The present study demonstrates that two different forms of the intracellular cholesterol esterification enzyme acyl-CoA:cholesterol acyltransferase (ACAT) are present in the nonhuman primate hepatocyte; one is similar to that originally cloned from human genomic DNA, here termed ACAT1, while a second gene product, termed ACAT2, is reported here. The primate ACAT2 gene product was cloned from an African green monkey liver cDNA library. Sequence analysis of an isolated, full-length clone of ACAT2 cDNA identified an open reading frame encoding a 526-amino acid protein with essentially no sequence similarity to the ACAT1 cDNA over the N-terminal 101 amino acids but with 57% identity predicted over the remaining 425 amino acids. Transfection of the cloned ACAT2 cDNA into two different mammalian cell types resulted in the production of abundant ACAT activity which was sensitive to ACAT inhibitors. Northern blot analysis showed that the ACAT2 mRNA was expressed primarily in liver and intestine in monkeys. In contrast, ACAT1 mRNA was expressed in almost all tissues examined. Topologic predictions from the amino acid sequence of ACAT2 indicates that it has seven trans-membrane domains in a configuration that places the putative active site of the enzyme in the lumen of the endoplasmic reticulum. This orientation of ACAT2 in the endoplasmic reticulum membrane, in addition to its expression only in liver and intestine, suggests that this enzyme may have as a primary function, the secretion of cholesteryl esters into apoB-containing lipoproteins.The intracellular formation of cholesteryl esters catalyzed by the action of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) 1 appears to be nearly ubiqitous in mammalian cells (1). Elucidation of the details of the structure and catalytic mechanism of ACAT and of the regulation of its activity have been stymied by the difficulty in isolating and purifying an active form of this membrane-associated enzyme. It has taken the isolation of a cDNA for ACAT from human genomic DNA, accomplished through functional complementation of mutant Chinese hamster ovary cells lacking ACAT activity, to initiate progress in understanding the biochemistry of ACAT function (2). The mRNA for this ACAT is expressed in most human tissues and cDNAs with nearly identical ACAT sequences have likewise been found in a variety of tissues from mouse, hamster, and rabbit (3-5).Several functions can be attributed to cholesterol esterification by ACAT. The enzyme appears to modulate the potentially toxic effects of cholesterol in cell membranes. By attaching a fatty acid to the free hydroxyl group of cholesterol, physical properties of the cholesterol molecule are changed and the solubility of esterified cholesterol in the lipids of the cell membrane is limited. Cholesteryl esters accumulate in lipid droplets in the cytoplasm, and maintenance of a balance between the free and esterified forms of cholesterol in a cell is believed to be a component of regulation of cholesterol signaling pathways (6...

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg ؋ apoE-knockout (KO) mice. Overexpression of hABCA1 in C57BL͞6 mice resulted in a unique anti-atherogenic profile characterized by decreased plasma cholesterol (63%), cholesteryl ester (63%), free cholesterol (67%), non-high density lipoprotein (HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and apoE (2.8-fold) levels. These beneficial changes in the lipid profile led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg mice. In marked contrast, ABCA1 overexpression had a minimal effect on the plasma lipid profile of apoE-KO mice and resulted in a 2-to 2.6-fold increase in aortic lesion area. These combined results indicate that overexpression of ABCA1 in C57BL͞6 mice on a high cholesterol diet results in an atheroprotective lipoprotein profile and decreased atherosclerosis, and thus provide previously undocumented in vivo evidence of an antiatherogenic role for the ABCA1 transporter. In contrast, overexpression of ABCA1 in an apoE-KO background led to increased atherosclerosis, further substantiating the important role of apoE in macrophage cholesterol metabolism and atherogenesis. In summary, these results establish that, in the presence of apoE, overexpression of ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism and reduces atherosclerosis in vivo, and indicate that pharmacological agents that will increase ABCA1 expression may reduce atherogenic risk in humans.

No abstract