Title: Postprandial lipemia enhances the capacity of large HDL2 particles to mediate free cholesterol efflux via SR-BI and ABCG1 pathways in type IIB hyperlipidemia
Abstract: Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodeling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular free cholesterol (FC) efflux, cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer, and selective hepatic CE uptake during the postprandial phase in subjects displaying type IIB hyperlipidemia (n = 16). Postprandial, large HDL2 displayed an enhanced capacity to mediate FC efflux via both scavenger receptor class B type I (SR-BI)-dependent (+12%; P < 0.02) and ATP binding cassette transporter G1 (ABCG1)-dependent (+31%; P < 0.008) pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; P < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB lipoproteins and with attenuated capacity (−17%; P < 0.02) of total HDL to deliver CE to hepatic cells. Postprandial lipemia enhanced SR-BI and ABCG1-dependent efflux to large HDL2 particles. However, postprandial lipemia is equally associated with deleterious features by enhancing formation of CE-enriched, triglyceride-rich lipoprotein particles through the action of CETP and by reducing the direct return of HDL-CE to the liver. Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodeling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular free cholesterol (FC) efflux, cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer, and selective hepatic CE uptake during the postprandial phase in subjects displaying type IIB hyperlipidemia (n = 16). Postprandial, large HDL2 displayed an enhanced capacity to mediate FC efflux via both scavenger receptor class B type I (SR-BI)-dependent (+12%; P < 0.02) and ATP binding cassette transporter G1 (ABCG1)-dependent (+31%; P < 0.008) pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; P < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB lipoproteins and with attenuated capacity (−17%; P < 0.02) of total HDL to deliver CE to hepatic cells. Postprandial lipemia enhanced SR-BI and ABCG1-dependent efflux to large HDL2 particles. However, postprandial lipemia is equally associated with deleterious features by enhancing formation of CE-enriched, triglyceride-rich lipoprotein particles through the action of CETP and by reducing the direct return of HDL-CE to the liver. Elevated postprandial hypertriglyceridemia is a characteristic metabolic disorder associated with increased cardiovascular risk (1Bansal S. Buring J.E. Rifai N. Mora S. Sacks F.M. Ridker P.M. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women.JAMA. 2007; 298: 309-316Crossref PubMed Scopus (1244) Google Scholar). After ingestion of dietary fat, an increase in plasma triglyceridemia is observed, reflecting transient accumulation of plasma triglyceride-rich lipoproteins (TRL) of intestinal and hepatic origin, namely, apoB48-containing chylomicrons (CM), apoB100-containing very low density lipoprotein (VLDL), and their remnants. The systemic accumulation of these particles represents a pro-atherogenic consequence of the postprandial period, as they can penetrate the arterial intima at sites of endothelial dysfunction, with retention by the extracellular matrix, thereby contributing to cholesterol accumulation and plaque formation (2Williams K.J. Molecular processes that handle–and mishandle–dietary lipids.J. Clin. Invest. 2008; 118: 3247-3259Crossref PubMed Scopus (138) Google Scholar). During the postprandial phase, plasma cholesteryl ester transfer protein (CETP) activity is enhanced as a result of an increase in circulating cholesteryl ester (CE) acceptors and/or CETP concentrations (3Guerin M. Egger P. Soudant C. Le Goff W. van Tol A. Dupuis R. Chapman M.J. Cholesteryl ester flux from HDL to VLDL-1 is preferentially enhanced in type IIB hyperlipidemia in the postprandial state.J. Lipid Res. 2002; 43: 1652-1660Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4Tall A. Sammett D. Granot E. Mechanisms of enhanced cholesteryl ester transfer from high density lipoproteins to apolipoprotein B-containing lipoproteins during alimentary lipemia.J. Clin. Invest. 1986; 77: 1163-1172Crossref PubMed Scopus (156) Google Scholar). On a quantitative basis, both CM and large VLDL-1 particles represent the preferential acceptors of CE from HDL among TRL during the postprandial phase (5Lassel T.S. Guerin M. Auboiron S. Chapman M.J. Guy-Grand B. Preferential cholesteryl ester acceptors among triglyceride-rich lipoproteins during alimentary lipemia in normolipidemic subjects.