Mechanism of Plasma Cholesteryl Ester Transfer in Hypertriglyceridemia

Transcription

1 Mechnism of Plsm Cholesteryl Ester Trnsfer in Hypertriglyceridemi Christopher J. Mnn, Frnces T. Yen, Andrew M. Grnt,* nd Bernrd E. Bihin Division oflipoprotein Metbolism nd Pthophysiology, Deprtment ofphysiology, Louisin Stte University Medicl Center, New Orlens, Louisin 7112; nd *Deprtment ofclinicl Biochemistry, Centre Hospitlier Universitire de Sherbrooke, Sherbrooke, Quebec JJH 5N4, Cnd Abstrct Plsm net cholesteryl ester (CE) trnsfer nd optimum cholesteryl ester trnsfer protein (CETP) ctivity were determined in primry hypertriglyceridemic ( = 11) nd normolipidemic ( = 15) individuls. The hypertriglyceridemic group demonstrted threefold greter net CE trnsfer leding to enhnced ccumultion of CE in VLDL. This incresed net trnsfer ws not ccompnied by chnge in CETP ctivity. In normolipidemi, but not in hypertriglyceridemi, net CE trnsfer correlted with VLDL triglyceride (r =.92, P <.1). In contrst, net CE trnsfer in hypertriglyceridemi, but not in normolipidemi, correlted with CETP ctivity (r =.73, P <.1). Correction of hypertriglyceridemi with bezfibrte reduced net CE trnsfer towrds norml nd restored the correltion with VLDL triglyceride (r =.9, P <.5) while suppressing the correltion with CETP ctivity. Tht net CE trnsfer depends on VLDL concentrtion ws confirmed by n increse of net CE trnsfer in normolipidemic plsm supplemented with purified VLDL. Supplementtion of purified CETP to normolipidemic plsm did not stimulte net CE trnsfer. In contrst, net CE trnsfer ws enhnced by ddition of CETP to both plsm supplemented with VLDL nd hypertriglyceridemic plsm. Thus, in norml subjects, VLDL concentrtion determines the rte of net CE trnsfer. CETP becomes rte limiting s VLDL concentrtion increses, i.e, in hypertriglyceridemi. (J. Clin. Invest : ) Key words: cholesteryl ester trnsfer protein * very low density lipoprotein - high density lipoprotein - crdiovsculr disese * reverse cholesterol trnsport Introduction In norml humn plsm, neutrl lipids, i.e., cholesteryl ester (CE)' nd triglyceride (Tg) re redistributed between lipopro- This work ws performed t the Deprtment of Physiology, Louisin Stte University Medicl Center, nd the Deprtment ofchemicl Pthology, Leeds Generl Infirmry, Leeds, Englnd. A portion of this pper hs been presented s n bstrct for the Americn Hert Assocition's 64th Scientific Session, Anheim, CA, Nov. 1991, nd ws published in bstrct form (1991. Arteriosclerosis. 11: 149.) Address reprint requests to Dr. C. J. Mnn, Deprtment ofphysiology, Louisin Stte University Medicl Center, 1542 Tulne Avenue, New Orlens, LA Receivedfor publiction 26 April 1991 nd in revisedform 1 August Abbrevitions used in this pper: CE, cholesteryl ester, CETP, CE trnsfer protein; E6, di-ethyl 4-nitrophenyl phosphte; LCAT, lecithin-cholesterol cyltrnsferse; Tg, triglyceride. J. Clin. Invest. The Americn Society for Clinicl Investigtion, Inc /91/12/259/8 $2. Volume 88, December 1991, teins by cholesteryl ester trnsfer protein (CETP) (1, 2; see ref. 3 for review). Kinetic dt (4) nd in vitro studies (5) indicte tht trnsfer of HDL CE, derived from the lecithin-cholesterol cyltrnsferse (LCAT) rection, to lower density, po B-contining lipoproteins is n importnt route for clernce ofhdl CE. However, the bsence of premture therosclerosis in subjects with mrkedly incresed HDL levels cused by CETP deficiency (null phenotype) (6, 7) suggests tht the ction of CETP could promote therogenesis. Low plsm HDL concentrtion, which is n importnt predictor of coronry hert disese risk (8-1), is commonly observed in hypertriglyceridemic subjects. Since HDL cholesterol concentrtion is inversely relted to plsm Tg levels (1 1, 12), the low HDL cholesterol levels in hypertriglyceridemi could result from n incresed rte of CE trnsfer from HDL to Tg-rich lipoproteins. Consistent with this nlysis, previous studies hve shown tht compred to norml, hypertriglyceridemic HDL re CE depleted nd Tg enriched, while VLDL re enriched with CE (13). In contrst, plsm incubtion studies hve shown tht in hypertriglyceridemic subjects with vsculr disese, the rte of net CE trnsfer from HDL to po B-contining lipoproteins is reduced (5). This observtion suggests tht the pob-contining lipoproteins of these subjects disply impired bility to ccept CE from HDL (5, 14). Recent investigtions, however, hve shown tht po B-contining lipoproteins of hypertriglyceridemic subjects ccept HDL CE t rte comprble with norml subjects (15). Also, net CE trnsfer hs been reported to be enhnced