Generation of hydrogen peroxide and failure of antioxidative responses in pancreatic islets of male C57BL/6 mice are associated with diabetes induced by multiple low doses of streptozotocin.

AIMS/HYPOTHESIS: We studied the impact of the reactive oxygen species hydrogen peroxide (H2O2) and antioxidative enzymes on the pathogenesis of diabetes induced by multiple low doses of streptozotocin (MLD-STZ). METHODS: We isolated the islets of C57BL/6 mice. For ex vivo analyses, mice had been injected with MLD-STZ. For in vitro analyses, islets were incubated with different concentrations of STZ, with either of the two moieties of STZ, methylnitrosourea and D-glucose, with H2O2 or with alloxan. Levels of H2O2 generation were measured by the scopoletin method. We assessed mRNA expression of Cu/Zn and Mn superoxide dismutase, catalase, and glutathione peroxidase (GPX) by semiquantitative polymerase chain reaction. GPX activity was measured spectrophotometrically. In vitro, beta cell function was assayed by measuring basal and D-glucose-stimulated release of immunoreactive insulin using an ELISA kit. RESULTS: Ex vivo, MLD-STZ significantly increased H2O2 generation in male but not in female mice. It also increased GPX activity and mRNA expression of catalase, Cu/Zn and Mn superoxide dismutase, and GPX in female but not in male mice. In vitro, STZ significantly stimulated H2O2 generation in islets of male mice only. In male islets, alloxan increased H202 generation at a highly toxic concentration, but D-glucose and methylnitrosourea did not. Both STZ and H2O2 dose-dependently inhibited the release of immunoreactive insulin after a D-glucose challenge. CONCLUSIONS/INTERPRETATION: The results indicate that H2O2 participates in the pathogenesis of MLD-STZ diabetes in male C57BL/6 mice, which do not up-regulate antioxidative enzymes in islets. Conversely, female mice are protected, probably due to an increment of several enzymes with the potential to detoxify H2O2.

Differential target molecules for toxicity induced by streptozotocin and alloxan in pancreatic islets of mice in vitro.

Streptozotocin (STZ) and alloxan (ALX) are potent diabetogens in different species of laboratory animals. Here, we describe differential in vitro effects of STZ and ALX on beta-cell molecules that are essential for glucose transport and metabolism, the glucose transporter 2 (GLUT2) and glucokinase (GK), respectively. Incubation of isolated pancreatic islets of C57 BL/6 mice with STZ or ALX for 30 min resulted in a concentration-dependent gradual loss of beta-cell function as determined by basal and D-glucose (D-G)-stimulated insulin release. ALX concentration-dependently reduced the mRNA expression of GLUT2 and GK and the effect on GLUT2 was more marked. STZ, in contrast, did not affect the mRNA expression of GLUT2 and GK, but concentration-dependently reduced the GLUT2 protein expression. Both STZ and ALX failed to affect the mRNA expression of proinsulin and of beta-actin. The deleterious effects of STZ and ALX were not due to beta-cell loss, because the total RNA yields and protein contents as well as the proinsulin mRNA expression in isolated islets of the differentially treated islets did not differ significantly from controls. Furthermore, islets that had been exposed to STZ or ALX responded to the non-glucose secretagogue arginine in a pattern comparable to that of solvent-treated cultures. When preincubating islet cultures with either D-G or its chemically closely related analogue 5-thio-D-glucose (5-T-G), different effects were obtained after treatment with either ALX or STZ. Thus, preincubation with 5-T-G protected the cultures from STZ-induced GLUT2 protein reduction, whereas D-G failed to do so. Preincubation with D-G, however, protected the cultures from ALX-induced reduction of GLUT2 and GK mRNA expression, whereas 5-T-G, at best, exerted a modest protection against ALX at a concentration of 1 mmol/l. Apparently, in vitro, GLUT2 protein is a key target molecule for STZ, while GLUT2 mRNA and GK mRNA are target molecules for ALX.