REGULATED EXPRESSION OF AN INSULIN-RESPONSIVE GLUCOSE TRANSPORTER (GLUT4) MINIGENE IN 3T3-L1 ADIPOCYTES AND TRANSGENIC MICE

Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficie nt to mediate differentiation-induced reporter gene expression in mous e 3T3-L1 preadipocytes. To locate the regulatory element(s) responsibl e for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected i nto mice and 3T3-L1 preadipocytes at relatively low copy number. In tr ansgenic mice the GLUT4 minigene exhibited a pattern of tissue-specifi c expression similar, but not identical, to that of the endogenous gen e. In 3T3-L1 cells expression of minigene mRNA occurred upon different iation into adipocytes, with kinetics virtually identical to that of e ndogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of t he endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocyte s with 8-bromo-cAMP, which represses transcription of the endogenous G LUT4 gene, also repressed expression of the GLUT4 minigene. However, i nsulin, which down-regulates transcription of the endogenous GLUT4 gen e, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for dire cting tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 1 4-kb GLUT4 minigene. However, the cis elements necessary for maximal t issue-specific expression and for insulin-induced down-regulation of e xpression are not located in the minigene.