C/EBPalpha activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes.
TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (-542/-371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPalpha (CCAAT/enhancer-binding protein alpha). Transient transfections of different mutant TGH promoter-luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions -470/-459 bp and -404/-390 bp are important for transcriptional activation. Co-transfection with C/EBPalpha cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPalpha increased TGH promoter activity. Ectopic expression of C/EBPalpha in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPalpha. This is the first evidence that C/EBPalpha participates directly in the regulation of an enzyme associated with lipolysis.
Wei
Enhui
E
Department of Biochemistry, CIHR Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Lehner
Richard
R
Vance
Dennis E
DE
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D002522:Chlorocebus aethiops; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction; D008049:Lipase / Q000235:genetics*; D051379:Mice; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism
D022763:CCAAT-Enhancer-Binding Protein-alpha; D014280:Triglycerides; D008049:Lipase