Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

Transcription

1 Moderte lcohol consumption increses cholesterol efflux medited by ABCA1 J. W. J. Beulens,*, A. Sierksm,* A. vn Tol, N. Fournier,** T. vn Gent, J-L. Pul,** nd H. F. J. Hendriks 1, * Netherlnds Orgniztion for Applied Scientific Reserch (TNO), Nutrition nd Food Reserch,* Zeist, The Netherlnds; Wgeningen University, Wgeningen, The Netherlnds; Deprtments of Biochemistry nd Cell Biology & Genetics, Ersmus University Medicl Center, Rotterdm, The Netherlnds; nd Lbortoire de Biochimie,** Fculté des Sciences Phrmceutiques, Châteny-Mlbry, Frnce Abstrct Moderte lcohol consumption increses HDL cholesterol, which is involved in reverse cholesterol trnsport (RCT). The im of this study ws to investigte the effect of moderte lcohol consumption on cholesterol efflux, using J774 mouse mcrophges nd Fu5AH cells, nd on other prmeters in the RCT pthwy. Twenty-three helthy men (45 65 yers) prticipted in rndomized, prtilly diet-controlled, crossover tril. They consumed four glsses of whisky (40 g of lcohol) or wter dily for 17 dys. After 17 dys of whisky consumption, serum cpcity to induce ABCA1-dependent cholesterol efflux from J774 mouse mcrophges ws incresed by 17.5% (P 0.027) compred with wter consumption. Plsm cpcity to induce cholesterol efflux from Fu5AH cells incresed by 4.6% (P 0.002). Pre -HDL, polipoprotein A-I (poa-i), nd lipoprotein A-I:A-II lso incresed by 31.6, 6.2, nd 5.7% (P 0.05), respectively, fter whisky consumption compred with wter consumption. Chnges of camp-stimulted cholesterol efflux correlted (r 0.65, P 0.05) with chnges of poa-i but not with chnges of pre -HDL (r 0.30, P 0.18). Cholesterol efflux cpcities from serum of len men were higher thn those from overweight men. In conclusion, this study shows tht moderte lcohol consumption increses the cpcity of serum to induce cholesterol efflux from J774 mouse mcrophges, which my be medited by ABCA1. Beulens, J. W. J., A. Sierksm, A. vn Tol, N. Fournier, T. vn Gent, J-L. Pul, nd H. F. J. Hendriks. Moderte lcohol consumption increses cholesterol efflux medited by ABCA1. J. Lipid Res : Supplementry key words ATP binding cssette trnsporter 1 reverse cholesterol trnsport J774 mcrophges Fu5AH cells pre high density lipoprotein body mss index Atherosclerosis is condition of the mjor rteries leding to crdiovsculr disese (CVD), in which progressive occlusion of the rteries occurs by the formtion of therosclerotic lesions. A mjor event in this process is the Mnuscript received 18 Mrch 2004 nd in revised form 18 My Published, JLR Ppers in Press, July 1, DOI /jlr.M JLR200 differentition of monocytes to mcrophges tht ccumulte lipoprotein-derived cholesterol to form fom cells (1). Epidemiologic studies hve shown tht moderte lcohol consumption is ssocited with decresed risk of CVD (2). Severl mechnisms, such s reduction in blood cogultion nd fibrinolysis, my be involved in the protective effect of moderte lcohol consumption on CVD (3, 4). However, more thn 50% of the protective ction of lcohol consumption is medited by n increse of HDL cholesterol (HDL-C) (5, 6). In previous studies, we hve shown tht moderte lcohol consumption results in n incresed blood HDL-C concentrtion (7 9). The functionl consequences of this increse in HDL-C concentrtion re not fully understood, but one of the protective ctions my involve n increse of proxonse ctivity, n HDL-ssocited enzyme tht my protect LDL ginst oxidtion (8, 10, 11). The role of HDL in reverse cholesterol trnsport (RCT) is nother proposed mechnism for these protective effects. RCT consists of the following steps: efflux of free cholesterol (FC) from peripherl tissue, esterifiction of FC by plsm LCAT, nd incorportion of cholesteryl esters (CEs) in the HDL prticle. HDL-CEs re clered from plsm by the liver for ctbolism vi severl pthwys (12). The first step in RCT, cholesterol efflux, cn be medited by three mechnisms (13, 14). The first mechnism is queous diffusion, in which cholesterol molecules desorb from the plsm membrne to be cptured by cceptors such s mture HDL prticles. The second mech- Abbrevitions: poa-i, polipoprotein A-I; BMI, body mss index; CE, cholesteryl ester; CETP, cholesteryl ester trnsfer protein; CVD, crdiovsculr disese; FC, free cholesterol; HDL-C, HDL cholesterol; LpA-I, lipoprotein A-I; PL, phospholipid; PLTP, phospholipid trnsfer protein; RCT, reverse cholesterol trnsport; SR-BI, scvenger receptor clss B type I; TC, totl cholesterol; TG, tricylglycerol. 1 To whom correspondence should be ddressed. e-mil: hendriks@voeding.tno.nl Copyright 2004 by the Americn Society for Biochemistry nd Moleculr Biology, Inc Journl of Lipid Reserch Volume 45, 2004 This rticle is vilble online t

