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Chylomicron remnant induction of lipid accumulation in J774 macrophages is associated with up-regulation of triacylglycerol synthesis which is not dependent on oxidation of the particles
Biochimica et Biophysica Acta 1631 (2003) 255 – 264 www.bba-direct.com
Chylomicron remnant induction of lipid accumulation in J774 macrophages is associated with up-regulation of triacylglycerol synthesis which is not dependent on oxidation of the particles Mariarosaria Napolitano a, Michael Avella b, Kathleen M. Botham b, Elena Bravo a,* a
Laboratorio di Metabolismo e Biochimica Patologica, Istituto Superiore di Sanita´, Viale Regina Elena 299, 00161 Rome, Italy b Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Received 7 October 2002; received in revised form 25 February 2003; accepted 26 February 2003
Abstract The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n 6 PUFA) or fish oil (rich in n 3 PUFA) were prepared in vivo and oxidised by incubation with CuSO4. The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants ( < 2-fold). Triacylglycerol synthesis, as measured by the incorporation of [3H]oleate and [3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n 6 PUFA as compared with n 3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Chylomicron remnant; Triacylglycerol; Cholesterol esterification; Polyunsaturated fatty acid, Macrophage
1. Introduction One of the earliest events in the development of atherosclerotic lesions is the accumulation of lipid-laden macrophages in the artery wall. Monocyte-macrophages, which have invaded the arterial tissue, scavenge lipoproteins from the subendothelial space in an unregulated manner, becoming so engorged with lipid that they take on a foamy appearance, and are termed foam cells [1]. There is a great deal of evidence to implicate low density lipoprotein (LDL)
* Corresponding author. Tel.: +39-06-4990-2730; fax: +39-06-49387149. E-mail address: [email protected] (E. Bravo).
in foam cell formation, but it is also clear that native LDL is relatively benign in this respect, and that oxidative modification of the particles is required to induce extensive intracellular lipid accumulation [2]. Recent work in our laboratory and others, however, has established that chylomicron remnants, the lipoproteins which carry lipids from the diet from the intestine to the liver, are also able to induce macrophages to form foam cells [3– 5], although compared to the extensive literature on the effects of LDL, relatively little is known about the processes involved. After absorption by intestinal cells, fat and cholesterol from the diet are incorporated into large triacylglycerol-rich chylomicrons, which are secreted into lymph and enter the blood via the thoracic duct. These particles rapidly undergo lipolysis by lipoprotein lipase in extrahepatic capillary beds,
1388-1981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1388-1981(03)00042-8
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depleting their triacylglycerol content and leaving chylomicron remnants, which deliver the cholesterol and the remaining triacylglycerol to the liver [6]. These remnant lipoproteins are now believed to be strongly atherogenic for a number of reasons [7]. Hyperchylomicronaemia leads to premature atherosclerosis, although these patients have low LDL levels [8], men with coronary heart disease have raised post-prandial chylomicron remnant concentrations [9], and remnant-like particles containing apolipoprotein (apo) E have been isolated from human atherosclerotic plaque [10]. Moreover, it has been demonstrated that chylomicron remnants are able to penetrate the arterial wall as efficiently as LDL [7,11 –13]. Previous in vitro studies in our laboratory have shown that chylomicron remnants induce the murine macrophage cell line, J774, to form foam cells [3], and we have also observed this effect with chylomicron remnant-like particles and macrophages derived from the human monocyte cell line, THP-1 [4]. In addition, Yu and Mamo [5] have reported similar findings with human monocyte-derived macrophages. A striking difference