Attenuation of expression of γ-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic β cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma -glutamylcysteine synthetase (gamma -GCS) heavy subunit ribozyme was stably transfected to MING cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 mug/10(6) cells/h in control cells (CC) to 1.48 mug/10(6) cells/h. This was associated with increased intracellular Ca2+ concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MING cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca2+ concentration. (C) 2000 Academic Press.