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Cholesterol esters and atherosclerosis– a game of ACAT and mouse Disruption of the gene encoding the cholesterol modifying enzyme ACAT2 in mice leads to a reduced capacity to absorb cholesterol and resistance to diet-induced hypercholesterolemia. This suggests that ACAT2 may be a good target for prevention of atherosclerosis. (pages 1341–1347)
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T
he discovery that cholesterol was a component of atherosclerotic plaques in the early 1900s (ref. 1) prompted the realization that hypercholesteroemia plays a major role in the development of atherosclerosis. In mammals, cholesterol exists as a sterol or in the form of fatty acid esters (cholesterol coupled through an ester bond to any of several long chain fatty acids). Most of the cholesterol that accumulates in the arterial plaques is in ester form, and is derived from infiltrated plasma lipoproteins by cells of the developing plaque, such as macrophages2. After a long search, the enzyme that performs intracellular cholesterol esterification, acylCoA:cholesterol acyltransferase (ACAT), was isolated3. This led to the subsequent identification of the ACAT family of acyltransferases, including ACAT1, ACAT2 and a triacylglycerol synthesizing enzyme, diacylglycerol acyltransferase (DGAT) (refs. 4-7). ACAT1 and ACAT2 were united with lecithin:cholesterol acyltransferase (LCAT) as the three known sources of cholesterol esters in animals. In this issue, Buhman et al.8 demonstrate that ACAT2, which is localized in intestine and liver in mice, monkeys and presumably man4,6,7,9, is a key cholesterol esterification enzyme necessary for the development of hypercholesterolemia. The authors observed that ACAT2-deficient mice have a reduced capacity to absorb cholesterol and are resistant to diet-induced hypercholesterolemia8. The data indicate that ACAT2 is required for the production of cholesteryl esters that are packaged into intestinal apoB-containing lipoproteins (chylomicrons). In ACAT2-deficient mice, the intestinal mucosal cells are unable to efficiently transport absorbed cholesterol to the rest of the body8. Disruption of ACAT2-mediated cholesterol ester formation in the intestine prevents cholesterol ester accumulation in liver, in bile as cholesterol gallstones, in blood within lipoprotein particles, and pre-
LAWRENCE L. RUDEL & GREGORY S. SHELNESS sumably in the aorta (although the latter was not documented in this first study)8. Thus, ACAT2, which is expressed in small intestine and liver but absent from arteries, is a viable target for prevention of hypercholesterolemia, atherosclerosis and their clinical manifestations (such as ischemia and myocardial infarction). What is the function of the cholesterol esterification process, and why are there several different enzymatic pathways for creating cholesterol esters? All mammalian cell membranes contain cholesterol. However, most mammalian cells, including those of the arterial wall, cannot degrade cholesterol. So when cellular cholesterol is no longer required as a metabolic intermediate or for membrane stabilization, it must either be released from the cell or stored in the cytosol. Physical chemists have taught us that addition of long chain fatty acids to cholesterol reduces its solubility in the phospholipid bilayer. Esterification therefore causes cholesterol to be moved out of the membrane into the cytoplasm, where it is stored as lipid droplets (Fig. 1). The presence of ACAT1 in the cell membrane limits the plasma membrane cholesterol concentration by inducing cytosolic lipid droplet formation. In instances in which cholesterol is in overabundance, such as in arterial macrophages during atherogenesis, ACAT1 may promote enough lipid storage to induce conversion of macrophages to lipid-laden foam cells (Fig. 1). The maintenance of cholesterol homeostasis in the body involves movement of cholesterol between peripheral tissues and liver—the only organ capable of catabolizing large amounts of cholesterol through conversion into bile acids. Transport of cholesterol from peripheral cells back to the liver, a process termed ‘reverse
NATURE MEDICINE • VOLUME 6 • NUMBER 12 • DECEMBER 2000
cholesterol transport’, requires another system of cholesterol-ester formation. Initially, phospholipids and cholesterol are transferred to high-density lipoproteins (HDL) that accumulate in the intimal layer of the arteries, a process thought to be mediated by the ATP-binding cassette A-1 (ABCA-1) transporter10 (Fig. 1). After transfer to HDL, cholesterol is esterified by the plasma-enzyme LCAT (Fig. 1). This enzyme is soluble in blood plasma and is mechanistically distinct from both ACAT1 and ACAT2 in that it uses phosphatidylcholine from HDL rather than acyl-CoA as the source of acyl chains for cholesterol esters. The cholesterol esters formed by LCAT maintain HDL particle structure by providing a source of core lipid, thereby supporting higher plasma HDL levels. The LCAT derived cholesterol ester of HDL is removed from plasma primarily by the liver through selective cholesterol-ester uptake11 and by whole HDL particle uptake12. In many species other than mice, the plasma cholesterol ester transfer protein (CETP) also contributes to reverse cholesterol transport by transferring cholesterol esters from HDL to very low-density lipoprotein particles and chylomicrons. These lipoproteins are rapidly cleared in the liver by receptormediated endocytosis. In liver, cholesterol esters taken into the hepatocytes are hydrolyzed to free cholesterol primarily in the lysosome (Fig. 1). Hepatic free cholesterol can be used intracellularly to maintain membrane function or, after re-esterification by ACAT2, for incorporation into cytosolic lipid storage droplets. ACAT2-derived cholesterol ester can also be incorporated into hepatic apoB containing very lowdensity lipoprotein, which are secreted into the plasma compartment. Some of the free cholesterol is also used for bile acid synthesis and for direct incorporation into bile micelles— mixtures of phospholipid, cholesterol and bile acids which are transported through the common duct into the in1313
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for treatment and prevention of atherosclerosis. Some studies suggest, however, that human liver may express lower levels of ACAT2 than mice9. Further primary research will be needed to determine whether mouse and human ACAT homologs have similar functions before clinical research can be considered.
