Attenuation of expression of gamma-glutamylcysteine synthetase by ribozymetransfection enhance insulin secretion by pancreatic beta cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutat hione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion a nd the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma -glutamylcysteine synthetase (gamma -GCS) heavy subu nit ribozyme was stably transfected to MING cells to reduce intracellular G SH concentration. In the presence of 10 mM glucose, ribozyme-transfected ce lls (RTC) increased insulin secretion from 0.58 mug/10(6) cells/h in contro l 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 r esults demonstrated that intracellular GSH concentration might influence in sulin secretion by MING cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increa sed intracellular Ca2+ concentration. (C) 2000 Academic Press.