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Differential effects of eicosapentaenoic acid on glycerolipid and apolipoprotein B metabolism in primary human hepatocytes compared to HepG2 cells and primary rat hepatocytes
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ELSEVIER
Biochimicaet BiophysicaActa 1256 (1995) 88-96
Biochi ~mic~a et BiophysicaA~ta
Differential effects of eicosapentaenoic acid on glycerolipid and apolipoprotein B metabolism in primary human hepatocytes compared to HepG2 cells and primary rat hepatocytes Yuguang Lin, Martin J. Smit, Rick Havinga, Henkjan J. Verkade, Roel J. Vonk, Folkert Kuipers * Groningen Institute for Drug Studies, Laboratory of Nutrition and Metabolism, Department of Pediatrics, UniL'ersity and Academic Hospital Groningen, Oostersinge159, 9713 EZ Groningen, The Netherlands
Received 18 August 1994; revised 13 December 1994; accepted 20 December 1994
Abstract We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or presence of 1 mM of EPA or OA. Synthesis and secretion of [3H]glycerolipid were determined after 1 h incubation with [3H]glycerol. Cellular and medium apoB abundance was semi-quantitatively estimated in human cells by Western blotting. The following observations were made. (1) Compared to control, OA induced a 7-fold increase in [3H]triacylglycerol (TG) synthesis in human hepatocytes and a 4-fold increase in rat hepatocytes and HepG2 cells. EPA enhanced [3H]TG synthesis about 2-fold in all three cell types although it stimulated [3H]diacylglycerol (DG) synthesis to an extent (i.e., 2.5- to 5-fold) similar to OA. (2) In contrast to OA, which stimulated VLDL-associated [3H]TG secretion 2.5- to 3-fold in the three cell types relative to control, EPA did not alter [3H]TG secretion in HepG2 and rat hepatocytes and suppressed [3H]TG secretion by 75% in primary human hepatocytes. (3) In primary human hepatocytes, both OA and EPA did not alter cellular apoB abundance but EPA decreased apoB secretion by 44% as compared to control. In contrast, both EPA and OA increased cellular and medium apoB abundance 2- to 2.5-fold in HepG2 cells, although medium apoB tended to be lower in EPA-treated cells. (4) EPA had no effect on the [3H]phosphatidylcholine (PC) synthesis in rat hepatocytes, but decreased [3H]PC synthesis by 30% in human hepatocytes and HepG2 cells. This study shows that, in primary human hepatocytes, EPA decreases TG secretion below control values by influencing VLDL particle assembly and/or secretion. There are both qualitative and quantitative differences in the effects of EPA and OA on the glycerolipid and apoB metabolism between primary human hepatocytes and both HepG2 and primary rat hepatocytes, the two most commonly used model systems to investigate hepatic lipid metabolism in vitro. Keywords: In vitro; Primary human hepatocyte; lcosapentaenoicacid; Oleic acid; Triacylglycerol;ApolipoproteinB; Phospholipid
1. Introduction The well-documented serum triacylglycerol (TG)-lowering effects of dietary fish [1] and fish oil [2] in humans has generated intensive research on the mechanism(s) responsible for this hypolipidemic action. The effect has been
Abbreviations:BSA, bovine serum albumin;DG, diacylglycerol;EPA, eicosapentaenoic acid; FCS, fetal calf serum; LDH, lactate dehydrogenase; OA, oleic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine;SDS, sodium dodecyl sulfatc; TG, triacylglycerol;(HP)TLC, (high performance) thin-layer chromatography; VLDL, very low density lipoprotein * Corresponding author. Fax: +31 50 696800. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0005-2760(95)00006-2
attributed to the presence of the n - 3 fatty acids [3], i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid, that are particularly abundant in fish oil. Fish oil and its isolated components are able to influence lipid metabolism in a number of ways, as reviewed by Harris [2]. It is generally accepted that interference with the secretion of very low density lipoproteins (VLDL) is one of the physiologically most important effects of fish oil. This action has been attributed to decreased hepatic triglyceride synthesis [4,5] and to inhibition of assembly a n d / o r secretion of newly synthesized apoB-containing lipoprotein particles [6]. Recently, it has been shown that, in rat hepatocytes, this latter effect may be related to an EPA-induced acceleration of intracellular apoB degradation [7]. Most of the
Y. Lin et al. /Biochimica et Biophysica Acta 1256 (1995) 88-96
studies on the mechanism(s) by which fish oil (components) affect VLDL secretion have been carried out in cell cultures of animal origin [5-7] or, as a model for the human liver, in HepG2 cells [8,9]. It is, however, well established that marked species differences in hepatic lipid metabolism exist between experimental animals and humans. For example, VLDL secreted by rat liver contains apoB-48 as well as apoB-100 whereas human VLDL only contains apoB-100. In addition, it has been reported that changes in lipid metabolism induced by hypolipidemic drugs, such as clofibrate and beclobric acid, are strikingly different between rat and human hepatocytes [10]. HepG2 cells, which are derived from a human hepatoma, have been reported to differ from normal liver cells in a number of ways [11]. It would be valuable, therefore, on one hand, to evaluate the effects of EPA on lipid metabolism in human hepatocytes in primary culture and, on the other hand, to compare the results in these cells with those obtained with HepG2 cells and rat hepatocytes in primary cultures, the latter two being the most commonly used cell systems in this area of research.
2. Materials and methods 2.1. Materials
Williams' E medium, glutamine, penicillin-streptomycin, fetal calf serum (FCS) and collagenase were purchased from Life Technologies, Paisley, UK. Insulin and dexamethasone came from Novo Nordisk Farma, Amsterdam, the Netherlands. Oleic acid (OA) (99% by capillary GC), eicosapentaenoic acid (98%), bovine serum albumin (BSA) (Fraction V, essentially fatty acid-free) and hydrated colloidal silica (Cab-O-Sil) (fumed silica, particle size 0.011 /xm) were purchased from Sigma Chemical Co., St. Louis, MO, USA. [2-3H]Glycerol (1 Ci/mmol), L-[4,53H]leucine (61 C i / m m o l ) and Enhance chemiluminescence Western blotting reagent were obtained from Amersham International, Amersham, UK. Cell culture plates were supplied by Costar, Cambridge, MA, USA. Thin-layer chromatography (TLC) plates and high performance thinlayer chromatography (HPTLC) plates were obtained from Merck, Darmstadt, Germany. Triglycerides GPO-PAP kit, sheep anti-human-apoB and anti-human-apoA-I antiserum were obtained from Boehringer Mannheim, Mannheim, Germany. All other chemicals and solvents were high purity commercial materials. 2.2. Preparation and culture o f hepatocytes
Human liver material was obtained from healthy liver transplant donors. Consent from legal authorities and donor families was obtained. Human hepatocytes were isolated from spare-parts when the donor livers were split to perform reduced-size liver transplantation in children. Age and sex of liver donors used for these studies were: a: 50
89
y, male; b: 46 y, female; c: 26 y, male; d: 6 y, male and e: 36 y, male. Livers were perfused with an organ preservation solution [12] at the time of harvesting and kept at 0 - 4 ° C until used for cell preparation (2-12 h). A wedgeshaped piece of 30-200 g with a single cut surface was used for hepatocyte isolation. Two to four major branches of the portal veins were cannulated with plastic cannulae for perfusion and other major vessels were tied off. The tissue was first perfused with 1000 ml calcium-free Hank's fluid containing glucose (10 mM) and Hepes (10 mM) at a flow rate of 90 m l / m i n to wash out the preservation fluid or blood and to warm the liver to 37 ° C. This was followed by perfusion of 1500 ml calcium-free Hank's fluid containing NaHCO 3 (25 mM) and glucose (10 mM) and 200 ml calcium containing Hank's buffer before 400 ml of enzyme solution (50 mg of collagenase/100 ml calcium containing Hank's buffer) was perfused with recirculation for 15 min. All solutions were pre-gassed with 95% 02 and 5% CO 2 and adjusted to pH 7.42. Rat hepatocytes were isolated by the method of Berry and Friend [13] from male Wistar rats of 180-220 g. Rats were maintained in a light and temperature controlled room and fed standard lab chow and water ad libitum until sacrificed. HepG2 cells were cultured in our laboratory and routinely replated weekly. The viability of both human and rat hepatocytes after isolation was examined by trypan blue exclusion (final concentration 0.2%). Cells were plated in 35 mm 6-well (pre-coated with collagen) plastic dishes at a density of 1.5-10 6 viable cells/well with 2 ml medium. When HepG2 cells were used for experiments, about 3 • 105 cells were seeded to each well of 6-well plate without collagen pre-coating. The culture medium consisted of Williams'E medium supplemented with 10% FCS, 2 mM glutamine, 1 0 0 / x g / m l penicillin, 1 0 0 / z g / m l streptomycin, 4 m U / m l insulin and 0.02 / z g / m l dexamethasone. After cells had attached to the plates, medium was renewed. Subsequently, medium was renewed every 24 h. Cells were incubated at 37°C in an atmosphere of 95% air and 5% CO z. 2.3. Experimental protocol
