OBJECTIVE-Abnormal expression of the hepatic gluconeogenic genes (glucose-6-phosphatase [G6Pase] and PEPCK) contributes to hyperglycemia. These genes are repressed by insulin, but this process is defective in diabetic subjects. Protein kinase B (PKB) is implicated in this action of insulin. An inhibitor of PKB, Akt inhibitor (Akti)-1/2, was recently reported; however, the specificity and efficacy against insulin-induced PKB was not reported. Our aim was to characterize the specificity and efficacy of Akti-1/2 in cells exposed to insulin and then establish whether inhibition of PKB is sufficient to prevent regulation of hepatic gene expression by insulin.
RESEARCH DESIGN AND METHODS-Akti-1/2 was assayed against 70 kinases in vitro and its ability to block PKB activation in cells exposed to insulin fully characterized.
RESULTS-Akti-1/2 exhibits high selectivity toward PKB alpha and PKB beta. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mu mol/l Akti-1/2, and this blocks insulin regulation of PEPCK and G6Pase expression. Our data demonstrate that only 5-10% of maximal insulin-induced PKB is required to fully repress PEPCK and G6Pase expression. Finally, we demonstrate reduced insulin sensitivity of these gene promoters in cells exposed to submaximal concentrations of Akti-1/2; however, full repression of the genes can still be achieved by high concentrations of insulin.
CONCLUSIONS-This work establishes the requirement for PKB activity in the insulin regulation of PEPCK, G6Pase, and a third insulin-regulated gene, IGF-binding protein-1 (IGFBP1); suggests a high degree of functional reserve; and identifies Akti-1/2 as a useful tool to delineate PKB function in the liver.