between foam cell induction by LDL and chylomicron remnants highlighted in all these studies is that the remnant particles do not require prior oxidation to bring about their effects. Other workers have found that chylomicrons and a lipoprotein fraction from human blood enriched in chylomicron remnants cause accumulation of triacylglycerol, rather than cholesteryl ester, in mouse macrophages [14,15]. It is clear, therefore, that at least some of the cellular events which occur when foam cell formation is induced by chylomicron remnants are different from those triggered by oxidised LDL. It has been established that chylomicron remnants are taken up and degraded by macrophages in experiments with both primary cells and cell lines, including human monocyte-derived [16], mouse peritoneal and rabbit alveolar [17,18] macrophages, the human monocyte cell line, THP1 [19], and the murine cell lines, J774 and P388D1 [17,20]. Few studies, however, have investigated the effects of this uptake on macrophage lipid metabolism, and how these might lead to the massive lipid accumulation associated with foam cell formation. It has been known for many years that dietary polyunsaturated fats (PUFA) decrease the risk of the development of atherosclerosis [21, 22]. Both n 6 (abundant in plant oils) and n 3 PUFA (found in fish oils) are effective in this respect, although they bring about their effects by different mechanisms [23]. Since the function of chylomicron remnants is the transport in the blood of lipids of dietary origin, their fatty acid composition varies according to that of the fat consumed in the diet [24]. We have demonstrated that the type of PUFA (n 3 or n 6) predominating in chylomicron remnants alters their uptake and metabolism in the liver [25 – 27], and this contributes to the different ways in which these dietary fats retard the disease process. These findings, together with the fact that
dietary PUFA are known to play a role in the regulation of gene expression [28], suggest that the type of PUFA in chylomicron remnants may influence their effects on macrophage lipid metabolism. In the present work, we have investigated the influence of chylomicron remnants on lipid metabolism in J774 macrophages. Effects on lipid accumulation in the cells, the synthesis of triacylglycerol, cholesterol, cholesteryl ester and phospholipid from radiolabelled precursors, and the activity and/or expression of mRNA for enzymes regulating cholesteryl ester synthesis and hydrolysis and triacylglycerol synthesis were studied. Enzymes investigated were: acyl Co-enzyme A:cholesterol acyltransferase (ACAT), which regulated cholesterol esterification; lysosomal cholesteryl ester hydrolase (lCEH), which hydrolyses cholesteryl ester taken up from lipoproteins in the lysosomes; neutral cholesteryl ester hydrolase (nCEH), responsible for the hydrolysis of cytosolic cholesteryl ester stores [29]; and acyl Co-enzyme A:diacylglycerol acyltansferase (DGAT), which catalyses the rate-limiting step in triacylglycerol synthesis [30]. As our previous studies have shown that macrophages form foam cells on exposure to oxidised, as well as native, chylomicron remnant-like particles [4], both types of particle were tested. In addition, in order to investigate the possibility that the PUFA composition of the remnants is important in their effects on macrophage lipid metabolism, experiments were carried out using particles derived from both corn (rich in n 6 PUFA) and fish (rich in n 3 PUFA) oil.
2. Materials and methods 2.1. Animals and materials Male rats of the Wistar strain (300 – 350 g body weight) were used to obtain chylomicrons for chylomicron remnant preparation. The animals were fed a standard pellet diet and allowed food and water ad libitum. Climatic conditions (temperature 21– 22 jC, relative air humidity 50 + 5%) and the light cycle (12 h light, 12 h dark)) were kept constant. The J774.2 murine macrophage-like cell line was supplied by the American Type Culture Collection (Rockville, MD, USA). [9,10 3H(n)]oleate (9.2 Ci/mmol), [1(3)-3H]glycerol, [4-14C]cholesteryl oleate (60 mCi/mmol) and L-3 phosphatidylcholine 1-palmitoyl-2-(1-14C) linoleoyl (60 mCi/mmol) were obtained from NEN Life Science Products Inc. (Boston, MA, USA), and [2-14C]-acetic acid sodium salt (specific activity: 57 Ci/mmol), [1,2 3H(n)]cholesterol (35 – 50 Ci/mmol), [1-14C]oleoyl-CoA (56 mCi/mmol), and [1a,2a(n)-[3H]cholesteryl oleate (30 – 60 Ci/mmol) were purchased from Amersham Pharmacia Biotech Inc. (Milan, Italy). Fetal bovine serum (FBS), glutamine, antibiotics and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Hyclone (Europe Ltd). Fatty acid-
