. Nephrol., 5, 960. Ballerman, B.J., Skorecki, K.L. and Brenner, B.M. (1984) Reduced glomerular angiotensin II receptor density in early untreated diabetes mellitus in the rat. Am.J. Physiol., 247, Fl 10– F116. Bank, N. and Aynedjian, H.S. (1990) Progressive increase in luminal glucose stimulate proximal sodium absorption in normal and diabetic rats. J. Clin. Invest., 86, 309–316. Bank, N. and Aynedjian, H.S. (1993) Role of EDRF (nitric oxide) in diabetic renal hyperfiltration. Kidney Int., 43, 1306–12. Bank, N., Lahorra, M.A.G., Aynedjian, H.S. and Schlondorff, D. (1988) Vasoregulatory hormones and the hyperfiltration of diabetes. Am. J. Physiol., 254, F202–F209. Bank, N., Mower, R, Aynedjian, H.S., Wilkes, B.M. and Silverman, S. (1989) Sorbinil prevents glomerular hyperperfusion in diabetic rats. Am. J. Physiol., 256, Fl000–6. Barthelmebs, M., Vailly, B., Grima, M., Velly, J., Stephan, D., Froehly, S. and Imbs, J.L. (1991) Effects of dopamine prodrugs and fenoldopam on glomerular hyperfiltration in streptozotocin-induced diabetes in rats.J. Cardiwasc Pharmacol., 18, 243–53. Benigni, A., Zoja, C., Corna, D., Orisio, S., Longaretti, L., Bertani, T. and Remuzzi, G. (1993) A specific endothelin subtype A receptor antagonist protects against injury in renal disease progression. Kidney Int., 44, 440–4. Beyer-Mears, A., Murray, F.T., Cruz, E., Rountree, J. and Sciadini, M. (1992) Comparison of sorbinil and ponalrestat (Statil) diminution of proteinuria in the BB rat. Pharmacology, 45, 285–91. Björck, S., Nyberg, G., Mulec, H., Granerus, G., Herlitz, H. and Aurell, M. (1986) Beneficial effects of angiotensin converting enzyme inhibition on renal function in patients with diabetic nephropathy. Br. Med. J., 293, 471–474. Blantz, R.C., Thomson, S.C., Peterson, O.W. and Gabbai, F.G. (1990) Physiologic adaptations of the tubologlomerular feedback system. Kidney Int., 38, 577–583.
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Bower, G., Brown, D.M., Steffes, M.W., Vernier, R.L. and Mauer, S.M. (1980) Studies of the glomerular mesangium and the juxtaglomerular apparatus in the genetically diabetic mouse. Lab. Invest., 43, 333–341. Braam, B., Mitchell, K.D., Koomans, H.A. and Navar, L.G. (1993) Relevance of the tubuloglomerular feedback mechanism in pathophysiology.J. Am. Soc. Nephrol., 4, 1257– 74. Brown, D.M., Steffes, M.W., Thibert, P., Azar, S. and Mauer, S.M. (1983) Glomerular manifestations of diabetes in the BB rats. Metabolism, 32, 131–135. Brown, S.A., Walton, C.L., Crawford, P. and Bakris, G.L. (1993) Long-term effects of antihypertensive regimens on renal hemodynamics and proteinuria. Kidney Int., 43, 1210–8. Bucala, R. and Vlassara, H. (1995) Advanced glycosylation end products in diabetic renal and vascular disease. Am. J. Kidney Dis., 26, 875–88. Cagliero, E., Maiello, E.M., Boeri, D., Roy, S. and Lorenzi, M. (1988a) Increased expression of basement membrane components in human endothelial cells cultured in high glucose. J. Clin. Invest., 82, 735–8. Cagliero, E., Maiello, M., Boeri, D. and Lorenzi, M. (1988b) High glucose increases transforming growth factor beta (TGF-ȕ) mRNA in endothelial cells: a mechanism for the altered regulation of basement membrane components? Diabetes, 37(Suppl. 1), 97A. Chang, W.P., Dimitriadis, E., Allen, T., Dunlop, M.E., Cooper, M. and Larkins, R.G. (1991) The effect of aldose reductase inhibitors on glomerular prostaglandin production and urinary albumin excretion in experimental diabetes mellitus. Diabetologia, 34, 225–31. Christlieb, A.R. and Boston, M.D. (1974) Renin, angiotensin and norepinephrine in alloxan diabetes. Diabetes, 23, 962–970. Cohen, A.J., McGill, P.D., Rossetti, R.G., Guberski, D.L. and Like, A.A. (1987) Glomerulopathy in spontaneously diabetic rat. Impact of glycemic control. Diabetes, 36, 944–951. Cohen, M.P., Hud, E. and Wu, V.Y. (1994) Amelioration of diabetic nephropathy by treatment with monoclonal antibodies against glycated albumin. Kidney Int., 45, 1673–9. Cooper, M.E., Allen, T.J., Macmillan, P., Bach, L., Jerums, G. and Doyle, A.E. (1988a) Genetic hypertension accelerates nephropathy in the streptozotocin diabetic rat. Am. J. Hypertension, 1, 5–10. Cooper, M.E., Allen, T.J., O’Brien, R., Clarke, B., Jerums, G. and Doyle, A.E. (1988b) Effects of genetic hypertension on diabetic nephropathy in the rat—functional and structural characteristics. J. Hypertension, 6, 1009–1016. Cooper, M.E., Allen, T.J., O’Brien, R.C., Papazoglou, D., Clarke, B.E., Jerums, G. and Doyle, A.E. (1990) Nephropathy in model combining genetic hypertension with experimental diabetes. Enalapril versus hydralazine and metoprolol therapy. Diabetes, 39, 1575–9. Cooper, M.E., Jerums, G. and Gilbert, R.E. (1997) Diabetic vascular complications. Clin. Exp. Pharmacol. Physiol., 24, 770–775. Correa-Rotter, R., Hostetter, T.H. and Rosenberg, M.E. (1992) Renin and angiotensinogen gene expression in experimental diabetes mellitus. Kidney Int., 41, 796– 804. Craven, P.A., Melhem, M.F. and DeRubertis, F.R. (1992) Thromboxane in the pathogenesis of glomerular injury in diabetes. Kidney Int., 42, 937–46.
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Tochino, Y. (1984) Breeding and characteristics of a spontaneously diabetic non-obese strain (NOD mouse) of mice. In Lesson from Animal Diabetes, edited by E. Shafrir and A. Renold. London: John Libbey. Tolins, J.P., Shultz, P.J., Raij, L., Brown, D.M. and Mauer, S.M. (1993) Abnormal renal hemodynamic response to reduced renal perfusion pressure in diabetic rats: role of NO. Am.J. Physiol., 265, F886–95. Trinder, D., Phillips, P.A., Stephenson, J.M., Risvanis, J., Aminian, A., Adam, W., Cooper, M. and Johnston, C.I. (1994) Vasopressin VI and V2 receptors in diabetes mellitus. Am. J. Pbysiol, 266, E217–23. van den Born, J., van Kraats, A.A., Bakker, M.A., Assmann, K.J., van den Heuvel, L.P., Veerkamp, J.H. and Berden, J.H. (1995) Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate. Diabetologia, 38, 161–72. Velasquez, M.T., Kimmel, P.L. and Michaelis, O.E.I.V. (1990) Animal models of spontaneous diabetic kidney disease. FASEB J., 4, 2850–9. Velasquez, T., Michaelis, O.E.I.V., Kimmel, P.L. and Bosch, J.P. (1993) Glomerular lesions in diabetic humans and spontaneously diabetic animals. In Lessons from Animal Diabetes, edited by E. Shafrir. London: John Libbey. Viberti, G.C., Mogensen, C.E., Groop, L.C. and Pauls, J.F. (1994) Effect of captopril on progression to clinical proteinuria in patients with insulin-dependent diabetes mellitus and microalbuminuria. European Microalbuminuria Captopril Study Group.JAMA, 271, 275–9. Vora, J.P., Anderson, S. and Brenner, B.M. (1994) Pathogenesis of diabetic glomerulopathy: the role of glomerular hemodynamic factors. In The kidney and hypertension in diabetes mellitus, edited by C.E. Mogensen. Boston: Kluwer Academic Publishers. Vora, J.P., Oyama, T.T., Thompson, M.M. and Anderson, S. (1995) Interactions of reninangiotensin (RAS) and kallikrein-kinin (KKS) systmes in the diabetic kidney. J Am. Soc. Nephrol, 6, 406 (Abstract). Wigness, B.D., Mauer, S.M., Rupp, W.M., Rohde, T.D., Steffes, M.W., Blackshear, P.J., Rucker, R.D., Jeraj, K. and Buchwald, H. (1982) A double balloon catheter technique for alloxan diabetogenesis in the dog. Surg. Gynecol. Obstet., 860–864. Wilkes, B.M., Mento, P.F. and Vernace, M.A. (1993) Angiotensin responsiveness in hyperfiltering and nonhyperfiltering diabetic rats. J. Am. Soc. Nephrol., 4, 1346–53. Yamamoto, T., Nakamura, T., Noble, N.A., Ruoslahti, E. and Border, W.A. (1993) Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy. Proc. Natl. Acad. Sci. USA, 90, 1814–8. Yang, C.W., Vlassara, H., Peten, E.P., He, C.J., Striker, G.E. and Striker, L.J. (1994) Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease. Proc. Natl. Acad. Sci. (USA), 91, 9436–40. Yokoyama, H., Myrup, B., Oturai, P. and Deckert, T. (1995) Heparin, a possible therapy for diabetic complications: the effect on mesangial and myomedial cells in vivo and in vitro, especially in relation to extracellular matrix. J. Diabetes Complications, 9, 97–103. Zatz, R., Dunn, B.R., Meyer, T.W. and Brenner, B. (1986) Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J. Clin. Invest., 77, 1925–30.
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Zatz, R., Meyer, T.W., Rennke, H.G. and Brenner, B.M. (1985) Predominance of hemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc. Natl. Acad. Sci (USA), 82, 5963–5967. Zhang, P.L., Mackenzie, H.S., Troy, J.L. and Brenner, B.M. (1994) Effects of an atrial natriuretic peptide receptor antagonist on glomerular hyperfiltration in diabetic rats. J. Am. Soc. Nepbrol., 4, 1564–70. Ziyadeh, F.N. (1994) Role of transforming growth factor beta in diabetic nephropathy. Exp. Nephrol., 2, 137. Ziyadeh, F.N. and Goldfarb, S. (1991) The renal tubulointerstitium in diabetes mellitus. Kidney Int., 39, 464–475.
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9. DIABETIC RETINOPATHY IN EXPERIMENTAL ANIMAL MODELS AND THEIR FEASIBILITY FOR UNDERSTANDING THE HUMAN DISEASE* SUBRATA CHAKRABARTI University of Western Ontario, London, Ontario, Canada
INTRODUCTION Animal experiments are desirable for the study of any disease process to replicate pathophysiological changes of a particular condition. Data from various controlled animal experiments may provide valuable perspectives for the development of preventive or interventional therapeutic approaches. In a condition like diabetic retinopathy, where the pathogenesis is poorly understood, animal experiments are of immense importance as they offer unique advantages by enabling sequential studies at the molecular, biochemical, functional and structural levels in order to pinpoint complex mechanisms in the dynamic development of the disease. Much of the knowledge regarding the pathogenesis of diabetic retinopathy stem from experiments in various animal models in which the abnormalities of diabetic retinopathy have been reproduced, This review will briefly discuss human diabetic retinopathy and address various abnormalities in animal models of diabetic retinopathy and how they may enter into the complex pathogenetic web of this disorder. Both diabetic and galactosemic animals have been used in the study of diabetic retinopathy. Most of the experiments have been carried out in diabetic dogs and rats. Galactosemic dogs and rats are important, as diabetic like lesions are produced in these models. They provide specific model systems for the study of polyol pathway as well other biochemical pathways activated secondary to hyperhexosemia. HUMAN DIABETIC RETINOPATHY Although diabetes mellitus was first described around 1000 BC, diabetic retinopathy was recognized relatively recently by Jager in 1855 following the discovery of the
ophthalmoscope (Frank, 1957). It is estimated that 5% of the world population Correspondence to: Dr. Subrata Chakrabarti, Department of Pathology, Dental Sciences Building, University of Western Ontario, London, Ontario, Canada N6A 5C1, Tel: (519) 663–3381, Fax: (519) 663–2930, E-mail:
[email protected] *Supported in part by a grant from Canadian Diabetes Association in honour of Florence Langlie.