2 nism of efflux involves the scvenger receptor clss B type I (SR-BI). SR-BI medites cellulr cholesterol efflux to mture HDL prticles in ddition to its role in selective lipid uptke (13, 14). In one of our previous studies (15), we showed tht moderte lcohol consumption incresed plsm cpcity to induce cholesterol efflux from Fu5AH rt heptom cells, which hve high levels of SR-BI. The third mechnism of cholesterol efflux involves the relese of FC to lipid-free or lipid-poor polipoproteins, prticulrly polipoprotein A-I (poa-i) nd pre -HDL, which is medited by ABCA1. This lipid efflux is the first stge of HDL biogenesis. Subsequently, FC is esterified by LCAT nd mture, sphericl -HDL prticles re formed (13). Efficient cholesterol efflux from mcrophges is criticl for the prevention of fom cell formtion, nd incresed cellulr ABCA1 levels my thus protect ginst therosclerosis (16). To the best of our knowledge, the effect of moderte lcohol consumption on cholesterol efflux medited by the ABCA1 receptor hs never been investigted. Therefore, we hve used the recently vlidted model of ABCA1-expressing J774 mcrophges (17) to study the effect of moderte lcohol intke on serum cholesterol efflux cpcity. SUBJECTS AND METHODS Subjects Twenty-four mle subjects ged yers, ll pprently helthy nd nonsmoking, were recruited from the Netherlnds Orgniztion for Applied Scientific Reserch (TNO) BIBRA Interntionl (Crshlton, Surrey, UK) dtbse of helthy humn volunteers, by dvertising in locl newsppers, nd by leflet drops to locl residentil res. A questionnire (self-report) ws used for informtion on lcohol intke, medicl history, nd fmily history of lcoholism. The questionnire ws checked by medicl investigtor during n interview with the volunteer, nd subsequently physicl exmintion ws performed. Subjects were considered helthy bsed on the vlues of the prestudy lbortory tests, their medicl history, nd the physicl exmintion. Subjects met the following inclusion criteri: consumption of between 10 nd 28 lcohol-contining beverges weekly, body mss index (BMI) between 20 nd 35 kg/m 2, nd no fmily history of lcoholism. A wide rnge of BMIs ws chosen to investigte whether the effect of moderte lcohol consumption on the outcome mesures is modified by level of obesity. The study ws conducted in ccordnce with the Declrtion of Helsinki South Afric Revision 1996 nd the Interntionl Conference of Hrmonistion Hrmonized Triprtite Guideline for Good Clinicl Prctice. Approvl to proceed with the study ws given by n independent medicl ethics committee, nd ll subjects provided written informed consent before prticiption. Study design The subjects entered rndomized, prtilly diet-controlled, crossover tril consisting of two periods of 17 dys. A rndom smple of 12 men were llocted to the sequence of consuming whisky (Fmous Grouse Scotch Whisky, 40 vol% lcohol) during dinner in the first period followed by drinking tp wter (control beverge) during dinner in the second period. The other 12 men consumed wter first, followed by whisky. The prticipnts nd stff dministering the protocol were not blinded to the tretment sequence. Four glsses (125 ml in totl) of ech beverge were consumed dily during dinner t TNO BIBRA. One glss ws tken before dinner, two glsses during dinner, nd one glss fter dinner. During the whisky period, lcohol intke equled 40 g/dy. The dily dinner contined 4,200 kj nd consisted of 21% of energy from protein, 38% of energy from ft, nd 41% of energy from crbohydrte. The menu consisted of 4-dy rottion. Dinner ws prepred ech dy by locl cterer using the sme source of ingredients throughout. Ech period ws preceded by 3 lcohol-free dys to prevent crryover effects. Subjects were sked to continue their norml eting hbits (except for the provided dinner) nd crry on with their norml everydy ctivities. Complince with the protocol ws checked by dily questionnire. Body weight ws mesured on the first nd lst dys of ech tretment period with the subjects wering indoor clothing, without shoes, wllet, nd keys. An lcohol breth test (Alcoholtest 7410; Dräger Nederlnd, Zoetermeer, The Netherlnds) ws performed during the whisky tretment to ensure tht subjects left the institute sfely. At the end of ech tretment period, fsting blood smples were collected in the morning. Blood ws tken from n ntecubitl vein nd collected in tube contining lithium-heprin to obtin plsm nd in tube contining gel nd clot ctivtor to obtin serum (Vcutiner Systems; Becton Dickinson, Plymouth, UK). To obtin plsm, the blood ws centrifuged for 20 min t 2,000 g nd 4 C between 15 nd 30 min fter collection. To obtin serum, the blood ws centrifuged for 15 min t 2,000 g nd 4 C between 15 nd 30 min fter collection. The plsm nd serum smples were stored t 80 C until nlysis. HDL ws seprted from the pob frction by precipittion of the ltter with polyethylene glycol. Serum totl nd HDL-C (cholesterol oxidse/peroxidse-midopyrine method) nd tricylglycerols (TGs; glycerol phosphte oxidse/peroxidse-midopyrine method), s well s unesterified cholesterol, TG, nd phospholipids (PLs) in HDL, were nlyzed enzymticlly. The coefficients of vrition within runs were 0.8% for totl cholesterol (TC), 1.5% for TG, nd 1.3% for HDL-C. CEs were clculted s the difference between HDL-FC nd totl HDL-C. LDL-C ws clculted by the eqution of Friedewld, Levy, nd Fredrickson (18). ApoA-I ws nlyzed in serum ccording to commercil nephelometric ssy on the Cobs Mir S (Roche). Lipoprotein A-I (LpA-I; contining poa-i but not poa-ii) concentrtions were determined by double rocket immunoelectrophoresis using the ssy kit provided by Sebi (Benelux N.V., Brussels, Belgium), ccording to the instructions of the mnufcturer. The stined nd dried gels were scnned nd rocket heights were determined. A stndrd curve ws mde bsed on the liner reltionship between rocket height nd concentrtion. LpA-I concentrtion ws determined s function of rocket height, wheres LpA-I:A-II ws clculted by subtrction (totl plsm poa-i minus LpA-I). Quntifiction of pre -HDL by crossed immunoelectrophoresis The crossed immunoelectrophoresis consisted of grose electrophoresis in the first dimension for seprtion of lipoproteins with pre nd mobility. Antigen migrtion from the first grose gel onto the second grose gel, contining got nti-humn poa-i ntiserum (3%, v/v), ws used to quntittively precipitte poa-i. The ntiserum ws monospecific for humn poa-i. Lipoprotein electrophoresis ws crried out on 1% (w/v) grose gels in brbitl buffer (50 mmol/l, ph 8.6). Plsm ws pplied t 3 l/well nd run in n LKB 2117 system (4 C for 2 h, 250 V for the first dimension). The trck of the first grose gel Beulens et l. Alcohol consumption nd cellulr cholesterol efflux 1717

3 ws excised nd nneled with melted grose to gel contining 3% (v/v) got nti-humn poa-i ntiserum tht ws cst on GelBond film (Phrmci). The plte ws run in n LKB 2117 system (4 C for 20 h, 50 V) in brbitl buffer. Unrected ntibody ws removed by extensive wshing in phosphte-buffered sline. The gel ws stined with Coomssie brillint blue R250 nd subsequently dried. Ares under the pre -HDL nd -HDL peks were scnned nd clculted using Scion softwre. The pre -HDL re cn be expressed s the percentge of the sum of the pre -HDL nd the -HDL res. The pre -HDL concentrtions re given in bsolute mounts (milligrms of poa-i present in pre -HDL per milliliter of plsm). These vlues were clculted from the percentge of poa-i present in pre -HDL nd the totl plsm poa-i concentrtion. Plsm cholesteryl ester trnsfer protein, LCAT, nd phospholipid trnsfer protein ctivity Plsm cholesteryl ester trnsfer protein (CETP) ctivity ws determined fter removl of VLDL nd LDL from ech smple s described previously (19, 20). The isotope ssy mesures the trnsfer of [1-14 C-olete]CE from lbeled LDL to n excess of unlbeled norml HDL. LCAT ws inhibited with dithiobis-2- nitrobenzoic cid. CETP ctivity ws clculted s the bidirectionl trnsfer between lbeled LDL nd HDL. There is strong correltion between the mesured CETP ctivities nd CETP mss (21). Plsm LCAT ctivity ws mesured using excess exogenous substrte s described (22). Plsm phospholipid trnsfer protein (PLTP) ctivity ws lso mesured with exogenous substrtes (20). Plsm smples were incubted with [ 3 H]phosphtidylcholine-lbeled liposomes nd n excess of HDL. Therefter, the liposomes were precipitted with mixture of NCl, MgCl 2, nd heprin (finl concentrtions of 230 mmol/l, 92 mmol/l, nd 200 U/ml, respectively). This method is not ffected by the PL trnsfer-stimulting ction of CETP (20). The mesured CETP, LCAT, nd PLTP ctivities re linerly relted to the mount of plsm used in ll incubtions. The plsm CETP, LCAT, nd PLTP ctivities obtined with these methods reflect the ctive mount of these proteins in plsm nd re not influenced by the endogenous plsm lipoproteins. For ech ssy, ll plsm smples were nlyzed in one run using the sme btch of substrtes. Plsm CETP, LCAT, nd PLTP ctivities were relted to reference pool of humn plsm obtined by mixing equl mounts of plsm, isolted t 4 C, from 250 helthy blood donors. All ctivities re