Experiments were conducted two days after seeding of cells. On the day of an experiment, BSA-fatty acid complexes were prepared: a sterile solution of 0.25 mM BSA in Williams' E medium with insulin, dexamethasone, penicillin and streptomycin at the concentrations mentioned above was adjusted to pH 10. Fatty acid was solubilized in chloroform in glass tubes and dried under nitrogen. The BSA-containing medium was mixed to the dried fatty acid while sonicating vigorously to produce a solution of 1 mM fatty acid/0.25 mM BSA. The pH was adjusted to 7.4, and media were warmed to 37 ° C before use. FCS-containing medium was removed from the wells and cells were washed two times with FCS-free medium
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1I.Lin et al. / Biochimica et BiophysicaActa 1256 (1995)88-96
and incubated in this medium for 2 h. Then the medium was changed to the medium (1 ml/well) containing fatty acid-BSA complexes or BSA alone as control and cells were incubated for 1-5 h. To determine the glycerolipid synthesis rates at indicated time points, [3H]glycerol (4.4 /xCi/well, 25 /xM) was added 1 h before termination of the experiments. All incubations were performed in triplicate and terminated by putting the culture plates on ice. Media were collected via suction and centrifuged at 10 000 rpm for 2 min to remove suspended cells. Cells were washed twice with phosphate-buffered saline (PBS) and harvested by using a rubber policeman in 2 ml PBS. Samples were frozen at - 2 0 ° C prior to analysis. Cells and media used for apoB determination were harvested as described below in subsection 2.7. 2.4. Assessment of glycerolipid synthesis
Cells were thawed and resuspended by passing through 26G X 1-inch (0.45 X 25 mm) needles. The cellular lipids were extracted with chloroform/methanol (1:2, v / v ) . Triacylglycerol (TG), diacylglycerol (DG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were separated by HPTLC. Plates were developed half-way with the solvent c h l o r o f o r m / e t h a n o l / w a t e r / t r i e t h y l a m i n e (30:34:2:35, v / v ) to separate the phospholipids. After drying of the plates, TG and DG were separated by developing by hexane/diethylether/acetic acid (80:20:1) to the top of the plate. TG, DG, PE and PC spots were visualized by spraying with 0.1% Rhodamine G and marked under ultraviolet light. The spots were scraped into vials and assayed for radioactivity by scintillation counting. 2.5. Assessment of TG mass
Cellular lipids were extracted and separated by thin layer chrom atography developed by hexane/diethylether/acetic acid (80:20:1) mixture. T G was extracted two times from the silica gel with 2.5 ml of chloroform. Pilot experiments with trihexadecanoylglycerol showed that the recovery of this procedure was 8 6 92% in three independent determinations. The TG extracts were dried under nitrogen, resuspended in 100 /xl isopropanol and TG mass was assayed according to the instruction of manufacturers of the Triglycerides GPO-PAP Kit, with glycerol as standard. 2.6. Assessment of [3H]TG secretion
Since previous studies [14-16] and own observations revealed that greater than 95% of [3H]TG secreted into the medium of rat and human hepatocytes in primary culture was recovered in the VLDL fraction (d < 1.006 g / m l ) , the [3H]TG in the media were used to estimate VLDL-[3H]TG secretion. In contrast to the primary hepatocytes, most of the TG secreted by HepG2 cells is associated with an LDL-like lipoprotein under basal conditions [17]. Before
lipids were extracted, 30 /xl of 2 mM triacylglycerol solution in chloroform was added to each sample as a carrier. TG was separated from other lipids by TLC using a similar procedure to the assay of cellular TG mass. Silica gel containing TG was scraped into vials and assayed for radioactivity via scintillation counting. 2.7. Western blotting of apoB and apoA-I
Medium samples (2 ml) of human hepatocytes and HepG2 cells were added 0.1 ml of a detergent buffer (containing 250 mM EDTA, 10% Triton X-100, 10% deoxycholic acid, 1.25 M NaC1, 0.5 M Tris-HCl, 1% sodium dodecyl sulfate (SDS) and freshly added 0.01 M dithiothreitol and 0.15% phenylmethylsulfonyl fluoride (PMSF)). The cell monolayer was solubilized by addition of 0.5 m l / w e l l of 10-times diluted detergent buffer. The cell lysates were pipetted into tubes and heated at 70 ° C for 30 min and diluted with an equal volume of a solution containing 0.01 M Tris-HC1 (pH 7.4) and 0.15 M NaC1. Cell lysates were centrifuged at 10 000 rpm for 2 min and the supernatant of the cell lysates were used for apoB determination ApoB in cell lysates was precipitated by adding antiapoB-serum at final dilution 1:200. The tubes were rotated overnight followed by adding 40 /zl of 12% ( w / v ) protein A beads and incubated for another 2 h. Protein A-antibody-apoB complexes were collected by centrifugation. ApoB and apoA-I contained in media were absorbed to Cab-O-Sil according to the methods previously described [18]. Control experiments showed that when the initial immunoprecipitation (cell lysate) or Cab-O-Sil absorption (media) was followed by a second immunoprecipitation of the supernatant, no additional apoB was recovered. After being washed two times with PBS, apoproteins were solubilized from the protein A beads or silica pellet with an extraction buffer comprising 2% SDS, 0.05 M Tris (pH 9.0), 6 M urea, 0.1% EDTA, 0.1% dithiothreitol, 0.13% E-aminocaproic acid and 0.05% glutathione in a 95°C water-bath for 10 min. ApoB and apoA-I were separated from other proteins by electrophoresis in 5% polyacrylamide gel containing 0.1% SDS. Subsequently, apoproteins were transferred to nitrocellulose membranes. Immunoblotting was carried out as follows: the membrane was immersed in a solution of Tris-buffered saline (20 mM Tris (pH 7.5) and 0.5 M NaCI) containing 5% dried milk for 1 h at room temperature to block non-specific binding; incubated with the primary antibody (1:5000) overnight; washed four times with Tris-buffered 5% milk solution following the incubation with secondary horseradish peroxidase conjugated antibody (1:10000) for 2 h and then washed as before. ApoB- or apoA-I-antibody complexes were visualized with Enhance chemiluminescence Western blotting reagent by exposure to Hyperfilm according to the manufacturer's instructions. Quantification of the bands was performed with an UltroScan XL densitometer.
Y. Lin et aL /Biochimica et Biophysica Acta 1256 (1995) 88-96 2.8. Miscellaneous methods Protein was measured according to Lowry et al. [19] using B S A as standard. Lactate dehydrogenase (LDH) activity in media and cells were determined as reported previously [20]. The leakage of L D H from cells was present as LDH-leakage %, which was calculated according to the formula: (LDH activity in m e d i u m / L D H activity in cell) × 100%. The [3H]leucine incorporation into the trichloroacetic acid-precipitable protein was investigated to determine the cellular protein synthesis. Hepatocytes were cultured for 5 h in the same medium as for TG synthesis measurement and [3H]leucine (2.5 /.~Ci/ml per well) was added 1 h before termination of the cell incubation.
2.9. Statistical analysis Data were normalized to the amount of cellular protein. Statistical differences were assessed using two-way analysis of variance. Values of P < 0.05 were considered significant.
3. Results 3.1. Effect o f fatty acids on cell viability Table 1 shows that cellular protein synthesis was not affected when human hepatocytes, HepG2 cells or rat hepatocytes were exposed to 1 m M O A or E P A for 5 h. The L D H activities in media were similar across all groups and were in agreement with a previous report concerning the viability of liver cell in culture [21]. These results indicate that under the conditions employed, incubation with fatty acids did not exert a toxic effect on the hepatocytes.
3.2. Effect o f fatty acids on TG synthesis and secretion Previous studies [22,23] have shown that rat hepatocytes incubated with 1 m M O A and 5 /xCi [3H]glycerol
synthesize [3H]TG in a linear fashion for only 1 to 2 h. Considering the fact that a trace amount of [3H]glycerol is rapidly catabolized by hepatocytes, [3H]glycerol was added 1 h before termination of the experiment and data are presented as accumulated [3H]TG synthesis and secretion at each indicated time point to compare the effects of O A and EPA. In all Jthe three cell types studied, the incorporation of [3H]glycerol into cellular TG was significantly greater with O A than with EPA. Control [3H]TG synthesis in human hepatocytes was lower than that in rat hepatocytes and H e p G 2 cells (Fig. 1). O A stimulated [3H]TG synthesis 7-fold as compared to the fatty acid-free controls in human hepatocytes in 5-h incubations, versus about 4-fold increases in rat hepatocytes and HepG2 cells. In contrast, E P A enhanced T G synthesis only 2-fold in these three cell types although the difference between human hepatocytes exposed to EPA and controls did not reach statistical significance (Fig. 1). O A induced a 2 . 5 - 3 - f o l d increase of [3H]TG secretion in the three cell preparations used (Fig. 2). In contrast, E P A did not affect [3H]TG secretion in HepG2 cells and rat hepatocytes and even suppressed its secretion by 75% in human hepatocytes. During 5-h incubations, O A caused an increase of cellular T G mass by (mean ___S.D.) 57.6 ___9.7, 51 _ 5.2 and 63.7 + 5.6 n m o l / m g cell protein over control (i.e. + 1 2 8 % , + 1 0 8 % and + 165%) in human hepatocytes, H e p G 2 cells and rat hepatocytes, respectively (HepG2 cells versus rat hepatocytes, P < 0.05). In contrast to OA, E P A did not have significant effects on cellular TG mass in either cell type (Fig. 3).
