EVIDENCE FOR DIFFERENTIAL ROLES OF THE RHO-SUBFAMILY OF GTP-BINDING PROTEINS IN GLUCOSE-INDUCED AND CALCIUM-INDUCED INSULIN-SECRETION FROM PANCREATIC BETA-CELLS

We utilized clostridial toxins (with known specificities for inhibitio n of GTPases) to ascertain the contribution of candidate GTPases in ph ysiologic insulin secretion from beta cells. Exposure of normal rat is lets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either to xin reduced glucose-or potassium-induced insulin secretion from rat is lets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose-or potassium-induced secretion. However, clostridial toxin C3 -exoenzyme (which ADP-ribosylates and inactivates only Rho) was withou t any effect on either glucose-or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose-or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, si nce no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan i n a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or L T) treatment also did not alter glucose-or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+](i)), suggesting that th ese GTPases are involved in steps distal to elevations in [Ca2+](i). R ecent findings indicate that: the carboxyl methylation of Cdc42 is sti mulated by only glucose, whereas that of Rap (Kowluru et al., J Clin I nvest 98: 540-555, 1996) and Rac (present study) are regulated by gluc ose or potassium. Together, these findings provide direct evidence, fo r the first time, that the Rho subfamily of GTPases plays a key regula tory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, wh ereas Rap and/or Rac may be required for a later step(s) in the stimul us-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin ). (C) 1997 Elsevier Science Inc.