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

Abstract

Glucokinase plays a key role in the regulation of glucose utilization in liver and its expression is strongly enhanced by insulin and modulated by venous pO(2). In primary rat hepatocytes, pO(2) modulated insulin-dependent glucokinase (GK) gene expression was abolished by wortmannin an inhibitor of phosphatidylinositol 3-kinase (PI3K). Transfection of vectors encoding the p110 catalytic subunit of PI3K or constitutively active protein kinase B (PKB) stimulated GK mRNA and protein expression. The transfection of GK promoter constructs together with expression vectors for p110 or constitutively active PKB revealed that the GK promoter region -87/-80 mediates the response to PI3K/PKB. Transfection experiments and gel shift assays show that this element is able to bind hypoxia-inducible factor-1 (HIF-1) in a hypoxia- and PKB-dependent manner. The ability of HIF-1alpha to activate the GK promoter was enhanced by hepatocyte nuclear factor-4alpha (HNF-4alpha), acting via the sequence -52/-39, and by the coactivator p300. Stimulation of the GK promoter by insulin was dependent on the intact -87/-80 region and maximal stimulation was achieved when HIF-1alpha, HNF-4, and p300 were cotransfected with the -1430 GK promoter Luc construct in primary hepatocytes. Maximal stimulation of GK promoter activity by insulin was inhibited when a p300 vector was used containing a mutation within a PKB phosphorylation site. Thus, a regulatory transcriptional complex consisting of HIF-1, HNF-4, and p300 appears to be involved in insulin-dependent GK gene activation.