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 ins ulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-indu ced increase in glucokinase mRNA seen in unhibited cells, indicating that t he phosphoinositide 3-kinase module has a key role. A ligand inducible prot ein 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, h eat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP 1, for eukaryotic initiation factor 4E-binding protein 1), a protein involv ed in the regulation of translation initiation. These effects were comparab le to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to trans duced hepatocytes resulted in an induction of glucokinase mRNA with kinetic s and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish t hat acute activation of PKB is sufficient to produce an insulin-like induct ion of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increas e in glucokinase mRNA brought about by insulin, suggesting a contributory r ole of the mitogen-activated protein kinase cascade.