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ILAR Journal V32(3) 1990
New Rat Models of Obesity and Type II Diabetes

Introduction

Following isolation of the mutant corpulent (cp) gene (Koletsky, 1973), rats containing it were initially bred into two standard strains: the LA/N and the SHR/N (Hansen, 1983). The progeny were then backcrossed more than 12 times to the parent strains to yield two congenic strains: LA/N-cp and SHR/N-cp.

The fully backcrossed, congenic strains of rats incorporating the corpulent gene are defined and stable inbred lines. The partially backcrossed strains, Jcr:LA-cp (five backcrosses) and SHHF/Mcc-cp (seven backcrosses), retain some genetic contribution, reflected in metabolism and disease expression, from the original Koletsky colony. The Jcr:LA-cp strain, corpulent (cp/cp) or lean (+/+ or +/?), is phenotypically indistinguishable from the LA/N-cp. There are, however, major metabolic differences between Jcr:LA-cp and other strains carrying the cp trait, including the La/N-cp. The Jcr:LA-cp also exhibits a unique pattern of atherosclerotic and myocardial lesions. This strain provides an unusual opportunity to study the metabolic aspects and mechanisms leading to spontaneous vascular and myocardial disease in an animal model. In particular, we have shown that this strain is a close model of the human syndrome, which is characterized by obesity, hyperlipidemia, insulin resistance, and a high risk for cardiovascular disease.

Genetics

The Jcr:LA-cp strain was derived from the LA/N-cp at the fifth backcross to the parent LA/N strain. Nucleus breeding stock of one male and three female rats was generously donated by C. T. Hansen of the Veterinary Resources Branch, National Institutes of Health, Bethesda, Maryland. These animals were used to establish the colony at the University of Alberta. The colony has been maintained as a closed outbred colony since establishment (approximately 25 generations). Nine defined sublines are maintained and selectively mated in rotation to minimize inadvertent bias in the breeding (Poiley, 1960).

Pathologic Findings

During our early studies on the Jcr:LA-cp rats, we discovered the presence of myocardial lesions in middle-aged (nine-month-old) rats. Scanning electron microscopy studies revealed the presence of early atherosclerotic lesions in cp/cp male rats (Russell and Amy, 1986a,b). In corpulent male rats, both the myocardial and vascular lesions develop in an age-dependent manner. Female rats are spared both types of lesions in a manner analogous to that seen in human females. At advanced ages (20+ months) the incidence of large raised intimal lesions on the aortic arch of cp/cp male rats reaches 100 percent. At such ages, cp/cp female rats also exhibit large raised intimal lesions. Lean rats do not develop significant vascular or myocardial lesions. The atherosclerotic lesions--including nuclear dropout, desquamation of endothelial cells, polygonal cells, large raised intimal lesions, and occlusive thrombi--are the same as those reported by other investigators in non-rat animal models of atherosclerosis, such as pigeons, swine, rabbits, and nonhuman primates. Examination of arteries by transmission electron microscopy has shown the raised lesions contain lipid, cellular debris, and cells in abnormal subendothelial positions (Heisler et al., 1987). These cells include smooth muscle cells migrating through the internal elastic lamina and endothelial cells in the subendothelial space. There are also macrophages adhering to the lesions and penetrating the endothelium. The lesions in the heart range from nodules of chronic inflammatory cells to areas of necrosis and mature scarred lesions. The incidence of myocardial lesions mirrors that for the vascular lesions. They appear to be of ischemic origin, a conclusion supported by the occasional finding of occlusive thrombi in major arteries of corpulent rats, including the coronary arteries.