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is affected by diabetes (Cantagallo, 1989). In the US, it is estimated that there are 50,000 people with blindness due to diabetes and 5,800 new cases are added each year (Mazze et al., 1985). Diabetic retinopathy is the leading cause of blindness among the people of occupational age (Benson et al., 1990; Javitt et al., 1988) Retinopathy accounts for a 25-fold increase in the incidence of blindness in patients with type I diabetes and a 2 to 3 fold increase in those with type II diabetes compared to age matched control populations (Report of the Second National Diabetes Research Conference, 1984). Duration of diabetes appears to be a major risk factor for the development of retinopathy. The incidence of retinopathy varies from nil with a diabetes duration of less than 5 years to 71% with diabetes of more than 10 years duration (Frank, 1991; Benson et al., 1990). The risk factors for the development of diabetic retinopathy include poor blood glucose control, hypertension, renal disease, pregnancy, tobacco and alcohol consumption (Blom et al., 1994). Although other modulatory factors are important, poor glycemic control is the primary pathogenetic factor leading to clinical retinopathy and other complications of diabetes (Santiago, 1993). Good blood glucose control by intensive insulin therapy delays the onset and progression of diabetic retinopathy (Diabetes Control and Complication Trial Research Group, 1993). Several clinical studies have shown that elevated glycated hemoglobin is an important predictive marker for the development of retinopathy (Klein et al., 1988; Diabetes Control and Complication Trial Research Group, 1993). Pathologic Features of Human Diabetic Retinopathy Human diabetic retinopathy is classified into various progressive stages, namely, non-proliferative (background) retinopathy, pre-proliferative (severe or advanced background) retinopathy and proliferative retinopathy. Background retinopathy consists of capillary microangiopathy, retinal hemorrhages and exudate, macular edema and soft exudate (cotton wool spots). Capillary microangiopathy encompasses loss of pericytes, basement membrane thickening, microvascular obstruction and permeability changes and microaneurysms. Cotton wool spots, representing microinfracts of the nerve fiber layer were once considered an important predictor of proliferative diabetic retinopathy. However, this has not been substantiated (Blom et al., 1994). Venous dilatation and beading, profuse retinal hemorrhages and exudate, widespread capillary nonperfusion and intraretinal microvascular abnormalities (IRMA) consisting of telengiactatic vessels shunting blood around areas of non-perfusion, are the characteristic features of preproliferative retinopathy. Patients with such lesions are prone to develop proliferative retinopathy. Neovascularization is the characteristic feature of the proliferative stage. New blood vessels form on the optic disc, within the retina, on the retinal surface or inside the vitreous. New vessels formation may be accompanied by bleeding and tractional retinal detachment. An outline of lesions in the various stages of diabetic retinopathy are presented in Figure 1.
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Figure 1 An outline of various lesions in different stages of diabetic retinopathy.
Pathogenesis of Human Diabetic Retinopathy The exact pathogenesis of diabetic retinopathy is not clear. Hyperglycemia appears to be a key initiating factor leading to several biochemical alterations in the target organs of diabetic complications (Frank, 1991; Williamson et al., 1993). As a result of sustained hyperglycemia, several secondary biochemical pathways and coagulation abnormalities are initiated. The secondary mechanisms include augmented polyol pathway activity, redox imbalances and oxidative stress, alterations of diacylglycerol (DAG)—protein kinase C (PKC) pathway and nonenzymatic glycation, (Brownlee et al., 1995; King et al., 1993; Williamson et al., 1993). These mechanisms have widely been studied in experimental animals and will be discussed in the section entitled ‘Retinal biochemical alterations in the animal models’. Along with the biochemical changes in the retina, the systemic coagulation abnormalities in the course of diabetes mellitus may impact on the functional and structural integrities of the retina. The final result is an imbalance between thrombus formation and dissolution, favoring the former (Ceriello, 1993). Increased plasma levels of fibrinogen, factor VII, VIII and von Willebrand factor have been demonstrated in diabetes (Coller et al., 1978; Ceriello et al., 1988; Osterman and van de Loo, 1986). Oxidative stress due to free radical generation in diabetes is correlated with hemostatic alterations (Collier et al., 1992). Non-enzymatic glycation of fibrin and platelet membrane further potentiate thrombotic activity (Ceriello, 1990, 1993). The biochemical and rheological alterations resulting from long term hyperglycemia lead to alterations in the gene expression of extracellular matrix proteins and other proteins, alterations of cellular synthetic activities and enzyme action, blood flow and permeability abnormalities, structural changes, cell death and the development of clinical diabetic retinopathy (Fukui et al., 1992; King et al., 1993; Porta, 1996). Development of background retinopathy sets the stage for subsequent development of retinal ischemia, upregulation and/or release of several vasoproliferative factors, leading to proliferative diabetic retinopathy (Forrester et al., 1993). The list of vasoproliferative factors include, fibroblast growth factors (FGFs), transforming growth factor ȕ (TGF-ȕ), tumor necrosis factor-Į (TNF-Į), insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), etc. (Forrester et al., 1993). Recently described vascular endothelial growth factor (VEGF) is most likely a key candidate responsible for neovascularization (Aiello et al., 1994). Increased levels of VEGF has been
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demonstrated in intraocular fluids of patients with neovascularization (Aiello et al., 1994). VEGF is further of importance as a permeability factor in background retinopathy (Aiello et al., 1994; Porta 1996). These factors stimulate endothelial cell proliferation and neovascularization of the ischemic retina. A detailed description of neovascularization factors and the mechanisms of neovascularization is however, beyond the scope of this review. DIABETIC RETINOPATHY IN ANIMALS Animal experiments have been importarnt in the understanding of the pathology and pathogenesis of human diabetic retinopathy. Both diabetic and galactosemic animals have been used as models for diabetic retinopathy. In addition non-diabetic animal models have been used to study phenomena such as neovascularization. In this review structural, biochemical and functional changes in diabetic retinopathy will be addressed. Retinal Structural Lesions in Animal Models Retinal structural lesions have been produced in both diabetic and galactosemic animals. Prevention of these changes by inhibition of specific biochemical pathways have provided indirect evidence for their role in the genesis of diabetic retinopathy. Diabetic dogs Histopathological characteristic features of background and proliferative diabetic retinopathy have repeatedly been produced in diabetic dogs (Engerman and Kern, 1995). Progressive early retinal lesions of diabetic retinopathy such as basement membrane thickening, pericyte loss, capillary occlusion, capillary leakage, loss of microvascular smooth muscle cells, as well as advanced retinopathic changes such as microaneurysms, intraretinal microvascular abnormalities and retinal neovascularization have consistently been produced in diabetic dogs (Engerman and Bloodworth, 1965; Hausler et al., 1964; Engerman and Kern, 1987; Gardiner et al., 1994) (Figure 2), Lesions of diabetic retinopathy develop and progress in this model irrespective of the methodologies used for the production of diabetes such as pancreatectomy, alloxan, growth hormones or whether diabetes is of idiopathic origin (Engerman and Kern, 1995; Engerman and Bloodworth, 1965; Hausler et al., 1964; Engerman and Kern, 1987, 1993). It is interesting to note that distribution of retinal lesions may vary in various retinal quadrants in the diabetic dog (Kern and Engerman, 1995). The exact reason for this variation is not known. However it is possible that local microenvironmental factors in various sectors of the retina may have a modulatory influence. The dog model has unequivocally demonstrated a definitive role of hyperglycemia in the development of diabetic retinopathy (Engerman et al., 1977; Engerman and Kern, 1987). Retinopathy was effectively prevented when strict blood glucose control was initiated shortly after onset of hyperglycemia. However, lesser effects were achieved, when strict blood glucose control was initiated after a longer period of preceding hyperglycemia (Engerman et al., 1977; Engerman and Kern, 1987). Virtuaily similar results were obtained in
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Figure 2 Trypsin digested retinal preparation from an alloxan diabetic dog showing saccular microaneurysm (arrow). (Courtesy Dr. T.S.Kern, Dept. Of Ophthalmology, University of Wisconsin, Madison).
human diabetic retinopathy (Diabetes Control and Complication Trial research group, 1993). Treatment with aldose reductase inhibitors (ARI), has failed to prevent the progression of retinopathy in this model (Engerman and Kern, 1993) suggesting activation of multiple biochemical mechanisms secondary to hyperglycemia and polyol pathway activation, which in themselves are not responsive to aldose reductase inhibition. Diabetic rats Although small laboratory animals do not develop advanced retinal lesions like diabetic dogs, except for rare microaneurysms, early microangiopathic lesions have repeatedly been produced in rats. These early lesions are important in the study the pathogenetic mechanisms and the development of preventive strategies. The streptozotocin (STZ) induced diabetic rat is the most extensively studied diabetic rat model. It shows early microangiopathic lesions such as basement membrane (BM) thickening, loss of capillary pericytes, acellular capillaries, endothelial cell proliferation and rare microaneurysms (Sosula et al., 1972; Babel et al., 1974; Struder et al., 1976; Sharma et al., 1985; Chakrabarti et al, 1987a). As in the diabetic dog, euglycemia achieved by pancreatic islet cell transplantation can prevent microangiopathic changes in the STZ diabetic rats (Chakrabarti et al, 1987; Sima et al., 1988). Although progressive intracellular advanced glycation end products (AGE) accumulation is associated with pericyte loss in STZ diabetic rats (Stitt et al., 1996), aminoguanidine, an inhibitor of non-enzymatic glycation, is only effective in preventing the late changes of diabetic retina, suggesting that AGE associated changes are time dependent (Hammes et al., 1991, 1995). On the other
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hand, antioxidant treatment have been shown to prevent increased endothelial cellpericyte ratio in long term diabetes without any effect of AGE accumulation (Hammes et al., 1997). In the STZ diabetic rats, increased permeability across the retinal pigment epithelium due to breakdown of the blood retinal barrier in association with structural alterations of the retinal pigment epithelium were observed. These changes are preventable by tight control of blood glucose by insulin therapy and syngeneic islet cell transplantation (Krupin et al., 1979; Grimes and Laties, 1980; Kernel and Arnqvist, 1983). The spontaneously diabetic BB/W rat shows basement membrane thickening, pericyte degeneration and loss, platelet microthrombi, retinal pigment epitheliopathy, thickening of the vitreoretinal border membrane (Blair et al., 1984; Sima et al., 1985; Chakrabarti et al., 1990; Heegaard, 1993) (Figure 3). Capillary basement membrane and Bruch’s membrane thickening in the diabetic BB-rat are associated with an absolute loss anionic sites, provided by the proteoglycans, resulting in a loss of the charge selective barrier function of the basement membrane, which is important in the pathogenesis of diabetes induced hyperpermeability (Chakrabarti et al., 1991). Similar to the STZ rat, retinal lesions in this animal were prevented by good blood glucose control by insulin or acarbose, an alpha glucosidase inhibitor, indicating the role of hyperglycemia in the pathogenesis of these lesions (Chakrabarti and Sima, 1989; Chakrabarti et al., 1993). ARI-treatment and myoinositol (Ml)-supplementation prevented BM thickening in the BB/W-rat in the superficial capillary bed but not in the deep capillary bed (Chakrabarti and Sima, 1989; Chakrabarti and Sima, 1992a). This interesting observation indicate that local microenvironmental factors such as oxygen tension and/or hemodynamic pressure may modulate the effects of hyperglycemia on the microvasculature. Several diabetic retinal lesions were demonstrated in other rat models of diabetes. The list include alloxan diabetes rats, rats with diabetes induced by pancreactectomy and by growth hormone, (Toussaint, 1966; Orloff et al., 1975; Musacchio et al., 1964; Agarwal et al., 1966). The lesions are of early microangiopathic nature and are similar to those in previously discussed models. Other diabetic animal models Some of the diabetic retinopathic changes have been demonstrated in diabetic monkeys following long term alloxan induced diabetes (Gibbs et al, 1969; Bresnick et al., 1976), as well as in spontaneously diabetic monkeys (Laver et al, 1994). However, very few studies have been carried out in diabetic monkeys and the usefulness of primates as models of diabetic retinopathy has yet to be established. The list of other animal models used in the study of diabetic retinopathy is impressive and include models such as diabetic carp (Yokote, 1973). However, in general these models do not offer any advantages over the diabetic dog or rat models. The body of data generated from small animal models of diabetes further confirm the role of hyperglycemia as the primary culprit and involvement of multiple secondary biochemical mechanisms responsible for the development of structural lesions in the retina in diabetes.
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Figure 3 Retinal capillary electronmicrographs from a non-diabetic BB/W rat (a) and an age matched 6 mo.diabetic BB/W rat (b). Please note basement membrane (arrow) thickening in b compared to a (ilm=internal limiting membrane).
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Galactosemic animal models Galactosemic animal models have a special role in the study of diabetic retinopathy. Galactosemic animals provide an opportunity to investigate effects of hyperhexosemia in isolation from hormonal alterations in diabetes. Augmented polyol pathway activity and other biochemical mechanisms, triggered secondary to hyperhexosemia, have been investigated in this model. Retinal structural lesions similar to those of diabetic retinopathy have been produced in the retina of galactosemic animals (Kern and Engerman, 1994; Engerman and Kern, 1984). Both galactosemic rats and dogs have been used. Early microangiopathic lesions such as basement membrane thickening were prevented by aldose reductase inhibitors in galactosemic rats (Frank et al., 1983; Robison et al., 1983; Robison et al., 1989; Robison et al., 1990; Robison et al., 1996; Robison et al., 1997). Chronically galactosemic rats, have been claimed to develop IRMA and microaneurysms (Robison et al., 1989; Robison et al, 1996; Robison et al., 1997). Some investigators, however have failed to find unequivocal microaneurysms in galactosemic rats (Engerman and Kern, 1995). Long term galactosemic rats, similar to diabetic animals further have been demonstrated to have increased VEGF expression, which is preventable by both ARI treatment and aminoguanidine (Frank et al., 1997). Galactosemic dogs produce advanced retinal structural lesions similar to diabetic dogs (Engerman and Kern, 1984; Kador et al., 1990). Treatment with aldose reductase inhibitors to prevent galactose induced retinal lesions have shown discordant results. Although complete prevention of retinal lesions in the dog were reported by some investigators (Kador et al., 1990, 1994), others have failed to demonstrate similar results (Engerman and Kern, 1993). The reason for these divergent results is not clear. Differences in the severity of galactosemia and different ARI agents used for treatment may be possible variables. Retinal Biochemical Alterations in Animal Models Studies in animal diabetes have shown that hyperglycemia is the primary insult in the pathogenesis of diabetic retinopathy (Engerman and Kern, 1995). Several secondary mechanisms may however be activated secondary to hyperglycemia. The pathways include augmented polyol pathway activity, altered redox state, PKC activation and non-enzymatic glycation (Williamson et al., 1993; King et al., 1993). Although these mechanisms may appear isolated and diverse at the outset, there are significant interactions and they may act synergistically to produce the effects of diabetes (Figure 4). This review will briefly comment on some of the salient features. Polyol pathway Augmented polyol pathway activity is a widely studied mechanism in diabetic complications and has been well established in the target organs of diabetic complications (Frank 1991). The key enzyme aldose reductase (AR) has a high Km, for glucose. Secondary to hyperglycemia, excessive cellular glucose, particularly in tissues where glucose uptake is insulin independent, can be reduced to sorbitol by the enzyme AR. Sorbitol is subsequently oxidized to fructose by sorbitol dehydrogenase (Kador and Kinoshita, 1985). The enzyme AR is present in the dog
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Figure 4 A schematic representation of various mechanisms triggered as a result of hyperglycemia and their possible interactions.