expressed in rbitrry units, corresponding to the percentges of the respective ctivities in the reference pool plsm; 100% is equivlent to the following ctivities: 216 nmol/ml/h for CETP, 65 nmol/ml/h for LCAT, nd 13.9 mol/ml/h for PLTP. The within-ssy coefficients of vrition for the ssys of plsm CETP, LCAT, nd PLTP ctivities mount to 2.7, 4.5, nd 3.5%, respectively. Cholesterol efflux from J774 cells Cholesterol efflux from J774 mouse mcrophge cells ws determined by the use of previously vlidted modifiction (17, 23, 24) of the generl technique described by Skr et l. (25). The current experiment is bsed on previous tests using normolipidemic humn serum to obtin n optiml dilution for serum nd ws crried out strictly ccording to the experimentl conditions described by Fournier et l. (17). Briefly, serum smples diluted to 1% were incubted t 37 C with the [ 3 H]cholesterollbeled cells pretreted for h with or without 0.3 mmol/l 8-(4-chlorophenylthio) camp (Sigm). To prevent intrcellulr cholesterol esterifiction, the ACAT inhibitor GW 447C88 ws dded into the medium during the lbeling period nd ll subsequent stges of the experiment. After 4 h of incubtion, the rdioctivity relesed to the medium ws expressed s the frction of the totl rdioctive cholesterol present in the well. The percentge of ABCA1-medited FC efflux ws clculted s the percentge of FC efflux from cells upregulted with camp minus the percentge of FC efflux from control J774 cells. This clcultion controls for the contribution of FC efflux from the queous diffusion mechnism nd yields dt tht re specific for the contribution of ABCA1 (ABCA1-dependent cholesterol efflux). All determintions were mde in triplicte. A stndrd pool of humn serum ws used to ssess the dily vrition in efflux experiments. The efflux vlues obtined with the stndrd pool of ll experiments re verged, nd this serves s the 100% vlue. Ech vlue of the experiments with the stndrd pool is clculted s percentge of the overll verge, nd this percentge is pplied to correct ll test vlues for tht dy. Cholesterol efflux from Fu5AH cells The cpcity of plsm to induce cholesterol efflux from Fu5AH cells ws mesured s described by de l Ller Moy, et l. (26). In short, Fu5AH cells were grown to confluency in the presence of [ 3 H]cholesterol. After removl of medium contining the lbeled cholesterol, the cells were llowed to equilibrte for 24 h. Subsequently, cholesterol efflux ws mesured in triplicte over 4 h in the presence of 20-fold-diluted plsm smples. Cholesterol efflux (rdiolbel present in the culture medium fter 4 h) is expressed s percentge of the rdioctivity initilly present in the cells (frctionl efflux). Dt were corrected for blnks, being the mount of lbel in the medium fter 4 h in the bsence of plsm. The within-ssy coefficient of vrition of the cholesterol efflux ssy is 5.5%. The frctionl cholesterol efflux using Fu5AH cells is minly ttributed to SR-BI, becuse this cell line hs high expression of SR-BI (27). Sttisticl nlysis Dt nlysis ws performed using the SAS sttisticl softwre pckge (SAS/STAT version 6.12; SAS Institute, Cry, NC). Dt were tested for normlity using the Shpiro-Wilk test nd by visully inspecting normlity plots. Tretment effects were evluted by ANOVA using the mixed-model procedure. The fctors tretment, moment, tretment order, BMI, nd the interction term of tretment nd BMI were included in the model. The effect of BMI on the outcome mesures ws obtined from the BMI fctor in the ANOVA model. Correltion coefficients to ssess the ssocition between (reltive chnges) outcome mesures were clculted ccording to Person or Spermn if vribles were not distributed normlly. Two-sided probbility vlues were considered sttisticlly significnt t P RESULTS Subjects Of the 24 subjects included in the study, 1 subject withdrew from the study t dy 21 for reson not relted to tretment. Therefore, only 23 subjects were included in the dt nlysis of this study. Chrcteristics of these subjects re shown in Tble 1. Averge body weight did not differ between the whisky nd wter tretment periods. The men breth lcohol concentrtion t 1 h fter dinner with whisky ws 0.43 g/l (rnge, g/l). Cholesterol efflux cpcity Tretment effects on the cholesterol efflux cpcity of serum or plsm re shown in Tble 2. Cholesterol efflux cpcity from Fu5AH cells ws incresed by 4.6% (P 1718 Journl of Lipid Reserch Volume 45, 2004

