Measurement of cholesterol bidirectional flux between cells and lipoproteins
2005; Elsevier BV; Volume: 47; Issue: 3 Linguagem: Inglês
10.1194/jlr.m500466-jlr200
ISSN1539-7262
AutoresFrancesca Zimetti, Ginny K. Weibel, MyNgan Duong, George H. Rothblat,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoWe developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum. We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum. The movement of cholesterol out of cells and onto extracellular lipoprotein acceptors represents the first step in the process of reverse cholesterol transport, a pathway by which excess cholesterol is removed from the peripheral tissues and delivered to the liver for excretion (1Glomset J.A. The plasma lecithin:cholesterol acyltransferase reaction.J. Lipid Res. 1968; 9: 155-167Google Scholar, 2Tall A.R. Wang N. Tangier disease as a test of the reverse cholesterol transport hypothesis.J. Clin. Invest. 2000; 106: 1205-1207Google Scholar, 3Rader D.J. Regulation of reverse cholesterol transport and clinical implications.Am. J. Cardiol. 2004; 92: 42J-49JGoogle Scholar). Many studies have probed this cholesterol efflux step and established that a number of mechanisms can mediate the efflux of free cholesterol (FC). Among these mechanisms are unmediated aqueous diffusion (4Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Cholesterol transport between cells and high density lipoproteins.Biochim. Biophys. Acta. 1991; 1085: 273-298Google Scholar) and pathways mediated by scavenger receptor class B type I (SR-BI) (5Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor B1 promotes high density lipoprotein-mediated cellular cholesterol efflux.J. Biol. Chem. 1997; 272: 20982-20985Google Scholar), ABCA1 (6Oram J.F. Yokoyama S. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.J. Lipid Res. 1996; 37: 2473-2491Google Scholar), and other ABC transporters, such as the recently discovered ABCG1 and ABCG4 (7Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoprotein.Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Google Scholar). Although there are multiple efflux mechanisms, the actual measurement of efflux is relatively simple and usually employs the quantitation of the release of radiolabeled FC from cells into the medium. However, because physiologically relevant acceptors, such as lipoproteins, contain both FC and cholesteryl ester (CE), there is a bidirectional movement of cholesterol between cells and lipoproteins. Whereas the measurement of cholesterol efflux is relatively simple, the quantitation of influx of lipoprotein cholesterol is much more complicated (8Rothblat G.H. de la Llera-Moya M. Favari E. Yancey P.G. Kellner-Weibel G. Cellular cholesterol flux studies: methodological considerations.Atherosclerosis. 2002; 163: 1-8Google Scholar). Influx involves the uptake of both FC and CE and is mediated by several mechanisms, including the unmediated uptake of lipoprotein FC via aqueous diffusion, the internalization of intact lipoprotein particles delivering both FC and CE, and the SR-BI-enhanced flux pathways of lipoprotein FC and selective CE uptake. These flux pathways become even more complex when cells are exposed to whole serum, in which individual lipoproteins and apolipoproteins differentially contribute to both the efflux and influx of cholesterol. When cells are incubated with isolated lipoproteins or whole serum, the net movement of cholesterol mass can result in the accumulation of cell cholesterol (influx), the depletion of cell cholesterol (efflux), or the bidirectional flux of cholesterol without a net change in cholesterol in either extracellular or intracellular compartments (exchange). Net efflux, net influx, and exchange can be determined by several factors, such as the level of cholesterol in the cells, the expression of receptors on the cell surface, and the concentration and composition of the