and rat retinal microvasculature and isolated microvessels from the dog retina show hexitol producing activity (Akagi et al., 1986; Kern and Engerman, 1985; Ludvigson and Sorenson, 1986; Chakrabarti et al.,1987). In diabetes increased retinal AR mRNA and increased AR immunoreactivity was demonstrated in the BB/W rat (Ghahary et al., 1991). As discussed in the previous section, some of the diabetes induced structural changes can be prevented by ARI treatment. However, unlike the ocular lens, the amount of sorbitol in the retina, accumulated secondary to polyol pathway activation, is very low. Furthermore by inhibiting sorbitol dehydrogenase, diabetes induced vascular dysfunctions in the retina can be prevented in spite of high sorbitol levels (Tilton et al., 1995; Williamson et al., 1993). Hence, it appears that, rather than direct accumulation of sorbitol, other consequences of the polyol pathway activation, may be of importance in the pathogenesis. Both glucose and fructose, the later produced as an end product of polyol pathway, are potent nonenzymatic glycators (Brownlee, 1995; Surez, 1989). Polyol pathway activation may further lead to increased NADH/NAD+ ratio resulting in a redox imbalance in the target organs of diabetic complications (Williamson et al., 1993). Redox imbalance Increased glycolysis in the tissues in diabetes as well as an augmented polyol pathway activity cause an increase in the NADH/NAD+ ratio (Williamson et al., 1993). This alteration is similar to that seen in hypoxia and has been termed ‘pseudohypoxia’. Increased NADH/NAD+ ratio may lead to alterations in lipid peroxidation, DAG synthesis and defective DNA repair (Williamson et al., 1993; Wolf et al., 1990). Redox imbalances in the target organs of diabetic complications may favor increased free radical generation, increased prostaglandin synthesis and decreased NO synthesis. Increased DAG synthesis further activates PKC in the retina (King et al., 1993; Williamson et al., 1993; Pugliese, 1991).
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DAG-PKC pathway Increased PKC activities are seen in several target organs of diabetic complications (King et al, 1993). High glucose levels lead to an increase in DAG synthesis which is a potent PKC activator (Inoguchi et al.t 1992). An increased NADH/NAD+ ratio also favors DAG synthesis (Williamson et al., 1993). Hyperglycemia induced DAG mediated PKC synthesis has been demonstrated in the retina (Craven et al., 1989; Lee et al., 1989). Recently, it was demonstrated that hyperhexosemia induces increased DAG levels and PKC activation in the retina and aorta of both the diabetic and galactosemic dogs (Xia et al., 1994) as well as decreased Na/K ATPase and Ca/ Mg ATPase activity in these tissues (Kern et al., 1994). Augmented PKC and decreased Na/K ATPase activity in the retina of the STZ diabetic rats can be prevented by a specific inhibitor of the ȕ2 isoform of PKC (Ishii et al., 1996; Kowluru et al., 1996). PKC is involved in several important vascular functions such as blood flow and permeability (Porte and Schwartz, 1996). Diabetes induced retinal blood flow alterations can be prevented by treatment with a specific inhibitor of ȕ2-isoform of PKC (Ishii et al., 1996). Furthermore, PKC is a regulator of several growth factors, such as VEGF, PDGF, EGF and IGF, which may be of importance in mediating the later effects of diabetic retinopathy such as endothelial proliferation and neovascularisation (Aiello et al., 1997; King et al., 1993). Non-enzymatic glycation Non-enzymatic modification of tissue proteins by physiologic hexoses in vivo is an important secondary mechanism in the pathogenesis of diabetic retinopathy (Brownlee, 1995; Vlassara, 1997). Glucose and fructose, generated as an end product, of polyol pathway activity take part in non-enzymatic glycation of proteins (Brownlee, 1995; Brownlee et al., 1988; Suarez, 1989). AGEs accumulate in the tissues as a function of time and sugar concentration. AGEs alter signal transduction pathways and the levels of soluble signals such as cytokines, hormones and free radicals and can directly affect protein functions in target tissues (Brownlee, 1995). In the vascular endothelial cells, AGE formation may affect gene expression of thrombomodulin and endothelin (ET)-l (Esposito et al., 1992; Nawroth et al., 1992) and modify growth factors such as bFGFs (Brownlee, 1995). In the microvasculature AGEs may interfere with extracellular matrix organization and matrix cell interaction (Haitoglou et al., 1992). AGEs may induce permanent abnormalities of extracellular matrix proteins and intracellular proteins, stimulate cytokines and reactive oxygen species through AGE receptors (Brownlee, 1995; Brownlee et al., 1988). Diabetes induced structural changes in the target organs of diabetic complications are prevented by aminoguanidine, an inhibitor of non-enzymatic glycation (Hammes et al., 1991). The concerted effects of these biochemical alterations may further cause inhibition of fatty acid oxidation, increased prostaglandin synthesis, inhibition of NO activity and possible alteration of vasoactive peptides such as ETs and growth factors expression (Williamson et al, 1993). PKC is an important regulator of ETs and several growth factors (King et al., 1993; Rubanyi and Polokof, 1993). In the STZ diabetic rat increased retinal ET-1 mRNA expression has been demonstrated (Lin et al., 1996). Increased immunocytochemically detectable ET-1 and ET-3 proteins
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have further been noted in the retina of diabetic BB/W-rat (Chakrabarti and Sima, 1997). Alterations of several growth factors such as VEGF, bFGF, IGF-I mRNA has been demonstrated in the retina of the STZ-diabetic rat (Lowe et al., 1995; Aiello et al., 1997). A detailed review of growth factors as they relate to the development of late changes of diabetic retinopathy is however beyond the scope of this article and several excellent reviews are available (King et al., 1993; Miller et al., 1991 ; Pfiffer et al., 1997; Sharp, 1995) A schematic representation of the biochemical mechanisms, triggered by hyperglycemia, and their interactions are given in Figure 4. These complex biochemical alterations cause functional and structural changes leading to clinical diabetic retinopathy. Retinal Functional Alteration in Animal Models Both the STZ diabetic rat and the BB/W rat have been used to study functional abnormalities such as retinal blood flow, permeability alteration and retinal electrophysiological changes. In human diabetes, retinal blood flow is initially decreased followed by an increase when background retinopathy is present (King et al., 1993). Retinal blood flow studies in STZ and in the BB/W rat have resulted in discordant data as to whether blood flow is increased or decreased (Shiba et al., 1991; Tilton et al., 1992). Variability in the duration and/or severerity of diabetes, various methods used in the detection of blood flow abnormalities may be responsible for such discrepancies. It has further been demonstrated that the alteration in blood flow is heterogenous in the retina of diabetic rats (Cringle et al., 1993). Treatment with various pharmaceutical agents such as insulin, acetyl-Lcarnitine, aminoguanidine, NO synthase inhibitor, antioxidant therapy, aldose reductase inhibitor therapy, sorbitol dehydrogenase inhibitors have been found to prevent, at least in part, retinal blood flow abnormalities (Pugliese et al., 1991; Tilton et al., 1995; Tilton et al., 1993; Tilton et al., 1989; Hasan et al., 1993; Williamson et al., 1991; Kunisaki et al., 1995; Clermont et al., 1994). Recently a specific inhibitor of the ȕ2 isoform of PKC has shown to prevent retinal circulatory abnormalities in the STZ-diabetic rats in parallel with inhibition of the increased PKC activities (Ishii et al., 1996). Increased vascular permeability is a characteristic feature of human diabetic retinopathy. Hard exudate seen in background retinopathy is formed as a result of increased permeability. Such changes have been demonstrated in association with increased retinal polyol accumulation both in the STZ rat and in the BB/W-rat (Williamson et al., 1985). Treatment with an ARI was able to prevent these changes (Williamson et al., 1987). VEGF has recently been established to be an important mediator of increased retinal vascular permiability, acting via a PKC dependant mechanism (Aiello et al, 1997). Breakdown of blood retinal barrier secondary to retinal pigment epitheliopathy were demonstrated both in the STZ diabetic rats and in the BB-rat (Grimes and Laties, 1980; Blair et al., 1984; Chakrabarti et al., 1990). In the BB/W-rat such changes were associated with increased AR immunoreactivity (Chakrabarti et al., 1990) in the retinal pigment epithelium. The retina in the STZ rat shows electrophysiological alterations consisting of abnormalities of a and b-waves of the electroretinogram, generated by the neuronal retina and oscillatory potentials contributed by the Müller cells, some of which were
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prevented by ARI treatment and proionyl-L-carnitine treatment. These data suggest an early neuronal alteration in the retina similar to human diabetes (Hotta et al., 1996; Hotta et al., 1997; Sakai et al., 1995; Segawa et al., 1988). A progressive alteration of c-wave amplitude, generated by the retinal pigment epithelium, were demonstrated in the STZ diabetic rats. These changes were prevented by both ARI treatment and myo-inositol supplementation (MacGregor and Matchinsky, 1985). In the BB/W rat prolongation of the latencies of the visual evoked potentials were demonstrated. This neuronal dysfiinction was associated with dystrophic changes in retinal ganglion cells and atrophy of the myelinated fibres of optic nerve and axoglial dysjunctions (Chakrabarti et al., 1991a; Sima et al., 1992; Kajimo et al, 1993). Treatment with an ARI was effective in preventing the visual evoked potential abnormalities and axoglial dysjunctions but not the axonal atrophy (Kajimo et al., 1993). Effect of Experimental Treatment Modalities on Retinal Changes Various treatment modalities, that have been carried out in animals to prevent the retinal lesions in diabetes, fall broadly within two groups. One group of treatment is directed to correct or reduce hyperglycemia by insulin injection, islet cell transplantation or adjuvant treatment by agents such as Į glucosidase inhibitors. Another group of therapies uses inhibitors of specific biochemical pathways. The data emerging from various treatments reveal the importance of specific biochemical abnormalities in the development of diabetic retinopathy. In addition these experiments provide the first step toward the development of adjuvant therapies for human diabetic retinopathy. The effects of specific treatments were discussed under each type of lesions, Overall it is evident that by correcting hyperglycemia, structural, functional or biochemical retinal lesion can be ameliorated in both human and in animal diabetes provided the treatment is initiated early in the disease process. The major specific treatments include ARI, aminoguanidine and PKC inhibitors. Using these blockers important pathogenetic mechanisms have been elucidated and confirmed. Animal models are therefore invaluable for further exploration of efficacy and development of specific therapies to prevent and/or treat diabetic retinopathy. What Have We Learned So Far from Animal Experiments? Data from various animal models have imparted on us an enormous body of knowledge regarding the pathogenesis of diabetic retinopathy. It is now clear that hyperglycemia is the key initiating event in the genesis of diabetic retinopathy (Engerman and Kern 1995; Williamson et al., 1993; Diabetes Control and Complication Trial research group, 1993). However development of retinal lesions is probably further influenced by individual genetic make-ups and environmental factors (e.g. smoking) (Blom et al., 1994; Forrester et al., 1993). Current data indicate activation of multiple interactive mechanisms such as augmented polyol pathway activity, nonenzymatic glycations, PKC alterations in the development of retinal lesions in diabetes. This may in part explain, the failure of some clinical trials with
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single interactive agents and the success of glycemic control in the prevention of diabetic complications (Diabetes Control and Complications Trial Research Group, 1993; Sorbinil Retinopathy Trial Research Group, 1990). Biochemical alterations lead to functional changes such as retinal blood flow and permeability defects. Subsequently structural changes develop in the microvasculature and clinical retinopathy is manifested. A simplified diagrammatic outline of the sequence of events leading to clinical diabetic retinopathy is given in Figure 5. Where We Go from Here? Animal models offer a unique advantage to study a disease process in a longitudinal fashion. Using the models of diabetic retinopathy further studies are needed to pinpoint biochemical abnormalities and their interrelationships in the development of early and late functional and structural abnormalities of retinopathy and to develop adjuvant treatment strategies. A largely underexplored area in diabetic retinopathy is altered gene expression. High blood glucose levels by itself, as well as through secondary mechanisms such as augmented polyol pathway, nonenzymatic glycation, PKC activation, perturbed Ca++ metabolism and altered ecosianoid production, may affect expression of several vasoactive genes such as ETs, NO synthase and VEGF (Aiello et al., 1997; Chakrabarti et al., 1997; Williamson et al., 1993; King et al., 1993). In a recent study it was shown that hyperhexosemia induced cataract formation and thrombotic occlusion of retinochoroidal vessels develop rapidly in a transgenic mice with human AR cDNA (Yamaoka et al., 1995). Diabetes induced biochemical changes may further alter the expression of extracellular matrix protein genes (Bucala et al., 1991; Caglerio et al., 1991; Kriesberg, 1992; Chojkier et al., 1989; Nakamura et al., 1995; Roy and Lorenzi, 1996). Using mRNA differential display techniques, several genes with diverse functions have been identified in the retinal capillary pericytes which are altered by exposure to high glucose levels (Aiello et al., 1994a). Increased expression of collagen IV and fibronectin mRNA has been demonstrated in human diabetic retinopathy, in the retina of galactosemic rats as well as in the cultured retinal capillary pericytes and endothelial cells in hyperhexosemic condition (Roy et al., 1996; Roy et al., 1994; Roy and Lorenzi, 1996; Mandarino et al., 1993). Altered gene expressions may play major roles in the genesis of structural lesions such as basement membrane thickening and neovascularization or vascular dysfunctions. Altered gene expressions secondary to hyperglycemia, insulin deficiency or systemic hyperinsulinism may conceptually be an important pathogenetic mechanisms in the development of diabetic retinopathy. Exploration and manipulation of genetic expressions is a potential important area of future research. A detailed analysis of such mechanisms will further open up possibilities of gene therapy along with good blood glucose control and other adjuvant therapies in the treatment of diabetic retinopathy.
