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.
Roth
Ulrike
U
Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Humboldtallee 23, D-37073 Göttingen, Germany.
Curth
Katja
K
Unterman
Terry G
TG
Kietzmann
Thomas
T
D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000096:biosynthesis* / Q000235:genetics; D051557:Hepatocyte Nuclear Factor 4; D051548:Histone Acetyltransferases; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007328:Insulin / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism*; D056921:Nuclear Receptor Coactivator 3; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D005941:Glucokinase; D051557:Hepatocyte Nuclear Factor 4; D051548:Histone Acetyltransferases; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007328:Insulin; D009687:Nuclear Proteins; D056921:Nuclear Receptor Coactivator 3; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D015534:Trans-Activators; D014157:Transcription Factors; C497444:Hif1a protein, rat