Insulin does not regulate the promoter of cholesteryl ester transfer protein (CETP) in HIRc/pCETP-CAT cells.
Cholesteryl ester transfer protein (CETP) is a plasma enzyme involved in cholesterol metabolism. As a potential target in the treatment of atherosclerosis, a number of studies have focused how this enzyme is regulated. It has been postulated that insulin may regulate CETP gene expression, and these effects may be mediated through CCAAT/enhancer binding protein alpha (C/EBPalpha). The present study examines the effects of insulin on the activity of the CETP promoter in rat fibroblasts expressing the human insulin receptor (HIRc). HIRc cells were stably transfected with a chimeric construct containing 3.2 kb of the CETP promoter attached to the bacterial chloramphenicol acyltransferase gene (pCETP-CAT) without significantly affecting the expression of the insulin receptor. CAT activity was 8-fold higher in cultured HIRc/pCETP-CAT in the presence of 100 mg/dL LDL cholesterol, than those cultured without cholesterol (p < 0.05). However, culturing these cells in the presence of 100 nM insulin did not result in any change in CAT activity when compared to control cells. In HIRc/pCETP-CAT cells transiently transfected with a construct that constitutively expressed C/EBPalpha protein, a 3-fold increase in CAT activity was observed when compared to cells transiently transfected with non-specific DNA (p < 0.05). However, no observable effect on the CETP promoter was observed in the presence of insulin. Thus, in HIRc/pCETP-CAT cells, we were unable to substantiate the hypothesis that insulin regulates CETP gene transcription. These results suggest that the effects of insulin on CETP expression regulation may be downstream of transcription.
MacLean
P S
PS
Department of Biochemistry, East Carolina University School of Medicine, Greenville, NC 27858, USA.
Barakat
H A
HA
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000494:pharmacology; D053480:Cholesterol Ester Transfer Proteins; D002934:Cinnamates; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006023:Glycoproteins; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics; D051381:Rats; D011972:Receptor, Insulin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D014162:Transfection
D000900:Anti-Bacterial Agents; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002934:Cinnamates; D006023:Glycoproteins; D007328:Insulin; D011993:Recombinant Fusion Proteins; D006921:Hygromycin B; C026273:hygromycin A; D002784:Cholesterol; D015500:Chloramphenicol O-Acetyltransferase; D011972:Receptor, Insulin