Title: Assessing the effects of LXR agonists on cellular cholesterol handling: a stable isotope tracer study
Abstract: The liver X receptors (LXRs) α and β are responsible for the transcriptional regulation of a number of genes involved in cholesterol efflux from cells and therefore may be molecular targets for the treatment of cardiovascular disease. However, the effects of LXR ligands on cholesterol turnover in cells has not been examined comprehensively. In this study, cellular cholesterol handling (e.g., synthesis, catabolism, influx, and efflux) was examined using a stable isotope labeling study and a two-compartment modeling scheme. In HepG2 cells, the incorporation of 13C into cholesterol from [1-13C]acetate was analyzed by mass isotopomer distribution analysis in conjunction with nonsteady state, multicompartment kinetic analysis to calculate the cholesterol fluxes. Incubation with synthetic, nonsteroidal LXR agonists (GW3965, T0901317, and SB742881) increased cholesterol synthesis (∼10-fold), decreased cellular cholesterol influx (71–82%), and increased cellular cholesterol efflux (1.7- to 1.9-fold) by 96 h. As a consequence of these altered cholesterol fluxes, cellular cholesterol decreased (36–39%) by 96 h. The increased cellular cholesterol turnover was associated with increased expression of the LXR-activated genes ABCA1, ABCG1, FAS, and sterol-regulatory element binding protein 1c. In summary, the mathematical model presented allows time-dependent calculations of cellular cholesterol fluxes. These data demonstrate that all of the cellular cholesterol fluxes were altered by LXR activation and that the increase in cholesterol synthesis did not compensate for the increased cellular cholesterol efflux, resulting in a net cellular cholesterol loss. The liver X receptors (LXRs) α and β are responsible for the transcriptional regulation of a number of genes involved in cholesterol efflux from cells and therefore may be molecular targets for the treatment of cardiovascular disease. However, the effects of LXR ligands on cholesterol turnover in cells has not been examined comprehensively. In this study, cellular cholesterol handling (e.g., synthesis, catabolism, influx, and efflux) was examined using a stable isotope labeling study and a two-compartment modeling scheme. In HepG2 cells, the incorporation of 13C into cholesterol from [1-13C]acetate was analyzed by mass isotopomer distribution analysis in conjunction with nonsteady state, multicompartment kinetic analysis to calculate the cholesterol fluxes. Incubation with synthetic, nonsteroidal LXR agonists (GW3965, T0901317, and SB742881) increased cholesterol synthesis (∼10-fold), decreased cellular cholesterol influx (71–82%), and increased cellular cholesterol efflux (1.7- to 1.9-fold) by 96 h. As a consequence of these altered cholesterol fluxes, cellular cholesterol decreased (36–39%) by 96 h. The increased cellular cholesterol turnover was associated with increased expression of the LXR-activated genes ABCA1, ABCG1, FAS, and sterol-regulatory element binding protein 1c. In summary, the mathematical model presented allows time-dependent calculations of cellular cholesterol fluxes. These data demonstrate that all of the cellular cholesterol fluxes were altered by LXR activation and that the increase in cholesterol synthesis did not compensate for the increased cellular cholesterol efflux, resulting in a net cellular cholesterol loss. The liver X receptor (LXR) is an oxysterol-sensing nuclear receptor responsible for transcriptional regulation of a number of genes involved in reverse cholesterol transport and therefore may be a molecular target for the treatment of cardiovascular disease (1Jaye M. LXR agonists for the treatment of atherosclerosis.Curr. Opin. Investig. Drugs. 2003; 4: 1053-1058Google Scholar, 2Tontonoz P. Mangelsdorf D.J. Liver X receptor signaling pathways in cardiovascular disease.Mol. Endocrinol. 