Animal models of non-insulin-dependent diabetes.

Animal Models of Non-Insulin-Dependent Diabetes Eleazar Shafrir Department of Biochemistry, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel

I. INTRODUCTION Some animal species are known to develop insulin-dependent diabetes mellitus (IDDM; type I diabetes) spontaneously as a result of an immune aberration targeted towards B-cell destruction. These models, particularly the BB rat and NOD mouse, have been intensively studied in the last two decades and have made an enormous contribution in the progress in the understanding of the autoimmune aetiology of this disease. Animal species with spontaneous or induced NIDDM (non-insulin-dependent diabetes mellitus)like syndromes (type I1 diabetes) are more numerous than those affected by IDDM. They are much more diverse in terms of the origin, development, manifestations, and pathogenesis of the disease in the absence of direct pancreatic injury. In many species, NIDDM is linked to obesity and, in fact, the diabetes and obesity are aspects which are so intertwined that their condition should be actually referred to as “diabesity”,l even if the diabetes and obesity occur sequentially. In other animals, the NIDDMlike syndrome is not associated with excessive weight gain. In some animal species, the primary diabetic manifestation is inappropriate cellular recognition of insulin, probably related to inadequate insulin receptor function. This may be compensated, at least in part, by profuse pancreatic insulin secretion. The animals may persist in this condition through life, being endowed with what may be defined as a “sturdy” or resilient pancreas. Other animal species are characterized by a “brittle”, labile pancreas. Their islets are not able to sustain the prolonged overtaxation and

they succumb to the hyperglycaemic stimulation consequently undergoing degradation and necrotic degeneration. Insulin secretion becomes erratic and compromised to the degree that the animals actually may lapse into a secondary IDDM-like state. Thus, both the periphery and the pancreas may be involved in the causation of diabetes. In addition, the occurrence, nature, and severity of complications differ in animals with IDDM and NIDDM. In the latter, there is a greater diversity of complications, some of them peculiar to a given species, which underscores the role of the genome in the susceptibility to a particular functional derangement in the face of hyperglycaemia and/or hyperinsulinaemia. Some complications appear to be related solely to hyperinsulinaemia, a phenomenon which is not observed in models of IDDM. It cannot be overstated how important is the availability of a large number of animals species which develop syndromes which model aspects of human NIDDM, a disease less dramatic in onset and severity than IDDM, but comprising as much as 95% of the total human diabetic population. It is now accepted that NIDDM is not a single entity but rather a group of diseases with a variety of mechanisms leading to insulin resistance, pancreatic 8-cell insufficiency, or other defects resulting in hyperglycaemia. The multitude of animal species with NIDDM is therefore an asset, since these species can provide the genetic and endocrinemetabolic basis for the subclassification and understanding of the variants of the NlDDM syndrome. Not all models which have been reported in the literature as manifesting a NIDDM-like syndrome will be

DiabetesIMetabolism Reviews, Vol. 8, No. 3, 179-208 (1992) & Sons, Ltd.

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described and discussed here. Those which have been included in this review exhibit a pattern of disease from which much can be learned and perhaps extrapolated for the understanding of certain aspects of the human disease. Many models which have been previously described in comprehensive reviewsT4 are no longer readily available or are investigated only in isolated colonies or single laboratories. Emphasis will be placed on animal species under current investigation, or those which may serve for novel experimental approaches. The groups of animals included in this review are listed in Table I and those less common and of historic-academic interest are listed in the footnote to the table.

Table 1. Animals with NIDDM-like Syndromes Group 1. Mice with long-lasting genetic diabesity C57BU6J obese (ob) KK mice (yellow agouti AY) and their hybrids NZO mice (polygenic) Group 2.

Mice with B-cell-losing diabesity C57BL/Ks diabetes mouse (db)

Group 3.

Rats with long-lasting genetic diabesity Zucker fatty (fa) Diabetic fatty (ZDFIDrt-fa) Wistar-Kyoto diabetic (WDF-TA-fa) Wistar-Kyoto fatty (WKYINdrt-fa)

Group 4.

Corpulent S H R N c p and LA/N-cp, hypertensive diabetic rat strains (alleles of fa suggested designation fa'p) SHRIN-cp with nephropathy SHIWMcc-cp with congestive heart failure Jcr:LA-cp with ischaemic cardiovascular disease

Group 5.

Animals with nutritionally induced NIDDM Psammomys obesus (sand rat), a gerbil on a regular laboratory diet Non-human primate Macaca mulatta on an ad libitum diet C57BL/6J mouse on a high caloric fat-disaccharide diet

Group 6.

Diabetes produced by selective breeding from normal pools GK rat bred by selection pressure on glucose intolerance Sucrose-induced Cohen rat NON mouse derived from the NOD mouse line

Group 7.

Animals with special NIDDM-like aspects WBN/Kob rats with spontaneous exocrine-endocrine lesion eSS rat with spontaneous late-onset NIDDM

11. MICE WITH GENETIC DIABESITY SYNDROMES Several strains of mice are known with mutations, either induced or spontaneous, which manifest both diabetes and obesity through most of their lifespan. The most extensively investigated representatives of this group are ob/ob mice, KK mice, and New Zealand obese (NZO) mice. Hyperphagia early in life is the common characteristic of these species and leads to excessive fat deposition, increased metabolic efficiency, and hyperinsulinaemia with peripheral and hepatic insulin resistance. The latter is manifested by increased hepatic glucose production. The longlasting insulin secretion compensates in part for the insulin resistance, so that the hyperglycaemia is usually moderate and the hepatic gluconeogenesis partially restrained. These animals do not exhibit ketosis despite the increased free fatty acid (FFA) turnover, since most of the FFAs are recirculated by the liver as plasma triglycerides and deposited in adipose tissue as a consequence of hyperinsulinaemia. A. ob/ob Mice

The C57BLl6J-ob mouse originated from the Jackson Laboratory in Bar Harbor, ME, U.S.A. Much of the original information on this species has been provided by C ~ l e m a n . The ~ , ~ ob gene was transferred from the stock of origin onto the B/6 genomic background and is located on chromosome 6. It is an autosomal recessive mutation with full penetrance. Obesity is prominent in these animals and most investigations

In addition to the animals listed above, other species studied intensively in the past, or in limited laboratories at present, can also be assigned to these groups. They are listed and reviewed elsewhere.' To mention a few: PBB/Ld mice (Group 1); Chinese hamsters of Upjohn colony, Djungarian hamsters of Dusseldorf colony, whitetailed South African rats Mysfromys albicaudafus (Group 2); spiny mice Acomys cahirinus, tuco-tuco rats Ctenornys talarus, Mongolian gerbils Meriones unguiculatus (Group 5); athymic nude nulnu mice, NZW rabbits, BHE rats Cdb (Group 7).

have focused on this particular aspect. B6 oblob mice may reach a body weight of 90 g and exhibit hyperinsulinaemia 10 to 50 times that of their non-obese litter-mates. Severe insulin resistance

ANIMAL MODELS OF NIDDM

is associated with the hyperinsulinaemia and is accompanied by increased gluconeogenesis, although the animals are only moderately hyperglycaemic. The activity of enzymes of both the glycolytic and the gluconeogenic hepatic pathways is increa~ed.~,'The insulin resistance at the hepatic level is evident by the failure to attenuate gluconeogenesis, even at extreme concentrations of endogenous insulin. The activity of regulatory enzymes of the lipogenesis pathway, especially the committed enzyme acetyl-CoA carboxylase, is markedly enhanced by insulin, in both the liver and the adipose tissue, promoting the conversion of glucose to lipids in the liver and peripheral tissues. Adipose tissue of oblob mice grows both through hypertrophy and through hyperplasia of adipocytes. Even auxiliary enzymes of lipogenesis such as glycerokinase, an enzyme enabling local utilization of glycerol for esterification of fatty acids to triglycerides, which is inactive in normal adipocytes, were reported to be enhanced in oblob mice as a result of hyperinsulinaemia.s It is of interest that in oblob mice, in common with other obese and hyperinsulinaemic animals, the lipogenic enzymes increase in activity concurrently with gluconeogenic enzymes, mainly phosphoenolpyruvate carboxykinase (PEPCK). The coexistence of increased hepatic lipogenesis and gluconeogenesis is contrary to the generally mutually exclusive behaviour of enzymes of these pathways in most physiopathological situations (fasting, IDDM). This constitutes a special feature of diabesity, in which the hepatic insulin resistance does not affect lipogenesis. It is possible that the lipogenic enzyme transcription message from the insulin receptor remains intact, whereas that for the suppression of PEPCK synthesis is not generated or not recognized at the postreceptor site. The sustained lipogenesis may somewhat attenuate the glycaemia, due to hyperphagic food intake or excessive hepatic production, by channelling the glucose into triglycerides. Enhanced lipogenesis also prevents ketosis, despite an increased FFA turnover, further facilitating fat accretion. Pancreatic B-cell hyperplasia and hypertrophy, as well as the content of insulin, are very high in 86 oblob mice. Recent morphometry has shown that the islet area increases two-fold at the age of 1month and 30-fold at the age of 6 months, compared with lean sibling^.^ The islet area increases more than the insulin content of the pancreas, most likely in response to the persistent hyperglycaemia. The population of A-, D-, and

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PP-cells had been reported to be reduced,1° but the changes in the relative insulin content of these cells may be dependent on the timecourse of the disease." Although increased B-cell degranulation has been observed, there appears to be continual B-cell regranulation and regeneration, thereby securing a lasting secretion of insulin. In fact, 8- to 9-week-old oblob mouse islets secrete four times the amount of insulin in response to glucose, compared with lean controls, and exhibit increased secretion at glucose levels lower than the lean islets.'* The oblob islets also show an exaggerated insulin response to acetylcholine and marked inhibition of secretion in the presence of norepinephrine.12 These pronounced alterations in the sensitivity to glucose and neurotransmitters suggest a predilection of the oblob islets to neurogenic effects on insulin secretion. Insulin resistance in oblob mice, as well as in other models with diabesity, may most probably be attributed to reduced insulin recognition and signal transduction by the insulin receptor and to decreased glucose transport. This has been demonstrated in the skeletal muscle of oblob mice by the decreased autophosphorylation of the psubunit of the insulin re~eptor.'~ In the liver, the receptors were found to be decreased in number but not in their specific insulin affinity.14 The primary defect in the oblob mice is of neuroendocrine origin, but the exact aberration and its site are not yet fully known. Its expression is a lack of satiety control at the hypothalamic and/or pituitary level. The first hint to this effect was provided by seminal parabiosis experi m e n t ~Normal . ~ ~ mice were saturated with satiety factor(s), derived from the blood of parabiosed oblob partners; they consequently ceased to eat and starved to death. The oblob mice continued to overfeed, being insensitive to the same factors. The hypothalamus-originating neurogenic overstimulation of insulin secretion was amply demonstrated by the Jeanrenaud group,16 whereas pituitary overproduction of a B-cell tropin was indicated by Beloff-Chain.17The role of hypothalamic peptides in neuroendocrine glucoregulation has been recently r e v i e ~ e d . l ~The *~~ levels of neuropeptide Y,a potent central appetite stimulant which mediates hyperphagia, were found to be high in animals with diabesity. The possibility was raised that the activity of this neuropeptide may be not suppressed by insulin in diabese animals as a result of inborn brain insulin resistance, and that this may be one of the reasons for the compensatory insulin oversecretion.

