Murray W. Huff,'." David B. Miller," Bernard M. Wolfe," Philip W. Connelly? and Cynthia G. Sawyez"

Transcription

1 Uptke of hypertriglyceridemic very low density lipoproteins nd their remnnts by HepG2 cells: the role of lipoprotein lipse, heptic triglyceride lipse, nd cell surfce proteoglycns Murry. Huff,'." Dvid B. Miller," Bernrd M. olfe," Philip. Connelly? nd Cynthi G. Swyez" Deprtments of Medicine nd Biochemistry nd the Robrts Reserch nstitute,* The University of estern Ontrio, London, Ontrio, Cnd N6A 5K8, nd The J. Alick Little Lipid Reserch Lbortory, St. Michel's Hospitl,' University of Toronto, Toronto, Ontrio, Cnd M5S 1A9 Abstrct Hypertriglyceridemic very low density lipoproteins (HTG-, Sf 6-4) re not tken up by HepG2 cells. However, ddition of bovine milk lipoprotein lipse (LPL) t physiologicl concentrtions mrkedly stimultes uptke. n the present study, we determined whether: ) LPL ctlytic ctivity is required for uptke, 6) LPL functions s lignd, nd c) cell surfce heptic triglyceride lipse (HL) nd/or proteoglycns re involved. ncubtion of HepG2 cells with HTG- plus LPL (8 ng/ml) incresed cellulr cholesteryl ester (CE) 3.5-fold nd triglyceride (TG) 6-fold. Het-inctivtion of LPL bolished the effect. Addition of tetrhydrolipsttin (THL, n LPL ctive-site inhibitor) LO HTG- + LPL, inhibited the cellulr increse in both CE nd TG by greter thn 9%. Co-incubtion of HTG- + LPL with heprin, heprinse, or hepritinse, blocked CE ccumultion by 7%, 48%, nd 95%, respectively, but hd no effect on the increse in cellulr TG. Pre-tretment of cells with 1 mm 4-methylumbelliferyl-~-11-xyloside, (P-xyloside) to reduce cell surfce proteoglycns inhibited the increse in CE induced by HTG- + LPL by 78%. HTG- remnnts, prepred in vitro nd isolted free of LPL ctivity, stimulted HepCP cell CE 2.8-fold in the bsence of dded LPL, process inhibited with THL by 66%. Addition of LPL (8 ng/ml) to remnnts did not further enhnce CE ccumultion. HepG2 cell HL ctivity, relesed by heprin, ws inhibited 95% by THL. The mount of HL ctivity nd immunorective mss, relesed by heprin, ws reduced 5-6% in P-xylosidetreted ce1ls.m These results indicte tht physiologicl coricentrtions of LPL promote HepCP cell uptke of HTG- primrily due to remnnt formtion nd tht LPL does not ply mjor role s lignd. HL ctivity nd cell surfce proteoglycns significntly enhnce the subsequent uptke of remnnts.-huff, M.., D. B. Miller, B. M. olfe, P.. Connelly, nd C. G. Swyez. Uptke of hypertriglyceridemic very low density lipoproteins nd their remnnts by HepG2 cells: the role of lipoprotein lipse, heptic triglyceride lipse, nd cell surfce proteoglycns.,j. 1,iflid &T. 199'7. 38: Supplementry key words hypertriglyceridemi heptocytes tetrhydrolipsttin P-xvloside polipoprotein E Very low density lipoproteins () re mcromoleculr complexes composed of neutrl lipid core of triglyceride nd cholesteryl ester nd contin polipoproteins (po) C, E, nd B-1. These lipoproteins re synthesized nd secreted by the liver into plsm where they interct with lipoprotein lipse (LPL), n enzyme present on endothelil cell surfces tht hydrolyzes triglyceride. The resulting remnnt is smller, enriched in cholesteryl ester, nd hs n ltered poprotein composition. Further lipolysis converts remnnts into intermedite density lipoproteins (DL) nd subsequently low density lipoproteins () (1). Plsm concentrtions of re elevted in subjects with Type TV hypertriglyceridemi. n contrst to normolipidemic, Type V hypertriglyceridemic (HTG-) is chrcterized by substntil metbolic heterogeneity, with bnorml spects being primrily confined to the lrger S 6-4 lipoprotein prticles (2-7). n these subjects, S 6-4 concentrtions re elevted due to n overproduction together with reduced frctionl ctbolic rte (4-7). Some S, 6-4 HTG- is clered directly, however, the mjority is converted to DL (S, 12-6) (6, 7). These remnnt lipoproteins re lmost quntittively clered from the circultion (6) by the liver. The mech- Abbrevitions:, very low density lipoproteins; HTG-., from subjects with Type V hyperlipoproteinemi;, low density lipoproteins; -, P-migrting ; po, polipoprotein; LRP, receptor-relted protein; LPL., lipoprotein lipse; HL, heptic triglyceride lipse; MEM, miniml Egle's medium; BSA, bovine serum lbumin; LPDS, lipoprotein-deficient serum; THL, tetrhydrolipsttin; P-xyloside, 4-methylumbelliferyl-~-~-xyloside; HSP(;, heprdn sulfte proteoglycns. 'To whom correspondence should he ddrrssed. Downloded from by guest, on October 3, Journl of Lipid Reserch Volume 38, 1997

2 nisms of how HTG- nd their remnnts my be tken up by heptocytes re not completely understood (1). ApoE, mjor polipoprotein constituent of these prticles, cts s lignd for clernce of remnnts by heptic receptors (8). There is evidence tht the receptor, the receptorrelted protein (LRP), nd/or unchrcterized recep tor(s) re involved in heptic uptke of remnnts (9-16). n previous studies, we found tht from hypertriglyceridemic subjects (HTG-) ws not tken up by cultured HepG2 cells unless co-incubted with bovine milk lipoprotein lipse (LPL) (17). Uptke resulted in cellulr ccumultion of cholesteryl ester nd triglycerides nd, in ddition, stimulted intrcellulr cyl-coa: cholesterol cyltrnsferse. These studies indicted tht lipolytic remodelling of HTG- by LPL is prerequisite for their recognition by heptic uptke processes. However, it is possible tht LPL itself could medite remnnt uptke. t hs been proposed tht LPL my provide high-ffinity recognition site for the heptic clernce of lipoproteins (1, 18). LPL cn ct s lignd for the LRP (1) nd enhnce binding of chylomicrons, norml,, Lp[], nd rbbit p- to fibroblsts, mcrophges, nd HepG2 cells (1, 19-29). t hs been reported tht LPL-medited binding of lipoproteins to the LRP is dependent on n intct C- terminl domin (residues ) nd the dimeric structure of LPL (2, 21, 27, 3), indicting tht the C- terminl domin functions both in binding to lipoproteins nd the LF (2, 27). Choi et l. (31) demonstrted specific interction of LPL with the mino- terminl region of pob of nd. The concept tht LPL my ct s lignd fcilitting remnnt clernce hs gined support from the finding tht LPL in pre-heprin plsm is ssocited with lipoproteins (32) nd significnt mount of this LPL is ssocited with triglyceride-rich lipoproteins nd their remnnts in hypertriglyceridemic subjects (33, 34). Consistent with these in vitro observtions, we hve demonstrted tht in Type lv subjects, n infusion of heprin to relese LPL into plsm resulted in mrked reduction in the concentrtion of Sf 6-4 lipoproteins. The remnnts were rpidly clered from plsm nd were not recovered in the DL or frction (35). t is not known whether the relesed LPL enhnced prticle clernce by incresing the rte of remnnt formtion or whether it lso cted s lignd, thereby enhncing heptic clernce. mportntly, it is not known whether the smll mount of LPL ssocited with triglyceride-rich lipoproteins nd their remnnts in pre-heprin plsm is cpble of functioning s lignd or bridge to the cell surfce. The interprettion of cell studies, which suggest tht LPL functions s lignd mediting lipoprotein uptke, is not cler. Although the binding of lipoproteins t 4 C ws enhnced 3- to 2-fold by LPL (1, 19-28), up tke nd degrdtion were only incresed 2- to 6-fold (19, 21-25, 28). n ddition, in most studies, high concentrtions of LPL were used: 1 to 2 ng/ml of medi (1, 19-28), concentrtions significntly higher thn the those reported to occur in pre-heprin plsm. Vilell et l. (33) hve reported tht the concentrtion of LPL in preheprin plsm is pproximtely 7 ng/ ml nd only bout 1-15% is ssocited with triglyceride-rich lipoproteins. Postprndilly, plsm LPL mss ssocited with triglyceride-rich lipoproteins nd their remnnts incresed pproximtely 2-fold (33, 34). n ddition, the mjor form of LPL in pre-heprin plsm is monomeric wheres the dimeric form is required to enhnce binding to the LRP nd medite cell uptke (2, 3). t hs not been firmly estblished whether the enhnced uptke by LPL requires ctlyticlly ctive enzyme. t hs been shown tht chylomicron binding to HepG2 cells is enhnced 3- to 4-fold by LPL nd tht lipolysis is not required to observe the effect (1). LPL either in its ntive form, het inctivted, or treted with prtil ctlytic inhibitors enhnced both the binding nd internliztion of norml humn '*'lbeled in fibroblsts (22, 25) nd rbbit z5lbeled p- in Hep3B cells (3). hether LPL-enhnced u p tke ffects intrcellulr cholesterol or triglyceride metbolism hs not been determined. n recently reported perfused rt liver studies, ctive LPL produced mrked stimultion of heptic chylomicron clernce, however, smll component of uptke does not pper to require ctlytic ctivity (36). Heptic triglyceride lipse (HL) is synthesized by heptocytes nd is locted on the surfce of heptocytes nd heptic endothelil cells (37). t plys role in the ctbolism of remnnts of both chylomicrons nd (38-4) by hydrolyzing these remnnts, thereby prepring them for heptocyte uptke. HL deficiency is chrcterized by n ccumultion in plsm of remnnt lipoproteins (38, 39). nfusion of nti-hl ntibodies into nimls delys remnnt lipoprotein ctbolism (37, 41-43). Hydrolysis of rt chylomicron remnnts by HL increses the exposure of poe (44). Rt heptom cells (McA-RH7777) trnsfected with humn HL demonstrted enhnced binding of rbbit p- compred to non-trnsfected cells (45). However, role for heptocyte cell surfce HL, under bsl conditions, in the cellulr uptke of humn HTG- hs not been clerly estblished. There is incresing evidence tht the uptke of remnnts of triglyceride-rich lipoproteins by heptocytes involve cell surfce proteoglycns. Severl groups of investigtors hve reported tht tretment of HepG2 cells Downloded from by guest, on October 3, 218 Huff et l. Role of LPL, HL, nd proteoglycns in HepGP cell uptke of 1319