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free bovine serum albumin (BSA) and Menhaden fish oil were supplied by Sigma Chemical Company (St. Louis, MO, USA). 2.2. Preparation and oxidation of chylomicron remnants Chylomicrons were collected from rats after cannulation of the thoracic duct as described by Lambert et al. [24]. Briefly, animals were fed a single dose of 1 ml corn oil or Menhaden fish oil containing a-tocopherol (4 mg/ml) as an antioxidant by stomach tube, and approximately 1 h later the rats were anaesthetised and the thoracic duct was cannulated. The body wall was then sutured, the animals were placed in restraining cages and chyle was collected for 15 –18 h in the presence of ampicillin (0.1 mg/ml). Chylomicrons were isolated by ultracentrifugation (6 105 g min at 12 jC in a fixed angle rotor) after layering the chyle under 0.9% NaCl (d 1.006 g/ml), and harvested from the top fraction by tube slicing. Chylomicron remnants were prepared by incubation of the chylomicrons in vitro with rat post-heparin plasma [3]. After the incubation, chylomicron remnants were isolated by ultracentrifugation (6 107 g min at 12 jC), and further purified by a second ultracentrifugation (3.2 107 g min at 12 jC) after layering under 0.9% NaCl (d 1.006 g/ml). The top fraction was collected by tube slicing, and the remnants were dialysed overnight against culture medium prior to addition to the cell cultures. Chylomicron remnants were oxidised by incubation with CuSO4 (10 AM) for 18 h at 37 jC followed by dialysis according to Grieve et al. [31]. The oxidised remnants were further dialysed overnight against the culture medium before addition to the cell cultures. 2.3. Culture of J774 cells J774.2 cells were maintained in DMEM supplemented with penicillin (100 U/ml), streptomycin (100Ag/ml), glutamine (2 mM) and FBS (10%) at 37 jC in a humidified atmosphere of 95% air/5% CO2. For the experiments, cells (passage 6 –10) were seeded into 16-mm dishes at a density of 5 104 cells/ml and used on the fifth day of culture. Cell viability was determined by Trypan blue exclusion. 2.4. Experiments with radiolabelled substrates The amount of [3H]oleate incorporated into triacylglycerol, cholesteryl ester and phospholipid was determined as previously described [3]. Cells were incubated for 4 h at 37 jC in 5% CO2 in serum-free medium containing 0.326 mM oleic acid and 6 ACi/ml [3H]-oleate bound to fatty acid-free BSA (2%). After incubation, the cells were washed twice with phosphate-buffered saline (PBS), pH 7.4, and the lipids were extracted, separated by TLC and the radioactivity in the bands corresponding to cholesteryl ester, triacylglycerol
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and phospholipid was determined by liquid scintillation counting as before [3]. Incorporation of [3H]glycerol into triacylglycerol and phospholipid was determined using a modification of the method described by Davis et al. [32]. Cells were incubated for 4 h at 37 jC in 95% air/5% CO2 in serum-free medium containing [3H]glycerol (4 ACi/ml, 20 AM). After incubation, cells were washed twice with PBS. [14C]-phosphatidylcholine (900 dpm) was added to each tube as an internal standard and the lipids were extracted and separated by TLC [3]. The incorporation of [14C]-acetate into cholesterol, triacylglycerol and phospholipid was measured as described previously [33], with minor modifications. Cells were incubated in serum-free medium containing [14C]-sodium acetate (4 ACi/ml, 500 AM) at 37 jC in 95% air/5% CO2 for 4 h. After this time, the medium was removed and the cells were washed with PBS, and the lipids were extracted and separated by TLC as in Ref. [3]. [3H]cholesterol (1000 dpm/tube) was used as recovery marker. In all radiolabelling experiments, after extraction of cellular lipids, the cells were dissolved in 1 M NaOH for measurement of cellular protein. 