Inhibition of lipoprotein lipase induced cholesterol ester accumulation in human hepatoma HepG2 cells

atherosclerosis ELSEVIER

Atherosclerosis 120 (1996) 101~114

Inhibition of lipoprotein lipase induced cholesterol ester accumulation in human hepatoma HepG2 cells Katherine Cianflone”, Rita Kohen Avramoglub, Cynthia Sawyez”, Murray W. Huff” “Robarts Research Institute, University Unit ,for the Prevention of’ Cardiovascular

bMcGill

of Western Ontario, London, Disease, McGill University,

Received4 April 1995;revision received9 August 1995;accepted9

ON, Canada Montreal, PQ. Canada August

1995

Abstract

It has been suggestedpreviously that lipoprotein lipasemay act as a ligand to enhancebinding and uptake of lipoprotein particles.In the presentstudy we have examinedthe capacity of bovine milk lipoprotein lipaseto induce intracellular accumulation of triglyceride and cholesterol ester by VLDL (S, 60-400) isolated from Type IV hypertriglyceridemicsubject(HTg-VLDL) in HepG2 cells,independentof its lipolytic activity. We have alsoattempted to elucidate the cellular receptor mechanismsresponsiblefor these effects. HTg-VLDL-mediated increasesin intracellular triglyceride and cholesterol ester were dependenton the presenceof an active lipase. Bovine milk lipoprotein lipase(LPL) increasestriglyceride massby 301% 2 28%(P < 0.0005)and cholesterolestermassby 176% k 12%(P < 0.0005).TheseHTg-VLDL-mediated increases in intracellular triglyceride and cholesterolesterdid not occur when heat-inactivatedlipasewas used.Rhizopus lipasecould replaceLPL and causeequivalent increasesin intracellular triglyceride and cholesterolester(472% & 61%(P < 0.005)and 202% + 25%(P < 0.025)respectively vs. control). HTg-VLDL treated with LPL and reisolatedalsocausedequivalentincreases(274% k 18%(P < 0.01) and 177% + 12% (P < 0.005) for triglyceride and cholesterolester). LDL also causedincreasesin intracellular cholesterolester(189% k 20% (P < 0.005)),althoughthree timesmore LDL cholesterolhad to be addedto achieve the sameeffect. TheseLDL-induced increaseswereeffectively blocked by monoclonalantibodiesdirected againstthe B,E receptor binding domainsof apo B ( - 97% _+ 13%(P < 0.0005)with anti-apo B 5Ell and - 68% 2 13%(P x 0.05) for anti-apo B BlB3) or by anti-B,E receptorantibodies( - 77% + 7% (P < 0.01)antibody C7). Thesesame antibodieshad little effect on the HTg-VLDL + LPL-inducedincreases in cholesterolester( + 210/r,,+ 15%and - 22% for 5El1, BlB3 and C7, respectively).Monoclonal anti-apoE antibodiesalsohad no effect on LDL-mediatedincreases in intracellularcholesterolester,but had a smalland significanteffect on VLDL-mediated increases in cholesterolester. However, heparin, which interferes with cell surface proteoglycan interaction, was very effective at blocking HTg-VLDL-mediated increasesin cholesterolesterin the presenceof LPL (- 86% k 8% P < 0.0005).Heparin was alsoeffective in the presenceof Rhizopus lipase( - 79%) or lipolyzed re-isolatedHTg-VLDL ( - 95%). Theseresults suggest that lipoprotein lipase may enhance the uptake process beyond its role in lipolytic remodelling but doesnot appearto be an absoluterequirement.In contrast, heparin had no effect on LDL-mediated cholesterol ester accumulation.Lactoferrin, which inhibits interaction with the low density lipoprotein receptor-relatedprotein * Corresponding author,CardiologyH7.35,RoyalVictoriaHospital,687PineAve. West,Montreal,PQ + 1 514 842 1231, ext 5426; Fax:

+ 1 514 982 0686. ’ CareerInvestigatorof the HeartandStroke Foundation

0021-9150/96/$15.00

0 1996 Elsevier

S’SDI 0021-9150(95)05690-X

Science

Ireland

Ltd.

of Ontario. All rights

reserved

H3A

1Al,

Canada.

Tel.:

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(LRP), was also very effective at inhibiting HTg-VLDL increases in intracellular cholesterol ester (- 95X k 6X, P c 0.01). However, there was no effect of either heparin or lactoferrin on HTg-VLDL-mediated triglyceride accumulation. Thus cell surface heparin sulphate may facilitate intracellular lipid acquisition by providing a stabilizing bridge with the lipoproteins and enhance uptake through receptor-mediated processessuch as LRP. Keywords:

Hepatocyte; Proteoglycan; VLDL; Lipoproteins

1. Introduction Very low density lipoproteins (VLDL) are hepatically derived lipoproteins that transport triglyceride and cholesterol to peripheral tissues. In normolipidemic human subjects, most of the VLDL is delipidated progressively, and 90% is converted to low density lipoproteins (LDL) [l]. In normolipidemic subjects, S, 60-400 is a minor fraction of the total VLDL. Type IV hypertriglyceridemia is characterized by increased levels of plasma VLDL due either to overproduction or to impaired catabolism of these lipoproteins, and has been associated with increased risk of cardiovascular disease [2]. The increase in plasma VLDL is primarily in the larger (S, 60-400) particles and a much smaller proportion of this material proceeds through the delipidation pathway to form LDL. A substantial proportion of these large VLDL particles (S, 60-400) appear to be directly catabolized and are not progressively delipidated [3,4]. Therefore, the aim of the present study is to examine potential clearance mechanisms of these particles directly by the liver. In vitro studies have demonstrated that a variety of cells are capable of metabolizing these particles, including macrophages, lymphocytes and HepG2 cells. HepG2 cells are a transformed human hepatoma cell line that have been widely used to study hepatic metabolism. HepG2 cells incubated with ‘251-labelled VLDL will bind VLDL [5]. However, the results are conflicting with respect to uptake and degradation of the particles [6]. Interpretation is further complicated by the dissociable nature of the radiolabelled lipids and many of the apolipoproteins that make up the VLDL particle. This laboratory has measured changes in the intracellular pools of triglyceride and cholesterol ester as a reflection of the uptake of hypertriglyceridemic VLDL (HTg-

VLDL)-derived lipids by macrophage and HepG2 cells, a process directly correlated to an increase in the incorporation of radiolabelled oleate into cholesterol ester and triglyceride [7-91. Hypertriglyceridemic Type IV VLDL added alone to HepG2 cells does not produce intracellular accumulation of triglyceride and cholesterol ester, nor does it increase the incorporation of [14C]oleate into triglyceride or cholesterol ester [8]. However, in the presence of bovine milk lipoprotein lipase (LPL), there is a three-fold increase in intracellular cholesterol ester and a seven-fold increase in intracellular triglyceride mass. These studies suggest, therefore, that lipolytic remodelling of Type IV VLDL is a prerequisite for their uptake by HepG2 cells [8]. Our previous work demonstrated that in 5774 macrophages lipoprotein-derived apo E was an important component of cholesterol ester accumulation induced by HTg-VLDL. In these cells, which do not produce apo E, Type III VLDL (containing E2) or E-poor VLDL caused significantly less cholesterol ester accumulation than E3-VLDL. Antibodies directed against the binding site of apo E were also effective at blocking cholesterol ester accumulation. In contrast, the same dosage of anti-apo E antibodies had little effect on VLDL-induced cholesterol ester accumulation in HepG2 cells [8]. Although the role of apo E in inducing cholesterol ester and triglyceride accumulation has been partially addressed in our previous publications [8,9], no attempt was made to address the receptor uptake mechanisms responsible and the present study addresses that question. Beisegel et al. [lo] proposed that lipoprotein lipase may act as a ligand and interact with the low density lipoprotein receptor-related protein (LRP) and enhance binding of triglyceride-rich lipoproteins [lo]. This hypothesis has been ex-

K. Cinnfione

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tended and lipoprotein lipase-mediated clearance of a number of lipoproteins including p-VLDL (from cholesterol-fed rabbits), chylomicron remnants, normolipidemic VLDL, LDL and Lp(a) has been proposed in a variety of cell types: macrophages, HepG2 and normal and LDL receptor negative fibroblasts [l 1 - 171. Although LPL substantially increases 4°C binding of “‘Ilipoproteins in these studies, the effects were not necessarily translated into equivalent effects on uptake and degradation when assayed at 37°C [13,14,16,17]. For example, LPL caused an 80% increase in binding of I ‘25-LDL at 4°C in normal human skin fibroblasts, but only a 31% increase in uptake when assayed at 37°C [14]. In the present studies, we have examined the capacity of bovine milk lipoprotein lipase to induce intracellular accumulation of triglyceride and cholesterol ester from Type IV hypertriglyceridemic VLDL (HTgVLDL) independent of its lipolytic capacity, and attempted to elucidate the cellular receptor mechanisms responsible. In particular, the major objective was to determine the contribution of the LDL (B,E) receptor. The results indicate that the LDL (B,E) receptor plays little role in uptake and that HTg-VLDL-induced cholesterol ester accumulation in HepG2 cells is mediated through proteoglycan interactions which may involve the LRP receptor. 2. Materials

and methods

2.1. Lipoprotein

isolation

Fresh plasma obtained from hypertriglyceridemic patients was generously provided by Dr. Bernard Wolfe, Outpatient Endocrinology Clinic at University Hospital, London, ON and by Dr. David Blank, Lipid Clinic/Metabolic Day Centre at the Royal Victoria Hospital, Montreal. Type IV hypertriglyceridemia was defined as a fasting hypertriglyceridemia with no floating chylomicronemia as defined by Schaefer and Levy [ 181. None of the patients were homozygous for E2 as determined by analytical isoelectric focusing gel electrophoresis [ 191. These studies were approved by the University of Western Ontario Health Sciences Standing Committee on Human Research and the Royal Victoria Hospital Ethics Commit-