3 TABLE 1. Plsm nd lipoprotein lipid concentrtions of donors Cholesterol Triglyceride Totl. HDL Totl mmol/l. mmol/, 6.92? t t t ?.75 Vlues re the men 2 SE in mmol/l from twelve subjects with Type N hypertriglyceridemi., S, > 2, ws prepred by ultrcentrifugtion. HDL cholesterol ws determined in the infrntnt fter ultrcentrifugtion nd precipittion of the pob-contining lipoproteins with dextrn sulfte-mgc1,. cholesterol ws clculted by difference. (25) nd norml nd FH-fibroblsts (19, 21, 22) with heprinse to deplete cell surfce heprn sulfte proteoglycns (HSPGs) inhibited the enhnced binding nd internliztion of norml humn or medited by LPL. However, no effect of diminished cell surfce HSPGs on or uptke in the bsence of LPL ws observed. Ji et l. (46, 47) hve demonstrted tht ddition of excess poe to - from cholesterol-fed rbbits enhnced its uptke by cultured heptocytes, process involving cell surfce HSPGs. The role of HSPGs in heptocyte interction with HTG- or their remnnts is unknown. The present studies, crried out in HepG2 cells, were designed to determine whether: 1) LPL, t physiologicl concentrtions, enhnces the uptke of humn Sf 6-4 HTG- nd HTG- remnnts, beyond its ctlytic function, 2) HL prticiptes in HTG- remnnt uptke, nd 3) cell surfce proteoglycns re involved in the uptke of HTG- remnnts without the ddition of exogenous LPL or poe. Subjects EXPERMENTAL PROCEDURES The lipid nd lipoprotein profiles of the ptients used in this study re shown in Tble 1. The Type V hypertriglyceridemic subjects were clssified ccording to the criterion of Schefer nd Levy (48) fter visits to the Outptient Endocrinology Clinic t University Hospitl, London, Ontrio, Cnd. These subjects ll presented with primry hypertriglyceridemi nd none displyed fsting chylomicronemi or hd known metbolic disorder such s hypothyroidism, morbid obesity, or renl dysfunction. n ddition, none of the subjects used in these experiments were being treted with lipid-lowering gents. The normolipidemic smples were obtined from helthy lbortory personnel. Prior to blood smpling ll subjects were required to fst for 12 h. These studies were pproved by the University of estern Ontrio Helth Sciences Stnding Committee on Humn Reserch nd ll subjects gve informed consent. Lipoprotein isoltion Lipoproteins were isolted from plsm essentilly s described previously (17). (S, 6-4) ws isolted by ultrcentrifugtion through buffer A (1.6 g/ ml density solution contining.195 M NCl, 1 mm Tris, ph 7.4, 1 mm EDTA, 1 p~ phenylmethne sulfonyl fluoride, 3 mm NN3,.1 mm merthiolte) in Beckmn 6 Ti rotor (Beckmn nstruments, Mississug, Ontrio) for 2 h t 4, rpm t 12 C using Beckmn L8 ultrcentrifuge. The S, 6-4 frction ws wshed through n equl volume of buffer A in Beckmn 7.1 Ti rotor spun t 4, rpm, 12 C for 16 h. ws isolted from the infrntnt, fter removl of nd DL, by ultrcentrifugtion for 16 h t 5, rpm in 6 Ti rotor in buffer A t d 1.63 g/ml. The ws wshed in d 1.63 g/ml density solution in 7.1 Ti rotor spun t 5, rpm, 12 C for 16 h. Lipoprotein-deficient serum (LPDS) ws isolted s described previously (1 7). All lipoprotein smples were nlyzed for protein content by modifiction of the Lowry method (49) nd for totl cholesterol nd triglyceride using dignostic kits from Boehringer Mnnheim Cnd, Lvl, Quebec (CHOD-PAP nd triglycerides without free glycerol, respectively). All lipoprotein smples were stored t 4 C nd used for tissue culture experiments within 1 week of isoltion. ApoE phenotypes were determined on ll smples used in these studies by nlyticl isoelectric focusing gel electrophoresis s described previously (4). The lipid compositions of nd smples used in these studies re listed in Tble 2. HepGZ cells HepG2 cells were cultured in 1-mm culture dishes (Flcon, Fisher Scientific, Ottw, Ontrio) in 1 ml of modified Egle s medi (MEM) with Erle s slts contining;.2% sodium bicrbonte, 1 mm sodium pyruvte,.3 mg/ml L-glutmine, 1% fetl bovine serum Downloded from by guest, on October 3, Journl of Lipid Reserch Volume 38, 1997