2.5. Assay of enzyme activities nCEH and lCEH activity in cellular homogenates was determined by measuring the release of [14C]oleate from the cholesteryl[1-14C]oleate substrate according to Martinez and Botham [34]. Cells were homogenised in Tris –HCl buffer, pH 7.4, or in 10 mM acetic acid/Na acetate buffer pH 4.6, for nCEH or lCEH, respectively. ACAT activity was determined according to Suckling et al. [35] using [14C]oleoylCoA as the substrate (19.9 nmol/ tube, specific activity 3200 dpm/nmol). Lipids were extracted with chloroform/methanol (2:1 v/v), and [3H]cholesteryl oleate (1500 dpm/tube) was added to estimate recovery. Lipids were separated by TLC [3] and radioactivity in cholesteryl ester was determined by scintillation counting. 2.6. Analytical methods Triacylglycerol and total cholesterol concentrations in chylomicron remnants and cell samples were measured enzymatically using kits supplied by Boehringer Mannheim (Germany), and protein levels in cell samples were measured by the method of Bradford [36]. Cell viability was determined using by Trypan blue exclusion. The extent of oxidation of chylomicron remnants before and after incubation with CuSO4 was determined from the content of the aldehydic products of lipid peroxidation, 4hydroxy-2(E)-nonenal (4-HNE) + malondialdehyde (MDA), assayed using the Bioxytech LPO-586 assay (Oxis International Inc., Portland, OR, USA) according to the manufacturer’s instructions.
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Table 1 Characteristics of native and oxidised chylomicron remnants Lipid
Corn remnants
TG (Amol/ml) TC (Amol/ml) ratio TG/TC 4HNE F MDA (nmol/Amol TG)
Fish remnants
Native
Oxidised
Native
Oxidised
6.3 F 0.5(4) 0.55 F 0.07(4) 11.1 F 1.6(4) 4.7 F 1.7(4)
4.9 F 0.6(3) 0.48 F 0.04(3) 10.4 F 2.3(3) 18.9 F 7.3(3)*
5.4 F 0.1(4) 0.62 F 0.17(4) 9.9 F 1.6(4) 4.0 F 1.2(4)
5.4 F 0.3(4) 0.60 F 0.87(4) 10.7 F 2.1(4) 9.0 F 1.8(4)*
Chylomicron remnants derived from corn and fish oil were prepared and oxidised as described in Materials and methods, and their content of triacylglycerol, total cholesterol and 4HNE F malondialdehyde was determined. Data shown are the mean F S.E. and the number of separate experiments performed is given in parentheses. * P < 0.05 vs. corresponding native remnants.
Total RNA was extracted from the macrophages by homogenisation in guanidinium thiocyanate buffer (4 M) followed by layering over a CsCl (5.7 M) sodium acetate (25 mM) cushion and centrifugation at 31,000 rpm for 21 h. The relative abundance of mRNA transcripts for ACAT1, hormone sensitive lipase (HSL) and DGAT was then determined using reverse transcriptase-polymerase chain reaction (RTPCR). First strand cDNA synthesis was performed using Hotstar Taq reverse transcriptase (Qiagen, Crowley, West Sussex, UK) and amplification was carried out using the following primers for the appropriate mouse genes: ACAT1: sense, 5V-CCATTGATCTATTCCCTTGTCC-3V, antisense, 5V-GAGTCCTTGGGTAGTTGTCTCG-3V; DGAT: sense, 5V-GTGGTGATGCTGATCCTGAGT-3V, antisense, GAGTATGATGCCAGAGCAAAC-3V; under the following PCR conditions: initial denaturation/activation at 95 jC for 15 min followed by 30 (ACAT1) or 32 cycles (DGAT) consisting of denaturation at 94 jC for 30 s, annealing at 56 jC for 1 min and extension at 72 jC for 1 min, followed by final extension at 72 jC for 10 min. The products (ACAT1, 301 bp; DGAT, 329 bp) were analysed by electrophoresis using agarose gels (1.2% w/v) containing ethidium bromide (0.5Ag/ml). The bands were quantified using optical density volume analysis, and normalised with the values obtained for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples in the same assay system (in separate tubes). No effect on GAPDH mRNA expression was found with any of the treatments used. Preliminary experiments were carried out for each gene to ensure all measurements were made on the linear part of the curve.