in other conditions ofhypertriglyceridemi, i.e., dysbetlipoproteinemi (16), nd postprndil lipemi (17, 18). To ddress this controversil issue, we investigted the mechnism of CETP-medited trnsfer of CE in homogeneous group of subjects with primry hypertriglyceridemi, nd in control group of normolipidemic subjects mtched for ge nd sex. In this pper, we report tht in primry hypertriglyceridemi, the rte of CE ccumultion in VLDL is incresed threefold compred to normolipidemi. This enhnced net CE trnsfer does not result from chnge in optimum CETP ctivity, but rther from n increse in VLDL concentrtion. Methods Mterils. [l,2(n)9h]cholesterol (sp ct, 45.6 Ci/mmol) ws obtined from Amershm Interntionl, Amershm, Buckinghmshire, Englnd. Phenyl-Sephrose CL4B nd DEAE Sephrose CL6B were from Phrmci Fine Chemicls, Uppsl, Sweden. Cholesterol (C-system CHOD-PAP), unesterified cholesterol (CHOD-PAP), nd triglyceride (GPO-PAP) enzymtic regents, stndrds, nd control ser were purchsed from Boehringer Mnnheim, London, Englnd. Apoprotein-mesuring kits (Orion Dignostic) were obtined from Unipth, Bsingstoke, Englnd. Di-ethyl 4-nitrophenyl phosphte (E6), heprin (porcine intestinl mucos, sodium slt), protinin, nd ll other regents were from Sigm Chemicl Co., Dorset, Englnd, or St. Louis, MO. Bezfibrte (2-[4-[2-[(4-chlorobenzoyl)mino]-ethyl]phenoxy]-2- methylpropnoic cid) ws obtined from Boehringer Mnnheim (formerly MCP Phrmceuticls), Livingstone, Scotlnd. Cholesteryl Ester Trnsfer in Hypertriglyceridemi 259

2 Subjects. This study involved the prticiption of informed, consenting dult humn subjects. Hypertriglyceridemic ptients (VLDL Tg > 1.5 mmol/liter or 13 mg/dl) ttending Leeds Generl Infirmry Lipid Clinic, Leeds, Englnd, were screened for secondry cuses of hyperlipidemi. Subjects with glucose intolernce, liver or kidney disese, thyroid dysfunction, or lcoholism were excluded. To ensure homogeneous popultion, those individuls with rised LDL (1.6 < d < 1.63 g/ml) cholesterol concentrtion (> 4.2 mmol/liter or 16 mg/dl) were lso excluded from the study. Of the I1 selected hypertriglyceridemic subjects, none hd tken lipid-ctive mediction in the previous four months, nd ech ws following lipid clinic dietry guidnce. 8 of these 11 subjects, dignosed with (n = 4) or hving fmily history of (n = 4) crdiovsculr disese, were restudied fter 6 wk tretment with bezfibrte (2 mg, three times per dy). The criterion for positive fmily history ws dignosis of crdiovsculr disese in first or second degree reltives: one before ge 5, two before ge 6, or three before ge normolipidemic control subjects (LDL cholesterol. 4.2 mmol/liter, nd VLDL Tg. 1.2 mmol/liter or 11 mg/dl) were mtched for ge nd sex. Smple preprtion. After n overnight fst, blood ws collected into tubes contining disodium EDTA (finl concentrtion, 1 mg/ml) t 4VC. Aprotinin (.14 IU/ml) nd E6 (di-ethyl 4-nitrophenyl phosphte) (2 mm, finl concentrtion) were dded to ll plsm smples to inhibit protese nd LCAT ctivities, respectively. Mesurement of lipids nd poproteins. Concentrtions of plsm nd lipoprotein totl cholesterol, unesterified cholesterol, nd triglyceride were mesured using enzymtic regents, nd re expressed s millimoles per liter using moleculr weights of 387 for cholesterol nd 885 for triglyceride. CE ws clculted s the difference between mesured totl nd unesterified cholesterol concentrtions. HDL totl nd unesterified cholesterol were determined in plsm fter precipittion of po B-contining lipoproteins with sodium heprin (25 U/ml plsm) nd MnCl2 (92 mm, finl concentrtion) t 4 C (19). To prevent interference of excess mngnese ions with these lipid determintions, the enzymtic regents were first supplemented with 8 mm EDTA. Concentrtions of poproteins Al nd B in plsm were determined by immunoturbidimetric methods. Protein ws determined by the method of Lowry et l. (2) using BSA s stndrd. Isoltion of lipoprotein frctions. VLDL nd LDL were obtined from individul plsm smples (1-1.5 ml) by sequentil ultrcentrifugtion. VLDL (d < 1.6 g/ml) ws isolted using rotor (TFT1 8.4; Sorvll Instruments Div., prt ofdupont Co., Newton, CT) t 31, g for 4 h 1 min t4c. LDL (1.6 < d < 1.63 g/ml) ws isolted by second centrifugtion t 265, g for 7 h 1 min t 4 C. Lipoproteins isolted for preprtive purposes were obtined by sequentil ultrcentrifugtion using rotor (Ti5O.2; Beckmn Instruments, Fullerton, CA) t 145, gnd 4C: VLDL (d < 1.6 g/ml, 18 h), LDL (1.6 < d < 1.63 g/ml, 24 h), HDL (1.63 < d < 1.21 g/ml, 48 h). Ech frction ws concentrted by subsequent flottion using rotor (SW4; Beckmn Instruments) t 