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The diabesity of oblob mice recedes with age. Plasma insulin levels tend to decline in the second year of life. Consequently, glucose tolerance and insulin resistance improve and there is a loss of adipose body weight. It is of interest that oblob mice are not known to develop major diabetic complications despite the marked insulinaemia, in contrast to other species. The role of the genome in this respect will be discussed later. The mechanisms modifying the expression of the ob and db genes have been recently reviewed and it has been suggested that shifts in activities of oestrogen- and androgen-dependent sulphotransferase enzymes may be involved.20

B. KK Mice The KK mouse group consists of several substrains. The original KK mice, which exhibit hyperphagia and moderate obesity, originate from Japan.21 They were crossed with the Bar Harbor C57BL/6J mice (the background mice of the ob mutant), inbred in the U.S.A. and Canada, and became known as T-KK hybrids or KKEiL mice.”*u Other hybrids were developed by inserting the yellow agouti (AY) gene from the yellow obese mice into the Japanese KK, and these have been designated as yellow KK or KKAY mice.24,25T-KK and yellow KK mice become diabetic when obese; Japanese KK mice become diabetic when maintained on a high energy diet.26The inheritance of the KK gene is supposedly dominant, but only with approximately 25% penetrance due to an association with a recessive modifier.27Obesity and hyperinsulinaemia are not as prominent as in oblob mice and are more marked in males than in femalesz3 Insulin resistance and body weight increase considerably at 2-3 months of age, peak at about 5 months, and return to normal at 9-12 months. This time-course may vary with the mutant strain. Hyperglycaemia is generally higher than in oblob mice, and hyperinsulinaemia may reach the level of > 1000 mu/l. The extent and duration of the diabetic symptoms vary with the strain, diet, and inbreeding. In common with oblob mice, hyperglycaemia and hyperinsulinaemia in the KK strains are associated with an elevation in the activity of the rate-limiting enzymes of both glycolysis and gluconeogenesis.28 The latter are even more pronouncedly increased.29The hepatic conversion of pyruvate to glucose is enhanced rather than suppressed by hyperinsulinaemia and at the same time, lipogenesis is also markedly induced.

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The endocrine pancreas of the KK mutants shows a markedly increased number of B-cells and insulin ~ o n t e n t . ~ There ~ , ~ ” is evidence of expanded Golgi apparatus and RER, increased leucine incorporation into cellular proteins, proliferation, and, possibly, transformation of extrainsular cells into B - ~ e l l s This . ~ ~ appears to compensate for the partial degranulation of B-cells in the face of insulin oversecretion, which does not, however, lead to cell necrosis. Additional evidence that the hyperglycaemia of KK mice is mainly due to insulin resistance comes from glucagon and glucocorticoid studies. Plasma glucagon levels are in the normal range and the release of glucagon is only slightly reduced by glucose.31The adrenal glands become enlarged and cortex activity may increase, but this seems to be a phenomenon secondary to hyperglycaemia and hyperin~ulinaemia.~~ Energy intake is all-important in terms of the severity of the diabetic syndrome and its progression. Food restriction greatly ameliorates the obesity and insulin resistance in KK mice. Treatment of yellow KK mice with a P,-receptor agonist BRL 26830A increased thermogenesis and the number of adipocyte insulin receptors, while reducing plasma insulin levels, obesity, and insulin r e ~ i s t a n c e .Treatment ~~ with metformin led to an increase in liver and skeletal muscle glycogen synthase activity in KK mice and increased muscle glycogen deposition, together with an improvement in glucose tolerance.M Among diabetes complications, microangiopat hi^^^ and g l ~ m e r u l a ? ~lesions ,~~ have been reported, and the lifespan of the KK mutants is definitely shorter compared with their nondiabetic siblings.23

C. NZO Mice The characteristics of diabesity in this strain has been recently described and reported38 and extensively reviewed.39The strain was developed from a mixed colony and exhibits a polygenic mode of inheritance. Body weight rises considerably during the first 10 weeks of life, subject to hyperphagia, and peaks at 12-14 months, together with the parallel manifestation of hyperglycaemia and hyperinsulinaemia. Although females gain more weight than males, this is ascribed to metabolic-endocrine rather than genetic reasons.4o Hepatic gluconeogenesis is increased in spite of hyperinsulinaemia, and insulin does not suppress the channelling of alanine carbons to glucose in


isolated, perfused liver of NZO The extent of hyperinsulinaemia varies with colonies of NZO mice, some of which may have been randomly bred, but generally it is lower than that in oblob or KK mice, while hyperglycaemia may be more prominent. Peripheral insulin resistance is pronounced and large amounts of exogenous insulin hardly affect the level of blood glucose. Soleus muscle in vitro shows a low basal 2deoxyglucose uptake and low insulin-induced transport s t i m ~ l a t i o n There . ~ ~ is reduced insulin binding to receptors both in muscles and in the liver," but it does not appear to be well correlated with the uptake defect. However, intraperitoneal implantation of albino mice islets resulted in reasonable diabetes control and retarded weight gain for several weeks.45 Adipose tissue growth is based both on hypertrophy and on hyperplasia of adipocytes,% and lipogenesis from glucose is increased in both the liver and adipose tissue. Adipose tissue resistance to insulin is present but it becomes substantial much later than that in muscle, as is the case in the development of obesity in NIDDM in general.47 It is of interest that the peritoneal adipose tissue in NZO mice retains a relatively greater proportion of fat than other depots compared with other obese mice. The ratio of total vs. abdominal fat in NZO mice is 2.5 vs. 3.5 in oblob mice.48 In humans, the excessive abdominal fat deposition is related to increased glucose intolerance, enhanced gluconeogenesis, and possibly cardiovascular disease. Thus, this species represents a potentially valuable model for the investigation of the role of fat distribution on general metabolic characteristics and on diabetes complications. As in other animals with diabesity, NZO mice show a much higher insulin content and secretion by the pancreas compared with a similar non-diabetic strain of New Zealand mice. The mechanism of insulin hypersecretion is somewhat puzzling. The isolated islets, in which an elevated activity of glycolytic enzymes was did not show an increased response to various s e c r e t a g o g ~ e s . The ~ ~ , ~responses ~ to glucose, tolbutamide, and cyclic AMP were low and the responses to glucagon and aminophylline were delayed, whereas arginine elicited a good insulin r e l e a ~ e . ~The ~ , ~ ~possibility of hypothalamic involvement did not receive support in a comparative study using gold-thioglucose VMH-lesioned mice, since glucose was effective in these mice in producing a strong insulin s e c r e t i ~ nIt. ~has ~

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been suggested that the relatively low in vitro response to glucose in NZO mice may be due to an impediment in B-cell glucose metabolism prior to the triose phosphate step, since glyceraldehyde effecteda potent insulin ~ e c r e t i o nAn . ~ attenuated ~ first phase of insulin release was a common pattern in the response to most secretagogues, indicating a disturbed stimulus-secretion coupling.50 Of interest is the application of an isletactivating 77 kD protein from Bordetella This protein is known to induce a lasting insulin secretion in diabetic dogs, rats, and KK mice through ribosylation of ADP on a regulatory guanine nucleotide-binding subunit of the islet membranal adenylate cyclase. A single injection of this protein produced a 5-day-long lowering of the blood glucose level and increased insulin secretion both in vivo and in isolated islets. However, neither the in vivo response to exogenous insulin nor the tissue sensitivity to insulin was improved. The authors concluded on the basis of these observations that tissue insulin resistance may be an independent defect in NZO mice. In contrast to oblob and KK mice, the concentration of plasma glucagon is considerably elevated in NZO mice, in spite of the prevailing hyperglycaemia and hyperin~ulinaemia,5~ which may contribute to insulin resistance and increased gluconeogenesis. Growth hormone levels are variable but suppressible by and are not likely to be involved in the resistance to insulin. No data are available on the adrenal function. NZO mice exhibit a number of kidney lesions not unlike human gl~merulonephritis;~~ however, it is doubtful whether these lesions are diabetesinduced, even though they may be potentiated by diabetes. Many substrains of non-diabese NZO mice manifest infiltration of several tissues by mononuclear but these autoimmune interactions are not specifically targeted to pancreatic islets. An autoimmune background of NZO diabesity is highly unlikely in view of the prevailing hyperinsulinaemia. NZO mice m a y thus be considered a model of non-immune NIDDM, and as such, the model requires further exploration of its early peripheral and hepatic insulin resistance leading to exaggerated gluconeogenesis, which occurs in the face of hyperinsulinaemia, despite a defect in first-phase insulin secretion.

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D. Diabetic C57BWKs db/db Mice The diabetes db gene mutation occurred spontaneously in the C57BL/KsJ strain of mice in Bar H a r b ~ r . ~It , ~is~ an autosomal recessive mutation on chromosome 4 with complete penetrance. The db gene can be coupled with the black coat colour misty gene (m)on chromosome 4 to allow early recognition of homozygotes of the mdb strains. The db/db mice are infertile, so heterozygous db/+ carriers are used to breed the mutants. C57BL/KsJ is an inbred strain distinct from the C57BL/6J strain, which serves as the recipient of the ob gene. A marked difference in outcome is evident on the Ks vs. 6J b a ~ k g r o u n d When .~~ the db gene is introduced into B6 mice, the diabetes is mild and resembles that manifested in the ob mutation. On the Ks background, the diabetes is severe and its time-course is bimodal. In the preweaning period of the dbldb mice, the events resemble those occurring in ob/ob mice:60 they become hyperphagic and hyperinsulinaemic while remaining normoglycaemic. After a few weeks their blood glucose gradually increases to very high levels, whereas their insulin levels and secretion capacity become greatly diminished. In recent generations of db/db mice bred in Bar Harbor, this bimodality has attenuated and the animals show early hyperinsulinaemia, hyperglycaemia, and deterioration in islet histopathology (E. Leiter, personal communication). Shifts in the onset and time-course of diabesity development may occur in mutant stocks in different localities. In the light of these observations, investigators should be encouraged not to depend entirely on published data and to establish their own reference values for animals maintained under their specific conditions. The response of the islets, whether this be long-lasting hypertrophy and hyperplasia (ob), or transient proliferation followed by atrophy and necrosis (db), appears to depend not so much on the particular gene, but on its interaction with background modifiers in the 6J or Ks genome. This was illustrated by the introduction of the ob gene to the Ks background, resulting in severe diabetes and B-cell d e g e n e r a t i ~ n . ~The ~ , ~exact ~ nature of the gene expression modification is not known and has been reviewed as mentioned above,2O but the observations described below point to the susceptibility of B-cell replication to hyperglycaemia as a possible effect. Experience to date with dbldb mice indicates