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 65-74Crossref PubMed Scopus (47) Google Scholar). This process favors CE enrichment of TRL and equally involves enrichment of HDL particles in TG; as such, they become a substrate for hepatic lipase. Concomitantly, during LPL-mediated lipolysis of postprandial TRL, an excess of surface components containing apolipoproteins, unesterified cholesterol, and phospholipids is generated and sequesters to HDL, thereby increasing the total circulating HDL pool and enhancing the remodeling of small HDL3 into large CE-rich HDL2 particles (6Zilversmit D.B. Atherogenic nature of triglycerides, postprandial lipidemia, and triglyceride-rich remnant lipoproteins.Clin. Chem. 1995; 41: 153-158Crossref PubMed Scopus (237) Google Scholar). Plasma HDL particles exert potent anti-atherogenic effects, including cellular cholesterol efflux and antioxidative and anti-inflammatory activities (7Kontush A. Chapman M.J. Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis.Pharmacol. Rev. 2006; 58: 342-374Crossref PubMed Scopus (609) Google Scholar). Cellular efflux of cholesterol is a critical component of the reverse cholesterol transport (RCT) pathway, which involves the centripetal movement of free cholesterol from peripheral tissues, including the vessel wall, to the liver (8Sviridov D. Nestel P. Dynamics of reverse cholesterol transport: protection against atherosclerosis.Atherosclerosis. 2002; 161: 245-254Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 9von Eckardstein A. Nofer J.R. Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 13-27Crossref PubMed Scopus (646) Google Scholar). This process is frequently cited as the primary mechanism by which HDL protects against atherosclerosis and by which it may induce plaque regression. Cellular cholesterol efflux represents the exit of free cholesterol (FC) from membrane to lipid-free or lipid-poor apoAI via an energy-mediated process through ABCA1, a transporter belonging to the ATP binding cassette family (10Oram J.F. Lawn R.M. ABCA1. The gatekeeper for eliminating excess tissue cholesterol.J. Lipid Res. 2001; 42: 1173-1179Abstract Full Text Full Text PDF PubMed Google Scholar). FC efflux from the plasma membrane also occurs toward mature HDL through ABCG1, another member of the ABC family (11Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Crossref PubMed Scopus (887) Google Scholar), and equally via the scavenger receptor-BI (SR-BI) (12Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Crossref PubMed Scopus (173) Google Scholar). The contribution of these pathways to overall cholesterol efflux is function of their tissue distribution and acceptor affinity. Passive diffusion also contributes to FC desorption from the plasma membrane; recently new evidence has been presented to support a key role of this process (13Adorni M.P. Zimetti F. Billheimer J.T. Wang N. Rader D.J. Phillips M.C. Rothblat G.H. The roles of different pathways in the release of cholesterol from macrophages.J. Lipid Res. 2007; 48: 2453-2462Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 14Duong M. Collins H.L. Jin W. Zanotti I. Favari E. Rothblat G.H. Relative contributions of ABCA1 and SR-BI to cholesterol efflux to serum from fibroblasts and macrophages.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 541-547Crossref PubMed Scopus (93) Google Scholar). Cholesterol associated with mature α-HDL can be returned to the liver via at least three pathways. First, HDL particles can deliver cholesterol to the liver by direct interaction with specific hepatic receptors, primarily via the SR-BI receptor, by a selective uptake process and to a lesser degree, via the LDL receptor when HDL particles contain apoE (12Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Crossref PubMed Scopus (173) Google Scholar, 15Ji Y. Wang N. Ramakrishnan R. Sehayek E. Huszar D. Breslow J.L. Tall A.R. Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile.J. Biol. Chem. 1999; 274: 33398-33402Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Second, it has been proposed that holo-HDL particles might be potentially endocytosed by hepatocytes, although the precise identity of the specific receptor(s) involved remains controversial (7Kontush A. Chapman M.J. Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis.Pharmacol. Rev. 2006; 58: 342-374Crossref PubMed Scopus (609) Google Scholar). Third, the CE content of HDL particles can be transferred to apoB-containing lipoproteins through the action of CETP, with ultimate uptake by specific hepatic receptors. This third pathway is responsible for up to 50% of RCT in humans (16Lewis G.F. Rader D.J. New insights into the regulation of HDL metabolism and reverse cholesterol transport.Circ. Res. 2005; 96: 1221-1232Crossref PubMed Scopus (828) Google Scholar). Postprandial studies are frequently conducted in healthy subjects but rarely in patients displaying elevated cardiovascular risk (17Lairon D. Lopez-Miranda J. Williams C. Methodology for studying postprandial lipid metabolism.Eur. J. Clin. Nutr. 2007; 61: 1145-1161Crossref PubMed Scopus (106) Google Scholar). Transient elevation of triglyceridemia represents a key marker of postprandial response and reflects the occurrence and accumulation of TRL. The impact of postprandial lipoprotein metabolism on the RCT pathway remains to be elucidated. Indeed, postprandial metabolism is associated with an intense intravascular remodeling of all classes of plasma lipoprotein particles. Thus, the postprandial phase is associated with both quantitative and qualitative modification of HDL subspecies that may influence their anti-atherogenic activities. In this context, we have previously observed that cholesterol efflux capacity via SR-BI to serum was increased during the postprandial phase in both normolipidemic and hyperlipidemic subjects (3Guerin M. Egger P. Soudant C. Le Goff W. van Tol A. Dupuis R. Chapman M.J. Cholesteryl ester flux from HDL to VLDL-1 is preferentially enhanced in type IIB hyperlipidemia in the postprandial state.J. Lipid Res. 2002; 43: 1652-1660Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Here we report that postprandial lipemia is associated with quantitative and qualitative modifications of large HDL particles that increase the capacity of these particles to mediate cholesterol efflux via both SR-BI and ABCG1 pathways but, conversely, reduce delivery of cholesteryl esters to hepatic cells. Sixteen males between 36 and 59 years of age (mean, 46 ± 7 years), who displayed a combined hyperlipidemia typical of the type IIB lipid phenotype with concomitant elevation of circulating levels of cholesterol (251 ± 7 mg/dl) and triglycerides (179 ± 14 mg/dl), were selected for the study. Subjects were on average overweight (body mass index, 27 ± 2 kg/m2), but none was obese or displayed the apolipoprotein E2/E2 genotype. Patients were excluded if they presented with diabetes mellitus, secondary causes of hyperlipimia, uncontrolled hypertension, or a history of major cardiovascular events. All patients were nonsmokers. They were without any lipid-lowering therapy, and their diet was stabilized (AHA step one diet or equivalent) during a six-week period before inclusion in the study. For each subject, a postprandial time course was performed following consumption of a solid mixed meal that provided a total of 1,200 kcal (14% protein, 38% carbohydrate, and 48% fat) as previously described (5Lassel T.S. Guerin M. Auboiron S. Chapman M.J. Guy-Grand B. Preferential cholesteryl ester acceptors among triglyceride-rich lipoproteins during alimentary lipemia in normolipidemic subjects.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 65-74Crossref PubMed Scopus (47) Google Scholar). Subjects were asked to abstain from alcohol and vigorous exercise for 24 h before the day of the test. Blood samples were obtained immediately before the test meal and at 2 h, 4 h, and 8 h after ingestion. Blood was collected by venipuncture from the antecubital vein into sterile EDTA-containing tubes (final concentration of EDTA, 1 mg/ml); plasma was separated immediately by low-speed centrifugation (2500 rpm) for 20 min at 4°C. The study was performed in accordance with the ethical principles set forth in the Helsinki Declaration. The study protocol and amendment were reviewed and approved by an ethics committee and met national institutional requirements. Written informed consent was obtained from all patients. Chylomicrons (Sf > 400) were isolated by centrifugation at 20,000 rpm for 45 min at 15°C using a SW41 Ti rotor in a Beckman XL70 ultracentrifuge (3Guerin M. Egger P. Soudant C. Le Goff W. van Tol A. Dupuis R. Chapman M.J. Cholesteryl ester flux from HDL to VLDL-1 is preferentially enhanced in type IIB hyperlipidemia in the postprandial state.J. Lipid Res. 2002; 43: 1652-1660Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Subfractions of triglyceride-rich lipoproteins [i.e., VLDL1 (Sf 60–400) and VLDL-2 (Sf 20–60)] were isolated from chylomicron-free plasma by nonequilibrium density gradient ultracentrifugation as previously described (18Guerin M. Le Goff W. Duchene E. Julia Z. Nguyen T. Thuren T. Shear C.L. Chapman M.J. Inhibition of CETP by torcetrapib attenuates the atherogenicity of postprandial TG-rich lipoproteins in type IIB hyperlipidemia.