4 TABLE 2. Composition of lipoproteins Lipoprotein n C/TG C/ Protein TG/Protein FFA/ Protein Type N " L.51 ND Type V Control Hydrolyzed in vitrob < 1.25?.26< ' < ? ND Vlues re expressed s mens 2 SD; C, cholesterol; TG, triglyceride; FFA, free ftty cid; ND, not determined. Rtios re weight rtios. " Sf 6-4. ' Sr 6-4 from six Type v hyperlipoproteinemic subjects were incubted with bovine milk lipse (1 mg cholesterol/unit enzyme) for 3 h nd the hydrolyzed lipoproteins were reisolted by ultrcentrifugtion s described in Experimentl Procedures. 'P <.5 compred to control. (FBS) (CN Biomedicl nc., Mississug, Ontrio), 1 U/ml of penicillin, 1 pg/ml streptomycin, nd.25 pg/ml Fungizone (CN). Cells were split (1:3) using trypsin-edta (CN) on 7-dy cycle up to mximum of 1 pssges. For ll of the experiments HepG2 cells were plted in 24-well (15 mm) culture pltes (Flcon) in 1 ml of MEM contining 1% FBS nd grown for 5 dys. Once the monolyers hd become pproximtely 8% confluent, the medi were replced with MEM contining 5% LPDS for 24 h prior to experiments. The pproprite concentrtions of lipoproteins were dded to duplicte dishes of cells nd were incubted for 5 h (olete incorportion studies) or 16 h (mss determintions) unless otherwise indicted. The mount of cell protein per dish verged.3 mg. Heprin, heprinse, hepritinse, nd p-xyloside tretment Heprin, 1 pg/ml of medi (Clbiochem, L Joll CA), heprinse, 4 U/ml (Sigm, heprinse, ctlogue number H-2519) or hepritinse, 4 U/ml (Sigm, heprinse 111, ctlogue number H-8891) were dded to cells 4 h prior to incubtions. The medi were chnged nd heprin, heprinse, nd hepritinse were dded t the sme concentrtions to medi contining 5% LPDS t the sme time s lipoproteins with or without LPL. Addition of the enzymes prevented regenertion of cell surfce HSPGs during the subsequent incubtions of cells with lipoproteins nd LPL (28). P-Xyloside (4-methylumbelliferyl-~-~-xyloside, Sigm) ws incubted with cells t concentrtion of 1 mm for 4 dys prior to nd during the ddition of lipoproteins with or without LPL (5). Determintion of cellulr lipid content nd cholesterol esterifiction n the present studies we hve used increses in the intrcellulr mss of cholesteryl ester s well s the incorportion of olete into cholesteryl esters s mesures of HTG- lipid uptke. The interprettion of cellulr uptke nd degrdtion studies of iodinted triglyceride-rich lipoproteins is complicted by the dissocible nture of the highly lbeled C poproteins, especilly under lipolytic conditions. n ddition, uptke nd degrdtion studies do not revel the intrcellulr consequences on lipid metbolism of prticle uptke. The determintion of cellulr free nd esterified cholesterol nd triglyceride mss ws essentilly s described previously (17). Upon completion of the experiment, cells were wshed twice in buffer B (.15 M NCl, 5 mm Tris, ph 7.4,.2% ftty cid-free bovine serum lbumin, BSA) nd 2 dditionl wshes with buffer B without BSA. Lipids were extrcted in situ using two 3- min incubtions with 1.O ml of hexnes-isopropnol 3 : 2 (v/v) nd the residul cell protein ws determined (49) fter digestion in.1 N NOH. Lipids were seprted by thin-lyer chromtogrphy nd eluted from the silic gel. The cholesteryl esters were determined fter sponifiction s their trimethylsilyl ether derivtives by gs-liquid chromtogrphy. Triglycerides were mesured colorimetriclly. The cellulr lipids mesured represent intrcellulr lipids, s ddition of 5 pg/ml of heprin to the wsh buffer did not ffect the results obtined (17). [ 4C] cholesterol, ['H cholesteryl olete, nd tri [ 14C] oleoylglycerol (Amershm, Okville, Ontrio) were used to ssess recovery. The incorportion of [ l-'*c]oleic cid into cellulr cholesteryl esters nd triglycerides ws determined s described previously (17). HepG2 cell heptic triglyceride lipse, polipoproteins E nd A- Confluent monolyers were incubted with MEM contining 5% LPDS in the presence or bsence of 1 pg/ml of heprin for 16 h. The conditioned medi were collected in sterile tubes nd stored t -8 C until ssyed. The cells were wshed 3 times with buffer B without BSA nd the cell protein ws determined. The Downloded from by guest, on October 3, 218 Huffet l. Role of LPL, HL, nd proteoglycns in HepC2 cell uptke of 1321

5 ctivity of H, in the conditioned medi ws determined using stble triolein-gum rbic emulsion s substrte, prepred s described previously ( 17). Duplicte 1-pl liquots of medi were dded to the incubtion buffer (1 M NCl,.4 M Tris,.5 g/rril ftty cid-free BSA, ph 8.9) nd 1 p1 of the substrte. Smples were incubted for 1 h t 37 C: nd the relese of oleic cid ws determined s described previously (1 7). HL ctivityws expressed in units/nlg of cell protein, where one unit of ctivity ws defined s 1 nmole of free ftty cid relesed per in1 of medi per h. Apolipoproteins E nd A- in medi were ssyed using modifictions of stndrd EJSA techniques s described previously ( 17). Bovine milk lipoprotein lipse Bovine skim milk LPL ws prepred s described previously (17). The enzyme ws in the dimeric form s ssessed by elution from heprin-sephrose (2). n most experiments involving the ddition of bovine milk LPL, the enzyme ws dded t.25 units of ctivity (1 unit is defined s 1 pmole of free ftty cid relesed per h) per nil of medi contining 5% LPDS nd lipoproteins s described previously (17). This ctivity corrcspondecl to 8 ng of',pl/ml of medi. Control dishes contined medi nd enzyme in the bsence of lipoproteins. The mount of milk LPL dded to the medi of HepG2 cells ws identicl to the mount of LPL, ctivity mesured in medi conditioned by J774 mcrophges (51). Also, this mount pproximtes the LPL ssocited with triglyceride-rich lipoproteins nd their remnnts in pre-heprin plsm (33). n one experiment, rnge of LPL concentrtions, from to 8 ng/ml, were studied. Het-inctivted LP, ws prepred by incubtion of the enzyme for 3 rnin t 56 C: in 2 M N(:1. This preprtion ws devoid of lipolytic ctivity s ssessed using ["C] triolein-gum rbic emulsion, serum-ctivted ssy described previously (17). n somc' experiments, the lipse inhibitor tetrhydrolipsttin (THL, Orlistt, Hoffmn L Roche) ws dissolved in dimethylsulfoxide (DMSO), diluted in medi, nd dded to cells such tht the finl concentrtion of DMSO ws 2 pl/ml of medi. Bsed on initil experiments in which rnge of THL concentrtions ws used (..5 to.5 p ~, ) subsequent experiments were perfimned with THL, concentrtions of'.5 pi. n the bsence of cells, THL inhibited the ctivity of LPL (8 ng/ml) by 75% t.1 p~ nd 96% t.5 p~ ssessed by mesuring free ftty cid relesed from HTG-VLD, into the medi, s described below. Remnnts of HTG- (hydrolyzed ) were prepred by incubting S, 6-4 ( mg of lipoprotein cholesterol) with 1 unit of LPL in 1 nil of MEM medi contining 1% LPDS nd 3% lbumin (w/w), t 37 C for 3 h. The density ws then immeditely rised to d 1.19 g/ml with buffer A t d 1.85 g/ml. These isoltion conditions remove LP, from the V.DL, surfce (52). The extent of lipolysis ws determined by mesuring the free ftty cids relesed, compred to the initil triglyceride content of. The percent triglyceride hydrolysis ws 5 i- 2.5%. Free ftty ;icid relesed nd the mount of free kitty cid ssocited with the reisolted lipoprotein were determincd using kit from ko Pure Chemicls (NEFA (; A<:S-A<:Ol), nimunocorp Sciences nc., Montrel, Qiiebec). To determirie the mount of LPL remining with the isoltctl hydrolyzed, the preprtion ws diluted with medi to 5 pg/ml of cliolesteol rid incubted t 37 C: tor 1 rid 2 h in the bsence ofcells. Free ftty cid relese ws mesured s described hove. No f'iirther lipolysis ws observed. Three hydrolyzed-v,d, preprtions were ssyed for the presence of LPL mss using sndwich ELSA described by Cdelis et l. (53). No LPL mss ws detected. A ~ ol~~me of 1 pl of hydrolyzed-vld, ws ssyed nd the litnit of detection wits 1 ng/well. Typiclly, 1 pl of reinnnt/ml medi ws dded to cells (5 pg, cholesterol/pl). Thereforc, if present, no imxe thn.5 ng of LPL/nil of tncdi would hl~ been dded to cells with the hydrolyzed- \'T2D11 preprtion. Detection of medi HL by immunoblotting Medi were obtdilied from HepG2 cells treted with P-xyloside nd incubted in the bsence of lipoproteins with or without heprin (1 pg/ml). The proteins were' seprted by electrophoresis on 1% polycrylmide (2.7% biscrylrnide) gels contining sodium dodecyl sulfte (SDS-PAGE) on Bio-Rd (Bio-Rd (:nd;i, Mississug, Ontrio) Mini-Proten 1 pprtus fix 1 h t 2 V. Proteins were then trnsferred to polyinylidene difluoride (P\DF) membrne ( Millipore mmobilon-p, Millipore Cnd, Mississug, Ontrio) t 4 C overnight iising buffer of.25 M Tris,.192 M glycine, 2% methnol, ph 8.3. Heptic lipse \vs detected by immunoblot using the Auror estern Blot chemiluminescence kit (CN Cnd, Mississug, Ontrio). The PVDF membrne ws blocked by iiicilbtion for 1 h t room temperture with.2%) Auror Blocking regent in PBS, ph 7.4,.1 % Tween-2. mmunoffiiiity-purified primry ntibody (1 pl in 1 ml of blocking buffer) ws incubted for 1 h with gittion, followed by two 5-min wshes with gittion with blocking buffer. The membrne ws then incubted with gittion with got nti-rbbit gg (1 p1 in 1 nil blocking buffer). The membrne ws then wshed three times (5 min ech with blocking buffer) followed by two 5-min wshes with Auror Assy buffer. The membrne ws incubted for 5 min t room temperture with 5 nil ofchemiluminescent detection solution (Auror), drined of excess Downloded from by guest, on October 3, Journl of Lipid Reserch Volume 38, 1997