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tee, and all subjects gave informed consent. Blood was collected into EDTA tubes (final concentration 0.15%) and plasma was obtained after centrifugation at 1000 x g for 30 min at 4°C. Plasma was layered under one-half to one volume of buffer A (1.006 g/ml density solution containing 0.195 M NaCl, 1 mM Tris, pH 7.4, 1 mM EDTA-Na2 and 3 mM NaN,). HTg-VLDL (S, 60-400) was isolated by ultracentrifugation in a Beckman 60Ti rotor for 2 h at 40 000 rev./min at 12°C [20]. This procedure does not isolate total VLDL but only the large, triglyceride-rich VLDL fraction and is defined HTg-VLDL. The collected HTg-VLDL fraction was washed through an equal volume of buffer A at 40 000 rev.,‘min at 12°C for 18 h. VLDL was sterile filtered (0.45 Llrn) and stored at 4°C. For LDL isolation, normolipidemic plasma was raised to a density of LI = 1.019 g/ml with density 1.350 g/ml KBr and ultracentrifuged at 40 000 rev./min at 12°C for 18 h. The infranate was increased to density 1.063 g/ml and spun at 40 000 rev.!min for 24 h at 12°C. LDL was dialysed against buffer B (0.15 M NaCl, 1 mM Tris, pH 7.4. 1 mM EDTA-Na,) sterile filtered and kept at 4°C. Lipoprotein-deficient serum (LPDS) was isolated from the plasma of healthy normolipidemic volunteers at a density > 1.21 g/ml, dialysed against buffer B, clotted with thrombin (200 units/ml), heat-inactivated at 56°C for 30 min, sterile filtered and stored at - 20°C. Lipolyzed HTg-VLDL was prepared by incubating 5.0 mg HTg-VLDL triglyceride with 1 unit of bovine milk lipoprotein lipase (LPL) in 2.0 ml of Minimum Essential Medium (MEM) containing IO mglml fatty acid-free albumin at 37°C for 4 h where 1 unit of lipoprotein lipase is defined as I l!mol of free fatty acid released per hour [8]. The lipolyzed particles were reisolated by ultracentrifugation in buffer ‘4 at density 1.019 g/ml at 40000 rev.:‘min for 18 h at 12°C under conditions reported to dissociate LPL from the VLDL [21]. All lipoprotein samples were analyzed for protein content by modified Lowry method [22]; total cholesterol and triglyceride mass were measured by calorimetric enzymatic kits (CHOD-PAP and triglyceride-without-freeglycerol respectively, from Boehringer Mannheim Canada, Laval. PQ).

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2.2. HepG2 cells

HepG2 cells obtained from the American Tissue Culture Collection (Rockville, MD) were routinely grown in MEM supplemented with 10% fetal calf serum and 10 IU/ml penicillin, 10 pg/ml streptomycin, 0.25 pgg/ml fungizone, 0.2% sodium bicarbonate, 1 mM sodium pyruvate in 100 mm culture dishes or 75 cm* flasks with 10 ml medium in a 37°C incubator with 5% CO,. Flasks were subcultured every 7-10 days with a split ratio of 1:3. The cells were dislodged from the culture flask with 0.25% trypsin-EDTA for 5 min at 37°C. For experiments, cells were plated out at a density of 1.3 x lo4 cells per cm’ in 17 mm dishes (24 well plates). Near confluency the cells were changed to a fetal calf serum-free medium supplemented with 5% LPDS for 18 h prior to experiments. The indicated concentrations of lipoproteins and all other additions were added simultaneously to the cells in triplicate and incubated for 24 h. At the end of the incubation period the cells were washed three times with 1 ml ice-cold buffer B (0.15 M NaCl, 50 mM Tris, pH 7.4, 0.2% BSA) and three additional washes with 1 ml of buffer B without BSA, and the cell lipids were extracted with 1 ml of hexane-isopropanol 3:2 (v/v). After 30 min the extracts were removed and the cells were re-extracted once with an additional 1 ml of hexane-isopropanol 3:2 (v/v) and the extracts were pooled. The soluble cell protein was dissolved in 1 ml of 0.1 N NaOH and was measured by a modified Lowry method [22] using bovine serum albumin (BSA) as a standard. 2.3. Determination

of cellular lipid content

Cell lipid extracts were evaporated under nitrogen and redissolved in a known volume of chloroform-methanol (2:l v/v). The extract was applied to Silica G thin layer chromatography (TLC) plate and developed in petroleum ether-diethyl etheracetic acid (84: 15: 1 v/v/v); the lipid spots were visualized by exposure to iodine vapour and identified by comparison to reference lipids. The spots corresponding to triglyceride and cholesterol ester were scraped into 12 x 75 test tubes for quantification. Recovery of triglyceride and cholesterol ester from TLC averaged 87% and 92%, respectively. The triglyceride was extracted from the

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silica gel with isopropanol and triglyceride mass of the extract was measured calorimetrically using triolein as a standard with a concentration curve from 0 to 90 pg triolein [23]. Cholesterol ester was extracted from the silica gel with isopropanol and measured by gas liquid chromatography [8] or by a calorimetric method using o-pthalaldehyde as described by Rude1 and Morris with a concentration curve from 0 to 5pg [24]. For gas liquid chromatography, Sa-cholestane (2 ug) was added as an internal standard, the sample was saponified in 0.5 N KOH in 90% ethanol at 70°C for 2 h and the cholesterol extracted following addition of water and petroleum ether to make two phases. The cholesterol was derivatized and assayed by gas liquid chromatography as described previously [8]. Essentially identical results were obtained by the two methods. 2.4. Lipases

Bovine milk lipoprotein lipase was partially purified by a modification of the method of Socorro and Jackson [25] as previously described [26] and added to the medium at 0.25 units/ml [8]. Fungal hpase from Rhizopus arrhizus and bacterial lipase from Pseudomonas species were obtained from Sigma (St. Louis, MO) and added to cell culture medium at 0.5 units/ml. Lipoprotein lipase was heat-inactivated at 56°C for 30 min in the presence of 1 M NaCl [27]. 2.5. Monoclonal

antibody preparation

Affinity purified human apo E and apo B monoclonal antibodies and Fab fragments were prepared as described previously [28]. The anti apo E antibodies used were: (i) lD7, an antibody that inhibits specific binding of apo E to the B,E receptor, and (ii) 6C5, an antibody that reacts with epitopes not involved with B,E receptor recognition [29]. The anti-apo B antibodies used in this study were: (i) 5Ell and BlB3, antibodies that specifically inhibit binding by apo B to the B,E receptor, and (ii) 1Dl and C4D1, antibodies that react with an epitope not involved in receptor recognition [28,30,31]. These antibodies were generously provided by Drs. Ross Milne and Yves Marcel, Ottawa Heart Institute, Ottawa, ON (lD1, lD7, 6C5 and 5Ell) and Dr. Elaine Krul,

