Dual Control of glut1 Glucose Transporter Gene Expression by Hypoxia and by Inhibition of Oxidative Phosphorylation

Abstract

glut1 gene expression and glucose transport are stimulated in a variety of cells and tissues in response to hypoxia. glut1 is also up-regulated by inhibitors of oxidative phosphorylation (such as azide) in the presence of oxygen. Here, we test the hypothesis that hypoxia stimulates glut1 gene expression independent of its inhibitory effect on oxidative phosphorylation. We examined the effect of cobalt chloride, a known stimulator of genes responsive to reduced oxygen concentration per se, on GLUT1 expression under normoxic conditions and compared the results with the response to azide. Exposure of a rat liver cell line (Clone 9) to 250 microM cobalt chloride increases GLUT1 mRNA content, which becomes evident at 2 h, reaches a maximal value of approximately 12-fold at 8 h, and remains elevated at approximately 8-fold at 24 h. GLUT1 mRNA was the only GLUT isoform expressed in control cells and in cells exposed to cobalt chloride or azide. The induction of GLUT1 mRNA by cobalt chloride is associated with a approximately 10-fold stimulation of cytochalasin B-inhibitable 3-O-methyl-D-glucose transport at 24 h. In contrast to the rapid decrease in cell ATP levels and the stimulation of glucose transport in response to azide, cell ATP content and glucose transport remained unaltered during the initial 1-h period of exposure to cobalt chloride. The effect of cobalt chloride on GLUT1 mRNA content is mimicked by Ni(II) or Mn(II) but not by Fe(II). Employing actinomycin D, we found no increase in the approximately 1.5-h half-life of GLUT1 mRNA in cobalt chloride-treated cells, suggesting that the effect of cobalt chloride on GLUT1 mRNA content is largely mediated at the transcriptional level; in contrast, GLUT1 mRNA half-life increased to >8 h in azide-treated cells. In transient transfections we found that approximately 6 kilobase pairs (kbp) of 5'-flanking region of the rat glut1 promoter confers both cobalt chloride- and azide-inducibility to a reporter gene. Deletion of approximately 2, 500 base pairs (bp) from the 5' end of the approximately 6-kbp DNA fragment results in a reduction of the response to cobalt chloride and a complete loss of the response to azide. A 666-bp DNA segment located approximately 6.0 kbp upstream of the transcription start site was found to be necessary for the increase in reporter gene expression in response to azide, whereas a 480-bp segment located at approximately -3.5 kbp mediated the response to cobalt chloride. The 480-bp segment is highly homologous to the previously reported mouse glut1 enhancer and contains several potential regulatory elements, including a hypoxia-inducible element; an additional hypoxia-inducible element is present in the 666-bp segment. Our results suggest that glut1 gene expression is regulated in a dual fashion by hypoxia per se and in response to inhibition of oxidative phosphorylation.