Abstract:To clarify whether oxidative stress is involved in the pathogenesis of islet lesions of diabetic animals, the effects of probucol (PB), an antioxidant and anti-hyperlipidemia agent, on the islets in streptozotocin (SZ)-induced diabetic APA hamsters in the acute and chronic phases of diabetes were examined. The control (CB group) and diabetic (SZ group) hamsters were treated with PB (1% in the diet) for 4 weeks from several days after SZ injection as the acute diabetic group, or 8 weeks from 6 weeks after SZ injection as the chronic diabetic group. Glucose tolerance test revealed that PB treatment decreased the high serum glucose level after glucose injection in the diabetic APA hamsters in the acute diabetic phase. Immunohistochemistry revealed that PB treatment significantly increased the percentage of the insulin positive area in the diabetic hamsters pancreata in both the acute and chronic phases. In addition, 4-hydroxy-2-nonenal (4HNE; an oxidative stress marker) positive cells were slightly reduced by PB treatment in the acute diabetic phase. Double-immunostaining for insulin and PCNA (proliferating cell nuclear antigen) revealed that elevation of the percentage of insulin and PCNA double-positive cells against insulinpositive cells was seen in the islets of PB-treated diabetic hamsters, but the difference was not significant compared with untreated diabetic hamsters (p=0.07). In semi-quantitative RT-PCR, the expression of two genes, Reg (Regenerating gene) and INGAP (islet neogenesis associated protein), in the diabetic APA hamsters was significantly increased compared to the control groups in both diabetic phases. PB treatment significantly reduced Reg expression in the chronic diabetic phase. These data suggest that PB treatment in SZinjected diabetic hamsters partially restored β-cell function through acting as an antioxidant and induced higher expression of Reg and INGAP genes in the pancreas of hamsters.

To clarify how Syrian hamsters of the APA strain (APA hamsters) keep a diabetic condition for a long period, the functional and histochemical changes in the pancreatic islets of diabetic APA hamsters were examined. By glucose tolerance test, no glucose-induced insulin secretion was seen in the diabetic APA hamsters. By immunohistochemistry, it was revealed that at 24 hr after SZ-injection, the number of islets had decreased and that remnant islets had become markedly smaller. The islets had hardly any insulin-immunoreactive cells and consisted of cells stained by anti-glucagon and somatostatin antibodies. One, three and six months after SZ-injection, a small number of cells with vacuolative changes, which were positive for PAS staining, were observed in most islets and the vacuolated cells were stained mainly by anti-insulin antibody. In addition, a number of PCNA-positive cells were observed, especially in the periphery of the vacuolated cells, while TUNEL-positive cells were not detected. This data suggests that beta-cells proliferating as a result of the replication of the resident beta-cells in islets had fallen into degeneration and necrosis by a stress, such as the glycogen deposition in hyperglycemia and hyperlipidemia. Consequently, secretion of insulin was maintained at low levels, which allowed the hamsters to live without insulin therapy in the diabetic condition for over 6 months.

We investigated the effect of probucol (PB) on atherosclerosis in streptozotocin (SZ)-induced diabetic-hyperlipidemic APA hamsters in three different stages, the early, middle and late stages of atherosclerosis. Male APA hamsters were injected intraperitoneally with SZ or vehicle alone (citrate buffer; CB) as a control at the age of 8 weeks. At 6 weeks after injection (WAI) of SZ or CB (the early stage), 14 WAI (the middle stage) and 26 WAI (the late stage), animals were assigned to PB treated- or non-treated groups (CBPB, SZPB, CB, SZ). After 8 weeks of PB administration with diet, the aorta was taken from each animal for assessment of atheromatous lesions and blood samples were subjected to serum biochemical analysis and the measurement of blood lipid peroxide (LPO). In the middle stage, PB treatment significantly decreased serum total cholesterol level, slightly decreased LPO, and also tended to reduce the lesion area, although no statistical difference was seen. There was no marked effect of PB treatment in the early and late stages. These findings suggest that single use of PB has little effect on atherosclerosis of a hyperglycemia-hyperlipidemia animal model.

Although it has been said that Syrian hamsters of the APA strain (APA hamsters) spontaneously develop glomerulosclerosis with age, more prominent and severe glomerulosclerosis with proteinuria as well as arteriosclerosis is induced in diabetic APA hamsters. In this study, in order to supply new information on APA hamsters, tests on renal function and histology were done on non-diabetic and streptozotocin (SZ)-induced diabetic APA hamsters (APA-N and APA-D, respectively), and the data were compared with those of normal Syrian (golden) hamsters (GOL). At 4, 8, 12, 20, and 32 weeks of age, the markers indicating renal function, serum urea nitrogen and creatinine levels and the urinary total protein level were measured and thereafter histological studies were done. Although there were no remarkable differences between APA-N and GOL in serum urea nitrogen and creatinine levels, APA-N excreted more urinary total protein from the early weeks of age. In APA-D, an apparent worsening in these markers indicating renal function was detected and diabetic nephropathy in this model was confirmed also in terms of renal function. In the histological studies, the major lesion observed in APA-D was diffuse glomerulosclerosis. This may mean that renal dysfunction in APA-D was mainly caused by the glomerular change and that it is similar to other experimental diabetic animals and human diabetic patients. These data show that the diabetic APA hamster is a desirable model of human diabetic nephropathy.