4 C. Isolted frctions were dilyzed ginst buffer contining 5 mm Tris, 15 mm NCl, 2 mm EDTA, ph 7.4, nd then stored t 4 C in the presence of protinin (.14 IU/ml). All preprtions were used within 48 h. Determintion of net CE trnsfer in plsm. Net CE trnsfer ws mesured s the chnge of CE concentrtion in VLDL nd LDL frctions fter in vitro incubtion of plsm, unless otherwise indicted. Fresh plsm smples (1-1.5 ml) cpped under nitrogen were incubted t 37 C for the indicted times, nd the rection ws terminted by chilling on ice. VLDL nd LDL frctions were isolted s described bove. Ech smple ws nlyzed twice nd ll lipid mesurements were in qudruplicte, unless otherwise indicted. Since CETP ctivity is negligible t 4 C (21), corresponding smples incubted t this temperture served s controls. Fig. 1 shows the linerity with time of net CE trnsfer to VLDL in normolipidemic nd hypertriglyceridemic plsm. Determintion ofoptimum CETP ctivity. CETP ctivity ws mesured under conditions tht optimize the unidirectionl movement of rdiolbeled CE between donor (HDL) nd cceptor (LDL), i.e., s Net CE trnsfier to VLDL nmol/ml Hypertriglyceridemi Normolipidemi Incubtion time h Figure 1. Net CE trnsfer to VLDL in normolipidemic nd hypertriglyceridemic plsm over time. Normolipidemic subject: cholesterol 4.9 mmol/liter (19 mg/dl), Tg.6 mmol/liter (53 mg/dl) nd HDL cholesterol 1.38 mmol/liter (53 mg/dl). Hypertriglyceridemic subject: cholesterol 8.1 mmol/liter (313 mg/dl), Tg 3.8 mmol/liter (336 mg/dl) nd HDL cholesterol 1.1 mmol/liter (43 mg/dl). Aliquots of fsting plsm (1.5 ml) were incubted t 37C for 1.5, 3, 5, nd 8 h in the presence of the LCAT inhibitor, E6 (finl concentrtion, 2 mm). VLDL (d < 1.6 g/ml) ws isolted from ech liquot using Sorvll TFT 8.4 rotor t 31, g for 4 h 1 min t 4VC. Net CE trnsfer ws determined s the ccumultion of CE in VLDL, reltive to VLDL from control liquot incubted t 4VC (men±sd, qudruplicte determintions). mesure of plsm CETP mss. 3H-CE-HDL3 (1.125 < d < 1.21 g/ml) were prepred ccording to Albers et l. (2). Plsm (5 JAl) ws incubted for 3 h t 37C with 3H-CE-HDL3 (25 nmol CE) nd unlbeled LDL (5 nmol CE) in buffer contining 5 mm Tris, 15 mm NCl, 2 mm EDTA, ph 7.4 (finl vol, 1 Ml). After incubtion, LDL ws removed by heprin/mnci2 precipittion, nd CETP ctivity ws determined s the loss of3h-ce from the HDL superntnt, compred to blnk incubted for the sme time without plsm. Ech plsm nd blnk ws ssyed in qudruplicte. Trnsfer of lbeled substrte did not exceed 25% per incubtion. Fig. 2 shows tht under the conditions described, movement of 3H-CE from donor to cceptor lipoproteins ws dependent on CETP mss (Fig. 2 A) nd time of incubtion (Fig. 2 B). Purifiction ofcetp. Humn plsm CETP ws purified 5-fold from blood bnk plsm (8 ml). Plsm (d = 1.21 g/ml) ws centrifuged in rotor (TiSO.2; Beckmn) t 145, g for 48 h t 4C. The cler middle zone, which contined > 7% oftotl plsm CETP ctivity, ws directly pplied to 45 X 2.5 cm column of phenyl-sephrose CL4B equilibrted in 1 mm Tris, 3 M NCl, ph 7.4. The column ws wshed with 5 bed vol ofbuffer contining 1 mm Tris, 15 mm NCl, ph 7.4. Bound mteril ws then eluted with distilled wter contining 3 mm sodium zide, ph 7.4. Frctions with CETP ctivity were pooled, dilyzed ginst 1 mm Tris, ph 7.4, nd then pplied to 25 X 1.5 cm column ofdeae Sephrose CL6B equilibrted with the sme buffer. Elution ws with liner grdient (6 ml) of-25 mm NCl in 1 mm Tris, ph 7.4. CETP-contining frctions, which eluted between 95 nd 13 mm NCl, were pooled nd concentrted by negtive pressure dilysis (Bio-Moleculr Dynmics, Beverton, OR). Buffers used in the purifiction contined 2 mm EDTA, nd ll procedures were t 4 C. The prtilly purified CETP (optimum CETP ctivity: 75 nmol CE trnsferred/h -mg protein) in buffer contining 1 26 C. J. Mnn, F. T. Yen, A. M. Grnt, nd B. E. Bihin

3 Smple vol jil Incubtion time h Figure 2. Reltionship of CETP ctivity ssy to CETP concentrtion (A) nd incubtion time (B). (A) Volumes of up to 1,d of normolipidemic plsm (-) or purified CETP (i, 2 Mg protein/ml) were incubted for 3 h t 37 C with 3H-CE-HDL3 (25 nmol CE) nd unlbeled LDL (5 nmol CE) in 5 mm Tris, 15 mm NCl, 2 mm EDTA, ph 7.4 in finl vol of 1 ul. (B) 5 Ml of normolipidemic plsm (-) or purified CETP (n) were incubted under the sme conditions s in A for the indicted times. After the incubtions, LDL were precipitted using heprin nd MnCl2. CETP ctivity ws determined from the loss of 3H-CE from the HDL superntnt, minus the loss from blnk smples incubted for the sme time without plsm or purified CETP. Points re expressed s men±sd of