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...

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ACAT (acyl coenzyme A: cholesterol acyltransferase, syn: sterol O-acyltransferase) catalyzes the esterification of free cholesterol (FC) by reaction with long-chain acyl CoA derivatives to form cholesterol esters (CE). In humans, ACAT enzymes are expressed from two genes (ACAT-1 and ACAT-2) and play important roles in cholesterol trafficking and regulating FC/CE ratios within cells. Various cholesterol-associated diseases such as gallstone formation appear to be associated with "abnormal" expression levels of these genes. This project developed quantitative methods to estimate the relative expression of ACAT-1 and ACAT-2 genes in various human tissues. Real time quantitative PCR after reverse transcription of total RNA (RT-qPCR) was used to quantify ACAT mRNAs, and Western Blots after SDS-PAGE was used to quantify ACAT proteins. β-actin was chosen as an endogenous reference ("housekeeping gene") to compare expression levels of both mRNA and protein in different samples.Chapters 2 and 3 address a number of technical issues, including the development of RT-qPCR assays that provide a realistic estimate of the molar ratio of ACAT-2/ACAT-1 mRNA in any sample.Assays for each ACAT isoform used TaqMan ® oligonucleotide probes to quantify PCR products, and were multiplexed with an assay for β-actin for the analysis of ACAT-1 and ACAT-2 mRNA abundance in human samples. Chapter 3 describes the adoption of a higher-throughput PCR machine and improved TaqMan® probe, and the development of strategies for comparing the results obtained by improved assay systems with assays using the original methods described in Chapter 2.Chapter 4 describes the assessment of six antisera for ability to detect ACAT proteins after SDS-PAGE and Western blot analysis of human liver preparations. Five ACAT-2 antisera gave complex and variable banding patterns and none were considered sufficiently well characterised for use in quantitative analysis. However, the ACAT-1 antiserum was highly specific and reproducibly detected a single protein of ~48 kDa, and was used for a survey ACAT-1 protein levels in 16 of the 17 human liver samples assayed for the survey of ACAT mRNA described in Chapter 2. Novel procedures are described for minimising errors when calculating mean ACAT-1/β-actin protein ratios from scans of band intensity in replicate Western blots. No correlation was found between ACAT-1 protein and ACAT-1 mRNA abundance. It was concluded that in human liver, ACAT-1 protein levels tend to fluctuate around a mean value that shows little relationship to ACAT-1 mRNA levels.iii As a fraction of total ACAT mRNA, ACAT-2 was on average about 10 times more abundant than ACAT-1 in duodenum (69% of total ACAT mRNA, n=10) than in liver (7% of total ACAT mRNA, n=17). These results demonstrated quantitatively that ACAT-1 was the predominant mRNA isoform in all human liver samples assayed, and that ACAT-2 was more abundant in 9 of the 10 human duodenal samples. ACAT-2 represented 11% of total ACAT mRNA in kidney (n=3), an organ not usually associated...