Changes in the phosphorylation state of the inhibitory guanine‐nucleotide‐binding protein Gi‐2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats

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Abstract
Treatment of intact, 32Pi‐labelled hepatocytes from lean Zucker rats with a range of agents including 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the α‐subunit of the inhibitory G protein of adenylate cyclase (αGi‐2). These agonist‐induced phosphorylations of αGi‐2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5′‐[β,γ imido]triphosphate (p[NH]ppG) to inhibit forskolin‐stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist‐induced phosphorylation of αGi‐2 and to p[NH]ppG‐mediated inhibition of adenylate cyclase. The basal level of αGi‐2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 ± 0.09 mol phosphate/mol αGi‐2 than in hepatocytes from lean animals which gave 0.54 ± 0.09 mol phosphate/mol αGi‐2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of αGi‐2 in the hepatocytes from lean animals but had little effect on the phosphorylation of αGi‐2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of αGi‐2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of αGi‐2. The resistance to protein‐kinase‐C‐mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine‐nucleotide‐mediated ‘Gi function’ seen in obese Zucker rats may be due to an inactivating phosphorylation of αGi‐2.

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