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that at about 1 month of age they are markedly hyperinsulinaemic and show hyperactive lipogenesis in the face of remarkable insulin resistance and gain considerable weight. The profuse insulin secretion initially compensates for the increased gluconeogenesis, but at the age of 2-3 months, the hyperglycaemia rises to 400-600 mg/dl (22-33 mmol/l) in spite of the six- to ten-fold increase in the circulating insulin levels (see Table 2&2 in ref. 1). After this peak, the hyperinsulinaemia recedes and the B-cells are depleted of insulin and show progressive degradation until collapse of insulin secretion. The mice lose weight, become ketotic, and do not survive longer than 10 months. Thus, the dbldb mice represent a striking example of a brittle pancreas and exemplify the importance of the genomic background in the course of the disease, particularly the susceptibility of the Bcells. Pancreas studies have shown that the basal insulin secretion in db/db mice is increased up to 4 months of age irrespective of the insulin content and that the response to glucose stimulation is also greater than that in non-diabetic siblings, but this stimulation gradually declines, parallel to the rising hyperglycaemia.61,62Incorporation of leucine into insulin and B-cell proteins is increased in 5-week-old mice, then decreases at 10-20 weeks to levels of non-diabetic controls.63 Glucose stimulation of leucine incorporation is also decreased at this time. Incorporation of thymidine into islets is increased even through the stages of falling insulin secretion, indicating an attempt to maintain insulin production and cell regeneration by increasing DNA synthesis.64Thymidine label was localized in the B-cells, not the A-cells, consistent with a selectively stimulated B-cell mitosis. With severe hyperglycaemia, DNA incorporation decreased. Light and electron microscopic s t ~ d i e s ~ have , ~ ~shown an expanded RER and Golgi apparatus in association with hyperglycaemia, degradation with compensatory hyperplasia, enlargement of B-cells, and necrosis of individual cells. There is also notable proliferation of ductal cells, which does not occur in other animals with diabetes. This may signify the mobilization of possible B-cell precursors to offset the ongoing degeneration of glucoseoverstimulated cells, but there is no overt evidence of neogenesis. Polyploidy, involving the genetic cell material, is prominent.66It possibly represents an adaptive B-cell response to stress, as degranulation was more extensive in polyploid than in diploid cells, suggesting a greater insulin release


capacity. These compensatory events in the Bcells do not seem to be so conspicuous in the current Ksldb mutants. Among other properties of the dbldb mice, the lack of suppression of glucagon secretion by glucose has been described;67glucagon oversecretion may contribute to the severity of diabetes as inferred from the observation that the injection of antiglucagon serum ameliorated the hyperglycaemia.68 The pattern of dbldb diabesity indicates a deleterious effect of long-standing hyperglycaemia on B-cell function, whether by direct toxicity or inability to sustain overstimulation, which consequently raises the possibility of prevention by dietary means. Simple diet restriction by pairfeeding with non-diabetic mice was shown to reduce the glycaemia and insulinaemia, but only delayed the onset of the hypoinsulinaemic stage.69 It was also observed that diet restriction reduced B-cell ~roliferation.6~ The dramatic results of Leiter et a1.70*71have demonstrated that the carbohydrate component of the diet is particularly detrimental: feeding an 83% protein, carbohydrate-free diet after weaning had a markedly beneficial effect on the survival of mice and the severity of diabetes. Obesity, hyperglycaemia, and insulin resistance were not abolished, but their onset was delayed and their magnitude diminished; furthermore, there was no islet degeneration at 6 months of age. In the absence of exogenous carbohydrate the gluconeogenesis from protein, the only source of endogenous glucose, was probably sufficiently adjusted not to exceed the vital needs inordinately, thereby sparing the 8-cells as well. Inclusion of as little as 8% of carbohydrate in the high protein diet, especially as sucrose or fructose, was already detrimental. Another protein-rich diet, administered by Chick and Like,72stimulated islet growth, B-cell replication, and thymidine incorporation into DNA, as well as insulin secretion. These results call for attention in devising regimens for the prevention of hyperglycaemia-induced pancreatic damage in animal and human NIDDM. There is always the consideration that high protein diets may be damaging to kidney function, but this may be true only when there is a preexisting, incipient nephropathy. In dbldb mice, there was no evidence of proteinuria after 4 months on the 83% protein diet, compared with the regular rodent Insulin resistance is very high in db/db mice, which do not respond either to exogenous insulin at 4 weeks of age58 or to the implantation of normal islets from co-isogeneic donors.74 The

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same islets were effective in streptozotocin (STZ)diabetic mice. At this point, it should be mentioned that islets which had been syngeneically implanted in the spleen of non-diabetic B/Ks mice performed poorly compared with the same islets implanted in B6 mice.75 This reiterates the importance of the genomic background for the function and survival of B-cells and their replicative capacity. Evidence that insulin is ineffective in suppressing the hepatic glucose production is provided by the higher than normal conversion of alanine to glucose despite hyperins~linaemia.~~ The synthesis rate of the regulatory enzyme of gluconeogenesis, PEPCK, which determines its activity, ceases to recognize insulin control. The hepatic PEPCK mRNA level, which in nondiabetic, or even in STZ-diabetic animals, is effectively reduced by physiological levels of insulin, requires enormous amounts of exogenous insulin (4 unitdl2 h per mouse for 2 days) to be This normalized in high protein-fed dbldb non-recognition of insulin is probably related to the deterioration in the signalling function of the insulin receptor, possibly as a result of the hyperinsulinaemia, failing to generate the stop message for PEPCK synthesis. The hepatic insulin receptors in dbldb mice were found to be decreased in number,13 and the muscle receptors diminished both in binding and in phosphorylation capacity.77Insulin receptors in fibroblast cultures from dbldb mice were also decreased in number and their response to insulin was impaired.78 These sporadic observations, which require more in-depth studies, hint that hyperinsulinaemia may be generally responsible for the lowering of receptor function. Insulin resistance does not encompass all metabolic activities: it does not affect either hepatic insulin-dependent glycolysis or lipogenic enzymes,’8,79 with the consequent result of hyperlipoproteinaemia. Adipose tissue lipogenesis is also enhanced, at least in the initial stages of diabesity. The glycolytic and lipogenic enzymes demonstrate increased activity, as does glycerokinase: and these factors are all involved in promoting fat accumulation. A hypothalamic defect in the dbldb mutants is suggested by the failure to respond to satiety signals, evident from the early emerging hyperphagia, even if hyperphagia per se may be a secondary rather than a primary phenomenon resulting from, rather than causing, hyperinsulinaemia. As mentioned before, this conclusion may

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be inferred from the results of food restriction,6’ which did not prevent obesity and hyperinsulinaemia. That hyperphagia signals are generated but not recognized was demonstrated by the striking results of parabiosis of dbldb and non-diabetic mice.E0 The non-diabetic mice died from starvation within 2-4 weeks, indicating that they had responded to the transmitted signals of the dbldb partners. This was also impressively demonstrated by parabiosis of dbldb with oblob mice: the latter stopped eating and lost weight up to death from ~tarvati0n.l~ However, when oblob mice were parabiosed with normal mice they lost weight autonomously. These experiments indicate that oblob mice, in contrast to dbldb mice, lack or produce defective satiety signals, but possess responsive satiety receptors. Further evidence on the hypothalamic defect of dbldb mice is supplied by the results of bilateral VMH lesion,a1 to which they respond with B-cell regranulation and amelioration of diabetes, including a reduction in gluconeogenesis. In contrast, normal VMHlesioned mice manifest hyperphagia, hyperinsulinaemia, obesity, and glucose intolerance. The catecholamines are elevated in the hypothalamus of dbldb mice;82 when reduced by hydroxydopamine a d m i n i s t r a t i ~ n a, ~body ~ weight loss and a fall in blood glucose levels occur. RenalE4and m i c r o v a ~ c u l adiabetic ~~ complications have been recorded in dbldb mice. The former were treated by diet restriction, by inhibiting intestinal carbohydrate breakdown with acarbose and other agents reducing the glycaemia, and showed a marked improvement.84 No cataracts on galactose feedingE6and no rise in osmotic fragility or lowering of Na/K ATPasedriven sodium pump in red blood cellsa7 were observed. However, neuropathy has been reported in dbldb miceEEdespite the presumed absence of the polyol pathway. On the other hand, the administration of statil, an aldose reductase inhibitor, prevented the appearance of galactitol and the reduction in conduction velocity in the sciatic nerve of galactose-fed dbldb mice?’ although a causal relationship remains to be proven. The possible scarcity of aldose reductase in dbldb mice may have a beneficial sparing effect on tissue NADPH concentration by promoting other NADPH-dependent enzyme systems, e.g. antioxidative glutathione reductase. In this context, the dbldb mice may be useful for studying the long-range consequences of diabetes distal to the polyol pathway.

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111. RATS WITH GENETIC DIABESITY SYNDROMES A. The Zucker fdfa Rats Fatty is an autosomal recessive mutation on rat chromosome 5. On the basis of relationships to the flanking genetic markers, f u is thought to be a rat homologue of the db gene on the mouse chromosome 4.’O The Zucker fatty rat has been extensively investigated for its insulin resistance associated with obesity. Recent reviews of Jeanrenaud,” Bray et ~ 1 . , 9and ~ Kava et comprise close to 1000 references. This rat was a chance result of a cross between Merck stock M and Sherman rats. The homozygotes develop early hyperphagia and extreme obesity by the general growth of all fat depots within 1 month (> 40% of the body weight), which makes it preferable for them to lie on their backs in the cages rather than to keep on their feet. The females are completely infertile and the males also have low fertility which may improve with hormonal support.94 Lean heterozygous f a / + rats are used for breeding and the incidence of obesity in the offspring is 25%. The obesity is accompanied by mild hyperglycaemia, striking hyperinsulinaemia, detectable as early as 2-3 weeks of age, and profound insulin resistance, all lasting throughout their lifespan. Ketosis is absent. The general aspects of their diabesity are reminiscent of the oblob mice. Among the differences is the notable general hyperlipoproteinaemia in the fulfu rats and, possibly, the causation of pancreas overstimulation. The fulfu rats have been hitherto used more as models of obesity than of NIDDM, perhaps because of their predominant weight gain and relatively mild hyperglycaemia. This may not be entirely justified since these animals have a marked glucose intolerance and insulin resistance which appears to be compensated by a robust and stable endogenous insulin output. In addition, a substrain of markedly hyperglycaemic fulfu rats, inbred by Peterson et u1.95p96 and referred to as Zucker diabetic fatty-ZDFIDrt-fa, offers many advantages for NIDDM investigation. Insulin hypersecretion is the most apparent cause of fulfu rat obesity, but several effector mechanisms have been postulated. The pancreatic islets of fulfu rats are hypertrophic and hyperplastic and sustain the mode of hypersecretion throughout life, showing a high rate of exocytosis and microtubule formation.97 However, fulfu rats

