The murine and human cholesterol 7alpha-hydroxylase gene promoters are differentially responsive to regulation by fatty acids mediated via peroxisome proliferator-activated receptor alpha

We determined if fatty acids can regulate the murine Cyp7a1 and human CYP7A1 gene promoters via peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor alpha (RXRalpha). In transfected cells, the murine Cyp7a1 gene promoter displayed markedly lower basal activity, but greater sensitivity to fatty acid- or WY 14,643-activated PPARalpha/RXRalpha when compared with the human CYP7A1 gene promoter. PPARalpha/RXRalpha can bind to a site (Site II) located within the region at nucleotides -158 to -132 of both promoters. Mutagenesis of the human CYP7A1 Site II element abolished the response to activated PPARalpha/RXRalpha. The murine Cyp7a1 gene promoter contains an additional PPARalpha/RXRalpha-binding site (Site I) located within nucleotides -72 to -57. Replacement of a single residue in human CYP7A1 Site I with that found in the murine Cyp7a1 Site I sequence enabled PPARalpha/RXRalpha binding, and this mutation resulted in reduced basal activity, but substantially improved the response to activated PPARalpha/RXRalpha in transfected cells. We conclude that fatty acids can regulate the cyp7a gene promoter via PPARalpha/RXRalpha. The differential response of the murine Cyp7a1 and human CYP7A1 gene promoters to PPARalpha activators is attributable to the additional PPARalpha/RXRalpha-binding site in the murine Cyp7a1 gene promoter.     

Microsomal triglyceride transfer protein is essential for hepatic secretion of apoB-100 and apoB-48 but not triglyceride

Despite a complete lack of microsomal triglyceride transfer protein (MTP), L35 rat hepatoma cells secrete triglyceride-containing lipoproteins, albeit at a rate 25% of that of parental FAO hepatoma cells, which express high levels of MTP. The inability to express MTP was associated with a complete block in the secretion of both apolipoprotein (apo)B-100 and apoB-48. Stable expression of a MTP transgene restored the secretion of both apoB-100 and apoB-48 in L35 cells, indicating that MTP is essential for the secretion of both forms of apoB. Treatment with the MTP inhibitor BMS-200150 reduced the secretion of triglyceride by 70% in FAO cells, whereas the inhibitor did not affect the secretion of triglycerides by L35 cells. Thus, in the presence of the MTP inhibitor, both cell types secreted triglycerides at similar rates. Essentially, all of the triglycerides secreted by L35 cells were associated with HDL containing apoA-IV and apoE but devoid of apoB-100 or apoB-48. These results suggest that these triglyceride-containing lipoproteins are assembled and secreted via a pathway that is independent of both apoB and MTP. Our findings support the concept that apoB and MTP co-evolved and provided a means to augment the secretion of triglyceride through the formation of lipoproteins containing large hydrophobic cores enriched with triglycerides.     

The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.