Behrooz A, Ismail-Beigi F
Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4951, USA.
J Biol Chem. 1997 Feb 28;272(9):5555-62. doi: 10.1074/jbc.272.9.5555.
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.
在多种细胞和组织中，缺氧会刺激葡萄糖转运蛋白1（GLUT1）基因表达及葡萄糖转运。在有氧存在的情况下，氧化磷酸化抑制剂（如叠氮化物）也会使GLUT1上调。在此，我们检验了这样一个假说：缺氧刺激GLUT1基因表达，与其对氧化磷酸化的抑制作用无关。我们研究了氯化钴（一种已知的可直接刺激对氧浓度降低有反应的基因的物质）在常氧条件下对GLUT1表达的影响，并将结果与对叠氮化物的反应进行比较。将大鼠肝细胞系（克隆9）暴露于250微摩尔/升氯化钴中会增加GLUT1信使核糖核酸（mRNA）含量，这在2小时时变得明显，在8小时时达到约12倍的最大值，并在24小时时保持在约8倍的升高水平。GLUT1 mRNA是对照细胞以及暴露于氯化钴或叠氮化物的细胞中唯一表达的GLUT异构体。氯化钴对GLUT1 mRNA的诱导与24小时时细胞松弛素B抑制的3 - O - 甲基 - D - 葡萄糖转运约10倍的刺激相关。与叠氮化物导致细胞三磷酸腺苷（ATP）水平迅速下降和葡萄糖转运受刺激相反，在暴露于氯化钴的最初1小时内，细胞ATP含量和葡萄糖转运保持不变。氯化钴对GLUT1 mRNA含量的影响可被镍（II）或锰（II）模拟，但不能被铁（II）模拟。使用放线菌素D，我们发现在氯化钴处理的细胞中，GLUT1 mRNA约1.5小时的半衰期没有增加，这表明氯化钴对GLUT1 mRNA含量的影响主要在转录水平介导；相反，在叠氮化物处理的细胞中，GLUT1 mRNA半衰期增加到超过8小时。在瞬时转染实验中，我们发现大鼠GLUT1启动子5'侧翼区域约6千碱基对（kbp）赋予报告基因对氯化钴和叠氮化物的诱导性。从约6 - kbp DNA片段的5'端缺失约2500碱基对（bp）会导致对氯化钴的反应降低以及对叠氮化物的反应完全丧失。位于转录起始位点上游约6.0 kbp处的一个666 - bp DNA片段被发现是报告基因表达因叠氮化物而增加所必需的，而位于约 - 3.5 kbp处的一个480 - bp片段介导了对氯化钴的反应。该480 - bp片段与先前报道的小鼠GLUT1增强子高度同源，并包含几个潜在的调控元件，包括一个缺氧诱导元件；在666 - bp片段中还存在另一个缺氧诱导元件。我们的结果表明，GLUT1基因表达受缺氧本身以及对氧化磷酸化抑制的双重调控。