K. Cianjone

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Washington University, St. Louis (BlB3 and C4Dl). Antibody to the LDL receptor was isolated as described above [32] from the monoclonal antibody-producing cell line C7 (American Tissue Culture Collection). 2.6. Other materials Lactoferrin and asialofetuin were obtained from Sigma and heparin (Hepalean) from Organon Canada Ltd. All tissue culture media and supplies were from Gibco (Burlington, ON) or Flow (Mississauga, ON).

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pg/ml LDL vs. 50 pg/ml HTg-VLDL cholesterol). In contrast to HTg-VLDL, there does not appear to be any LPL enhancement of LDL-mediated increases in intracellular cholesterol ester. We then examined the capacity of various monoclonal antibodies to inhibit the uptake of the HTg-VLDL, thus inhibiting the intracellular triglyceride and cholesterol ester increase. We have previously shown that anti-apo E antibodies had little effect on preventing cholesterol ester accumulation in HepG2 cells [8], in contrast to ug CHOL

2.7. Statistic-al unalyses A11 results are the average of triplicate determinations for each experiment and are expressed per mg soluble cell protein + standard error of the mean. Significance was measured by unpaired Student’s t-test.

101-114

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3. Results

As shown in Fig. 1, when HTg-VLDL (large triglyceride-rich VLDL S, 60-200) alone is added to HepG2 cells, there is no increase in intracellular cholesterol ester (Fig. 1, top panel) or triglyceride (Fig. 1, bottom panel) even after 24 h of incubation. However, in the presence of 0.25 units/ml of bovine milk LPL, intracellular triglyceride increased to 301% + 28% of control (P < 0.0005) and intracellular cholesterol ester increased to twice the control level (176% + 12% vs. control, P < 0.0005). This increase in intracellular triglyceride and cholesterol ester is concentration-dependent as shown previously [8], and a concentration of 50 pug/ml produced a maximal increase in both intracellular cholesterol ester and triglyceride. As also shown in Fig. 1, heat-inactivation of LPL blocks the cellular increase in both cholesterol ester and triglyceride. Incubation of HepG2 cells with LDL also causes increases in intracellular cholesterol ester up to 189% + 20% of control (P < 0.005). However, much more lipoprotein cholesterol must be added to achieve the same maximal effect as with 50 pgg/ml of VLDL + LPL (concentration curve not shown), and in all subsequent experiments, three times more LDL cholesterol was added to the cells (150

TRIGLYCERIDE

(ugimg

cell

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400

300

Fig. 1. Effect of VLDL and LDL on intracellular accumulation of cholesterol ester and triglyceride in HepG2 cells: HepG2 cells were incubated alone (control) or with 50 pg VLDL cholesterol/ml medium (m) or 150 pg LDL cholesterol/ml medium ( q ) + LPL (0.25 IU/ml medium) or heat-inactivated LPL (hiLPL) for 24 h. Intracellular cholesterol ester (top panel) and triglyceride (lower panel) are expressed as pgjrng cell protein f standard error of the mean. The number of experiments for each addition is indicated in parentheses. *P < 0.05, **P < 0.005, ***P < 0.0005 significantly different from control.

106

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P)

100 HVLDL

q LDL

1

Fig. 2. Effect of anti-apo E monoclonal antibodies on intracel. Mar accumulation of cholesterol ester in HepG2 cells: HepG2 cells were incubated with 5Opg VLDL cholesterol/ml medium + LPL (0.25 U/ml) (S) or 150 pug LDL cholesterol/ml medium (0) and 600 pg/mI monoclonal IgG 6C5 (or 100 pg/ml Fab fragments monoclonal I D7) for 24 h. Intracellular cholesterol ester is measured as pg choIesterol/mg cell protein and results are expressed as ‘%,inhibition k standard error of the mean compared with stimulation with lipoprotein alone (VLDL/LPL or LDL). *P < 0.05 compared with incubation without antibody. The number of experiments for each addition is indicated in parentheses.

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terol ester caused by incubation of the HepG2 cells with LDL (Fig. 2). In addition, no change in HTg-VLDL-induced intracellular triglyceride accumulation was seen with any of the antibodies (data not shown), suggesting that lipolysis by LPL was not hindered in the presence of the antibodies and that the fatty acids which were released caused the triglyceride accumulation in the HepG2 cells. We also tested the effectiveness of monoclonal anti-apo B antibodies on the cholesterol ester increases caused by HTg-VLDL in the presence of LPL and LDL. As shown in Fig. 3, monoclonal antibodies specific to the apo B epitope that binds to the B,E receptor, 5Ell and BlB3 were able to effectively inhibit LDL-mediated cholesterol ester increases by 97% f 13% (P < 0.005) and 68% f 13% (P < 0.05 vs. LDL alone). Antibodies that bound to the apo B at non-B,E receptor binding regions (monoclonal 1Dl and C4Dl) were ineffective at reducing LDL-induced increases in cholesterol ester. In contrast to LDL, none of these monoclonal antibodies had even % change