Significance limits were determined using the paired Student’s t test.
3. Results 3.1. Characteristics of native and oxidised chylomicron remnants Our previous work has demonstrated that the fatty acid composition of chylomicron remnants prepared by the methods used in this study reflects that of the oil fed prior to collection of the chyle, and shows little variation from preparation to preparation [37]. Thus, in remnants derived from corn oil, the ratio of n 3:n 6 PUFA was 0.03, while that in particles derived from fish oil was 1.33, representing an increase of approximately 44-fold [37]. The lipid composition of four types of remnants tested (native and oxidised remnants derived from corn or fish oil) did not differ significantly in terms of their content of triacylglycerol, total cholesterol and triacylglycerol: total cholesterol ratio (Table 1). The content 4HNE + malondialdehyde, decomposition products of PUFA peroxidation, was determined in chylomicron remnants before and after incubation with CuSO4 (10 AM) for 18 h as a measure of the extent of oxidation of the particles [38]. Prior to incubation, the values obtained for corn and fish oil remnants were similar, and a significant increase was observed after incubation in both cases.
Table 2 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on triacylglycerol and total cholesterol levels in J774 macrophages Lipid
TG (nmol/mg protein) TC (nmol/mg protein)
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
65.0 F 3.6(4) 104.1 F 46.3(4)
410.6 F 46.3(4)** 175.6 F 43.7(4)
288.9 F 83.9(4)* 154.1 F 54.8(4)
57.6 F 2.7(3) 154.1 F 13.8(3)
101.9 F 9.5(3)* 127.5 F 47.0(3)
89.2 F 12.8(3)* 159.6 F 41.9(3)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 8 h, and the triacylglycerol and total cholesterol content of the cells was then determined. Data shown are the mean F S.E. and the number of separate experiments (with separate chylomicron remnant preparations) performed is given in parentheses. * P < 0.05 vs. corresponding control cells. ** P < 0.01 vs. corresponding control cells.
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Table 3 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the incorporation of [3H]oleate into cellullar lipids by J774 macrophages Lipid
TG CE PL
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
2.16 F 0.21(3) 0.206 F 0.092(3) 6.03 F 1.23(3)
8.78 F 1.50(3)* 0.158 F 0.053(3) 3.35 F 0.17(3)
5.61 F 2.1(2) 0.082 F 0.025(2) 2.66 F 0.44(2)
1.74 F 0.09(4) 0.167 F 0.007(4) 3.56 F 0.09(4)
2.40 F 0.31(4)* 0.176 F 0.020(4) 4.87 F 1.10(4)
2.60 F 0.45(4)* 0.205 F 0.014(4) 4.30 F 0.37(4)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 4 h in the presence of [3H]oleate (6 ACi/ml), and the incorporation of radioactivity into triacylglycerol, cholesteryl ester and phospholipid was determined. Data are expressed as nmol [3H]oleate/h/mg cell protein incorporated into each lipid and are the mean F S.E. (three or four experiments) or the mean F range (two experiments). The number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells.
