INSULIN-RESPRONSIVE GLUCOSE TRANSPORT IN ADIPOSE TISSUE

The overall goal of this research proposal is to examine the insulin and glucose-dependent regulation of the facilitative glucose transport systems found in rat epididymal adipose tissue and primary isolated adipocytes. We have recently observed that streptozotocin (STZ)-induced diabetes results in a specific decrease in the expression of the insulin- responsive (GLUT4) glucose transporter mRNA in rat adipose tissue without significant effect on GLUT1 expression. In these studies, we will ultimately address the molecular mechanisms responsible for the regulation of adipose glucose transporter gene expression. This will be accomplished by detailed analysis of GLUT4 promoter function and identification of cis-regulatory elements. In addition, we propose to characterize adipocyte-specific DNA binding factors responsible for the hormonal/metabolic and tissue-specific regulation of this gene. To address these issues, the following specific aims will be addressed. 1) In comparison to in vivo regulation of adipose tissue GLUT4, we will examine the effect of in vitro insulin and glucose treatments in isolated primary adipocytes from control, STZ-diabetic and insulin-treated STZ- diabetic rats in terms of altered basal and insulin-stimulated glucose transport activity and relative expression of glucose transporter protein and mRNA levels. 2) We will determine whether the hormonal/metabolic regulation of GLUT4 mRNA levels in adipose tissue and isolated adipocytes results from transcriptional events by nuclear run-on analysis. 3) To identify sequences important for the adipocyte-specific and hormonal/metabolic-dependent transcriptional regulation of this gene, fusions between the GLUT4 promoter region and a CAT reporter gene will be used in transient transfection assays. 4) Based upon the information obtained in specific aims 2 and 3, several promoter constructs will be tested for their ability to produce appropriate tissue-specific and hormonal/metabolic-dependent regulation in transgenic mice. 5) The specific binding of known transcription factors, which have consensus DNA sequences in the GLUT4 control region, will be assessed along with their effect on GLUT4 gene expression. In future studies, the functional DNA sequences determined in specific aims 3 and 4 will be used in mobility shift and DNase 1 footprint assays to identify trans-factors in nuclear extracts which affect adipocyte-specific GLUT4 expression.