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 148-154Crossref PubMed Scopus (36) Google Scholar). HDL subfractions were isolated from chylomicron-free plasma by isopycnic density gradient ultracentrifugation (19Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.J. Lipid Res. 1981; 22: 339-358Abstract Full Text PDF PubMed Google Scholar). The quantification of preβ-HDL in plasma was performed as previously described (20Mweva S. Paul J.L. Cambillau M. Goudouneche D. Beaune P. Simon A. Fournier N. Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity.Eur. J. Clin. Invest. 2006; 36: 552-559Crossref PubMed Scopus (24) Google Scholar) The relative abundance of the human apoAI among the α- or preβ-HDL species was determined by scanning reflectance densitometry (Quantity One software; Bio-Rad). The amount of preβ-HDL was expressed as the percentage of total apoAI (% apoAI, relative concentration) and as absolute concentration (mg/l apoAI) by multiplying its percentage by plasma apoAI levels. The lipid contents of plasma and isolated lipoprotein fractions, total protein, and apoAI were quantified with an Autoanalyzer (Konelab 20). Reagent kits from Roche Diagnostics and ThermoElectron were used for determination of total cholesterol and triglyceride levels, respectively. The levels of unesterified cholesterol and phospholipids were determined with commercial reagent kits (Wako Diagnostics). Cholesteryl ester mass, calculated as (TC − FC) × 1.67, represents the sum of the esterified cholesterol and fatty acid moieties (19Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.J. Lipid Res. 1981; 22: 339-358Abstract Full Text PDF PubMed Google Scholar). The Bicinchoninic acid assay reagent (Pierce) was used for protein quantification. ApoAI was determined using immunoturbidimetric assays (ThermoElectron reagents and calibrators). Determination of endogenous CE transfer from HDL to apolipoprotein B-containing lipoproteins was assayed by modification of the method of Guerin et al. as previously described (21Catalano G. Duchene E. Julia Z. Le Goff W. Bruckert E. Chapman M.J. Guerin M. Cellular SR-BI and ABCA1-mediated cholesterol efflux are gender-specific in healthy subjects.J. Lipid Res. 2008; 49: 635-643Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies. Identification of preferential cholesteryl ester acceptors in human plasma.Arterioscler. Thromb. 1994; 14: 199-206Crossref PubMed Google Scholar). Cholesteryl ester transfer was determined after incubation of whole plasma (500 µl) from individual subjects at 37°C or 0°C for 3 h in the presence of radiolabeled HDL (25 µg HDL-CE) and iodoacetamide (final concentration, 1.5 mmol/l) for inhibition of lecithin cholesterol acyltransferase (LCAT). Following incubation, plasma lipoproteins were fractionated by isopycnic density gradient ultracentrifugation, and the radioactive content of each isolated lipoprotein fraction was quantified by liquid scintillation spectrometry with a Trilux 1450 (Perkin Elmer). The CETP-dependent CE transfer was calculated from the difference between the radioactivity transferred at 37°C and 0°C. The rate of CE transfer was calculated from the known specific radioactivity of radiolabeled HDL-CE and expressed as μg CE transferred/h/ml plasma (22Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies. Identification of preferential cholesteryl ester acceptors in human plasma.Arterioscler. Thromb. 1994; 14: 199-206Crossref PubMed Google Scholar). Fu5AH cells were maintained in Eagle's MEM containing 5% new-born calf serum, Raw264.7 cells were maintained in DMEM supplemented with 10% fetal bovine serum, and CHO-K1 cells (wild-type and hABCG1, mouse ABCA1 or Cla1 transfected cells) were maintained in Ham's F-12 medium containing 10% fetal bovine serum. All media were supplemented with 1% L-glutamine and 0.75% penicillin-streptomycin. Lipid efflux assays using Fu5AH, Raw264.7, CHO-K1, and CHO-hABCG1, CHO-mouse ABCA1, or CHO-Cla1 cells were performed as described previously (21Catalano G. Duchene E. Julia Z. Le Goff W. Bruckert E. Chapman M.J. Guerin M. Cellular SR-BI and ABCA1-mediated cholesterol efflux are gender-specific in healthy subjects.J. Lipid Res. 2008; 49: 635-643Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 23Catalano G. Julia Z. Frisdal E. Vedie B. Fournier N. Le Goff W. Chapman M.J. Guerin M. Torcetrapib differentially modulates the biological activities of HDL2 and HDL3 particles in the reverse cholesterol transport pathway.