2003; 17: 985-993Google Scholar, 3Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat. Med. 2002; 8: 1243-1248Google Scholar). LXR is a member of the nuclear receptor family that regulates cellular cholesterol efflux (4Sparrow C.P. Baffic J. Lam M.H. Lund E.G. Adams A.D. Fu X. Hayes N. Jones A.B. MacNaul K.L. Ondeyka J. et al.A potent synthetic LXR agonist is more effective than cholesterol loading at inducing ABCA1 mRNA and stimulating cholesterol efflux.J. Biol. Chem. 2002; 277: 10021-10027Google Scholar) and whole body cholesterol excretion (5Ploäsch T. Kok T. Bloks V.W. Smit M.J. Haringa R. Chimini G. Groen A.K. Kuipers F. Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1.J. Biol. Chem. 2002; 277: 33870-33877Google Scholar) and is endogenously activated by various oxysterols (6Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Google Scholar), including 24(S),25-epoxycholesterol and 22(R)-hydroxycholesterol, an intermediate in steroid hormone production (7Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.Nature. 1996; 383: 728-731Google Scholar, 8Fu X. 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Cellular cholesterol metabolism and handling has been examined in terms of its synthesis by measuring the fraction of newly synthesized cholesterol (11Holleran A.L. Lindenthal B. Aldaghlas T.A. Kelleher J.K. Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes.Metabolism. 1998; 47: 1504-1513Google Scholar, 12Kelleher J.K. Kharroubi A.T. Aldaghlas T.A. Shambat I.B. Kennedy K.A. Holleran A.L. Masterson T.M. Isotopomer spectral analysis of cholesterol synthesis: applications in human hepatoma cells.Am. J. Physiol. 1994; 266: E384-E395Google Scholar), in terms of catabolism by measuring levels of bile acids through biochemical assays or HPLC (13Sniderman A.D. Zhang Z. Genest J. Cianflone K. Effects on apoB-100 secretion and bile acid synthesis by redirecting cholesterol efflux from HepG2 cells.J. Lipid Res. 2003; 44: 527-532Google Scholar, 14Levy J. Budai K. Javitt N.B. Bile acid synthesis in HepG2 cells: effect of cyclosporine.J. Lipid Res. 1994; 35: 1795-1800Google Scholar), in terms of influx by measuring the radioactivity of absorbed [3H]cholesteryl oleate (15Brissette L. Charest M.C. Falstrault L. Lafond J. Rhainds D. Tremblay C. Truong T.Q. Selective uptake of cholesteryl esters from various classes of lipoproteins by HepG2 cells.Biochem. Cell Biol. 1999; 77: 157-163Google Scholar, 16Xu X.X. Tabas I. Lipoproteins activate acyl-coenzyme A:cholesterol acyltransferase in macrophages only after cellular cholesterol pools are expanded to a critical threshold level.J. Biol. Chem. 1991; 266: 17040-17048Google Scholar), and in terms of efflux by measuring specific radioactivity in labeled cells (17Phillips M.C. Johnson W.J. Rothblat G.H. Mechanisms and consequences of cellular cholesterol exchange and transfer.Biochim. Biophys. Acta. 1987; 966: 223-276Google Scholar, 18Czarnecka H. Yokoyama S. Regulation of cellular cholesterol efflux by lecithin:cholesterol acyltransferase reaction through nonspecific lipid exchange.J. Biol. Chem. 1996; 271: 2023-2028Google Scholar, 19Johnson W.J. Manlberg F.H. Chacka 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, 20Sviridov D. Pyle L.E. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Google Scholar). However, prior methods address only the unidirectional flow of cholesterol and are not capable of measuring the bidirectional flow of cholesterol. A comprehensive analysis of cholesterol turnover by assessing the bidirectional flow of cholesterol is critical when examining LXR activation. Therefore, the focus of this study was to investigate the kinetics of cholesterol synthesis and transport in/out of HepG2 cells after treatment with several classes of natural and synthetic LXR agonists, such as the nonsteroidal synthetic compounds GW3965, SB742881, and T0901317, the steroidal synthetic agonist N,N-dimethly-3β-hydroxy-cholenamide (DMHCA), and the natural steroidal agonists 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. HepG2 cells were chosen for this study because this cell line is often used for cholesterol metabolic studies and has the constitutive ability to synthesize and secrete lipoproteins (21Javitt N.B. Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids.FASEB J. 1990; 4: 161-168Google Scholar, 22Dixon J.L. Ginsberg H.N. Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells.J. Lipid Res. 1993; 34: 167-179Google Scholar). However, it is acknowledged that HepG2 cells differ from primary human hepatocytes in their production of bile acids, a catabolic product of cholesterol (23Einarsson C. Ellis E. Abrahamsson A. Ericzon B. Bjorkhem I. Axelson M. Bile acid formation in primary human hepatocytes.World J. Gastroenterol. 2000; 6: 522-525Google Scholar). LXR-regulated genes involved in reverse cholesterol transport and lipogenesis were also investigated to assess the temporal correlation of cholesterol flux with gene activation. Additionally, a HMG-CoA reductase inhibitor, atorvastatin, was used to examine the dynamic fidelity of the kinetic analysis and to compare its effects on cellular cholesterol handling with those observed with several classes of LXR agonist. We used [1-13C]sodium acetate as a precursor for cholesterol synthesis, and mass isotopomer distribution analysis (MIDA) (24Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis: a technique for measuring biosynthesis and turnover of polymers.Am. J. Physiol. 1992; 263: E988-E1001Crossref Google Scholar, 25Hellerstein M.K. Kletke C. Kaempfer S. Wu K. Shackleton C.H. Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans.Am. J. Physiol. 1991; 261: E479-E486Google Scholar) of the 13C-labeled cholesterol molecule allowed for the precursor pool enrichment and fraction of newly synthesized cholesterol to be calculated. These parameters were then used to obtain cholesterol flux information using a two-compartment kinetic model. HepG2 cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), high glucose (25 mM), sodium bicarbonate (45 mM), and GlutaMax® (2 mM). Cells were seeded at 5 × 105 per dish into 60 mm dishes 2 days before the start of the experiment. To avoid depleting the cells of the tracers or LXR agonists (whose concentration decreases by 60% within 24 h) and essential growth supplements, the cells were replenished with fresh medium and the tracer at appropriate concentrations every 24 h. Cellular toxicity was measured using the ToxiLight nondestructive cytotoxicity kit (No. LT17-217; Cambrex Bio Science, Inc., Rockland, ME). Total apolipoprotein B (apoB) secreted by the cells to the culture medium was analyzed using ELISA kit A70102 (AlerChek, Inc., Portland, ME). To measure cholesterol synthesis and to model cholesterol flux kinetics, HepG2 cells were seeded in 60 mm dishes at an optimal seeding density to become 80% confluent by day 2 after seeding. On the third day, cells were given fresh medium with [1-13C]sodium acetate (10 mM) (Cambridge Isotope Laboratories, Inc., Andover, MA.), 99% isotope purity. The control groups received vehicle (0.1% DMSO, ethanol, or methanol), and the treatment group received GW3965, T0901317, or SB742881 (1 μM in DMSO), 22(R)-hydroxycholesterol, 24(S),25-epoxycholsterol, or DMHCA (1 μM in ethanol), or atorvastatin (1 μM in methanol). The cells were supplemented with medium containing LXR agonists or atorvastatin and [1-13C]sodium acetate every 24 h for up to 4 days. The LXR agonist concentrations used were typical of efficacious plasma concentrations observed in hamsters after oral administration of drug at 10 mg/kg body weight (unpublished observation). Cholesterol handling was also examined in the absence of serum with or without exogenous apoA-I. In these studies, cells were given 10% FBS on day 1 followed by serum-free medium with apoA-I on day 2. The treated group received GW3965 (1 μM) on both day 1 and day 2, and [1-13C]acetate was administered to all groups on day 2. Therefore, treatment was for 48 h, whereas acetate incorporation was for 24 h. Total cholesterol, free cholesterol, cholesteryl ester, and other neutral sterols were extracted as described previously (11Holleran A.L. Lindenthal B. Aldaghlas T.A. Kelleher J.K. Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes.Metabolism. 1998; 47: 1504-1513Google Scholar) with some modifications. Briefly, at the end of the treatment, cells were washed twice with ice-cold PBS. The cell culture medium was removed and stored at 4°C until analyzed. Cells were scraped into 2 ml of PBS and centrifuged at 1,000 rpm for 5 min. The PBS supernatant was discarded, and the cell pellets were saved at −80°C until analyzed. Cholesterol was extracted from the cell pellets twice with hexane-isopropanol (3:2, v/v), and 5α-cholestane was added as an internal recovery standard. The dried extract was saponified by adding freshly prepared 1 N KOH ethanolic solution (3 ml). Total cholesterol was derivatized as described previously (26Batta A.K. Salen G. Batta P. Tint G.S. Alberts D.S. Earnest D.L. Simultaneous quantitation of fatty acids, sterols and bile acids in human stool by capillary gas-liquid chromatography.J. Chromatogr. B. 2002; 775: 153-161Google Scholar). Recoveries for cholesterol and cholesteryl esters were assessed using cholesterol and cholesteryl oleate standards and found to range from 84% to 92%. An HP 5973 mass selective detector coupled with an HP 6890 gas chromatograph equipped with an HP-5 column (95% dimethylsiloxane) was used to measure cholesterol concentration and enrichment. The GC-MS apparatus was operated using splitless mode for higher sensitivity. In the scan-acquisition mode, dwell times were optimized for monitoring the internal standard peak at m/z 217 and the 13C-labeled cholesterol species from m/z 367–382. The fraction of newly synthesized cholesterol in the cell and medium compartments and the fractional enrichment of the cholesterol precursor pool were obtained using MIDA (27Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.Am. J. Physiol. 1999; 276: E1146-E1170Google Scholar). An in-house program that uses an unbiased simulation algorithm (Monte Carlo) was used to obtain the percentage of precursor 13C enrichment and the percentage of newly synthesized cholesterol. The atom percentage excess (APE), a standard parameter used to measure 13C enrichment, was converted to percentage of newly synthesized cholesterol using the validated technique of Hellerstein and colleagues (24Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis: a technique for measuring biosynthesis and turnover of polymers.Am. J. Physiol. 1992; 263: E988-E1001Crossref Google Scholar, 25Hellerstein M.K. Kletke C. Kaempfer S. Wu K. Shackleton C.H. Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans.Am. J. Physiol. 1991; 261: E479-E486Google Scholar). A multicompartment kinetic analysis was performed using SAAM II software, version 1.1.1 (SAAM Institute, Inc., Seattle, WA). This software allows for a simple and direct analysis of tracer kinetics using a compartment model approach. A precursor-product model was developed with a tracer (labeled) and a tracee (unlabeled) system. The notation F(destination, source) represents the total cholesterol flux measured as μg/h/mg cell protein. F(0,1) represents the cholesterol catabolic flux (i.e., bile acid synthesis, steroid synthesis, or other irreversible loss of cholesterol from the cells), F(1,2) represents the total cholesterol flux from the medium to the cells, and F(2,1) represents the total cholesterol flux from the cells to the medium. The administration of the [1-13C]acetate precursor was treated as a function of the endogenous flux U(1) equivalent to the cholesterol synthesis flux (Fig. 1), corresponding to the fraction of precursor enrichment. In the experiments in which equilibrium was not impaired significantly [untreated controls, 22(R)-hydroxycholesterol], the size of the cell and medium cholesterol pools (pool 1 and pool 2, respectively) were considered constant, and in experiments in which the equilibrium was disturbed [GW3965, T0901317, SB742881, 24(S),25-epoxycholesterol, DMHCA groups (see Figs. 2, 3 below)], the actual cholesterol content of cells as well as medium was used in the SAAM II compartment analysis, as explained previously (28Pont F. Duvillard L. Verges B. Gambert P. Development of compartmental models in stable-isotope experiments: application to lipid metabolism.