lipoproteins to which cells are exposed.Although efflux studies have been very useful in identifying the different pathways involved in the movement of cholesterol out of cells, one criticism of this approach is that the assay does not allow the determination of bidirectional flux. In this study, we have developed an assay that, based on a combination of mass and isotopic determinations, permits the simultaneous quantitation of both cholesterol efflux and influx when cells are exposed to isolated lipoproteins or whole serum. With this approach, we have determined the contribution to cholesterol flux of different serum fractions and whole serum. Also, we have measured the bidirectional flux of cholesterol when either Fu5AH rat hepatoma cells or murine peritoneal macrophages (MPMs) are exposed to sera or isolated lipoproteins.EXPERIMENTAL PROCEDURESMaterialsTissue culture plasticware was obtained from Falcon (Lincoln, NJ). RPMI 1640 and MEM were purchased from Mediatech Cellgro (Herndon, VA). FBS, calf serum (CS), gentamycin, DNase I, sodium cholate, and the HMG-CoA reductase inhibitor Mevinolin were purchased from Sigma-Aldrich (St. Louis, MO). BSA was obtained from Serological Corp. (Norcross, GA). [1,2 3H]cholesterol was purchased from Perkin-Elmer Analytical Sciences (Boston, MA). The ACAT inhibitor CP113,818 was kindly provided by Pfizer Pharmaceuticals (Groton, CT). The SR-BI inhibitor BLT-1 (for block lipid transport-1) was obtained from ChemBridge (San Diego, CA).Serum lipids and lipoprotein analysisHuman HDL3 and LDL were obtained by sequential ultracentrifugation as described previously (for LDL, d = 1.019–1.063; for HDL, d = 1.063–1.21) (9Hatch F.T. Lees R.S. Practical methods for plasma lipoprotein analysis.Adv. Lipid Res. 1968; 6: 1-68Google Scholar). Reconstituted high density lipoprotein (rHDL) particles, containing 100:1 (mol/mol) 1-palmitoyl-2-oleoylphosphatidilcholine/apolipoprotein A-I were prepared using a cholate dialysis technique (10Matz C.E. Jonas A. Reaction of human lecithin cholesterol acyltransferase with synthetic micellar complexes of apolipoprotein A-I, phosphatidylcholine, and cholesterol.J. Biol. Chem. 1982; 257: 4541-4546Google Scholar). Acetylated low density lipoprotein (acLDL) was obtained by modification of LDL with acetic anhydride, as described previously (11Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.Proc. Natl. Acad. Sci. USA. 1979; 76: 333-337Google Scholar). The concentration of all of the lipoproteins used during the experiment was expressed as μg protein/ml. Human serum was collected with approved consent from healthy normolipidemic individuals and pooled. The apolipoprotein B (apoB)-depleted serum was obtained from the whole serum by precipitating the apoB-containing lipoproteins with a polyethylene glycol solution as described previously (12Asztalos B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. Rothblat G.H. Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux.J. Lipid Res. 2005; 46: 2246-2253Google Scholar). Briefly, whole serum was incubated for 20 min with a 20% polyethylene glycol (PEG) 6000 solution. After spinning samples at 10,000 rpm, the supernatant was collected and stored at 4°C.Cell cultureRat Fu5AH hepatoma cells were maintained in MEM containing 5% CS. Cells at 90% confluence were trypsinized and plated at a density of 6 × 105/well on 12-well plates. Cell number was determined using the Z1 Coulter Particle Counter (Hialeah, FL). MPMs were isolated from B6CF31 mice. Mice were injected intraperitoneally with 0.5 ml of a 10% solution of Brewer Thioglycollate Medium (Difco Laboratories, Detroit, MI). Three days later, macrophages were harvested by lavage of the peritoneal cavity with PBS. After spinning and resuspension in MEM-HEPES, cells were counted and plated in 10% RPMI at a density of 7 × 105 cells/well on 12-well plates. All media were supplemented with gentamycin.Preparation