(ug CHOL

IN CE/mg

cell

P)

ioo~ocDL~

the marked inhibition in 5774 macrophages [9]. However, 5774 macrophages do not secrete apo E, whereas apo E is secreted by HepG2 cells. We therefore felt it was important to evaluate the effect of high doses of anti-apo E antibody sufficient to block both lipoprotein bound and cell secreted apo E on HTg-VLDL-induced cholesterol ester accumulation. As shown in Fig. 2, an antiapo E antibody specific for the epitope on apo E that binds to the B,E receptor, lD7, was able to inhibit the HTg-VLDL stimulated increase in intracellular cholesterol ester marginally but significantly (- 27% + 3%, P < 0.05 vs. HTg-VLDL). Overall, however, the results were not different to those obtained with monoclonal antibodies against an epitope on apo E not in the B,E receptor binding region (6C5). Even when the molar ratio of antibody IgG to VLDL apo E was increased to approximately 40:1, there was no further inhibition of cholesterol ester accumulation. Moreover, neither of these antibodies had any effect on the increases in intracellular choles-

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Fig. 3. Etfect of anti-apo B monoclonal antibodies on intracellular accumulation of cholesterol ester in HepG2 cells: HepG2 cells were incubated with 5Oilg VLDL cholesterol/ml medium + LPL (0.25 IU/mI medium) (a) or I5Ojtg LDL cholesterol/ ml medium ( q ) and 600 jlg/rnI monoclonal IgG for 24 h. Intracellular cholesterol ester is measured as /cg cholesterol/mg cell protein and results are expressed as ‘%) inhibition + standard error of the mean compared with incubation with lipoprotein alone (VLDL+ LPL or LDL). *P i 0.05, **P < 0.005 compared with incubation without antibody. The number of experiments for each addition is indicated in parentheses.

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effects on the HTgmarginal inhibitory VLDL + LPL-induced increases in cholesterol ester (Fig. 3‘). Similarly, none of the anti-apo B antibodies had any effect on the increased intracellular triglyceride levels seen in the presence of HTg-VLDL + LPL (results not shown). In light of the results of Beisegel et al. [lo] and. more recently, other investigators [l 1- 171, we wondered whether LPL acts as both a lipolytic enzyme and as a ligand to mediate cellular interaction. We therefore attempted to differentiate between lipolysis and ligand-cellular interaction of LPL as a mediator of intracellular increases in triglyceride and cholesterol ester. As shown in Fig. 4, HTg-VLDL was lipolysed with several different lipases and intracellular triglyceride and cholesterol ester increases were measured. HTg-VLDL was lipolyzed with LPL in vitro as described in Materials and methods and the VLDL reisolated using conditions that have been reported to dissociate LPL activity from VLDL [21]. This laboratory has shown previously that no LPL could be detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [8]; however, we cannot exclude the possibility that small amounts of LPL may be associated with reisolated VLDL. Lipolyzed reisolated VLDL readily caused substantial increases in both triglyceride and cholesterol ester (274% f 18’s, P < 0.05, and 177% + 12X, P < 0.005, respectively). Similarly, HTg-VLDL added to the medium in the presence of a 0.50 units/ml of Rhizopus arrhizus lipase, a structurally unrelated fungal lipase [33], also increased intracellular triglyceride and cholesterol ester content by 472% + 61”/0 (P < 0.005) and 202% & 25% (P < 0.025) respectively. Both results suggest that lipolysis is necessary, but that there is not an absolute requirement for LPL as a specific ligand for cellular uptake. In contrast, however, the results obtained with a bacterial lipase from Pseudomonasspecies, also structurally unrelated to LPL [34], are not as clear. Although the lipase is clearly active, and causes marked increases in intracellular triglyceride as shown in Fig. 4b (522% + 99X, P < 0.025) there is no increase whatsoever in intracellular cholesterol ester (Fig. 4a, 97% + 18%, P NS). Attempts to reisolate HTg-VLDL by ultracentrifugation fol-

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ug CHOL IN CE/mg cell P

TRIGLYCERIDE (ugimg cell P)

“:I:~--~

Fig. 4. Effect of different lipases on VLDL-induced intracellular accumulation of cholesterol ester and triglyceride in HepG2 cells: HepG2 cells were incubated with SO/lg VLDL cholesterol/ml medium and various lipases for 24 h. LPL, lipoprotein lipase (0.25 units/ml medium); RhiLip, fungal Rhi~-opus arrhtus Iipase (0.5 units/ml medium); PseuLip, bacterial fsr~dorrtonas species lipase (0.5 units/ml medium); Lip-RI, LPL lipolyzed re-isolated VLDL. Intracellular cholesterol ester (a) (top panel) and triglyceride (0) (lower panel) are expressed as iLg/rng cell protein f standard error of the mean where the number of experiments for each addition is indicated in parentheses. *P < 0.05. **P < 0.025; ***P < 0.005 significantly ditferent from control.

lowing treatment with this lipase in vitro resulted in a total loss of VLDL. Uptake of LDL was also completely abolished by this enzyme (data not shown). The phospholipase activity of this lipase may have destroyed the integrity of the VLDL particle resulting in no accumulation of cholesterol ester intracellularly. This emphasizes the necessity of an intact particle in order to mediate uptake of the lipolyzed VLDL by the HepG2 cells.

