In vivo effects of peroxovanadium compounds in BB rats.

Peroxovanadium compounds, each containing an oxo ligand, one or two peroxo anions, and an ancillary ligand in the inner coordination sphere of vanadium, were synthesized, crystallized and characterized by 51V NMR as > 95% pure. They markedly decreased plasma glucose in insulin-deprived diabetic BB rats, with a nadir occurring between 60 and 100 min after intravenous, intraperitoneal or subcutaneous administration. Plasma glucose was reduced after oral administration in insulin-treated and in insulin-deprived BB rats. When compared to sodium orthovanadate, peroxovanadium compounds exhibited a markedly greater potency on a molar basis, and in relation to their toxicity. The in vivo potency can be predicted by the degree of phosphotyrosine phosphatase inhibition observed in vitro. These are the first agents other than insulin that can acutely and markedly reduce plasma glucose in hypoinsulinemic diabetic BB rats.

Hypoglycemic effects of peroxovanadium compounds in Sprague-Dawley and diabetic BB rats.

Highly purified peroxovanadium (pV) compounds, each containing an oxo ligand, one or two peroxo anions, and an ancillary ligand in the inner coordination sphere of vanadium, were shown to decrease plasma glucose markedly in both normal Sprague-Dawley and insulin-deprived diabetic BB rats. Maximal decreases in plasma glucose were at 60-100 min after intravenous, intraperitoneal, or subcutaneous administration. Synergism between these compounds and insulin was observed. Whereas parenterally administered orthovanadate or vanadyl sulfate did not induce hypoglycemia before inducing acute mortality, pV compounds effected hypoglycemia at doses much lower than those inducing acute mortality. When administered subcutaneously over a period of 3 days to insulin-deprived diabetic BB rats, pV compounds, but not vanadate, caused a significant decrease in plasma glucose concentrations and prevented the appearance of ketosis in these animals. Thus, pV compounds are the first agents other than insulin that acutely and markedly reduce plasma glucose in hypoinsulinemic diabetic BB rats.

Predictive value of lymphocyte antibodies for the appearance of diabetes in BB rats.

Lymphocyte antibodies have been described in autoimmune disorders, including insulin-dependent diabetes mellitus (IDDM). We have developed a quantitative method to measure autoantibodies directed against T-lymphocytes, based on two-color fluorescence labeling of Wistar mononuclear cells and analysis of fluorescence by flow cytometry. The lymphocyte antibody levels were determined retrospectively in the serum of 73 BB and 18 Wistar rats. We demonstrated the binding of the lymphocyte autoantibodies of both CD4+ and CD8+ T-cells. Lymphocyte antibodies were present in 90% of the BB rats at diabetes onset, compared with 11% of the Wistar rats. At 75 days of age, 83% of the BB rats, which later became diabetic, were positive for the lymphocyte antibodies, compared with 15% of their littermates who maintained a normal glucose tolerance. In all cases, the antibodies were of the immunoglobulin M isotype. We conclude that lymphocyte antibodies are present before diabetes onset and, using this method, that their presence can predict the development of diabetes with a sensitivity of 83% and a specificity of 85% in BB rats.

Prevention of adoptive transfer in BB rats by prophylactic insulin treatment.

Prophylactic insulin can prevent diabetes in the BB rat. We evaluated its use to prevent adoptive transfer of diabetes by activated splenocytes. ConA-activated spleen cells from acutely diabetic BB rats were divided into two equal aliquots and injected intravenously in paired diabetes-prone BB rat littermates. At the time of cell injection, subcutaneous insulin injections (15 U.kg-1.day-1) were started in one of each pair (n = 21) of littermates, and the control littermates (n = 21) were injected with saline. The incidence of diabetes, observed 35 days after cell injection, was 95% in control rats compared with 29% in insulin-treated rats (P less than 0.05). To confirm the absence of diabetes, insulin was stopped in all nondiabetic insulin-treated rats at 63 days of age. An OGTT was performed at 65 days of age: 4 rats were glucose intolerant. All rats received comparable numbers of ConA-activated splenocytes. At the time the rats were killed, 3 insulin-treated rats had a completely normal morphology and a normal glucose tolerance. All control rats had insulitis whether they were diabetic or not. No significant difference in any mononuclear subset was observed in relation to insulin treatment. We conclude that prophylactic insulin can prevent adoptive transfer of diabetes in the BB rat without inducing changes in the mononuclear cell subsets.

Metabolic time course and immunologic concomitants of adoptive transfer of type I diabetes in the BB rat.