of BLT-1 solutionTo prepare a stock solution, BLT-1 was dissolved in DMSO at a concentration of 10 mM. The day before the treatment with cells, BLT-1 was diluted in 4% BSA-containing medium at a concentration of 200 μM and stored at 4°C. Just before the addition to cells, BLT-1 was diluted in medium to a final concentration of 10 μM.Measurement of cholesterol bidirectional fluxThe assay to measure cholesterol efflux and influx uses a combination of isotopic and mass determinations. Cells were labeled for 24 h with [3H]cholesterol in medium supplemented with 2.5% CS. After an equilibration period in 0.2% BSA-containing medium, a set of cells was harvested before the incubation with cholesterol acceptors (time zero) and the cholesterol content of cell lysates was measured as described below. Total counts were determined as well, and the specific activity (SA) at time zero (T0 SA) (cpm/μg cholesterol) was calculated. The remaining cells were incubated with either serum or isolated lipoproteins for 8 h, at the end of which cholesterol SA was again determined (T8 SA). Because the medium volume can affect the quantitation of cholesterol influx, in all experiments, 0.5 ml of medium was used in the flux period. To inhibit de novo cholesterol synthesis, the HMG-CoA reductase inhibitor Mevinolin was added to the efflux medium at a concentration of 5 μg/ml (13Nordskog B.K. Reagan Jr., J.W. St. Clair R.W. Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux.J. Lipid Res. 1999; 40: 1806-1817Google Scholar). Furthermore, to prevent cholesterol esterification, 2 μg/ml of the ACAT inhibitor CP113,818 was added during labeling, equilibration, and the flux stages of the experiment. For the determination of efflux, the following parameters were established: 1) T0 cholesterol content; and 2) fractional efflux. The efflux of cell cholesterol mass was calculated as fractional efflux × T0 cholesterol content, as described previously (14de la Llera Moya M. Atger V. Paul J.L. Fournier N. Moatti N. Giral P. Friday K.E. Rothblat G.H. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux: relationships between serum components and efflux, esterification and transfer.Arterioscler. Thromb. Vasc. Biol. 1994; 14: 1056-1065Google Scholar). For influx determinations, the following terms were used: 1) T0 SA; 2) T8 SA; 3) SA factor (T0 SA/T8 SA); 4) theoretical mass (T0 cholesterol content − the mass of cholesterol effluxed); and 5) calculated mass (SA factor × theoretical mass). Influx was determined as the calculated mass − theoretical mass. Cholesterol efflux and influx are expressed as μg cholesterol/8 h/mg protein. The net cholesterol movement resulting from these two opposite fluxes are expressed as an influx-to-efflux ratio (I/E). I/E < 1 indicates net cholesterol efflux, whereas I/E > 1 reflects net cholesterol influx. Cholesterol exchange without significant net flux is reflected by I/E = 1.Protein and cholesterol determinationAt the end of the experiment, cell monolayers were washed with MEM-HEPES medium and lysed in 0.5 ml of a 1% sodium cholate solution in water supplemented with 10 U/ml DNase. A total of 125 μl of a reaction buffer containing 0.5% Triton X-100, 0.5 M potassium phosphate (pH 7.4), 0.25 M NaCl, and 1% sodium cholate was added to the cell lysates, and plates were shaken for 30 min at room temperature. Care must be taken to ensure complete solubilization of the cell monolayer. After heating samples at 60°C for 30 min, to inactivate enzymes that could compete with the enzymatic cholesterol assay, cholesterol was measured fluorimetrically using the Amplex Red Cholesterol Assay Kit (Molecular Probes, Eugene, OR) as described by the manufacturer. Because the experiments were conducted in the presence of an ACAT inhibitor, the cholesterol present in the cells at T0 was unesterified. However, the enzymatic kit assay for cholesterol included a cholesterol