Multiple Subdiabetogenic Doses Research Articles

Anti-interleukin 2 receptor antibody attenuates low-dose streptozotocin-induced diabetes in mice

Recent evidence indicates that activated T cells and macrophages play an important role in the induction of insulitis and diabetes in certain strains of mice treated with multiple subdiabetogenic doses of streptozotocin. In the present study, we treated C57BL/6J mice with five daily doses of 40 mg/ml streptozotocin and examined the prophylactic effect of an anti-interleukin 2 receptor monoclonal antibody (PC61). In mice treated with streptozotocin, interleukin 2 receptor-positive mononuclear cells were shown to infiltrate into the islets and soluble interleukin 2 receptors in the sera were significantly increased compared with control mice. The administration of PC61 to the mice attenuated the insulitis, and diminished interleukin 2 receptor-positive cells from islets and soluble interleukin 2 receptors in the sera. Moreover, the administration of PC61 significantly reduced the development of hyperglycaemia shown in these mice (12.8 +/- 1.1 mmol/l vs 18.5 +/- 0.7 mmol/l, p less than 0.005). As judged by flow cytometric analysis, this antibody did not cause any changes in either spleen cell counts or T cell subsets. Interleukin 2 receptors were expressed on a minor population of spleen cells regardless of treatment with PC61 (STZ + normal rat IgG: 2.1 +/- 0.3%, STZ + PC61: 2.4 +/- 0.3%). Even after stimulation of spleen cells with concanavalin A or alloantigen, interleukin 2 receptor expression was not significantly different between the two groups. Our studies suggest that interleukin 2 receptor-positive activated T cells or macrophages are important in the development of multi-low-dose streptozotocin diabetes and that an anti-interleukin 2 receptor antibody can attenuate this process.

Class II MHC antigens in vivo induced by the diabetogen streptozotocin in BALB/c mice

Immunologic reactions are involved in the pathogenic pathway of hyperglycemia development induced by multiple subdiabetogenic doses of streptozotocin (STZ) in mice. Recently, we have reported that STZ, a nitrosurea derivative, can act in vivo as hapten for T lymphocytes, as assayed by applying the popliteal lymph node assay. In subsequent experiments, induction of Class II major histocompatibility (MHC) antigens by STZ were evaluated in BALB/c mice. After subcutaneous injection of STZ a remarkable increment of Class II-positive cells was visible in multiple organs. Ia antigens were detected by a peroxidase method on cryo-cut sections using a monoclonal antibody with specificity for I-Ad. A monoclonal antibody with specificity for Iak was used as control. Definite increases of I-Ad-binding cells were found in pancreatic, liver, kidney and heart tissue, but brain tissue remained unaffected. These Class II MHC alterations occurred on interstitial cells of the monocyte lineage, renal tubular cells and lymphoid cell infiltrates of the endocrine pancreas, but not on beta cells. Intense Ia-positivity at the pancreatic islet pole and at intra-insular sites was not necessarily associated with hyperglycemia development.

Observations on offspring of mice made diabetic with streptozocin.

My objective was to determine the effect of streptozocin (STZ)-induced diabetes in male inbred mice on nonfasting blood glucose levels and body weights of offspring. Hyperglycemia was induced in CBA/H male mice by either multiple subdiabetogenic doses (MD) of STZ (5 doses/day of 50 mg STZ/kg body wt) or by a single high sublethal (SD) dose (200 mg STZ/kg body wt). Females were made diabetic by the multiple low-dose procedure. The following matings were set up: SD males with normal (NOR) females; MD males with NOR females; NOR males with MD females; MD males with MD females. Controls were matings of NOR males with NOR females. Among the first cohort of litters was born one female from the cross of an SD male with an NOR female who became spontaneously hyperglycemic at 5 wk of age; the female progeny of this cross had significantly lower body weights. All other progeny groups were normoglycemic (up to 5 wk) and had normal body weights. Test progeny weaned in the second and subsequent cohorts of litters were also normoglycemic. The major effect in this progeny group was on body weight; diabetic fathers (particularly MD males) mated with NOR females produced offspring with significantly higher juvenile body weights than the controls (increase of approximately 0.5 g). These body-weight distributions also appeared more homogeneous than the more variable body-weight distribution of the controls. In contrast, MD mothers (mated with NOR males) produced second-cohort offspring with significantly lower average body weights (decrease of approximately 1-2 g) than the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment with anti-T-lymphocyte antibodies prevents induction of insulitis in mice given multiple doses of streptozocin.

