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TCDD suppresses insulin‐responsive glucose transporter (GLUT‐4) gene expression through C/EBP nuclear transcription factors in 3T3‐L1 adipocytes
Author(s) -
Liu Phillip ChinChen,
Matsumura Fumio
Publication year - 2006
Publication title -
journal of biochemical and molecular toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.526
H-Index - 58
eISSN - 1099-0461
pISSN - 1095-6670
DOI - 10.1002/jbt.20120
Subject(s) - chloramphenicol acetyltransferase , transactivation , glut4 , transfection , glucose transporter , microbiology and biotechnology , reporter gene , chemistry , transcription factor , glucose uptake , response element , adipose tissue , 3t3 l1 , gene , gene expression , biology , promoter , insulin , biochemistry , adipocyte , endocrinology
TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3‐L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between −78 and −273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel‐shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [ 32 P]‐labeled probe containing C/EBP response element equivalent of the −265 to −242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBPα and an increase in that of C/EBPβ among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBPα, C/EBPβ, or C/EBPδ through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBPα or C/EBPβ. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3‐L1 cells. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:79–87, 2006; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20120