Lipid Abnormalities

We have extensively studied the lipid abnormalities of the Jcr:LA-cp rat that are present only in cp/cp animals. Our work has revealed that the hyperlipidemia is primarily due to elevated concentrations of large (622A) triglyceride-rich very low density lipoprotein (VLDL; Dolphin et al., 1987). This lipoprotein contains both apolipoproteins B₁ and B_h. There are also increased levels of a phospholipid-rich high density lipoprotein (HDL). The result is a hypertriglyceridemia that is more severe in females (512 mg/100 ml) than in males (282 mg/100 ml) at 3 months of age. Both sexes show a modest hypercholesterolemia: 74 and 84 mg/100 ml for males and females, respectively. There are smaller elevations in apolipoproteins B and E in the corpulent rats, and apolipoprotein A-1 levels are approximately tripled. The levels of intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and HDL cholesterol are significantly lower in the corpulent males compared to females. Thus, the lipid abnormalities, although very significant, do not correlate well with the vascular and myocardial disease, as the more severely affected females do not develop vascular or myocardial disease until advanced ages. This finding in Jcr:LA-cp rats mimics the human case as shown by major epidemiological studies.

As the rats age beyond 3 months, the hyperlipidemia increases markedly in the cp/cp female but not in the male. By 9 months of age, the cp/cp females have triglyceride concentrations over 1,000 mg/100 ml, total cholesterol of 150 mg/100 ml, and phospholipids of 340 mg/100 ml (Dolphin et al., 1990). In cp/cp male rats, chronic food restriction to 12 g/day (approximately 60 percent of the intake of a lean male rat) results, at 12 months of age, in markedly decreased triglycerides (106 versus 215 mg/ 100 ml), esterified cholesterol (88 versus 108 mg/100 ml), and apolipoproteins B and E. Apo A-1 is unchanged. The food-restricted animals are smaller but retain a corpulent phenotype. The incidence of myocardial lesions is significantly reduced by the food reduction.

Postheparin lipid clearance and apparent hepatic secretion rate following Triton WR1339 inhibition of lipoprotein lipase have been determined (Russell et al., 1989d). The concentrations of cholesterol and cholesteryl esters are not significantly altered by either treatment. Triglycerides undergo rapid postheparin clearance in corpulent rats of both sexes. The apparent hepatic secretion rate is markedly higher in corpulent than in lean male rats. The rate in corpulent females is still higher, reflecting their higher triglyceride concentrations. The relative secretion rate of C:48 triglyceride molecular species is lower than that of the C:50 to C:56 molecular species (where C:48, for example, refers to triacylglycerols with 48 acyl carbon atoms). In contrast, the longer-chain fatty acids (C:52-C:56 triglyceride molecular species) have higher rate constants and are cleared more rapidly by postheparin lipase activity. These results indicate that the VLDL hyperlipidemia in the corpulent rat is due to hepatic hypersecretion of VLDL and not to a defect in lipoprotein lipase.

Insulin Resistance

The other major metabolic abnormality in the corpulent rat is a complex disturbance of insulin and glucose metabolism. This is most probably linked to the hyperlipidemia in some manner, but the relationship is not a direct one. The abnormality is most evident in the islets of Langerhans of the pancreas, which undergo an extreme age-dependent hyperplasia (Ahuja et al., 1987). Studies using immunocytochemical staining and quantitative image analysis showed that cp/cp male rats have larger islets than lean rats as early as 1 month of age. The volume density of islets rose to over 20 percent at 9 months of age. At advanced ages, the architecture of the islets becomes disrupted with fusing of adjacent islets and extensive fibrosis. The hyperplasia is largely of B cells, although A cells are also increased in corpulent rats. Fasting glucose concentrations are not significantly raised in the cp/cp rats. However, fasting insulin levels are markedly elevated, increasing rapidly up to 6 months of age (350 versus 24 mU/L). Glucagon concentrations are only mildly elevated.

