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Papers In Press, published online ahead of print November 16, 2003
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Department of Pathology, Wake Forest University, Winston-Salem, NC 27157
Corresponding Author: lrudel{at}wfubmc.edu
ACAT1 and ACAT2 are enzymes responsible for the formation of cholesteryl esters (CE) 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 thereby defining potential benefit for selective inhibition. 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 non-polar core of cytoplasmic lipid droplets and was strongly fluorescent. The strength of the fluorescent signal increased linearly over an 8-hour incubation and was correlated with two independent measures of ACAT activity. The NBD-CEs could be subsequently hydrolyzed demonstrating that NBD-cholesterol undergoes a cycle of esterification and hydrolysis similar to that of cholesterol. Several compounds were found to have greater inhibitory activity towards 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 reduction of plasma lipoprotein cholesterol concentrations.
Revised on October 27, 2003
Accepted on November 12, 2003
Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescent assay: evidence defining uniqueness for individual ACATs
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