Analysis of acyl coenzyme A: cholesterol acyltransferase (ACAT) expression in human tissues

Abstract

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...