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 853-860Google Scholar, 29Millar J.S. Lichtenstein A.H. Dolnikowski G.G. Ordovas J.M. Schaefer E.J. Proposal of a multicompartmental model for use in the study of apolipoprotein E metabolism.Metabolism. 1998; 47: 922-928Google Scholar, 30Branswig S. Kerksiek A. Sudhop T. Luers C. Bergmann K.V. Berthod H.K. Carbamazepine increases atherogenic lipoproteins: mechanism of action in male adults.Am. J. Physiol. 2002; 282: H704-H716Google Scholar). Using the actual values of cellular and medium cholesterol allowed for the linear cumulative two-compartmental model to be investigated more explicitly. Replenishment of the cells with fresh medium including drug and tracer was accounted for in the model to provide a more accurate calculation.Fig. 2Total cellular and culture medium cholesterol after treatment with nonsteroidal liver X receptor (LXR) agonists. Total cholesterol is shown in HepG2 cells (A) and in the culture medium (B) for up to 96 h of treatment with GW3965 (1 μM), T0901317 (1 μM), or SB742881 (1 μM). Data are presented as means ± SEM (n = 3). * P < 0.01, ** P < 0.001, control versus treated groups.View Large Image Figure ViewerDownload (PPT)Fig. 2Total cellular and culture medium cholesterol after treatment with nonsteroidal liver X receptor (LXR) agonists. Total cholesterol is shown in HepG2 cells (A) and in the culture medium (B) for up to 96 h of treatment with GW3965 (1 μM), T0901317 (1 μM), or SB742881 (1 μM). Data are presented as means ± SEM (n = 3). * P < 0.01, ** P < 0.001, control versus treated groups.View Large Image Figure ViewerDownload (PPT)Fig. 3Total cellular and culture medium cholesterol after treatment with steroidal LXR agonists. Total cholesterol is shown in HepG2 cells (A) and in the culture medium (B) for up to 96 h of treatment with 22(R)-hydroxycholesterol (1 μM), 24(S),25-epoxycholesterol (1 μM), or N,N-dimethly-3β-hydroxy-cholenamide (DMHCA; 1 μM). Data are presented as means ± SEM (n = 3). * P < 0.05, control versus treated groups.View Large Image Figure ViewerDownload (PPT)Fig. 3Total cellular and culture medium cholesterol after treatment with steroidal LXR agonists. Total cholesterol is shown in HepG2 cells (A) and in the culture medium (B) for up to 96 h of treatment with 22(R)-hydroxycholesterol (1 μM), 24(S),25-epoxycholesterol (1 μM), or N,N-dimethly-3β-hydroxy-cholenamide (DMHCA; 1 μM). Data are presented as means ± SEM (n = 3). * P < 0.05, control versus treated groups.View Large Image Figure ViewerDownload (PPT) Cellular protein was measured using the Bio-Rad DC protein assay (Bio-Rad Laboratories, South San Francisco, CA) according to the manufacturer's instructions. Total RNA was isolated from HepG2 cells using the Qiagen RNeasy 96 kit (No. 74181; Qiagen, Valencia, CA). cDNA was then synthesized with random hexamer primers using the Superscript-III first-strand synthesis system for RT-PCR (No. 18080-051; Invitrogen Corp., Carlsbad, CA). Gene-specific quantitative PCR mixes were prepared using 50 ng of cDNA, 0.4 μM forward primer, 0.4 μM reverse primer, and 0.2 μM FAM-labeled fluorogenic probe (Applied Biosystems, Foster City, CA) to a reaction volume of 25 μl. Quantitative PCR was performed on an ABI PRISM® 7700 sequence detection system. The comparative cycle threshold method was used to quantitate gene expression in cells. Data are reported as fold change from control. All assays were performed in quadruplicate. The following primer