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• W1977432493 abstract "The liver X receptors (LXRs) have been shown to affect lipoprotein plasma profile, lipid metabolism, and reverse cholesterol transport (RCT). In the present study, we investigated whether a short-term administration of the synthetic LXR agonist T0901317 (T0) to mice may affect RCT by modulating the capacity of plasma to promote cellular lipid efflux. Consistent with previous data, the pharmacological treatment of mice caused a significant increase of macrophage-derived [3H]cholesterol content in plasma, liver, and feces and resulted in improved capacity of plasma to promote cellular cholesterol release through passive diffusion and scavenger receptor class B type I (SR-BI)-mediated mechanisms. Differently, plasma from treated mice possessed similar or reduced capacity to drive lipid efflux via ABCA1. Consistent with these data, the analysis of plasma HDL fractions revealed that T0 caused the formation of larger, lipid-enriched particles. These results suggest that T0 promotes in vivo RCT from macrophages at least in part by inducing an enrichment of those HDL subclasses that increase plasma capacity to promote cholesterol efflux by passive diffusion and SR-BI-mediated mechanisms. The liver X receptors (LXRs) have been shown to affect lipoprotein plasma profile, lipid metabolism, and reverse cholesterol transport (RCT). In the present study, we investigated whether a short-term administration of the synthetic LXR agonist T0901317 (T0) to mice may affect RCT by modulating the capacity of plasma to promote cellular lipid efflux. Consistent with previous data, the pharmacological treatment of mice caused a significant increase of macrophage-derived [3H]cholesterol content in plasma, liver, and feces and resulted in improved capacity of plasma to promote cellular cholesterol release through passive diffusion and scavenger receptor class B type I (SR-BI)-mediated mechanisms. Differently, plasma from treated mice possessed similar or reduced capacity to drive lipid efflux via ABCA1. Consistent with these data, the analysis of plasma HDL fractions revealed that T0 caused the formation of larger, lipid-enriched particles. These results suggest that T0 promotes in vivo RCT from macrophages at least in part by inducing an enrichment of those HDL subclasses that increase plasma capacity to promote cholesterol efflux by passive diffusion and SR-BI-mediated mechanisms. 8-(4-chlorophenylthio) cyclic AMP liver X receptor reverse cholesterol transport scavenger receptor class B type I sterol-regulatory element binding protein-1 T0901317 The liver X receptors (LXRs) belong to the family of nuclear hormone receptors that act as transcription factors for several classes of genes (1.Lund E.G. Menke J.G. Sparrow C.P. Liver X receptor agonists as potential therapeutic agents for dyslipidemia and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1169-1177Crossref PubMed Scopus (118) Google Scholar). The two isoforms, LXRα and LXRβ, differ in distribution and function, the former being highly expressed in liver, intestine, and macrophages, where it is involved in lipid metabolism (2.Millatt L.J. Bocher V. Fruchart J.C. Staels B. Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis.Biochim. Biophys. Acta. 2003; 1631: 107-118Crossref PubMed Scopus (78) Google Scholar), whereas the latter is ubiquitous and present also in the central nervous system (3.Whitney K.D. Watson M.A. Collins J.L. Benson W.G. Stone T.M. Numerick M.J. Tippin T.K. Wilson J.G. Winegar D.A. Kliewer S.A. Regulation of cholesterol homeostasis by the liver X receptors in the central nervous system.Mol. Endocrinol. 2002; 16: 1378-1385Crossref PubMed Scopus (0) Google Scholar).The role of LXR in atherosclerosis and reverse cholesterol transport (RCT) is no longer in doubt: the induction of genes such as ABC transporters A1 and G1, sterol-regulatory element binding protein-1 (SREBP-1), cholesteryl ester transfer protein, by regulating the absorption of cholesterol in intestine, the conversion of cholesterol in bile acids, lipid efflux, and the synthesis of apolipoproteins, has been shown to protect the arterial wall (4.Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar, 5.Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. Mangelsdorf D.J. et al.Identification of macrophage liver X receptors as inhibitors of atherosclerosis.Proc. Natl. Acad. Sci. USA. 2002; 99: 11896-11901Crossref PubMed Scopus (370) Google Scholar, 6.Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers.Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1144) Google Scholar).LXR activation occurs upon the binding of specific agonists with the receptors: this event promotes the formation of a heterodimer with the retinoid X receptor and the binding to promoter sequences on different target genes (2.Millatt L.J. Bocher V. Fruchart J.C. Staels B. Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis.Biochim. Biophys. Acta. 2003; 1631: 107-118Crossref PubMed Scopus (78) Google Scholar). Natural ligands are represented by oxysterols, such as 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, 27-hydroxycholesterol, and 24,25-epoxycholesterol (7.Janowski 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: 728-731Crossref PubMed Scopus (1440) Google Scholar), whereas synthetic compounds have been developed recently. Among them, the nonsteroidal compound T0901317 (T0) has been demonstrated to increase HDL plasma levels (4.Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar) and to suppress the development of atherosclerotic lesions (8.Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Crossref PubMed Scopus (290) Google Scholar) in mice. The tertiary amine GW3965 showed protective properties toward the formation of atherosclerotic plaques in mice, not necessarily related to changes in HDL cholesterol concentration (9.Joseph S.B. McKilligin E. Pei L. Watson M.A. Collins A.R. Laffitte B.A. Chen M. Noh G. Goodman J. Hagger G.N. et al.Synthetic LXR ligand inhibits the development of atherosclerosis in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 7604-7609Crossref PubMed Scopus (770) Google Scholar).The atheroprotective role of LXR has clearly emerged in studies of genetically modified animals: in mice deficient for both isoforms, aortic lipid deposition is highly increased (10.Schuster G.U. Parini P. Wang L. Alberti S. Steffensen K.R. Hansson G.K. Angelin B. Gustafsson J.A. Accumulation of foam cells in liver X receptor-deficient mice.Circulation. 2002; 106: 1147-1153Crossref PubMed Scopus (156) Google Scholar), whereas the deletion of bone marrow LXR in mice increases their susceptibility to atherosclerosis (5.Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. Mangelsdorf D.J. et al.Identification of macrophage liver X receptors as inhibitors of atherosclerosis.Proc. Natl. Acad. Sci. USA. 2002; 99: 11896-11901Crossref PubMed Scopus (370) Google Scholar). In particular, the expression of LXR in macrophages has been recognized to be mandatory for the antiatherosclerotic activity (11.Levin N. Bischoff E.D. Daige C.L. Thomas D. Vu C.T. Heyman R.A. Tangirala R.K. Schulman I.G. Macrophage liver X receptor is required for antiatherogenic activity of LXR agonists.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 135-142Crossref PubMed Scopus (2) Google Scholar).LXR stimulation is also involved in the modulation of the RCT, the process by which excess cholesterol is re-moved from extrahepatic tissues and transported back to the liver (12.Barter P.J. Rye K.A. Molecular mechanisms of reverse cholesterol transport.Curr. Opin. Lipidol. 1996; 7: 117-123Crossref PubMed Scopus (171) Google Scholar). This observation is supported by several lines of evidence: LXR agonists, by upregulating ABCA1 and ABCG1 expression in cell culture, may stimulate cel-lular cholesterol efflux, the first step of RCT (13.Schwartz K. Lawn R.M. Wade D.P. ABC1 gene expression and apoA-I-mediated cholesterol efflux are regulated by LXR.Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar, 14.Wang N. Ranalletta M. Matsuura F. Peng F. Tall A.R. LXR-induced redistribution of ABCG1 to plasma membrane in macrophages enhances cholesterol mass efflux to HDL.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1310-1316Crossref PubMed Scopus (183) Google Scholar). The capacity of LXR to modulate RCT in vivo has been dem-onstrated directly by Rader's group (15.Naik S.U. Wang X. Da Silva J.S. Jaye M. Macphee C.H. Reilly M.P. Billheimer J.T. Rothblat G.H. Rader D.J. Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo.Circulation. 2006; 113: 90-97Crossref PubMed Scopus (312) Google Scholar), who showed a stimulated mobilization of cholesterol in different mouse models administered with GW3965. However, despite the large amount of data presented and the convincing data, no explanation for the potential mechanism involved in the LXR effect was provided.Several works revealed that treatment of mice with T0 or GW3965 induced a significant increase in circulating HDL (4.Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar, 8.Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Crossref PubMed Scopus (290) Google Scholar, 16.Miao B. Zondlo S. Gibbs S. Cromley D. Hosagrahara V.P. Kirchgessner T.G. Billheimer J. Mukherjee R. Raising HDL cholesterol without inducing hepatic steatosis and hypertriglyceridemia by a selective LXR modulator.J. Lipid Res. 2004; 45: 1410-1417Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Because these lipoproteins have a well-established role in cholesterol efflux, representing the physiological lipid acceptor, we wondered whether LXR may promote RCT through the increase in the efflux potential of plasma. To address this question, we treated mice with increasing doses of T0. Once it was confirmed that LXR stimulation in vivo caused the promotion of cholesterol mobilization from macrophages to plasma, liver, and feces, we investigated whether plasma from T0-treated mice possessed a higher capacity to stimulate cholesterol release from cultured cells.EXPERIMENTAL PROCEDURESAnimalsIn the first experiment, 15 week old male BALB/c mice (Charles River, Calco, Italy) were treated by oral gavage with T0 (Alexis Biochemicals, Lausen, Switzerland), 10 mg/kg/day, dissolved in propylene glycol-Tween 80 (4:1) or vehicle only for 8 days. Mice received standard diet and water ad libitum.In the second experiment, 15 week old male BALB/c mice were treated with T0, 100 mg/kg/day, or vehicle only, as described above.All experiments were conducted in conformity with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and performed with the approval of the Ethical Committee for Animal Experiments of the University of Parma.Measurement of macrophage RCT in vivoOn day 6 of pharmacological treatment, [3H]cholesterol-labeled J774 macrophages prepared as described previously (17.Zhang Y. Zanotti I. Reilly M.P. Glick J.M. Rothblat G.H. Rader D.J. Overexpression of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages to feces in vivo.Circulation. 2003; 108: 661-663Crossref PubMed Scopus (371) Google Scholar) were intraperitoneally injected into mice, which were successively separated into individual cages. On day 8, animals were euthanized and blood and liver were collected. Plasma was separated and counted in a β-counter. Livers were extracted by the Bligh and Dyer method (18.Iverson S.J. Lang S.L. Cooper M.H. Comparison of the Bligh and Dyer and Folch methods for total lipid determination in a broad range of marine tissue.Lipids. 2001; 36: 1283-1287Crossref PubMed Scopus (517) Google Scholar) and counted by liquid scintillation addition. Feces were collected at 24 and 48 h after the injection of radiolabeled cells and extracted by the Bligh and Dyer method.Gene expression analysis by real-time PCRThe isolation of total RNA from liver was achieved using NucleoSpin RNA II according to the manufacturer's instructions (Macherey-Nagel, Duren, Germany). Reverse transcription was done using the cDNA archive kit (Applied Biosystems, Foster City, CA). The resulting cDNA was used for real-time quantitative PCR in the ABI Prism 7000 sequence detection system (Applied Biosystems). The specific primers and TaqMan probes for murine ABCA1 and 18S were obtained from Applied Biosystems (Assays-on-Demand Gene Expression Products and TaqMan Rodent GAPDH Control Reagents). To control for variations in the amount of DNA available for PCR in the different samples, gene expression of the target sequence was normalized in relation to the expression of an endogenous control, 18S.Evaluation of plasma efflux potentialMice, treated as described above, were euthanized on day 8; blood was collected from the heart, recovered in plastic tubes, and anticoagulated with 3.8% sodium citrate (one part citrate to nine parts blood). Plasma was isolated by low-speed centrifugation and stored at −80°C until use. Aliquots of plasma were used as acceptors in cholesterol efflux experiments. To study the specific mechanism of lipid efflux, we used different cell types. To characterize scavenger receptor class B type I (SR-BI)-mediated efflux, we tested plasma activity on Fu5AH rat hepatoma cells treated or not with BLT-1, which selectively blocks receptor activity (19.Nieland T.J. 