Fig. 1 A model for ACAT2 function. From the intestinal lumen, free cholesterol (FC) is absorbed into the enterocyte, accompanied by ACAT2-catalyzed esterification to form cholesterol ester (CE). This CE is incorporated into chylomicron particles. CE in chylomicrons is trafficked through lymphatic fluid and plasma to the liver (dark brown area) where it enters the hepatocyte through receptor-mediated uptake. In the hepatocyte, CE is hydrolyzed to FC by lysozomal cholesterol esterase (CE’ase). This FC, along with phospholipids (PL), can be used for bile acid (BA) synthesis, secretion and incorporation into bile micelles. The micelles are secreted into the intestinal lumen where the micellar FC mixes with dietary FC for subsequent absorption into the enterocyte. In the hepatocyte, FC can also be esterified back to CE by ACAT2 for storage in lipid droplets or for incorporation into VLDL particles, which are subsequently secreted into the plasma compartment. In the arterial wall, macrophages accumulate CE in lipid droplets after ACAT1-catalyzed cholesterol esterification. Some of the CE can be hydrolyzed by a cytosolic cholesterol esterase (CE’ase) to FC, which can then be discharged from cell and bind to HDL particles. Much of the FC picked up by HDL is esterified by LCAT (in the plasma compartment) to form CE, and this CE is returned to liver for whole particle uptake and/or selective CE uptake into the hepatocyte.
testine during fat digestion (Fig. 1). The cholesterol secreted in micelles subsequently mixes in the intestinal lumen with dietary cholesterol and a portion gets absorbed into the mucosal cells. Most of the absorbed cholesterol is esterified by ACAT2 for incorporation into chylomicron particles, so that 75– 80% of newly absorbed cholesterol transported into the body in chylomicrons enters as cholesterol ester13. Chylomicron cholesterol esters are rapidly and selectively removed from plasma by the liver 14. ACAT2 has a role in the enterohepatic recirculation of cholesterol, as revealed by the reduced intestinal cholesterol absorption efficiency in ACAT2-deficient mice8. The findings of Buhman et al.8 provide important information for the role of cholesterol acyltransferase en1314
zymes in cholesterol metabolism. LCAT provides cholesterol esterification to fix cholesterol in the cores of HDL particles for reverse cholesterol transport, and ACAT1 provides membrane integrity in cells throughout the body. In cases of hypercholesterolemia, the presence of ACAT1 in arterial wall macrophages appears to induce the foam cell formation associated with atherosclerosis. ACAT2 participates in cholesterol movement from the intestine to the liver, in regulation of cholesterol metabolism within the hepatocyte, and in cholesterol ester secretion and transport in plasma lipoproteins. These findings elucidate the role of ACAT2 in development of hypercholesterolemia and indicate that this enzyme may be prove to be a viable target
1. Anitschkow, N. Über die atherosclerose der aorta beim kaninchen and überderin entstehungsbedingungen. Beitraege zur pathologischen Anatomie und zur allgemeinen Pathologie 59, 308–348 (1914). 2. Smith, E.B. The relationship between plasma and tissue lipids in human atherosclerosis. Adv. Lipid Res. 12, 1–49 (1974). 3. Chang, C.C.Y., Huh, H.Y., Cadigan, K.M. & Chang, T.Y. Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J. Biol. Chem. 268, 20747–20755 (1993). 4. Anderson, R.A. et al. Identification of a form of acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J. Biol. Chem. 273, 26747–26754 (1998). 5. Cases, S. et al. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc. Natl. Acad. Sci. USA 95, 13018–13023 (1998). 6. Cases, S. et al. ACAT-2, A second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization. J. Biol. Chem. 273, 26755–26764 (1998). 7. Oelkers, P., Behari, A., Cromley, D., Billheimer, J.T. & Sturley, S.L. Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes. J. Biol. Chem. 273, 26765–26771 (1998). 8. Buhman, K. K., et al. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice. Nature Med. 6, 1341–1347 (2000). 9. Chang, C.C.Y. et al. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J. Biol. Chem. 275, 28083–28092 (2000). 10. Oram, J.F. & Vaughan, A.M. ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Curr. Opin. Lipidol. 11, 253–260 (2000). 11. Williams, D.L. et al. Scavenger receptor BI and cholesterol trafficking. Curr. Opin. Lipidol. 10, 329–339 (1999). 12. Huggins, K.W., Burleson, E.R., Sawyer, J.K., Kelly, K. & Rudel, L.L. Determination of the tissue sites responsible for the catabolism of large high density lipoprotein in the African green monkey. J. Lipid Res. 41, 384–394 (2000). 13. Klein, R.L. and Rudel, L.L. Cholesterol absorption and transport in thoracic duct lymph lipoproteins of nonhuman primates. Effect of dietary cholesterol level. J. Lipid Res. 24, 343–356 (1983). 14. Goodman, D.S. The metabolism of chylomicron cholesteryl ester in the rat. J. Clin. Invest. 41, 1886–1896 (1962).
Arteriosclerosis Research Program, Department of Pathology Wake Forest University School of Medicine 300 South Hawthorne Road Winston-Salem, NC 27103 USA Email:
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NATURE MEDICINE • VOLUME 6 • NUMBER 12 • DECEMBER 2000