and probe sets were used: hABCA1 forward, 5′-GCTCCCGGAGTTGTTGGAAA-3′; hABCA1 reverse, 5′-GTATAAAAGAAGCCTCCGAGCATC-3′; hABCA1 probe, 6FAM-TTTAACAAATCCATTGTGGCTCGCCTGT-TAMRA; hABCG1 forward, 5′-AGCATCATGAGGGACTCGGT-3′; hABCG1 reverse, 5′-GGAGGCCGATCCCAATGT-3′; hABCG1 probe, 6FAM-CTGACACACCCTGCGCATCACCTCG-TAMRA; hFAS forward, 5′-ACCTGGGCGCGGACTAC-3′; hFAS reverse, 5′-CGATGACGTGGACGGATACTT-3′; hFAS probe, 6FAM-ACCTCTCCCAGGTATGCGACGGG-TAMRA. For the detection of human sterol-regulatory element binding protein 1c (SREBP1c), HMG-CoA reductase, scavenger receptor class B type I (SR-BI), SREBP2, low density lipoprotein receptor (LDLR), and cholesterol 7α-hydroxylase (CYP7A1), commercial primer/probe sets Hs00231674_ml, Hs00168352_ml, Hs00194092_ml, Hs00190237_ml, Hs00181192_ml, and Hs00167982_ml, respectively, were used (Assays by Demand; Applied Biosystems). All statistical tests were performed using Prism software (Graphpad Software, San Diego, CA), and a value of P < 0.05 was considered significant. When necessary, nonlinear regression fitting was used to verify the trends in the time-course study. Student's t-test or ANOVA with Newman-Keuls posthoc test was used to determine the level of significance between the treatment groups. In the control (without LXR agonist) experiments, total cholesterol (esterified and nonesterified) remained constant in both the HepG2 cells and the medium for the entire course of the study (96 h). However, in LXR agonist-treated cells, there was a reduction in cellular cholesterol from 56.8 ± 3.9 μg/mg cell protein at 24 h in the control group to 47.8 ± 1.7, 37.8 ± 0.5, 32.3 ± 2.0, and 31.4 ± 0.8 μg/mg cell protein in the GW3965-treated group at 24, 48, 72, and 96 h, respectively. Similar temporal decreases in cellular cholesterol were also observed in the T0901317 and SB742881 treatment groups (Fig. 2A). Consequently, there was an increase in the cell medium cholesterol concentration from 25.2 ± 1.7 μg/ml at 24 h in the control group to 28.0 ± 1.7, 33.3 ± 1.7, 34.0 ± 1.2, and 38.0 ± 01.7 μg/ml in the GW3965-treated group at 24, 48, 72, and 96 h, respectively. Similar temporal increases in medium cholesterol were also observed in the T0901317 and SB742881 groups (Fig. 2B). In contrast, there was no change in cellular cholesterol after treatment with 22(R)-hydroxycholesterol. However, cellular cholesterol decreased to 41.6 ± 2.2 and 36.5 ± 4.4 μg/mg cell protein after treatment with 24(S),25-epoxycholesterol and decreased to 49.0 ± 2.5 and 42.6 ± 5.2 μg/mg cell protein after treatment with DMHCA at 72 and 96 h, respectively (Fig. 3A). The medium cholesterol did not change significantly and remained ∼35 μg/ml in the 22(R)-hydroxycholesterol-, 24(S),25-epoxycholesterol-, and DMHCA-treated groups (Fig. 3B). Cellular free and esterified cholesterol pools were examined at 24 and 48 h to determine whether the different pools contributed to overall cholesterol handling by the cells. Both concentration (Fig. 4A, C) and 13C enrichment (Fig. 4B, D) of cellular free cholesterol and cholesteryl esters were assessed at 24 and 48 h in control and GW3965-treated cells. Although the enrichment of esterified cholesterol was decreased initially at 24 h in the treated versus control cells, the enrichment of both free cholesterol and esterified cholesterol was higher in the treated versus control cells by 48 h (Fig. 4D). The total cholesterol pool and enrichment appear to reflect the rapid turnover of free cholesterol rather than the slow turnover of esterified cholesterol. Therefore, for the purposes of this study, kinetic modeling of the total cholesterol pool rather than separate free and esterified cholesterol pool modeling was performed. To obtain kinetic data for mathematical modeling, [1-13C]acetate precursor for cholesterol synthesis was added to HepG2 cells and [1-13C]acetate incorporation into intracellular cholesterol after LXR treatment with two distinct structural classes; nonsteroidal and steroidal