3.3. Effect o f fatty acids on other glycerolipid synthesis [3H]Glycerol was also used to monitor the changes of biosynthesis of DG, PE and PC (Fig. 4). Both fatty acids caused a 2.5- to 5-fold increase versus control in [3H]glycerol incorporation into DG in all three cell systems. In contrast to OA, which did not significantly alter cellular [3H]PC or [3H]PE content, E P A lowered [3H]PC
Table 1 Protein synthesis and LDH leakage of cells incubated with BSA alone or BSA-bound fatty acids Human hepatocytes HepG2 cells Protein synthesis (dpm • 10- 4/mg cell protein) Control EPA OA LDH leakage (% cell LDH) Control EPA OA
91
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7.37 + 1.21 7.37 ± 1.04 8.02 ± 0.91
14.35 ± 0.03 15.33 ± 1.45 19.09 ± 1.31
4.49 ± 0.15 4.78 ± 0.28 4.54 ± 0.56
6.01 ± 1.94 6.18 ± 1.80 6.11 ± 0.92
5.07 ± 0.38 5.79 ± 0.13 6.44 ± 0.09
6.11 ± 1.72 6.59 ± 2.14 7.09 ± 0.34
Human hepatocytes, HepG2 cells and rat hepatocytes were incubated for 5 h with 0.25 mM BSA in the absence (control) or presence of 1 mM EPA or OA. Cellular protein synthesis was estimated by adding [3H]leucine (2.5 b~Ci/ml) to the media 1 h before termination of the experiment. Labeled trichloroacetic acid-precipitable proteins were quantitated by scintillation counting. LDH was measured as described in Section 2. LDH in medium is expressed as % of the amount of cellular LDH. Data are expressed as mean + S.E. of three separate experiments, each one performed in triplicate. No statistically significant differences (P > 0.05) were found among fatty acids or fatty acid-free treatment groups.
92
~ Lin et al, / Biochimica et Biophysica Acta 1256 (1995) 88-96
content by 30% in human hepatocytes and HepG2 cells ( P < 0.01), but not in rat hepatocytes. EPA did not affect [3H]PE content in human hepatocytes but lowered the -e--
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Fig. 1. Time course of the effect of OA and EPA on [3H]TG synthesis. Human hepatocytes (upper panel), HepG2 cells (middle panel) and rat hepatocytes (bottom panel) were incubated in Williams' E medium containing 1 mM OA or EPA complexed with 0.25 mM BSA or BSA alone (control) for ] to 5 h. [3H]glycerol was added to the media (4.4 /xCi/ml, 25 /zM final concentration) 1 h before the indicated time points. Cellular lipid was extracted and purified by HPTLC, [3H]TG was determined by scintillation counting. Data represent the cumulative [3H]TG synthesis as the mean + S.E. of three (HepG2 cell: two) separate experiments (human hepatocytes isolated from the livers of donors a, b, and c) with triplicate incubation. Error bars smaller than the symbol size are not shown. * Shows that statistical differences are OA > EPA and control ( P < 0,05). § Shows OA > EPA > control ( P < 0.05).
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Fig. 2. Time course of the effects of OA and EPA on [3H]TG secretion by human hepatocytes (upper panel), HepG2 cells (middle panel) and rat hepatocytes (bottom panel). Cell incubation and data presentation are as described in the legend of Fig. 1. Medium TG was extracted and purified by TLC. [3H]TG was determined by scintillation counting. Error bars smaller than the symbol size are not shown. * Shows OA is significantly different from EPA and control ( P < 0.05). § Shows EPA is significantly less than control ( P < 0.05).
[3H]PE content by 50% in HepG2 cells and increased [3H]PE content by 300% in rat hepatocytes.
3.4. Effect of fatty acids on apoB secretion Cell and medium total apoB abundances were semiquantitatively determined in primary human hepatocytes and HepG2 cells. Fig. 4 demonstrates that there are distinct
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Fig. 3. Time course of the effects of OA or EPA on cellular TG mass accumulation. Human hepatocytes (upper panel), HepG2 cells (middle panel) and rat hepatocytes (bottom panel) were incubated as described in the legend of Fig. 1. Cellular TG was extracted and purified by TLC. TG mass was analyzed as described in Section 2. Data are presented as mean_+ S.E. Error bars smaller than symbol size are not shown. * Shows OA is significantly different from EPA and control ( P < 0.05). § Shows statistical difference between AO and control ( P < 0.05).
differences between the effects of OA and of EPA in human hepatocytes, as well as between human hepatocytes and HepG2 cells. In human hepatocytes, both fatty acids did not alter cellular apoB amount, while EPA decreased medium apoB content by 44%. In marked contrast, in HepG2 cells both EPA and OA increased cell and medium
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Fig. 4. Differential effects of OA or EPA on [3H]glycerolipids synthesis other than TG. Human hepatocytes, HepG2 cells and rat hepatocytes were incubated as described in Fig. l. [3H]DG, [3H]PE and [3H]PC were purified by HPTLC and determined by scintillation counting. The cumulative [3H]glycerolipid synthesis during 5-h incubation with EPA or OA were divided by those obtained from cells incubated with BSA alone to obtain the relative values. Data are presented as mean +S.E. Significant differences at level of P < 0.05 among OA (a), EPA (b) and control (c) are shown in each panel. The absolute control values (dpm. 1 0 - 4 / r a g cell protein, mean_+S.E.) for human, HepG2 and rat cells are (1) [3H]DG: 4.6_+1.6, 5.4_+2.0 and 5.6_+1.5; (2) [3H]PC: 6.7_+0.5, 16.9_+ 5.2 and 6.6-+1.4; (3) [3H]PE: 1.7_+0.2, 7.5-+1.1 and 0.9_+0.1, respectively.
E Lin et al. / Biochimica et Biophysica Acta 1256 (1995) 88-96
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apoB abundances 2- to 2.5-fold as compared to fatty acid-free control incubation. ApoB content was about 20% lower in medium of EPA-treated HepG2 cells than in those of OA-treated cells, but this difference did not reach statistical significance. ApoA-I secretion both by human hepatocytes and HepG2 cells was not altered by OA or EPA (data not shown).
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Fig. 5. lmmunoblot detection of cell and medium apoB abundances. Human hepatocytes (isolated from liver donor d and e) and HepG2 cells were incubated with 0.25 mM BSA in the absence (control) or presence of 1 mM OA or EPA for 5 h. Cellular or medium apoB samples were treated as described in Section 2. ApoB samples corresponding to same amount of cellular total protein were chromatographed on a 5% polyacrylamide gel. ApoB was visualized by Western blot exposed to Hyperfilm. ApoB abundances were quantified by densitometry. Values were divided by those obtained in the medium from cells incubated with BSA alone to obtain the relative values. Data are presented as mean + S.E. of two separate experiments with triplicate incubation. * Shows significant difference versus OA and control ( P < 0.01). § Shows significant difference versus EPA and OA ( P < 0.01).