qudruplicte determintions. mm Tris, 15 mm NCl, 2 mm EDTA, ph 7.4, ws liquoted nd stored t -7 C. No LCAT ctivity (22) ws detectble in this frction. Sttisticl nlyses. Sttisticl significnce of the differences between two mens ws determined by two-tiled Student's t test for pired or unpired smples s indicted. Correltions were nlyzed using liner Person correltion coefficient. Results Incresed net CE trnsfer in hypertriglyceridemic subjects does not result from chnges in CETP ctivity. Compred to the normolipidemic group, hypertriglyceridemic subjects showed significntly higher VLDL Tg nd significntly lower HDL cholesterol (Tble I). Two prmeters were determined to ssess neutrl lipid redistribution between lipoprotein subfrctions. Net CE trnsfer ws mesured s the chnge of CE concentrtion in VLDL nd LDL frctions following whole plsm incubtions. Plsm optimum CETP ctivity ws mesured under conditions tht minimize the reltive effects of donor nd cceptor lipoproteins. The ltter prmeter provides mesure of CETP mss (23). In the hypertriglyceridemic group, net CE trnsfer ws enhnced compred to normolipidemic subjects leding to threefold incresed ccumultion of CE in VLDL (Tble I). Net CE trnsfer to LDL ws not significntly incresed in hypertriglyceridemi. For both normolipidemic nd hypertriglyceridemic subjects, net CE trnsfer to VLDL ws ccompnied by n equivlent molr net Tg trnsfer from VLDL (dt not shown). There ws no sttisticlly significnt increse in optimum CETP ctivity in hypertriglyceridemic plsm. These dt suggested tht net trnsfer of neutrl lipids is ccelerted in hypertriglyceridemi, but does not result from n increse in plsm CETP mss. Net CE trnsfer correltes with plsm VLDL triglyceride concentrtion only in the normolipidemic group. The observtion tht CETP ctivity is norml in hypertriglyceridemi led us to question the bility of lipoprotein neutrl lipid levels to modulte the rte of net CE trnsfer. Since net CE trnsfer to LDL ws smll, nd not sttisticlly different between the two groups studied, we nlyzed potentil reltionships of net CE trnsfer with lipoprotein CE donor (HDL) nd cceptor (VLDL) concentrtions. A positive liner correltion (r =.92, P <.1) between net CE trnsfer nd VLDL Tg ws found in the normolipidemic group (Fig. 3 A) but not in the hypertriglyceridemic group (Fig. 3 B). The correltion observed in normolipidemic subjects is consistent with dt reported previously in 15 other normolipidemic controls (24). No sttisticlly significnt correltion could be found in either group, between net CE trnsfer nd HDL CE concentrtion (Fig. 3, C nd D). These results suggested tht in normolipidemi, the concentrtion of CE cceptor (VLDL), rther thn CE donor (HDL), determines the rte of net CE trnsfer. For the hypertriglyceridemic group, however, the bsence of significnt correltions between net CE trnsfer nd either cceptor or donor lipoprotein lipid concentrtions indicted tht the rte of net CE trnsfer is independent of plsm lipid levels in these subjects. Net CE trnsfer correltes with CETP ctivity only in the hypertriglyceridemic group. We next questioned whether in hypertriglyceridemi the rte of net CE trnsfer is relted to CETP mss. Fig. 4 shows tht, in contrst with the normolipidemic group (Fig. 4 A), the hypertriglyceridemic group (Fig. 4 B) displyed positive significnt correltion between net CE trnsfer nd optimum CETP ctivity. Tken together, these observtions led us to hypothesize tht: () the rte of net CE trnsfer in normolipidemi is limited by Tg-rich cceptor-lipoprotein concentrtion; nd (b) in hypertriglyceridemi, enhnced net CE trnsfer s result of rised VLDL concentrtion, is limited by the norml CETP mss in these subjects. Supplementtion of normolipidemic plsm with norml VLDL increses net CE trnsfer. To test whether net CE trnsfer is determined by VLDL concentrtion, we creted conditions of hypertriglyceridemi in vitro by supplementing Tble L Plsm Lipids, Apolipoproteins, nd CE Trnsfer Mesurements ofnorml nd Hypertriglyceridemic Subjects Normolipidemic Hypertriglyceridemic Number of subjects Sex (Mle/Femle) 11/4 8/3 Age (yr) 48.2±7.8* 51.6±7.1 Cholesterol (mmol/liter) 5.2±.7 6.1±.6* Triglycerides (mmol/liter) 1.19± ±.97 VLDL-Triglycerides (mmol/liter).64± ±.63 HDL-Cholesterol.93± ±.7 (mmol/liter) LDL-Cholesterol (mmol/liter) 1.41± ±.52 Apo Al (mg/ml) 1.28±.22 Apo B (mg/ml).88± ± ±.17" (nmol/ml * 6 h) to VLDL Net CE trnsfer 11.1± ±52.4 to LDL 15.7± ±68.5 Optimum CETP Activity - (nmol/ml h) 141.3± ±56.4 * Men±SD. I P <.1; t P < (unpired t test)..1; 11 P <.5 vs. normolipidemic Cholesteryl Ester Trnsfer in Hypertriglyceridemi 261