6 fluid, nd exposed to Kodk XAR X-ry film for up to 5 min. Bnds corresponding to HL were quntitted using the JAVA Video Anlysis Softwre,Jndel Scientific. An nti-humn heptic lipse peptide ntibody ws prepred in New Zelnd hite rbbits by stndrd techniques. Briefly, synthetic peptide corresponding to mino cids 466 to 477 of humn heptic lipse (EKSKTSKRKR) (54) ws coupled to ovlbumin using glutrldehyde. Anti-peptide ntibody ws isolted by immunoffinity chromtogrphy using peptide-keyhole limpet hemocynin conjugted to Bio-Rd Affi-Gel 1.! 1 f c 5 j i j 16 5 s % 6 4 * 2 $ Sttisticl nlyses " The dt were nlyzed using n unpired Student's t-test. A CHOLESTERYL ESTER P <.5 vs NO ADDTONS NO ADDTONS *LPL TRGLYCERDE C L RESULTS The effect of HTG- (Sr 6-4) on the cholesteryl ester content of HepG2 cells is shown in Fig. lk ncubtion of HTG- (5 pg/ml of cholesterol) for 16 h did not cuse significnt ccumultion of cellulr cholesteryl esters compred to cells not exposed to lipoproteins. Coincubtion of HTG- with 8 ng/ml of bovine milk lipse resulted in 3.5fold increse in cellulr cholesteryl ester compred to control cells or cells incubted with HTG- in the bsence Of LpL ('<.5)' HTG- did not cuse nycellulrccumultion oftriglyceride unless co-incubted with LPL (Fig. 1B). HTG- plus LPL incresed cellulr triglyceride &fold Over control or incubtions in the bsence of LpL (f' <.5).HTG- plus LPL enhnced the incorportion of [ ''C]~lete into both cholesteryl ester nd t,.iglyceride in the Sme proportions s the increse in cellulr mss of these l i p ids (dt not shown). To determine whether the LPL-enhnced uptke of HTG- ws relted to the extent of lipolysis, (Sr 6-4) tht hd been incubted with LPL for 3 h in the bsence of cells nd reisolted by ultrcentrifugtion ws incubted with HepG2 cells. hen identicl mounts of lipoprotein cholesterol were dded, hydrolyzed cused the sme ccumultion of cholesteryl esters s HTG- co-incubted with LPL (Fig. 1). The reisolted hydrolyzed did not contin ny lipolytic ctivity, however, the preprtion contined significntly more free ftty cid thn did the pre-hydrolyzed (Tble 2). These ftty cids comprised only.85% of the initil triglyceride present nd 7 "d 6 5 R -1 P <.5 vs NO ADDTONS 5 p -p 4 3 p 8> " P 2 1 NO ADDTONS Fig. 1. Effect of lipoprotein lipse on the esterified cholesterol nd triglyceride content of HepG2 cells incubted with Type V (HTG-) S6-4 or hydrolyzed HTG-. Fifty pg of HTGVADL(S,6-4) cholesterol/ml of medi (n = 8) ws incubted with HepG2 cells in the bsence or presence of.25 units (8 ng) of bovine milk LPL/ml of medi. Hydrolyzed () ws prepred by inc"htinghtg-with bovinemilk LpL ( l mgof cholesterol with 1 unit of LPL ctivity/ml of medi) for 3 h nd then reisolting by ultrcentrifugtion.fifty pg of cholesterol/ mi (n = 6) ws incubted with HepG2 cells. The cellulr cholesteryl ester (A) nd triglyceride ( R ) were determined s described in Experimentl Procedures. Results re expressed.men 5 S E n refers to the number of experiments for ech condition. totlled 8 pg/ml of medi when dded to cells with the hydrolyzed. Previously, we hd estblished tht ddition of 325 pg of oleic cid, complexed to lbumin, to HTG- in the bsence of LPL would not stimulte cholesteryl ester ccumultion or the incorportion of ["'Clolete into cholesteryl ester (17). Similrly, ddition of olete/lbumin to the hydrolyzed did not enhnce cellulr cholesteryl ester ccumultion bove tht observed with hydrolyzed lone (dt not shown). LPL (8 ng/ml of medi) ws dded to h y drolyzed HTG- to determine whether cholesteryl ester ccumultion would be enhnced. As shown in Huffet l. Role of LPL, HL,nd proteoglycns in HepG2 cell uptke of 1323 Downloded from by guest, on October 3, 218 LPL-enhnced HTG- nd HTG- remnnt uptke by HepGf cells E

7 n 5 w O E w 1 - E 8 v w 6 t- v) w 4 - >. 2 k o w -1 CHOLESTERYL ESTER NO - - ADDTONS +H LPL + H LPL Fig. 2. Effect of het-inctivted lipoprotein lipse on the esterified cholesterol content of HepGP cells incubted with Type N (HTG-) (S, 6-4) or hydrolyzed HTG-. Fifty pg of HTG- VLD. (S, 6-4) or hydrolyzed HTG- cholesterol/ml of medi (n = 4) ws incubted with HepC2 cells in the bsence or presence of 8 ng of ntive or het inctivted hovine milk LPL/ml of medi. Het-inctivted LPL (H LPL) ws prepred by incubtion t 56'C for 3 min in 2 M NCl s described in Experimentl Procedures. Results re expressed s men -C SE. Fig. 1, no effect of dded LPL ws observed. The timecourse curves for the ccumultion of both cellulr cholesteryl ester nd triglyceride, from 2 to 16 h, were similr for hydrolyzed plus LPL, hydrolyzed lone, nd plus LPL (dt not shown). Addition of 1-fold less LPL (.8 ng/ml) to HTG- incresed cellulr cholesteryl ester content 1.2- fold over no dditions. At 4 nd 8 ng/ml, LPL produced increses of cellulr cholesteryl ester of 1.4- nd 1.7-fold, respectively, over the levels observed t 8 ng/ ml of LPL. For hydrolyzed HTG-, no effect of dded LPL ws observed until 4 nd 8 ng/ml of LPL were dded. This produced increses of cholesteryl ester of 1.4- nd 1.7- fold over,.8, nd 8 ng/ml of LPL (dt not shown). Effect of het-inctivted LPL nd tetrhydrolipsttin on HTG- nd remnnt uptke LPL ws het-inctivted by incubtion for 3 min t 56OC, which completely inhibited its ctlytic ctivity. hen dded to cells with HTG-, it ws unble to stimulte cholesteryl ester ccumultion over control cells (Fig. 2). Addition of het-inctivted LPL to hydrolyzed did not enhnce cellulr cholesterol ester ccumultion beyond tht observed for hydrolyzed. Cellulr triglyceride ccumultion ws lso completely inhibited in the presence of the het- inctivted enzyme (dt not shown). As it is not known whether het-inctivtion of LPL preserves the conformtion of the enzyme required for binding to cells, we used the ctive-site inhibitor THL to further investigte the role T of LPL ctlytic ctivity in the uptke of HTG- by HepG2 cells. As shown in Fig. 3, co-incubtion of HTG- with 8 ng/ml of LPL nd.5 p~ THL inhibited cholesteryl ester ccumultion by 91%. THL lso inhib ited LPL-induced HTG- triglyceride ccumultion by 95%. THL hd no effect on the lck of HTG- uptke in the bsence of LPL (not shown). THL lone hd no effect on cellulr cholesteryl esterifiction, cholesteryl ester or triglyceride concentrtions (Fig. 3). Effect of het-inctivted LPL nd tetrhydrolipsttin on uptke n contrst to HTG-, norml humn (15 pg/ml of cholesterol) resulted in 2.5-fold in E 8-6- b w k >. K 2- w k n " " w NO THL - ADDTONS +THL n n 4-1 m 2 2 >. t- TRGLYCERDE NO ADDTONS THL p<.2 vs NO ADD b p<.2 vs T b +THL Fig. 3. The effect of tetrhydrolipsttin on the esterified cholesterol nd triglyceride content of HepGP cells incubted with Type V (HTC-) (S, 6-4) in the presence or bsence of LPL. Fifty pg of HTG- (S, 6-4) cholesterol/ml of medi (n = 7) ws incubted with HepG2 cells in the bsence or presence of.25 units (8 ng) of bovine milk LPL/ml of medi. Tetrhydrolip sttin (THL) dissolved in DMSO ws dded to cells (.5 ~ LM) t the sme time s LPL s described in Experimentl Procedures. A cholesteryl ester; R: triglyceride. Result? re expressed s the men? SE. Results for THL plus without LPL were the Sme s for No Additions nd THL lone. Downloded from by guest, on October 3, Journl of Lipid Reserch Volume 38, 1997