3.2. The effect of chylomicron remnants on lipid accumulation in J774 macrophages J774 macrophages were incubated with or without native or oxidised chylomicron remnants derived from corn or fish oil for 4 h, and the triacylglycerol and total cholesterol content of the cells was then determined. The results are shown in Table 2. All four types of remnants tested caused significant increases in triacylglycerol levels in the cells. Remnants derived from corn oil, however, had a markedly greater effect than fish oil remnants, and this was the case with both native and oxidised particles (compared to control cells; corn oil remnants, native 6.3, oxidised 4.4; fish oil remnants, native 1.8, oxidised 1.6). The total cholesterol content of the macrophages was not significantly changed after incubation with native or oxidised fish oil remnants, and although the mean values obtained in experiments with both native and oxidised corn oil remnants were higher than the control values, because of the relatively large variation between individual experiments, these differences did not reach statistical significance. 3.3. Effect of chylomicron remnants on the incorporation of radiolabelled substrates into cellular lipids in J774 macrophages In order to investigate the effects of chylomicron remnants on lipid synthesis in J774 cells, their effects on the
incorporation of radiolabelled substrates into cellular lipids were determined. The influence of native or oxidised corn oil remnants on the incorporation of [3H]oleate into triacylglycerol, cholesteryl ester and phospholipid by the macrophages is shown in Table 3. The results showed that the native remnants caused a significant increase in incorporation into triacylglycerol over control cells incubated without remnants, and the mean incorporation was also increased by oxidised remnants. In the case of fish oil remnants, both native and oxidised particles caused a small but significant rise in incorporation into triacylglycerol (Table 3). The increases found with the native ( 1.35) and oxidised ( 1.49) fish oil remnants, however, was less marked than that seen with native ( 4.1) and oxidised ( 2.6) corn oil remnants (Table 3). In contrast to the results obtained with triacylglycerol, incorporation of [3H]oleate into cholesteryl ester and phospholipid was not significantly changed by any of the four types of remnants, although in experiments with native and oxidised corn oil particles there was a tendency towards a decrease in the amount of radiolabel found in phospholipid in comparison to control cells (Table 3). The incorporation of [3H]glycerol into triacylglycerol and phospholipid showed a generally similar pattern to that observed with [3H]oleate (Table 4). The amount of radiolabel incorporated into triacylglycerol was significantly increased in macrophages incubated with native and oxidised corn and fish oil remnants, as compared to those incubated in the absence of lipoproteins, with particles derived from corn oil (native 10.3, oxidised 14.5) having
Table 4 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the incorporation of [3H]glycerol into cellullar triacylglycerol and phospholipid by J774 macrophages Lipid
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
TG PL
0.13 F 0.03(3) 2.23 F 0.13(3)
1.34 F 0.27(3)** 0.77 F 0.23(3)**
1.93 F 0.54(2) 0.84 F 0.14(2)
0.13 F 0.03(4) 2.81 F 0.59(4)
0.57 F 0.12(4)* 3.90 F 0.95(4)*
0.40 F 0.11(4)* 2.47 F 0.70(4)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 4 h in the presence of [3H]glycerol (4 ACi/ml), and the incorporation of radioactivity into triacylglycerol and phospholipid was determined. Data are expressed as nmol [3H]glycerol/ h/mg cell protein incorporated into each lipid and are the mean F S.E. (three or four experiments) or mean F range (two experiments). The number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells. ** P < 0.01 vs. corresponding control cells.
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Fig. 1. Native ( – o – ) or oxidised ( – . – ) fish oil chylomicron remnants (5 – 20 Ag cholesterol/ml) were incubated with J774 macrophages for 4 h in the presence of [14C]acetate and the incorporation of radioactivity into (A) cholesterol, (B) triacylglycerol, and (C) nonesterified fatty acids was determined. Each point is the mean from four separate experiments (with separate chylomicron remnant preparations). Error bars show the S.E.