4 2 A 1 Net CE. trnsfer - to VLDL nmol/ml.6h 2r NORMOLUPIDEMIA C b. I 9 U Ir--.33 NS [9 1[ U VLDL Tg * U HDL CE HYPERTRIGLYCERIDELIA plemented with CETP. Consistent with tht observed in VLDL-enriched plsm (Fig. 6), CETP supplementtion of hypertriglyceridemic plsm further incresed the initilly high 5 B rte of net CE trnsfer (Tble II). Frms Tken together, these dt show tht VLDL concentrtion LtEJ NS * limits net CE trnsfer in normolipidemic conditions. CETP becomes rte limiting only in the presence of n excess of Tg- 25. rich lipoprotein. Effect on net CE trnsfer ofsupplementing normolipidemic plsm with purified norml HDL or LDL. To define the roles ofhdl nd LDL in determining the rte of net CE trnsfer, we supplemented plsm with incresing concentrtions of these lipoproteins isolted from the sme subject (Fig. 7). Fig. 7 A * shows tht net CE trnsfer to VLDL ws not enhnced by mmol/l 5 D supplementtion of HDL to: () normolipidemic plsm; (b) plsm enriched to 5 mmol/liter VLDL Tg; or (c) VLDLenriched plsm with twofold incresed CETP ctivity. Fig. 7 B shows tht supplementtion with LDL tended to increse net CE trnsfer. However, this increse reched sttisticl signifir.-.24 * cnce (P <.5, two-tiled, pired t test) only t the highest NS, concentrtion of LDL (12.4 mmol/liter cholesterol) in plsm 25. enriched with both VLDL (5 mmol/liter VLDL Tg) nd CETP (twofold CETP ctivity). These dt therefore support the notion tht neither HDL nor LDL is primry determinnt ofnet CE trnsfer. Correction of hypertriglyceridemi with bezfibrte sup-. presses the correltion of net CE trnsfer with CETP ctivity.51 nd restores correltion with plsm VLDL triglyceride. To rmol/l further test our hypothesis, we investigted the effect ofcorrect- Figure 3. Correltions of plsm net CE trnsfer to VLDL with ing hypertriglyceridemi on the reltionships of net CE trnsfer VLDL Tg (A nd B) nd HDL CE (C ndi D) in normolipidemic nd with CETP ctivity, nd with donor nd cceptor lipoprotein hypertriglyceridemic subjects. levels. 8 of the 11 hypertriglyceridemic subjects, considered to require lipid lowering drug bsed on dignosis or strong fmily history of crdiovsculr disese, were treted with bezfi- concentrtions of brte. Six weeks fter tretment, both plsm nd VLDL Tg normolipidemic plsm with incresing VLDL isolted from normolipidennic subject. Fig. 5 shows were significntly reduced nd HDL cholesterol ws signifiofvldl concentrtion. cntly incresed (Tble III). Net CE trnsfer ws reduced to net CE trnsfer to VLDL s function For VLDL Tg within the norml rnge (i.e., < 1.2 mmol/liter hlf the pretretment level s consequence of significntly or 11 mg/dl), net CE trnsfer increised with VLDL Tg con- less ccumultion of CE in the VLDL frction (Tble III). Belevels, net CE trnsfer zfibrte did not lter plsm CETP ctivity. These dt sup- centrtion. Beyond norml VLDL tended to plteu s the rte pprc)ched plsm optimum port the concept tht net CE trnsfer cn be ltered without CETP ctivity. These dt show tht supplementtion of nor- chnge in plsm CETP mss. molipidemic plsm with excess norml VLDL cn induce In further nlysis, we compred the correltions of net CE threefold increse in net CE trnsferr, consistent with the en- trnsfer to VLDL with CETP ctivity, VLDL Tg, nd HDL CE hncement of net CE trnsfer observed in hypertnglycencdemic subjects. Supplementtion of VLDL-enriched normolipidemic plsm or hypertriglyceridemic plsm with CETPfurther increses net CE trnsfer. To test tht CETP concentrtion limits net CE trnsfer in conditions of excess Tg-rich cceptor, normolipidemic plsm (VLDL Tg =.38 mmol/liter, 34 mg/dl) nd the sme plsm enriched with norml VLDL (VLDL Tg = 5 mmol/liter, 44 mg/dl) were supplemented with prtilly purified CETP. Fig. 6 shows tht ddition of CETP enhnced net CE trnsfer in VLDL-enriched plsm, but not in normolipidemic plsm. The inset of Fig. 6 demonstrtes tht the optimum CETP ctivities in both VLDL-rich nd norml plsm incresed linerly with the ddition of CETP. Thus, the inbility of CETP to increse net CE trnsfer in norml plsm is consistent with this protein being in excess in normolipidemic subjects. To further test the hypothesis tht CETP is rte limiting in hypertriglyceridemi, hypertriglyceridemic plsm were sup- 262 C. J. Mnn, F. T. Yen, A. M. Grnt, nd B. E. Bihin 2 Net CE trnsfer to VLDL 15 nmol/ml.6h 1 5 A NORMOLIPIDEUIA e * U. B 5 r HYPERTRIGLYCERIDEUIA 4k * S 3k 2 too [rug CETP ctivity nmol/ml.h Figure 4. Correltions of plsm net CE trnsfer to VLDL with plsm optimum CETP ctivity in normolipidemic (A) nd hypertriglyceridemic (B) subjects.

