Abstract
Prolonged treatment of 3T3-L1 adipocytes decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of promoter-reporter gene constructs that contained 2900 or 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or 78 base pairs of 5'-flanking region was not. Nuclear extract from 3T3-L1 adipocytes protected the region from -707 to -681 in the GLUT4 5'-flanking region from DNase I digestion. Using an oligonucleotide probe that corresponded to this footprinted region, two major protein-DNA complexes were identified by a gel mobility shift assay. Southwestern analysis identified four protein bands with molecular masses from 38 to 46 kDa that bound to the insulin-responsive region probe. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by insulin treatment, confirming that this sequence is necessary for the repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a distinct mechanism of gene regulation by insulin.
Highlights
Alterations in GLUT4 expression have been found in many models of insulin resistance [4]
Like the endogenous GLUT4 gene [14], expression of promoter-reporter constructs containing at least 785 bp of 5Јflanking region of the GLUT4 gene is decreased by insulin treatment in 3T3-L1 adipocytes, whereas expression of constructs containing 641 bp or less of 5Ј-flanking region is not altered by treatment with insulin
Gel mobility shift assays using a probe with a block mutation of bases Ϫ683 to Ϫ678 showed the same pattern as that for the wild-type sequence, further delineating the DNA-binding sequence to bases Ϫ707 to Ϫ684
Summary
Insulin acutely stimulates an increase in GLUT4 activity at the plasma membrane, it has been shown to decrease GLUT4 gene expression in the 3T3-L1 adipocyte [14], an in vitro model of tissue adipocytes. This effect suggests that the decreased expression of GLUT4 found in adipose tissue in obesity and type 2 diabetes mellitus might be due to suppression of GLUT4 expression by the hyperinsulinemia that occurs in both of these conditions. Of insulin on GLUT4 expression to identify the cis-acting elements in the GLUT4 gene that mediate this response and the trans-acting factors that interact with these elements
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