8 crese in cellulr cholesteryl ester compred to control cells (P<.5) (Fig. 4). This ws similr to the extent of cholesteryl ester ccumultion observed with 5 pg/ ml of HTG- cholesterol plus LPL. Coincubtion of LPL (8 ng/ml) with norml incresed cellulr cholesteryl ester 1.3-fold over tht found with in the bsence of LPL (P<.5). Norml incresed cellulr triglyceride only 1.4-fold over control reflecting the much lower triglyceride content of (Tble 2). plus LPL incresed cellulr triglyceride 1.'l-fold (P <.5). The modest enhncement of -derived cholesteryl ester ccumultion by LPL ws prtilly blocked when the het-inctivted enzyme ws used. The ddition of THL to plus LPL completely CHOLESTERYL ESTER b p p.2 vs NO ADDTONS.5 vs + LPL A T NO NORMAL ADDTONS 2 +HLPL +THL +LpL +THL TRGLYCERDE p.5 v NO ADDTONS n 8 E b p c.5 vs + LPL e 18 U n A g 12 A -Y v NO NORMAL ADDTONS +THL *LPL *LpL +THL Fig. 5. Effect of lipoprotein lipse or lipoprotein lipse nd tetrhydrolipsttin on the esterified cholesterol content of HepC2 cells incubted with Type V (HTG-) ( S, 6-4) or hydrolyzed HTG-. Fifty pg of HTG- (S, 6-4) (n = 6), or 5 pg of hydrolyzed ( ) cholesterol/ml of medi (n = 6) ws incubted with HepG2 cells in the bsence or presence of.25 units (8 ng) of bovine milk LPL/ml of medi. Tetrhydrolipsttin (THL) dissolved in DMSO ws dded to cells t concentrtion of.5 p ~ Results. re expressed s men f SE. NO ADD'S, no dditions. blocked the LPL-stimulted cellulr cholesteryl ester ccumultion. n the bsence of LPL, THL hd no effect on -induced cellulr cholesteryl ester ccumultion (Fig. 4) or cholesterol esterifiction (dt not shown) indicting tht THL did not influence -receptor-medited endocytosis. Consistent with previous studies (55), these results indicte tht the LPL-enhnced uptke of ws result of lipolytic modifiction of. nhibition of HepGZ cell HL by tetrhydrolipsttin 1 2 NO ADD'S b p<o.oos vs NO ADD'S p<.2 vs + LPL +HLPL *THL *THL Fig. 4. Effect of lipoprotein lipse, het- inctivted lipoprotein lipse, nd tetrhydrolipsttin on the esterified cholesterol nd triglyceride content of HepG2 cells incubted with norml. One hundred fifty pg of HTG- ( S, 6-4) cholesterol/ml of medi (n = 5 ) ws incubted with HepC2 cells in the bsence or presence of.25 units (8 ng) of bovine milk LPL/ml of medi. Het- inctivted LPL (H LPL) ws prepred by incubtion t 56OC for 3 min in 2 M NC. Tetrhydrolipsttin (THL) dissolved in DMSO m 3 dded to cells t the sme time s LPL. The cellulr cholestelyl ester (A) nd triglyceride (B) were determined s described in Experimentl Procedures. Results re expressed s men f SE; n refers to the number of experiments for ech condition. THL ws dded to hydrolyzed HTG- in the b sence of LPL (Fig. 5). Unexpectedly, we observed tht cholesteryl ester ccumultion ws inhibited by 66% (P <.2). Addition of THL to hydrolyzed HTG in the presence of LPL inhibited cholesteryl ester ccumultion by 5% (P <.2) (Fig. 5). The lck of effect of THL on -induced CE ccumultion (Fig. 4) suggests tht the lipolytic ctivity inhibited by THL is specific for HTG- remnnts. As hydrolyzed HTG- did not hve ny ctive LPL ssocited with it, these experiments indicte tht THL inhibited lipse involved in HTG- remnnt uptke other thn LPL, most likely heptic triglyceride lipse. As shown in Tble 3, HepG2 cells secrete significnt mount of HL ctivity into the medi over 16 h, n mount incresed 5-fold by the ddition of 1 pg/ ml heprin. Addition of.5 p~ THL to the HL ssy inhibited ctivity by %. n one experiment, HL ws relesed from HepG2 cells with heprin nd incubted with HTG- nd hydrolyzed HTG-. Huflet l. Role of LPL, HL, nd proteoglycus in HepG2 cell uptke of 1325 Downloded from by guest, on October 3, 218 O n CHOLESTERYL ESTER z,

9 ~~ TABLE 3. Heptic lipw ctivity, heptic lipse irnmunorccrivic, poe nd poa- relesed hy heprin from HepC2 cclls with or withotit pretretment with 8-xylosicte ~:<ln~lol (:ell\ px~losidt.-trctctl (:t.ll~ Hcprin f kpirin Vriblr Mccliiiiii Medium Krlrshlr - _.._ - nrrrol fifa/h/ncg rrll protrin Heptic lipse ctivity t 16.5 mr[i/rng d l protriri Heptic lipse immu norectivi ty 4i t 19.5 pg/iizg wll pmtriii Apolipoprotein E 15.6 % t.85h 9.4 t.8: t.79 pg/rng r.14 protein < Apolipoprotein A- -.A3 t t.oi 2.35 t t.8 Vlues represent heptic triglyceride lipse ctivity (n = X), heptic lipse immunorectivity (ti = 4), nd mss of poe nd poa- (n = 8) nd re expressed s men t SE. Ten pg of heprin/ml dded. Vlues represent the ctivity or inss in the mrdi plus the ctivity or t~~ss relesed by heprin. P <.1 compred to control medium without heprin. 1 <.1 compred to P-xyloside medium without heprin. P <.3 compred to control medium plus hepr-in. P <.5 compred to P-xyloside medium without heprin. The free ftty cid relesed from HTG- ws 2-fold over bckground, wheres the net free ftty cid relesed from the remnnt ws 5-fold higher thn tht for HTG-. Relese of free ftty cids from both lipoproteins ws inhibited by THL. Collectively, these results indicte tht HL plys n importnt role in enhncing HTG- remnnt uptke in HepG2 cells. Effect of heprin, heprinse, hepritinse, nd P-xyloside on HepG2 cell uptke of HTG- Heprin dded t 1 pg/ml inhibited the cholesteryl ester ccumultion induced by HTG- in the presence of LPL by 7% (P <.1) (Fig. 6). Heprin hs severl potentil mechnisms to explin this effect other thn blocking interction of lipoproteins with cell surfce proteoglycns, such s binding to poe (4, 5). Therefore, we investigted the effects of pretreting HepC2 cells with heprinse or hepritinse (4 units/ ml ech) followed by incubtion with HTC- plus LPL in the presence of ech enzyme. As shown in Fig. 6A, heprinse or hepritinse significntly reduced cellulr cholesteryl ester ccumultion by 48% (P <.1) nd 95% ( P <.1), respectively. Heprin, heprinse, nd hepritinse did not reduce the increse in cellulr triglyceride (Fig. 6B). This indictes tht lipolysis by LP,, cellulr uptke of free ftty cids, nd their subsequent re-esterifiction re not influenced by these tretments nd tht their effects re restricted to blocking the uptke of the cholesteryl ester-contining remnnt lipoproteins. To further estblish the importnce of cell surfce proteoglycns, n lternte pproch ws investigted in which glycosminoglycn synthesis ws blocked. Cells were pretreted for 4 dys with P-xyloside, compound tht cn substitute for the protein core moiety during proteoglycn synthesis, significntly reducing the ppernce of proteoglycns t the cell surfce (5,56). n P-xyloside-treted cells, cholesteryl ester ccumultion ws inhibited by 78% (P <.1) fter incubtion of HTG- plus LPL (Fig. 7A). Cellulr ccumultion of cholesteryl ester fter incubtion with hydrolyzed HTG- in the bsence of LPL ws inhibited by 8% (P <.1) in P-xyloside-treted cells. The reduction in cellulr cholesteryl ester by P-xyloside ws similr for intct HTG- coincubted with LPL or hydrolyzed HTG- coincubted with or without LPL (Fig. 7A) indicting tht ny LPL-enhnced binding ofthe remnnt to cell surfce proteoglycns ws not rte-limiting for uptke. P-Xyloside hd no effect on cellulr ccumultion of triglycerides fter incubtion with HTG- plus LPL, hydrolyzed lone, or in the presence of LPL (dt not shown). P-Xyloside lone hd no effect on cellulr cholesteryl ester or triglyceride concentrtions (dt not shown). P-Xyloside hd no effect on the smll increse in cellulr cholesteryl ester fter incubtion with (Fig. 7B). However, P-xyloside inhibited the dditionl increse in cholesteryl ester induced by in the presence of LPL. Effect of P-xyloside on HepG2 cell-secreted HL, poe, nd poa- t is known tht HL is bound to heptocyte cell surfce proteoglycns (41) nd HepGZ cells secrete he- Downloded from by guest, on October 3, Journl of Lipid Reserch Volume 38, 1997

