The overall goal of this research is to examine the tissue-specific and hormonal/metabolic regulation of the GLUT4 facilitative glucose transporter gene in adipose tissue, heart and skeletal muscle. Previous studies have established that GLUT4 gene expression is transcriptionally regulated in insulin-deficient states such as fasting and streptozotocin (STZ)-induced diabetes. Recent evidence suggests that overexpression of GLUT4 enhances insulin sensitivity in both normal and genetically diabetic transgenic mice. Using transgenic mice, we have also demonstrated that the regulatory sequences required for tissue-specific and hormonal/metabolic regulation are located within 1 kb of the major transcription start site of the human GLUT4 gene. By deletion analysis carried out in transgenic mice, we have shown that the regulatory regions conferring skeletal muscle-specific expression do not overlap with regions responsible for adipose tissue- and cardiac specific expression. Further, the regions responsible for regulated expression in STZ-induced diabetes do not coincide with the putative skeletal muscle-specific elements. Within the putative skeletal muscle-specific element we have identified an authentic MEF2 binding site which may be responsible in part for gene expression. We propose to further define the regulatory region(s) and DNA binding transcription factors responsible for GLUT4 gene expression in order to determine the molecular mechanisms responsible for tissue specificity and hormonal/metabolic regulation. To accomplish these goals, the following specific aims will be addressed: 1 To further define the regulatory regions within the human GLUT4 promoter regions are responsible for regulated gene expression, we will generate lines of transgenic mice carrying fusions between various deletions of the human GLUT4 promoter regions and a CAT reporter gene. 2. To begin determining the transcription factors responsible for GLUT4 gene expression, we will initially focus on the role of MEF2. We will assay nuclear extracts from adipose tissue, heart and skeletal muscle for MEF2 binding activity and identify the tissue- specific expression of MEF2 isoforms. 3. We will used isolated adipocytes from transgenic mice to determine the molecular mechanisms underlying the time dependent decrease in GLUT4 transcription rate that occurs in these cells. Understanding this mechanism will provide useful insight into the mechanisms by which GLUT4 gene expression occurs in vivo.

R01DK047894

1997-09-17

2001-08-31