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 insulindependent 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 proteinkinase B (PKB) stimulated GK mRNA and protein expression. The

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... ansfection 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-1␣ to activate the GK promoter was enhanced by hepatocyte nuclear factor-4␣ (HNF-4␣), 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-1␣, 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.