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: 15422-15427Crossref PubMed Scopus (180) Google Scholar), and COS-7 cells, control or transiently transfected with SR-BI. Plasmid cDNA containing SR-BI or plasmid cDNA alone was transiently transfected in this cell line with FuGENE 6 as the carrier using an established procedure (20.Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and scavenger receptor BI (SR-BI), shows that both receptors mediate high density lipoprotein-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Cultures of J774 mouse macrophages incubated in the presence or absence of 0.3 mM 8-(4-chlorophenylthio) cyclic AMP (cpt-cAMP; Sigma) were used as a model for ABCA1-mediated or passive diffusion efflux, respectively. Quantification of cholesterol efflux was performed as described previously (21.Zanotti I. Poti F. Favari E. Steffensen K.R. Gustafsson J.A. Bernini F. Pitavastatin effect on ATP binding cassette A1-mediated lipid efflux from macrophages. Evidence for LXR-dependent and LXR-independent mechanisms of activation by cAMP.J. Pharmacol. Exp. Ther. 2006; 317: 1-7Crossref PubMed Scopus (30) Google Scholar), using a time zero set of cells to calculate total [3H]cholesterol content in the monolayer. Fractional efflux was calculated as cpm [3H] in the medium/[3H] at time zero × 100.Measurement of plasma lipids and lipoproteinsPlasma HDL cholesterol levels were determined using a standard enzymatic technique with a Roche Diagnostics Integra 400 autoanalyzer. Lipoproteins were analyzed by gel filtration on a 10/30 Superose 6B column (fast-protein liquid chromatography; Amersham Pharmacia Biotech) at 0.5 ml/min in phosphate-buffered saline containing 0.1 mM EDTA and 0.02% sodium azide. Total cholesterol, phospholipid, and triglyceride concentrations were measured in the collected fractions using standard enzymatic techniques.Two-dimensional gel electrophoresisPlasma HDL subclasses were separated by two-dimensional electrophoresis, in which agarose gel electrophoresis was followed by nondenaturing polyacrylamide gradient gel electrophoresis and subsequent immunoblotting (22.Favari E. Lee M. Calabresi L. Franceschini G. Zimetti F. Bernini F. Kovanen P.T. Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein.J. Biol. Chem. 2004; 279: 9930-9936Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In the first dimension, plasma (5 μl) was run on a 0.5% agarose gel; agarose gel strips containing the separated lipoproteins were then transferred to a 3–20% polyacrylamide gradient gel. Separation in the second dimension was performed at 30 mA for 4 h. Fractionated HDLs were then electroblotted onto a nitrocellulose membrane and detected with an anti-apolipoprotein A-I antibody.Statistical analysisResults were analyzed by Student's t-test with the use of GraphPad Prism software. Efflux data are reported as means of triplicate assays ± SD.RESULTSIn the first part of this study, we aimed to confirm the impact of LXR stimulation on macrophage-specific RCT in vivo. Different from the previous report (15.Naik S.U. Wang X. Da Silva J.S. Jaye M. Macphee C.H. Reilly M.P. Billheimer J.T. Rothblat G.H. Rader D.J. Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo.Circulation. 2006; 113: 90-97Crossref PubMed Scopus (312) Google Scholar), we treated animals with the LXR synthetic agonist T0 at 10 or 100 mg/kg/day for 8 days. The former dose was shown to be efficient at reducing atherosclerosis development (8.Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Crossref PubMed Scopus (290) Google Scholar), whereas the latter significantly affected HDL quality and quantity in mice (23.Cao G. Beyer T.P. Yang X.P. Schmidt R.J. Zhang Y. Bensch W.R. Kauffman R.F. Gao H. Ryan T.P. Liang Y. et al.Phospholipid transfer protein is regulated by liver X receptors in vivo.J. Biol. Chem. 2002; 277: 39561-39565Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar).Indeed, mice that received LXR agonist at both doses showed a higher content of macrophage-derived [3H]cholesterol in plasma (Fig. 1A , B). The hepatic content of [3H]cholesterol was higher in mice treated with 100 mg/kg/day T0 than in untreated mice (Fig. 1D), whereas at the lower dose there was no significant difference (Fig. 1C). Importantly, mice treated with T0 excreted significantly more radioactivity in the feces compared with untreated mice (Fig. 1E, F).As reported previously in the literature, the stimulation of LXR in vivo can affect lipoprotein plasma profile (4.Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar, 8.Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Crossref PubMed Scopus (290) Google Scholar, 15.Naik S.U. Wang X. Da Silva J.S. Jaye M. Macphee C.H. Reilly M.P. Billheimer J.T. Rothblat G.H. Rader D.J. Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo.Circulation. 2006; 113: 90-97Crossref PubMed Scopus (312) Google Scholar, 16.Miao B. Zondlo S. Gibbs S. Cromley D. Hosagrahara V.P. Kirchgessner T.G. Billheimer J. Mukherjee R. Raising HDL cholesterol without inducing hepatic steatosis and hypertriglyceridemia by a selective LXR modulator.J. Lipid Res. 2004; 45: 1410-1417Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and hepatic gene expression (2.Millatt L.J. Bocher V. Fruchart J.C. Staels B. Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis.Biochim. Biophys. Acta. 2003; 1631: 107-118Crossref PubMed Scopus (78) Google Scholar). Consistent with these data, in our study we observed an increase in HDL plasma concentration in mice treated with 100 mg/kg/day T0 compared with untreated mice (average ± SD: 110.5 ± 2.1 vs. 41.0 ± 9.9 mg/dl; P < 0.05) and a 2.8-fold increase in hepatic Abca1 mRNA expression, as evaluated by quantitative PCR (data not shown). Because HDLs are the physiological acceptors of cholesterol, we wondered whether the observed increase of RCT, and especially its first step, cholesterol efflux, could be related to a potentiated capacity of plasma to accept cholesterol from cells. Plasma from mice treated with T0 were tested in in vitro cholesterol efflux experiments in different cellular models to specifically verify whether SR-BI, passive diffusion, and/or ABCA1-mediated processes were affected. The first experiment was performed on Fu5AH rat hepatoma cells, which express high levels of SR-BI and have been used extensively to characterize this efflux pathway (22.Favari E. Lee M. Calabresi L. Franceschini G. Zimetti F. Bernini F. Kovanen P.T. Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein.J. Biol. Chem. 2004; 279: 9930-9936Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 24.Asztalos 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: 2246-2253Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 25.Fournier N. Francone O. Rothblat G. Goudouneche D. Cambillau M. Kellner-Weibel G. Robinet P. Royer L. Moatti N. Simon A. et al.Enhanced efflux of cholesterol from ABCA1-expressing macrophages to serum from type IV hypertriglyceridemic subjects.Atherosclerosis. 2003; 171: 287-293Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Here, we demonstrate that treatment of mice with T0 dose-dependently increased cholesterol release to plasma, increasing from 4.7 ± 0.6% in cells exposed to plasma from control mice to 7.4 ± 2.2% (P < 0.05) and 9.3 ± 0.2% (P < 0.001) in cells exposed to plasma from mice treated with T0 at 10 and 100 mg/kg/day, respectively. To confirm the role of SR-BI in explaining the increased efflux capacity of T0-treated plasma, we repeated the experiment on Fu5AH cells in the presence or absence of BLT-1, which has been demonstrated to selectively and efficiently block SR-BI activity (19.Nieland T.J. 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: 15422-15427Crossref PubMed Scopus (180) Google Scholar). As shown in Fig. 2 (left panel), although plasma from mice treated with T0 promoted a more efficient release of cholesterol when SR-BI was fully functional, when cells were pretreated with BLT-1 this difference disappeared. To further support the role of this transporter in driving T0-treated plasma efflux capacity, the experiment was repeated in COS-7 cells transfected or not with SR-BI. Whereas in basal conditions plasma from T0-treated mice possessed only a slightly increased efflux potential compared with control plasma, when SR-BI was expressed the difference was significant (Fig. 2, right panel).Fig. 2.Efflux potential of plasma from untreated or T0-treated mice: the role of scavenger receptor class B type I (SR-BI). Fu5AH cells, treated or not with BLT-1 (left panel), and COS-7 cells, control or transiently transfected with SR-BI (right panel), were radiolabeled with [3H]cholesterol, equilibrated in an albumin-containing medium, and exposed to 2.5% plasma for 4 h. Efflux was expressed as cpm in medium/cpm at time zero × 100. Values shown are means of triplicate assays ± SD. ** P < 0.01 versus Fu5AH cells not treated with BLT-1or versus SR-BI-expressing COS-7 