Fatty acyl CoA and cholesterol are the substrates for cholesteryl ester synthesis by acyl coenzyme A:cholesterol acyltransferase (ACAT). Two ACAT genes have been identified; ACAT1 is expressed ubiquitously while ACAT2 is primarily expressed in intestine and liver. We tested effects of different free fatty acids (FFAs) on ACAT1 and ACAT2 expression and activity in HepG2 human hepatocytes and THP1 human macrophages. Incubation of oleic acid, arachidonic acid, or eicosapentaenoic acid, but not 25-hydroxycholesterol, induced ACAT1 mRNA levels 1.5--2-fold in HepG2, with no affect on ACAT2 mRNA. FFA had no affect on ACAT1 mRNA in THP1 cells. To determine if FFAs affect ACAT1 or ACAT2 posttranscriptionally, cells were labeled with [(3)H]cholesterol in the presence of the different FFAs for 1--5 h. Both HepG2 and THP1 cells showed the greatest cholesteryl ester production with oleic acid. This was also confirmed by the observation that more [(3)H]oleic acid incorporated into CE compared to [(3)H]eicosapentaenoic acid, even though there was no difference in the total uptake of these FFAs. In ACAT-deficient SRD4, CHO cells stably transfected with human ACAT1 or ACAT2, ACAT1 expressing cells showed a strong preference for oleic acid while ACAT2 expressing cells utilized unsaturated FFAs. Acyl CoA substrate specificity was further tested in microsomes isolated from these cells as well as HepG2 and THP1. THP1 and ACAT1 cells utilized oleoyl CoA preferentially. In contrast, HepG2 and ACAT2 microsomes utilized linolenoyl CoA as well. We conclude that FFAs increase ACAT1 mRNA levels in a cell specific manner, and furthermore that the ACAT reactions exhibit differential FFA utilization.
Saccharomyces cerevisiae transcribes two genes, ARE1 and ARE2, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 and ARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from the ARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded by ARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1 promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2 are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold while ARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in a rox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2 transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.
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