Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity (2009)

ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein. ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein. endoplasmic reticulum trans-Golgi network 2-methyl-β-cyclodextrin In mammalian cells, the cholesterol content in various membranes varies from the highest in the plasma membrane to the lowest in the endoplasmic reticulum (ER) membrane. Excess cellular cholesterol is converted to cholesteryl esters or is removed from cells by cellular cholesterol efflux (1Maxfield F.R. Tabas I. Role of cholesterol and lipid organization in disease.Nature. 2005; 438: 612-621Crossref PubMed Scopus (969) Google Scholar, 2Ikonen E. Cellular cholesterol trafficking and compartmentalization.Nat. Rev. Mol. Cell Biol. 2008; 9: 125-138Crossref PubMed Scopus (996) Google Scholar). The conversion of free cholesterol to cholesteryl ester is catalyzed by the enzyme ACAT. Two ACAT isoforms exist in mammals: ACAT1 and ACAT2 (3Buhman K.F. Accada M. Farese Jr, R.V. Mammalian acyl-CoA:cholesterol acyltransferases.Biochim. Biophys. Acta. 2000; 1529: 142-154Crossref PubMed Scopus (174) Google Scholar, 4Rudel L.L. Lee R. Cockman T. Structure, function, and regulation of ACAT.Curr. Opin. Lipidol. 2001; 12: 121-127Crossref PubMed Scopus (208) Google Scholar, 5Chang T.Y. Li B.L. Chang C.C. Urano Y. Acyl-coenzyme A:cholesterol acyltransferases.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E1-E9Crossref PubMed Scopus (318) Google Scholar). ACAT1 is ubiquitously expressed in various tissues, while ACAT2 is mainly expressed in intestinal enterocytes and in hepatocytes (5Chang T.Y. Li B.L. Chang C.C. Urano Y. Acyl-coenzyme A:cholesterol acyltransferases.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E1-E9Crossref PubMed Scopus (318) Google Scholar, 6Parini P. Davis M. Lada A.T. Erickson S.K. Wright T.L. Gustafsson U. Sahlin S. Einarsson C. Eriksson M. Angelin B. et al.ACAT2 is localized to hepatocytes and is the major cholesterol-esterifying enzyme in human liver.Circulation. 2004; 110: 2017-2023Crossref PubMed Scopus (168) Google Scholar, 7Song B.L. Wang C.H. Yao X.M. Yang L. Zhang W.J. Wang Z.Z. Zhao X.N. Yang J.B. Qi W. Yang X.Y. et al.Human acyl-CoA:cholesterol acyltransferase 2 gene expression in intestinal Caco-2 cells and in hepatocellular carcinoma.Biochem. J. 2006; 394: 617-626Crossref PubMed Scopus (39) Google Scholar). Macrophages play key roles in atherosclerosis. Under hyperlipidemic conditions, macrophages continuously internalize denatured/modified LDL. Cholesterol derived from internalized lipoproteins is converted to cholesteryl esters by ACAT1, the major ACAT isozyme in macrophages (8Miyazaki A. Sakashita N. Lee O. Takahashi K. Horiuchi S. Hakamata H. Morganelli P.M. Chang C.C. Chang T.Y. Expression of ACAT1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1568-1574Crossref PubMed Scopus (130) Google Scholar). Chronic exposure to denatured/modified LDL causes macrophages to become foamy in appearance; foamy macrophages are hallmarks of early atherosclerotic lesions. In advanced lesions, in addition to cholesteryl esters, free (unesterified) cholesterol also accumulates in macrophages. The excessive buildup of free cholesterol in membranes can cause cellular toxicity, especially in macrophages (9Warner G.J. Stoudt G. Bamberger M. Johnson W.J. Rothblat G.H. Cell toxicity induced by inhibition of acyl coenzyme A:cholessterol acyltransferase and accumulation of unesterified cholesterol.J. Biol. Chem. 1995; 270: 5772-5778Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 10Tabas I. Consequences of cellular cholesterol accumulation: basic concepts and physiological implications.J. Clin. Invest. 2002; 110: 905-911Crossref PubMed Scopus (525) Google Scholar). In various cell types examined, ACAT1 is mainly located in the tubular ER (11Chang T.Y. Chang C.C.Y. Cheng D. Acyl-coenzyme A:cholesterol acyltransferase.Annu. Rev. Biochem. 1997; 66: 613-638Crossref PubMed Scopus (440) Google Scholar); in mouse macrophages and other cell types examined, 5–15% of the total immunoreactive ACAT1 signal is also present in ER-derived, perinuclear structure(s) near the trans-Golgi network (TGN) and the endocytic recycling compartment (12Khelef N. Soe T.T. Quehenberger O. Beatini N. Tabas I. Maxfield F.R. Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1769-1776Crossref PubMed Google Scholar). In human macrophages, when maintained in normal medium, the ACAT1 signal is mainly with the tubular ER; however, adding modified LDL to the growth medium of these cells caused up to 30–40% of the total ACAT1 immunoreactive signals to become associated with small, ER-derived vesicles 80–100 nm in diameter (13Sakashita N. Miyazaki A. Takeya M. Horiuchi S. Chang C.C.Y. Chang T.Y. Takahashi K. Localization of human acyl-coenzyme A:cholesterol acyltransferase-1 in macrophages and in various tissues.Am. J. Pathol. 2000; 156: 227-236Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These studies suggest that in macrophages, ACAT1 can be associated with ER-derived vesicles, especially when cells are grown in cholesterol-rich conditions. These ACAT1-positive, ER-derived structures had not previously been isolated in vitro. In the current work, we used OptiPrep gradient ultracentrifugation to isolate various ACAT1-positive membrane fractions and evaluated the ACAT enzyme activities in these fractions. FBS, RPMI1640 medium, protease inhibitor cocktail, SDS, 2-methyl-β-cyclodextrin (mβCD), paraformaldehyde, BSA, saponin, primulin, and PMA were from Sigma. Penicillin-streptomycin solution was from Invitrogen. DTT and SuperSignal Chemiluminescent substrate were from Pierce. ProLong Antifade kit, anti-rabbit IgG Alexa Fluor 488, and anti-mouse IgG Alexa Fluor 568 were from Molecular Probes. The rabbit anti-human ACAT1 antibodies (DM10 and DM102) and the mouse anti-human ACAT1 monoclonal antibody were as previously described (14Chang C.C.Y. Chen J. Thomas M.A. Cheng D. Del Priore V.A. Newton R.S. Pape M.E. Chang T.Y. Regulation and immunolocalization of acyl-coenzyme A: cholesterol acyltransferase in mammalian cells as studied with specific antibodies.J. Biol. Chem. 1995; 270: 29532-29540Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 15Chang C.C. Lee C.Y. Chang E.T. Cruz J.C. Levesque M.C. Chang T.Y. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.J. Biol. Chem. 1998; 273: 35132-35141Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Monoclonal antibodies against syntaxin 6, calnexin, BiP, and GM130 were from BD Biosciences. Monoclonal antibody against sodium/potassium ATPase (Na/K ATPase) was from Upstate USA Inc. Iodixanol solution (OptiPrep) used to prepare density gradient was from Axis-Shield. Pansorbin beads for the immunoadsorption experiment were from Calbiochem. [3H]oleyl-CoA was prepared as described previously (15Chang C.C. Lee C.Y. Chang E.T. Cruz J.C. Levesque M.C. Chang T.Y. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.J. Biol. Chem. 1998; 273: 35132-35141Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The ACAT1 specific ACAT inhibitor K-604 was a generous gift from Kowa Co. Ltd. (Tokyo, Japan) (16Ikenoya M. Yoshinaka Y. Kobayashi H. Kawamine K. Shibuya K. Sato F. Sawanobori K. Watanabe T. Miyazaki A. A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels.Atherosclerosis. 2007; 191: 290-297Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). LDL (density of 1.019–1.063 g/ml) and acetylated LDL were prepared from fresh human plasma as previously described (8Miyazaki A. Sakashita N. Lee O. Takahashi K. Horiuchi S. Hakamata H. Morganelli P.M. Chang C.C. Chang T.Y. Expression of ACAT1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1568-1574Crossref PubMed Scopus (130) Google Scholar). Aggregated LDL was prepared by vortex mixing LDL at maximal speed for 2 min as described previously (17Huang W. Ishii I. Zhang W-Y. Sonobe M. Kruth H.S. PMA activation of macrophages alters macrophage metabolism of aggregated LDL.J. Lipid Res. 2002; 43: 1275-1282Abstract Full Text Full Text PDF PubMed Google Scholar). Human monocytic cell line THP-1 was from ATCC. THP-1 cells were maintained in 150 mm dishes in medium A (RPMI1640 medium plus 10% FBS, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin sulfate). THP-1 cells treated with 100 nM PMA in medium A for 7 days were used for experiments described here. Human monocytes were isolated from blood of healthy volunteers. Informed written consent was obtained from all volunteers in accordance with protocols approved by the Kumamoto University Hospital Review Board. Monocyte-derived macrophages, grown in medium A in 150 mm dishes for 9 days, were used. Cholesterol-rich macrophages were prepared