5 Net CE trnsfer to VLDL mol/ml.h f% 2Cu 15C 1 I- 5 k threefold fster thn in normolipidemic individuls. In either group, CE did not ccumulte in LDL. Our findings lso show tht optimum CETP ctivity, which provides mesure of CETP mss (23) is not concomitntly incresed in hypertriglyceridemic plsm. This ltter finding is in greement with the observtion tht hypertriglyceridemic subjects hve norml CETP mss s mesured by rdioimmunossy (23). Investigtion of the fctors determining net CE trnsfer in normolipidemic nd hypertriglyceridemic subjects provided mechnism to explin the dissocition between rte of net CE trnsfer nd CETP mss. Correltion nlysis indicted tht direct reltionship exists between plsm VLDL concentrtion nd net CE trnsfer in normolipidemic subjects. The nlysis further suggested tht, in contrst to norml subjects, net CE trnsfer in hypertriglyceridemi is determined by CETP mss. A possible explntion for these different correltions ws tht in normolipidemi, CETP is in excess nd VLDL concentrtion (i.e., vilbility of Tg) limits the rte of net CE trnsfer, while in hypertriglyceridemi, where VLDL concentrtion is incresed, net CE trnsfer is dependent on CETP mss. fu VLDL Tg mmol/l Figure 5. Effect on net CE trnsfer of incresing concentrtions of norml VLDL in whole normolipidemic plsm. Fsting plsm ws obtined from two subjects: subject 1 (m)-cholesterol 5.4 mmol/liter (29 mg/dl), Tg 1.2 mmol/liter (16 mg/dl), nd HDL cholesterol 1.9 mmol/liter (42 mg/dl); subject 2 (.)-cholesterol 3.3 mmol/liter (128 mg/dl), Tg.54 mmol/liter (48 mg/dl), nd HDL cholesterol 1.21 mmol/liter (47 mg/dl). Plsm CETP ctivities were 216 nd 143 nmol/ml * h, respectively. l-ml liquots from subjects 1 nd 2 were incubted t 37 C for 2 h with or without incresing mounts of VLDL isolted from subject 1. The LCAT inhibitor, E6 (2 mm, finl concentrtion) ws included in the ssy nd the finl volume ws djusted to 1.2 ml. Net CE trnsfer ws mesured s the loss of CE from the d > 1.6 g/ml (HDL + LDL) frction. Corresponding smples incubted t 4 C served s controls. Ech point represents the men±sd of qudruplicte determintions. in the eight subjects before nd fter tretment. The significnces ofthese correltions before tretment were not different from those observed in the 11 originl subjects (Figs. 3 nd 4). Fig. 8 shows the correltions fter bezfibrte tretment. The correltion between net CE trnsfer nd optimum CETP ctivity did not rech sttisticl significnce (Fig. 8 A), contrsting with the significnt correltion observed prior to tretment. Concomitntly, sttisticlly significnt correltion between net CE trnsfer nd plsm VLDL Tg concentrtion ws observed fter bezfibrte tretment (Fig. 8 B). The lck of correltion between net CE trnsfer nd HDL CE observed before bezfibrte, persisted fter therpy (Fig. 8 C). This showed tht correction of hypertriglyceridemi with bezfibrte suppresses the reltionship of net CE trnsfer with CETP ctivity, nd restores the reltionship with plsm VLDL Tg. These dt therefore further support the hypothesis tht Tg-rich lipoprotein concentrtion determines the rte of net CE trnsfer in normolipidemi, while CETP limits net CE trnsfer in the presence of high VLDL concentrtions. Discussion The dt presented here show tht in plsm from primry hypertriglyceridemic subjects, net CE trnsfer to VLDL is u.sLI1LLIU.f/ Net CE trnsfer to VLDL 2/ nmol/ml.h 15 - CETP - 5 "o VLDL TS -.38mmol/1 8so CETP dded Figure 6. Effect on net CE trnsfer of CETP supplementtion to plsm enriched (m) or not (e) with norml VLDL. Using the sme subjects s in Fig. 5, fsting 1-ml plsm liquots from subject 2 were enriched or not with VLDL from subject 1, then supplemented or not (first point) with incresing concentrtions of purified (5-fold) CETP in finl vol of 1.4 ml. These smples were incubted t 37 C for either 4 h (normolipidemic plsm) or 2 h (VLDL-enriched plsm). All smples contined LCAT inhibitor (2 mm E6, finl concentrtion) nd corresponding smples incubted t 4 C served s controls. Net CE trnsfer ws mesured s loss of CE from the d > 1.6 g/ml (HDL + LDL) frction. Points represent men±sd of qudruplicte determintions. (Inset): Effect ofcetp supplementtion on plsm optimum CETP ctivity. CETP ctivity ws determined in ll bove smples (5 gl) s described in Fig. 2 except incubtion time ws 2 h. The optimum (unidirectionl) CE trnsferred, expressed in nnomoles per milliliter. h, ws clculted from HDL 3H-CE specific ctivity. pg Cholesteryl Ester Trnsfer in Hypertriglyceridemi 263