a considerably greater effect than those derived from fish oil (native 4.4, oxidised 3.1). In addition, when [3H]glycerol was used, effects on incorporation into phospholipid were seen, with native and oxidised corn remnants causing a decrease, while native fish oil remnants caused an increase. The effects of native and oxidised fish oil remnants on endogenous fatty acid, triacylglycerol and cholesterol synthesis in the macrophages were also investigated using [14C]acetate (Fig. 1). Cholesterol synthesis (Fig. 1A) was unaffected, but the formation of triacylglycerol (Fig. 1B) and fatty acid (Fig. 1C) was decreased by 40 –50%. There were no significant differences in the effects of native and oxidised particles. 3.4. Effect of chylomicron remnants on the activity of enzymes involved in cholesteryl ester synthesis and hydrolysis in J774 macrophages J774 macrophages were incubated in the presence or absence of native or oxidised chylomicron remnants derived from corn or fish oil, and the activities of ACAT, nCEH and lCEH were determined. The results are shown in Table 5. The activity of ACAT was increased by native, but not oxidised, corn oil remnants, while neither native nor oxidised fish oil remnants had any significant effect. nCEH
activity, on the other hand, was increased by both native and oxidised corn oil remnants, while the two types of fish oil remnants again had no effect. There was no change in the activity of lCEH after incubation of the cells with any of the four types of remnants under investigation. 3.5. Effect of chylomicron remnants on the expression of mRNA for enzymes involved in cholesteryl ester and triacylglycerol synthesis in J774 macrophages Recent studies have demonstrated that there are two forms of ACAT, designated ACAT1 and ACAT2. However, ACAT1 has been found in most tissues, while ACAT2 appears to be confined to the liver and intestine [39]. In the present study, therefore, the effects of chylomicron remnants on the expression of mRNA for ACAT1 in the J774 macrophages were measured. The results showed that the level of mRNA for this enzyme was increased by about 60% after incubation of the cells with native corn oil remnants. Oxidised corn oil remnants or native or oxidised fish oil remnants, in contrast, did not have any significant effect (Table 6). DGAT catalyses the terminal and sole committed step in triacylglycerol synthesis, and thus is important in the regulation of the process [30]. The amounts of DGAT
Table 5 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the activities of ACAT, nCEH and lCEH in J774 macrophages Enzyme
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
ACAT nCEH lCEH
4.32 F 1.25(4) 3.63 F 0.55(4) 36.7 F 14.7(3)
7.82 F 0.74(4)* 5.67 F 0.97(4)* 45.0 F 11.3(3)
3.21 F 0.90(4)yy 5.15 F 0.25(3)* 37.2 F 13.4(3)
5.20 F 0.72(5) 4.42 F 0.43(5) 39.5 F 10.3(4)
4.15 F 0.83(5) 4.94 F 0.58(5) 34.0 F 4.5(4)
4.83 F 0.94(5) 5.00 F 0.87(5) 42.5 F 12.1(4)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 8 h, and ACAT, nCEH and lCEH activity in the cells was then determined. Data are expressed as nmol product formed /h/mg cell protein and are the mean F S.E. The number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells. yy P < 0.01 vs. corresponding native remnants.
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Table 6 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the expression of mRNA for ACAT1 and DGAT in J774 macrophages Gene
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
ACAT1 DGAT
0.65 F 0.22(3) 0.24 F 0.09(3)
1.03 F 0.33(3)* 0.36 F 0.13(3)
0.95 F 0.26(3) 0.30 F 0.06(3)
0.67 F 0.19(4) 0.38 F 0.17(3)
0.82 F 0.22(4) 0.26 F 0.9(3)
0.83 F 0.12(4) 0.25 F 0.06(3)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 8 h, and the relative abundance of mRNA transcripts for ACAT1 and DGAT in the cells was determined by RT-PCR. the bands were quantified by optical density analysis and the values were normalised using those obtained for GAPDH in the same system. Data shown are the mean F S.E. and the number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells.
mRNA found in the macrophages were relatively low, and no changes were detected after incubation in the presence of either native or oxidised corn or fish oil remnants.
