Modulation of glucokinase expression by hypoxia-inducible factor 1 and upstream stimulatory factor 2 in primary rat hepatocytes.

Glucokinase (GK) is the key enzyme of glucose utilization in liver and is localized in the less aerobic perivenous area. Until now, the O2-responsive elements in the liver-specific GK promoter are unknown, and therefore the aim of this study was to identify the O2-responsive element in this promoter. We found that the GK promoter sequence -87/-80 matched the binding site for hypoxia inducible factor 1 (HIF-1) and upstream stimulatory factor (USF). In primary rat hepatocytes we could show that venous pO2 enhanced HIF-1alpha and USF-2a levels, both of which activated GK expression. Furthermore, transfection experiments revealed that the GK sequence -87/-80 mediated the HIF-1alpha- or USF-2-dependent activation of the GK promoter. The binding of HIF-1 and USF to the GK-HRE was corroborated by electrophoretic mobility shift assay (EMSA). However, the maximal response to HIF-1alpha or USF was only achieved when constructs with the -87/-80 sequence in context with a 3'-36 bp native GK promoter sequence containing a hepatocyte nuclear factor 4 (HNF-4) binding site were used. HIF-1alpha and HNF-4 additively activated the GK promoter, while USF-2 and HNF-4 together did not show this additive activation. Thus, HIF-1 and USF may play differential roles in the modulation of GK expression in response to O2.

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.

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.

Hypoxia-inducible factor-1 and hypoxia response elements mediate the induction of plasminogen activator inhibitor-1 gene expression by insulin in primary rat hepatocytes.

The expression of the plasminogen activator inhibitor-1 (PAI-1) gene is enhanced by insulin both in vivo and in various cell types. Because insulin exerts a number of its biologic activities via the phosphatidylinositol 3-kinase and protein kinase B (PI3K/PKB) signaling pathway, it was the aim of the present study to investigate the role of the PI3K/PKB pathway in the expression of the PAI-1 gene and to identify the insulin responsive promoter sequences. It was shown that the induction of PAI-1 mRNA and protein expression by insulin and mild hypoxia could be repressed by the PI3K inhibitor wortmannin. Overexpression of a constitutively active PKB led to induction of PAI-1 mRNA expression and of luciferase (Luc) activity from a gene construct containing 766 bp of the rat PAI-1 promoter. Mutation of the hypoxia response elements (HRE-1 and HRE-2) in rat PAI-1 promoter, which could bind hypoxia inducible factor-1 (HIF-1), abolished the induction of PAI-1 by insulin and PKB. Insulin and the constitutive active PKB also induced Luc expression in cells transfected with the pGl3EPO-HRE Luc construct, containing 3 copies of the HRE from the erythropoietin gene in front of the SV40 promoter. Furthermore, insulin and the active PKB enhanced all 3 HIF alpha-subunit protein levels and HIF-1 DNA-binding activity, as shown by electrophoretic mobility shift assays (EMSAs). Thus, the insulin-dependent activation of the PAI-1 gene expression can be mediated via the PI3K/PKB pathway and the transcription factor HIF-1 binding to the HREs in the PAI-1 gene promoter.

Activation of glucokinase gene expression by hepatic nuclear factor 4alpha in primary hepatocytes.

Glucokinase (GK) is a key enzyme for glucose utilization in liver and shows a higher expression in the perivenous zone. In primary rat hepatocytes, the GK gene expression was activated by HNF (hepatic nuclear factor)-4alpha via the sequence -52/-39 of the GK promoter. Venous pO2 enhanced HNF-4 levels and HNF-4 binding to the GK-HNF-4 element. Thus, HNF-4alpha could play the role of a regulator for zonated GK expression.