6 Tble II. Effect on Net CE Trnsfer to VLDL ofsupplementing Plsm from Hypertriglyceridemic Subjects (n = 3) with Prtilly Purified CETP Nonsupplemented plsm CETP-supplemented plsm Net CE trnsfer CETP Net CE trnsfer CETP to VLDL ctivity to VLDL ctivity nmol/ml. h Subject Men±SD 13±29 212±49 235±56* 644±12W 1 ml of blood ws obtined from overnight-fsted hypertriglyceridemic subjects (men plsm cholesterol, 4.82±1.37 mmol/liter, VLDL Tg 4.74±1.26 mmol/liter, HDL cholesterol.67±.25 mmol/liter, LDL cholesterol 2.26±.51 mmol/liter). Net CE trnsfer nd CETP ctivity were mesured s described in Fig. 6 on ntive plsm (left pnel) nd on the sme plsm supplemented with CETP (44,g) to increse plsm CETP ctivity threefold (right pnel). * P <.1; * P <.5 vs. respective mesurement in nonsupplemented plsm (pired t test). The possibility tht VLDL concentrtion limits net CE trnsfer in normolipidemi ws verified by the observtion tht supplementtion of plsm with purified VLDL incresed net CE trnsfer. Conversely, the hypothesis tht CETP is rte limiting in hypertriglyceridemi ws verified by supplementtion of 25r 2z Net CE trnsfer to VLDL 15 nmol/ml.h 1 5 A HDL cholesterol mmol/l B LDL cholesterol mmol/i VIDL nd CElP-rich plsm VLDL-rich plsm Norml plsm Figure 7. Effect of plsm HDL (A) nd LDL (B) concentrtions on net CE trnsfer to VLDL. Fsting plsm () ws obtined from normolipidemic subject-cholesterol 4.3 mmol/liter (166 mg/dl), Tg.61 mmol/liter (54 mg/dl), HDL cholesterol 1.2 mmol/liter (46 mg/dl), CETP ctivity 193 nmol/ml * h. Plsm liquots (1 ml) were supplemented with incresing concentrtions ofutologous HDL (A) or LDL (B) s indicted, nd incubted t 37 C for 4 h. Seprte liquots of plsm were enriched to 5 mmol/liter VLDL Tg either without (e) or with (,) ddition of purified CETP (twofold ctivity of originl plsm), then supplemented with incresing concentrtions ofhdl or LDL nd incubted t 37 C for 2 h. All smples (1.4 ml, finl vol) contined 2 mm E6 s LCAT inhibitor. After incubtion, net CE trnsfer ws mesured s the increse ofce in the d < 1.6 g/ml frction (VLDL). Mens±SD re shown for qudruplicte determintions except where SD brs re conceled by the symbols. Supplementtion of HDL or LDL did not significntly (pired t test, two tiled) lter net CE trnsfer except t the highest concentrtion of LDL in plsm enriched with both VLDL nd CETP (P <.5). Tble III. Plsm Lipids, Apolipoproteins, nd CE Trnsfer Mesurements ofeight Hypertriglyceridemic Subjects before nd during Bezfibrte Therpy Week of Tretment 6 Cholesterol (mmol/liter) 6.±.6* 5.7±.7 Triglycerides (mmol/liter) 3.91± ±.63* VLDL-Triglycerides (mmol/liter) 2.91± ±.54* HDL-Cholesterol (mmol/liter).9± ±.23t LDL-Cholesterol (mmol/liter) 3.27± ±.7 Apo Al (mg/ml).99± ±.14t Apo B (mg/ml) 1.13±.12.98±.21 Net CE trnsfer (nmol/ml- 6 h) to VLDL 364.4± ±96.1' to LDL 3.8± ±83.3 Optimum CETP ctivity - (nmol/ml h) 159.9± ±7.2 * Men+SD. ' P <. 1; * P <.2; 1 P <.5 vs. week (pired t test). norml nd VLDL-enriched plsm with purified CETP. Incresing the mss of CETP in normolipidemic plsm hd no effect on net CE trnsfer to VLDL. In contrst, the sme quntity of CETP dded to VLDL-enriched plsm incresed net CE trnsfer more thn twofold. The rte-limiting effect of CETP ws lso demonstrted directly in hypertriglyceridemi. Incresing the mss ofcetp in plsm from hypertriglyceridemic subjects significntly enhnced the initilly high rte of net CE trnsfer. Further evidence for this interprettion is found in dt obtined from hypertriglyceridemic subjects treted with bezfibrte. Correction of hypertriglyceridemi reduced net CE trnsfer towrds norml levels but did not ffect optimum CETP ctivity. After this tretment, liner correltion between net CE trnsfer nd VLDL concentrtion ws restored s in the normolipidemic group, while the preexisting correltion between net CE trnsfer nd optimum CETP ctivity ws no longer significnt. Our investigtions therefore indicte tht in normolipidemi, the concentrtion of Tg-rich lipoproteins in plsm provides the driving force for net CE trnsfer, while in hypertriglyceridemi, the enhnced rte of net trnsfer is limited by CETP mss. This suggests tht, for hypertriglyceridemic subjects, dietry (23) or genetic (25) fctors ffecting CETP expression could influence the individul vrition in net CE trnsfer. We hve demonstrted in this study tht stimultion of net CE trnsfer to the sme extent s tht observed in