Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescence assay
2004; Elsevier BV; Volume: 45; Issue: 2 Linguagem: Inglês
10.1194/jlr.d300037-jlr200
ISSN1539-7262
AutoresAaron T. Lada, Matthew A. Davis, Carol R. Kent, James Chapman, Hiroshi Tomoda, Satoshi Ōmura, Lawrence L. Rudel,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoAcyl 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. 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. In tissues, cholesterol is esterified by the enzyme acyl CoA:cholesterol acyltransferase (ACAT). The two identified forms, termed ACAT1 and ACAT2, along with acyl-CoA:diacylglycerol acyltransferase 1, (DGAT1) make up the ACAT gene family (1Farese Jr., R.V. Acyl CoA:cholesterol acyltransferase genes and knockout mice.Curr. Opin. Lipidol. 1998; 9: 119-123Crossref PubMed Scopus (35) Google Scholar, 2Anderson R.A. Joyce C. Davis M. Reagan J.W. Clark M. Shelness G.S. Rudel L.L. Identification of a form of acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates.J. Biol. Chem. 1998; 273: 26747-26754Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 3Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. Erickson S.K. Farese R.V. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13018-13023Crossref PubMed Scopus (886) Google Scholar, 4Cases S. Novak S. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Welch C.B. Lusis A.J. Spencer T.A. Krause B.R. Erickson S.K. Farese R.V. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.J. Biol. Chem. 1998; 273: 26755-26764Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 5Oelkers P.M. 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. 1998; 273: 26765-26771Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). ACAT1 and ACAT2 are more than 50% similar in primary amino acid sequence; however, significant differences in membrane topology have been identified (6Joyce C.W. Shelness G.S. Davis M.A. Lee R.G. Skinner K. Anderson R.A. Rudel L.L. ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane.Mol. Biol. Cell. 2000; 11: 3675-3687Crossref PubMed Scopus (101) Google Scholar). The two enzymes also differ in their tissue distribution, with ACAT2 protein present only in the liver and intestine, whereas ACAT1 is more ubiquitously expressed (2Anderson R.A. Joyce C. Davis M. Reagan J.W. Clark M. Shelness G.S. Rudel L.L. Identification of a form of acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates.J. Biol. Chem. 1998; 273: 26747-26754Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 4Cases S. Novak S. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Welch C.B. Lusis A.J. Spencer T.A. Krause B.R. Erickson S.K. Farese R.V. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.J. Biol. Chem. 1998; 273: 26755-26764Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 5Oelkers P.M. 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. 1998; 273: 26765-26771Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Furthermore, in the liver and intestine, where both enzymes are present, the cellular location of the two enzymes is distinct. In nonhuman primates, ACAT2 was found in hepatocytes and ACAT1 was localized to Kupffer cells (7Lee R.G. Willingham M.C. Davis M.A. Skinner K.A. Rudel L.L. Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.J. Lipid Res. 2000; 41: 1991-2001Abstract Full Text Full Text PDF PubMed Google Scholar). In the primate intestine, ACAT1 was found in goblet cells, macrophages, and Paneth cells, whereas ACAT2 was localized to the apical third of the mucosal cells (7Lee R.G. Willingham M.C. Davis M.A. Skinner K.A. Rudel L.L. Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.J. Lipid Res. 2000; 41: 1991-2001Abstract Full Text Full Text PDF PubMed Google Scholar). These differences in the cellular localization of ACAT1 and ACAT2 suggest different functions of the two enzymes. ACAT2 is thought to function in intestinal cholesterol absorption and transport in chylomicrons and in providing cholesteryl esters (CEs) for VLDL assembly in the liver. ACAT1 appears to function to maintain free cholesterol (FC) balance in cells that accumulate and store extra cholesterol as CEs. Studies in mice support these roles for ACAT1 and ACAT2. ACAT1-deficient mice lack CEs in macrophages and in the adrenal cortex but have normal cholesterol absorption and hepatic cholesterol esterification (8Meiner V.L. Cases S. Myers H.M. Sande E.R. Bellosta S. Schambelan M. Pitas R.E. McGuire J. Herz J. Farese R.V. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals.Proc. Natl. Acad. Sci. USA. 1996; 93: 14041-14046Crossref PubMed Scopus (242) Google Scholar). ACAT2-deficient mice have reduced cholesterol absorption and were resistant to diet-induced hypercholesterolemia by a high-fat, high-cholesterol diet (9Buhman K.F. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (297) Google Scholar). In mice fed a fat- and cholesterol-enriched diet, ACAT1 deficiency did not protect against atherosclerosis (10Accad M. Smith S.J. Newland D.L. Sanan D.A. King Jr., L.E. Linton M.F. Fazio S. Farese Jr., R.V. Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1.J. Clin. Invest. 2000; 105: 711-719Crossref PubMed Scopus (210) Google Scholar, 11Yagyu H. Kitamine T. Osuga J. Tozawa R. Chen Z. Kaji Y. Oka T. Perrey S. Tamura Y. Ohashi K. Okazaki H. Yahagi N. Shionoir F. Iizuka Y. Harada K. Shimano H. Yamashita H. Gotoda T. Yamada N. Ishibashi S. Absence of ACAT1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.J. Biol. Chem. 2000; 275: 21324-21330Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), and in one study, ACAT1 deficiency apparently led to increased atherosclerosis, possibly as a result of FC toxicity in macrophages (12Fazio S. Major A.S. Swift L.L. Gleaves L.A. Accad M. Linton M.F. Farese R.V. Increased atherosclerosis in LDL-receptor-null mice lacking ACAT1 in macrophages.J. Clin. Invest. 2001; 107: 163-171Crossref PubMed Scopus (217) Google Scholar). In contrast, in apolipoprotein E-deficient mice, ACAT2 deficiency protected against atherosclerosis (13Willner E.L. Tow B. Buhman K.F. Wilson M. Sanan D.A. Rudel L.L. Farese R.V. Deficiency of acyl CoA:cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice.Proc. Natl. Acad. Sci. USA. 2003; 100: 1262-1267Crossref PubMed Scopus (154) Google Scholar). Numerous ACAT inhibitors have been identified. However, the different functions and potential roles in atherosclerosis development suggest a need for specific inhibitors of ACAT2. Current methods for assaying ACAT activity are laborious and time-consuming, involving the use of radioactive substrates in live cells or in incubations of cell homogenates or microsomes with the isolation of the radioactive CE products by TLC and subsequent quantification. To facilitate the identification of potential ACAT-specific inhibitors, we have developed a more rapid and high-throughput cell-based assay. The assay uses 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol), a fluorescent sterol analog in which the NBD moiety replaces the terminal segment of the alkyl tail of cholesterol. NBD-cholesterol has been shown to mimic native cholesterol absorption in hamsters (14Sparrow C.P. Patel S. Baffic J. Chao Y-S. Hernandez M. Lam M-H. Montenegro J. Wright S.D. Detmers P.A. A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro.J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar), intracellular lipid transport (15van Meer G. Stelzer E.H. Wijnaendts-van-Resandt R.W. Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.J. Cell Biol. 1987; 105: 1623-1635Crossref PubMed Scopus (306) Google Scholar, 16Koval M. Pagano R.E. Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick type A fibroblasts.J. Cell Biol. 1990; 111: 429-442Crossref PubMed Scopus (102) Google Scholar), and esterification in vivo and by cultured cells (14Sparrow C.P. Patel S. Baffic J. Chao Y-S. Hernandez M. Lam M-H. Montenegro J. Wright S.D. Detmers P.A. A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro.J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar). The relative fluorescence of NBD-cholesterol is dependent on its environment. When in a polar environment, NBD-cholesterol is weakly fluorescent, whereas in a nonpolar environment, it is strongly fluorescent (17Chattopadhyay A. London E. Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes.Biochim. Biophys. Acta. 1988; 938: 24-34Crossref PubMed Scopus (145) Google Scholar, 18Fery-Forgues S. Fayet J.P. Lopez A. Drastic changes in the fluorescence properties of NBD probes with the polarity of the medium: involvement of a TICT state?.J. Photochem. Photobiol. 1993; 70: 229-243Crossref Scopus (182) Google Scholar, 19Mukherjee S. Chattopadhyay A. Samanta A. Soujanya T. Dipole moment change of NBD group upon excitation using solvatochromic and quantum chemical approaches: implications in membrane research.J. Phys. Chem. 1994; 98: 2809-2812Crossref Scopus (106) Google Scholar). This property of NBD-cholesterol was used to measure ACAT activity in cells stably expressing ACAT1 or ACAT2. These cells readily form lipid droplets rich in CE, providing the opportunity to compare the effects of potential inhibitors separately on the two enzymes. Identification of inhibitors specific for either enzyme should facilitate the study of ACAT activity in tissues that contain both enzymes and in the longer term will assist in the development of compounds that can be used to selectively inhibit ACAT enzymes in vivo, helping to define the roles of ACAT1 and ACAT2 in atherosclerosis. All cell lines were maintained at 37°C in 5% CO2 in Ham's F-12 medium supplemented with 1% Eagle's vitamins, penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% heat-inactivated FBS. AC29 cells, a Chinese hamster ovary cell line with no ACAT activity, was a generous gift from T-Y. Chang (Dartmouth College of Medicine, Hanover, NH). For transfections, AC29 cells were grown to 50–75% confluence on 100 mm dishes. Transfections were carried out with FuGENE (Boehringer Mannheim Biochemicals, Indianapolis, IN) and African green monkey ACAT1 or ACAT2 cDNA in pOPSRV1 plasmid (Stratagene, La Jolla, CA) at a 3 μl:1 μg FuGENE/DNA ratio. Cells were selected with geneticin (G418), and monoclonal populations expressing ACAT1 or ACAT2 were isolated. AC29 cells were also transfected with human ACAT1 or ACAT2 in a similar manner. NBD-cholesterol (Molecular Probes, Eugene, OR) was solubilized in ethanol for a stock solution of 1 mg/ml. AC29 cells and AC29 cells stably expressing African green monkey ACAT1 or ACAT2 were seeded onto 35 mm culture dishes. The next day, 2 ml of medium containing 1 μg/ml NBD-cholesterol in ethanol (final ethanol concentration, 0.1%) was added, and cells were incubated for 2 h at 37°C in a CO2 incubator. To prepare cells for microscopy, dishes were washed three times with PBS and fixed with 1 ml of 3.7% formaldehyde in PBS for 20 min at room temperature. Dishes were washed and drained, and two drops of p-phenylenediamine (1 mg/ml) in glycerol was added as a mounting medium to each dish. Cells were examined using a Zeiss Axioplan microscope using a neofluor (numerical aperture, 1.3) oil immersion objective (63×) with green channel filters (488 nm excitation, 540 nm emission). High-performance TLC was used to separate free and esterified NBD-cholesterol. Cellular isopropanol lipid extracts were dried under nitrogen and brought up in 20 μl of chloroform. Ten microliters was spotted onto a 10 × 20 TLC plate (Whatman, Inc., Clifton, NJ) using a Camag Linomat IV plate spotter, and lipids were separated using a 50:50:1 hexane-ether-acetic acid solvent system. Stock NBD-cholesterol was run as a standard to identify the free NBD-cholesterol peak. Plates were scanned in a Camag TLC Scanner II with a mercury lamp set at 469 nm excitation wavelength with a 560 nm filter for emission. Peaks were quantitated using the TLC Evaluation Software CATS version 3.19 from Camag. A total of 30,000 cells per well were plated on Falcon 96-well culture plates and allowed to recover overnight. Assays were done with cells at least 80% confluent. Cells were incubated in medium containing 1 μg/ml NBD-cholesterol for 2–6 h as indicated in the figure legends. NBD-cholesterol was added from a 1 mg/ml stock in ethanol, and ethanol concentrations did not exceed 0.1%. AC29 cells incubated with NBD-cholesterol or ACAT-expressing cells incubated with NBD-cholesterol and the ACAT inhibitor CP113 were used to determine background fluorescence attributable to free NBD-cholesterol, as indicated in