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 268-275Crossref PubMed Scopus (58) Google Scholar). It has been previously demonstrated that Fu5AH cells highly express SR-BI and that such expression correlates with the rate of HDL-mediated cellular free cholesterol efflux (24Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux.J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). We recently confirmed these observations (25Dreux M. Dao Thi V.L. Fresquet J. Guérin M. Julia Z. Verney G. Durantel D. Zoulim F. Lavillette D. Cosset F.L. et al.Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.PLoS Pathog. 2009; 5: e1000310Crossref PubMed Scopus (101) Google Scholar). Equally, in Raw264.7 cells, the expression of ABCA1 is induced by up to 70-fold by stimulation with 8Br cAMP and leads to a concomitant induction of cellular free cholesterol efflux to apoAI (26Zheng P. Horwitz A. Waelde C.A. Smith J.D. Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein AI and HDL.Biochim. Biophys. Acta. 2001; 1534: 121-128Crossref PubMed Scopus (23) Google Scholar). After plating, cells were labeled by incubation with [3H]cholesterol (1 μ Ci/ml) in culture medium for 48 h. Subsequently, Fu5AH cells were incubated for 24 h in serum-free medium supplemented with BSA (0.5%). After equilibration, cholesterol acceptors (2.5% diluted plasma or 10 μg phospholipid/ml of isolated HDL subfractions) were added in serum-free medium and incubated with cells for 4 h at 37°C. The day after cell plating, cells were loaded and labeled with acetylated LDL (50 μg/ml) and 0.5 μ Ci/ml [3H]cholesterol for 24 h in serum-free DMEM containing glucose (4.5 g/l) and BSA (0.2%) (DGGB, DMEM-Glutamine-Glucose-BSA). After incubation, Raw264.7 cells were incubated with DGGB in the absence or presence of cAMP (0.3 mM) for 16 h to induce ABCA1 expression. Cholesterol acceptors (2.5% diluted plasma) were added to Raw264.7 cells in serum-free DMEM for 4 h at 37°C in the presence or absence of 0.3 mM 8-Br cAMP. The ABCA1-dependent efflux was calculated as the difference between fractional cholesterol efflux to cells in the presence or absence of 8-Br cAMP. Two days after plating, cellular cholesterol was labeled by incubation of cells with culture medium and 1 μ Ci/ml [3H]cholesterol for 24 h. Equilibration of the label was performed for 90 min in serum-free medium and BSA (0.1%). After equilibration of labeling, acceptors (2.5% diluted plasma, 2.5% diluted apoB-depleted plasma or 5 μg PL/ml of isolated HDL subfractions) were added to the cells in serum-free medium containing BSA (0.1%) for 4 h at 37°C. The ABCG1-dependent efflux was calculated as the difference between efflux to hABCG1-transfected CHO-K1 cells and efflux to wild-type CHO-K1 cells. The ABCA1-dependent efflux was calculated as the difference between efflux to mouse ABCA1-transfected CHO-K1 cells and efflux to wild-type CHO-K1 cells. The Cla1-dependent efflux was calculated as the difference between efflux to Cla1-transfected CHO-K1 cells and efflux to wild-type CHO-K1 cells. All efflux experiments were performed in triplicate for each sample. Fractional cholesterol efflux was calculated as the amount of the label recovered in the medium divided by the total label in each well (radioactivity in the medium plus radioactivity in the cells). The background cholesterol efflux obtained in the absence of any acceptor was subtracted from the efflux values obtained with the test samples. The capacity of HDL subfractions, HDL2 or HDL3, to mediate free cholesterol efflux was expressed as % of cholesterol efflux per mole of acceptor particle. Molar concentration of HDL particle was calculated as the sum of molar concentration of individual lipid and protein as previously described (27Chancharme L. Therond P. Nigon F. Lepage S. Couturier M. Chapman M.J. Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 810-820Crossref PubMed Scopus (61) Google Scholar, 28Nobecourt E. Jacqueminet S. Hansel B. Chantepie S. Grimaldi A. Chapman M.J. Kontush A. Defective antioxidative activity of small dense HDL3 particles in type 2 diabetes: relationship to elevated oxidative stress and hyperglycaemia.Diabetologia. 