DIABETES-INDUCED ALTERATIONS IN THE EXPRESSION, FUNCTIONING AND PHOSPHORYLATION STATE OF THE INHIBITORY GUANINE-NUCLEOTIDE REGULATORY PROTEIN GI-2 IN HEPATOCYTES

Levels of the G-protein α-subunits α-G(i)-2, α-G(i)-3 and the 42 kDa, but not the 45 kDa, form of α-G(s) were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in α-G(i) subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of G(s) were increased. G-protein β subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of G(i)-3, caused an increase in G(s) transcripts and decreased transcript number for G(i)-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, α-G(i)-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than α-G(i)-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of α-G(i)-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of α-G(i)-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.