4 TABLE 1. Chrcteristic Chrcteristics of the volunteers included in the dt nlysis (n 23) Vlue Age (yers) 52 (5) [45 65] Body weight (kg) 81.4 (11.5) [ ] BMI (kg/m 2 ) 26.7 (3.0) [ ] Hemoglobin (mmol/l) 8.8 (0.6) [ ] TG (mmol/l) 1.2 (0.7) [ ] TC (mmol/l) 5.6 (0.9) [ ] HDL-C (mmol/l) 1.4 (0.4) [ ] LDL-C (mmol/l) 3.6 (0.7) [ ] Alkline phosphtse (U/l) 56 (13) [39 88] Asprgine minotrnsferse (U/l) 25 (5) [18 41] Alnine minotrnsferse (U/l) 28 (10) [14 51] -Glutmyltrnsferse (U/l) 26 (12) [13 49] BMI, body mss index; HDL-C nd LDL-C, HDL nd LDL cholesterol; TC, totl cholesterol; TG, tricylglycerol. Dt re expressed s mens, (SD), nd [rnge] ) fter whisky consumption, nd cholesterol efflux cpcity from J774 cells without stimultion of camp lso incresed fter whisky consumption by 3.3% (P 0.014). After stimultion with camp, cholesterol efflux cpcity using J774 cells ws incresed by pproximtely twice s much (6.3%; P 0.001) fter consumption of whisky compred with wter. This resulted in 17.5% (P 0.027) increse fter consumption of whisky compred with wter of the ABCA1-dependent cholesterol efflux in J774 cells. Individul serum ABCA1-dependent cholesterol efflux cpcities re shown in Fig. 1. Blood lipid nd lipoprotein profiles nd relted fctors Tretment effects on serum lipid profiles nd relted fctors re shown in Tble 3. A significnt tretment effect on HDL-C ws found, indicting 7.4% increse of HDL-C fter whisky consumption. The HDL-PL frction incresed by 9.3% (P ) fter whisky consumption compred with wter consumption. No tretment effects on TC, LDL-C, VLDL-C, nd TG were found (Tble 3). Pre -HDL incresed by 31.6% (P 0.026), serum poa-i concentrtion incresed by 6.2% (P 0.001), nd concentrtion of LpA-I:A-II incresed by 6.0% (P 0.015). A borderline significnt (P 0.069) effect of moderte lcohol consumption ws observed on LpA-I concentrtion, suggesting n increse of 6.5% fter whisky consumption compred with wter consumption. LCAT ctivity ws incresed significntly by 5.8% fter whisky consumption compred with wter. No tretment effects were found on CETP nd PLTP ctivity. BMI effect The effect of BMI on cholesterol efflux, lipid nd lipoprotein profiles, nd relted fctors is shown in Tbles 4, 5. Tble 4 shows tht cholesterol efflux cpcities using J774 were higher in len men (BMI 27) thn in overweight men (BMI 27). Also, the cholesterol efflux cpcity using Fu5AH cells tended to be lower in overweight men. However, ABCA1-dependent cholesterol efflux cpcity ws not different between len nd overweight men. Tble 5 shows tht HDL-C, HDL-PL, poa-i, nd LpA-I were higher in len men thn in overweight men, wheres LCAT ctivity ws 9.2% higher in overweight men. VLDL-C nd TG tended to be lower in len men thn in overweight men. No significnt interction between BMI nd lcohol consumption hs been found for ny of the efflux nd lipoprotein vribles. Correltions The chnges of camp-stimulted cholesterol efflux cpcity from J774 cells induced by whisky consumption correlted with chnges of poa-i (r 0.65, P ), HDL-C (r 0.55, P 0.009), HDL-PL (r 0.56, P 0.008), nd CETP ctivity (r 0.49, P 0.021). A borderline significnt (P 0.054) correltion (r 0.42) ws found between whisky-induced chnges in cholesterol efflux cpcity using camp-treted J774 cells nd whiskyinduced chnges of cholesterol efflux cpcity using Fu5AH cells. Chnges of cholesterol efflux cpcity from Fu5AH cells correlted (r 0.52, P 0.012) with chnges of HDL-PL. Chnges of cholesterol efflux cpcity from camptreted J774 cells did not correlte significntly with chnges of pre -HDL (r 0.30, P 0.18). However, whisky-induced chnges of pre -HDL did correlte with chnges in PLTP ctivity (r 0.65, P ), poa-i (r 0.47, P 0.030), nd HDL-PL (r 0.54, P 0.010). Chnges induced by whisky consumption in serum poa-i lso correlted with chnges in HDL-C (r 0.65, P ), LpA- I:A-II (r 0.61, P ), nd HDL-PL (r 0.66, P ). DISCUSSION In the present study, we investigted the effect of moderte lcohol consumption on cholesterol efflux cpcity of serum or plsm by using two different cell systems, providing informtion on both ABCA1- nd SR-BI-medited cholesterol efflux. We showed n increse of 6.3% in TABLE 2. Cholesterol efflux cpcities fter consumption of whisky or wter in 23 helthy middle-ged men Efflux Wter Whisky Percent Chnge P Fu5AH cholesterol efflux J774 cholesterol efflux ( camp) J774 cholesterol efflux ( camp) ABCA1-dependent efflux Efflux cpcity vlues re expressed s men percentges SEM. ANOVA with tretment, tretment order, period, BMI, nd BMI-tretment interction s fixed fctors in the model. Beulens et l. Alcohol consumption nd cellulr cholesterol efflux 1719