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Figure 5 An outline of putative sequence of events leading to clinical diabetic retinopathy.
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Kern, T.S., Kowluru, R.A. and Engerman, R.L. (1994) Abnormalities of retinal metabolism in diabetes or galactosemia: ATPases and glutathione. Invest. Ophthalmol. Vis. Sci., 35(7), 2962–2967. Kernal, A. and Arnqvist, H. (1983) Effect of insulin treatment on the blood retinal barrier in the rats with streptozotocin induced diabetes. Arch. Ophthalmol., 101, 968–70. King, G.L., Oliver, F.J., Inoguchi, T., Shibat and Banskota, N.K. (1993) Abnormalities of the vascular endothelins in diabetes. In Diabetes Annual, edited by S.M.Marshall, P.D.Home, K.G.M.M.Alberti and L.P.Krall, pp. 107–126. Elsevier. Klein, R., Klein, B.E.K., Moss, S.E., Davis, M.D. and Demet, D.L. (1988) Glycosylated hemoglobin predicts the incidence and progression of diabetic retinopathy. JAMA, 260, 2864–71. Kowluru, R.A., Jirousek, M.R., Engerman, R.L. and Kern, T.S. (1996) In the STZ rat PKC and Na/K ATPase alteration was prevented by specific B-II PKC inhibitor. Diabetes, 45(Suppl. 2), 16A. Kreisberg, J.I. (1992) Biology of disease: hyperglycemia and microangiopathy. Direct regulation by glucose of microvascular cells. Lab. Invest., 67, 416–426. Krupin, T., Waltman, S.R., Scharp, D.W., Oestrich, C, Fieldman, S.L., Becker, B., Ballinger, W.F. and Lacy, P.E. (1979) Ocular fluorophotometry in streptozotocin diabetes meliitus in the rat: Effect of pancreatic islet isograft. Invest. Ophthalmol. Vis. Sci., 18, 1185–90. Kunisaki, M., Bursell, S.E., Clermont, A.C., Ishii, H., Ballas, L.M., Kirousek, M.R., Umeda, F., Nawata, H. and King, G.L. (1995) Vitamin E presents diabetes-induced abnormal retinal blood flow via the diacylglycerol-protein kinase C pathway. Am. J. Physiol., 269(2 Pt 1), E239–246. Laver, N., Robison, W.G. Jr. and Hansen, B.C. (1994) Spontaneously diabetic monkeys as a model for diabetic retinopathy. ARVO Abstract Invest. Ophthalmol. Vis. Sci., 35 (Suppl. 1), 1733. Lee, T.-S., Saltsman, K.A., Ohashi, H. and King, G.L. (1989) Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism for the development of diabetic vascular complication. Porc. Natl. Acad. Sci. USA, 86, 5141–45. Lin, U.W., Duh, E. and Jian, Z. (1996) ET-1 expression have been demonstrated in the retina and heart of STZ diabetic rats. Diabetes, 45(SuppL), 48A. Lowe, W.L., Jr., Florkiewicz, R.Z., Yorek, M.A., Spanheimer, R.G. and Albrecht, B.N. (1995) Regulation of growth factor mRNA levels in the eyes of diabetic rats. Metabolism, 44,1038. Ludvigson, M.A. and Sorenson, R.L. (1980) Immunohistochemical localization of aldose reductase. II. Rat eye and kidney. Diabetes, 29, 450–59. MacGregor, L.C. and Matchinsky, F.M. (1985) Treatment with an aldose reductase inhibitor or with myo-inositol arrests detorioretion of electroretinogram of diabetic rats. J. Clin. Invest., 76, 887–89. Mandarino, L.J., Sundarraj, N., Finlayson, J. and Hassell, J.R. (1993) Regulation of fibronectin and liminin synthesis by retinal capillary endothelial cells pericytes in vitro. Exp. Eye. Res., 57, 609–21. Miller, J.W., Adamis, A.P. and Aiello, L.P. (1997) Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy. Diabetes Metab. Rev., 13(1), 37– 50.
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Musacchio, I., Palermo, N. and Rodriguez, R. (1964) Microaneurysms in the retina of diabetic rats. Lancet, 1, 146. Mazze, R.S., Sinnock, P., Deeb, L. and Brimberry, J.L. (1985) An epidemiological model for diabetes mellitus in the United States: five major complications. Diab. Res. Clin. Prac., 1, 185–91. Nakamura, T., Ebihara, I., Fukui, M., Tomino, Y. and Koida, H. (1995) Effect of a specific endothelin-A receptor antagonist on mRNA levels for extracellular matrix components and growth factors in diabetic glomeruli. Diabetes, 44, 895–99. Nawroth, P.P., Stern, D. and Bierhaus, A. (1992) AGE-Albumin stimulierte Endothelzellenein in-Vitro Modell diabetischer Spätschäden. Diabetes Stoffweechs, 1 (Suppl. 1), 153A. Orloff, M., Lee, S., Charters, A., Granbort, D.E., Storock, G. and Knox, D. (1975) Long term studies of pancreas transplantation in experimental diabetes mellitus. Ann. Surg., 182, 198. Osterman, H. and Vande Loo, J. (1986) Factors of the hemostatic system of the diabetic patients. A survey of controlled studies. Hemostasis, 16, 386–416. Pfeiffer, A., Spranger, J., Meyer-Schwickerath, R. and Schatz, H. (1997) Growth factor altera-tions in advanced diabetic retinopathy: A possible role of blood retina barrier breakdown. Diabetes, 46(Suppl. 2), S26-S30. Porta, M. (1996) Endothelium: the main actor in the remodelling of the retinal microvasculature in diabetes. Diabetologia, 39, 739–44. Porte, D. Jr. and Schwartz, M.W. (1996) Diabetes complications: why is glucose potentially toxic? Science, 272, 699–700. Pugliese, G., Tilton, R.G. and Williamson, J.R. (1991) Glucose-induced metabolic imbalances in the pathogenesis of diabetic vascular disease. Diabetes Metab. Rev., 7, 35– 59. Report on the Second National Diabetes Research Conference: Progress and promise in diabetes research. Eye complications (1984) Public Health Service, NIH Publication No. 84–661, pp. 21–86. Robison, W.G. Jr., Kador, P.F. and Kinoshita, J.H. (1983) Retinal capillaries: basement membrane thickening by galactosemia prevented with aldose reductase inhibitor. Science, 221, 1177–1179. Robison, W.G., Laver, N.M., Jacot, J.L., Chandler, M.L., York, B.M. and Glover, J.P. (1997) Efficacy of treatment after measurable diabetic like retinopathy in galactose-fed rats. Invest. Ophthalmol. Vis. Sci., 38(6), 1066–1073. Robison, W.G., Laver, N.M., Jacot, J.L., Glover, J.P., Basso, M.D., Blouin, P. and Hohman, T.C. (1996) Diabetic-like retinopathy ameliorated with the aldose reductase inhibitor WAY-121, 509. Invest. Ophthalmol. Vis. Sci., 37(6), 1149–1156. Robison, W.G. Jr., Nagata, M., Laver, N., Hohman, T.C. and Kinoshita, J.H. (1989) Diabetic like retinopathy in rats prevented with an aldose reductase inhibitor. Invest. Ophthalmol. Vis. Sci., 30, 2285–2292. Robinson, G.W. Jr., Tillis, T.N., Laver, N. and Kinoshita, J.H. (1990) Diabetes related histopathologies of rat retina prevented with an aldose reductase inhibitor. Exp. Eye Res,, 50, 355–366. Roy, S. and Lorenzi, M. (1996) Early biosynthetic changes in the diabetic like retinopathy of galactose fed rats. Diabetologia, 39, 735–738.
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Roy, S., Maiello, M. and Lorenzi, M. (1994) Increased expression of basement membrane collagen in human diabetic retinopathy. J. Clin. Invest., 93, 438–42. Roy, S., Cagliero, S. and Lorenzi, M. (1996) Fibronectin overexpression in retinal microvessels of diabetic patients. Invest. Ophthalmol. Vis. Sci., 37, 258–266. Rubanyi, G.M. and Polokoff, M.A. (1994) Endothelins: molecular biology, biochemistry, pharmacology, physiology and pathophysiology. Pharmacological Reviews, 46, 325–414. Sakai, H., Tani, Y, Shirasawa, E., Shirao, Y. and Kawasaki, K. (1995) Development of electroretinographic alterations in streptozotocin-induced diabetes in rats. Ophtbalmic Res., 7, 57–63. Santiago, J.V. (1993) Lessons from the diabetes control and complication trial. Diabetes, 42, 1549–54. Segawa, M., Hirata, Y, Fujimori, S. and Kada, K. (1988) The development of electroretinogram abnormalities and possible roles of polyol pathway activity in diabetic hyperglycemia and galactosemia. Metabolism, 37, 454–59. Sharma, N.K., Gardiner, T.A. and Archer, D.B. (1985) A morphological and autoradiographic study of cell death and regeneration in the retinal microvasculature of normal and diabetic rats. Am. J. Ophthalmol., 100, 51. Sharp, P.S. (1995) The role of growth factors in the development of diabetic retinopathy. Metabolism, 44(Suppl. 4), 72–75. Shiba, T., Bursell, S.E., Clermont, A., Sportsman, R., Heath, W. and King, G.L. (1991) Protein kinase C (PKC) activation is a causal factor for the alteration of retinal blood flow in diabetes of short duration. Invest. Ophthalmol, Vis. Sci. (suppl.), 32, 785. Sima, A.A.F., Chakrabarti, S., Garcia-Salinas, R. and Basu, P.K. (1985) The BB rat—an authentic model of human diabetic retinopathy. Current Eye Res., 4, 1087–92. Sima, A.A.F., Chakrabarti, S., Tze, W.J. and Tai, J. (1988) Pancreatic islet allograft prevents basement membrane thickening in diabetic retina. Diabetologia, 31, 175–181. Sima, A.A.F., Zhang, W.-X., Cherian, P.V. and Chakrabarti, S. (1992) Impaired visual evoked potential and primary axonopathy of the optic nerve in the diabetic BB/W rat. Diabetologia, 35, 602–07. Sorbinil Retinopathy Trial Research Group (1990) A randomized trial of sorbinil, an aldose reductase inhibitor, in diabetic retinopathy. Arch. Ophthalmol, 108, 1229–31. Sousula, L., Beaumont, P., Hollows, F.C. and Jonson, K.M. (1972) Dilatation and endothelial cell proliferation of retinal capillaries in streptozotocin diabetic rats: Quantitative electron microscopy. Invest. Ophthalmol., 11, 926–35. Stitt, A.W., Li, Y.M., Gardiner, T.A. and Vlassara, H. (1996) A progressive intracellular AGE accumulation in association with pericyte loss has recently been demonstrated in STZ rats. Diabetes, 45(Suppl. 2), 15A. Studer, P.P., Muller, W.A. and Reynold, A.E. (1976) Alteration of retinal capillaries by long term streptozotocin diabetes. In Current Topics in Diabetes Research. Int. Diab. Fed. Abst. Amsterdam: Excerpta Medica 125. Suarez, G. (1989) Non enzymatic browning of proteons and the sorbitol pathway. Prog. Clin. Biol. Res., 304, 141–62. Tilton, R.G., Chang, K., Pugliese, G., Eades, D.M., Province, M.A., Sherman, W.R., Kilo, C. and Williamson, J.R. (1989) Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors. Diabetes, 38, 1258–1270.
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Tilton, R.G., Chang, K., Allison, W. and Williamson, J.R. (1992) Comparable diabetes induced increases in retinal blood flow assessed with conventional versus molecular (3HDesmethylimiprimine) microspheres. Invest. Ophthalmol. Vis. Sci. (suppl.), 33, 1048. Tilton, R.G., Chang, K., Hasan, K.S., Smith, S.R., Petrash, J.M., Misko, T.P., Moore, W.M., Currie, M.G., Corbett, J.A., McDaniel, M.L. and Williamson, J.R. (1993) Prevention of diabetic vascular dysfunction by guanidine. Inhibition of nitric oxide synthase versus advanced glycation end-product formation. Diabetes, 42(2), 221–232. Tilton, R.G., Chang, K., Nyengaard, J.R., Van den Enden, M., Ido, Y. and Williamson, J.R. (1995) Inhibition of sorbitoi dehydrogenase. Effects on vascular and neural dysfunction in streptozocin-induced diabetic rats. Diabetes, 44, 234–242. Toussaint, D. (1966) Lesions retiniennes au cours de diabete alloxanique chez le rat. Bull. Soc. Belge Ophthalmol., 143, 648. Vlassara, H. (1997) Recent progress in advanced glycation end products and diabetic complications. Diabetes, 46(Suppl. 2), S19-S25. Williamson, J.R., Chang, K., Rowold, E., Marvel, J., Tomlison, M., Sherman, W.R., Tilton, R.G. and Kilo, C. (1985) Sorbinil prevents diabetes induced increased vascular permeability but does not alter collagen cross linking. Diabetes, 34, 1460–67. Williamson, J.R., Chang, K., Tilton, R.G., Prater, C, Jeffrey, J.R., Weigel, C, Sherman, W.R., Eades, D.M. and Kilo, C. (1987) Increased vascular permeability in spontaneously diabetic BB/W rats and in rats with mild versus severe streptozotocin induced diabetes: prevention by aldose reductase inhibition and castration. Diabetes, 36, 813–21. Williamson, J.R., Chang, K., Allison, W.S., Tilton, R.G., Orfalian, Z. and ArrigoniMartelli, E. (1991) Acetyl-L-carnitine, but not L-carnitine attenuates diabetes-induced increases in vascular 131I-BSA clearance. Invest. Ophthalmol. Vis. Sci., 32, 1029. Williamson, J.R., Chang, K., Frangos, M., Hasan, K.S., Ido, Y, Kawamura, T, Nyengaard, J.R., Van Den Enden, M., Kilo, C. and Rilton, R.G. (1993) Perspectives in Diabetes. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes, 42, 801–813. Wolf, B.A, Williamson, J.R., Easom, R.A., Chang, K., Sherman, W.R. and Turk, J. (1990) Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels. J. Clin. Invest., 87, 31–38. Xia, R, Inoguchi, T., Kern, T.S., Engerman, R.I., Oates, P.J. and King, G.L. (1994) Characterization of the mechanism for the chronic activation of DAG-PKC pathway in diabetes and hypergalactosemia. Diabetes, 43, 1122–1129. Yamaoka, T., Nishimura, C., Yamashita, K., Itakura, M., Yamada, T., Fujimoto, J. and Kokai, Y (1995) Acute onset of diabetic pathological changes in transgenic mice with human aldose reductase cDNA. Diabetologia, 38, 255–61. Yokote, M. (1973) Retinal and renal microangiopathy in carp with spontaneous diabetes mellitus. Adv. Metabol. Disorder, (Suppl. 2), 399–404.