esterase and allowed the measurement of all cellular cholesterol. This permitted the quantitation of any CE incorporated from lipoproteins. The amount of cholesterol in each well was measured by comparison with a cholesterol standard curve included in each experiment. An aliquot of the cell lysates was also taken to measure cell protein by a modified Lowry method (15Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). The total [3H]cholesterol present in cell lysates was measured by scintillation counting.Statistical analysisAll of the experiments were conducted in triplicate, and the data represent means of multiple experiments or multiple assays. The error bars represent standard deviations. All statistical analyses were performed with GraphPad (San Diego, CA) Prism software.RESULTSWe first investigated whether the direction of net cholesterol flux depends on the type and composition of the extracellular acceptors. Fu5AH cells were exposed to isolated lipoproteins or serum for 8 h, and cholesterol efflux, cholesterol influx, and the I/E were measured. Rat Fu5AH hepatoma cells express high levels of SR-BI and thus can be viewed as a cellular system in which SR-BI-mediated bidirectional flux of cholesterol is of major importance (16Yancey P.G. Kawashiri M. Moore R. Glick J.M. Williams D.L. Connelly M.A. Rader D.J. Rothblat G.H. In vivo modulation of HDL phospholipid has opposing effects on SR-BI- and ABCA1-mediated cholesterol efflux.J. Lipid Res. 2004; 45: 337-346Google Scholar). As shown in Table 1, when cells were exposed to rHDL, there was significant cholesterol efflux (5.29 ± 0.36 μg cholesterol/mg protein) in the absence of influx, as expected, because these particles contain no cholesterol. With HDL3, cholesterol efflux and influx values were similar (2.14 ± 0.45 and 1.86 ± 0.73 μg cholesterol/mg protein, respectively), with the resulting I/E of 0.87 ± 0.52, indicating that cholesterol was exchanged between HDL and the hepatoma cells without resulting in net cholesterol flux. Furthermore, rHDL was a more efficient cholesterol acceptor than HDL3 when the two particles were used at similar protein concentrations. Incubation of cells for 8 h with 100 μg/ml LDL resulted in influx being 4.7-fold greater than efflux, producing net cholesterol movement into cells (I/E of 4.65 ± 0.24). Net influx was also observed upon exposure of cells to 2.5% human serum, producing an I/E of 2.15 ± 0.29.TABLE 1.Cholesterol efflux, cholesterol influx, and I/E in Fu5AH cells exposed to lipoproteins or serumParameterrHDL, 25 μg/ml (n = 6)HDL3, 25 μg/ml (n = 9)Human Serum, 2.5% (n = 9)LDL, 100 μg/ml (n = 9)Cholesterol efflux (μg/8 h/mg protein)5.29 ± 0.362.46 ± 0.455.76 ± 0.944.16 ± 0.37Cholesterol influx (μg/8 h/mg protein)−0.42 ± 0.031.86 ± 0.7312.32 ± 1.8319.42 ± 2.67I/E−0.08 ± 0.000.87 ± 0.522.15 ± 0.294.65 ± 0.24I/E, influx-to-efflux ratio; rHDL, reconstituted high density lipoprotein. Cells were labeled with [3H]cholesterol for 24 h in 2.5% serum-containing medium. After an equilibration period in 0.2% BSA-containing medium, apolipoprotein A-I/phospholipid rHDL (25 μg/ml), HDL3 (25 μg/ml), human serum (2.5%), and LDL (100 μg/ml) were added for 8 h. ACAT inhibitor (2 μg/ml) was added during the entire experimental period, and 5 μg/ml Mevinolin was added during the flux period. Cholesterol efflux, influx, and I/E were calculated as described in Experimental Procedures. Data are expressed as averages ± SD. Open table in a new tab It has been proposed that SR-BI enhances aqueous diffusion, facilitating the bidirectional movement of FC, and this flux follows the concentration gradients between cell membranes and extracellular particles (17Yancey P.G. de la Llera-Moya M. Swarnakar S. Monzo P. Klein S.M. Connelly M.A. Johnson W.J. Williams D.L. Rothblat G.H. HDL phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor-BI (SR-BI).J. Biol. Chem. 2000; 275: 36596-36604Google