5 Fig. 1. Individul serum ex vivo ABCA1-dependent cholesterol efflux cpcities fter consumption of whisky (40 g lcohol/dy) or wter for 17 dys of 23 helthy mle subjects. camp-stimulted cholesterol efflux cpcity from J774 cells. Consequently, the cpcity of serum to induce ABCA1-dependent cholesterol efflux incresed by 17.5%. The cpcity of plsm to induce cholesterol efflux from Fu5AH cells showed less pronounced increse of 4.6%. Effects on both cholesterol efflux cpcities were ssocited with concomitnt chnges of other prmeters in the RCT pthwy. The study ws prtilly diet-controlled nd performed ccording to rndomized crossover design. No importnt devitions from the study protocol occurred, nd no significnt chnges of body weight were found during the study. It is therefore unlikely tht the results of this study re confounded by crryover effects or by chnges in diet or body weight. In this study, serum nd plsm used for the cholesterol efflux experiments were diluted to 1% to pproch the composition of the interstitil fluid tht comes in direct contct with the cells. Cholesterol efflux cpcities mesured in this study were of the sme order TABLE 3. of mgnitude s those described previously (15, 17, 28). Nevertheless, ny extrpoltion of the ex vivo cholesterol efflux cpcity to the in vivo sitution should only be done with cution. However, the consistency between our results of cholesterol efflux cpcity nd relted prmeters mesured in vivo indicte tht in this cse this extrpoltion is not unresonble. The findings of this study, however, do not gree with those of Mrmillot et l. (29), who showed 21% decrese of cholesterol efflux from mouse J774 mcrophges induced by plsm of mle Wistr-Furth rts fter chronic lcohol consumption. Aprt from the species difference, the rts were fed diet contining s much s 36% of energy from lcohol, which is much higher thn the moderte dose of lcohol ( 10 energy %) in our study. This is nother reson why the results of these studies cnnot be directly compred. A similr inhibition of cholesterol efflux hs been reported in study with in vitro fibroblsts by Avdulov et l. (30) using high lcohol concentrtion. In humn intervention studies with moderte lcohol consumption, the effect on cholesterol efflux hs only been investigted using Fu5AH heptom cells. These studies showed increses in cholesterol efflux of 5 7% (15, 31), which is in ccordnce with the findings of our current study using this cell system (4.6%). The impct of moderte lcohol consumption on camp-stimulted (6.3%) nd prticulrly on ABCA1-dependent cholesterol efflux cpcity in J774 cells (17.5%) ws greter thn the effects observed using Fu5AH cells. Our results show tht both mechnisms my ply role in the crdioprotective effect of moderte lcohol consumption but suggest tht ABCA1-medited cholesterol efflux my be more relevnt pthwy in this respect. Besides the incresed cholesterol efflux cpcity, other prmeters involved in RCT were lso incresed by moderte whisky consumption (pre -HDL, HDL-C, HDL-PL, poa-i, LpA-I, LpA-I:A-II, nd LCAT ctivity). Moreover, Blood lipid profiles nd relted fctors fter consumption of wter or whisky in 23 middle-ged men Vrible Wter Whisky Percent Chnge P TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) VLDL-C (mmol/l) TG (mmol/l) HDL-PL (mmol/l) HDL-TG (mmol/l) Pre -HDL (mg/ml poa-i) Totl poa-i (mg/ml) LpA-I (g/l) LpA-I:A-II (g/l) LCAT ctivity (A.U.) b PLTP ctivity (A.U.) b CETP ctivity (A.U.) b Vlues shown re mens SEM. poa-i, polipoprotein A-I; CETP, cholesteryl ester trnsfer protein; LpA-I, lipoprotein A-I; PLTP, phospholipid trnsfer protein. ANOVA with tretment, tretment order, period, BMI, nd BMI-tretment interction s fixed fctors in the model. b A.U., rbitrry units, corresponding to the percentges of the respective ctivities in the reference pool plsm; 100% is equivlent to the following ctivities: 216 nmol/ml/h for CETP, 65 nmol/ml/h for LCAT, nd 13.9 mol/ml/h for PLTP Journl of Lipid Reserch Volume 45, 2004

6 TABLE 4. Cholesterol efflux cpcities in 12 len nd 11 overweight middle-ged men Efflux Len Overweight Percent Difference P Fu5AH cholesterol efflux J774 cholesterol efflux ( camp) J774 cholesterol efflux ( camp) ABCA1-dependent efflux Efflux cpcity vlues re expressed s men percentges SEM. ANOVA with tretment, tretment order, period, BMI, nd BMI-tretment interction s fixed fctors in the model. TABLE 5. totl poa-i correlted significntly with camp-stimulted cholesterol efflux cpcity, which is in greement with the concept tht cholesterol efflux from mcrophges my be medited by cholesterol-poor poa-i (32, 33). On the other hnd, the increse of cholesterol efflux cpcity did not correlte significntly with chnges of pre -HDL. This my simply be ttributble to the reltively lrge vrince in the cholesterol efflux cpcity