Original Article Characterization of a Protein Kinase B Inhibitor In Vitro and in Insulin-Treated Liver Cells Lisa Logie, 1 Antonio J. Ruiz-Alcaraz, 1 Michael Keane, 2,3 Yvonne L. Woods, 2 Jennifer Bain, 2 Rudolfo Marquez, 3 Dario R. Alessi, 1 and Calum Sutherland 1 OBJECTIVE—Abnormal expression of the hepatic gluconeo- genic genes (glucose-6-phosphatase [G6Pase] and PEPCK) con- tributes 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 PKB. Complete inhibition of PKB activity is achieved in liver cells incubated with 1–10 mol/l Akti-1/2, and this blocks insulin regulation of PEPCK and G6Pase expression. Our data demon- strate that only 5–10% of maximal insulin-induced PKB is re- quired to fully repress PEPCK and G6Pase expression. Finally, we demonstrate reduced insulin sensitivity of these gene promot- ers 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. Diabetes 56:2218–2227, 2007 P rotein kinase B (PKB) is a member of the AGC family of protein kinases (1–3). In mammals, there are three isoforms (PKB, PKB, and PKB) (1). PKB is activated following induction of phosphatidylinositol 3 (PI3) kinase activity and the resultant generation of the lipid second messengers PI 3,4,5 trisphosphate and PI 3,4 bisphosphate (4). These lipids bind to the PH domain of PKB, altering its confor- mation and permitting access to upstream protein kinases (5). Phosphoinositide-dependent protein kinase-1 phos- phorylates PKB at Thr 308 (6), and a second phosphoryla- tion (at Ser 473 ) occurs through the action of an alternative kinase, such as the rapamycin-insensitive mTOR complex 2 (TORC2) (7). Therefore, most growth factors, including platelet-derived growth factor, epidermal growth factor, and insulin, which are potent activators of PI3 kinase, also strongly induce PKB in cells. One of the first substrates of PKB to be characterized was GSK3, as part of the insulin signaling pathway that regulates glycogen metabolism (8). Since then, multiple potential substrates of PKB have been proposed including the proapoptotic protein Bad (9,10), the tuberous sclerosis complex (TSC)2 gene product (11), the Rab-GAP AS160 (12), proline-rich Akt substrate of 40 kDa (PRAS40) (13), and the key forkhead transcription factor subfamily, fork- head box class O (FOXO). PKB phosphorylates FOXO on several residues, promoting its inactivation and exclusion from the nucleus (14 –16). A growing number of insulin- inhibited genes are proposed to be targets of FOXO. These include glucose-6-phosphatase (G6Pase), PEPCK, and the insulin-like growth factor– binding protein-1 (IGFBP1) (17). All three genes are completely repressed in liver cells exposed to insulin (18) or in intact liver following feeding (19). This gene regulation requires PI3 kinase (20 –22) and phosphoinositide-dependent protein kinase (PDK)1 (19) activity and can be recapitulated by overexpression of active PKB (23). Meanwhile, overexpression of FOXO will induce insulin-responsive DNA sequences within these gene promoters (24 –27). These data suggest that insulin turns off these gene promoters by activating the PI3 kinase–PDK1-PKB pathway to inhibit FOXO. However the importance of PKB and/or FOXO in the regulation of these genes has been questioned (18,28). For example, overex- pression of dominant-negative PKB does not block insulin action on PEPCK (29) or G6Pase (22) and inhibitors of mTOR will block insulin regulation of IGFBP1 but not PKB or FOXO (21,26), while inhibitors of GSK3 (also down- stream of PKB) will inhibit these genes without regulating FOXO activity (30,31). It is also suggested that insulin can From the 1 Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland; the 2 Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee, Scotland; and the 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@dundee.ac.uk. Received for publication 17 March 2007 and accepted in revised form 5 June 2007. Published ahead of print at http://diabetes.diabetesjournals.org on 11 June 2007. DOI: 10.2337/db07-0343. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0343. Akti, Akt inhibitor; BP1-TIRE, binding protein 1–thymine-rich insulin re- sponse element; FOXO, forkhead box class O; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PDK, phosphoinosit- ide-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. © 2007 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2218 DIABETES, VOL. 56, SEPTEMBER 2007