The present study for the first time documents the marked effects of EPA on lipid metabolism in primary cultures of human hepatocytes, which allows a better understanding of the underlying mechanism(s) of the hypolipidemic effects of fish oil in humans. In addition, the comparison of the responses to exogenous fatty acids in human hepatocytes on the one hand and HepG2 and rat hepatocytes on the other hand clearly demonstrates divergent metabolic responses in the different cell types. The most important finding of this study is that, as compared to fatty acid-free control, EPA suppresses [3H]TG secretion by 75% and apoB by 44% in human hepatocytes, while in HepG2 cells and rat hepatocytes EPA affects [3H]TG secretion only when compared to OA. This indicates that, in primary human hepatocytes, EPA leads to 'true inhibition' of VLDL production. Recent studies [5] demonstrated that EPA also suppresses TG secretion to a level below the fatty acid-free control in rabbit hepatocytes. One of the long-standing arguments concerning the effects of EPA is whether or not it affects VLDL secretion by reducing cellular TG synthesis. Some earlier reports, using HepG2 cells [8], rat hepatocytes [4,15] or rabbit hepatocytes [5], showed that the incorporation of [3H]glycerol into both secreted TG and cellular TG was reduced by EPA. However, other studies using the same cell models (HepG2 cells [9,23] or rat hepatocytes [6]) indicated that EPA decreases [3H]TG secretion when compared to OA despite the fact EPA is as effective as OA in stimulating cellular TG synthesis. The reasons for these divergent results are not clear and may be related to different experimental conditions employed. In the present study, we incubated EPA with human hepatocytes, HepG2 cells and rat hepatocytes under the same experimental conditions and were able to show that, compared to OA, EPA was significantly less effective in stimulating cellular TG synthesis in these three cell types. Our data therefore indicate that, compared to OA, the EPA-induced decline in VLDL secretion may, at least in part, be related to the reduction in cellular TG synthesis. It has recently been demonstrated also that EPA stimulates TG synthesis to a lesser extent than OA in cultured normal human fibroblasts [24]. In contrast to its lower ability to stimulate [3H]TG synthesis, EPA stimulated cellular [3H]DG synthesis to a
Y. Lin et al. /Biochimica et Biophysica Acta 1256 (1995) 88-96
similar extent as did OA (Fig. 4). Other researchers also reported that, in HepG2 cells, EPA caused a 200% increase of incorporation of [3H]glycerol into cellular DG over that of fatty acid-free control [9]. The elevated [3H]DG levels in EPA-treated cells suggest that conversion of DG to TG is reduced in the presence of EPA, in agreement with the finding that EPA decreases the activity of acylcoenzyme A:l,2-diacylglycerol acyltransferase in isolated rat liver microsomes [25]. However, it should be realized that by using [3H]glycerol for this purpose only de novo synthesized D G / T G is determined. Though EPA caused a 2-fold increase of cellular [3H]TG synthesis in HepG2 cells and rat hepatocytes, the cellular TG mass was not significantly altered. As cellular TG mass reflects the balance between TG synthesis and its degradation and secretion, this suggests that accelerated cellular TG synthesis must be coupled to an increase of its degradation. A previous study has shown that EPA increases cellular fatty acid /3-oxidation [26], but further studies are needed in this field. Despite the fact that EPA and fatty acid-free treated human hepatocytes contained similar amounts of cellular TG, the former secreted much less [3H]TG than the latter did (Fig. 2). This indicates that the inhibitory effect of EPA on the [3H]TG secretion can probably not be explained by the reduction of cellular TG availability in human hepatocytes. It should be emphasized, however, that measurement of [3H]TG secretion only, as performed in our study, may mask potential alterations in specific activities of intracellular TG pool(s) involved in VLDL secretion. Due to the relatively small amounts of cells used and short-term nature of these experiments, mass TG in medium could not be quantitated. On the other hand, it is known that both in rat hepatocytes [6] and in HepG2 cells [8] the effects of EPA (1 mM) on [3H]TG and TG mass secretion are very similar. Inhibition of apoB secretion by EPA (Fig. 5) may account for the reduced [3H]TG secretion, as apoB is required for VLDL assembly and its subsequent secretion. The simultaneous inhibition of [3H]TG and apoB secretion suggests that less VLDL particles are secreted by human hepatocytes in the presence of EPA. As cellular apoB abundance was not altered, the reduction of VLDL secretion cannot be attributed to lack of apoB availability in cells. It may be that EPA-containing TG is not suitable for VLDL assembly a n d / o r influences VLDL structure, subsequently leading to impaired VLDL secretion. The particles not secreted are probably rapidly degraded, since no apoB accumulation in human hepatocytes was observed. It has recently been reported that apoB degradation is increased in rat hepatocytes treated with EPA [7] and in livers of rats consuming diets containing fish oil [27]. In addition, our data imply that in human hepatocytes, the regulation of cellular TG and apoB synthesis are uncoupled, as OA stimulated TG synthesis 7-fold and caused TG mass to increase by 128% without altering cellular apoB abundance (Figs. 1, 3 and 5). The fact that
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OA increased [3H]TG secretion without a coincident increase of apoB secretion in human hepatocytes suggests that, in the short term, human hepatocytes regulate TG secretion via altering apoB-containing lipoprotein assembly. ApoB availability may be rate-limiting in VLDL secretion in this situation. When HepG2 cells were incubated with OA or EPA for 5 h, cellular apoB abundances were increased 2.5-fold, suggesting that HepG2 cells increase apoB synthesis in response to exogenous fatty acids. Using a semiquantitative method, we found that the amount of apoB in medium was slightly, non-significantly, lower ( - 2 0 % ) in the presence of EPA compared to OA. This is in line with previous observations [8] using concentrations of 1 mM of these fatty acids. These data show that the regulation of apoB synthesis and secretion in HepG2 cells is distinctly different from that in primary human hepatocytes. In addition, in primary human hepatocytes, OA stimulated [3H]TG secretion by increasing the efficiency of TG packaging into apoB-containing lipoprotein (VLDL) as discussed above, while in HepG2 cells OA stimulated both [3H]TG and apoB secretion, indicating increased secretion of VLDL particle numbers. Taken together, these data suggest that the regulation of VLDL assembly/secretion in HepG2 cells may be different from that in primary human hepatocytes. Since cellular PC synthesis is obligatory for the secretion of VLDL particles by rat hepatocytes [28] and newly made PC is preferred for assembly into lipoproteins [29], the influence of EPA on PC synthesis were also examined. Although EPA decreased cellular [3H]PC contents in either cell type as compared to OA, no relation between cellular PC synthesis and TG secretion was observed among these cell types. Furthermore, EPA strikingly promoted PE synthesis compared to OA or fatty acid-free control in rat hepatocytes (Fig. 4). It may be that polyunsaturated fatty acids are preferentially incorporated into PE in rat [30]. The alternative explanation is that the increase of PE synthesis reflects the adaptation of metabolism caused by inhibition of cellular TG synthesis in the presence of EPA. This phenomenon was not observed in human hepatocytes or in HepG2 cells, which suggests that species differences exist in EPA-induced phospholipid metabolism between rat and human hepatocytes and HepG2 cells. In a long-term culture of rat hepatocytes with EPA, it has been shown that EPA-associated peroxidation events are cytotoxic. EPA-associated cytotoxicity is characterized in a time- and dose-dependent increase of cell LDH-leakage [31]. However, the diminution of [3H]TG secretion and apoB secretion observed in human hepatocytes in the present study are unlikely due to a non-specific cytotoxic effect of EPA, as the cell viability (Table 1) and apoA-I (data not shown) secretion were not affected in the presence of EPA. In summary, in human hepatocytes, EPA potently inhibits [3H]TG secretion by suppressing VLDL particle assembly a n d / o r secretion. EPA and OA induced strikingly different effects on cellular lipids and apoB
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metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes both in quantity and quality.
Acknowledgements Y.L. is supported by a grant from the Royal Netherlands Academy of Arts and Sciences. H.J.V. is supported by grant NWO-KWO(No. 900-716-028). F~K. is an established investigator of the Netherlands Heart Foundation. This study was performed in collaboration with the Human Liver Group, Groningen.
References [1] Von Lossonczy, T.O., Ruiter, A., Bronsgeest-Schoute, H.C., Van Gent, C.M. and Hermus, R.J.J. (1978) Am. J. Clin. Nutr. 31, 1340-1346. [2] Harris, W.S. (1989) J. Lipid Res. 30, 785-807. [3] Nestel, P.L. (1990) Annu. Rev. Nutrl 10, 149-168. [4] Graham, A., Zammit, V.A. and Brindley, D.N. (1988) Biochem. J. 249, 727-733. [5] Benner, K.G., Sasaki, A., Gowen, D.R., Weaver, A. and Connor, W.E. (1990) Lipids 25, 534-540. [6] Lang, C.A. and Davis, R.A. (1990) J. Lipid Res. 31, 2079-2086. [7] Wang, H. Chen, X. and Fisher, E.A. (1993) J. Clin. Invest. 91, 1380-1389. [8] Wong, S.H. Fisher, E.A. and Marsh, J.B. (1989) Arteriosclerosis 9, 836-841. [9] Homan, R., Grossman, J.E. and Pownall, H.J. (1991) J. Lipid Res. 32, 231-241. [10] Blaauboer, B.J., Van Holsteijn, C.W.M., Bleumink, R., Mennes, W.C., Van Delt, F.N.A.M., Yap, S.H., Van Pelt, J.F., Van Iersel, A.A.J., Timmerman, A. and Schmid, B.P. (1990) Biochem. Pharmacol. 40, 521-528.