mesure. However, becuse pre -HDL ws mesured in vivo nd cholesterol efflux ws mesured ex vivo fter 4 h of incubtion, serum proteins involved in the cycle of poa-i between nscent nd mture HDL species such s LCAT, CETP, or PLTP my influence the cholesterol efflux process (17, 34). LCAT, which incresed significntly fter whisky consumption in this study, my contribute to the pre -HDL concentrtion (17, 34). On the other hnd, dditionl formtion of pre -HDL through PLTP my lso occur (35, 36), s we found reltively strong correltion (r 0.65) between chnges of pre -HDL nd PLTP ctivity in this study. Even using the highly diluted serum, these events could tke plce during the 4 h incubtion period nd my led to ttenution of the correltion between pre - HDL nd cholesterol efflux cpcity. Finlly, the chemicl composition of pre -HDL my lso ply role. This composition is not entirely defined but is likely to contin both precursors nd products of ABCA1-medited cholesterol efflux (34). Thus, becuse the lipid content of pre - HDL my influence the structure nd functionl properties of poa-i (37), these vritions my ffect cholesterol efflux cpcity nd therefore ttenute its correltion with pre -HDL concentrtion. We lso found significnt positive correltions between cholesterol efflux cpcity both from J774 nd Fu5AH cells nd HDL-PL. The ltter correltion is in ccordnce with erlier studies tht reported tht SR-BI-rich cells re highly sensitive to HDL-PL concentrtions (38, 39). The correltion between cholesterol efflux cpcity from camp-treted J774 cells nd HDL-PL ws lso found in ptients with primry hypertriglyceridemi by Brites et l. (40). Recent results of Yncey et l. (28) using plsm from humn poa-i trnsgenic mice, however, re not entirely consistent with these dt. This my be ttributble to the reltively extreme chnges in HDL composition resulting from overexpression in these trnsgenic mice or to species difference. Furthermore, this study shows tht cholesterol efflux cpcities of serum, HDL-C, HDL-PL, poa-i, nd LpA-I were significntly lower in obese thn in len subjects. VLDL-C nd TG tended to be higher nd LCAT ctivity ws significntly higher in obese thn in len subjects. Although the differences in our study re less pronounced, our findings show the sme pttern s the results of Ss- Blood lipid profiles nd relted fctors in 12 len nd 11 overweight middle-ged men Vrible Len Overweight Percent Difference P TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) VLDL-C (mmol/l) TG (mmol/l) HDL-PL (mmol/l) HDL-TG (mmol/l) Pre -HDL (mg/ml poa-i) Totl poa-i (mg/ml) LpA-I (g/l) LpA-I:A-II (g/l) LCAT ctivity (A.U.) b PLTP ctivity (A.U.) b CETP ctivity (A.U.) b Vlues shown re mens SEM. ANOVA with tretment, tretment order, period, BMI, nd BMI-tretment interction s fixed fctors in the model. b A.U., rbitrry units, corresponding to the percentges of the respective ctivities in the reference pool plsm; 100% is equivlent to the following ctivities: 216 nmol/ml/h for CETP, 65 nmol/ml/h for LCAT, nd 13.9 mol/ml/h for PLTP. Beulens et l. Alcohol consumption nd cellulr cholesterol efflux 1721

7 hr et l. (41), who studied the pthwy of cholesterol efflux from humn fibroblsts in len nd obese subjects. They found somewht lrger difference between len nd obese subjects, but this my be ttributble to methodologicl differences, such s nother study popultion or the use of nother cell system (fibroblsts). Syvänne et l. (42), using the Fu5AH cell system, observed decresed cholesterol efflux cpcity in ptients with noninsulin-dependent dibetes mellitus (7%) or coronry rtery disese (7%) of the sme mgnitude s our findings in obese subjects. To the best of our knowledge, this is the first study to show tht moderte lcohol consumption increses ABCA1-dependent cholesterol efflux cpcity of humn serum mesured with J774 mcrophges. This increse of cholesterol efflux cpcity using J774 cells ws lrger thn the increse using Fu5AH cells, suggesting tht the ABCA1 trnsporter is more relevnt for the effect of moderte lcohol consumption on HDL-C thn the SR-BI. At the sme time, moderte lcohol consumption incresed other prmeters of the RCT pthwy, such s totl poa-i, LpA-I:A-II, pre -HDL, HDL-C, nd LCAT ctivity. The increse in serum cholesterol efflux cpcity from J774 cells correlted with the increse of totl poa-i, but the correltion with pre -HDL did not rech sttisticl significnce. Altogether, these findings suggest tht moderte lcohol consumption my stimulte erly steps in the RCT pthwy medited by ABCA1. The uthors thnk the tem tht conducted this study nd the volunteers for their enthusistic prticiption. Dr. Pieter H. E. Groot nd Leo M. Scheek re thnked for the preprtion of the got nti-humn polipoprotein A-I (poa-i) ntiserum used in the quntittion of pre -HDL. Leo M. Scheek lso performed the PLTP nd LCAT ctivity ssys, nd Frh Sdeghi Nirki mesured CETP ctivities. Cor Groffen mesured plsm cpcities to induce cholesterol efflux from Fu5AH cells. Dr. Geesje Dlling-Thie performed the ssy of totl plsm poa-i. 