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Characterization of a Protein Kinase B Inhibitor In Vitro and in Insulin-Treated Liver Cells

¹ Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland
² Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee, Scotland
³ Division of Biological Chemistry, University of Dundee, Dundee, Scotland

Address correspondence and reprint requests to Dr. Calum Sutherland, Pathology and Neurosciences, University of Dundee, Ninewells Hospital, Dundee, Scotland, U.K., DD1 9SY. E-mail: c.d.sutherland{at}dundee.ac.uk

Abbreviations: Akti, Akt inhibitor; BP1-TIRE, binding protein 1–thymine-rich insulin response element; FOXO, forkhead box class O; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PDK, phosphoinositide-dependent protein kinase; PI3, phosphatidylinositol 3; PKB, protein kinase B; PRAS40, proline-rich Akt substrate of 40 kDa; smMLCK, smooth muscle myosin light-chain kinase; TSC, tuberous sclerosis complex

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 and PKBß. Complete inhibition of PKB activity is achieved in liver cells incubated with 1–10 µmol/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.

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