LXR agonists over a 96 h period were monitored (Fig. 5). The GW3965, T0901317, and SB742881 treatment groups showed an increase in the rate of cholesterol labeling, reflecting an increase in the rate of cholesterol synthesis (Fig. 5A). However, the natural LXR ligand [22(R)-hydroxycholesterol] had no effect on the rate of cholesterol labeling, whereas the steroidal LXR agonists [24(S),25-epoxycholesterol, DMHCA] appeared to slightly reduce the rate of cholesterol labeling, reflecting a decrease in the rate of cholesterol synthesis (Fig. 5B), consistent with earlier observations (6Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Google Scholar). Furthermore, as a positive control for the inhibition of cholesterol synthesis, a HMG-CoA reductase inhibitor (atorvastatin) was examined (Fig. 5A). A decrease in cholesterol synthesis in the atorvastatin-treated cells was consistent with the significantly lower cholesterol APE observed in these cells.Fig. 5Cellular cholesterol 13C enrichment time course. Cellular cholesterol enrichment was measured in HepG2 cells during a 96 h incubation with GW3965 (1 μM), T0901317 (1 μM), SB742881 (1 μM), or atorvastatin (1 μM) (A) and 22(R)-hydroxycholesterol (1 μM), 24(S),25-epoxycholesterol (1 μM), or DMHCA (1 μM) (B). Data are presented as means ± SEM (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, control versus treated groups.View Large Image Figure ViewerDownload (PPT) A Monte Carlo simulation was used to generate the isotopomer distribution data for a given cholesterol precursor 13C enrichment and for a given fraction of newly synthesized cholesterol. The cholesterol 13C precursor enrichment percentage (P = 45%) calculated in the present study was similar to two validated methods for measuring precursor enrichment: MIDA (P = 47%) and isotopomer spectral analysis (P = 49%). Although these methods estimate the precursor fractional 13C enrichment differently, the underlying principle is the same. The calculated cholesterol precursor enrichment (p) and the percentage of newly synthesized cholesterol (f) for different treatment conditions at 24 h are shown in Table 1. The cholesterol precursor enrichment in the GW3965 group was increased rapidly and was stable throughout the study (37 ± 2%, 42 ± 1%, 43 ± 1%, and 45 ± 1% at 2, 4, 8, and 24 h, respectively). All treatment groups had similar precursor enrichments as their control groups at 24 h. The cholesterol precursor enrichment did not exceed 50% for the various experimental conditions when using [1-13C]sodium acetate in the incubation medium, reflecting the steady state of the precursor pool. The newly synthesized cholesterol in HepG2 cells at 24 h increased from 19 ± 1% in the control group to 28 ± 1%, 26 ± 1%, and 24 ± 1% (P < 0.01) after GW3965, T0901317, and SB742881 treatment, respectively, and decreased from 17 ± 1% to 2 ± 0% (P < 0.001) after atorvastatin treatment. However, newly synthesized cholesterol was not altered significantly [22(R)-hydroxycholesterol, 24(S),25-epoxycholesterol] or decreased (DMHCA) versus control at 24 h (Table 1). No significant differences were observed in the percentage of newly synthesized cholesterol under serum-free conditions.TABLE 1Percentage of precursor enrichment and newly synthesized cholesterol in HepG2 cellsMediumTreatmentpf%10% FBSControl45 ± 119 ± 110% FBSGW396545 ± 028 ± 1aP < 0.001, control versus treated groups.10% FBST090131745 ± 126 ± 1aP < 0.001, control versus treated groups.10% FBSSB74288147 ± 224 ± 1aP < 0.001, control versus treated groups.10% FBSControl43 ± 217 ± 110% FBS22(R)-Hydroxycholesterol42 ± 118 ± 210% FBS24(S),25-Epoxycholesterol43 ± 115 ± 210% FBSDMHCA43 ± 014 ± 0aP < 0.001, control versus treated groups.Serum-freeControl38 ± 128 ± 2Serum-freeGW396538 ± 031 ± 1Serum-free + apoA-IControl40 ± 022 ± 1Serum-free + apoA-IGW396538 ± 140 ± 0aP < 0.001, control versus treated groups.10% FBSControl40 ± 017 ± 110% FBSAtorvastatin40 ± 12 ± 0aP < 0.001, control vers