10. FETAL MALFORMATIONS IN DIABETES ULF J.ERIKSSON Department of Medical Cell Biology, Uppsala Universtty, Biomedical Center, P.O. Box 571, S-751 23 Uppsala, Sweden
Maternal diabetes during pregnancy is associated with a high risk of fetal maldevelopment (Pedersen, 1977; Freinkel, 1980; Mills, 1982). There is an increased incidence of congenital malformations and growth disturbances in the offspring of the diabetic mother (Becerra et al., 1990; Hanson et al., 1990). The exact etiology of both of these types of developmental alterations is currently unclear. During recent years, however, diabetic embryopathy has been suggested to share etiologic features with several different complications of diabetes. In particular, the pathogenesis of retinopathy, neuropathy, and nephropathy has been implicated to possess similarities with the teratogenic process of diabetic pregnancy. This is due to the putative involvement of sorbitol accumulation, inositol depletion, and excess radical oxygen species (ROS) in the diabetes-induced organ complications (Greene et al., 1987; Baynes, 1991; Bravenboer et al., 1992; Cameron and Cotter, 1993; Wolff, 1993; Kilic et al., 1994; Vinson et al., 1994), as well as in embryonic dysmorphogenesis (Baker and Piddington, 1993; Eriksson et al., 1995). It has been pointed out that the intrauterine exposure of the embryonic tissue to the maternal diabetic blood is similar to the exposure of the retina, neural tissue and kidneys to the blood of the diabetic individual. Both the embryonic and adult tissues are hence challenged in a similar way by the diabetic environment with perturbed levels of nutrients (glucose, lipids, ketone bodies, and branched chain amino acids) as well as altered endocrine and paracrine agents. There is one apparent difference, however, with regard to the time required for the lesions to appear. The time span for the induction of the embryonic damage in diabetic pregnancy appears to be remarkably short in comparison with the considerable time needed for the introduction of the organ complications of diabetic individuals. In particular, the pivotal teratological insult of several of the major congenital malformations takes place in the first six weeks of human pregnancy (Mills et al., 1979), and has been identified to occur on gestational days 6–10 in rodent pregnancy (Eriksson et al., 1989ab).
Correspondence to: Associate Professor Ulf J. Eriksson, Department of Medical Cell Biology, Uppsala University, Biomedical Center, P.O. Box 571, SE-751 23 Uppsala, Sweden. Telephone: +46 18 471 41 29, Faximile: +46 18 55 07 20, E-mail:
[email protected]
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It is the aim of this review to try to identify teratologically relevant metabolic changes in the embryo caused by the maternal diabetes in early pregnancy (section on Metabolic changes in embryos). Also, alterations secondary to the exposure of the embryo to the diabetic milieu—“ teratologic mechanisms”— will be reviewed (section on Teratological processes and synthesis of teratological knowledge), and possible modes of preventive treatment will also be considered (section on Modes of intervention). METABOLIC CHANGES IN EMBRYOS Hyperglycemia One primary consequence of maternal diabetes is embryonic hyperglycemia, both extracellular and intracellular (Sussman and Matschinsky, 1988; Eriksson and Fredén, 1988). The uptake of glucose by the embryo is dependent on the ambient glucose level, Sussman and Matschinsky showed that the glucose concentration in the offspring closely mirrors the maternal glucose level in day-10 and day-11 embryos of normal and diabetic rats, suggesting no upper limit for glucose uptake by the embryonic tissue (Sussman and Matschinsky, 1988). Furthermore, the GLUT-1 protein, identified as the major constitutive glucose transporter in embryos, is equally abundant in embryos exposed to hyperglycemia as in control embryos, indicating no down-regulation of the transport capability in an environment with high glucose concentration (Takao et al., 1993; Trocino et al., 1994). The hyperglycemic state has been associated with increased risk for embryo-fetal dysmorphogenesis (Karlsson and Kjellmer, 1972), as evidenced by the increased incidence of malformations in the offspring of mothers with elevated HbAlc levels in early human pregnancy (Leslie et al., 1978; Jovanovic et al., 1980; Miller et al., 1981; Persson et al., 1982; Ylinen et al., 1984; Olofsson et al., 1984; Mø1sted Pedersen and Kühl, 1986; Greene et al, 1989), and similar findings in rodent diabetic pregnancy (Baker et al., 1981; Eriksson et al., 1982; Giavini et al., 1986; Eriksson et al., 1989ab; Styrud et al., 1995). In whole embryo culture, increased glucose concentration is teratogenic to the early post-implantation embryo (Cockroft, 1977; Sadler, 1980b; Garnham et al., 1983), and causes high-amplitude mitochondrial swelling (Yang et al., 1995), increased activity of superoxide dismutase (SOD) (Eriksson and Borg, 1991), increased expression of MnSOD (Forsberg et al., 1996) and ECM proteins (Cagliero et al., 1993), decreased activity of embryonic catalase (Cederberg and Eriksson, 1997), decreased activity and expression of the ratelimiting enzyme in the synthesis of glutathione, Ȗ-GCS (Trocino et al., 1995), as well as increased rate of DNA mutations (Lee et al., 1997). Increased glucose concentration also inhibits the development of embryonic neural crest cells in vitro (Suzuki et al., 1996) whereas more developed neural crest cells, the mandibular (pre) chondrocytes, are only marginally affected in their chondrogenic development in vitro, unless they are derived from offspring of diabetic rats (Styrud and Eriksson, 1990). In addition, it has been shown that an increased ambient concentration of glucose (Zusman et al., 1985; Diamond et al., 1990; De Hertogh et al., 1991) during
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Figure 1 (a) An embryo cultured in 10 mmol/1 glucose with normal appearance with closed neural tube. (b) An embryo cultured in 30 mmol/1 glucose for 24 hours, showing multiple malformations, including malrotation, and growth retardation.
in vitro culture of preimplantation rodent embryos causes growth retardation. The growth and development of the embryoblast (the inner cell mass) of the rodent conceptus appears to be especially sensitive to the hyperglycemic environment (De Hertogh et al., 1991). It appears, therefore, that cxcess glucose has a direct teratogenic effect in embryos. This effect is conveyed by alterations in various cellular processes, and depends on the type of tissue and the state of embryogenesis. We will now examine metabolic consequences of increased extra—and intracellular glucose concentrations in the embryos, consequences with a teratological significance (Figure 1). Of interest, however, in this context is the evidence pointing to the possible existence of other teratogens than excess glucose in diabetic pregnancy—the clinical Diabetes In Early Pregnancy multicenter study which failed to demonstrate a clear association between malformation rate and maternal glucose levels in early human pregnancy (Mills et al., 1988), the experimental finding of other teratogens associated with congenital malformations in diabetic rat pregnancy than high maternal glucose levels (Styrud et al., 1995). The repeated demonstration in vitro of the dysmorphogenic effect of diabetic serum (Deuchar, 1977; Sadler, 1980a; Rashbass et al., 1988; Styrud and Eriksson, 1992; Menegola et al., 1995), a teratological influence that remains despite normalized glucose levels (Buchanan et al., 1994; Wentzel and Eriksson, 1996), also support the existence of other teratogens than increased glucose levels.
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Increased Glucose Transport—Decreased Inositol Concentration The primary effect of exposure to high concentration of extracellular glucose is increased transport of this hexose into the embryonic cells. Increased influx of glucose yields a diminished cellular uptake of inositol in embryos, as noted by Weigensberg and collaborators (1990), which may be a consequence of competition for the transport mechanism between glucose and inositol. Decreased uptake of inositol in the face of ambient hyperglycemia is not restricted to embryonic cells, but has been demonstrated in several in vivo and in vitro systems, also in cells from adult animals. Furthermore, the fact that decreased inositol concentration per se is teratogenic has been demonstrated in rat embryo culture in vitro, where administration of the metabolic competitor scyllo-inositol elicits embryonic dysmorphogenesis, and re-supplementation of inositol to the compromised embryos normalizes embryonic development (Strieleman et al., 1992, 1993). The mechanism by which decreased cellular concentration of inositol causes developmental disturbance in the embryo is not clear. Evidently, decreased amount of inositol available for the synthesis of phosphatidylinositol could induce decreased production, and possibly decreased quantity, of this phospholipid. This would, in turn, decrease the concentration of the phosphoinositides (IP3, IP2, IP), which are used in the intracellular signal transduction pathways, and thereby extensively affect embryonic developmental processes dependent upon this signal system. One particular enzyme whose activity may be decreased as a result of a diminished stimulation by phosphatidylinositol is the key enzyme in the metabolism of triglycerides and phospholipids, phospholipase A2. A decrease of phospholipase A2 activity would then diminish the production of free arachidonic acid, the fatty acid most often occupying the middle position of the glycerol backbone of these lipids, and have profound effects on the metabolism of prostaglandins and leukotrienes (cf. B2). The levels of phosphatidylinositol in day-11 embryos were not found to be afFected by maternal diabetes (Simán and Eriksson, 1997c), which may indicate that the changes described above only pertain to a selected cell population which is too small to influence the mean value of the whole embryo. Alternatively, this finding may indicate that the flux in the inositol pathway is decreased, although the steady state concentrations are not. Increased Flux in the Polyol Pathway The increased intracellular concentration of unphosphorylated glucose yields increased glycolytic flux, but also exceeds the (high) Km of the aldose reductase enzyme and thereby activates the polyol pathway with subsequent accumulation of sorbitol and fructose (Eriksson et al., 1986; Hod et al., 1986; Sussman and Matschinsky, 1988; Eriksson et al., 1989c; Hashimoto et al., 1990). No teratogenic effect has been directly associated with the polyol pathway. Thus, diminishing the flux of glucose in the shunt by administering aldose reductase inhibitors in vivo or in vitro does not alter the incidence of fetal malformations or glucose-induced embryopathy, despite significant lowering of the sorbitol concentration in the offspring (Eriksson et al., 1986; Hod et al., 1986).