by incubating macrophages with medium A containing 100 µg/ml of aggregated LDL, 100 µg/ml of acetylated LDL, or 250 µM of mβCD containing 16 µM cholesterol (mβCD/cholesterol complex) for 48 h. SDS (at 10% final) and DTT (at 0.1 M final) were added to OptiPrep fractions, immunoadsorbed samples, or whole cell lysates. After incubation at 37°C for 30 min, the solubilized proteins were analyzed by10% SDS-PAGE and subjected to immunoblotting as described previously (14Chang C.C.Y. Chen J. Thomas M.A. Cheng D. Del Priore V.A. Newton R.S. Pape M.E. Chang T.Y. Regulation and immunolocalization of acyl-coenzyme A: cholesterol acyltransferase in mammalian cells as studied with specific antibodies.J. Biol. Chem. 1995; 270: 29532-29540Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). THP-1 and human macrophages grown in 6-well tissue culture plate with medium A with or without various additional cholesterol sources for 48 h were rinsed with PBS three times and dried at room temperature, then treated with 2 ml/well of hexane:2-propanol (3:2). The total cholesterol and free cholesterol contents present in the lipid extracts were determined by using the cholesterol determination kit following the manufacturer's instruction (Wako Chemical, Tokyo, Japan). The cholesteryl ester values were calculated by subtracting the free cholesterol values from the total cholesterol values. Cellular protein dissolved in 0.1 N sodium hydroxide was determined by using a BCA Protein Assay kit (Pierce) with BSA as a standard. Immunoadsorption was employed to isolate ACAT1-positive membranes using the procedure briefly described below: 30 µL of precleaned pansorbin beads were incubated with 6 µg of rabbit polyclonal ACAT1 antibodies DM102 or nonimmunized rabbit IgG in buffer for 2 h at After the beads were incubated with OptiPrep in buffer containing 100 sodium for h at After in a the was isolated and cultured in a 6-well tissue culture plate with were with A containing BSA and and were with in 0.1 M buffer for 30 After the were with and incubated with antibodies for min, then incubated with anti-rabbit Alexa Fluor and anti-mouse Alexa Fluor 568 for min and and as using or at a of using an with and of at 30 were at the ACAT1 signal and various markers as were calculated by using the was by were were at grown in a 150 mm plate were buffer containing M buffer and protease inhibitor and were using a tissue The was the of a 9 OptiPrep gradient in buffer and was by ultracentrifugation in a at for 2 equal fractions were from to the the OptiPrep was added to the to in The was at the of a OptiPrep gradient and at for 2 h. equal fractions were from to OptiPrep membrane was used for ACAT enzyme activity determination using the in the cellular cholesterol associated with the membrane as the enzyme substrate (15Chang C.C. Lee C.Y. Chang E.T. Cruz J.C. Levesque M.C. Chang T.Y. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.J. Biol. Chem. 1998; 273: 35132-35141Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, Chang T.Y. A for of cell and human ACAT activity Lipid Res. Full Text PDF PubMed Google Scholar). In in membrane was subjected to ACAT activity determination using both the assay and the reconstituted In the the cholesterol present in the reconstituted vesicles as the enzyme substrate (15Chang C.C. Lee C.Y. Chang E.T. Cruz J.C. Levesque M.C. Chang T.Y. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.J. Biol. Chem. 1998; 273: 35132-35141Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, Chang T.Y. A for of cell and human ACAT activity Lipid Res. Full Text PDF PubMed Google Scholar). briefly the membrane was treated with the and M ACAT solubilized in was reconstituted vesicles by the treated a large of vesicles with (at assay the assay was by adding µL of solution containing of and of fatty BSA, and the was incubated at 37°C for up to 30 The enzyme activity was with 30 The ACAT inhibitor K-604 was employed at of µM (16Ikenoya M. Yoshinaka Y. Kobayashi H. Kawamine K. Shibuya K. Sato F. Sawanobori K. Watanabe T. Miyazaki A. A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels.Atherosclerosis. 