[11] Moshage, H., and Yap, S.H. (1992) J. Hepatol. 15, 404-413. [12] Kalayoglu, M, Sollinger, H.W., Stratta, R.J., D'Alesandro, A.M., Hoffman, R.M., Pirsch, J.D. and Belzer, F.O. (1988) Lancet 1, 617-619. [13] Berry, M.N. and Friend, D.S. (1969) J. Cell Biol. 43, 506-520. [14] Davis, R.A., Engelhorn, S.C., Pangburn, S.H., Weinstein, D.B. and Steinberg, D. (1979) J. Biol. Chem. 254, 2010-2016. [15] Nossen, J.O., Rustan, A.C., Gloppestad, S.H., Mlilbakken, S. and Drevon, C.A. (1986) Biochim. Biophys. Acta 879, 56-65. [16] Kosykh, V.A. Preobrazhensky, S.N., Fuki, I.V., Zaikina, O.E., Tsibulsky, V.P., Repin, V.S. and Smirnov~ V.N. (1985) Biochim. Biophys. Acta 836, 385-389. [17] Thrif, R.N., Forte, T.M. Cahoon, B.E. and Shore, V.C. (1986) J. Lipid Res. 27, 236-250. [18] Vance, D.E., Weinstein, D.B. and Steinberg, D. (1984) Biochim. Biophys. Acta 792, 39-47. [19] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [20] Smit, M.J., Duursma, A.M. and Bouma, J.M.W. (1986) lsr. J. Vet. Med. 42, 260-270. [21] Hoshino, M, Ohiwa, T., Hayakawa, T., Kamiya, Y., Tanaka, A., Hirano, A., Kumai, T., Katagiri, K., Miyaji, M. and Takeuchi, T. (1993) Scand. J. Gastroenterol. 28, 833-838. [22] Nossen, J.O., Rustan, A.C., Barnard, T. and Drevon, C.A. (1984) Biochim. Biophys. Acta 803, 11-22. [23] Wong, S. and Nestel, P.J. (1987) Atherosclerosis 64, 139-146. [24] Miller, M., Motevalli, M., Westphal, D. and Kwiterovich, P.O. Jr. (1993) Lipids 28, 1-5. [25] Rustan, A.C., Nossen, J.O., Christiansen, E.N. and Drevon, C.A. (1988) J. Lipid Res. 29, 1417-1426. [26] Christensen, E., Hagve, T. and Christophersen, B.O. (1986) Biochim. Biophys. Acta 879, 313-321. [27] Ribeiro, A., Mangeney, M., Cardot, P., Lorette, C., Rayssiguier, Y., Chambaz, J. and Bereziat, G. (1991) Eur. J. Chem. 196, 499-507. [28] Yao, Z. and Vance, D.E. (1988) J. Biol. Biochem. 263, 2998-3004. [29] Vance, J.E. (1989) Biochim. Biophys. Acta 1006, 59-69. [30] Valtersson, C., Filipsson, L. and Dallner, G. (1986) J. Lipid Res. 27, 731-741. [31] Mikkelsen, L., Hansen, H.S., Grunet, N. and Dich, J. (1993) Biochim. Biophys Acta 1166, 99-104.
ELSEVIER
Biochimicaet BiophysicaActa 1256 (1995) 88-96
Biochi ~mic~a et BiophysicaA~ta
Differential effects of eicosapentaenoic acid on glycerolipid and apolipoprotein B metabolism in primary human hepatocytes compared to HepG2 cells and primary rat hepatocytes Yuguang Lin, Martin J. Smit, Rick Havinga, Henkjan J. Verkade, Roel J. Vonk, Folkert Kuipers * Groningen Institute for Drug Studies, Laboratory of Nutrition and Metabolism, Department of Pediatrics, UniL'ersity and Academic Hospital Groningen, Oostersinge159, 9713 EZ Groningen, The Netherlands
Received 18 August 1994; revised 13 December 1994; accepted 20 December 1994
Abstract We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or presence of 1 mM of EPA or OA. Synthesis and secretion of [3H]glycerolipid were determined after 1 h incubation with [3H]glycerol. Cellular and medium apoB abundance was semi-quantitatively estimated in human cells by Western blotting. The following observations were made. (1) Compared to control, OA induced a 7-fold increase in [3H]triacylglycerol (TG) synthesis in human hepatocytes and a 4-fold increase in rat hepatocytes and HepG2 cells. EPA enhanced [3H]TG synthesis about 2-fold in all three cell types although it stimulated [3H]diacylglycerol (DG) synthesis to an extent (i.e., 2.5- to 5-fold) similar to OA. (2) In contrast to OA, which stimulated VLDL-associated [3H]TG secretion 2.5- to 3-fold in the three cell types relative to control, EPA did not alter [3H]TG secretion in HepG2 and rat hepatocytes and suppressed [3H]TG secretion by 75% in primary human hepatocytes. (3) In primary human hepatocytes, both OA and EPA did not alter cellular apoB abundance but EPA decreased apoB secretion by 44% as compared to control. In contrast, both EPA and OA increased cellular and medium apoB abundance 2- to 2.5-fold in HepG2 cells, although medium apoB tended to be lower in EPA-treated cells. (4) EPA had no effect on the [3H]phosphatidylcholine (PC) synthesis in rat hepatocytes, but decreased [3H]PC synthesis by 30% in human hepatocytes and HepG2 cells. This study shows that, in primary human hepatocytes, EPA decreases TG secretion below control values by influencing VLDL particle assembly and/or secretion. There are both qualitative and quantitative differences in the effects of EPA and OA on the glycerolipid and apoB metabolism between primary human hepatocytes and both HepG2 and primary rat hepatocytes, the two most commonly used model systems to investigate hepatic lipid metabolism in vitro. Keywords: In vitro; Primary human hepatocyte; lcosapentaenoicacid; Oleic acid; Triacylglycerol;ApolipoproteinB; Phospholipid
1. Introduction The well-documented serum triacylglycerol (TG)-lowering effects of dietary fish [1] and fish oil [2] in humans has generated intensive research on the mechanism(s) responsible for this hypolipidemic action. The effect has been
Abbreviations:BSA, bovine serum albumin;DG, diacylglycerol;EPA, eicosapentaenoic acid; FCS, fetal calf serum; LDH, lactate dehydrogenase; OA, oleic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine;SDS, sodium dodecyl sulfatc; TG, triacylglycerol;(HP)TLC, (high performance) thin-layer chromatography; VLDL, very low density lipoprotein * Corresponding author. Fax: +31 50 696800. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0005-2760(95)00006-2
attributed to the presence of the n - 3 fatty acids [3], i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid, that are particularly abundant in fish oil. Fish oil and its isolated components are able to influence lipid metabolism in a number of ways, as reviewed by Harris [2]. It is generally accepted that interference with the secretion of very low density lipoproteins (VLDL) is one of the physiologically most important effects of fish oil. This action has been attributed to decreased hepatic triglyceride synthesis [4,5] and to inhibition of assembly a n d / o r secretion of newly synthesized apoB-containing lipoprotein particles [6]. Recently, it has been shown that, in rat hepatocytes, this latter effect may be related to an EPA-induced acceleration of intracellular apoB degradation [7]. Most of the
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studies on the mechanism(s) by which fish oil (components) affect VLDL secretion have been carried out in cell cultures of animal origin [5-7] or, as a model for the human liver, in HepG2 cells [8,9]. It is, however, well established that marked species differences in hepatic lipid metabolism exist between experimental animals and humans. For example, VLDL secreted by rat liver contains apoB-48 as well as apoB-100 whereas human VLDL only contains apoB-100. In addition, it has been reported that changes in lipid metabolism induced by hypolipidemic drugs, such as clofibrate and beclobric acid, are strikingly different between rat and human hepatocytes [10]. HepG2 cells, which are derived from a human hepatoma, have been reported to differ from normal liver cells in a number of ways [11]. It would be valuable, therefore, on one hand, to evaluate the effects of EPA on lipid metabolism in human hepatocytes in primary culture and, on the other hand, to compare the results in these cells with those obtained with HepG2 cells and rat hepatocytes in primary cultures, the latter two being the most commonly used cell systems in this area of research.