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oversecrete insulin even when pair-fed with their lean controls,98and the stimulus for hypersecretion in the absence of overfeeding is present in isolated B-cells for as long as 21 days, suggesting that the oversecretion is a separate, genetically determined f e a t ~ r e .The ~ ~dissociation ,~~~ between hyperphagia and hyperinsulinaemia is supported by long-term food restriction which does not prevent the hyperinsulinaemia, weight gain, and the induction of insulin-dependent enzymes in the liver and adipose tissue, instrumental in the enhanced lipogenesis and triglyceride accretion.101,102Destruction of B-cells by STZ and supplementation with exogenous insulin at different levels did not prevent weight gain and increased food c o n s u m p t i ~ n . 'All ~ ~ this ~ ~ ~does ~ not negate the well-documented role of hypothalamic dysfunction in the stimulation of insulin s e c r e t i ~ n ~with l , ~ ~the ~ additional involvement of a pituitary B-cell tropin.'& As mentioned earlier, the nature of the abnormality in the brain neuropeptide physiology is not fully known. There is evidence that fulfu rats have a lower than normal brain insulin contentlo7 and low insulin binding capacity in the arcuate hypothalamic nucleus,1o* and that the brain is less sensitive to exogenous insulin.lo9The defective hypothalamic response of the fulfu rat to insulin may elicit compensatory hyperinsulinaemia with subsequent resistance in other tissues. The signal for the increased insulin secretion may be through a direct effect of the central nervous system on the vagus nerve."* Which of these abnormalities is primary or secondary remains to be elucidated. As reviewed recently by York"' and reported by Bray et u1.,112 adrenalectomy or adrenal suppression in fulfu rats (which have enlarged adrenal glands and elevated corticosterone in the circulation) lowers the food intake, depresses lipogenesis and prevents fat accumulation (probably by diminishing the insulinaemia), and improves the insulin response of peripheral tissues. However, results to the contrary have also been presented, showing that adrenalectomy in suckling fulfu rats did not prevent the emergence of ~ b e s i t y . " ~ Insulin resistance in fulfu rats is not related to the extent of hyperinsulinaemia, as inferred by a comparison with VMH-lesioned rats in which resistance to insulin was not present under similar conditions of obesity and plasma insulin elevation.'ll In f d f u rats, insulin resistance and/or impediment in glucose transport were found in several tissues in vitro. In clamp experiments,

187

normalization of glucose metabolism was achieved only at insulin concentrations three- to four-fold higher than those in the lean control^."^ Liver insulin receptor activity was reported to be both decrea~edl'~ and increased,l16 and these findings require resolution. However, the binding of insulin to hepatocytes was found to be decreased without alteration of binding affinity and the removal of insulin was reduced by a similar extent.'17 In muscle the receptor activity is decrea~ed,ll~ whereas * ~ ~ ~ in adipose tissue it is increased,l19 but insulin sensitivity of adipose tissue decreased with age and size. In the liver insulin resistance is evident from unrestrained gluconeogenesis and glycogenolysis and increased PEPCK activity,'20 so that the postprandial hyperglycaemia in fulfa rats is to be attributed more to the lack of suppression of gluconeogenesis than to the retarded glucose removal.'*' Hepatic lipogenesis remains enhanced.lZ2 The question of whether insulin resistance precedes or follows the hyperinsulinaemia remains unresolved. Results of a longitudinal study have indicated that at 21 days insulin-stimulated 2-deoxyglucose uptake by the diaphragm of fulfu rats was found to be enhanced relative to controls; at 31 days it was similar and at 70 days it was reduced compared with controls. On the other hand, the mRNA of the insulin-dependent Glut-4 glucose transporter remained unchanged.lZ3Since hyperinsulinaemia was present at all times, it was concluded that muscle insulin resistance might not be the primary aetiological event, but might be secondary to other pathogenic factors. However, in the ZDFIDrt-fa rat, which is more hyperglycaemic and less insulinaemic, the expression of the Glut-4 transporter was unchanged in white muscle but substantially reduced in red muscle as a result of hypergly~aemia.~~~ The exocrine pancreas amylase activity in fu/fa rats is of interest. It is known that insulin is required for regular exocrine function, particularly for amylase synthesis and secretion, which is inhibited in insulin deficiency. The activity of this enzyme is also reduced in falfu rat^^^^,^^^ and in oblob mice,lZ7which indicates that not only lack of insulin prevents the expression of amylase mRNA, but also insulin resistance extends to the proximal acinar cells of the pancreas and precludes the induction of amylase synthesis. This also occurs in other conditions of insulin resistance, as exemplified by laboratory rats on high fat diets.lZ8Thus, both insulin deficiency and insulin

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resistance may leave an acinar insulin receptor lipogenic enzymes in the liver or adipose tissue. inactive and the amylase gene u n e ~ p r e s s e d . ' ~ ~ With respect to HDL-cholesterol, it should be A distinctive feature of fulfu rats is hyperlipoborne in mind that in rodents this lipoprotein is proteinaemia, encompassing all lipoprotein responsible for both the forward and the reverse classes, but predominantly the very low density transport of cholesterol. lipoprotein (VLDL)'30,131 synthesized mainly from As mentioned above, inbreeding for ten the dietary carbohydrate. Adipose tissue capacity generations of a substrain of fulfu rats, with higher to take up the VLDL-camed triglycerides is plasma glucose levels than in most other colonies, enhanced, as evident from the elevated activity resulted in a cohort of animals with non-fasting of the insulin-dependent lipoprotein lipase which plasma glucose levels in males of >175 mg/dl facilitates their uptake. This occurs early in life, (10 mmol/l) at 7 weeks, rising to >400 mg/dl even before the rats can be identified as obese. (22 mmol/l) at 10 weeks of age.96The hyperlipoThese observations have led to the suggestion proteinaemia in these ZDFIDrt-fa rats is as that the high lipoprotein lipase activity is interconpronounced as in the "regular" fulfu rats, but the weight gain and insulin levels are somewhat nected with hyperphagia in such a way as to lower. These rats are promising for the exploration ensure the filling of adipose tissue, thereby of eventual complications under the condition maintaining the high capacity of this enzyme in of hyperglycaemia, having as an asset all the spite of insulin r e ~ i s t a n c e . ~ ~ Recent ~ , ' ~ ~work accumulated background knowledge on the "regsuggests that the obese phenotype exerts a ular'' fulfu rats. In fact, preliminary observations coordinated control of the lipid storage-related on neuropathy have demonstrated decreased enzymes in adipose tissue at the transcriptional motor conduction velocity and morphological level, which is amplified by the hyperinsulinaechanges in tibial and sciatic nerves.143 Furthermia.134The gene expression of lipogenic enzymes more, a novel aspect, important for the underand of Glut-4 transporter in young fulfu rats standing of the aetiology of NIDDM, was discois greatly increased after weaning to a high vered in these rats: the pancreatic islet high K,, carbohydrate diet and is also correlated with facilitative glucose transporter Glut-2 was found hyperins~1inaemia.l~~ Conversely, muscle lipoto be underexpressed, suggesting the presence of protein lipase is d e ~ r e a s e d , 'in ~ ~accord with the a compensatory mechanism in hypersecreting Breciprocal behaviour of this enzyme in adipose cells. Assuming that NIDDM develops as a result and muscle tissues as in fasting or during insulin of the failure of B-cells to cope with insulin abundance. The marked hyperlipoproteinaemia resistance by increasing insulin secretion, the suggests that the liver is an important site of fatty reduced glucose entry into the B-cell, at the acid synthesis in fulfu rats, from which there is ambient hyperglycaemia of 20 mmolll, and the increased transport through plasma to adipose decreased stimulation of insulin output may be tissue, which serves mainly as the storage depot. factors involved in the causation of diabetic It is intriguing that the marked hyperinsulinahypergly~aemia.'~,'~~ emia with hyperglycaemia does not appear to lead to severe complications in fulfu rats. Controversial reports are available on h y p e r t e n ~ i o n . ~ ~ ~ , ' ~ ~ B. Wistar Diabetic Kyoto (WDFiTa-fa) and Recently some diabetes-related kidney lesions139 Wistar-Kyoto Fatty (WKYhJDrt-fa) Rats with intraglomerular fibronectin accumulation140 These rats were developed in Japan to provide have been reported. One of the treatments a model of adult-onset NIDDM and they were recently tested in fulfu rats was oral vanadate initially named WKY fatty.146To this end, the admini~trati0n.l~~ Plasma insulin decreased conlean heterozygote 13M fulfu rat was crossed with siderably. Glucose levels also fell, mainly due to the glucose-intolerant WKY non-fatty rat. The enhanced peripheral disposal rather than to history of crosses and backcrosses and of breeding inhibition of hepatic production. The impaired of these strains is summarized by Kava et ~ 1 . sensitivity of the cardiac muscle was particularly and Peterson et ~ 1 . 'They ~ ~ are now referred to as improved. Treatment with HMG-CoA inhibitor WDFITa-fa and WKYINDrt-fa. They are less obese lovastatin was effective in reducing the hypertriand hyperlipidaemic than the parent fulfu rats, glyceridaemia and high-density lipoprotein but more glucose- and insulin-intolerant in terms (HDL)-cholestero1.l4 The fall in triglycerides was of peripheral and hepatic re~istance.'~~ Glucose mainly due to the lowering of hepatic VLDL levels may rise to 300 mg/dl (17 mmol/l) in secretion and did not inhibit the activity of

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l ~ ~


association with a prominent 8-cell hypertrophy. A pancreatic defect, usually not present in other NIDDM models, of abnormal response to arginine was also described.150WDF-Ta-fa rats are sexually dimorphic in their spontaneous diabetes expression. Males are more hyperphagic and exhibit more readily the NIDDM-like syndrome. However, the predisposition to diabetes exists in females as well; when placed on a sucrose-rich regimen they show diabetic characteristics similar to those of males kept on a regular rodent ~ h o ~Several . ~ colonies ~ ~ ,of ~these ~ rats ~ have been established and investigated comparatively in the U.S.A. These substrains have exhibited several diversities in their metabolic-endocrine responses including a propensity to hypertension. This is an advantage for NIDDM investigation, since the diversity itself represents a syndrome with a wide spectrum of characteristics.

