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

Abstract

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 30min of the addition of 4-hydroxytamoxifen and to stay high for 8h, 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 hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics 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 that acute activation of PKB is sufficient to produce an insulin-like induction 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 increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.

The Transcription Factors HIF-1 and HNF-4 and the Coactivator p300 Are Involved in Insulin-regulated Glucokinase Gene Expression via the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway

“…The involvement of PKB in regulating GK expression also has been shown by transducing primary rat hepatocytes with an adenovirus vector allowing an 4-hydroxytamoxifen-activable expression of PKB. The 4-hydroxytamoxifen treatment of the transduced cells resulted in a GK mRNA induction similar to the insulin-induced mRNA accumulation (53). This regulation does not appear to be hepatocyte-specific, since GK gene expression also is stimulated by insulin in pancreatic ␤ cells via involvement of the insulin receptor type B, PI3K, and PKB (54).…”

“…To our knowledge these studies provide the first report that insulin is able to activate the liverspecific GK promoter through the hypoxia response element in the proximal GK promoter and that interactions between HIF-1, HNF-4, and p300 contribute to this effect. with another study in rat hepatocytes showing that the insulininduced increase in GK mRNA abundance was completely abolished by PI3K inhibitors such as wortmannin and LY294002 (53). The involvement of PKB in regulating GK expression also has been shown by transducing primary rat hepatocytes with an adenovirus vector allowing an 4-hydroxytamoxifen-activable expression of PKB.…”

Berberine ameliorates hyperglycemia in alloxan-induced diabetic C57BL/6 mice through activation of Akt signaling pathway

“…Inactivation or the activation mutation of glucokinase gene have been linked to the development of maturity-onset diabetes of the young (MODY), Type 2 (MODY2) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) [31][32][33]. Patrick B et al have demonstrated that the activation of Akt signal pathway increases glucokinase gene expression [7]. In our study, we found that berberine markedly increased glucokinase expression and its activity as well as the activation of Akt signal pathway in diabetic liver, which contributed to glycogen synthesis resulting in lower of blood glucose.…”

“…It induces GLUT translocation to plasma membrane [23] and regulates downstream factors including GSK-3β and glucokinase involved in glycogen synthesis [7,24,25]. The impaired Akt phosphorylation occurs in liver of diabetic individual [10].…”

“…It is believed that insulin-stimulated Akt activation promotes glycogen synthesis via inactivation of glycogen synthase kinase-3β (GSK-3β). In addition, glucokinase, another downstream factor of Akt found recently is a rate-limiting enzyme to catalyze glycogen synthesis [7]. In hepatocytes, the activation of Akt increases glucokinase gene expression and glycogen synthesis to maintain the euglycemia [8].…”