A transcriptional repressor regulates mouse GLUT4 gene expression during the differentiation of 3T3-L1 cells

GLUT4, the major glucose transporter in adipose tissue, is expressed during the differentiation of 3T3-L1 cells from preadipocytes to adipocytes. We previously examined the mouse GLUT4 promoter activity up to -590 bp, and demonstrated that the 5'-flanking region of the GLUT4 gene between -200 and -100 bp contains sequences that act as a repressor in preadipocytes, but not in adipocytes. Here we examine in detail the activity of this repressor in 3T3-L1 cells. Transient transfections indicated that the region extending from -125 to -112 bp functions as a repressor element only in preadipocytes. In electrophoretic mobility shift assay (EMSA), this GLUT4 repressor element (G4RE) generated specific bands with nuclear extracts from preadipocytes, but not from adipocytes. Southwestern blot analysis identified a protein of approximately 96 kDa from preadipocytes that bound to the G4RE site. Mutation of the G4RE site, which abolished the protein/DNA complex formation by EMSA, increased GLUT4 promoter activity only in preadipocytes. These results suggest that the G4RE site and its binding protein may regulate GLUT4 gene transcription during adipocyte differentiation.