the figure legends. After incubation, medium was removed, and the cells were washed two times with cold balanced salt solution (BSS). Plates were read from the bottom using a Tecan GENios fluorescent plate reader equipped with 485 nm excitation and 535 nm emission filters. After measurement, cellular protein was digested through incubation with 25 μl of 0.4 N NaOH for 2 h. Protein content was determined by the method of Lowry et al. (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). CE fluorescence was calculated by subtracting the background from the total fluorescence, and these values were normalized by cellular protein. To compare measures of ACAT activity using fluorescence and [14C]oleate incorporation into CEs, cells were seeded onto Falcon 12-well culture plates. When confluent, medium containing 1 μg/ml NBD-cholesterol and 1 μCi/ml [14C]oleate was added, and cells were incubated for 6 h. To determine background fluorescence, cells were incubated with 1 μg/ml NBD-cholesterol, 1 μCi/ml [14C]oleate, and 2 μg/ml CP113. Plates were washed two times with cold BSS and read on a fluorescent plate reader as described above. Cellular lipids were extracted through an overnight incubation with 1 ml of isopropanol. The isopropanol lipid extract was dried down under nitrogen, brought up in 50 μl of chloroform, and spotted onto a TLC plate. Lipids were separated using a solvent system of 140:60:2 hexane-ether-acetic acid. The CE band was scraped, and radioactivity was determined by scintillation counting. To solubilize cellular protein, 0.5 ml of 1 N NaOH was added to each well. A 100 μl aliquot was taken for protein determination by the method of Lowry et al. (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Both fluorescence and radioactivity values were normalized by dividing by cellular protein. ACAT activity was assayed in either cell homogenates or microsomes. Microsomes were prepared from liver samples from wild-type, ACAT1−/− (8Meiner V.L. Cases S. Myers H.M. Sande E.R. Bellosta S. Schambelan M. Pitas R.E. McGuire J. Herz J. Farese R.V. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals.Proc. Natl. Acad. Sci. USA. 1996; 93: 14041-14046Crossref PubMed Scopus (242) Google Scholar), and ACAT2−/− (9Buhman K.F. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (297) Google Scholar) mice as previously described (7Lee R.G. Willingham M.C. Davis M.A. Skinner K.A. Rudel L.L. Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.J. Lipid Res. 2000; 41: 1991-2001Abstract Full Text Full Text PDF PubMed Google Scholar). To prepare cell homogenates, cells were grown in 35 mm dishes, and when near confluence (>80%), the cell monolayer was scraped, suspended in BSS, and sonicated. An aliquot was taken for protein determination by the method of Lowry et al. (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and 50 μg of whole cell or microsomal protein was used to determine ACAT activity as previously described (7Lee R.G. Willingham M.C. Davis M.A. Skinner K.A. Rudel L.L. Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.J. Lipid Res. 2000; 41: 1991-2001Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, protein was mixed with 1 mg of BSA and 50 nmol of free cholesterol in 45% (w/v) β-cyclodextrin and incubated for 30 min. Then, 30 nmol of [14C]oleoyl-CoA was added and incubated for 10 min at 37°C in a shaking water bath. The reaction was stopped by the addition of 2:1 chloroform-methanol, phases were split, and an aliquot of the organic phase was subjected to TLC. The CE band was scraped and counted for 14C radioactivity. AC29 cells stably expressing African green monkey ACAT1 or ACAT2 were used to test the effects of a variety of compounds on the activity of the two enzymes. Four compounds were synthesized by Dr. Chapman at the University of South Carolina, and we term these compounds 1A, 1B, 1C, and 1D. Pyripyropene A (PPPA) was isolated by Dr. Satoshi Omura of the Kitasato Institute (21Omura S. Tomoda H. Kim Y. Nishida H. Pyripyropenes, high potent inhibitors of acyl-CoA cholesterol acyltransferase produced by Aspergillus fumigatus.J. Antibiot. 1993; 46: 1168-1169Crossref PubMed Scopus (146) Google Scholar, 22Tomoda H. Kim Y. Nishida H. Masuma R. Omura S. Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. I. Production, isolation, and biological properties.J. Antibiot. 1994; 47: 148-153Crossref PubMed Scopus (117) Google Scholar). ACAT1 and ACAT2 cells were plated on the same 96-well plate and allowed to recover overnight. A stock solution of each compound was made to a concentration of 10 mg/ml in DMSO, and an equal volume of DMSO was added to all cells (0.05% final DMSO concentration). Cells were preincubated for 20 min with each compound over the concentration range 0.0001–20 μg/ml, with three wells for each concentration. After preincubation, the medium was removed and replaced with 100 μl of medium containing 1 μg/ml NBD-cholesterol and the same concentration of the compound as in the preincubation. Cells were incubated for 6 h, and the fluorescence was determined as described above. Three wells each of ACAT1 and ACAT2 cells were incubated in a similar manner in the presence of 2 μg/ml CP113 to determine the background fluorescence for each cell line. Fluorescence values were normalized to cellular protein mass and plotted versus the log of the concentration of compound. Prism 3 software was used for the curve fit of the data using a sigmoidal dose-response curve, and IC50 values were calculated from the curve fit. The localization of the fluorescent signal of NBD-cholesterol was compared in AC29 cells, which do not express ACAT, and AC29 cells stably expressing ACAT2 (ACAT2 cells). Cells were incubated with 1 μg/ml NBD-cholesterol for 120 min and then viewed by fluorescence microscopy. In AC29 cells, the fluorescent signal was weak and diffuse, indicative of membrane localization (Fig. 1A), whereas in ACAT2 cells, there was a strong fluorescent signal localized to cellular lipid droplets (Fig. 1B). Similar results were seen with ACAT1 cells (data not shown). These results agree with the known property of NBD-cholesterol to be weakly fluorescent in a more polar environment (cell membrane) and strongly fluorescent in a nonpolar environment (neutral lipid droplet). To demonstrate that NBD-cholesterol is esterified by ACAT, AC29, ACAT1, and ACAT2 cells were incubated with 1 μg/ml NBD-cholesterol with or without the ACAT inhibitor CP113. In preliminary studies, it was determined that 1 μg/ml NBD-cholesterol produced maximum fluorescence in ACAT-expressing cells (data not shown). After incubation, cellular lipids were extracted and separated by high-performance TLC, and fluorescent bands were scanned and quantified. In AC29 cells, only one fluorescent band was visible, corresponding to free NBD-cholesterol (Table 1). When ACAT1 and ACAT2 cells were incubated with NBD-cholesterol, two distinct peaks were visible, corresponding to free and esterified NBD-cholesterol, indicating that the fluorescent compound was esterified by these cells. The addition of the ACAT inhibitor CP113 eliminated the esterified NBD-cholesterol band, further demonstrating that it was a product of ACAT activity.TABLE 1Esterification of NBD-cholesterolCell TypeTreatmentFree CholesterolCholesteryl EsterAC29NBD617 ± 22–NBD + CP113513 ± 24–ACAT1NBD566 ± 96354 ± 53NBD + CP113575 ± 43–ACAT2NBD671 ± 40458 ± 19NBD + CP113598 ± 37–AC29, ACAT1, and ACAT2 cells were incubated with 1 μg/ml NBD-cholesterol ± 2 μg/ml CP113 for 2 h. After incubation, cellular lipids were extracted and separated by high-performance TLC. The plates were scanned using a fluorescent plate reader, and peaks corresponding to free NBD-cholesterol and NBD-cholesteryl ester were quantitated as described in Methods. The quantitated values are expressed as arbitrary units. The data are expressed as means ± SD (n = 3). Open table in a new tab AC29, ACAT1, and ACAT2 cells were incubated with 1 μg/ml NBD-cholesterol ± 2 μg/ml CP113 for 2 h. After incubation, cellular lipids were extracted and separated by high-performance TLC. The plates were scanned using a fluorescent plate reader, and peaks corresponding to free NBD-cholesterol and NBD-cholesteryl ester were quantitated as described in Methods. The quantitated values are expressed as arbitrary units. The data are expressed as