cells exposed to plasma from vehicle-treated mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The capacity of plasma to drive the efflux that occurs via passive diffusion was evaluated in J774 macrophages. In this system, plasma from T0-treated mice produced an increased efflux over that of control mice (Fig. 3); however, upon cell treatment with cpt-cAMP, the increase in T0 plasma acceptor capacity was less pronounced (Fig. 3). Consequently, the contribution of ABCA1 to total efflux, calculated as the difference between the percentage cholesterol efflux from cells upregulated with cpt-cAMP and the percentage cholesterol efflux from control J774 cells, was diminished.Fig. 3.Efflux potential of plasma from untreated or T0-treated mice: passive diffusion and ABCA1-mediated efflux. J774 cells were radiolabeled with [3H]cholesterol, equilibrated in an albumin-containing medium in the presence or absence of 0.3 mM 8-(4-chlorophenylthio) cyclic AMP (cpt-cAMP), and exposed to 2.5% plasma for 4 h. Efflux was expressed as cpm in medium/cpm at time zero × 100. * P < 0.05, ** P < 0.01, *** P < 0.001 versus cells exposed to plasma from vehicle-treated mice. Error bars represent ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Together, these results suggest that LXR stimulation in vivo may cause the qualitative modifications of plasma lipoprotein profile that altered its efflux potential. Another possibility was that plasma of treated animals could contain sufficient concentrations of drug or an active metabolite to directly stimulate cells to release cholesterol. To rule out this possibility, we performed an experiment with Fu5AH, a cell model in which T0 plasma was more active than in controls, radiolabeling with [3H]cholesterol and exposing to 2.5% plasma from untreated or T0-treated mice for 4 h. Finally, lipid efflux was promoted to normal mouse plasma for 4 h. If plasma from T0-treated mice contained active compounds that directly affected cell efflux capacity, we would expect higher efflux from cells equilibrated in the presence of T0 plasma. On the contrary, in both conditions, we found the same amount of radiolabeled cholesterol in the cell medium (9.1 ± 0.8% vs. 8.9 ± 1.0%; n = 3), suggesting that 4 h of incubation with plasma did not directly affect cell function.Plasma capacity to stimulate lipid release through a specific mechanism has been shown to depend strictly on the presence of specific particles that act as cholesterol acceptors (26.Yancey P.G. Bortnick A.E. Kellner-Weibel G. de la Llera-Moya M. Phillips M.C. Rothblat G.H. Importance of different pathways of cellular cholesterol efflux.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 712-719Crossref PubMed Scopus (435) Google Scholar). Therefore, we evaluated the effect of T0 on lipoprotein plasma profile. For this purpose, fast-protein liquid chromatography and two-dimensional gel electrophoresis were carried out on samples of plasma obtained from untreated or T0-treated mice. Cholesterol and phospholipids were higher in all fractions of plasma from animals treated with 100 mg/kg/day T0 (Fig. 4), whereas triglycerides were decreased slightly (Fig. 4). Consistent lipid profiles were obtained using the lower dose of drug (10 mg/kg/day) (data not shown).Fig. 4.Elution profiles of untreated (closed circles) and T0-treated (open triangles) mouse plasma by fast-protein liquid chromatography. Mouse plasma (500 μl) was applied on a 30/10 Superose 6B column. The concentrations of cholesterol, triglycerides, and phospholipids in each fraction are indicated on the y axis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Two-dimensional gel electrophoresis revealed that HDLs from mice treated with T0 were larger and almost completely lacked the preβ-fraction compared with untreated mice (Fig. 5).Fig. 5.HDL subclasses in untreated and T0-treated mice. HDL subclasses were separated by two-dimensional electrophoresis and transferred onto a nitrocellulose membrane, on which lipoproteins were detected with an anti-apolipoprotein A-" @default.
• W1977432493 created "2016-06-24" @default.
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• W1977432493 date "2008-05-01" @default.
• W1977432493 modified "2023-10-16" @default.
• W1977432493 title "The LXR agonist T0901317 promotes the reverse cholesterol transport from macrophages by increasing plasma efflux potential" @default.
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