10 ptic lipse into the medi, n effect enhnced by heprin (Tble 3). Hydrolysis of HTG- by LPL nd the remnnt by HL my fcilitte binding of poe, secreted by heptocytes nd present on heptocyte cell surfces bound to HSPGs (5,57),to remnnt lipoproteins. U p tke of P- from cholesterol-fed rbbit5 by rt heptocytes is enhnced by mrked over-expression of HL or poe, fter cellulr trnsfection of either gene (45, 47). Therefore, we investigted whether the inhibition of cellulr cholesteryl ester ccumultion by HTG plus LPL by P-xyloside ws due in prt to reduced presence of HL nd/or poe on the cell surfce. CHOLESTERYL ESTER n - f n " NO ADD'S CHOLESTERYL ESTER E p<o.oo p<o.o vs - C b V NO ADD'S +&XYLO +B-XYLo *LPL +E-XYLO ~ b! - - ) E w fn w ji Le A p<o.oi vs NO ADD'S p<o.o vs b 8- h ; - - z D 4-2 fn - 2o No ADD'S + HEP No ADD'S pq.4 vs NO ADD'S p<o.ol vs p<.2 V HEP'ASE HEP'TASE Pco.1 + HEP C n U cn - " - P vs NO ADD'S +&XYLO C > TRGLYCERDE O A b C b f n CHOLESTERYLESTER r NO ADD'S 8-XYLO Fig. 7. The effect of -xyloside on the esterified cholesterol nd triglyceride content of HepC2 cells incubted with (A) Type N (HTC-) (Sr 6-4). hydrolyed HTG- or ( 5) in the presence or bsence o f LPL. Fifty pg of HTG- (S,6-4) cholesterol (n = 5 ), 5 pg of hydrolyied HTC- (-) c h e lesterol (n = 5) or 15 pg of norml cholesterol/ml of medi (n = 4) were incubted with HepG2 cells in the bsence or presence of.25 units (8 ng) of bovine milk LPL/ml of medi. HepC2 cells were preincubtecl with or without P-xyloside ( 1 mm) for 4 dys. The medi were chnged nd the sme concentrtion of en7yme c dded to the medi long with lipoproteins, with or without LPL s described in Experimentl Procedures. Results re expressed s the men? SE. Vlues for P-xyloside dded without lipoproteins were the sme s N o Additions. NO ADD'S, no dditions; P-XYLO, -xyloside. + + HEP'ASE HEP'TASE Fig. 6. The effect of heprin, heprinse, nd hepritinse on the esterified cholesterol nd triglyceride content of HepG2 cells incubted with Type N (HTC-) S 6-4 in the presence or bsence of LPL. Fifty pg of HTG- (Sr 6-4) cholesterol/ ml of medi (n = 7) ws incubted with HepGP cells in the bsence or presence of.25 units (8 ng) of bovine milk LPL/ml of medi. HepC2 cells were preincubted with heprin (1 pg) heprinse or hepritinse (4 units/ml of medi) for 4 h. The medi were chnged nd the sme concentrtions of heprin or enzyme were dded to the medi long with lipoproteins with or without LPL s described in Experimentl Procedures. A esterified cholesterol; B triglyceride. Results re expressed s the men 2 SE. Results for heprin, heprinse, or hepritinse dded without lipoproteins or LPL were the sme s N o Additions. N O ADD'S, no dditions; HEP, heprin; HEP'ASE, heprinse; HEP'TASE, hepritinse. As shown in Tble 3, heptic lipse ctivity secreted into the medi over the 16 h incubtion ws low nd did not differ from tht secreted by cells pre-incubted with Pxyloside. However, when 1 pg/ml of heprin ws dded, P-xyloside-treted cells relesed 58% less HL ctivity thn untreted cells (P <.3). As determined by immunoblotting, heptic lipse mss secreted into the medi ws unffected by P-xyloside (Fig. 8 ). Addition of heprin to P-xyloside-treted cells resulted in 55% (P<.1) decrese in HL relesed. The decrese in HL ctivity nd mss in P-xyloside-treted cells is quntittively similr to the decrese in cellulr choles- HuJset l. Role of LPL, HL, nd proteoglycns in HepG2 cell uptke of 1327 Downloded from by guest, on October 3, 218 -F 141

11 HEPARN (-1 (+ (-1 (+) CONTROL p-xylosde Fig. 8. mmunoblot nlysis of medi HL. HepG2 cells were incuhted for 4 dys in the presence or bsence of P-xyloside. Cells were then incubted in the bsence or presence of heprin (O pg/ml) for 6 h. The medi were seprted by SDS-PAGE, blotted, nd the HL ws detected with humn HL ntipeptide ntibody. DSCUSSON Lipoprotein kinetic studies in Type lv hypertriglyceridemic subjects hve estblished tht significnt proportion of the remnnts derived from (S, 64) re clered from the circultion prior to conversion to (6, 7). The liver is likely responsible for the mjority of this clernce by mechnisms tht re now beginning to be understood. A smll mount of LPL is present in plsm, some of which is ssocited with triglyceride-rich lipoproteins nd their remnnts (33). n the present experiments, HepG2 cells were used s model to explore the role of physiologicl concentrtions of LPL nd HL in mediting the interction of HTG- (S, 6-4) nd their remnnts with heptocytes. n prticulr, we wnted to determine whether, in ddition to its ctlytic function, LPL would lso function s lignd. n previous studies we estblished tht HTG- were not tken up by HepG2 cells unless co-incubted with LPL (17). The present studies clerly show tht remnnt formtion by ctlyticlly ctive LPL is required for HepG2 cell uptke s ssessed by cellulr cholesteryl ester ccumultion. However, LPL does not ply significnt role s lignd in mediting the uptke of HTG remnnts. LPL hs been shown to enhnce the binding, uptke, 1328 Journl of Lipid Reserch Volume 38, 1997 Downloded from by guest, on October 3, 218 teryl ester induced by HTG- plus LPL, hydrolyzed HTG-, or hydrolyzed HTG- plus LPL. This suggests role for HL in enhncing the uptke of HTG remnnts by HepG2 cells. HepC2 cells secreted poe into the medi over 16 h of incubtion, n mount incresed by 22% ( P<.1) in the presence of 1 pg/ ml of heprin (Tble 3). P-Xyloside decresed the mount of poe secreted into the medi by 4% ( P <.1), in the presence or bsence of 1 pg/ml of heprin. ApoA- secretion ws not ffected by heprin or Pxyloside (Tble 3). nd degrdtion of rdiolbeled triglyceride-rich lipoproteins in vriety of cells (19-28). t hs been postulted tht LPL concentrtes lipoproteins t the cell surfce by fcilitting their binding to cell surfce proteoglycns which then enbles them to interct with cellulr receptors including the LRP nd/or the receptor (1,19,21-26). However, in most studies high concentrtions of LPL hve been used (greter thn 1 ng/ml), the mjority using between 5 nd 2 ng/ml. n humn pre-heprin plsm, the concentrtion of LPL is less thn 7 ng/ml (33) which, on molr bsis, is mny-fold less thn the concentrtion of triglyceride-rich lipoproteins in plsm. The results of the present study show tht t physiologicl concentrtions, LPL enhnces the interction of HTG- with HepG2 cells, nd tht ctlyticlly ctive enzyme is required. Het-inctivted LPL could not enhnce intrcellulr cholesteryl ester ccumultion. However, s it is likely tht het inctivtion results in LPL conformtionl chnges tht my influence the lipoprotein binding nd cell surfce binding functions of LPL, we used the specific lipse inhibitor THL. THL completely inhibited the ctlytic ctivity of LPL nd completely blocked the bility of LPL to enhnce HTG-induced HepG2 cell cholesteryl ester ccumultion. t is known tht THL binds to Ser152 of pncretic lipse (58), one of the residues of the ctlytic trid. Due to the close structurl homology between pncretic lipse nd LPL, it is ssumed by nlogy tht THL binds to Ser134 of bovine LPL (59). Lookene, Skottov, nd Olivecron (59) demonstrted tht THL only binds to the dimeric form of bovine LPL nd tht THL binding to LPL does not pper to chnge the enzyme conformtion or ffect its binding to heprin. THL increses, rther thn decreses, LPL s interction with lipoprotein surfces. THL does not ffect the binding of LPL to the LRP or to P- (2). Collectively this indictes tht, in our experiments, the inhibition of LPL-enhnced cellulr interction with HTG- by THL is relted entirely to inhibition of ctlytic ctivity nd not to the loss of LPL s bility to bind to proteoglycns, the LRP, or lipoproteins. The reson tht our findings differ from those of Chppell et l. (21,22) nd Mulder et l. (25,26) is not redily pprent. These investigtorsshowed tht either het-inctivted LPL (26) or LPL tht hd been inhibited by PNPDC or PMSF (22) did not ffect the degrdtion of iodinted norml. Their results my be relted to the high concentrtions of LPL used (.1 pg/ ml nd 3.4 pg/ml, respectively), the use of fibroblsts rther thn HepC2 cells, the use of iodinted lipoproteins, or incomplete inhibition of LPL. Residul ctive LPL my hve been sufficient to stimulte lipoprotein lipolysis nd subsequent cellulr degrdtion (22). The effect of LPL-induced cellulr lipoprotein uptke