C. Spontaneously Hypertensive, Diabetic, Corpulent SHWN-cpand LAIN-cp Rat Strains These mutants with NIDDM were developed from two congenic strains, LA/N and SHR/N, at the National Institutes of Health (NIH), Bethesda, MD, USA. Both strains were obtained by introducing the cp gene of the Koletzky corpulent SHR strain, which carries an allele of the f d f a rat. The SHR/N inbred strain derives from the hypertensive Okamoto strain bred at Kyoto University. The substrain code N, which denotes the Genetic Resource Laboratory at the MH, signifies the origin and distinction of this strain from other SHR strains. Details on the development; the number of backcrosses which led to the development of the substrain, which vary in susceptibility to various complications and the nomenclature of these rats have been reported by and by Hansen.l% Greenhouse et In addition to SHRIN-cp, which manifests NIDDM with mainly renal complications,lS two important substrains were developed by backcrossing the cp mutation. When reintroduced into the SHR/N strain, a substrain denoted SHHF/Mcccp was obtained which exhibits congestive heart failure (CHF) on a hypertension background.*56 A further transfer to the inbred LAIN, which was derived from a cross between Albany/N and a hooded, not well characterized strain, produced a substrain denoted Jcr:LA-cp which manifests ischaemic heart disease on a hyperinsulinoemic b a c k g r 0 ~ n d . The l ~ ~ SHRIN-cp and LAn-cp strains

189

differ in the specific chromosome locus. For reproduction, congenic heterozygotes have to be used since the corpulent homozygotes (cp/cp) are not fertile. The mode of inheritance is autosomal recessive. Lean rats consist of two-thirds heterozygotes (cpI+) and one-third homozygotes (+/+). Mating yields a lean/corpulent ratio of 3:l recognized at 5 weeks of age. The important feature of the evolved cp substrains is the difference in the extent of hyperlipidaemia, obesity, hyperinsulinaemia, insulin resistance, and hypertension, as well as the propensity to specific complications. This fact demonstrates that even small differences in the genome are decisive for the selective susceptibility of the affected tissue and for the morphological and metabolic-endocrine impact of the lesions resulting from the genetic-environmental influences on the nature of the NIDDM derangement. Since all these strains are allellic with the fu gene, it has been proposed that the SHR rat group should be designated as cpfa or fucP.158Mating fa and SHHFIMcc-cp heterozygotes has indeed produced obese offspring, which indicates that fa and cp mutations are at the same locus. However, this does not prove that they are the same mutation (S. A. McCune and J. C. Russel, personal communications) and it is felt that these substrains are either different or specifically influenced by gene modifiers.

D. The SHIUN-cp Rat, Metabolic Properties, and Renal Complications Details on this rat and its sister strains, used for comparisons, are available e l ~ e w h e r e . ' ~ ~ - l ~ ~ The LAIN-cp males are normotensive, normoglycaemic, hyperinsulinaemic, and hyperlipoproteinaemic, whereas the SHRIN-cp males are mildly hypertensive, hyperlipoproteinaemic, and hyperinsulinaemic, and show islet cell hyperplasia. They are glucose-intolerant in response to a glucose load or on sucrose- or fructose-rich diets. Glucose intolerance is expressed in both sexes. Insulin receptor malfunction is suggested by decreased insulin binding by liver membranes163 and increased gluconeogenesis, although no decrease in the receptor tyrosine kinase activity has been detected. Post-receptor effects cannot be excluded. The rat corpulence (a term preferred by the authors to o b e ~ i t y l ~is~ evident l ~ ~ ) both as adipocyte hypertrophy and as hyperplasia. Its extent is lower than in f d f u rats, but it is associated with reduced thermogenesis.la Plasma concentrations of insulin counter-regulatory hor-

190

mones, such as corticosterone, glucagon, growth hormone, and somatostatin, are also elevated. Females are somewhat smaller in size, but exhibit higher hepatic lipogenesis and triglyceridaemia, even though the insulin levels are similar to those of males. The corpulence and glucose intolerance subside in both sizes with age. A characteristic feature and the predominant diabetes complication of SHWN-cp rats is early proteinuria and g l ~ m e r u l o p a t h y land ~ ~ inner ear hair The renal changes are consistent with both diabetic nephropathy and inflammation, and may be abetted by hypertension. Male and female rats show similar morphological changes of segmental, diffuse, and nodular intercapillary mesangial expansion. Long-term sucrose diet magnifies the glomerular changes, increases the proteinuria, and decreases the GFR compared with obese male LAIN-cp rats which do not develop glomerulopathy. The sucrose diet also accentuates the diabetic changes and hypertension in both the corpulent and the lean SHRIN-cp males.

E. The SHHFIMcc-cp Rat with Congestive Heart Failure

SHAFRIR

There is a sexual dimorphism in the expression of CHF as well as of other NIDDM elements, such as hyperglycaemia, hyperinsulinaemia, insulin resistance, and hyperlipoproteinaemia. Males die earlier of CHF than females and both earlier than lean litter-mates. The latter may also develop overt CHF and all those that died of CHF were heterozygotes for the rp gene. Plasma insulin clearance is slow. There are hints that the insulin resistance, hyperinsulinaemia, and hyperlipidaemia appear to be more closely related to CHF than to hypertension, and that those which carry the c p gene are more insulin-resistant than normal rats. Clinical symptoms of SHHFIMcc-cp rat CHF parallel those of human CHF. The rats develop pronounced cardiomegaly, oedema, hydrothorax, ascites, dyspnoea, and visceral hyperaemia. Plasma levels of ANF, aldosterone, norepinephrine, and renin follow a pattern similar to that of human CHF. Ultrastructural observations reveal degenerative changes in the cardiac tissue similar to human dilated cardiomyopathy, suggesting degradation of myocytes. Renal lesions are also present in SHHFIMccc p rats and are more pronounced in males than in females. These lesions resemble diabetic diffuse intercapillary sclerosis and are similar to those of SHRIN-cp rats, but are not severe prior to the onset of CHF.

It is now well known that obesity, hyperlipoproteinaemia, glucose intolerance, hyperinsulinaemia, and hypertension constitute a cluster of abnormalities associated with cardiovascular disease in humans.16s167 This cluster has often been referred to as ”syndrome X” or “plurimetabolic F. The Jcr: LA-cp Rat with Ischaemic syndrome”, because of an incomplete congruence Cardiovascular Disease with NIDDM or diabesity. Animals which may This strain, originally derived from backserve as models of this syndrome have been crossed LAIN-cp rats, is a closed outbred strain recently reviewed168and some of these belong to with only about 3% genetic contribution from the cp group. The cardiovascular disease may be expressed as atherosclerosis or as diabetes-related the obese SHR. As r e p ~ r t e d ” ~ ,and ’ ~ ~extensively cardiomyopathy. Many diabese animals, e.g., reviewed by Russell,172 these rats are insulinresistant, VLDL-hyperliproteinaemic but not oblob mice and falfa rats, which manifest several hypertensive and develop early atherosclerotic elements of this cluster, do not express cardiac complication^.^^^ The SHHFIMcc-cp rat has a lesions of the major blood vessels. Such lesions do not appear in the Zucker falfa or SHRIN-cp genomic background appropriate for the rats under similar conditions. The lesions were expression of cardiomyopathy dramatically preappraised morphologically and classified in four senting as congestive heart failure (CHF). This strain, maintained by McCune and c o l l e a g ~ e s , 1 ~ ~ , stages, ~ ~ ~ according to the development of damage: from chronic inflammatory cell infiltration (stage exhibits CHF in 100% of animals together with l),through myocytolysis and debris removal (stage hypertension (10Oo/o) and obesity (25%).The CHF trait has been maintained for more than 15 2), focal infiltration after completed scavenging process (stage 3), to well-developed collagen bands generations and potentiated by inbreeding. The with inflammatory cells, representing scars at mode of inheritance of the hypertension and CHF various stages of maturity (stage 4). These stages is probably multifactorial, but it is certain that are assumed to represent the evolution and repair the presence of the cp gene is essential for the of the initial ischaemic lesions. expression of CHF.


191

Some myocardial lesions have been observed carry an overt genetic defect; however, they may in SHRIN-cp rats, but they were very small and be considered as carriers of a genetic trait which did not progress beyond stage 2. These rats also limits the capacity of their metabolic system to had perivascular fibrosis, absent in the Jcr: LAadjust to the high nutrient intake. Several factors cp rats, which may be related to the hypertension. may be responsible: inability to provide sufficient The advanced lesions of stages 3 and 4 were seen amounts of insulin for dissimilation of the surplus substrate, constraints in the cellular metabolism only with high frequency in the Jcr: LA-cp rats, of the substrate, development of resistance to the presenting a picture of widespread cardiovascular available insulin, or an excessive sympathetic damage. In comparison with falfa rats, the outflow response with hypertension. Several Jcr: LA-cp strain showed a markedly higher models of this type of NIDDM evolution are insulin response to GIP and arginine and greater known, although only some are reviewed here. islet hyperplasia, as well as insulin resistance. Male Jcr: L-cp rats show the greatest incidence of myocardial lesions with occlusive thrombi in B. The Sand Rat (Psammomys obesus) the coronary arteries, which supports the premise The native habitat of this animal is the that these lesions are of an ischaemic, atherosclerdesert areas of North Africa and the eastern otic nature. At 9 months of age, there is almost Mediterranean, where it subsists on a herbivorous 100°/~incidence of atherosclerotic lesions in the diet. Its common name stems from the fact that major blood vessels. When the rats were subjected diabetes was discovered when these animals were to severe food restrictions, there was a marked collected from the sandy shores of the Nile delta reduction in stage 2 and 4 lesions, and those and transferred to the USA. The diabetes appeared which exercised strenuously by running on an a d libitum diet of regular rodent chow. In 6OOC-10000 m/day had no lesions at all.173Confact, the sand rat is a gerbil and should be referred sumption of 4 g/day of ethanol prevented the appearance of stage 4,but not of stage 2 1 e ~ i o n s . l ~ ~to as Psammomys. Psammomys exhibits diabesity when overfed relative to its energy intake in its All these treatments resulted in a pronounced native habitat, and the spectrum of abnormalities decrease in insulin resistance and in plasma may range from hyperinsulinaemia with insulin insulin levels. Nifedipine, a Ca2+channel antagonresistance accompanied by weight gain, through ist, also prevented the formation of stage 4 lesions hyperglycaemia, hypoinsulinaemia, collapse of and reduced stage 2 lesions without affecting insulin secretion, and ketosis. For details on the insulin concentrations or insulin resistance. stages of Psammomys diabesity and a synopsis of In view of the possibility that the severity early studies the reader is referred to publications and frequency of the lesions may be related to listed in ref. 1. hyperinsulinaemia, Russell et ~ 1 . ' proposed ~ ~ Most studies on Psammomys were performed that high plasma insulin induces the primary with the first generation of the imported animals endothelial damage, which in the face of hyperlipibecause of the difficulty in establishing and daemia progresses to atherosclerosis and large maintaining a long-lasting and reproducible colintimal injury. These vascular lesions are associated ony. Adler and c o - ~ o r k e r s 'have ~ ~ succeeded in with thrombus formation and vasospasm (possibly establishing a durable Psammomys colony in alleviated by nifedipine) and consequently are Jerusalem based on animals domesticated from responsible for areas of myocardial ischaemia, the Dead Sea area and existing now for more thereby shortening the lifespan of the animals. than 20 years. Initially the colony was maintained on a free-choice diet of salt bush Atriplex halimus, the native staple, and a limited ration of pelleted IV. NUTRITIONALLY INDUCED rodent chow. Later two diets were used, a NIDDM diabetogenic diet containing 60% carbohydrate and 10% fat, and a maintenance diet consisting of a regular rodent chow repelleted with 30% of In contrast to animals with genetically predefinely ground straw. The animals are transferred termined diabetes, which exhibit their derangeto the respective diets after weaning. On the ments spontaneously, with diet constituting only high energy diet, most of the animals become an ancillary factor, several non-diabetic animal hyperglycaemic within a few weeks,176 whereas species may acquire NIDDM when exposed to on the low energy diet they remain normoglycaenergy-abundant diets. They do not appear to