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8 Rothblt Induction of cellulr cholesterol efflux to lipidfree polipoprotein A-I by camp. Biochim. Biophys. Act. 1438: de l Ller Moy, M. M., V. Atger, J. L. Pul, N. Fournier, N. Motti, P. Girl, K. E. Fridy, nd G. Rothblt A cell culture system for screening humn serum for bility to promote cellulr cholesterol efflux. Reltions between serum components nd efflux, esterifiction, nd trnsfer. Arterioscler. Thromb. 14: Ji, Y., B. Jin, N. Wng, Y. Sun, M. L. Moy, M. C. Phillips, G. H. Rothblt, J. B. Swney, nd A. R. Tll Scvenger receptor BI promotes high density lipoprotein-medited cellulr cholesterol efflux. J. Biol. Chem. 272: Yncey, P. G., M. A. Kwshiri, R. Moore, J. M. Glick, D. L. Willims, M. A. Connelly, D. J. Rder, nd G. H. Rothblt In vivo modultion of HDL phospholipid hs opposing effects on SR- BI- nd ABCA1-medited cholesterol efflux. J. Lipid Res. 45: Mrmillot, P., M. N. Ro, Q. H. Liu, S. J. Chirtel, nd M. R. Lkshmn Effect of dietry omeg-3 ftty cids nd chronic ethnol consumption on reverse cholesterol trnsport in rts. Metbolism. 49: Avdulov, N. A., S. V. Chochin, U. Igbvbo, nd W. G. Wood Cholesterol efflux to high-density lipoproteins nd polipoprotein A-I phosphtidylcholine complexes is inhibited by ethnol: role of polipoprotein structure nd coopertive interction of phosphtidylcholine nd cholesterol. Biochemistry. 39: Senult, C., D. Betoulle, G. Luc, P. Huw, D. Rigud, nd F. Fumeron Beneficil effects of moderte consumption of red wine on cellulr cholesterol efflux in young men. Nutr. Metb. Crdiovsc. Dis. 10: Hr, H., nd S. Yokoym Interction of free polipoproteins with mcrophges. Formtion of high density lipoproteinlike lipoproteins nd reduction of cellulr cholesterol. J. Biol. Chem. 266: Yncey, P. G., J. K. Bielicki, W. J. Johnson, S. Lund-Ktz, M. N. Plgunchri, G. M. Annthrmih, J. P. Segrest, M. C. Phillips, nd G. H. Rothblt Efflux of cellulr cholesterol nd phospholipid to lipid-free polipoproteins nd clss A mphipthic peptides. Biochemistry. 34: Brrns, A., B. Jsprd, R. Brbrs, H. Chp, B. Perret, nd X. Collet Pre-bet HDL: structure nd metbolism. Biochim. Biophys. Act. 1300: Lie, J., R. de Crom, M. Juhiinen, T. vn Gent, R. vn Hperen, L. Scheek, H. Jnsen, C. Ehnholm, nd A. vn Tol Evlution of phospholipid trnsfer protein nd cholesteryl ester trnsfer protein s contributors to the genertion of pre bet-high-density lipoproteins. Biochem. J. 360: Pussinen, P. J., M. Juhiinen, nd C. Ehnholm ApoA-II/ poa-i molr rtio in the HDL prticle influences phospholipid trnsfer protein-medited HDL interconversion. J. Lipid Res. 38: Sprks, D. L., P. G. Frnk, S. Brschi, T. A. Neville, nd Y. L. Mrcel Effect of polipoprotein A-I lipidtion on the formtion nd function of pre-bet nd lph-migrting LpA-I prticles. Biochemistry. 38: Atger, V., M. M. de l Ller, M. Bmberger, O. Frncone, P. Cosgrove, A. Tll, A. Wlsh, N. Motti, nd G. Rothblt Cholesterol efflux potentil of ser from mice expressing humn cholesteryl ester trnsfer protein nd/or humn polipoprotein A-I. J. Clin. Invest. 96: Fournier, N., M. M. de l Ller, B. F. Burkey, J. B. Swney, J. Pterniti, Jr., N. Motti, V. Atger, nd G. H. Rothblt Role of HDL phospholipid in efflux of cell cholesterol to whole serum: studies with humn poa-i trnsgenic rts. J. Lipid Res. 37: Brites, F. D., C. D. Bonvit, C. De Geitere, M. Cloes, B. Delfly, M. J. Yel, J. Fruchrt, R. W. Wikinski, nd G. R. Cstro Altertions in the min steps of reverse cholesterol trnsport in mle ptients with primry hypertriglyceridemi nd low HDL-cholesterol levels. Atherosclerosis. 152: Sshr, T., P. Nestel, N. Fidge, nd D. Sviridov Cholesterol trnsport between cells nd high density lipoprotein subfrctions from obese nd len subjects. J. Lipid Res. 39: Syvänne, M., G. Cstro, C. Dengremont, C. De Geitere, M. Juhiinen, C. Ehnholm, S. Michelgnoli, G. Frnceschini, J. Khri, nd M. R. Tskinen Cholesterol efflux from Fu5AH heptom cells induced by plsm of subjects with or without coronry rtery disese nd non-insulin-dependent dibetes: importnce of LpA-I: A-II prticles nd phospholipid trnsfer protein. Atherosclerosis. 127: Beulens et l. Alcohol consumption nd cellulr cholesterol efflux 1723