The metabolic and cellular immune changes during adoptive transfer of type I diabetes mellitus in the BB rat were examined. Concanavalin A (Sigma Chemical Co, St Louis) stimulation of acutely diabetic BB rat splenocytes increased the Ia-positive cells, but no other lymphocyte subset. Each spleen cell preparation was divided into two and injected into two separate recipients. Thirty-day-old diabetes-prone BB rats received these splenocytes intravenously (62 +/- 5 x 10(6) cells, n = 30) or buffer alone (controls, n = 14). Seventy-seven percent of the cell-injected rats became diabetic before 60 days of age, 15 +/- 1 days after injection. They were glucose intolerant two to three days before onset, with normal fasting glucose. All controls maintained normal glucose tolerance. The morphology revealed intense insulitis in all the rats that became diabetic, and its absence in all the controls. Eighty-three percent of the spleen cell preparations produced the same outcome in both recipients. The cell-injected rats had an increase in lymphocyte counts eight days after injection compared with the controls. The most affected subsets were the pan T cells (OX19+) and helper T cells (W3/25+). While the rats that ultimately became diabetic had a decrease of their lymphocyte subsets to control levels between eight and 14 days, the injected rats that maintained normal glucose tolerance maintained elevated T cells. We conclude that (1) adoptive transfer of diabetes occurs in the presence of an increase of the helper T (W3/25+) lymphocytes after spleen cell injections; (2) glucose intolerance precedes by two to three days fasting hyperglycemia; and (3) while the lymphocyte counts are increased in all recipients of splenocyte preparations, these counts decrease rapidly only in the rats that develop diabetes, possibly by entrapment of lymphocytes in the insulitis.

Pharmacokinetic profile of cyclosporine A and G and their effects on cellular immunity and glucose tolerance in male and female Wistar rats.

This study examined the effects of the widely used immunosuppressor cyclosporine A and of one of its derivatives, cyclosporine G, on glucose tolerance, cellular immunity, and renal and hepatic function, in relation to their pharmacokinetic profile in Wistar rats. After 3 weeks of daily cyclosporine A doses of 10 mg/kg body weight plasma cyclosporine levels were higher in male than in female rats. This was associated in male rats with marked decrease in lymphocyte subsets, affecting particularly the OX19+ T cells, with glucose intolerance, and an increase in plasma creatinine. The female rats had none of these effects. After 3 weeks of daily cyclosporine G doses of 10 mg/kg body weight, plasma cyclosporine levels were higher in male than female animals, and higher than with cyclosporine A in both sexes. Similar cellular immune effects and glucose intolerance were seen in male rats, but of a lesser magnitude than with cyclosporine A. No increase in creatinine was seen, but rats of both sexes treated with cyclosporine G had elevated plasma bilirubin. We conclude that (1) both cyclosporine A and G can cause glucose intolerance, (2) the cyclosporine plasma levels are higher in male than in female rats and with cyclosporine G than with cyclosporine A, for the same oral dose, (3) the absence of glucose intolerance, nephrotoxicity, and cellular immune changes in female rats treated with cyclosporine A is related to their lower cyclosporine levels, and (4) cyclosporine G is less nephrotoxic than cyclosporine A, but more hepatotoxic.

Major histocompatibility complex gene product expression on pancreatic beta cells in acutely diabetic BB rats.

Type I diabetes mellitus was induced in young, diabetes-prone BB rats by the passive transfer of concanavalin A-activated T lymphocytes from the spleens of acutely diabetic BB rats. The pancreas of the recipients was examined 1-2 days after the onset of glycosuria by immunocytochemistry by means of monoclonal antibodies for determining whether 1) Class I and/or II major histocompatibility gene complex (MHC) products were expressed on beta cells and 2) the mononuclear cell infiltrates were represented by T cells. Marked expression of Class I MHC gene products was evident on beta cells. In contrast, Class II MHC gene products were not identified on normal-appearing beta cells. Dendritic cells dispersed throughout the acinar and interstitial pancreas were markedly increased in number. The mononuclear cell infiltrate contained few cells (1-15%) recognized by a pan-T cell marker. Although it is possible that this passive transfer model might differ considerably from the spontaneously occurring diabetic state in the rat, this study suggests that 1) Class I, rather than Class II, MHC gene expression may be pivotal to beta-cell injury in diabetic rats, and 2) non-T cells may constitute an effector cell population central to beta-cell necrosis in Type I diabetes mellitus.