Within the last ten years, significant progress has been made which extends our understanding of the glucose transport process, often the rate-limiting step in glucose metabolism. The two most important discoveries during this period include the translocation mechanism which underlies insulin-stimulated glucose transport and the identification of multiple isoforms. Catabolite repression of metabolism (adaptive regulation) has been widely studied in procaryotes and is recognized as an important and global regulatory system. Despite this, nutrient control in eucaryotes has been under investigation only recently with few studies focusing exclusively on the glucose transporter. With the 3T3-Ll adipocyte cell line as a model for adipose, we will explore the regulation of the constitutive glucose transporter, GLUT 1. The objective of this proposal is to define the mechanisms by which transport is regulated by changes in glucose availability. This objective will be met by addressing the following Specific Aims. First, we will characterize the enhanced 'basal' transport activity as a function of glucose starvation. We will test the hypothesis that enhanced activity is due to elevated GLUT 1 protein, regulated at the translational level. Secondly, we will define the mechanism of down-regulation of starvation-induced transport. We will test the hypothesis that glucose represses transport activity by a protein-synthesis independent mechanism using metabolic inhibitors, subcellular fractionation, and immunodetection. Finally, we will define the relationship between the glucose-regulated protein, GRP 78, and GLUT 1. These unique studies will test the hypothesis that GRP 78 retards GLUT 1 processing when the transporter is inappropriately glycosylated. This will be accomplished by analyzing co-precipitation of GRP 78 and GLUT 1 in glucose-deprived cells. Together, these studies will define the mechanisms unique to adipocytes which may act in vivo to regulate constitutive transport in response to fluctuations in circulating glucose.
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