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The progress to diabetes is nutrition-dependent emic for several months, although a proportion of them may become insulin-resistant and hyperand is not gradual or consecutive. Some animals may transfer abruptly from Group A to Group C insulinaemic. Marquie's group177 in France has also established a colony of Psammomys derived or D, and some may transfer and remain in Group from Algeria, in which similar progression to B for a long period. Therefore, these groups should diabetes has been observed. It is of interest that not be regarded as representing consecutive stages the animals maintained in France have shown of progress into ketotic diabetes. Attempts are diabetic macroangiopathic complication^.^^^ In currently being made to isolate defined substrains the Jerusalem Psammomys colony, ongoing catarof Psammomys with and without a propensity to actogenesis is mainly o b ~ e r v e d , ~lo~osely ~ , ~ ~ ~hypergly~aemia.'~~ A further important characterrelated to the extent of hyperglycaemia. Neuroistic of Psammomys is the capacity to revert from pathy, manifested as hyperalgesia and measured the diabesity Group B or C to Group A on dietary by pain threshold by a paw pressure test and restriction. In Group D, the ketotic diabetes may motor nerve conduction velocity were markedly also be ameliorated by food re~triction:~ but reduced and correlated inversely with plasma there is no return to Group A. glycohaemoglobin level. It should be borne in mind that the PsarnrnoThe progression from incipient to fullymys Groups B and C are characterized by a fledged diabetes in Psammomys was described by striking peripheral and hepatic insulin resistance investigating more than 100 animals removed at with gluconeogenesis not suppressible by the random from the colony.lS1 The animals formed pronounced rise in endogenous insulin.18' At groups whose characteristics are reminiscent, in this stage of hyperinsulinaemia, the hepatic several aspects, of the progress of diabetes lipogenesis and adipose tissue lipoprotein lipase described in the genetically predetermined dbldb activity are greatly enhanced, which contributes mice (see Table 20-4 in ref. 1).Group A has been to both hypertriglyceridaemia and increased fat defined as normoglycaemic and normoinsulinatora age.'^^,'^ The importance of sustained hepatic emic, and Group B as hyperinsulinaemic with lipogenesis may be related to the fact that in considerable weight gain mostly in superscapular Psammomys, in contrast to other rodents but in white adipose tissue forming a "hump"; these common with humans, the liver is the predomianimals had hypertriglyceridaemia, but were still nant site of lipogenesis. There is negligible fatty normoglycaemic. In Group C, the plasma glucose acid synthesis in adipose tissue, which serves level increased considerably despite the continumainly as a fat depot.Ia3 ing hyperinsulinaemia, which reached a peak at To better define the nature of the nutritionthis stage; weight gain and hypertriglyceridaemia induced insulin resistance, we have recently increased, but there was marked pancreatic B-cell studied the binding of insulin by purified liver degranulation. Group D was characterized by a and muscle insulin receptors in Psamrnomys, further increase in glycaemia, weight loss, a taking the laboratory albino rat as a reference. decline in insulin levels, and B-cell necrosis. In Insulin binding, both by the purified hepatic Group E, comprising only a few animals, there receptors and by isolated plasma membranes of was ketosis associated with almost complete Bthe normoinsulinaemic Group A Psammomys, as cell loss. The animals did not persist in this stage well as the actual receptor number, was very low for more than a few days. This "pyramidal" timecompared with those of the albino rat185*l E 6 , course of rise and decline in insulin secretionla2 indicating a species characteristic of a diabetesis similar to that observed in dbldb mice, as are prone desert rodent. Consistent with this finding, the functional and histopathological changes in the clearance of insulin by the perfused liver of the B-cells. Psammomys was also about five times slower than However, when comparing Psammomys with by the liver of albino rat.lS7 Since insulin is dbldb mice, several important differences are to oversecreted in response to the affluent nutrition, be emphasized. Psammomys is not hyperphagic; these findings indicate that when hyperinsulinaethat is, it merely responds to food availability. mia appears on a calorie-rich diet, it may be at The progress to diabetes does not occur in all least in part due to the delayed removal and animals; a substantial proportion, nearly 3Oo/o, degradation by the liver. Slower insulin degraremain normoglycaemic and normoinsulinaemic dation was also reported in WDFITa-fa rats and (Group A) on the "free choice" diet but this in NZO mice, and may be a common feature proportion may decrease on the high energy diet.

ANIMAL MODELS OF NIDDM of the condition of hyperinsulinaemia, which probably suppresses the hepatic insulin receptors. The insulinaemia may be further potentiated by the fact that pancreatic islets of Psammomys respond very well to glucose stimulation and the glucose threshold for insulin release is even lower than in other rodents.188J89These observations suggest that the hyperinsulinaemia in Psamrnomys, as well as in other nutritional NIDDM-prone species, may be promoted both by profuse secretion and by sluggish removal. The hyperinsulinaemia may then be an element both precipitating the subsequent resistance and resulting from it. To investigate such a possibility we have determined the activity of the hepatic insulin receptor tyrosine kinase (TK), the enzyme elicited by the binding of insulin to the receptor which is thought to be instrumental in the transduction of insulin signals to intracellular metabolic systems. TK activity, measured by phosphorylation of an exogenous substrate, was very low compared with that of the albino rat r e c e p t ~ r . ' ~ ~To ,'~~ demonstrate TK activation by insulin, ATP concentrations were required in large excess over those of albino rat receptor TK, indicating that the K, of this enzyme for ATP is much higher than that of other species. The autophosphorylation on the insulin receptor was also very low compared with that of the albino rat, whether investigated by in vitro incubation with insulin, or after an intraportal injection of a bolus of insulin shortly before liver removal. These findings further support the contention that the low insulin binding is due to the small number of hepatic receptors rather than to the low binding capacity of the Psammomys receptor. In Psammomys Groups B and C, TK activity was found to be reduced to between half and a third of that of Group A (Table 11). This decrease in activity proved to be reversible by a 50% dietary restriction which placed the animals on straw-rich chow for 1-2 weeks. Hyperinsulinaemia was most probably responsible for the deterioration of receptor TK activity. When Psamrnomys of Group C recovered completely from the hyperglycaemia and hyperinsulinaemia and returned to the levels of Group A, TK activity also reverted to normal for the species. However, when the recovery was incomplete and Psammomys became normoglycaemic but remained hyperinsulinaemic (Group B), the activity of TK, though decreased, did not revert completely to that of Group A. It should be added that the correction

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of TK activity was associated with the return to normal of other hepatic regulatory enzymes of intermediary metabolism, such as pyruvate kinase, NADP-malate dehydrogenase, and PEPCK (Table 11). The fact that hyperinsulinaemia is detrimental to the insulin-receptor function has been shown in several situation^.^^'^^ In particular, transgenic mice with a multiple dose of insulin gene had pronounced hyperinsulinaemia and also manifested insulin resistance and insulin receptor f a i 1 ~ r e . l ~ ~ It may be concluded that Psarnmomys in the desert is a healthy gerbil, well adjusted to subsistence on a low caloric density food and endowed, through evolutional selection, with an endocrine-metabolic pattern appropriate for survival. The dietary affluence unmasks a predisposition to diabesity, hyperinsulinaemia, deterioration of cellular signalling, and pancreatic breakdown. This is a paradox of plenty, where existence is secured possibly by a "thrifty gene",193 but survival is endangered by transition to conditions of lasting satiety. This is just the opposite to the case of organisms accustomed to affluence when subjected to starvation. It is possible that the central site of this bidirectional regulation is the insulin receptor, though this hypothesis needs further substantiation. The great asset of animals like Psammomys is the possibility of applying various strategies for reversal of the nutritioninduced NIDDM. A further advantage is modelling for the mechanisms leading to the metabolicendocrine changes provoking NIDDM and obesity, hyperinsulinaemia and insulin resistance in calorically underprivileged human populations emerging into caloric abundance, e.g., the Australian Aborigines and the American Indians, to name but a few.

B. NIDDM in Non-human Primates Diabetes is known to occur in several primate species. In some, it is associated with autoimmune phenomena and amyloid d e p ~ s i t i o n , ' and ~ ~ in others, it appears without any immune element. Among the latter are some closed colonies with ad libitum access to One of the best researched, long-standing colonies is that of the rhesus monkeys, Macaca mulatta, maintained by Hansen and co-workers.19g-zwClearly, the amount of food available and consumed by these monkeys is by far greater than that in their native habitat, and in addition, the animals have a low energy expenditure due to their sedentary mode of life;

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Table 11. Effect of Diet Restriction on the Activity of Hepatic Insulin Receptor Tyrosine Kinase (TK) and of Glucoregulatory Enzymes in Psamrnomys oberus

Group

Plasma

Plasma

glucose (mmol/l)

insulin (mu/l)

P-enolphosphate carboxykinase (nmol/min per mg protein)

NADP-malate dehydrogenase (nmol/min per mg protein)

TK

(pmol/min per mg protein)

Animals kept ad libitum on a mixed salt-bush and regular chow diet A (12)

c (8)

3.9 k 0.3 15.3 f 1.5

158 f 11 316 f 21*

37 5 6 314 2 48

115 2 13 273 2 18*

18.5 ? 2.1 8.1 5 1.0*

102 f 9 t 180 2 16

19.8 5 2.0t 14.9 & 3.8

Recovered from stage C diabetes on half diet ration for 1-2 weeks C to A (8) C to B (6)

4.3 f 0.6 4.5 f 0.8

46 2 7 181 f 29

140 f 16t 202 f 18t

Values are means f SE for the number of animals indicated in parentheses. Insulin receptor tyrosine kinase activity was measured after purification on wheat germ agglutinin columns by phosphorylation of an external substrate. *Differencefrom Group A Psammomys significant at p < 0.02 at least. tDifference from Group C Psammomys significant at p < 0.05 at least. Unpublished experiments similar to those performed in refs 73, 185, and 186.

consequently, the NIDDM evolving in these conditions may be classified as nutrition-induced diabesity . The macaques in this colony develop NIDDM gradually. In longitudinal studies spanning over many years, several phases of the development of the disease were identified according to metabolic-endoaine and body weight change^.^^^,^^ Phase I comprises monkeys more than 10 years old (fasting glucose level of -60 mg/dl, 3.3 mmol/l), and Phases 2-3 comprise older monkeys, both lean and obese, with no appreciable deviations from normal in blood glucose or insulin even during an intravenous tolerance test. Significant hyperinsulinaemia was noted in macaques of Phase 4, which were all overweight and showed fasting insulin values of 160 vs. 40 munitll in Phase 1. Body weight peaked in Phase 5 at 19 kg. Plasma insulin peaked at Phase 6 at 415 munit/l and declined at Phase 7 despite continuing hyperglycaemia, indicating the impending loss of secretion. Overt diabetes, weight loss, and B-cell failure occurred in all monkeys of Phase 8 (10-20 years old). In Phase 9, the euglycaemic clamp procedure revealed significantly increased hepatic gluconeogenesis. The monkeys also showed increases in VLDL-triglyceride levels coupled with a decrease in HDL, in correlation with hyperinsulinaemia.200These studies correlate with the observations recorded in relation to Psammomys and underscore the role of nutrition in the expression of a latent predisposition to diabesity

-

-

in animal species accustomed to survive in the wild on a modest regimen, and subsequently offered unlimited access to food.

