REGULATION OF GLUT1 GLUCOSE TRANSPORTER EXPRESSION

多种细胞暴露于氧化磷酸化抑制剂 或缺氧条件导致糖酵解ATP的显着增强 合成. 对于葡萄糖渗透性高的细胞， 在维持糖酵解增强的葡萄糖需求中， 没有限制。 然而，对于许多细胞来说，葡萄糖进入基底膜下， 条件是葡萄糖消耗的限速， 只有当葡萄糖转运本身 也增强了。 我们最近在克隆9号中验证了这一假设 细胞，一种“非转化”大鼠肝细胞系，其中内部 葡萄糖浓度是外部培养基中存在的葡萄糖浓度的10%，并且 已经发现，在氰化物存在下孵育这些细胞， 叠氮化物导致葡萄糖转运的显著双相刺激 （由GLUT-1介导）。 运输刺激的“早期”阶段（0-2 小时）完全由翻译后机制介导，而 在响应的“后期”阶段（8-24小时）， 葡萄糖转运的增强与 GLUT-1含量及其编码mRNA的变化。 我们还显示了 GLUT-1 mRNA的诱导是对氧化应激抑制的反应， 磷酸化是由GLUT-1基因的增强和 转录和GLUT-1 mRNA周转率下降， 在缺氧条件下孵育时也获得GLUT-1 mRNA的诱导 条件 本申请的目的是研究 在“晚期”GLUT-1基因表达增强的基础 对氧化磷酸化抑制的反应阶段。 具体而言，我们将： 1)研究正在进行的蛋白质合成 刺激GLUT-1基因转录所必需的 抑制氧化磷酸化，并鉴定DNA 在GLUT-1基因的5 '-侧翼区的序列，介导这一点 反应; 2)检验蛋白质与特定蛋白质相互作用的假设 GLUT-1 mRNA的区域介导GLUT-1 mRNA的稳定， 氧化磷酸化的抑制;和 3)检查GLUT-1中较高的分数增量 mRNA含量与GLUT-1含量的比较反映了调节 在翻译水平上起作用的机制。 这些研究的结果应有助于更好地了解 适应性细胞中葡萄糖转运的调节机制 细胞对葡萄糖需求增加的反应，并应提供新的 深入了解这一重要的潜在的新型生理调节剂 运输过程。

Exposure of a variety of cells to inhibitors of oxidative phosphorylation or to hypoxic conditions result in a marked enhancement of glycolytic ATP synthesis. For cells that are highly permeable to glucose, the increase in glucose requirement to sustain enhanced glycolysis in principle poses no limitation. For many cells, however, glucose entry under basal conditions is rate-limiting for glucose consumption, and a sustained simulation of glycolysis is possible only if glucose transport itself is also augmented. We have recently tested this hypothesis in Clone 9 cells, a "nontransformed" rat liver cell line in which the internal glucose concentration is 10% of that present in the external medium, and have found that incubation of these cells in the presence of cyanide or azide leads to a striking biphasic stimulation of glucose transport (mediated by GLUT-1). The "early" phase of transport stimulation (0-2 hours) is mediated entirely by post-translational mechanisms, whereas during the "late" phase of the response (8-24 hours) the further enhancement of glucose transport is associated with substantial increase in GLUT-1 content and in its encoding mRNA. We have additionally shown that the induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation is mediated by both an enhancement of GLUT-1 gene transcription and a decrease in GLUT-1 mRNA turnover, and that a marked induction of GLUT-1 mRNA is also obtained upon incubation under hypoxic conditions. The goal of the present application is to investigate mechanisms that underlie the enhancement of GLUT-1 gene expression during the "late" phase of the response to inhibition of oxidative phosphorylation. Specifically, we will: 1) Investigate the possibility that on-going protein synthesis is necessary for the stimulation of GLUT-1 gene transcription in response to inhibition of oxidative phosphorylation, and to identify the DNA sequences in the 5'-flanking region of the GLUT-1 gene that mediate this response; 2) Test the hypothesis that interaction of protein(s) with specific regions of GLUT-1 mRNA mediate the stabilization of GLUT-1 mRNA following inhibition of oxidative phosphorylation; and 3) Examine the hypothesis that the higher fractional increment in GLUT-1 mRNA content compared to that of GLUT-1 content reflects regulatory mechanisms operative at the translational level. The results of these studies should lead to a better understanding of mechanisms underlying the regulation of glucose transport in the adaptive response to increased cellular demand for glucose and should provide new insights into potential novel physiological regulators of this important transport process.