2. Materials and methods 2.1. Materials
Williams' E medium, glutamine, penicillin-streptomycin, fetal calf serum (FCS) and collagenase were purchased from Life Technologies, Paisley, UK. Insulin and dexamethasone came from Novo Nordisk Farma, Amsterdam, the Netherlands. Oleic acid (OA) (99% by capillary GC), eicosapentaenoic acid (98%), bovine serum albumin (BSA) (Fraction V, essentially fatty acid-free) and hydrated colloidal silica (Cab-O-Sil) (fumed silica, particle size 0.011 /xm) were purchased from Sigma Chemical Co., St. Louis, MO, USA. [2-3H]Glycerol (1 Ci/mmol), L-[4,53H]leucine (61 C i / m m o l ) and Enhance chemiluminescence Western blotting reagent were obtained from Amersham International, Amersham, UK. Cell culture plates were supplied by Costar, Cambridge, MA, USA. Thin-layer chromatography (TLC) plates and high performance thinlayer chromatography (HPTLC) plates were obtained from Merck, Darmstadt, Germany. Triglycerides GPO-PAP kit, sheep anti-human-apoB and anti-human-apoA-I antiserum were obtained from Boehringer Mannheim, Mannheim, Germany. All other chemicals and solvents were high purity commercial materials. 2.2. Preparation and culture o f hepatocytes
Human liver material was obtained from healthy liver transplant donors. Consent from legal authorities and donor families was obtained. Human hepatocytes were isolated from spare-parts when the donor livers were split to perform reduced-size liver transplantation in children. Age and sex of liver donors used for these studies were: a: 50
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y, male; b: 46 y, female; c: 26 y, male; d: 6 y, male and e: 36 y, male. Livers were perfused with an organ preservation solution [12] at the time of harvesting and kept at 0 - 4 ° C until used for cell preparation (2-12 h). A wedgeshaped piece of 30-200 g with a single cut surface was used for hepatocyte isolation. Two to four major branches of the portal veins were cannulated with plastic cannulae for perfusion and other major vessels were tied off. The tissue was first perfused with 1000 ml calcium-free Hank's fluid containing glucose (10 mM) and Hepes (10 mM) at a flow rate of 90 m l / m i n to wash out the preservation fluid or blood and to warm the liver to 37 ° C. This was followed by perfusion of 1500 ml calcium-free Hank's fluid containing NaHCO 3 (25 mM) and glucose (10 mM) and 200 ml calcium containing Hank's buffer before 400 ml of enzyme solution (50 mg of collagenase/100 ml calcium containing Hank's buffer) was perfused with recirculation for 15 min. All solutions were pre-gassed with 95% 02 and 5% CO 2 and adjusted to pH 7.42. Rat hepatocytes were isolated by the method of Berry and Friend [13] from male Wistar rats of 180-220 g. Rats were maintained in a light and temperature controlled room and fed standard lab chow and water ad libitum until sacrificed. HepG2 cells were cultured in our laboratory and routinely replated weekly. The viability of both human and rat hepatocytes after isolation was examined by trypan blue exclusion (final concentration 0.2%). Cells were plated in 35 mm 6-well (pre-coated with collagen) plastic dishes at a density of 1.5-10 6 viable cells/well with 2 ml medium. When HepG2 cells were used for experiments, about 3 • 105 cells were seeded to each well of 6-well plate without collagen pre-coating. The culture medium consisted of Williams'E medium supplemented with 10% FCS, 2 mM glutamine, 1 0 0 / x g / m l penicillin, 1 0 0 / z g / m l streptomycin, 4 m U / m l insulin and 0.02 / z g / m l dexamethasone. After cells had attached to the plates, medium was renewed. Subsequently, medium was renewed every 24 h. Cells were incubated at 37°C in an atmosphere of 95% air and 5% CO z. 2.3. Experimental protocol
Experiments were conducted two days after seeding of cells. On the day of an experiment, BSA-fatty acid complexes were prepared: a sterile solution of 0.25 mM BSA in Williams' E medium with insulin, dexamethasone, penicillin and streptomycin at the concentrations mentioned above was adjusted to pH 10. Fatty acid was solubilized in chloroform in glass tubes and dried under nitrogen. The BSA-containing medium was mixed to the dried fatty acid while sonicating vigorously to produce a solution of 1 mM fatty acid/0.25 mM BSA. The pH was adjusted to 7.4, and media were warmed to 37 ° C before use. FCS-containing medium was removed from the wells and cells were washed two times with FCS-free medium
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and incubated in this medium for 2 h. Then the medium was changed to the medium (1 ml/well) containing fatty acid-BSA complexes or BSA alone as control and cells were incubated for 1-5 h. To determine the glycerolipid synthesis rates at indicated time points, [3H]glycerol (4.4 /xCi/well, 25 /xM) was added 1 h before termination of the experiments. All incubations were performed in triplicate and terminated by putting the culture plates on ice. Media were collected via suction and centrifuged at 10 000 rpm for 2 min to remove suspended cells. Cells were washed twice with phosphate-buffered saline (PBS) and harvested by using a rubber policeman in 2 ml PBS. Samples were frozen at - 2 0 ° C prior to analysis. Cells and media used for apoB determination were harvested as described below in subsection 2.7. 2.4. Assessment of glycerolipid synthesis
Cells were thawed and resuspended by passing through 26G X 1-inch (0.45 X 25 mm) needles. The cellular lipids were extracted with chloroform/methanol (1:2, v / v ) . Triacylglycerol (TG), diacylglycerol (DG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were separated by HPTLC. Plates were developed half-way with the solvent c h l o r o f o r m / e t h a n o l / w a t e r / t r i e t h y l a m i n e (30:34:2:35, v / v ) to separate the phospholipids. After drying of the plates, TG and DG were separated by developing by hexane/diethylether/acetic acid (80:20:1) to the top of the plate. TG, DG, PE and PC spots were visualized by spraying with 0.1% Rhodamine G and marked under ultraviolet light. The spots were scraped into vials and assayed for radioactivity by scintillation counting. 2.5. Assessment of TG mass
Cellular lipids were extracted and separated by thin layer chrom atography developed by hexane/diethylether/acetic acid (80:20:1) mixture. T G was extracted two times from the silica gel with 2.5 ml of chloroform. Pilot experiments with trihexadecanoylglycerol showed that the recovery of this procedure was 8 6 92% in three independent determinations. The TG extracts were dried under nitrogen, resuspended in 100 /xl isopropanol and TG mass was assayed according to the instruction of manufacturers of the Triglycerides GPO-PAP Kit, with glycerol as standard. 2.6. Assessment of [3H]TG secretion
Since previous studies [14-16] and own observations revealed that greater than 95% of [3H]TG secreted into the medium of rat and human hepatocytes in primary culture was recovered in the VLDL fraction (d < 1.006 g / m l ) , the [3H]TG in the media were used to estimate VLDL-[3H]TG secretion. In contrast to the primary hepatocytes, most of the TG secreted by HepG2 cells is associated with an LDL-like lipoprotein under basal conditions [17]. Before
lipids were extracted, 30 /xl of 2 mM triacylglycerol solution in chloroform was added to each sample as a carrier. TG was separated from other lipids by TLC using a similar procedure to the assay of cellular TG mass. Silica gel containing TG was scraped into vials and assayed for radioactivity via scintillation counting. 2.7. Western blotting of apoB and apoA-I
Medium samples (2 ml) of human hepatocytes and HepG2 cells were added 0.1 ml of a detergent buffer (containing 250 mM EDTA, 10% Triton X-100, 10% deoxycholic acid, 1.25 M NaC1, 0.5 M Tris-HCl, 1% sodium dodecyl sulfate (SDS) and freshly added 0.01 M dithiothreitol and 0.15% phenylmethylsulfonyl fluoride (PMSF)). The cell monolayer was solubilized by addition of 0.5 m l / w e l l of 10-times diluted detergent buffer. The cell lysates were pipetted into tubes and heated at 70 ° C for 30 min and diluted with an equal volume of a solution containing 0.01 M Tris-HC1 (pH 7.4) and 0.15 M NaC1. Cell lysates were centrifuged at 10 000 rpm for 2 min and the supernatant of the cell lysates were used for apoB determination ApoB in cell lysates was precipitated by adding antiapoB-serum at final dilution 1:200. The tubes were rotated overnight followed by adding 40 /zl of 12% ( w / v ) protein A beads and incubated for another 2 h. Protein A-antibody-apoB complexes were collected by centrifugation. ApoB and apoA-I contained in media were absorbed to Cab-O-Sil according to the methods previously described [18]. Control experiments showed that when the initial immunoprecipitation (cell lysate) or Cab-O-Sil absorption (media) was followed by a second immunoprecipitation of the supernatant, no additional apoB was recovered. After being washed two times with PBS, apoproteins were solubilized from the protein A beads or silica pellet with an extraction buffer comprising 2% SDS, 0.05 M Tris (pH 9.0), 6 M urea, 0.1% EDTA, 0.1% dithiothreitol, 0.13% E-aminocaproic acid and 0.05% glutathione in a 95°C water-bath for 10 min. ApoB and apoA-I were separated from other proteins by electrophoresis in 5% polyacrylamide gel containing 0.1% SDS. Subsequently, apoproteins were transferred to nitrocellulose membranes. Immunoblotting was carried out as follows: the membrane was immersed in a solution of Tris-buffered saline (20 mM Tris (pH 7.5) and 0.5 M NaCI) containing 5% dried milk for 1 h at room temperature to block non-specific binding; incubated with the primary antibody (1:5000) overnight; washed four times with Tris-buffered 5% milk solution following the incubation with secondary horseradish peroxidase conjugated antibody (1:10000) for 2 h and then washed as before. ApoB- or apoA-I-antibody complexes were visualized with Enhance chemiluminescence Western blotting reagent by exposure to Hyperfilm according to the manufacturer's instructions. Quantification of the bands was performed with an UltroScan XL densitometer.