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A consequence of increased flux in the polyol pathway is consumption of NADPH and production of NADH, the second change affects the redox potential, whereas the first change is likely to diminish the cellular antioxidant status, since reduction of glutathione from its oxidized state (GSSG) to the reduced state (GSH) requires NADPH. Thus, increased polyol pathway activity increases sorbitol and fructose levels and may diminish GSH concentration in the cell. On the other hand, in no experimental system has it been proven that sorbitol accumulation per se is associated with congenital malformations, therefore these changes may be viewed as predisposing, rather than directly inducing, the developmental disturbances in the embryogenesis. Increased Glycolytic Flux Yields a Crabtree Effect Another consequence of hyperglycemia is increased glycolytic flux, a state which in embryos leads to a Crabtree effect, i.e. a redistribution of ADP and phosphate from the mitochondria to the phosphorylating activity of the glycolysis in the cytosol. As a consequence of this redistribution, there is a relative lack of substrates for the oxidative phosphorylation in the mitochondria where all the components of the electron transport chain are fully reduced and the ATP production and oxygen consumption are decreased. This is a State 4 respiration stage of the mitochondria, a state which enables an increased leakage of reactive oxygen species (ROS) from the reduced electron transport chain, (Yang et al., 1995, 1997a), a leakage which may be of significance since the endogenous levels of ROS scavenging enzymes are low in embryos (El-Hage and Singh, 1990). Indeed, in isolated neuroepithelial tissue from rat embryos Yang and coworkers were able to simultaneously demonstrate a Crabtree effect and secure evidence for a production of superoxide, thereby illustrating the teratogenic potential of this pathway (Yang et al., 1997a). Hyperketonemia Increased ambient ketone body concentration is also a recognized teratogen in vitro (Horton and Sadler, 1983; Lewis et al.t 1983; Horton et al., 1985; Sheenan et al., 1985; Moore et al., 1989) and in vivo (Styrud et al., 1995). The disturbance can be blocked by SOD addition in vitro (Eriksson and Borg, 1993), suggesting that the effect of hyperketonemia is partly mediated via ROS excess (Yang et al., 1997b; Forsberg et al., 1998a). In support of this notion is the finding of swollenmito chondria in the neuroepithelium of high ketone body exposed embryos (Horton and Sadler, 1985; Yang et al., 1995), of similar appearance as those mitochondrial changes found in high glucose exposed embryos (Yang et al,. 1995). Another important finding is the stage dependence of the teratogenic effect of ȕ-hydroxybutyrate excess—younger embryos are markedly more susceptible than older (Hunter et al., 1987; Moore et al., 1989; Shum and Sadler, 1990). In a previous study Hunter and collaborators found a decreased flux in the pentose phosphate shunt as a teratologically important effect of the increased ketone body concentration (Hunter et al., 1987). This would result in lowered production of NADPH and ribose moieties, thus at the same time altering the redox potential, GSH production, and nucleotide synthesis of the embryonic cells. Indeed,
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supplementation of ribose to the culture medium diminished the embryonic dysmorphogenesis caused by high ȕ-hydroxybutyrate concentration in older (Hunter et al., 1987), but not in younger (Shum and Sadler, 1990) embryos. The possibility of a double effect of the ketone body, both affecting the oxidative status and the ribose production, seems to be a valid hypothesis in view of the observations, and a combined treatment with ribose and an antioxidant may prove, or disprove, this point. There is also some evidence for a direct teratogenic effect of the ketone body itself (Yang et al., 1997b), but how those observations should be combined with a relative or absolute ROS excess is not clear at present. Increased Concentration of Branched Amino Acids Branched chain amino acids also have a teratological role in diabetic pregnancy. This is suggested by findings of in vitro teratogenicity of Į-ketoisocaproate, a metabolite of leucine (Eriksson and Borg, 1993), and an in vivo association of increased branched chain amino acid concentration with disturbed embryonic morphogenesis (Styrud et al., 1995). Addition of Į-ketoisocaproate to the embryo culture medium also causes mitochondrial swelling of a type similar to that in embryos exposed to high glucose and ketone body concentration (Yang et al., 1995). Furthermore, the in vitro teratogenicity can be diminished by SOD addition to the culture medium, implicating ROS excess involvement in the process (Eriksson and Borg, 1993). TERATOLOGICAL PROCESSES Excess Radical Oxygen Species (ROS) The first direct evidence in favour of an involvement of oxidative processes in the teratogenicity of diabetic pregnancy was published in 1991 (Eriksson and Borg, 1991). We could show that addition of the scavenging enzymes SOD, catalase or glutathione peroxidase to the culture medium protected rat embryos from dysmorphogenesis elicited by high glucose concentration in vitro (Eriksson and Borg, 1991). We found in subsequent work that teratological concentrations of ȕhydroxybutyrate or branched chain amino acids can be blocked by addition of SOD to the culture medium (Eriksson and Borg, 1993). Furthermore, serum from rats given high doses of streptozotocin is teratogenic in vitro, an effect that can be diminished by addition of SOD and N-acetylcysteine (NAC) to the culture medium (Wentzel et al., 1997). In a study of the early development of cranial neural crest cells, it was shown that high glucose inhibited, and NAC normalized, the migration and proliferation of these cells, but that somite cells of non-neural crest origin were not affected by either treatment (Suzuki et al., 1996). This finding supports the notion of varied susceptibility among different embryonic cell populations to the developmental effects of high ambient glucose concentration, as well as a role for ROS excess in the execution of these effects. Further support is offered by the demonstration that exposure of embryos for ROS excess (in the absence of high glucose concentration) causes embryonic dysmorphogenesis in vitro (Jenkinson et al., 1986).
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There is also in vivo evidence of an involvement of ROS. Thus, treatment with several different ROS scavengers has been successful in blocking the teratogenic effect of a maternal diabetic state (cf. Dl). And, in addition, a decreased capacity of embryos to scavenge radicals is associated with maternal diabetes, yielding lowered Į-tocopherol (vitamin E) concentration in day-11 embryos and in the liver of day-20 fetuses (Simán and Eriksson, 1997a), as well as lowered catalase activity in embryos of a malformation-prone rat strain (Cederberg and Eriksson, 1997). From the previous reasoning it follows that several lines of evidence point to a state of embryonic ROS excess as a pivotal process in the induction of embryonic dysmorphogenesis in diabetic pregnancy. The nature of this excess is slightly enigmatic, however. While it is clear that antioxidative treatment diminishes teratogenesis of a diabetes-like environment both in vivo and in vitro (cf. D), direct measurements of ROS in early embryonic cells exposed to a diabetes-like milieu have yielded conflicting results (Trocino et al., 1995; Forsberg et al., 1998a). This may indicate that the teratologically important process is not increased ROS production per se but rather decreased ROS antioxidative defence capacity. This notion is supported by the demonstrated decreased activity of Ȗ-GCS, the rate limiting enzyme in the production of glutathione, GSH, in embryos subjected to high glucose concentration in vitro (Trocino et al., 1995). Decreased GSH levels have also been reported, both in the high-glucose exposed embryos (Trocino et al., 1995), and in day 11 embryos of diabetic rats (Menegola et al., 1996), adding further support to the idea of decreased antioxidative defence capacity as an integral element in the diabetes-induced dysmorphogenesis. In addition, the ROS excess and/or diminished antioxidative capacity may only be present in a particular cell population in the conceptus—and possibly only during a limited gestational period, thereby making the identification of this change difficult. One cell population of specific interest in this context are the neuroepithelial cells, the cells that consistently display mitochondrial swelling in a diabetic milieu (Yang et al., 1995). In particular, the neural crest cells are of interest (Davis et al., 1990), since they have demonstrated ROS scavenger restrainable developmental disturbances in a diabetes-like environment in vivo (Simán et al., 1997) and in vitro (Suzuki et al., 1996). In summary, however, despite difficulties in demonstrating ROS in embryos acutely exposed to high glucose, the combined data yields the notion that the teratological process in diabetic pregnancy does involve excess oxygen radicals at some, relatively late, stage. Altered Metabolism of Arachidonic Acid and Prostaglandins Disturbed metabolism of arachidonic acid and prostaglandins has been found in previous studies of experimental diabetic pregnancy. Thus, Goldman and associates reported that addition of arachidonic acid to the culture medium blocked the embryonic dysmorphogenesis elicited by high glucose concentration (Goldman et al., 1985), a finding which has been repeated (Pinter et al., 1986), and expanded (Wentzel and Eriksson, 1998) in subsequent studies. Furthermore, intraperitoneal injections of arachidonic acid to pregnant diabetic rats diminished the rate of neural tube damage (Goldman et al., 1985), thereby indicating a disturbance of the
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arachidonic acid cascade as a consequence of a diabetic environment. Addition of prostaglandin E2 (PGE2) to the culture medium also blocks glucose-induced teratogenicity in vitro (Baker et al., 1990; Wentzel and Eriksson, 1998), as well as maldevelopment of embryos cultured in diabetic serum (Goto et al., 1992). Measurements of PGE2 have indicated that this prostaglandin is decreased in embryos of diabetic mice during the period of neural tube closure (Piddington et al., 1996) as well as in the yolk sac of embryos of diabetic women (Schoenfeld et al., 1995). In a recent study, culture of rat embryos with inhibitors of cyclooxygenase, COX, displayed embryonic dysmorphogenesis of similar type as that caused by high glucose culture, a maldevelopment that could be blocked with arachidonic acid and PGE2 supplementation (Wentzel and Eriksson, 1998). These effects may indicate that a restriction of the principal substrate for the prostaglandin biosynthesis, arachidonic acid, is exerted by a diabetes-like environment, and that this restriction causes embryonic maldevelopment via diminished PGE2 production. This may implicate an inhibition of the rate-limiting enzyme, phospholipase A2, or a decreased uptake or an inhibition of earlier biosynthetic steps in the production of this polyunsaturated fatty acid. Our previous studies have shown, however, that the uptake of arachidonic acid by embryonic yolk sacs is increased in a hyperglycaemic environment (Engström et al., 1991). This finding would preclude an outright deficiency of arachidonic acid in the conceptus of diabetic pregnancy, a result supported by the demonstration of similar concentration of arachidonic acid in high glucose cultured embryos in vitro (Pinter et al., 1988). The changes in embryogenesis demonstrated in the present study do not seem to depend on a decreased availability of arachidonic acid, but there are, evidently, a number of studies suggesting a role for a disturbed arachidonic acid-prostaglandin metabolism in embryos exposed to a diabetic environment, although the exact metabolic localisation of the disturbance has not been determined. A second major finding in the study was the diminishing effect exerted by SOD and NAC on the COX inhibitor-induced dysmorphogenesis, analogous to the effect of the antioxidants on glucose-induced embryonic maldevelopment (Wentzel et al., 1997). This result, together with the finding of diminished glucose-induced embryopathy by addition of arachidonic acid and PGE2, suggest a cross-talk between teratogenic effects caused by a decreased prostaglandin synthesis and ROS excess in embryos subjected to a diabetic environment. Production of Reactive Oxo-Aldehydes It was recently shown that embryos cultured in high glucose accumulate the oxoaldehyde, 3-deoxyglucosone (3-DG), a reactive glycating agent, and a precursor of advanced glycosylated endproducts (AGE), (Eriksson et al., 1997a). Rat embryos exposed to high glucose levels in vitro showed severe dysmorphogenesis and increased concentration of 3-DG compared to low glucose cultured controls. Exogenous 3DG added to the medium of low glucose culture yielded increased embryonic malformation rate and 3-DG concentration similar to those of high glucose cultured embryos. Addition of superoxide disinutase (SOD) to the culture medium decreased the malformation rates of embryos exposed to either high glucose or high 3-DG levels, but did not decrease the high embryonic 3-DG concentrations
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Figure 2 A tentative scheme for the embryonic effects of a diabetic environment.
caused by either agent. Our results implicate the potent glycating agent 3-DG as a teratogenic factor in diabetic embryopathy. In addition, the anti-teratogenic effect of SOD administration appears to occur downstream of 3-DG formation, suggesting that 3-DG accumulation leads to superoxide-mediated embryopathy. Induction of DNA Damage Lee and collaborators transferred mouse embryos with a DNA mutation reporter transgene to normal and diabetic pseudo pregnant recipients, and let them develop in utero until day 15–16 of pregnancy. The embryos were harvested, and the subsequent analysis showed that exposure to a diabetic environment yields increased rate of nuclear DNA damage (Lee et al., 1995). In a recent series, Lee and collaborators have expanded the initial observations to study the DNA mutation rate in high glucose cultured mouse embryos, and in day-11 embryos of diabetic rats, both of which are carrying the reporter transgene (Lee et al., 1997). The result showed a clear increase of nuclear DNA damage in both types of embryos, most pronounced in the embryos of the diabetic rats. It is clear that both high glucose exposure in vitro and a diabetic environment in vivo cause an increased rate of DNA mutations in embryos. The forces effecting this damage are not known at present, but the possibility of excess ROS and/or increased glycation activity (3-DG excess) heralding an attack on the DNA is both likely and attractive. Alterations in Gene Expression A number of genes have shown to be altered in embryos subjected to high glucose in vitro and diabetes in vivo, although a clearcut pattern is not visible as yet. It was previously shown that the diabetes-like condition induced increased expression of
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the ECM genes Bl-laminin and fibronectin (Cagliero et al., 1993). These genes are also altered in placentae of diabetic rats (Forsberg et al., 1998b). Also, the MnSOD gene is overexpressed in embryos exposed to a diabetes-like environment (Forsberg et al., 1996). In a recent study it was shown that the developmentally crucial gene Pax-3 shows reduced expression in the offspring of diabetic mice (Phelan et al., 1997). SYNTHESIS OF TERATOLOGICAL KNOWLEDGE There is a multitude of changes occurring as consequence of embryonic exposure to a diabetes-like environment (Figure 2). From the previous discussion it is apparent that there must be several metabolic pathways that share the teratological responsibility, and that they converge toward ROS excess, a feature distal in the teratological process. Whether the postulated ROS excess is a general feature, or only present in specific cell populations is not known. Likewise, it is not clear if this condition exists during a restricted time window during embryogenesis, and, lastly, the nature of the ROS excess is not known—it could be a lack of antioxidant capacity, rather than an outright increase in ROS production. MODES OF INTERVENTION In vitro Treatment The first evidence of a protective effect of anti-oxidative treatment was found in early 90s when addition of SOD, citiolone (SOD inducer), catalase, and glutathione peroxidase to the culture medium showed to be beneficial for the embryonic development, despite the presence of high glucose concentration (Eriksson and Borg, 1991). This was followed by studies of SOD addition to cultures with high glucose, high ȕ-hydroxybutyrate high Į-ketoisocaproic acid, and combinations of these, all of which showed to block disturbed embryogenesis in vitro (Eriksson and Borg, 1993). Adding GSH ester and NAC protects embryos in high glucose culture (Trocino et al., 1995; Wentzel et al., 1997; Wentzel and Eriksson, 1998), the latter agents also oppose in vitro teratogenicity elicited by diabetic serum (Wentzel et al., 1997), and blocks glucose-induced alterations in neural crest cell development (Suzuki et al., 1995), thereby supporting the role of GSH depletion as a component of the ROS induced teratogenic process. Also, SOD addition to culture medium with high 3-deoxyglucosone concentration causes diminished embryo damage, which shows the relatively late appearance of the ROS excess in the induction of the dysmorphogenesis (Eriksson et al., 1997a). And, also the addition of a SOD transgene has proven to be efficient in preventing high-glucose-induced malformations (Eriksson et al., 1997b). There are a couple of other agents that block the embryonic dysmorphogenesis in vitro. In embryos exposed to high ȕ-hydroxybutyrate concentrations, ribose supplementation of the culture medium diminished the rate of embryonic dysmorphogenesis (Hunter et al., 1987), although this effect was stage-dependent (Shum and Sadler, 1990). Addition of the pyruvate transport inhibitor cyanohydroxycinnamic acid (CHC) diminishes glucose-induced disturbance in embryo development (Eriksson and Borg, 1993), as well as mitochondrial swelling (Yang et
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al., 1998). The first agent that was proven to inhibit glucose-induced embryonic dysmorphogenesis in vitro was arachidonic acid (Goldman et al., 1985), a result subsequently repeated (Pinter et al., 1986; Pinter et al., 1988; Wentzel and Eriksson, 1998), and extended to prostaglandin addition, in particular PGE2 (Baker et al., 1990; Goto et al., 1992; Wentzel and Eriksson, 1998). It was also shown that PGE2 addition diminishes the dysmorphogenesis exerted by diabetic serum in vitro (Goto et al., 1992). A positive effect of inositol addition on glucose-induced embryo damage in vitro has also been reported (Baker et al., 1990; Hashimoto et al., 1990; Hod et al., 1990; Strieleman and Metzger, 1993), whereas treatment with various aldose reductase inhibitors have been largely unsuccessful both in lowering the elevated embryonic sorbitol concentration as well as diminishing the embryonic rate of maldevelopment (Eriksson et al., 1989c; Hashimoto et al., 1990; Hod et al., 1990). All these results have paved the way for the in vivo trials with various agents, aimed at blocking the complex teratogenic effect of diabetic pregnancy on embryogenesis. In vivo Treatment Several different treatments have shown to be beneficial for embryonic and fetal development in experimental diabetic pregnancy, this is, in addition to the obvious treatment with exogenous insulin (Eriksson et al, 1982). Thus, administration of the antioxidant butylated hydroxytoluene (BHT) to pregnant diabetic rats diminishes the rate of fetal dysmorphogenesis (Eriksson and Simán, 1996), and, also diminishes the mitochondrial swelling encountered in the neuroepithelial cells of the embryos (Yang et al., 1998). Likewise, administration of vitamin E diminishes the rate of fetal malformations (Simán and Eriksson, 1997a), in particular the neural crest related damages (Simán et al, 1997), and the rate of embryonic dysmorphogenesis (Viana et al., 1996; Sivan et al., 1996; Simán and Eriksson, 1997a; Loeken, 1997) in the offspring of diabetic rodents. Furthermore, dietary supplement of vitamin C to pregnant diabetic rats diminishes fetal malformation rate (Simán and Eriksson, 1997b), as does supplementation of lipoic acid (Potashnik et al., 1997), whereas administration of GSH monoester diminishes embryonic developmental damage (Akazawa et al., 1997). In addition, mice expressing an SOD transgene show less malformations and growth disturbances in diabetic pregnancy than non-transgenic diabetic mice (Hagay et al., 1995). Administration of arachidonic acid to pregnant rodents diminishes the fetal malformation rate (Goldman et al., 1985), and blocks to some extent the embryonic dysmorphogenesis (Reece et al., 1996) in offspring of diabetic rats. Nobody has attempted to supply PGE2 to pregnant diabetic animals. Administration of exogenous inositol to pregnant diabetic animals diminishes the embryonic dysmorphogenesis (Akashi et al., 1991; Reece et al., 1997a), whereas the supplement of aldose reductase inhibitors to pregnant diabetic rats does not manage to block gross fetal malformations (Eriksson et al., 1986), although recent experimental efforts indicate a beneficial effect of aldose reductase inhibitor supplementation to pregnant diabetic rats on the severity of the congenital cataract in the offspring (Dorner and Berg, personal communication).