B. The C57BW6J Mouse as a Diet-induced and Stress-related Model of NIDDM The B6 oblob mice, although only moderately hyperglycaemic, may become significantly hyperglycaemic when stressed.6 This may originate from their genomic background, the non-obese, non-diabetic BL/6J mice, which are sensitive to adrenergic stimulation produced by an appropriate diet.201 This would suggest that an altered sympathetic response is linked to impaired glucose tolerance in these animals. Latent genetic factors, responsible for this effect, may become expressed in BL/6J mice on a high caloric density diet, since this mouse strain was shown to be somewhat insulin-resistant with a weak first phase of insulin r e l e a ~ e . ~ ~ ~ , ~ ~ ~ As reviewed recently by Surwit and Kuhn,ZD4 this hypothesis was tested by feeding a 36% fat, 37% sucrose, and 21% protein diet, which resulted after 6 months in an average weight of 55g in BL/6J male mice vs. 40 g in control A/J mice. The same mice on a regular diet weighed only 28 g. There was an accentuation of mesenteric fat deposition in the BL/6J mice. Fasting levels of glucose became elevated prior to hyperinsulinaemia, suggesting insulin resistance. The role of neural factors was indicated by greater hypergly-


caemia after exposure to epinephrine or stress. Other mice strains, when introduced to the same diet, failed to show such a response. The male BL/6J mice developed hypertension on the experimental diePo5 which was eliminated by ganglionic blockade, suggesting a sympathetic origin. There was no hyperphagia or elevation in circulating corticosterone. A rise in insulin, occurring later, did not have an effect on hypertension. These experiments illustrate that animals showing no overt disturbances in glucoregulation may have hidden vulnerabilities, which become evident upon challenge from high energy nutrition. In this particular case, the diabesity did not result from an oblob-like hyperphagia, but rather from differential metabolic handling of the specific diet. While obesity does not necessarily result in fully-fledged NIDDM, it may be attributed to an additional factor present in the lean state and expressed when the animal becomes obese. This may be, for example, due to a defect in the sympathetic nervous system causing an overstimulation of the outflow in obesity or due to a specific dietary intake. The authors undertook several genetic strategies to demonstrate that insulin resistance and hyperglycaemia are controlled by different genetic factors, and that insulin sensitivity and impaired pancreatic activity may be different phenomena. They propose that insulin resistance is not simply a function of body weight and may be inherited independently, and also maintain that the lean BL/6J male mouse is a far better model of human NIDDM than the diabese rat or mice mutants with multiple endocrine aberrations. The application of their findings to the human development of NIDDM is strengthened by the fact that many elements of the mouse genome are conserved in humans.

V. DIABETIC ANIMALS PRODUCED BY SELECTIVE INBREEDING OF NORMAL POPULATIONS A. The Goto-Kakizaki (GK) Rat

Most of the animals with diabetes described here were discovered and/or inbred after a chance observation of inappropriate hyperglycaemia or another anomaly related to diabetes. A working hypothesis was constructed which proposed that NIDDM can be selected from normal rats by repetitive inbreeding at the cut-off point of upper

195

10% of normal glucose tolerance areas.*06Indeed, a diabetic pattern became discernible after the tenth generation of such animals from a Wistar rat pool which had been maintained on a regular rodent chow. The sister-brother inbreeding was continued up to 35 generations, at which time there was no further increase in glucose intolerance and the characteristics of the colony became stable. This experiment, described in more detail elsewhere by Suzuki et dZo7 indicates that the inheritance of NIDDM is polygenic. All offspring of the GK rats are similarly affected and hyperglycaemia is present at the end of the first 2 weeks. The authors’ concept of “multifactorial inheritance” is best understood as an overexpression, potentiation, or defective integration of factors dependent on several genes related to endocrine and metabolic pathway functions as well as the formation and function of islet cells. GK rats are non-obese. Their impaired glucose tolerance neither improves with age nor deteriorates with further breeding, and does not proceed to a ketotic stage. They present increased activity of the hepatic glycolytic and gluconeogenic pathways, indicating insulin resistance, which is further evident from non-suppresion of the hepatic glucose production in clamp studies. Their pancreatic islets become deformed in shape at 2 months of age, but do not increase in total area. This is related to the deposition of fibrous, nonamyloid material in the islets. The percentage of A-cells is higher and that of B-cells lower compared with control rats of the same stock. At 6 months of age, the 8-cells diminish in number and partially degranulate in association with a decrease in the insulin and glucagon contents. The islet DNA content is relatively low. Few Bcells remain in the aged GK rats, but no lympocytic infiltration has been observed throughout their lifespan. The in vitro responses of the isolated islets of GK rats were studied in the Stockholm colony208 and certain peculiarities were detected: for example, there was a diminished insulin response to glucose and glyceraldehyde but not to other secretagogues, and glucose cycling was increased in the GK islets compared with controls, suggesting a link between the impaired insulin release and abnormal glucose metabolism. Stimulation of insulin secretion by glucose was also found to be decreased in a perfused pancreas preparation in the Paris GK rat colony.20gAlthough the basal insulin secretion was higher than that in controls, glucose did not elicit a substantial increase in

196

insulin output. There was a paradoxical tendency to a decrease in insulin release relative to the duration of exposure to glucose and resumption of release on glucose withdrawal. Neuropathy and glomerulopathy are present despite mild hyperglycaemia.210 The nerve conduction velocity is low at 2 months and the total fascicular area, visualized by electron microscopic morphometry, is not decreased at 6 months, although the number of myelinated nerve fibres is significantly reduced.211 Such a reduction could not be detected in the young rats despite the retarded conduction velocity. Nerve levels of glucose, sorbitol, and fructose were found to be increased, rising gradually with age, while the levels of myoinositol decreased. The ultrastructural changes in the kidney develop very slowly and are manifested primarily by thickening of the glomerular basement membrane, first discernible at 12 weeks.

B. The Cohen Sucrose-induced Diabetic Rat Similar selection and potentiation of the diabetic trait in a normal rat pool were obtained by Cohen et d 2 1 2 by placing the animals on a synthetic 72% sucrose-copper-poor diet for 2 months. A “two-way selection” was used, in which rats with the highest and lowest responses to a glucose tolerance test were mated with the offspring returned to the diabetogenic diet after weaning. After four to five generations, a diabetic line with plasma glucose of -18mmol/l was obtained as opposed to the downward selection line which remained normoglycaemic even on a sucrose-rich diet. The diabetic rats are non-obese but hyperinsulinaemic and insulin-resistant with a hepatic enzyme pattern typical of insulin resistance.213They gain less weight with age than the non-diabetic controls and become insulindeficient but remain non-ketotic. The Cohen rats exhibit many complications-renal, retinal, skeletal, reproductive, and fetal-extensively described elsewhere.’14

B. NON Mice These non-obese, “non-diabetic“ mice were bred in Japan from the same CTS line from which the NOD mice were derived, but they do not exhibit autoimmune a e t i o l ~ g y The .~~~ haplotype of NON mice is H-2b, distinct from the H-29 of NOD mice. Details on the MHC features of NON

SHAFRIR

mice,216 and immunological differences between NON and NOD mice217 have been recently described. Nine-week-old male NON mice are normoglycaemic in the fasting state, but display impaired tolerance to a glucose load (IGT), and their postload insulin/glucose ratio is lower than that in females.215The acute phase insulin response is absent in the perfused pancreas and its insulin and insulin mRNA content is about one-third that of controls.218 However, pancreas secretion capacity is durable since islet hyperplasia occurs with a rise in insulin secretion after VMH lesion with gold-thioglucose. Thus, the NON mouse may be defined as an animal with IGT due to an “intinsic” B-cell defect in insulin secretion and synthesis capacity, but responsive to B-cell proliferation triggered by hyperphagia or direct hypothalamic stimulation. It should also be considered that NON mice fed on an American laboratory chow responded with hyperinsulinaemia and became obese at 20 weeks of age (E. Leiter, personal communication). They also develop glomerular lesions with lipid depo~ i t i o n The . ~ ~ pathogenesis ~ and genomic background of such nephropathy may be of interest because of its occurrence in a mildly hyperglycaemic animal.

