Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes.Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role.A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture.The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB.The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I ; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation.These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepa-Abbreviations used : GSK3, glycogen synthase kinase 3 ; HA, haemagglutinin ; MAP, mitogen-activated protein ; mTOR, mammalian target of rapamycin ; OHT, 4-hydroxytamoxifen ; PDEs, phosphodiesterases ; PEPCK, phosphoenolpyruvate carboxykinase ; PHAS-I, phosphorylated, heat and acid stable regulated by insulin protein (also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein-1) ; PI-3K, phosphoinositide 3-kinase ; PKB, protein kinase B (also termed cAkt) ; SREBP, sterol-regulatory-element-binding protein.