2007; 191: 290-297Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We treated macrophages with aggregated LDL or with other cholesterol to cell in vitro. When THP-1 cells were treated with various cholesterol for 48 cellular free cholesterol content from to or (mβCD/cholesterol complex) cellular cellular cholesteryl ester content from to or or (mβCD/cholesterol complex) protein when human macrophages grown in medium A were treated with aggregated LDL, cellular cholesterol content from to cholesteryl ester content from to protein After 48 h of aggregated LDL the protein content of ACAT1 was in the THP-1 macrophages but not in the human macrophages We employed to isolate the membranes in THP-1 macrophages. The results showed that in conditions, of the ACAT1 signal in the fractions the ER protein and the protein syntaxin 6 were In incubating cells with aggregated LDL, of the ACAT1 signal and the signal to fractions a of the syntaxin 6 signal also to the fractions in the of the membranes were when the THP-1 macrophages were treated for 2 days with 100 µg/ml of acetylated LDL or with We the ACAT enzymatic activities in of the OptiPrep membrane fractions in by using the assay in assay cholesterol associated with membrane as the enzyme The results lower showed that in cells grown in conditions, the ACAT activities in various membrane fractions were in cells treated with various cholesterol the membranes higher ACAT activity than the membranes We also that incubating the THP-1 macrophages with aggregated LDL for h is to produce the membranes with elevated ACAT activity the increase in ACAT activity in the membranes be due to in cholesterol content in these we the experiment in and the ACAT activity in by using both the assay and the reconstituted the assay in cholesterol present in the reconstituted lipid vesicles as the enzyme The results showed that the ACAT activities in the membranes elevated when cholesterol was used as the enzymatic that the in ACAT activity in these membranes is not caused by an increase in cholesterol content in these the ACAT1 enzyme activity ACAT1 protein we the experiment in from OptiPrep fractions and that equal of ACAT activity in and their ACAT1 protein content by The results showed that at equal ACAT1 protein mass, the membrane fractions significantly higher ACAT enzymatic activity than the membrane fractions We also that treating cell of macrophages grown in medium A with high ACAT enzyme activity in but to produce the membranes that density not treating THP-1 macrophages with aggregated LDL and with a specific ACAT inhibitor the ER-derived while the ACAT activities in these fractions were not these that is the increase in cellular not cellular cholesteryl that the membranes with elevated ACAT activity and that the of cholesterol cellular We employed to in ACAT1 in THP-1 and human macrophages The results show that in cells grown in medium ACAT1 signal was mainly located in the ER however, in cells treated with aggregated LDL, a of ACAT1 signal in the We using ACAT1 antibodies or antibodies against syntaxin 6 protein mainly localized in the of J.B. J. S. 6 in trans-Golgi network Biol. 1997; PubMed Scopus Google Scholar). The results showed that in cells grown in medium the ACAT1 signal and the In the cholesterol-rich macrophages, of the ACAT1 signal with the The ACAT1 and syntaxin 6 are in In cholesterol-rich cells, of the ER protein was also to the and with the syntaxin 6 signal not experiments showed that in THP-1 macrophages grown in medium A with or without aggregated LDL, the ACAT1 signals with the ER signal not the in THP-1 macrophages also in human macrophages, we incubated human macrophages in medium A or in medium A plus aggregated LDL for 2 days, then the ACAT1 by The results showed that in medium the ACAT1 signal did not significantly with the syntaxin 6 in medium with aggregated LDL these signals The calculated ACAT1 and syntaxin 6 are in After cholesterol the of the ER and the syntaxin 6 also not these suggest that in cholesterol-rich THP-1 macrophages or human macrophages, a of ACAT1 be located near more for a of ACAT1 with a specific of we purified the ACAT1-positive, membranes in THP-1 macrophages by OptiPrep gradient we then subjected the purified fractions to a OptiPrep gradient in After the the ACAT1-positive fractions in the fractions. The fractions also the syntaxin 6, the ER BiP, and the plasma membrane but they the GM130 in the fractions the These fractions high ACAT enzymatic activity in lower After the we analyzed the lipid contents present in various OptiPrep fractions. The