VI. RATS WITH SPECIAL ASPECTS OF NIDDM-LIKE DIABETES A. Diabetes Involving Endocrine and Exocrine Pancreas A Wistar rat (WBN/Kob) with a spontaneous fibrotic lesion in both the endocrine and the exocrine pancreas has been described by Mori et al. 220,221 and recently reviewed in A recessive gene appears to be responsible for the lesion, which affects most males of the colony at the age of 1 year. The onset of diabetes is accelerated by a high energy diet, although the cumulative incidence in males reaches 100%. The hyperglycaemia and other features of the disease are associated with weight loss rather than obesity and the animals do not appear to be insulinresistant, since they respond well to exogenous insulin. The characteristic features of the lesion in the pancreas are decreases in A- and B-cells, insulin and glucagon contents, and amylase activity. With age, the decrease in B-cells, compared with A-


cells, is more prominent. Insulinopenia, weight loss, and hyperglycaemia develop slowly in association with multifocal fibrosis of the whole pancreas. Infiltration by inflammatory cells is noted along with fibrous exudation and deposition of a haemosiderin around the pancreatic ducts at the age of 3 months together with enlargement of interlobular lymph nodes. However, the immune background, if any, and the morphology are markedly distinct from those observed in BB rats or NOD mice although T-cell lymphopenia was observedzzz. Insulin treatment is not required for survival. The pancreas becomes atrophic at advanced age with extensive fat infiltration. Females exhibit only mild fibrotic changes, which may be potentiated by ovariectomy. Ocular, renal, and neural lesions, typical of diabetes, have been observed. Peripheral opacity of the lens is evident at the age of 15 months with progress to a full cataract in all males at 24 months. Albuminuria becomes extensive at about 1 year of age. Morphologically, thickening of the glomerular basement membrane and enlargement of mesangial areas are seen. Apart from a reduction in nerve conduction velocity, there is a marked decrease in the density and diameter of myelinated fibres of the sciatic nerve together with increased activity of the polyol pathway. The animal is of interest since the gradual loss of both endocrine and exocrine pancreas, with generalized pancreatitis, possibly of autoimmune aetiology, is a not infrequent finding in dogs, cats, and other domestic animal^.^^^,^^^ 8. The eSS Rat with Spontaneous, Lateonset NIDDM

The eSS rats, bred in Argentina, are characterized by a slow onset of glucose intolerance, more severe in males, which becomes conspicuous at about 1 year of age.zz5~zz6 There is a loss of Bcells due to islet disruption by fibrotic lesions confined to the endocrine pancreas. No overt hyperglycaemia precedes the islet lesion, although impaired glucose tolerance is observed at 2 months. This age- and diet-dependent NIDDM may be due to prolonged inbreeding. The colony development of the eSS rat and the basically polygenic inheritance pattern of the strain, together with its morphological and metabolic characteristics, have been extensively described.2z7 The rats are slightly corpulent, hyperinsulinaemic, and insulin-resistant. They are also hyperlipidaemic, with rises in both

197

triglyceride and cholesterol levels. Caloric restriction attenuates the progress of the disease. On the other hand, a high energy diet accelerates the onset of the disease and makes it more conspicuous in terms of metabolic abnormalities, chronic complications, and shortened lifespan of the animals. These observations demonstrate that beyond their genetic defect, eSS rats are unable to adapt to hyperalimentation, even if they are not overtly obese. The islets of 6-month-old eSS rats show a disrupted pattern with an increase in interstitial tissue, vacuolization, and the formation of groups separated by fibrous deposits. There is no lymphocyte infiltration or other evidence of autoimmunity. An ongoing regenerative process involving endocrine tissue integrating with the ductal epithelium may be observed. This suggests a compensatory hyperplasia (nesidioblastosis). At 1 year of age, the islets become smaller and scarcer; the insulin content diminishes; and marked scarring and necrosis are evident. The A-, D- and PP-cells are irregularly distributed through the islets. The exocrine pancreas is completely normal. At 18 months, there is a markedly diminished Bcell mass and almost total islet exhaustion. Chronic complications occur in the eSS rats, primarily in the kidneys, which exhibit focal, interstitial, and pyelic inflammatory infiltrates. With age, the glomeruli become markedly lesioned, showing a diffuse hypertrophy of mesangial tissue, thickening of basement membrane, and a reduction in capillary lumen. Tubular disruption proceeding to necrosis can also be 6 months and observed. Proteinuria starts at pronouncedly increases by year 1. In the nerves of 18-month-old animals, there are alterations in the myelin structure consisting of a split sheath next to the axons. Lens alterations are also apparent, with capsular and subcapsular cataracts in both eyes as well as total bilateral cataracts.

-

VII. CONCLUSION The animal species reviewed here represent a medium for investigation of endocrine, metabolic, and structural alterations occurring in the diverse forms of the human NIDDM syndrome (see comparative synopsis in Table 111). None of the selected models manifests the full spectrum of functional or degenerative changes reported in humans. However, each one offers access to

(2) Metabolic-endocrine anomalies

Obesity, hyperinsulinaemia, insulin resistance with neuroendocrine dysregulation; no response to satiety signals; defective thermogenesis; decreased insulin receptor function Obesity promoted by high energy nutrition; insulin resistance with hyperglycaemia Obesity, insulin resistance; insulinaemia less severe than in ob mice

Hyperinsulinaemia with obesity, then hypoinsulinaemia and ketosis; hyperphagia and faulty satiety signals; resistance with low insulin receptor response; carbohydrate diet detrimental Obesity, hyperphagia, hyperinsulinaemia, and resistance similar to their gene homologues, ob mice; obesity develops even on restricted diets; probable brain insulin resistance; hyperlipidaemia

(1) Species

C57 BL/6J ob mice

KK mice

NZO mice

C57BL/KS db mice

Zucker fa and ZDRt-fn rats

Neuroendocrine effects as in ob mice; insulin regulation of lipogenesis vs. gluconeogenesis; intrinsic islet mechanisms causing hypersecretion; antidiabetic drugs (ZDF/Drt-fu)

Effect of genomic and nutritional modifiers on B-cell replication and survival; drugs mitigating insulin resistance and receptor function

Selectivity of tissue insulin resistance; body fat distribution, islet glucose metabolism, and stimulussecretion coupling

Renal lesions not necessarily related to diabetes

Renal and vascular lesions; neuropathy not necessarily related to polyol pathway

Muscle and liver insulin resistance; effects of antidiabetic drugs

Neuroendocrine factors; satiety and hypothalamic insulin responses; thermoregulation

(5) Research suitability

Vascular microangiopathy; nephropathy

Minor complications, infertility

(4)

Lesions and complications

Insulin hypersecretion Renal changes; possible persists in isolated islets; hypertension; infertility separate gene or neurogenic factor effects

Labile islets; hypertrophy and enhanced replication followed by cell degeneration

Loss of first phase release but persistent oversecretion; impaired islet glucose metabolism

Changes as in ob mice; oversecretion does not lead to cell necrosis

Profuse and lasting insulin oversecretion, hyperplasia, and hypertrophy; sensitivity to neurogenic stimuli

(3) Pancreatic function

Table 111. Synopsis of the Characteristics of Animals with NIDDM-like Syndromes

Genomic predisposition; preventive measures, including Ca2+ channel blockade; role of hyperinsulinaemia

Genomic characteristics leading to hypertension on high energy nutrition; hyperinsulinaemia and hypertension Insulin secretion abnormalities; drug effects in moderate, insulin-resistant diabetes

Ischaemic coronary lesions with atherogenesis and thrombi

Initial oversecretion with Cataracts; cell hyperplasia followed macroangiopathy; kidney by necrotic degeneration lesions; neuropathy manifested as hyperalgesia

Not described

Nephropathy and neuropathy

As above

Lasting insulin oversecretion

Islet deformation and secretion abnormality; gradual 8-cell loss

Obesity; hyperinsulinaemia and resistance; no hypertension; hyperlipidaemia; sexual dimorphism Overnutrition-evoked hyperinsulinaemia resistance, obesity, and hyperlipidaemia lapsing into hypoinsulinaemia, severe hyperglycaemia, weight loss, and ketosis Susceptibility to high energy diet evoking hypertension, catecholamine secretion; hyperinsulinaemia and obesity Non-obese, mildly hyperglycaemic; insulinresistant, non-ketotic

Jcr:L A - c ~ rats

Psnmmomys obesus gerbils (sand rats)

C57BL/6J mice

GK rats

Continued on next page.

Ovemutrition-induced insulin resistance; low insulin degradation; low islet secretion threshold; reversible insulin effect on receptor function

As above; role of Ca2+ channel blockade in CHF alleviation

Congestive heart failure (also in heterozygotes) and nephropathy

As above

Interrelation of hyperinsulinaemia, hypertension, and nutrition on kidney lesions; genomic predisposition to these lesions; preventive measures

Severe and diffuse nodular glomerular changes

Obesity; hyperinsulinaemia and resistance; hypertension; hyperlipidaemia; sexual dimorphism

Suitabilities similar to Zucker f a but greater colony diversity; hyperlipidaemia and diabetes

Nephropathy

SHHFIMCC-cp rats

Long-lasting secretion Corpulence, with hyperplasia persists hyperinsulinaemia, and resistance accentuated by sucrose diet; rise in counter-regulatory hormones; hypertension; hyperlipidaemia

SHRIN-cp rats

B-cell hypertrophy and hyperplasia; abnormal response to secretagogues

Hyperphagia, obesity, and insulin resistance similar to f a rats; more pronounced lipidaemia and diabetes

WDF-TA/fu and WKYINDRt-fa rats

Nephro- retino-, and osteopathy, testicular degeneration

Fatty glomerular lesions

Cataracts, renal and neural lesions

Cataracts, neuropathy, tubular and glomerular kidney pathology

Defective first phase and stimulated release

Mild oversecretion despite partial insulin deficiency

Non-obese, h yperglycaemia, transiently hyperinsulinaemic, then overtly diabetic Non-obese, inborn insulin synthesis deficit; no autoimmune involvement; develop obesity on high energy diet Disappearance of Non-obese, gradual hypoinsulinaemia due to both B- and A-cells fibrotic, inflammatory (immune?) exo- and endocrine pancreas destruction Non-obese, slow onset of Fibrotic lesions, islet vacuolization, end-stage IGT,then B-cell loss hypoinsulinaemia; changes accelerated on high energy diet; hyperlipidaemia

NON mice

WBN/Kob rats

eSS rats

Cohen sucroseinduced rats

Lesions and complications

Pancreatic function

Metabolic-endocrine anomalies

Species

Table 111. Continued

Deleterious responses to hyper-alimentation without obesity; origin and pattern of islet changes

Co-existence of exo- and endocrine pancreas injury; preventive (immunosuppressive?) measures

Intrinsic islet defect in insulin production and secretion; kidney injury in mild diabetes

Interrelation of high sucrose and low Cuz+ on diabetes induction

Research suitability

0 0

N

ANIMAL MODELS OF NIDDM tissues a n d cells not readily available in larger mammals and provides an opportunity for testing of preventive strategies. A further advantage is the possibility of conducting the investigations within a time-frame not feasible in longitudinal human research. Since h u m a n NIDDM is a cluster of diverse diseases leading to insulin resistance and/or islet exhaustion by different mechanisms, the various animal models provide a valuable means for the investigation of the multiple causes of these abnormalities. The importance of the dietary and environmental factors, as well as of genomic modifiers, further extends the suitability of the animals for the study of diabetic complications. One must bear in mind, however, that these animal models are not miniaturized replicas

201 10. Baetens D, Stefan Y, Ravazolla M, Malaisse-Lagae

11.

12.

13.

14.

of the total human disease, and they should be used for the study a n d extrapolation of certain defined aspects of similarity with human NIDDM.

Acknowledgement Sincere thanks are due to Dr Edward H. Leiter, Jackson Laboratory, Bar Harbor, ME, U.S.A., for helpful comments on the manuscript.

15. 16.

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