hypertriglyceridemi ws chieved by supplementtion of norml VLDL to norml plsm. Altertions in VLDL composition re therefore not required for these prticles to increse net CE trnsfer. This does not exclude tht the reported enrichment of hypertriglyceridemic VLDL with unesterified cholesterol (13, 26) could modulte the bility of such prticles to ccumulte CE (27). The observtion tht VLDL concentrtion drives net CE trnsfer provides mechnism to explin the correltions between plsm Tg concentrtion nd lipoprotein core-lipid compositionl bnormlities in hypertriglyceridemi, s well s the reversion of these compositionl chnges towrds nor- 264 C. J. Mnn, F. T. Yen, A. M. Grnt, nd B. E. Bihin

7 5Oor A Df Net CE 4 trnsfer to VLDL 3 nmol/ml.6h 2 1 S L=.58 NS B r=.9 Ip<.51 [ NS Figure 8. Correltions of net CE trnsfer to VLDL with optimum CETP ctivity (A), VLDL Tg (B), nd HDL CE (C) in hyper- CETP ctivity VL DL Tg HDL CE triglyceridemic subjects (n = 8) fter 6 wk of nmol/ml.h mol/l mmol/i bezfibrte therpy. iii.67 S S ml fter reduction of VLDL concentrtion by dministrtion of bezfibrte (13). Although plsm HDL CE concentrtion did not correlte with net CE trnsfer in either the normolipidemic or the hypertriglyceridemic group, significnt inverse correltion (r = -.69, P <.1) ws observed when dt from both groups were pooled. Thus, ccelerted net CE trnsfer s result of incresed VLDL concentrtion could explin differences in HDL CE concentrtion between, but not within, the two groups. It must lso be considered tht determintion of net CE trnsfer in incubted plsm reflects only prtilly the physiologicl trnsfer of neutrl lipid. Indeed, the role of fctors, e.g., lipolysis which hs been shown to enhnce CETP ctivity (28), is not tken into ccount in such n ssy. Therefore, the possibility remins tht other prmeters contribute in vivo to the regultion of net CE trnsfer. In greement with the present study, incresed plsm net CE trnsfer hs been reported in other conditions of hypertriglyceridemi. Compred to fsting norml subjects, net CE trnsfer ws enhnced threefold in dysbetlipoproteinemi (type III hyperlipidemi) (16), nd two to threefold during postprndil lipemi (17, 18). In both conditions, the enhnced net CE trnsfer ws ttributed to combined stimultory effect of hypertriglyceridemi nd incresed ctivity of CETP (16, 18). Our conclusions, however, re not consistent with the interprettion tht net CE trnsfer is reduced or unchnged in hypertriglyceridemi (5). This discrepncy most likely results from the fct tht hypertriglyceridemic groups studied in the previous investigtion were smll nd included subjects with secondry hypertriglyceridemi, s well s individuls receiving lipid-ctive drugs or with high plsm cholesterol. In this report, we clrify the mechnisms through which potentilly independent therogenic risk fctor, i.e., CETP, ffects lipoprotein core composition in hypertriglyceridemi. This nlysis led us to propose tht the driving force for net CE trnsfer is VLDL. As VLDL concentrtion increses, CETP becomes rte-limiting. Thus, in hypertriglyceridemi, net CE trnsfer is limited by CETP nd could therefore be ffected by fctors modulting CETP concentrtion. We postulte tht the reltive excess of CETP in plsm of overnight fsted normolipidemic subjects my provide the reserve to stisfy greter need for trnsfer of neutrl lipids during the postprndil stge. Acknowledgments The uthors re deeply indebted to Dr. Pul S. Roheim for his criticl review of the mnuscript, mny vluble suggestions nd continul support. Thnks re extended to Drs. Jen Dvignon, Ir Goldberg, nd Alfredo Lopez for providing the plsm from subjects presented in Tble II. We lso wish to thnk Dr. Chrles Sloop for his ssistnce in preprtion of the figures. This reserch ws supported by George S. Bel Fellowship from the Americn Hert Assocition-Louisin, Inc. (wrded to C. J. Mnn), nd by grnts from the Ntionl Institutes of Helth (HL-25596) nd Leeds Western Are Helth Authority (LW74). References 1. Morton, R. E., nd D. B. Zilversmit Inter-reltionship of lipids trnsferred by the lipid-trnsfer protein isolted from humn lipoprotein-deficient plsm. J. Biol. Chem. 258: Albers, J. J., J. H. Tollefson, C.-H. Chen, nd A. Steinmetz Isoltion nd chrcteriztion of humn plsm lipid trnsfer proteins. Arteriosclerosis. 4: Tll, A. R Plsm lipid trnsfer proteins. J. Lipid Res. 27: Brter, P. J., G. J. Hopkins, nd G. D. 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