fractions in ACAT enzyme activities were also in cholesterol and in cholesteryl esters we purified these ACAT1-positive vesicles by immunoadsorption using Pansorbin beads containing ACAT1 specific After the were analyzed by The results show that when ACAT1 antibodies were the membrane ACAT1, the ER proteins and and syntaxin 6, but did not When the nonimmunized rabbit IgG was employed as a of the markers were present in the These results suggest that the highly purified ACAT1-positive membrane is associated with the ER proteins as as the protein syntaxin the of in we human macrophages in normal or in cholesterol-rich incubating with 100 µg/ml of aggregated LDL for 48 and used these cells to experiments as described The results showed that when cells were grown in medium a of ACAT1 as as the ER and the syntaxin 6 were in density fractions In cholesterol-rich conditions, more ACAT1 protein and more ER protein in the fractions, and the ACAT1 protein present in these fractions higher enzyme activity in human and mouse macrophages also ACAT2 N. Miyazaki A. Chang C.C.Y. Morganelli P. Chang T.Y. O. E. Hakamata H. M. H. et of ACAT2 in human and mouse in and in Invest. PubMed Scopus Google Scholar). We immunoblotting and that the membranes the ACAT1 signal but not the ACAT2 signal not we employed an inhibitor K-604 ACAT1 ACAT2 by more than at of the ACAT1 activity without the ACAT2 The results showed that K-604 used at µM of the ACAT activity associated with the membranes These results that the ACAT enzyme activity in these membranes mainly from ACAT1, not We immunoadsorption using the as the The results showed that the ACAT1-positive membranes from cells in cholesterol-rich were associated with the syntaxin 6 lower while membranes from cells grown in medium A a of syntaxin 6 These results our results obtained in THP-1 macrophages. ACAT1 is located in the ER and ER-derived vesicles (12Khelef N. Soe T.T. Quehenberger O. Beatini N. Tabas I. Maxfield F.R. Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1769-1776Crossref PubMed Google Scholar, N. Miyazaki A. Takeya M. Horiuchi S. Chang C.C.Y. Chang T.Y. Takahashi K. Localization of human acyl-coenzyme A:cholesterol acyltransferase-1 in macrophages and in various tissues.Am. J. Pathol. 2000; 156: 227-236Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). We had previously that loading macrophages with denatured LDL, is an increase in the ER-derived, ACAT1-positive vesicles (13Sakashita N. Miyazaki A. Takeya M. Horiuchi S. Chang C.C.Y. Chang T.Y. Takahashi K. Localization of human acyl-coenzyme A:cholesterol acyltransferase-1 in macrophages and in various tissues.Am. J. Pathol. 2000; 156: 227-236Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). to the of in the current we that in macrophages, cholesterol as a in the of the ER-derived vesicles. We isolated these ER-derived vesicles in and showed that the activity of ACAT1 present in these vesicles is the increase in activity is not due to an increase in cholesterol content associated in these These results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles that elevated ACAT1 enzyme activity without having to produce more ACAT1 protein. we not the for the of these ER-derived vesicles. We the following as a for that ER membranes be of various with protein and lipid O. B. reticulum of cells and its organization and Rev. 2001; PubMed Scopus Google Scholar, Y. and 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In various cell tubular ER and ER-derived structures with the tubular ER the of cholesterol in cells, especially in the macrophages, the of small, ER-derived vesicles, by an ER the cholesterol-rich of the ER to from the of the the ER from with ACAT1 present in the small, ER-derived vesicles cholesterol the accumulation of free cholesterol in cholesterol-rich the is with the by et (12Khelef N. Soe T.T. Quehenberger O. Beatini N. Tabas I. Maxfield F.R. Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1769-1776Crossref PubMed Google that the of the ACAT1-positive, ER-derived structure(s) with the cholesterol-rich and endocytic recycling a for localized of cholesterol content of the causes the ACAT1 in the membranes to be more than the ACAT1 in the ER membranes also is that the ER membrane an ACAT1 inhibitor that the membranes an ACAT1 We are these We of the for the of We are also to H. and for of