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We next examined the process by which the lipolyzed HTg-VLDL is taken up by the HepG2 cells. As shown in Fig. 5, asialofetuin was tested for the capacity to inhibit the HTg-VLDL-induced increases in cholesterol ester and triglyceride. It has been suggested that the asialoglycoprotein receptor in liver may mediate removal of chylomicron remnants through interaction with galactosyl residues, a process which is inhibited by asialofetuin [35]. Competition with asialofetuin inhibited the HTg-VLDL cholesterol ester increase by 52% f 22% (P < 0.05). There

120 (1996) ug CHOL

IOIin CE/mg

114 cell

P

16 14 12 10 a 6 4 2 0 TRIGLYCERIDE

(ugimg

ceil

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1,200 ug CHOL

IN CE/mg

cdl

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14 800

TRIGLYCERIDE

(ugimg

cell

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500

300 200

100 0 Pl G&@

Fig. 5. Inhibition of intracellular accumulation of cholesterol ester and triglyceride in HepG2 cells: HepG2 cells were incubated with 5Olg VLDL cholesterol/ml medium and LPL (0.25 ) or I50 pg LDL cholesterol/ml medium ( q ) and asialofetuin (IO mg/ml) as indicated for 24 h. Intracellular cholesterol ester (top panel) and triglyceride (lower panel) are expressed as jcg/mg cell protein + standard error of the mean where the number of experiments for each addition is indicated in parentheses. *P < 0.05, significantly different from incubation with HTg-VLDL + LPL alone.

Fig. 6. Effect of heparin on intracellular accumulation of cholesterol ester and triglyceride in HepG2 cells: HepG2 cells were incubated with 5Opg VLDL cholesterol/ml medium and the indicated lipases (same concentrations as in legend to Fig. 4) (B) or I50 pg LDL cholesterol/ml medium (a) with or without heparin (IO W/ml) for 24 h. Intracellular cholesterol ester (top panel) and triglyceride (lower panel) are expressed as pgjmg cell protein k standard error of the mean, where the number of experiments for each addition is indicated in parentheses. *P c 0.025, **P < 0.0005, significantly different from incubation without heparin.

was no effect of asialofetuin on LDL-mediated increases in cholesterol ester. There was no decrease with asialofetuin on intracellular triglyceride accumulation caused by the action of LPL on HTg-VLDL, indicating that lipolysis and fatty acid uptake were not affected (Fig. 5, lower panel). The effect of heparin on intracellular accumulation of triglyceride and cholesterol ester was examined as shown in Fig. 6. In the presence of heparin (10 IU/ml), the cholesterol ester increases

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caused by incubation with HTg-VLDL + LPL were completely blocked ( - 86% + 8”S, P < 0.0005, Fig. 6, upper panel). Heparin was also very effective at blocking the increases in cholesterol ester caused by reisolated lipolyzed VLDL and HTg-VLDL -t- Rhizopus lipase ( - 79% and - 95%, respectively). In contrast there was no effect of heparin on the intracellular accumulation of triglyceride (Fig. 6, lower panel). At the concentration used, heparin had no significant effect on the LDL-mediated increases in cholesterol ester ( - 23% + 17%, P NS). We then examined the potential involvement of the B,E receptor and the LRP receptor on HTgVLDL mediated increases in intracellular cholesterol ester and triglyceride. An anti-LDL receptor monoclonal antibody (C7) was added to inhibit uptake of HTg-VLDL and LDL via the B,E receptor. As shown in Fig. 7, monoclonal antibody C7 almost completely blocked the increase in LDL-induced intracellular cholesterol ester accumulation (- 77% * 7%, P < 0.01). In marked contrast, there was only a small but sigug CHOL

IA CE/mg

cell

P

--~~-

167-----

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vg,

r ,’,* ,’/ e...;L;,:

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Fig. 7. Erect of anti-LDL-receptor monoclonal antibodies and lactoferrin on intracellular accumulation of cholesterol ester in HepG2 cells: HepG2 cells were incubated with 5Opg VLDL cholesterol/ml medium + LPL (0.25 IU/ml medium) ( q ) or 15Opg LDL cholesterol/ml medium (III) and 600 pg/ml monoclonal C7 IgG or 2 mgjml lactoferrin for 24 h. Intracellular cholesterol ester is expressed as pg cholesterol/mg cell protein -t standard error of the mean, where the number of experiments is indicated in parentheses for each addition. *P < 0.025, **P < 0.01 as compared with incubation with HTgVLDL + LPL or LDL alone.

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nificant effect on the HTg-VLDL mediated increases in cholesterol ester ( - 22% + lO’/o, P < 0.025). There was no effect on intracellular triglyceride levels (data not shown). Other investigators [36,37] have demonstrated that the LRP is a multifunctional receptor binding x-2-macroglobulin, lactoferrin and chylomicron remnants. We have assessed the effects of lactoferrin on HTg-VLDL intracellular accumulation of triglyceride and cholesterol ester. Lactoferrin was very effective at inhibiting the HTg-VLDL-mediated increases in intracellular cholesterol ester with an almost complete blockage of the cholesterol ester increase ( - 9.5”/;1 + 6% P < O.Ol), but had no effect on the increase in intracellular triglyceride levels (data not shown). 4. Discussion

