In HIT-T15 insulinoma B-cells incubated in presence of [P-32]NAD, we identified by autoradiography and immunoblotting ADP-ribosylation (ADP-R) of the trimeric G-protein G alpha(s) and G alpha(olf) subunits (45 kDa) induced by cholera toxin in M1 (120,000g) and M2 (70,000g) subcellular fractions containing plasma membranes, insulin granules, and mitochondria. This ADP-R indicates that these two fractions contain functionally competent G alpha subunits for adenylyl cyclase activation. Prolonged exposure of HIT-T15 cells to high glucose (25 mM instead of 6 mM) specifically reduced the ADP-R in G alpha(s) and G alpha(olf) subunits in the M1 fraction only, despite the clear increase of their accumulation in this compartment. A similar alteration in the ADP-R of the M1-associated G alpha(s) and G alpha(olf) subunits was observed in pancreatic islets isolated from fasted and fed rats. These results may explain, at least in part, the undesirable effects of sustained hyperglycemia on the cAMP-dependent process of insulin secretion in diabetes.