12 (other thn secreted iodides) on cellulr cholesterol metbolism (cellulr cholesteryl ester content or cholesterol esterifiction) ws not reported. n further ttempt to determine whether LPL could function s lignd nd medite the uptke of HTG- remnnts by HepG2 cells, remnnts were prepred invitrowithlplsuch tht5% Ofthetriglycerides were hydrolyzed. These remnnts, free of LPL ctivity, induced the sme cellulr cholesteryl ester ccumultion s ntive HTG- incubted in the presence of LPL, indicting tht LPL ws not required for remnnt uptke fter initil lipolysis. At 5- nd 1- fold higher concentrtions of LPL (4 nd 8 ng/ml, representing rtio of LPL to lipoprotein cholesterol of 1 :625), modestly stimulted HTG- remnnt u p tke (1.4- to 1.7-fold). Although the LPL ws ctive, it is possible tht the stimulted uptke ws independent of ctlytic ctivity. This is consistentwith recent liver perfusion studies in which high concentrtions of ctlyticlly inctive LPL (LPLtolipoprotein cholesterol 1 : 1) stimulted heptic chylomicron uptke %fold (36). t hs been shown tht the dimeric form of LPL is required for mediting the interction of lipoproteins with the cell surfce (2, 3) nd tht the presence of monomers inhibits dimeric LPL-stimulted uptke of rbbit p- by Hep3B cells (3). The mjor form of LPL in pre-heprin plsm is monomeric (33). n the present studies, dimeric LPL ws used t concentrtions tht would pproximte the mounts ssocited with triglyceride-rich lipoproteins nd their remnnts (33). As we were unble to show tht ctive dimeric LPL (8 ng/ ml) could enhnce cellulr cholesteryl ester ccumultion by HTG- remnnts, it is unlikely tht the inctive monomer would be ny different. hether sufficient LPL exists in humn plsm to function s lignd fcilitting remnnt heptic uptke remins n importnt unnswered question. On the other hnd, these experiments reveled tht lipolytic ctivity other thn LPL ws importnt for remnnt uptke (Fig. 4). As shown in Tble 3, HepG2 cells secrete ctive HL tht is inctivted by THL. These findings indicte tht heptocyte HL ctivity hs n importnt function in remnnt uptke s mesured by cellulr cholesterol esterifiction nd cholesteryl ester ccumultion. Previous studies hve demonstrted tht HL is importnt in the ctbolism of remnnt lipoproteins. Remnnt lipoproteins ccumulte in plsm of ptients with HL deficiency (38-4), observtions consistent with niml studies in which nti-hl ntibodies resulted in incresed plsm remnnts due to decresed heptic clernce (37, 41-43) or the trgeted inctivtion of the HL gene in mice resulted in delyed chylomicron clernce (6). Ji et l. (45) hve shown tht rt heptom cells (MA-RH7777) trnsfected with the humn HL gene secrete high levels of the enzyme which enhnces the binding nd uptke of rbbit P- 3-fold over nontrnsfected cells. n recent studies of Krp et l. (61), ddition of exogenous HL (1 ng/ml) to chylomicrons nd rbbit p- stimulted uptke in HepGP cells. Our findings clerly show tht inhibition of bsl ctivity of HL in HepG2 cells significntly inhibits the bility of humn HTG- remnnts to induce cellulr cholesteryl ester ccumultion. t is importnt to note tht HL ctivity cnnot substitute for LPL ctivity s HTG- without prior exposure to LPL fil to induce ny cellulr increse cholesteryl ester. This is consistent with the concept tht lrge triglyceride-rich lipoproteins re poor substrtes for HL unless previously exposed to LPL (41, 62). Our demonstrtion of role for HL in HTG- remnnt uptke in HepG2 cells could explin our inbility to demonstrte tht LPL plys significnt role beyond its ctlytic function. n contrst to FH-fibroblsts, HL is present on the cell surfce of HepG2 cells nd, in ddition to its ctlytic function, it my lso provide lignd function. Addition of LPL to HTG- remnnts produced no further enhncement of cellulr cholesteryl ester ccumultion (Figs. 1, 5). t is possible tht the HL, lredy present on the cell surfce, ws sufficient to medite uptke. Approximtely 3% of remnnt-induced cholesteryl ester ccumultion ws not inhibitble by THL, suggesting tht some uptke of remnnts occurs independent of HL ctlytic ctivity. hether this uptke is independent of HL or reflects role for HL beyond its ctlytic function, s discussed below, remins to be elucidted. Cell surfce proteoglycns re importnt prticipnts in the cellulr ctbolism triglyceride-rich lipoproteins in the presence of LPL (19, 21, 22, 25, 26) nd ctbolism of poe-enriched rbbit P- in the bsence of LPL (46, 47, 63). The current studies indicte tht this is lso true for cellulr uptke of humn HTG- coincubted with LPL or HTG- remnnts, but not ntive HTG-. Tretment of cells with heprinse, hepritinse, or p-xyloside to deplete cell surfce proteoglycns blocked cholesteryl ester ccumultion induced by HTG- plus LPL or HTG- remnnts. The increse in cellulr triglyceride ccumultion ws not inhibited, indicting tht depletion of cell surfce proteoglycns hd no effect on the ctivity of LPL or HL, but only blocked the uptke of the cholesteryl ester contined in the remnnt. For HSPGs to fcilitte uptke, remnnt formtion is required; however, fter initil lipolysis, the HSPG requirement is observed in the bsence of LPL. Depletion of cell surfce proteoglycns blocked cellulr ccumultion of cholesteryl esters induced by hydrolyzed isolted free of LPL ctivity. Studies in MA-RH7777 cells hve demonstrted tht heprinse does not ffect the binding of rbbit p- Downloded from by guest, on October 3, 218 Huffet l. Role of LPL, HL, nd proteoglycm in HepG2 cell uptke of 1329

13 unless these cells hve been trnsfected with poe3 or the P- ws supplemented with exogenous poe (46,47,63). The results of the present study show tht the requirement of HSPGs for enhncing HTG- remnnt uptke does not depend on exogenous LPL or exogenous poe over nd bove tht present on the lipoprotein nd/or secreted from HepG2 cells. Studies presented in this pper indicte tht one of the mechnisms whereby cell surfce proteoglycns niedite the cellulr cholesteryl ester ccumultion cused by HTG- remnnts is by nchoring secreted HL. P-Xyloside significntly reduced the mount of H, iictivity nd immunorectivity relesed from HepG2 cells with heprin. This result is consistent with the studies of Ji et l. (4.5) who demonstrted tht overexpression of HL by rt McA-RH7777 heptom cells significntly enhnced rbbit P- uptke, process inhibited by hepr-inse. However, role for proteoglycn-bound HL, AS not pprent in non- trnsfected cells s heprinse hd no effect on P-VLD, uptke under bsl conditions. Our experiments with THL nd P-xyloside show tht HL on HepG2 cell surtces significntly enhnces HTG- remnnt uptke nd tht the role of cell surfce proteoglycns in rernntit uptke is, in prt, to bind secreted HL. Hydrolysis of chylomicrons nd VLD, by LPL (17,5 1, 64,65) nd their remnnts by HL (44) gretly increses the exposure of poe epitopes, Fcilitting enhnced binding to specific ntibodies nd lipoprotein receptors. n ddition, hydrolysis of reinnnt surfce lipids by HL my fcilitte the cquisition of cell surfce poe. ApoE is secreted by heptocytes nti is boimd to cell surfce HSPGs (5, 57). Addition of exogenous poe to rbbit P- (46, 63, 66) or humn (64) or the incubtion of rbbit P- with rt heptom cells over-expressing poe (47) enhnces uptke by either the, receptor or the L,. n the present studies, P-xyloside significntly decresed the rnoun t of poe present on the cell surfce nd relesed into the medi. This indictes tht nother mechnism whereby cell surfce HSPGs medite cellulr cholesteryl ester ccumultion cused by HTG-VLD, remnnts is the binding of secreted poe. Although our experiments demonstrted tht HL lipolytic ctivity ws required for HTG- remnnt uptke, we did not determine whether HL plyed role beyond its ctlytic function. t is possible tht HSPGs bind nd internlize remnnts fter interction with HL, s suggested previously (45). Alterntively, internliztion of remnnts s prt of remnnt-h,-hspg complex could occur fter hydrolysis, where HL functioris s bridge between the remnnt nd HSPGs, s suggested recently by Shfi et l. (41). Obunike et l. (24) hve suggested tht LP, my senre similr fiinc- tion in the uptke of nd by mcrophges, cells tht secrete LPL but not HL. HL, could lso ficilitte HTG-VLD, remnnt binding nd internliztion by mediting binding to the LRP. Others hw denioiistrted tht excess LPL bound to norml VLD, (21 ) or rbbit P- (1, 2) medites binding to tlie LRP on FH-fibroblsts. Addition of excess, exogenous, ctlyticlly inctive HL to chylomicrons or rbbit - hs been shown to stimulte uptke in Hep3b cells, n effect greter thn tht observed for n equivlent mount of LPL (61). Therefore, fter lipolysis, HSPG bound HL could lso serve to concentrte the reirinnt t the cell surfce, llowing the trnsfer of cell surfiicr bound poe to the remnnt prior to uptke by LRP or the receptor. n vivo, lrge HTG- (S, 6-4) prticles re clered directly from the circultion (6, 7). n ddition, most of the remnnts formed from these lrge VLD. re clered lri-orn plsm without conversion to. LPL is ble dissocite from endothelil cell surfces diuing lipolysis, some of which ssocites with triglyceriderich lipoproteins nd their remnnts (33). The prrscnt studies suggest tht LPL, t physiologicl concentrtions, does not ply significnt role in mediting HTG- nd HTG- remnnt uptke beyond its ctlytic function. On the other hnd, we provide evidence tht H. ctivity significntly enhnces uptke of' the HTG-VL,DL remnnt rid tht cell surfce proteoglycns ply n importnt role in the interction of HT(:- VLD, remnnts with HepG2 cells. 'hether the importnce of heprn sulfte proteoglycns lies in nchoring secreted heptic lipse nd secreted poe or h binding the remnnt prior to either internliztion directly or trnsfer to cellulr receptors remins to be clrified.m e thnk Sndr Kleinstiver for expert Lechnicl ssistnce nd Dr. A. Evns nd John ylie for performing the heptic triglyceride lipse ctivity nd imniunoblot nlyses. e lso thnk Dr. Pierre Julin for performing the LP, ELSA ssys. This work ws supported by Medicl Reserch Council of <:nd Grnt (MA814) to M.. H. M. M'. H is Creer nvestigtor of the Hert rid Stroke Foundtion of Ontrio. D.B.M. is the recipient of Hert nd Stroke Foundtion of Cnd Reserch Fellowship. Mnnusrrifit rmriurd 24 Sqitrmbur 1996 curd in rroisrd form 3 fi~brur)) 19% REFERENCES Hvel, K. J., nd J. P. 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Hvekes The binding of humn lipoprotein lipse treted by the humn heptom cell line HepG2. Biochim. Biophys. Act. 181: illims, S. E.,. noue, H. Trn, G.,. Fry, M.. Pldet, P. H. verius, J. M. Llouel, D. A. Chppell, nd D. K. Stricklnd The crboxyl-terminl domin of lipoprotein lipse binds to the low density lipoprotein receptor-relted protein/2- mcroglobulin receptor (LRP) nd medites binding of norml very low density lipoproteins to LRP.J. Biol. Chem. 269: illims, K. J., G. M. Fless, K. A. Petrie, M. L. Snyder, R.. Broci, nd T. L. Swenson Mechnisms by which lipoprotein lipse lters cellulr metbolism of lipoprotein[], low density lipoprotein nd nscent lipoproteins. J. Biol. Chem. 267: illnow, T. E., J. L. Goldstein, K. Orth, M. S. Brown, nd J. Herz Low density lipoprotein receptor-relted Downloded from by guest, on October 3, 218 Huffet ul. Role of LPL, HL, nd proteoglycns in HepG2 cell uptke of 1331

15 protein nd gp33 bind similr lignds, including plsminogen ctivtor- inhibitor complexes nd lctoferrin, n inhibitor of chylomicron remnnt clernce.,/. Biol. Ch. 267: Krpp, A., H. J. Zhng, D. Ginzinger, M. S. Liu, A. Lindberg, G. Olivecron, M. R. Hyden, nd U. Beisiegel Structurl fetures in lipoprotein lipse necessry for the medition of lipoprotein uptke into cel1s.j. Lifiid RES. 36: Choi, S. Y., P. Sivrm, D. E. lker, L. K. Curtiss, D. G. Gretch, S. L. Sturley, A. D. Attie, R. J. Deckelbum, nd. J. Goldberg Lipoprotein lipse ssocition with lipoproteins involves protein-protein interction with polipoprotein B. J. Biol. Chem Goldberg,. J., J. J. Kndel, C. B. Blum, nd N. H. Ginsberg Assocition of plsm lipoproteins with postheprin lipse ctivities. J. Clin. nvest. 78: Vilell, E., J. Joven, M. Fernndez, S. Vilro, J. D. Brunzell, T. Olivecron, nd G. Bengtsson-Olivecron Lipoprotein lipse in humn plsm is minly inctive nd ssocited with cholesterol-rich lipoproteins.,/. Lipid Rm Krpe, F., T. 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Connelly, G. F. Mguire, J. A. Little, nd R. A. Hegele P- in heptic lipse deficiency induces poe-medited cholesterol ester ccumultion in mcrophges. ArtPriosrlPr. Thromb. 13: Shfi, S., S. E. Brdy, A. Bensdoun, nd R. J. Hvel Role of heptic lipse in the uptke nd processing of chylomicron remnnts in rt liver. J. Lipid Res. 35: Goldberg,. J., N. A. Le, J. R. Pterniti, H. N. Ginsberg, F. T. Lindgren, nd. V. Brown Lipoprotein metbolism during cute inhibition of heptic triglyceride lipse in the cynomolgus mnkey.j. Clin. nvest. 7: Dggy, B. P., nd A. Bensdoun Enrichment of polipoprotein B-48 in the density clss following in vivo inhibition of heptic lipse. Biochim. Biophys. Act. 877: Brsemle, D. L., K. Cornely-Moss, nd A. Bensdoun Heptic lipse tretment of chylomicron remnnts increses exposure of polipoprotein E. J. Lipids Res Ji, Z. S., S. J. Luer, S. Fzio, A. Bensdoun, J. M. Tylor, nd R.. Mhley Enhnced binding nd uptke of remnnt lipoproteins by heptic lipse-secreting heptom cells in culture. J. 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Huff Evidence tht cholesteryl ester nd triglyceride ccumultion in 5774 mcrophges induced by very low density lipoprotein sub- frctions occurs by different mechnisms../. Lipid &.s Goldberg,. J., N. A. Le, H. N. Ginsberg, R. M. Kruss. nd F. T. Lindgren Lipoprotein metbolism during cute inhibition of lipoprotein lipse in the cynomolgus monkey.,/. Clin. nvest. 81: Cdelis, F., P. Julien, J. P. Vlet, Y. Deshies, M. R. Ven Murthy, nd P. Lupien A monoclonl ntibody ginst humn lipoprotein lipse inhibiting heprin binding without ffecting ctlytic ctivity. Biochem. Cell Riol Sthnke, G., R. Sprengel, J. Augustin, nd H. ill Humn heptic triglyceride lipse: cdna cloning, mino cid sequence nd expression in cultured cell line. Dif fmentition. 35: Avirm, M., E. Biermn, nd A. Chit Modifiction of low density lipoprotein lipse or heptic lipse induces enhnced uptke nd cholesterol ccumultion in cells. J. Bid. Ch Shimdd, K., nd T. Ozw Modultion of glycosminoglycn production nd ntithrombin 111 binding by cultured ortic endothelil cells treted with 4-methylumbelliferyl-bet-D-xyloside. Artetiosclerosis. 7: Hmilton, R. L., J. S. ong, L,. S. Guo, S. Krisns, nd R. J. Hvel Apolipoprotein E locliztion in rt heptocytes by immunogold lbeling of cryothin sections. J Lipid &.s. 31: Hdvry, P.,. Sidler,. Meister,. Vetter, nd H. olfer The lipse inhibitor tetrhydrolipsttin binds covlently to the puttive ctive site serine of pncretic lipse. J. Biol. Chem Lookene, A., N. Skottov, nd G. Olivecron nterctions of lipoprotein lipse with the ctive site inhibitor tetrhydrolipsttin (Orlistt@). bhr.,. Bioch Homnics, G. E., H. V. de Silv, J. Osd, S. H. Zhng, H. ong, J. Borensztjn, nd N. Med Mild dyslipi- Downloded from by guest, on October 3, Journl of Lipid Reserch Volume 38, 1997