Project Summary GLUT4 is the isoform principally responsible for insulin-mediated glucose uptake in mammalian tissues. Glucose homeostasis is sensitive to changes in GLUT4 levels. Modulation of GLUT4 levels is therefore an attractive molecular target for therapeutic intervention in insulin-resistant states, including diabetes mellitus. A straightforward approach to enhance GLUT4 expression is to increase the transcription rate of the gene. GLUT4 gene expression is transcriptionally regulated in physiologic states such as insulin-deficiency and exercise, and it is likely that a pharmacological intervention can be developed to enhance Glut4 gene transcription. To reach this goal, we must first understand the molecular basis for transcriptional regulation of the GLUT4 gene. Using transgenic mice, we have shown that cis-elements regulating the human Glut4 promoter are located within 895 bp immediately 5'of the transcription initiation site. This region contains two major regulatory domains, referred to as Domain I and the MEF2 domain. These elements function cooperatively to support regulated expression of GLUT4. The MEF2 domain binds isoforms of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors, while Domain I binds GEF (GLUT4 Enhancer Factor), a transcriptional activator identified and cloned in our laboratory. MEF2 isoforms and GEF form a protein complex in vivo;however, the function of this complex in regulating gene transcription is not known. We hypothesize that both the tissue-specific and the hormonal and/or metabolic regulation of the GLUT4 gene are carried out through these two regulatory domains and their cognate binding proteins. The primary goal of this proposal is to understand the molecular mechanisms of the tissue-specific and hormonal/metabolic regulation of GLUT4 gene transcription. To achieve these goals, we propose the following specific aims: 1) To understand the basis of cooperation between GEF and MEF2 proteins for DNA binding;2) To understand the mechanisms by which GEF and MEF2 proteins regulate the GLUT4 promoter;3) ) To determine the nature of the metabolic signal(s) that regulates GLUT4 gene transcription in vivo. Preliminary evidence suggests that changes in lipid metabolism and/or cAMP signaling target GLUT4 promoter function. Completion of the aims of this proposal will determine how these signals are transmitted.