REGULATION OF GLUT1 GLUCOSE TRANSPORTER EXPRESSION

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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.