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CONCLUSIONS The study of etiologic factors in the pathogenesis of congenital malformations in diabetic pregnancy has revealed a series of competing and synergistic processes. The first, and possibly the most important, conclusion is that we are studying a multifactorial process where the diabetic state simultaneously induces alterations in a series of teratologically capable pathways. These pathways are intertwined, and several of them seem to result in an imbalance of the ROS metabolism, yielding a ROS excess in teratologically sensitive cell population(s), an imbalance which delivers the final insult to the developmental processes and causes the congenital malformations. This notion is of importance for the therapeutical design in the embryopathy field. Blocking the ROS excess may be a valid way to diminish the disturbed development caused by the diabetic environment. ACKNOWLEDGEMENTS The author gratefully acknowledges the support from The Swedish Medical Research Council (Grants no., 12X–7475, 12X–109), The Family Ernfors Fund, The Novo Nordisk Foundation, The Swedish Diabetes Association, and The Juvenile Diabetes Foundation International. REFERENCES Akazawa, S., Sakamaki, H., Ishibashi, M., Izumino, K., Abiru, N., Kondo, H., Takino, H., Yamasaki, H., Yamaguchi, Y., Kondo, T. and Nagataki, S. (1997) Glutathionedependent antioxidant system in diabetes-induced embryopathy. Diabetologia, 40 (Suppl. 1), A 229 (Abstract). Akashi, M., Akazawa, S., Akazawa, M., Trocino, R., Hashimoto, M., Maeda, Y., Yamamoto, H., Kawasaki, E., Takino, H., Yokota, A. and Nagataki, S. (1991) Effects of insulin and myo-inositol on embryo growth and development during early organogenesis in streptozocininduced diabetic rats. Diabetes, 40, 1574–1579. Baker, L., Egler, J.M., Klein, S.M. and Goldman, A.S. (1981) Meticulous control of diabetes during organogenesis prevents congenital lumbosacral defects in rats. Diabetes, 30, 955– 959. Baker, L., Piddington, R., Goldman, A., Egler, J. and Moehring, J. (1990) Myo-inositol and prostaglandins reverse the glucose inhibition of neural tube fusion in cultured mouse embryos. Diabetologia, 33, 593–596. Baker, L. and Piddington, R. (1993) Diabetic embryopathy: a selective review of recent trends. J. Diab. Comp., 7, 204–212. Baynes, J.W. (1991) Role of oxidative stress in development of complications in diabetes. Diabetes, 40, 405–412. Becerra, J.E., Khoury, M.J., Cordero, J.F. and Erickson, J.D. (1990) Diabetes mellitus during pregnancy and the risks for specific birth defects: a population-based casecontrol study. Pediatrics, 85, 1–9. Bravenboer, B., Kappelle, A.C., Hamers, F.P.T., van Buren, T., Erkelens, D.W. and Gispen, W.H. (1992) Potential use of glutathione for the prevention and treatment of
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270
INDEX
Acarbose, 6 Accelerated atherosclerosis, 60 ACE inhibition, 206 ACE inhibitors, 210, 215 Acellular capillaries, 13, 109, 233 Acellularity, 12 Acetyl-L-carnitine, 17, 114 Acetylcholine, 102 Acetylcholine transferase, 18 Advanced glycation, 11 N-Acetylcysteine, 17 Advanced glycation end-products (AGEs), 44, 50, 108, 212 albumin, 56 binding proteins, 57 immunohistochemical detection, 52 receptors, 44, 57 Afferent arteriolar resistance, 205 Albuminuria, 109, 204, 214, 215 Aldose reductase (AR), 1, 86, 109, 110, 253 gene, 86 inhibitors, 49, 138, 139, 140, 190, 212, 232, 254, 260 pathway, 207 transgenic animals, 73 Allogenic islet cells, 145 Alloxan, 132, 202 cytotoxic effect, 132 in dogs, 132, 204 in rabbits, 132, 137 in rats, 132 Alloxan-diabetic dogs, 3, 204
Alloxan-diabetic rats, 14 Alloxan induced diabetes, 234 Amadori adduct, 45, 46 Amadori products, 108, 212 Amine oxidases, 54 Aminoguanidine (AG), 4, 13, 18, 44, 53, 108, 140, 212, 233 Amyloid deposition, 81 Angiogenic growth factors, 12 Angiotensin converting enzyme (ACE), 4, 101 Angiotensin II, 101 Angiotensin II receptors, 206 Angiotensin II receptor ontagonist, 6, 101 Anterior horn cells, 141 ‘Anti-adhesins’, 209 Apoptosis, 192 AR activity, 15 AR inhibitors, 1, 9 Arachidonate, 17 Arachidonic acid (ARA), 111, 257, 261 ARI-treatment, 233, 236 Ascorbate, 55 Ascorbic acid, 98, 101, 102 Atherosclerosis, 101 ATPase activity, 13, 15, 20 Atrial natriuretic peptide (ANP), 1, 206 Autonomic neuropathy, 149 Autoxidation, 98, 103 of free glucose, 54 Autoxidative glycosylation, 46, 108 Axo-glial dysjunction, 15, 16, 150, 239 271
272 INDEX
Axon, 108 diameter, 143 dwindling, 103 -to-myelin ratio, 147 size, 142 swelling, 19 Axonal atrophy, 14 Axonal calibre, 170 Axonal degeneration, 148, 151 Axonal sequestration, 150 Axonal transport, 145, 167, 171 Axoplasmic transport, 189 Axotomy, 192 ȕx-cells, 10 ȕx-cell dysfiinction, 78 ȕX FGF, 12 ȕn isoform of PKC, 207 ȕX isoform specific PKC inhibitor, 106 Bl-laminin, 259 Baboons, 10, 136 Background retinopathy, 20 Basal lamina, 149 Basement membrane thickening, 12, 230, 231, 233, 234 Basic fibroblast growth factor (bFGF), 209 BB rats, 12, 14, 15, 20, 204 BB/W rats, 5, 79, 149, 233, 238 BBZ/WORDR rat, 20 BHE/cdb rat, 9 Bladder dysfunction, 150 Blindness, 230 Blood/nerve barrier, 17 Blood-retinal barrier, 12, 233 Bovine growth hormone (bGH), 83 Brain-derived neutrophic factor (BDNF), 167 Branched amino acids, 255 Butylated hydroxytoluene (BHT) 17, 102, 261 CC57BL/Ks mice, 148 Calcitonin gene-related peptide (CGRP), 170, 174 Calcium adenosine triphosphatase, 185 Calcium-ATPase function, 114, 194 Calcium channel antagonist, 187
Calcium channel blockers, 140, 210 Calcium current density, 151, 189 Calcium homeostasis, 184 Calcium signaling, 184 Calmodulin, 18, 79, 82 Ca/Mg ATPase, 237 Candidate genes, 73 Capillary acellularity, 13 Capillary microangiopathy, 230 Capillary nonperfusion, 230 Capillary occlusion, 231 Capillary pericytes, 233 Capsaicin, 172 Caractogenesis, 11 Carboxymethylation, 57 Cardiomyopathy, 8 L-Carnitine, 111, 114 ȕx-Carotene, 101, 102 Carotenoids, 101 Cataracts, 10, 88 Catecholamine, 98 CEL, 46 Ceruloplasmin, 101 c-fos, 170 Chinese hamster (Cricetulus griseus), 5, 13, 19 Chromosome, 16, 77 Ciliary neurotrophic factor (CNTF), 192 CML, 46, 46, 48, 50, 53 Cohen diabetic rats, 1, 8, 13 Collagen crosslinking, 52 Collagen fluorescence, 110 Conduction block, 15 Congenital malformations, 250, 253 Continuous subcutoneous infusion, 143 Corpus cavernosum, 102, 103 ‘Cotton wool’ spots, 10 C-peptide, 18 Crabtree effect, 254 Cranial neural crest, 256 Creatinine clearance, 1, 6 Crosslinking of tissue proteins, 44 Cyclooxygenase, 116 Cyclooxygenase pathway, 102 Cysteine, 101 Cytokine, 71, 75, 208, 238 Cytokine expression, 74 Cytokine production, 18
INDEX 273
Cytosolic calcium, 184 Cytotoxicity, 55 DA rat, 204 DAG, 237 DAG-PKC pathway, 237 db/db mice, 5, 12, 14, 19, 20, 148 Deferoxamine, 105 Degu (Octodon degus), 12 Demyelination, 14, 19, 152 motor neuropathy, 19 paranodal, 151, 152 segmental, 19, 148, 151, 152 3-Deoxyfructose, 46 3-Deoxyglucosone (3-DG), 46, 258, 260 Diabetic dogs, 136, 231 Diabetic embroypathy, 250 Diabetic monkey, 234 Diabetic nephropathy (DN), 202 Diabetic neuropathy, 188 Diabetic Ren2 rats, 212 Diabetic retinopathy, 227 Diacylglycerol (DAG), 1, 15, 106, 207, 230 Dimethylthiourea, 102 Distal myenteric parasympathetic nerves, 150 Distal symmetric polyneuropathy, 149, 150 DNA damage, 258 DNA fragmentation, 134 DNA mutations, 258 Dorsal root ganglia (DRG), 141, 167, 189 Electrortinogram, 239 Embryonic catalase, 251 Embryonic hyperglycemia, 251 Endoneurial blood flow, 17, 139 Endoneurial capillary density, 114 Endoneurial hypoxia, 101, 112 Endoneurial oxygen tension, 103 Endothelial cell-pericyte ratio, 233 Endothelial function, 101 Endothelial proliferation, 12, 109, 231, 233 Endothelial prostacyclin, 16 Endothelin, 207 Endothelin-1, 101, 113 Endothelin receptor antagonist, 214
Endothelium-dependent relaxation, 102, 113, 190 End stage renal failure, 202 Epidermal growth factor (EGF), 209, 231 eSS rat, 9 Essential fatty acids, 98, 111, 114, 116 ET-1, 238 ET-3, 238 ETA/ETB antagonist, 102 Evening primrose oil, 111, 114 Ewes, 136 Exogenous NGF, 171 Extracellular matrix (ECM), 208 Extracellular matrix proteins, 46 Factor VIII, 230 Fatty acid, 99, 102 Fatty acid oxidation, 238 Fatty Wistar diabetic rat (WKY/N-cp), 151 Fenton reaction, 99, 105, 132 Ferritin, 101 Fetal maldevelopment, 250, 261 Fiber atrophy, 88 Fiber size, 142 Fibroblasts, 170 Fibroblast growth factors (FGFs), 209, 230 Fibronectin, 174, 259 Fish oil, 113 Foot ulceration, 176 Free radicals, 98 Free radical scavengers, 101 Free radical theory of aging, 50 Fructose, 11, 138, 185 Fructose-3-phosphate, 11 Fucose, 16 fed rat, 110 Galactitol, 86, 109 Galactose, 11, 86 Galactose-fed dogs, 13 Galactose-fed rats, 13 Galactosemic animal models, 234 Gangliosides, 20 Gastric circular muscle function, 187 Gene expression, 101, 259 GFR, 205 GK rats, 8, 19, 151
274 INDEX