Our results are consistent with a two-stage uptake process for HTg-VLDL by HepG2 cells. Lipolysis of the particles appears to be a necessary first step, since in the absence of lipolysis, without LPL or another lipase present, there is no cellular increase in triglyceride and cholesterol ester. In the presence of active lipolysis, there is an increase in both intracellular triglyceride and cholesterol ester in the HepG2 cells. The greater part of the increase in intracellular triglyceride would appear to be simply a function of the extracellular fatty acid concentration as demonstrated previously in both HepG2 cells and J774 macrophages [7--91. As long as there is lipolysis, the free fatty acids produced in the medium are transported across the cell membrane and result in increases in intracellular triglyceride accumulation. In fact there was a tendency for the cellular triglyceride even to increase slightly in the presence of heparin, perhaps due to heparin stabilizing the LPL activity or stimulating hepatic lipase activity [38,39] and generating even greater amounts of extracellular fatty acids. Although lipolysis of these particles releases fatty acids, not all of the particle triglyceride is hydrolyzed. Following lipolysis, these particles can be reisolated by ultracentrifugation at d < 1.019 and are therefore still triglyceride-rich particles. When reisolated VLDL is then added to cells, uptake proceeds in the absence of added

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LPL. The increase in cellular triglyceride is due to uptake of free fatty acid following hydrolysis either by hepatic triglyceride lipase or as part of the remnant itself. Nevertheless, in the presence of active lipase, none of the manoeuvres designed to affect uptake of the HTg-VLDL particle had any effect on triglyceride accumulation. In contrast, cholesterol ester accumulation is dependent on particle uptake. As previously demonstrated by Evans et al. [8], uptake of lipolyzed VLDL results in cellular influx of cholesterol (and remaining triglyceride) through lysosomal degradation of the particle, and as a consequence there is an increase in acyl-CoA cholesterol-acyltransferase activity. We have previously suggested that cholesterol ester and triglyceride accumulation occur by different mechanisms in 5774 macrophages [9]. In 5774 cells, apo E clearly played a crucial role in mediating cholesterol ester accumulation. What, then, is the ligand interacting with this extracellular matrix in the present studies with HepG2 cells? Both lipoprotein lipase and apo E are obvious candidates. We have tried to differentiate between LPL-mediated lipolysis effects and effects independent of lipolytic activity. Clearly, with the heat-inactivated LPL, there was no lipolysis and no VLDLmediated intracellular increase in TG or CE. However, it has been shown using specific monoclonal antibodies that heat-inactivated LPL is a monomer, not a dimer [40], which not only destroys its enzymatic activity [27] but interferes with its capacity to act as a bridge between VLDL and the LRP receptor [41]. Thus we may have lost both effects. Similarly with the reisolated VLDL, although recentrifugation has been shown clearly to destroy LPL activity, and dissociate LPL from the particle, we cannot rule out the possibility that small amounts of inactive enzyme are present and that heparin addition results in binding to the remaining LPL and inhibits its ability to act as a ligand. Nonetheless, our data do not suggest an exclusive role for lipoprotein lipase interaction since a non-structurally related lipase (R/&opus avrhizus) [33] is just as effective at increasing cholesterol ester accumulation, and both can be inhibited by heparin. However, the possibility that heparin can influence the binding of both lipases

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to the HepG2 plasma membrane cannot be ruled out. Although monoclonal antibodies to apo E had only small but significant effects on inhibition of HTg-VLDL-mediated increases in cholesterol ester, apo E may also be involved in the process. Apo E is found in high concentrations in the space of Disse [42]. Previous studies have shown that apo E secreted from HepG2 cells can associate with exogenous chylomicron remnants [43,44] and enhance uptake of rabbit /j’-VLDL enriched in apo E [45]. A number of recent papers have suggested that apo E secreted by the liver is localized to the cell surface [46,47], possibly to glycosaminoglycans, and may even be linked to glycosaminoglycans intracellularly and transported to the cell surface as a complex [47]. This cell surface apo E may participate in a secretion-recapture process for hepatic endocytosis of triglyceride-rich lipoproteins and remnants [48]. This cell surface apo E is sensitive to heparin, heparinase I and III but not hyaluronic acid or chondroitin sulfate [47]. It has also been shown that heparin, heparin sulfate and heparanase interfered with the uptake of apo E supplemented triglyceride emulsions and apo E supplemented /3-VLDL [42,49], and that hyaluronic acid and chondroitin sulfates are ineffective [42]. In the present studies, excess amounts of anti-apo E antibody were added to the culture medium during incubation in order to bind the VLDL-associated apo E, medium free apo E and cell surface bound apo E. Leblond and Marcel have demonstrated that this same antibody (lD7) can bind the cell surface proteoglycan bound apo E in HepG2 cells [46]. However, these monoclonal antibodies are known to bind at sites near the heparin binding epitopes of apo E [28,30], and one possible explanation for the lack of effect is that under our experimental conditions (in the presence of VLDL and LPL) the cell surface bound apo E may not be sterically accessible to the antibodies. Our results indicate clearly that HepG2 cell-mediated uptake of LDL and HTg-VLDL occurs by distinct pathways. In the case of HTg-VLDL, a series of experiments were consistent with the model that the partially lipolyzed particle interacts with cell surface proteoglycans before internalization. Asialofetuin is a ligand for the galac-

