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 insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible 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 into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing 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 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene.
Ezaki
O
O
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, MD 21205.
Flores-Riveros
J R
JR
Kaestner
K H
KH
Gearhart
J
J
Lane
M D
MD
D016475:3T3 Cells; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002454:Cell Differentiation; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D005786:Gene Expression Regulation; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D015342:DNA Probes; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA