BODY CHOLESTEROL

Body Cholesterol

Abstract

cholesterol acyltransferase (ACAT) catalyzes the synthesis of cholesteryl esters from cholesterol and long-chain fatty acids. The two ACAT enzymes, ACAT1 and ACAT2, lack sterol regulatory elements in their promoters and have not been thought to be transcriptionally regulated by cellular cholesterol. However, Cynomolgus monkeys respond to high-cholesterol diet with increased hepatic ACAT2 mRNA expression. Also, a decrease in hepatic ACAT2 mRNA expression has been observed during statin treatment in humans. Thus, we hypothesized that cholesterol may exert transcriptional regulation on the human ACAT2 gene. To test this, we studied two human hepatoma cell lines (HuH7 and HepG2) under conditions of cholesterol loading or depletion and analyzed ACAT gene expression, enzymatic activity, and cellular cholesterol mass. We show a dose-dependent increase of ACAT2 mRNA expression, an increased enzymatic activity of ACAT2, and increased esterified cholesterol mass upon cholesterol loading. These results suggest that human ACAT2 is transcriptionally regulated by cholesterol.

Body Cholesterol

Introduction

cholesterol acyltransferase (ACAT) is an integral membrane protein located in the rough endoplasmatic reticulum that catalyzes the synthesis of cholesteryl esters from cholesterol and long-chain fatty acids. There are two genes encoding the two ACAT enzymes, ACAT1 and ACAT2, also known as steroyl O-acyltransferases 1 and 2 (Soat1 and Soat2). In humans, ACAT1 is present in most tissues; whereas ACAT2 is confined to enterocytes and hepatocytes. The precise role(s) of ACAT1 and ACAT2 in the homeostasis of cholesterol is not yet fully understood. Based on animal studies, and on the cellular localization of the two ACAT enzymes it has been suggested that they both convert free cholesterol into cholesteryl esters in response to excess intracellular cholesterol. ACAT2-derived cholesteryl esters may also be incorporated into hepatic apoB-containing lipoproteins and secreted into plasma (J.G. Hengstler, 2005).

Sterol regulatory elements (SREs), present within the promoters of many cholesterol-regulated genes, have not been identified within the ACAT1 and or the ACAT2 promoters. In addition, a previous study by Matsuda et al. showed that cholesterol loading of HepG2 cells had no effect on the ACAT1 mRNA expression. These findings would be consistent with the concept that the main mode of sterol-dependent regulation of ACAT is by allosteric and not by transcriptional control. However, Cynomolgus monkeys fed a high-cholesterol diet express increased hepatic ACAT2 mRNA levels, and patients treated with statins have a dose-dependent decrease in ACAT2 expression (in review). These findings indicate that a sterol-regulated transcription of the human ACAT2 gene may occur. In order to gain more insight into a possible transcriptional regulation by cholesterol of the human ACAT2 gene, we studied its expression in HuH7 and HepG2 cells. Also, we wanted to appraise the use of these two cell lines as model systems in the study of ACAT and evaluate whether cell differentiation could influence its expression (C. Pramfalk, 2005).

Methodology

Materials

HuH7 and HepG2 cells were purchased from American Type Culture Collection (Manassas, Virginia). All cell culture reagents were purchased from GIBCO (Paisley, Scotland). All chemicals used in this paper were purchased from Sigma (St. Louis, MO), unless specified ...