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Published on: August 6, 2020
Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescence assay: individual ACAT
Aaron T Lada1, Matthew Davis, Carol Kent
1Department of Pathology, Arteriosclerosis Research Program, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
Abstract:
Acyl CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2 are enzymes responsible for the formation of cholesteryl esters in tissues. While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACAT1 and ACAT2 have separate functions. In this study, NBD-cholesterol was used to screen for specific inhibitors of ACAT1 and ACAT2. Incubation of AC29 cells, which do not contain ACAT activity, with NBD-cholesterol showed weak fluorescence when the compound was localized in the membrane. When AC29 cells stably transfected with either ACAT1 or ACAT2 were incubated with NBD-cholesterol, the fluorescent signal localized to the nonpolar core of cytoplasmic lipid droplets was strongly fluorescent and was correlated with two independent measures of ACAT activity. Several compounds were found to have greater inhibitory activity toward ACAT1 than ACAT2, and one compound was identified that specifically inhibits ACAT2. The demonstration of selective inhibition of ACAT1 and ACAT2 provides evidence for uniqueness in structure and function of these two enzymes. To the extent that ACAT2 is confined to hepatocytes and enterocytes, the only two cell types that secrete lipoproteins, selective inhibition of ACAT2 may prove to be most beneficial in the reduction of plasma lipoprotein cholesterol concentrations.
Insights
Researchers identified specific inhibitors for Acyl CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2 enzymes. Selective ACAT2 inhibition may reduce plasma lipoprotein cholesterol levels.
Area of Science:
- Biochemistry
- Enzymology
- Lipid Metabolism
Background:
- Acyl CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2 are key enzymes in cholesteryl ester formation.
- Distinct cellular localization of ACAT1 and ACAT2 in the liver and intestine suggests separate physiological roles.
- Understanding ACAT1 and ACAT2 functions is crucial for metabolic disease research.
Purpose of the Study:
- To screen for specific inhibitors of ACAT1 and ACAT2 using NBD-cholesterol.
- To investigate the functional differences between ACAT1 and ACAT2.
- To evaluate the potential of selective ACAT2 inhibition for managing plasma lipoprotein cholesterol.
Main Methods:
- Utilized NBD-cholesterol as a fluorescent probe to detect ACAT activity in transfected AC29 cells.
- Quantified ACAT activity using fluorescence in lipid droplets and two independent assays.
- Screened compound libraries for selective ACAT1 and ACAT2 inhibitors.
Main Results:
- NBD-cholesterol fluorescence correlated with ACAT activity in cells expressing ACAT1 or ACAT2.
- Identified compounds with differential inhibitory effects on ACAT1 versus ACAT2.
- Discovered a novel compound exhibiting specific inhibition of ACAT2.
Conclusions:
- Selective inhibition of ACAT1 and ACAT2 demonstrates their unique structures and functions.
- ACAT2, primarily in hepatocytes and enterocytes, is a potential therapeutic target.
- Targeting ACAT2 may offer a strategy to lower plasma lipoprotein cholesterol concentrations.