The importance of T-lymphocytes in the induction of insulitis and hyperglycemia in certain strains of mice treated with multiple subdiabetogenic doses of streptozocin has been a matter of controversy. To understand the role of T-lymphocytes, we treated thymectomized BALB/c ByJ mice with five daily doses of streptozocin (45 mg/kg) and determined the effect of treatment with monoclonal antibodies against T-lymphocyte subsets on the development of diabetes and insulitis. Hyperglycemia (mean glucose of 321 +/- 29 vs. 167 +/- 15 mg/dl in controls) and insulitis were induced in BALB/c ByJ mice given streptozocin. Thy1.2+, L3T4, and Lyt2+ cells were all identified within the islets of diabetic mice. There was a relative paucity of L3T4+ cells and an overabundance of Lyt2+ cells compared with the frequency of these cells found in lymphatic tissues or peripheral blood. Treatment with anti-L3T4 or anti-Lyt2 monoclonal antibodies caused a reduction in splenic T-lymphocyte subsets and attenuated the hyperglycemia to 212 +/- 14 and 197 +/- 16 mg/dl (P less than .001 and .01), respectively, compared with controls and prevented the insulitis induced by streptozocin. Our studies support the hypothesis that an immune response is important to the development of multi-low-dose streptozocin diabetes and indicate that treatment with monoclonal antibodies against the L3T4+ or Lyt2+ T-lymphocyte subsets can attenuate this process.

Multiple low-dose streptozotocin-induced diabetes in the mouse: further evidence for involvement of an anti-B cell cytotoxic cellular auto-immune response.

Anti-B cell auto-immunity may play a role in the pathogenesis of diabetes in mice resulting from multiple subdiabetogenic doses of the pancreatic B cell toxin, streptozotocin. In the present study we have investigated the cytotoxic anti-B cell response in these mice. A major role for B lymphocytes, macrophages, or their products in the cytotoxic response originally detected in vitro was eliminated by passing splenocytes from the mice treated with multiple subdiabetogenic doses of streptozotocin over a nylon wool column. The removal of the adherent cells enhanced the cytotoxicity against a rat insulinoma cell line in vitro by that expected due to enrichment of T-lymphocytes by approximately two-fold. The induction of diabetes after multiple subdiabetogenic doses of streptozotocin is strain dependent. Mice of five strains were immunized with rat insulinoma cells, but only splenocytes from the two strains susceptible to multiple subdiabetogenic doses of streptozotocin demonstrated a significant cytotoxic response against the rat insulinoma cells in vitro. Mice pre-immunized with either the rat insulinoma cells or with syngeneic islets labelled in vitro with the hapten trinitrophenol developed hyperglycaemia more rapidly than control mice after multiple subdiabetogenic doses of streptozotocin. In the latter experiment the control mice immunized with complete Freund's adjuvant alone also became hyperglycaemic after a modified multiple subdiabetogenic dose of streptozotocin that did not cause diabetes in non-immunized mice. In mice pre-treated with either adjuvant or cyclophosphamide and then given a modified multiple subdiabetogenic dose of streptozotocin (35 mg/kg X 5 rather than 40 mg/kg) the degree of hyperglycaemia was reduced and there was no protective effect of cyclophosphamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl-homocysteine-thiolactone-induced increase of superoxide dismutase counteracts the effect of subdiabetogenic doses of streptozocin.

Acetyl-homocysteine-thiolactone (CYT) is an organic thio compound that exerts free radical scavenger activity and increases superoxide dismutase (SOD) activity. Administration of 32 mg CYT/kg body wt/day/30 days in rats increased SOD activity in erythrocytes by 126%, and in pancreatic islets by 202%. Treatment affected only the Cu-Zn fraction of the enzyme. Transmission electron microscope observations showed that the damage to the pancreatic beta cells induced by single or multiple subdiabetogenic doses of streptozocin (STZ) (45 mg/kg body wt) was attenuated in animals treated with CYT. This protective effect was not observed with 65 mg of STZ. The experimental results seem to support the hypothesis that pancreatic beta cells are particularly vulnerable to the effect of oxygen radicals and that the cytotoxic effect of STZ is related to free radical-induced peroxidation.

Diabetes induction by subdiabetogenic doses of streptozotocin in BALB/cBOM mice. Noninvolvement of host B-lymphocyte functions.