Intravenous glucose tolerance testing revealed a serious glucose intolerance in cp/cp male rats (Russell et al., 1987). The rate constant for glucose removal, k, is 0.94 x 10-2min^-1 in cp/cp males compared to 2.06 x 10-2min^-1 in lean males. Occasional animals show prolonged glucose concentrations over 500 mg/100 ml in response to the 0.5 g/kg glucose load. On average, little further insulin response is possible following glucose challenge, but values over 1,000 mU/L are seen. Female cp/cp rats show more moderate fasting insulin levels (144 mU/L) and better glucose tolerance (k = 2.29 x 10-2min-1). Thus, the insulin resistance is mild in the female but severe in the male rat--leading to glucose intolerance and at least a borderline type II diabetes. However, there is no evidence of glycosuria in the cp/cp males eating a normal diet of rat chow. The insulin resistance thus correlates strongly with the vascular and myocardial disease. This finding has lead us to postulate that the hyperinsulinemia may be an initiating factor, perhaps through direct effects on the endothelial cell. There is evidence (Steiner, 1986) that insulin is potentially toxic to the endothelial cell. (We also are prepared to consider that hyperglycemia is a key factor.) The development of lesions would then require the presence of at least a certain degree of hyperlipidemia. The cp/cp male may thus have concentrations of triglycerides, cholesterol, or an apolipoprotein above the required threshold and therefore develops lesions, while the female does not develop lesions.

Comparison with Fatty Zucker Rat

We have made detailed comparisons between the fatty Zucker and Jcr:LA-cp rats at six months of age (Amy et al., 1988). This study was performed using identical techniques on the two strains. Both strains, when obese, exhibited a VLDL hyperlipidemia with elevated triglycerides and moderate elevations of plasma cholesterol concentrations. However, the hyperlipidemia was more moderate in the fa/fa rat than in the cp/cp rat, and plasma lipid concentrations were lower in the Zucker than in the comparable Jcr:LA-cp. Obese rats of each sex from both strains had elevated fasting insulin levels. These levels were higher in the Jcr:LA-cp rats, and the cp/cp males had markedly higher insulin levels than all others. Oral glucose tolerance testing showed a major glucose intolerance only in the cp/cp male rats. The fa/fa male and female rats had an exaggerated insulin response to the 1.0 g/kg oral glucose challenge that was almost identical to that of the cp/cp females. The cp/cp male integrated response was almost twice as large. Most strikingly, neither male nor female fa/fa rats developed vascular lesions, as seen with scanning electron microscopy, or myocardial lesions. This study confirms the strong association between insulin resistance and cardiovascular disease. The findings emphasize the unique character of the corpulent strains of rats and suggest that the fa gene and the precise defect it creates are different from that of the cp gene.

Further studies have shown that the cp/cp rat is hypergastrinemic and has increased numbers of gastrin secreting cells in the stomach (Pederson and Russell, unpublished data, 1989). The lean Jcr:LA-cp rat also shows increased numbers of gastrin-secreting cells compared to the lean (fa/fa) Zucker. Studies of the isolated perfused pancreas have revealed the cp/cp male rat to have a grossly exaggerated response to the infusion of either GIP or arginine. The rat's insulin secretion rate in response to these agents exceeded 15,000 mU/min, compared with 2,000 mU/min for lean males or females of either strain.

Obesity

The origin of obesity in the cp/cp rat is a major question. The trait is deeply embedded in the animals, and they retain a corpulent phenotype in the presence of severe food restriction and exercise. One possibility is that the cp/cp rat lacks the ability to initiate thermogenesis. We have conducted studies of the ability of the Jcr:LA-cp rat to engage in thermogenesis under rigorous experimental conditions (Lee et al., 1987). Our results showed that nonshivering thermogenesis stimulated by norepinephrine was present in the cp/cp rat at the same level as the +/+ rat. There was no difference in cold tolerance between the genotypes at any age. Similarly both genotypes showed the same thermogenic response to aminophylline. Thus, the obesity is not likely caused by a deficiency in thermogenesis at the end-organ level.

There are significant differences between the adipocytes and preadipocytes of corpulent and lean rats. The cp/cp rat showed a lower rate of preadipocyte replication than the +/+ rat, although the preadipocyte pool was significantly larger. The preadipocytes of cp/cp rats were resistant to hormonally induced differentiation, but did differentiate when cocultured with mature adipocytes. In contrast, the adipocytes from +/+ rats were unable to induce differentiation of cocultured preadipocytes (Shillabeer et al., 1990). These results, although not definitive, demonstrate major metabolic differences between the genotypes.