Y. Lin et aL /Biochimica et Biophysica Acta 1256 (1995) 88-96 2.8. Miscellaneous methods Protein was measured according to Lowry et al. [19] using B S A as standard. Lactate dehydrogenase (LDH) activity in media and cells were determined as reported previously [20]. The leakage of L D H from cells was present as LDH-leakage %, which was calculated according to the formula: (LDH activity in m e d i u m / L D H activity in cell) × 100%. The [3H]leucine incorporation into the trichloroacetic acid-precipitable protein was investigated to determine the cellular protein synthesis. Hepatocytes were cultured for 5 h in the same medium as for TG synthesis measurement and [3H]leucine (2.5 /.~Ci/ml per well) was added 1 h before termination of the cell incubation.
2.9. Statistical analysis Data were normalized to the amount of cellular protein. Statistical differences were assessed using two-way analysis of variance. Values of P < 0.05 were considered significant.
3. Results 3.1. Effect o f fatty acids on cell viability Table 1 shows that cellular protein synthesis was not affected when human hepatocytes, HepG2 cells or rat hepatocytes were exposed to 1 m M O A or E P A for 5 h. The L D H activities in media were similar across all groups and were in agreement with a previous report concerning the viability of liver cell in culture [21]. These results indicate that under the conditions employed, incubation with fatty acids did not exert a toxic effect on the hepatocytes.
3.2. Effect o f fatty acids on TG synthesis and secretion Previous studies [22,23] have shown that rat hepatocytes incubated with 1 m M O A and 5 /xCi [3H]glycerol
synthesize [3H]TG in a linear fashion for only 1 to 2 h. Considering the fact that a trace amount of [3H]glycerol is rapidly catabolized by hepatocytes, [3H]glycerol was added 1 h before termination of the experiment and data are presented as accumulated [3H]TG synthesis and secretion at each indicated time point to compare the effects of O A and EPA. In all Jthe three cell types studied, the incorporation of [3H]glycerol into cellular TG was significantly greater with O A than with EPA. Control [3H]TG synthesis in human hepatocytes was lower than that in rat hepatocytes and H e p G 2 cells (Fig. 1). O A stimulated [3H]TG synthesis 7-fold as compared to the fatty acid-free controls in human hepatocytes in 5-h incubations, versus about 4-fold increases in rat hepatocytes and HepG2 cells. In contrast, E P A enhanced T G synthesis only 2-fold in these three cell types although the difference between human hepatocytes exposed to EPA and controls did not reach statistical significance (Fig. 1). O A induced a 2 . 5 - 3 - f o l d increase of [3H]TG secretion in the three cell preparations used (Fig. 2). In contrast, E P A did not affect [3H]TG secretion in HepG2 cells and rat hepatocytes and even suppressed its secretion by 75% in human hepatocytes. During 5-h incubations, O A caused an increase of cellular T G mass by (mean ___S.D.) 57.6 ___9.7, 51 _ 5.2 and 63.7 + 5.6 n m o l / m g cell protein over control (i.e. + 1 2 8 % , + 1 0 8 % and + 165%) in human hepatocytes, H e p G 2 cells and rat hepatocytes, respectively (HepG2 cells versus rat hepatocytes, P < 0.05). In contrast to OA, E P A did not have significant effects on cellular TG mass in either cell type (Fig. 3).
3.3. Effect o f fatty acids on other glycerolipid synthesis [3H]Glycerol was also used to monitor the changes of biosynthesis of DG, PE and PC (Fig. 4). Both fatty acids caused a 2.5- to 5-fold increase versus control in [3H]glycerol incorporation into DG in all three cell systems. In contrast to OA, which did not significantly alter cellular [3H]PC or [3H]PE content, E P A lowered [3H]PC
Table 1 Protein synthesis and LDH leakage of cells incubated with BSA alone or BSA-bound fatty acids Human hepatocytes HepG2 cells Protein synthesis (dpm • 10- 4/mg cell protein) Control EPA OA LDH leakage (% cell LDH) Control EPA OA
91
Rat hepatocytes
7.37 + 1.21 7.37 ± 1.04 8.02 ± 0.91
14.35 ± 0.03 15.33 ± 1.45 19.09 ± 1.31
4.49 ± 0.15 4.78 ± 0.28 4.54 ± 0.56
6.01 ± 1.94 6.18 ± 1.80 6.11 ± 0.92
5.07 ± 0.38 5.79 ± 0.13 6.44 ± 0.09
6.11 ± 1.72 6.59 ± 2.14 7.09 ± 0.34
Human hepatocytes, HepG2 cells and rat hepatocytes were incubated for 5 h with 0.25 mM BSA in the absence (control) or presence of 1 mM EPA or OA. Cellular protein synthesis was estimated by adding [3H]leucine (2.5 b~Ci/ml) to the media 1 h before termination of the experiment. Labeled trichloroacetic acid-precipitable proteins were quantitated by scintillation counting. LDH was measured as described in Section 2. LDH in medium is expressed as % of the amount of cellular LDH. Data are expressed as mean + S.E. of three separate experiments, each one performed in triplicate. No statistically significant differences (P > 0.05) were found among fatty acids or fatty acid-free treatment groups.
92
~ Lin et al, / Biochimica et Biophysica Acta 1256 (1995) 88-96
content by 30% in human hepatocytes and HepG2 cells ( P < 0.01), but not in rat hepatocytes. EPA did not affect [3H]PE content in human hepatocytes but lowered the -e--
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Fig. 1. Time course of the effect of OA and EPA on [3H]TG synthesis. Human hepatocytes (upper panel), HepG2 cells (middle panel) and rat hepatocytes (bottom panel) were incubated in Williams' E medium containing 1 mM OA or EPA complexed with 0.25 mM BSA or BSA alone (control) for ] to 5 h. [3H]glycerol was added to the media (4.4 /xCi/ml, 25 /zM final concentration) 1 h before the indicated time points. Cellular lipid was extracted and purified by HPTLC, [3H]TG was determined by scintillation counting. Data represent the cumulative [3H]TG synthesis as the mean + S.E. of three (HepG2 cell: two) separate experiments (human hepatocytes isolated from the livers of donors a, b, and c) with triplicate incubation. Error bars smaller than the symbol size are not shown. * Shows that statistical differences are OA > EPA and control ( P < 0,05). § Shows OA > EPA > control ( P < 0.05).
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Fig. 2. Time course of the effects of OA and EPA on [3H]TG secretion by human hepatocytes (upper panel), HepG2 cells (middle panel) and rat hepatocytes (bottom panel). Cell incubation and data presentation are as described in the legend of Fig. 1. Medium TG was extracted and purified by TLC. [3H]TG was determined by scintillation counting. Error bars smaller than the symbol size are not shown. * Shows OA is significantly different from EPA and control ( P < 0.05). § Shows EPA is significantly less than control ( P < 0.05).
[3H]PE content by 50% in HepG2 cells and increased [3H]PE content by 300% in rat hepatocytes.
3.4. Effect of fatty acids on apoB secretion Cell and medium total apoB abundances were semiquantitatively determined in primary human hepatocytes and HepG2 cells. Fig. 4 demonstrates that there are distinct
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differences between the effects of OA and of EPA in human hepatocytes, as well as between human hepatocytes and HepG2 cells. In human hepatocytes, both fatty acids did not alter cellular apoB amount, while EPA decreased medium apoB content by 44%. In marked contrast, in HepG2 cells both EPA and OA increased cell and medium
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Fig. 4. Differential effects of OA or EPA on [3H]glycerolipids synthesis other than TG. Human hepatocytes, HepG2 cells and rat hepatocytes were incubated as described in Fig. l. [3H]DG, [3H]PE and [3H]PC were purified by HPTLC and determined by scintillation counting. The cumulative [3H]glycerolipid synthesis during 5-h incubation with EPA or OA were divided by those obtained from cells incubated with BSA alone to obtain the relative values. Data are presented as mean +S.E. Significant differences at level of P < 0.05 among OA (a), EPA (b) and control (c) are shown in each panel. The absolute control values (dpm. 1 0 - 4 / r a g cell protein, mean_+S.E.) for human, HepG2 and rat cells are (1) [3H]DG: 4.6_+1.6, 5.4_+2.0 and 5.6_+1.5; (2) [3H]PC: 6.7_+0.5, 16.9_+ 5.2 and 6.6-+1.4; (3) [3H]PE: 1.7_+0.2, 7.5-+1.1 and 0.9_+0.1, respectively.
E Lin et al. / Biochimica et Biophysica Acta 1256 (1995) 88-96
94
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apoB abundances 2- to 2.5-fold as compared to fatty acid-free control incubation. ApoB content was about 20% lower in medium of EPA-treated HepG2 cells than in those of OA-treated cells, but this difference did not reach statistical significance. ApoA-I secretion both by human hepatocytes and HepG2 cells was not altered by OA or EPA (data not shown).