Multiple subdiabetogenic doses of streptozotocin induce an insulin-dependent progressive hyperglycemia in genetically susceptible strains of mice. We have shown previously that T-cell-dependent autoimmune mechanisms play an obligatory role in this model of diabetogenesis by demonstrating that athymic nude mice and lethally irradiated euthymic mice are selectively resistant to diabetes induction and that the susceptibility can be reconstituted by grafting thymus in nude mice or by giving B-cell-depleted splenic lymphocytes to the irradiated mice. In this report we investigate more directly the possible role of host B-cell functions in the induction of hyperglycemia. Mice were rendered selectively deficient in functional B lymphocytes by repeated injections of a polyclonal antiserum against mouse IgM, starting immediately after birth. These B-cell-suppressed mice had no detectable ability to produce antibodies against a test antigen but appeared to have normal levels of T cells. When treated with multiple low doses of streptozotocin, they developed progressive hyperglycemia in a manner indistinguishable from control mice with normal B-cell functions. These results suggest that host B cells, in contrast to host T cells, are not etiologically involved in the development of diabetes induced by multiple subdiabetogenic doses of streptozotocin.

Induction of insulin-dependent diabetes by streptozotocin. Inhibition by estrogens and potentiation by androgens.

Susceptibility of mice to experimental insulin-dependent diabetes as induced by multiple subdiabetogenic doses of streptozotocin has been shown to be strongly gender-dependent, males being much more susceptible than females. We examined this gender difference further in two strains of genetically susceptible mice to determine whether exogenous steroid sex hormones can both suppress the high susceptibility of males and potentiate the low susceptibility of females. Our results show that, in both BALB/cBOM and C57BL/6 mice, exogenous estrogens can suppress the high susceptibility of males. Conversely, the normally streptozotocin-resistant females become as highly susceptible as males after the administration of androgens. The inhibitory effect of estrogens and the potentiating effect of androgens can be demonstrated after the hormones are given to the mice either chronically (in slow-release capsules implanted at a subcutaneous site), or immediately prior to streptozotocin injection. These observations are consistent with the view that the critical factor that determines the susceptibility of mice to the hyperglycemic effects of streptozotocin is not the absolute concentration of androgens per se, but rather the relative overall level of androgens over estrogens in the recipient animal. Several alternative mechanisms for the effect of sex hormones on diabetogenic sensitivity are discussed.

Diabetes induced with multiple subdiabetogenic doses of streptozotocin: lack of protection by exogenous superoxide dismutase

Diabetes induced with multiple subdiabetogenic doses of streptozotocin: lack of protection by exogenous superoxide dismutase

Diabetes induced with multiple subdiabetogenic doses of streptozotocin: lack of protection by exogenous superoxide dismutase.

The addition of exogenous superoxide dismutase (SOD) was examined as a possible means of protecting B-cells of mice against either the immediate or delayed toxicity caused by multiple injections of low doses of streptozotocin (Sz). Three different routes of SOD administration (i.p. and i.v. injection and continuous s.c. infusion) and several different doses and schedules were tried. In addition, a long-acting derivative of SOD was synthesized and tested. Despite the observation of a modest delay in the onset of diabetes in one experiment, no protective effect of SOD on the progressive elevation of blood glucose concentrations was evident in the majority of studies. Moreover, a loss in pancreatic insulin content and a tripling of pancreatic glucagon content occurred in all mice treated with low dosages of Sz, irrespective of whether or not either SOD or a long-acting derivative of SOD was administered. Finally, in parallel experiments in vitro, this enzyme was ineffective in protecting isolated rat islets from the acute toxicity of exposure to Sz on glucose-stimulated insulin release.

Studies of streptozotocin-induced insulitis and diabetes.

Multiple small injections of streptozotocin produce a delayed, progressive increase in plasma glucose in mice within 5-6 days after the injections, in association with pronounced insulitis and induction of type C viruses within beta cells. Multiple subdiabetogenic doses of streptozotocin in rats and multiple injections of another beta cell toxin, alloxan, in mice did not induce insulitis although hyperglycemia followed the injection of larger quantities of both agents. In mice, the prior injection of 3-O-methyl-D-glucose (3-OMG) or nicotinamide attenuated the diabetic syndrome produced by streptozotocin; however, 3-OMG was more protective. Rabbit antimouse lymphocyte serum, alone, provided partial protection but, when given together with either 3-OMG or nicotinamide, effectively prevented the streptozotocin-induced diabetic syndrome. Cessation of these preventive treatments was followed by the appearance of insulitis and diabetes. These findings suggest that multiple injections of streptozotocin induce, in susceptible hosts, the triad of direct beta cell cytotoxicity, virus induction within beta cells, and cell-mediated autoimmune reaction. These factors, acting separately or in concert, appear to induce a destructive insulitis and severe diabetes. The relative importance of each component and the factors governing host susceptibility remain to be clarified.