Glomerular basement membrane thickening (GBM), 204, 204 Glomerular filtration rate (GFR), 1, 106 Glomerular hypertension, 1 Glomerular hypertrophy, 88, 204, 214, 215 Glomerular volume, 210 Glomerulopathy, 5 Glomerulosclerosis, 83 Glomerulosclerosis lipohyaline exudative changes, 8 Glucagon, 194 Glucokinase (GK), 78, 79, 79 Glucokinase (GK) gene, 78 Gluconeogenesis, 135 Glucose, 98 Glucose autooxidation, 17 Glucose-6-phosphate, 78 Glucose transporters, 78, 79, 194, 251, 253 Glucosidase inhibitor, 6, 239 GLUT 1, 79, 251 GLUT 1, 79 GLUT 3, 79 GLUT 4, 79 overexpression, 81 GLUT 5, 79 Glutathione, 17, 101, 251, 254 peroxidase, 101, 255 redox cycle, 101, 109 Glycated hemoglobin (HbAlc), 45 Glycative stress, 48 Glycerophospholipid composition, 114 Glycogenolysis, 135 Glycogenosomes, 149 Glycosaminoglycans, 3 Glycosuria, 149 Glycosylation end products, 185 Glycoxidation, 1, 108, 110 products, 46, 48, 50 Glyoxal (GD), 46 G protein, 13, 190 Growth-associated protein 43 (GAP-43), 174 Growth disturbances, 250 Growth factors, 208 Growth hormone (GH), 83 Growth retardation, 137
Haber-Weiss reaction, 99 Hemagglutinin influenza virus, 74 Heparan sulfate synthesis, 4 Heparin, 212 Heparin-binding growth factor, 209 High affinity NGF receptors, 170, 174 Hodge pathway, 45 H reflex, 178 hrNGF treatment, 172 Human aldose reductase (hAR), 83, 86 Human growth hormone releasing factor (hGHRF), 83 Hyaluronan acid derivatives, 3 Hydrogen peroxide, 98, 99 Hydrogen peroxide generation, 134 Hydroquinone, 98 ȕx-Hydroxybutyrate, 255, 260 Hydroxyl radical, 98 Hyperfiltration, 205 Hyperketonemia, 254 Hyperlipidemia, 8, 98, 135 Hyperhagia, 136 Hypertension, 7, 210 Hypoinsulinemia, 111 Hypoxia, 16 Hypoxia-Ischemia, 16 Hypoxic conduction failure, 101 IDDM, 44, 71 IFN-Ȗx, 76 IFN-Ȗx, 74 Inositol depletion, 250 Insulin, 18 Insulin-like growth factor-1 (IGF-1), 12, 18, 82, 83, 192, 209, 231, 238 Insulinoma, 73 Insulin promoter, 73 Insulin receptor, 78, 79, 82 Insulin-receptor substrate-1 (IRS-1), 79,82 Insulin resistance, 7, 9, 78, 185 Insulin resistant, 8 Insulin therapy, 137, 148, 209 Insulin treatment, 142 Insulitis, 5, 74, 77 Interferon (IFN), 74 Interleukin (IL)-l, 74, 170 Interleukin (IL)-2, 74
INDEX 275
Intraglomerula pressure, 205 Ischaemia-reperfusion effects, 98 Islet amyloid polypeptide (IAAP), 78, 79 gene, 81 Islet transplantation, 4, 212 Keratopathy, 12 Ketoamine fructoselysine (FL), 46 Ȗ-Ketoisocaproate, 255 Ȗ-Ketoisocaproic acid, 260 Ketone bodies, 49 Ketonuria, 149 Kidney hypertrophy, 1 Kilhams’s rat virus, 149 Kimmelstiel-Wilson glomerusclerosis, 3, 204 Kinin antagonist, 206 KK mice, 6, 13 KKAY mice, 7 LCM-virus, 74 Lens crystallins, 45, 48 Lewis rat, 204 Linoleic acid, 111 Ȗ-Linolenic acid (GLA), 111 Lipid peroxidation, 52, 140 Lipoic acid, 261 Ȗ-Lipoic acid, 103 Long chain fatty acids, 114 Low protein diet, 214 L-type calcium channel antagonist, 188, 190 Lycopene, 101 Lysine residues, 48 Macrophage chemoattractants, 60 Maillard reaction, 44, 45 Major histocompatibility (MHC) antigen, 75, 76 Malondialdehyde, 109 Matrix metalloproteinases (MMPs), 208 Maturational defect, 149 Maturity onset of diabetes of the young (MODY), 79 Medium neurofilament protein (NF-M), 170 Membrane phospholipids, 185
Mesangial expansion, 88, 109, 204 Mesangial matrix, 3 Mesangial proliferation, 6 Mesangial volume, 210 Mesangium, 1 Mesenteric smooth muscle, 185 Metal-catalyzed oxidation, 56 Metal ions, 59 Methionine sulfoxide, 56 4-Methylcatechol, 176 Methylglycoxal (MGO), 46 Methylguanidine, 55 MHC-1 antigen, 76 Microalbuminuria, 50 Microaneurysms, 13, 230, 233, 234 MMP-1, 208 MMP-3, 208 Monkeys, 136, 234 Mononeuropathy, 149 Motoneurons, 167 Motor nerve conduction velocity (MNCV), 88, 136, 171 M wave, 178 Myelin wrinkling, 19 Myelinated fiber atrophy, 239 Myoinositol, 1, 11, 14, 20, 138 Myoinositol supplement, 16 Na+/K+ ATPase, 15, 114, 115, 139, 185, 189, 194, 237 Na+ permeability, 150 NADH/NAD+ ratio, 237 NADPH, 109, 190, 254 Neovascularization, 231 Nephropathy, 1, 101 Nerve blood flow, 101, 102, 109, 112, 115 Nerve conduction velocity (NCV), 14, 112, 148 Nerve fibre diameter, 137 Nerve fiber regeneration, 16 Nerve growth factor (NGF), 18, 167, 172, 190 Nerve growth factor receptor, 115 Neural crest cells, 251 Neural vascular resistance, 140 Neurofilament synthesis, 170 Neuropathy, 14
276 INDEX
Neurotrophic factors, 167, 190 Neurotrophin 3 (NT-3), 167 Neurotrophin 4/5 (NT-4/5), 167 NGF-like immunoreactivity, 172, 174, 176 NGF protein, 172 NIDDM, 44, 78 Nitric ocide (NO), 17, 101, 185, 190 pathway, 113 production, 115 synthase, 55, 190 inhibitor, 108, 115 Nitric ocide synthase, 1, 207 Nitrol-L-arginine methyl ester, 55 NO activity, 238 Nociceptive fiber, 170 NOD (non-obese diabetic) mice, 5, 19, 73, 77, 204 Node of Ranvier, 15 NON mice, 5 Nonenzymatic glycation, 18, 86, 237 Non-proliferative retinopathy, 230 Noradrenergic antagonists, 140 Norepinephrine, 105, 186 Nuclear factor-KB (NF-KB), 58, 101, 108 Nutritive blood flow, 103 NZO mice, 7 Obese Zucker rat, 204 ob/ob mice, 5 OLETF rat, 10, 19 Oncogenic gene, 71 Organic radicals, 56 Osmotic stress, 15 ȕ-Oxidation, 114, 115 Oxidative damage, 51 Oxidative defenses, 58 Oxidative stress, 1, 16, 17, 44, 46, 50, 58, 98, 101, 109, 116, 230 Oxygen free radicals, 11 Pancreatic transplantation, 145, 205, 233 Paranodal demyelinization, 19, 150 Paranodal swelling, 19, 151 Pax-3, 259 Pentose phosphate shunt, 255 Pentosidine, 46, 46, 50, 53 Pericyte degeneration, 12
Pericyte ‘ghosts’, 13 Pericyte loss, 13, 109, 231 Perineurial vessels, 105 Peripheral insulin resistance, 20 Peroneal nerves, 141, 152 Peroxyl radical, 99 PGE2, 260 Phosphoinositides, 253 Phosophoinositide metabolism, 139 Phospholipase A2, 253 PKC, 207, 237 activity, 3, 13 agonist, 16 PKC ȕ-selective inhibitor, 207 Platelet aggregation, 113 Platelet derived growth factor (PDGF), 209, 231 Platelet thrombosis, 13 Polydipsia, 136 Polyol dehydrogenase, 11 Polyol hypothesis, 138 Polyol pathway, 1, 6, 10, 11, 13, 14, 15, 86, 108, 109, 189, 230, 236, 253 Polyuria, 136, 149 Potassium-sensitive ATP channels, 78 Pro-proliferative retinopathy, 230 Preprotachykinin-A (PPT-A), 170, 174 Primary axonal pathology, 149 Probucol, 17, 101 Probucol analogue, 118 Proliferative retinopathy, 230 Propionyl-L-carnitine, 17 Prostacyclin effect, 16 Prostaglandin, 111, 257 Prostaglandin E2, 257 Prostaglandin synthesis, 238 Prostanoids, 102 Prostanoid analogues, 140 Protein catabolism, 135 Protein kinase A, 188 Protein kinase C (PKC), 1, 106, 188, 189, 230 Proteinuria, 1, 4 Proteoglycan, 3, 233 Proximal tubule, 205 Psammomys obesus (sand rat), 9, 12, 19 Pyridinoline, 52 Pyrraline, 46, 53
INDEX 277
Radiation-induced cataracts, 50 Radical oxygen species (ROS), 250, 255 RAGE (receptor for AGE), 57 Reactive oxygen radicals, 1 Reactive oxygen species, 17,50, 52, 98, 254 Redox imbalances, 230, 237 Reich granules, 149 Renal failure, 202 Renin-angiotensin system, 101, 140, 206, 210 Retinal blood flow, 115, 238 Retinal detachment, 10 Retinal hemorrhages, 230 Retinal microaneurysms, 10 Retinal pigment epithelium (RPE), 12, 233 Retinopathy, 10, 12, 114, 227, 231 Retrograde axonal transport, 167 Rhesus macaques, 12 ROS scavengers, 256 R-R interval, 150 Rubidium ion uptake, 139 Salt restriction, 214 Scavenger-type receptors, 57 Schiff’s base, 108 Schwann cells, 108, 170 Scyllo-inositol, 253 Segmental glomerulosclerosis, 6 Serum factor(s), 190 SHHF/Mcc-cp rats, 8 SHR/N-cp rats, 8 Skeletal growth, 143 Smooth muscle, 187 SOD, 99 Sodium: calcium exhcnage pump, 186 Sodium permeability, 15 Soft exudate, 230 Somatostatin analogue, 212 Sorbinil, 13 Sorbitol, 11, 20, 138, 185, 236 accumulation, 250 dehydrogenase, 109, 236 SPARC, 209 Spiny mice (Acomys canirinus), 9 Spontaneously diabetic dog, 10, 152 Spontaneously diabetic guinea pig, 10, 12 Spontaneousiy hyperinsulinemic, 5
Spontaneously hypertensive rat (SHR), 7, 204, 210 S-S bond, 99 Streptozotocin, 132, 202 in dogs, 134 in guinea pigs, 134 in mice, 134 in monkeys in rats, 134 STZ-diabetic rat, 1, 1, 4, 14–16, 49, 187, 137, 202, 233, 238 Substance P, 174 Sugar cataracts, 49 Superoxide, 58, 98, 99 Superoxide dismutase (SOD), 17, 99, 251, 258 Sural nerves, 141, 152 SV40 T-antigen, 73 SV40 transgenic mice, 83 Systemic amyloidosis, 145 Taurine, 15 TCR (T cell receptor), 74 TCRĮ gene, 77 Thiol, 98 Thromboxane, 111 Thromboxane synthetase inhibition, 214 Thromoxane Az, 214 Tibial nerves, 141 TIMP-1, 208 TIMP-2, 208 T-lymphocytes, 74 Tocopherol, 55 Ȗ-Tocopherol, 101, 102, 256 Transforming growth factor-ȕ (TGF-ȕ), 55, 208, 230 Transgenes for glucokinase, 73 Transgenes for glucose transporters, 73 Transgenic AR overexpressing rats, 16 Transgenic knockout, 79 Transgenic mice, 71, 86 Transgenic mice overexpressing AR, 11 Transgenic technology, 71 Transition metal ions, 101, 105 trkA, 18, 174, 176 trkB, 176 trkC, 176
278 INDEX
Tromboxane A2, 17 Tubular hypertrophy, 207 Tuco tuco (Ctenomis talarus), 9 Tumor necrosis factor (TNF), 74 Tumor necrosis factor-Ȗ (TNF-Ȗ), 58, 209, 230 Type IV collagen, 210, 215 Ubiquinone, 101 Urinary albumin excretion, 106, 110, 204 Vagal nerve dysfunction, 15 Vagus nerve, 150 Vasa nervorum, 103, 106 Vasoactive hormones, 207 Vasoactive prostanoids, 111 Vasoconstricting eicosanoids, 185 Vasodilators, 140 Vasoproliferative factors, 230 VEGF, 12, 234, 239 Viral antigens, 73, 75 Viral infections, 73 Vitamin D, 174 Vitamin E, 17, 55, 101, 261 Voltage-activated calcium channels, 184 von Willibrand factor, 106, 230 Wallerian degeneration, 19 Wbn/Kob rat, 9, 151 WBN/Kob rats, 19, 151 WDF/Ta-Fa rats, 7 WKY fatty rats, 19 White tailed rat (Mystromys albicandatus), 9 Wolff pathway, 46 Zucker fa/fa rats, 6, 7