Scholar, 18Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. Cell cholesterol efflux: integration of old and new observations provides new insights.J. Lipid Res. 1999; 40: 781-796Google Scholar). Thus, increasing cell cholesterol content should create a gradient that facilitates the movement of cholesterol out of cells. The initial cholesterol content in Fu5AH cells was increased by incubation with 100 μg/ml LDL added during the labeling period (Fig. 1). In a representative experiment, FC content was 20.4 ± 1.15 μg cholesterol/mg protein for cholesterol-normal cells and 36.5 ± 4.78 μg cholesterol/mg protein after cholesterol enrichment. HDL3, LDL, and human serum were then added to the culture medium for 8 h, and cholesterol efflux and influx were measured. Cholesterol efflux and influx values for normal cells were similar to those presented in Table 1. The fractional release of cholesterol from enriched cells was similar to that obtained with cholesterol-normal cells. However, because the cellular FC pool was expanded by ∼40%, the mass of FC released to all acceptors was increased, as has been described (19Johnson W.J. Mahlberg F.H. Chacko G.K. Phillips M.C. Rothblat G.H. The influence of cellular and lipoprotein cholesterol contents on the flux of cholesterol between fibroblasts and high density lipoprotein.J. Biol. Chem. 1988; 263: 14099-14106Google Scholar). Although the cholesterol mass released was increased, no significant change in cholesterol influx between normal and enriched cells was observed. Thus, the I/E obtained with enriched Fu5AH cells was reduced compared with that in cholesterol-normal cells.In vivo, cells are exposed to a mixture of lipoproteins present in serum. For this reason, further experiments were performed to measure the bidirectional flux of cholesterol upon exposure of cells to serum. Figure 2 shows how the mass of cholesterol undergoing efflux or influx, and the I/E, changed when Fu5AH cells were incubated with increasing concentrations of human serum, ranging from 2.5% to 12.5%. We also established the contribution of the apoB-depleted serum and the apoB-containing lipoproteins to both efflux and influx. To obtain the apoB-depleted serum, apoB-containing lipoproteins of whole serum were precipitated with PEG, as described in Experimental Procedures. The contribution of the apoB-containing lipoproteins was calculated by subtracting the flux obtained with PEG supernatant (i.e., HDL) from that obtained with serum. The cholesterol efflux to whole serum (Fig. 2A) increased in a dose-dependent manner, from 5.33 ± 0.16 μg cholesterol/mg protein with 2.5% serum to 11.23 ± 0.13 μg cholesterol/mg protein for the highest dose of serum (12.5%). Cholesterol influx from serum also increased in a dose-dependent manner, ranging from 11.15 ± 0.89 μg FC/mg protein with 2.5% serum to 27.24 ± 0.92 μg FC/mg protein at 12.5% serum. At every serum concentration, the mass of cholesterol incorporated into cells was greater than that released from cells, and this was reflected by I/E > 1, which increased linearly with increasing serum concentrations (Fig. 2B). After isolating the apoB-depleted fraction, the HDL- and apoB-containing lipoprotein contributions to efflux and influx were calculated. As shown in Fig. 2C, the apoB-depleted fraction was responsible for values ranging from 55% to 79% of the total amount of cholesterol released from cells (compare Fig. 2C with 2A). Furthermore, for every concentration of apoB-depleted serum, the I/E was somewhat 1, with ratios ranging from 3.00 to 3.92, indicating that time did not affect the direction of the net flux, at least over 12 h.Fig. 3.Cholesterol efflux, cholesterol influx, and I/E with Fu5AH cells exposed to human serum for 4, 8, and 12 h. Cells were labeled with [3H]cholesterol for 24 h in 2.5% serum-containing medium. After a 1 h equilibration period in 0.2% BSA-containing medium, 