2005; 48: 529-538Crossref PubMed Scopus (178) Google Scholar). The protein moiety was considered to consist of two apolipoproteins, apoAI and apoAII, and the molecular weight of the protein moiety in each HDL subfraction was calculated using mass content of apoAI and apoAII converted to molarity (29Chapman M.J. Comparative analysis of mammalian plasma lipoproteins.Methods Enzymol. 1986; 128 (Review): 70-143Crossref PubMed Scopus (270) Google Scholar, 30Kontush A. Chantepie S. Chapman M.J. Small, dense HDL particles exert potent protection of atherogenic LDL against oxidative stress.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1881-1888Crossref PubMed Scopus (349) Google Scholar). Optimal plasma dilutions of HDL-PL concentrations were determined on the basis of dose response curves for the release of free cholesterol from each cellular model as previously described (23Catalano G. Julia Z. Frisdal E. Vedie B. Fournier N. Le Goff W. Chapman M.J. Guerin M. Torcetrapib differentially modulates the biological activities of HDL2 and HDL3 particles in the reverse cholesterol transport pathway.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 268-275Crossref PubMed Scopus (58) Google Scholar). In addition, we used the release of labeled cellular cholesterol to quantify efflux; this approach has been shown to accurately reflect net mass transfer of cholesterol from cells to extracellular acceptors under our experimental conditions (23Catalano G. Julia Z. Frisdal E. Vedie B. Fournier N. Le Goff W. Chapman M.J. Guerin M. Torcetrapib differentially modulates the biological activities of HDL2 and HDL3 particles in the reverse cholesterol transport pathway.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 268-275Crossref PubMed Scopus (58) Google Scholar). In vitro selective HDL-CE uptake was performed as previously described (31Le Goff W. Settle M. Greene D.J. Morton R.E. Smith J.D. Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis.J. Lipid Res. 2006; 47: 51-58Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). HepG2 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine, and 0.75% penicillin-streptomycin. HepG2 or Fu5AH cells were plated in 24-well tissue culture plates (106 cells/well). Two days after plating, cells were washed three times with PBS and once with serum-free medium. Cells were subsequently incubated in the presence of 3H-CE labeled HDL (60 µg protein/ml) diluted in serum-free medium at 37°C for 5 h. At the end of incubation, the medium was removed, and cells were washed four times with PBS and incubated in the presence of an excess of unlabeled HDL (100 µg protein/ml) for 30 min. Cells were then washed four times with PBS and solubilized with 200 µl of NaOH 0.2N for 15 min at room temperature with gentle mixing. The protein content in each well was determined. The radioactive content of 100 µl of each cell lysate was measured by liquid scintillation counting. Selective uptake was calculated from the known specific radioactivity of radiolabeled HDL-CE and expressed in µg HDL-CE/µg cell protein. Experimental data were analyzed using the SAS software (SAS/STAT User's Guide, Version 8; SAS Institute Inc., Cary, NC). Postprandial lipemia was quantified by calculating the area under the curve (AUC) and the incremental AUC (iAUC) for plasma triglyceride and lipoprotein subfractions. The incremental AUC represents the increase in area in response to the test meal relative to lipid or lipoprotein concentrations determined before meal intake. Repeated-measure ANOVA was performed to assess changes in plasma lipid levels, lipoprotein concentrations, CETP activity, cellular free cholesterol efflux, and in HDL-CE, selective uptake during the postprandial phase. Results were considered statistically significant at P < 0.05. Values are given as means ± SEM. Following consumption of the solid mixed meal, postprandial hypertriglyceridemia reached a peak (+90%; P < 0.0001) 4 h after meal intake (178.8 ± 14.4 mg/dl and 341.2 ± 26.4 mg/dl before and 4 h after meal intake, respectively) (supplementary Fig. I). Postprandial variations in plasma TG levels reflected transient accumulation of circulating triglyceride-rich lipoproteins, mainly in the form of plasma chylomicrons-TG (32.6 ± 4.8 mg/dl and 162.3 ± 19.0 mg/dl before and 4 h after meal intake, respectively; P < 0.0001) and VLDL-1-TG (76.5 ± 7.1 mg/dl and 109.0 ± 8.6 mg/dl before and 4 h after meal intake, respectively; P < 0.0001). Interestingly, 4 h postprandially, we observed a significant reduction (178.5 ± 6.5 mg/dl and 142.1 ± 8.7 mg/dl before and 4 h after