means ± SD (n = 3). Preliminary experiments were carried out to determine the background fluorescence in cells attributable to free NBD-cholesterol. The total fluorescence within cells is the sum of free and esterified NBD-cholesterol, and the fluorescence attributable to esterified NBD-cholesterol can be calculated by subtracting the background fluorescence from the total fluorescence. Two methods to determine the background fluorescence were compared. NBD-cholesterol was incubated with AC29 cells (with no ACAT activity) or with ACAT2 cells and the ACAT inhibitor CP113. Similar low amounts of fluorescence were obtained using the two methods (data not shown). In addition, the low fluorescence levels in AC29 cells were the same with or without the addition of CP113, demonstrating no measurable ACAT activity in these cells (data not shown). Therefore, either cells that do not express ACAT (AC29 cells) or cells stably transfected with ACAT after treatment with the ACAT inhibitor CP113 can be used to determine background fluorescence. Figure 2shows the increase in fluorescence over time in ACAT1 and ACAT2 cells incubated with NBD-cholesterol. Because the background fluorescence attributable to free NBD-cholesterol was subtracted, the resulting fluorescence values represent esterified NBD-cholesterol and the extent of the increase with time is an indicator of ACAT activity. Although this increase was not linear over 48 h (Fig. 2A, C), it was linear through the first 8 h for both ACAT1 and ACAT2 cells (Fig. 2B, D). Based on these data, further incubations with NBD-cholesterol were done for 6 h maximum to remain within the range of linearity. To determine if esterified NBD-cholesterol could be hydrolyzed, ACAT1 or ACAT2 cells were first incubated with NBD-cholesterol for 6 h to load the cells with NBD-CEs. To determine background attributable to free NBD-cholesterol, ACAT1 or ACAT2 cells were incubated with NBD-cholesterol and CP113. After the loading period, the fluorescence was 2.2-fold higher in ACAT1 cells and 1.8-fold higher in ACAT2 cells than in the cells in which ACAT was inhibited (Fig. 3), demonstrating the increase in fluorescence when NBD-cholesterol is esterified. Both groups of cells were then incubated with hydrolysis medium consisting of 10% FBS and CP113 for 18 h. In cells loaded with NBD-CEs, the fluorescence decreased during the hydrolysis period, indicating that the cells hydrolyzed NBD-CEs. In addition, the fluorescence in both sets of cells decreased below the free NBD-cholesterol background from the loading period and the fluorescence in the medium increased (data not shown), indicating that cells effluxed NBD-cholesterol. Thus, the results shown in Table 1 and Fig. 3 demonstrate that NBD-cholesterol undergoes the esterification and hydrolysis cycle typical of native cholesterol in cells expressing ACAT1 or ACAT2. The fluorescence measure of ACAT activity was correlated with two independent measures of ACAT activity, i.e., [14C]oleate incorporation into CEs in live cells and a broken-cell assay using incorporation of [14C]oleate from radioactive oleoyl-CoA into CEs. For this study, AC29 cells and six stable cell lines with different levels of ACAT2 activity were incubated with both NBD-cholesterol and [14C]oleate. In these dishes, both fluorescence and [14C]oleate incorporation in CEs was measured. In addition, parallel dishes from each cell line were harvested for the broken-cell ACAT assay. The fluorescence values correlated well with both [14C]oleate incorporation into CE (r = 0.94) (Fig. 4A)and ACAT activity measured in the broken cell assay (r = 0.94) (Fig. 4B), indicating that the increase in fluorescence upon esterification of NDB-cholesterol is an accurate measure of cellular ACAT activity. The fluorescent ACAT assay was then used to identify ACAT1- and ACAT2-specific inhibitors. AC29 cells stably transfected with either ACAT1 or ACAT2 were used to separately compare the effects of various compounds on the two enzymes. PPPA selectively inhibited ACAT2 without affecting cell integrity, demonstrating that the compound is fully available to t
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