tose-recognizing lectin asialoglycoprotein receptor (ASPG), a binding site abundant in hepatocytes [35]. It is also known that asialoglycoprotein can interact with proteoglycans, and in this way it may also interfere with VLDL interaction with proteoglycans [50], although the present experiments cannot differentiate between these two mechanisms. Similarly heparin, which was the most effective, blocked cholesterol ester accumulation regardless of whether LPL or fungal lipase was used to lipolyze the VLDL. A number of recent studies [l I- 171 have also suggested that lipoprotein interaction with the extracellular matrix in the presence of lipoprotein lipase is key to increased binding of lipoprotein particles. This has been shown in a wide variety of cells: endothelial. HepG2, LDL-receptor negative human skin fibroblasts and (as a negative control) hamster glycosaminoglycan-deficient Chinese ovary cells. A number of different lipoproteins ~ normal human VLDL, rabbit ,&-VLDL, LDL and Lp(a) -- have been used as ligands in these studies. Our results are consistent with and extend these studies by demonstrating the effects of HTgVLDL-proteoglycan interaction on the subsequent metabolic events that occur at the cellular level, that is, the accumulation of intracellular triglyceride and cholesterol ester. In fact, it has been suggested that cell surface proteoglycans may play important roles in gene expression, growth factor response [51-571 and liver-specific protein synthesis [58860]. Heparin and heparin sulphate are the most abundant glycosaminoglycans in the liver extracellular cell matrix [61] and are produced by HepG2 cells [58]. The mechanism whereby cell surface proteoglycans facilitate lipoprotein uptake is not completely understood [49]. In the case of LDL, clearance is through the B,E receptor pathway since cholesterol ester accumulation could be effectively blocked with anti apo B and anti-LDL receptor antibodies. However, these had little effect on HTg-VLDL-mediated increases in triglyceride or cholesterol ester. This is consistent with previous findings in human skin fibroblasts and 5774 macrophages that apo B is not the primary determinant for HTg-VLDL uptake [9,30,62]. We found no effect of LPL on enhancement of LDL-mediated effects on choles-

terol ester accumulation. This is in contrast to previous studies in HepG2 cells which have demonstrated a 20- to 30-fold increase in 4°C LDL binding in the presence of LPL [I2 - 14,171. There may be several explanations for these differences: when LDL uptake and degradation were examined at 37°C in these studies, the effects of LPL were not as marked and, secondly, the concentrations of LPL used in these studies were much higher than those used here. Similarly heparin. which strongly inhibits the HTg-VLDL effect, had only marginal effects on the LDL-mediated increases in cholesterol ester, suggesting that there is no interaction with cell surface proteoglycans. It should be noted that the concentrations of heparin used in this study are much lower than those known to interfere with LDL-B,E receptor interaction [63]. In a similar manner, it has been reported that heparinase or heparatinase, which cleaves cell surface proteoglycans, or heparin, which interferes with interaction with cell surface proteoglycans, has little effect on the binding and uptake of LDL alone in hepatocytes [64], HepG2 cells [12] and FH fibroblasts [17]. Lipolytic remodelling of the HTg-VLDL may therefore permit the association of the lipoprotein particles with HepG2 proteoglycans or cell-associated apo E-proteoglycan complexes. Lipoprotein lipase may enhance this process beyond its role in lipolytic remodelling. but LPL does not appear to be an absolute requirement. Mulder et al. have suggested that interaction of normal human “51-VLDL and LPL with proteoglycans enhances uptake primarily through the B,E receptor [13]. Our results suggest that only a portion of the uptake may be mediated through the B,E receptor, since the monoclonal antibody C7 effectively inhibited LDL-mediated increases in intracellular cholesterol ester but had only a small effect on HTg-VLDL-mediated increases. Chappell et al. also suggested that through interaction with cell surface proteoglycans. LPL induces catabolism of normal VLDL in B,E receptor-deficient fibroblasts [l 11. In HepG2 cells, Ji et al. demonstrated that cell surface proteoglycans enhanced both the binding and the internalization of apo E enriched rabbit I-VLDL in the absence of lipoprotein lipase [49]. In both reports it was

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suggested that the proteoglycans were obligatory participants in the process, but that this did not exclude a two-step process where the proteoglycan enhances subsequent uptake via LRP. Our results are consistent with proteoglycan-LRP involvement since lactoferrin, an inhibitor of the LRP receptor [36], was very effective at preventing HTg-VLDL-mediated cholesterol ester increases. It should be pointed out that it has been reported recently that lactoferrin in fact also binds heparin sulfate proteoglycans [65] and chondroitin sulfate proteoglycans, although this interaction is not sufficient to inhibit lipoprotein remnant uptake [50,66]. Thus, cell surface heparin sulfate may facilitate lipoprotein uptake and lipid acquisition by providing a reservoir of cell surface-associated apo E to mediate the uptake of VLDL particles and/or by providing a stabilizing bridge with the LPL-lipoprotein particles and enhance uptake through receptor-mediated processes. What are the implications of this process? Lipoprotein lipase is normally cleared by the liver [67], although in fasting plasma the greater part of LPL in plasma is inactive and associated with LDL and HDL [68]. Postprandially, however, there is an increase in the amount of active LPL free in plasma, and an increased proportion is associated with triglyceride-rich lipoproteins [68,69]. These circumstances, based on our results, might lead to increased uptake of HTg-VLDL in the liver and increased triglyceride and cholesterol ester delivery to the liver. Acknowledgements

This work was supported by a Medical Research Council of Canada grant to MWH (MT 8014). This work was conducted while KC was present as a visiting scientist with Dr Murray Huff at the Robarts Research Institute, London, ON. References [I] Packard CJ, Munro herd J. Metabolism ride-rich very low hypertriglyceridemic

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