The body fat content of the cp/cp (> 32 percent) is higher, and that of the +/+ rats (< 2 percent) is lower than that seen in other rat strains. Our results show that mRNA levels for hepatic glycerophosphate dehydrogenase and enzyme activity are correlated. These levels in turn are both suppressed in +/+ rats and increased in cp/cp rats (G. Shillabeer, Department of Medicine, University of Calgary, unpublished data, 1990). This effect may be related to the insulin resistance and is undoubtedly involved in the processes leading to increased triglyceride and VLDL synthesis and release.

Alteration of Metabolism in cp/cp Rat

Besides mechanistic studies, we have also conducted a number of studies designed to test whether the metabolic and/or disease processes can be altered. Such an approach addresses questions about the nature of the metabolic defect(s) and, if successful, may provide insight into the roles played by the various metabolic disturbances in the atherosclerotic process.

In one study, four-week-old male and female cp/cp rats were fed a normal low-fat laboratory chow supplemented with either 10 percent olive oil or 10 percent red fish (Sebastes marinus) oil ad libitum for a period of two months (Dolphin et al., 1988). Red fish oil reduces serum triacylglycerols by 54 percent in males and by 45 percent in females after two months. VLDL levels were reduced by 44 percent in males and 39 percent in females. HDL lipid mass was significantly reduced in both sexes (by 27 percent in males and 49 percent in females). However, the levels remained above those of +/+ male and LE (Long Evans) rats of the same age. Olive oil feeding had no significant effect upon VLDL levels in either sex but significantly increased the particle diameter. HDL levels were unaffected. Both oils markedly reduced serum LDL levels in rats of both sexes. These results show that the Jcr:LA-cp rat responds to dietary fish oil containing low levels of omega-3 fatty acids in a similar manner to other animal models and humans, and they suggest that HDL levels are more quickly modulated than VLDL levels by the fish oil. Insulin resistance is not affected by oil feeding (J. C. Russell, unpublished data, 1989). Long-term experiments are under way to explore the effect of these changes on both pancreatic morphology and vascular/ myocardial lesions.

Male +/+ and cp/cp rats were exposed to low-dose (0.5 percent w/v) or high-dose (4 percent w/v) ethanol in their drinking water from 1 month to 12 months of age (Russell et al., 1989a). The total lipid profile of the rats showed only limited changes with ethanol consumption. Ethanol consumption was associated with elevated fasting glucose concentrations in both lean and corpulent rats and a strong decrease in fasting insulin levels and pancreatic B cell volume density in the hyperinsulinemic corpulent rats. The relative frequency of myocardial nodules of chronic inflammatory cells was increased in both lean and corpulent ethanol-consuming rats. In contrast, old organized lesions (scars) were absent in the hearts of ethanol-consuming corpulent rats. The results do not support the concept that ethanol consumption has any major effect on serum lipids or lipoproteins in these rats. However, ethanol consumption, in the corpulent rats, is associated with a reduction in insulin resistance and islet cell hyperplasia with an associated decreased incidence of ischemic myocardial lesions.

Corpulent and lean male rats were induced, through mild food restriction, to run intensively (6,000 m/day) from 6 weeks to 6 months of age and compared to pair-fed and ad libitum-fed control animals (Russell et al., 1989b). The food restriction, especially when coupled with running, lowered all classes of lipids in the whole serum of corpulent rats. The greatest changes in lipid concentration was in the VLDL fraction, although, all lipids in the sera of the running rats fell to approximately one-third that of the freely eating sedentary rats (e.g., triglycerides from 169 + 9 to 49 + 02 mg/100 ml). The food restriction caused a significant drop in fasting insulin levels of corpulent rats (from 350 + 115 to 75 + 53 mU/L). The running caused a marked decrease in B-cell hyperplasia in corpulent rats (from 196 - 157 to 37 + 23 x 10^-3 I. tm2/islet), which confirmed the improvement in insulin and glucose metabolism. Histological examination of the hearts showed a significant decrease in the frequency of myocardial lesions in the food-restricted corpulent rats and an absence of lesions in the running rats. The results indicate that intensive physical activity can largely correct the lipid abnormalities and insulin resistance of this strain, and the changes are associated with inhibition of the disease process. However, moderate food restriction has similar effects, and the greater effects seen with intensive running may simply reflect a more severe relative metabolic restriction in the presence of the exercise.