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Fig. 5. lmmunoblot detection of cell and medium apoB abundances. Human hepatocytes (isolated from liver donor d and e) and HepG2 cells were incubated with 0.25 mM BSA in the absence (control) or presence of 1 mM OA or EPA for 5 h. Cellular or medium apoB samples were treated as described in Section 2. ApoB samples corresponding to same amount of cellular total protein were chromatographed on a 5% polyacrylamide gel. ApoB was visualized by Western blot exposed to Hyperfilm. ApoB abundances were quantified by densitometry. Values were divided by those obtained in the medium from cells incubated with BSA alone to obtain the relative values. Data are presented as mean + S.E. of two separate experiments with triplicate incubation. * Shows significant difference versus OA and control ( P < 0.01). § Shows significant difference versus EPA and OA ( P < 0.01).
The present study for the first time documents the marked effects of EPA on lipid metabolism in primary cultures of human hepatocytes, which allows a better understanding of the underlying mechanism(s) of the hypolipidemic effects of fish oil in humans. In addition, the comparison of the responses to exogenous fatty acids in human hepatocytes on the one hand and HepG2 and rat hepatocytes on the other hand clearly demonstrates divergent metabolic responses in the different cell types. The most important finding of this study is that, as compared to fatty acid-free control, EPA suppresses [3H]TG secretion by 75% and apoB by 44% in human hepatocytes, while in HepG2 cells and rat hepatocytes EPA affects [3H]TG secretion only when compared to OA. This indicates that, in primary human hepatocytes, EPA leads to 'true inhibition' of VLDL production. Recent studies [5] demonstrated that EPA also suppresses TG secretion to a level below the fatty acid-free control in rabbit hepatocytes. One of the long-standing arguments concerning the effects of EPA is whether or not it affects VLDL secretion by reducing cellular TG synthesis. Some earlier reports, using HepG2 cells [8], rat hepatocytes [4,15] or rabbit hepatocytes [5], showed that the incorporation of [3H]glycerol into both secreted TG and cellular TG was reduced by EPA. However, other studies using the same cell models (HepG2 cells [9,23] or rat hepatocytes [6]) indicated that EPA decreases [3H]TG secretion when compared to OA despite the fact EPA is as effective as OA in stimulating cellular TG synthesis. The reasons for these divergent results are not clear and may be related to different experimental conditions employed. In the present study, we incubated EPA with human hepatocytes, HepG2 cells and rat hepatocytes under the same experimental conditions and were able to show that, compared to OA, EPA was significantly less effective in stimulating cellular TG synthesis in these three cell types. Our data therefore indicate that, compared to OA, the EPA-induced decline in VLDL secretion may, at least in part, be related to the reduction in cellular TG synthesis. It has recently been demonstrated also that EPA stimulates TG synthesis to a lesser extent than OA in cultured normal human fibroblasts [24]. In contrast to its lower ability to stimulate [3H]TG synthesis, EPA stimulated cellular [3H]DG synthesis to a
Y. Lin et al. /Biochimica et Biophysica Acta 1256 (1995) 88-96
similar extent as did OA (Fig. 4). Other researchers also reported that, in HepG2 cells, EPA caused a 200% increase of incorporation of [3H]glycerol into cellular DG over that of fatty acid-free control [9]. The elevated [3H]DG levels in EPA-treated cells suggest that conversion of DG to TG is reduced in the presence of EPA, in agreement with the finding that EPA decreases the activity of acylcoenzyme A:l,2-diacylglycerol acyltransferase in isolated rat liver microsomes [25]. However, it should be realized that by using [3H]glycerol for this purpose only de novo synthesized D G / T G is determined. Though EPA caused a 2-fold increase of cellular [3H]TG synthesis in HepG2 cells and rat hepatocytes, the cellular TG mass was not significantly altered. As cellular TG mass reflects the balance between TG synthesis and its degradation and secretion, this suggests that accelerated cellular TG synthesis must be coupled to an increase of its degradation. A previous study has shown that EPA increases cellular fatty acid /3-oxidation [26], but further studies are needed in this field. Despite the fact that EPA and fatty acid-free treated human hepatocytes contained similar amounts of cellular TG, the former secreted much less [3H]TG than the latter did (Fig. 2). This indicates that the inhibitory effect of EPA on the [3H]TG secretion can probably not be explained by the reduction of cellular TG availability in human hepatocytes. It should be emphasized, however, that measurement of [3H]TG secretion only, as performed in our study, may mask potential alterations in specific activities of intracellular TG pool(s) involved in VLDL secretion. Due to the relatively small amounts of cells used and short-term nature of these experiments, mass TG in medium could not be quantitated. On the other hand, it is known that both in rat hepatocytes [6] and in HepG2 cells [8] the effects of EPA (1 mM) on [3H]TG and TG mass secretion are very similar. Inhibition of apoB secretion by EPA (Fig. 5) may account for the reduced [3H]TG secretion, as apoB is required for VLDL assembly and its subsequent secretion. The simultaneous inhibition of [3H]TG and apoB secretion suggests that less VLDL particles are secreted by human hepatocytes in the presence of EPA. As cellular apoB abundance was not altered, the reduction of VLDL secretion cannot be attributed to lack of apoB availability in cells. It may be that EPA-containing TG is not suitable for VLDL assembly a n d / o r influences VLDL structure, subsequently leading to impaired VLDL secretion. The particles not secreted are probably rapidly degraded, since no apoB accumulation in human hepatocytes was observed. It has recently been reported that apoB degradation is increased in rat hepatocytes treated with EPA [7] and in livers of rats consuming diets containing fish oil [27]. In addition, our data imply that in human hepatocytes, the regulation of cellular TG and apoB synthesis are uncoupled, as OA stimulated TG synthesis 7-fold and caused TG mass to increase by 128% without altering cellular apoB abundance (Figs. 1, 3 and 5). The fact that
95
OA increased [3H]TG secretion without a coincident increase of apoB secretion in human hepatocytes suggests that, in the short term, human hepatocytes regulate TG secretion via altering apoB-containing lipoprotein assembly. ApoB availability may be rate-limiting in VLDL secretion in this situation. When HepG2 cells were incubated with OA or EPA for 5 h, cellular apoB abundances were increased 2.5-fold, suggesting that HepG2 cells increase apoB synthesis in response to exogenous fatty acids. Using a semiquantitative method, we found that the amount of apoB in medium was slightly, non-significantly, lower ( - 2 0 % ) in the presence of EPA compared to OA. This is in line with previous observations [8] using concentrations of 1 mM of these fatty acids. These data show that the regulation of apoB synthesis and secretion in HepG2 cells is distinctly different from that in primary human hepatocytes. In addition, in primary human hepatocytes, OA stimulated [3H]TG secretion by increasing the efficiency of TG packaging into apoB-containing lipoprotein (VLDL) as discussed above, while in HepG2 cells OA stimulated both [3H]TG and apoB secretion, indicating increased secretion of VLDL particle numbers. Taken together, these data suggest that the regulation of VLDL assembly/secretion in HepG2 cells may be different from that in primary human hepatocytes. Since cellular PC synthesis is obligatory for the secretion of VLDL particles by rat hepatocytes [28] and newly made PC is preferred for assembly into lipoproteins [29], the influence of EPA on PC synthesis were also examined. Although EPA decreased cellular [3H]PC contents in either cell type as compared to OA, no relation between cellular PC synthesis and TG secretion was observed among these cell types. Furthermore, EPA strikingly promoted PE synthesis compared to OA or fatty acid-free control in rat hepatocytes (Fig. 4). It may be that polyunsaturated fatty acids are preferentially incorporated into PE in rat [30]. The alternative explanation is that the increase of PE synthesis reflects the adaptation of metabolism caused by inhibition of cellular TG synthesis in the presence of EPA. This phenomenon was not observed in human hepatocytes or in HepG2 cells, which suggests that species differences exist in EPA-induced phospholipid metabolism between rat and human hepatocytes and HepG2 cells. In a long-term culture of rat hepatocytes with EPA, it has been shown that EPA-associated peroxidation events are cytotoxic. EPA-associated cytotoxicity is characterized in a time- and dose-dependent increase of cell LDH-leakage [31]. However, the diminution of [3H]TG secretion and apoB secretion observed in human hepatocytes in the present study are unlikely due to a non-specific cytotoxic effect of EPA, as the cell viability (Table 1) and apoA-I (data not shown) secretion were not affected in the presence of EPA. In summary, in human hepatocytes, EPA potently inhibits [3H]TG secretion by suppressing VLDL particle assembly a n d / o r secretion. EPA and OA induced strikingly different effects on cellular lipids and apoB
96
Y. Lin et al. /Biochimica et Biophysica Acta 1256 (1995) 88-96
metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes both in quantity and quality.
Acknowledgements Y.L. is supported by a grant from the Royal Netherlands Academy of Arts and Sciences. H.J.V. is supported by grant NWO-KWO(No. 900-716-028). F~K. is an established investigator of the Netherlands Heart Foundation. This study was performed in collaboration with the Human Liver Group, Groningen.
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