2.5% human serum was added for 4, 8, and 12 h. Experimental conditions were as described in Table 1. Cholesterol efflux (solid line), cholesterol influx (dashed line), and I/E (numbers) were calculated as described in Experimental Procedures. Data are expressed as means ± SD (n = 3).View Large Image Figure ViewerDownload (PPT)Cholesterol movement in Fu5AH cells is mainly attributable to a high expression of SR-BI; however, other pathways can be involved. To quantitate the contribution of SR-BI and other mechanisms to the bidirectional flux, we incubated Fu5AH cells with BLT-1, a low molecular weight compound capable of inhibiting lipid transport mediated by SR-BI (20Nieland T.J.F. Penman M. Dori L. Krieger M. Kirchhausen T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI.Proc. Natl. Acad. Sci. USA. 2002; 99: 15422-15427Google Scholar). Fu5AH cells were treated with 10 μM BLT-1 for 2 h before cholesterol acceptors were added. The results of cholesterol efflux and influx, in the absence or presence of BLT-1, are shown in Fig. 4A, B. BLT-1 inhibited HDL3-mediated efflux by 54%, indicating that approximately half of the efflux from Fu5AH cells to HDL3 occurred via SR-BI (Fig. 4A). Cholesterol influx was inhibited by 87% in BLT-1-exposed cells (Fig. 4B). Exposing cells to LDL after BLT-1 treatment caused a reduction in cholesterol efflux of 67% (Fig. 4A), and the effect on influx was significant but modest (20%; Fig. 4B). In the presence of human serum as an acceptor, BLT-1 inhibition of efflux was 57%; the reduction in influx was only 20%. The apoB-depleted portion of the serum gave a flux pattern similar to that of isolated HDL3, with ∼50% of the efflux mediated by SR-BI and a much greater fraction of influx linked to SR-BI (∼90%). I/E values in the presence or absence of BLT-1 are shown in Table 2. With isolated HDL3 and apoB-depleted serum, inhibition of SR-BI reduced influx to a greater extent than efflux, resulting in a reduction in I/E. However, this pattern was reversed with serum and LDL, in which case the inhibition of SR-BI resulted in a greater reduction in efflux. This caused increases in I/E.Fig. 4.Cholesterol efflux (A) and cholesterol influx (B) in BLT-1 (for block lipid transport-1)-pretreated Fu5AH cells exposed to different acceptors. Cells were labeled with [3H]cholesterol for 24 h in 2.5% serum-containing medium. During a 2 h equilibration period in 0.2% BSA-containing medium, cells were incubated in the absence (open bars) or the presence (closed bars) of BLT-1 (10 μM). For the following 8 h, HDL3 (25 μg/ml), LDL (100 μg/ml), human serum (2.5%), and its apoB-depleted fraction were added to the medium. Experimental conditions were as described in Table 1. Cholesterol efflux and influx were calculated as described in Experimental Procedures. The numbers above the bars indicate the percentage inhibition of efflux (A) or influx (B) after BLT-1 treatment. Data are expressed as means ± SD (n = 3).View Large Image Figure ViewerDownload (PPT)TABLE 2.I/E with Fu5AH cells preincubated with BLT-1 and exposed to different acceptorsAcceptor−BLT-1+BLT-1HDL30.93 ± 0.340.26 ± 0.44LDL4.59 ± 0.4011.21 ± 0.85Human serum2.48 ± 0.224.52 ± 0.39Apolipoprotein B-depleted serum0.71 ± 0.090.06 ± 0.10BLT-1, block lipid transport-1. Experimental conditions were as described for Fig. 4. Open table in a new tab These bidirectional flux data were obtained with Fu5AH hepatoma cells expressing high levels of SR-BI, which mediates large amounts of cholesterol movement in and out of cells. We extended the assay to cholesterol-normal and cholesterol-enriched MPMs by incubation with 100 μg/ml acLDL (Fig. 5). The initial cholesterol content of cholesterol-normal cells was 21.84 ± 1.85 μg cholesterol/mg protein, and that in cholesterol-enriched cells was 40.20 ± 4.04 μg cholesterol/mg protein. Macrophages were
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