Treatment of corpulent male rats with clofibrate resulted in a marked decrease in plasma cholesterol and a modest decrease in triglycerides (Russell et al., 1989c). Plasma glucose levels, insulin levels, and glucose tolerance were unaffected. There was no effect on the frequency of myocardial lesions. The role of the sympathetic nervous system in the differences between genotypes was studied through measurement of tissue catecholamine levels and monamine oxidase activities. No significant differences were found between corpulent and lean rats. Long-term treatment with 6-OH dopamine caused effective chemical sympathectomy, but had no effect on myocardial lesion frequency. These results suggest that vasospasm induced by neurogenic mechanisms does not play a role in generation of the myocardial lesions. We have shown that the vascular smooth muscle of corpulent male rats, but not female or lean rats, is hyperreactive to norepinephrine. There is also a defect in the endothelium-dependent relaxation mechanism that is confined to corpulent males (J. C. Russell, unpublished data, 1989). Long-term treatment with nifedepine, a calcium channel antagonist, was highly effective in preventing myocardial lesions (Russell et al., 1990). This result was accompanied by a marked decrease in plasma triglyceride concentration that was confined to molecular species with 52 or more fatty acyl carbon atoms.

Recent results (Lopaschuck and Russell, unpublished data, 1990) show that the isolated working heart of cp/cp rats, both male and female, is highly sensitive to calcium and functions only in the presence of less than 1.75 mM calcium. The cp/cp rat heart has an absolute requirement for insulin, although the ability to readily metabolize free fatty acids is normal. These results indicate some further fundamental metabolic abnormalities in the Jcr:LA-cp strain.

Comparison of Strains Incorporating the cp Gene

A major comparative study of the five strains incorporating the cp gene has been completed. The hearts of cp/cp male rats of the other strains have all been compared to the Jcr:LA-cp rat using histological techniques. The LA/N-cp and WKY/N-cp do not develop myocardial lesions at a significant frequency. The SHR/N-cp rat does not develop the large ischemic lesions seen in the Jcr:LA-cp rat, but has a high incidence of perivascular fibrosis with associated Anitskow cells. The SHR/N-cp is sensitive to high-sucrose diets and may be induced to develop ischemic lesions by such feeding. The SHHF/Mcc-cp rat resembles the SHR/N-cp rat in having only very small ischemic lesions and perivascular fibrosis in addition to its characteristic cardiomyopathic cell loss.

Summary

Our studies have established the presence of spontaneous vascular and myocardial disease in the cp/cp rat. The major associated metabolic abnormalities--hyperlipidemia and insulin resistance--have been characterized. However, the specific hormone, enzyme, or receptor defect(s) leading to insulin resistance and/or hyperlipidemia is not known. Further studies of both aspects of metabolism in the cp/cp rat are required to elucidate the mechanisms involved. We have also been able to modulate the metabolic abnormalities and prevent the myocardial lesions through experimental manipulation of the rats. This opens the possibility of new approaches to the study of atherogenesis. When the studies proposed have been completed, the cp/cp rat strains will constitute a well-characterized model for both mechanistic and pharmacological studies on atherosclerosis and its sequelae as well as obesity and type II diabetes.

Cardiovascular disease is a major cause of death and morbidity and is closely associated with the common human syndrome of obesity, hyperlipidemia, and non-insulin-dependent diabetes. A fundamental understanding of the origin of this syndrome is essential to the prevention and treatment of the single most common cause of death in Western societies.

References

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