title: Characterization of a protein kinase B inhibitor in vitro and in insulin-treated liver cells.
abstract: 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.
  
  
  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 PKBalpha and PKBbeta. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mumol/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.
authors.affiliation: Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Logie
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Ruiz-Alcaraz
authors.forename: Michael
authors.initials: M
authors.lastname: Keane
authors.forename: Yvonne L
authors.initials: YL
authors.lastname: Woods
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bain
authors.forename: Rudolfo
authors.initials: R
authors.lastname: Marquez
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Calum
authors.initials: C
authors.lastname: Sutherland
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017930:Genes, Reporter; D005943:Gluconeogenesis / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000502:physiology*; D008113:Liver Neoplasms; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C501521:Akt-I-1,2 compound; D001596:Benzylamines; D004791:Enzyme Inhibitors; D007328:Insulin; D011810:Quinoxalines; D051057:Proto-Oncogene Proteins c-akt