Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR2

Abstract

ACAT (also called sterol o -acyltransferase) catalyzes the esterification of cholesterol by reaction with longchain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to ␤ -actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n ‫؍‬ 17), ACAT-1 transcripts were on average 9-fold (range, 1.7-to 167-fold) more abundant than ACAT-2 , whereas in duodenal samples (n ‫؍‬ 10), ACAT-2 transcripts were on average 3-fold (range, 0.39-to 12.2-fold) more abundant than ACAT-1 . ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine. Since its initial discovery in rat liver (1, 2), ACAT activity has been detected in a diverse range of tissues and cell types (3) and is known to play an important role in cell biology and in the pathogenesis of important lipid-related diseases such as atherosclerosis (3) and cholesterol gallstones (4). For example, ACAT has been shown to play a pivotal functional role in the intestinal absorption of cholesterol, the hepatic secretion of VLDL, the biosynthesis of steroid hormones, the production of cholesteryl esters in macrophages (foam cells) in atheroma, and the secretion of biliary cholesterol. From a biochemical and physiological perspective, ACAT is one of the central enzymes regulating plasma and biliary cholesterol concentrations. An excess of plasma cholesterol can lead to atherosclerosis, and an increased secretion of cholesterol in bile can predispose individuals to cholesterol gallstones. Not surprisingly, ACAT has been the focus of considerable research during the past decade because of its obvious pharmacological relevance.Two ACAT genes are known in humans. The first ACAT gene, cloned by Chang and colleagues (5) in 1993, is now termed ACAT-1 . The second, ACAT-2 , was cloned in 1998 (6-8). The two genes have 47% overall nucleotide identity (5, 8) and encode specific ACAT proteins that have 43% amino acid sequence identity and 63% similarity. These landmark studies (5-8) using gel-based detection of ACAT mRNA (RT-PCR and Northern blot analysis) provide evidence that ACAT-2 is expressed primarily in liver and inAbbreviations: PBMC, peripheral blood mononuclear cells; RTqPCR, RT treatment of RNA followed by quantitative real-time PCR.1 The official name fo...

Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy

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ACAT2 Is Localized to Hepatocytes and Is the Major Cholesterol-Esterifying Enzyme in Human Liver

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TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2

“…Tgif1 is highly expressed in the human liver and weakly expressed in the small intestine ( 31 ), whereas Acat2 has the opposite tissue expression ( 2,3,28 ). Because Tgif1 functions as a repressor of SOAT2 in both hepatic and intestinal cells, it may, at least partly, be responsible for the different tissue expression levels of SOAT2 in humans.…”

“…Caco2 cells were grown in DMEM supplemented with 20% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin; the media were replaced every second day. Cells were maintained in 75 cm 2 cell culture fl asks and passaged when reaching ‫ف‬ 90% confl uence. For all cell experiments, cells were plated out on 6-well tissue culture plates so that they reached ‫ف‬ 70% confl uence after 24 h (i.e., ‫ف‬ 700,000 cells/well).…”