4. Discussion Although elevated plasma LDL cholesterol levels are known to be a major factor in the development of atherosclerotic lesions containing foam cells engorged with cholesteryl esters [1,2], it is now recognised that plasma triacylglycerol levels are an independent risk factor for the disease [40]. Hypertriglyceridemia results when the clearance of chylomicron remnants from the blood is delayed post-prandially, and this has been found to lead to premature atherosclerosis development in the absence of raised LDL concentrations [8]. Previous work from our group has shown that the fatty acid composition of chylomicron remnants derived from corn oil (enriched in n 6 PUFA, mainly linoleic acid) or fish oil (enriched in n 3 PUFA, mainly eicosapentaenoic and docosahexaenoic acids) prepared by the methods used here reflects that of the parent oils, so that the ratio of n 3:n 6 PUFA was increased by 44-fold in fish as compared to corn oil remnants [37], although the overall distribution of lipids between triacylglycerol, total cholesterol and phospholipid was not changed. In the present study, oxidation of corn and fish oil remnants was found to cause a significant increase in their content of 4HNE + MDA, which is commonly used as an index of lipid peroxidation [38] (Table 1). As the triacylglycerol/total cholesterol ratio did not differ significantly in the four different types of remnants (native and oxidised corn, native and oxidised fish) (Table 1), the amount of triacylglycerol added to the cells was similar at each concentration of remnant cholesterol tested. Thus, as well as investigating the influence of chylomicron remnants on lipid metabolism in J774 macrophages, we have been able to observe the effects of changes in the ratio of n 3:n 6 PUFA, and in the oxidation state of the particles. Studies in our laboratory and others have demonstrated that chylomicron remnants cause lipid accumulation in macrophages [3– 5]. In general, triacylglycerol-rich lipoproteins, such as VLDL or h-VLDL, have been found to cause
the accumulation of triacylglycerol, rather than cholesterol [14,15,41], and preliminary experiments in our laboratory have demonstrated that chylomicron remnant-like particles have this effect in THP-1 macrophages [4] and unpublished results. This pattern was also observed in the present study, with cellular triacylglycerol levels being raised considerably after incubation of J774 macrophages with chylomicron remnants, while total cholesterol levels were not significantly changed (Table 2). It is well established that LDL must be chemically or oxidatively modified before it is able to induce macrophages to form foam cells [2]. Like other triacylglycerol-rich lipoproteins such as VLDL [46], however, chylomicron remnants have been found to cause foam cell formation without prior modification of the particles, and the present findings with J744 macrophages (Table 3) confirm this. In contrast, the type of PUFA in the particles has a clear effect on the amount of triacylglycerol accumulated, with remnants derived from corn oil causing a four- to sixfold increase over the levels found in control cells, while those derived from fish oil induced a rise of less than twofold (Table 3). In addition, the amount of total cholesterol in the cells tended to be increased in cells incubated with remnants derived from corn (increase observed in three out of four experiments), but not fish oil, although because of the relatively large variation between individual experiments this change did not reach significance. Thus, enrichment of chylomicron remnants with n 3 as compared to n 6 PUFA leads to decreased lipid accumulation in macrophages. Although it is known that macrophages take up and degrade chylomicron remnants [16 – 20], little information is available about the subsequent effects on intracellular lipid metabolism. In the present study, investigation of the effects of chylomicron remnants on lipid synthesis in the macrophages using the radiolabelled precursors [3H]oleate or [3H]glycerol showed that triacylglycerol synthesis was increased in the presence of the particles, and this is in agreement with a previous report [42]. Our results demonstrate further, however, that corn oil remnants cause a more marked rise than fish oil remnants (Tables 3 and 4). These results are consistent with our findings on triacylglycerol accumulation in the cells (Table 2), and suggest that remnants enriched with n 3 as compared to n 6 PUFA cause a lower level of accumulation because they stimulate
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triacylglycerol synthesis to a lesser extent. The PUFA composition of remnants also had differential effects on phospholipid synthesis, with a decrease observed with corn oil particles, and an increase with those derived from fish oil (Table 4). This is consistent with the differences in the effects of corn and fish oil remnants on phospholipid synthesis in the liver observed in our earlier work [25]. These results suggest that the delivery of n 6 PUFA to the cells leads to the diversion of the fatty acid and glycerol substrates into triacylglycerol synthesis at the expense of phospholipid formation, while remnant-derived n 3 PUFA tends to be shared between the two pathways. One drawback of the radiolabelling experiments with [3H]oleate and [3H]glycerol is that the dilution of the intracellular substrate pools by fatty acids and glycerol derived from the remnants taken up by the cells may interfere with the results. The changes in triacylglycerol and phospholipid synthesis observed with the two different labels, however, were similar overall, suggesting that any such dilution effect did not influence the overall pattern of the findings. Furthermore, the up-regulation of triacylglycerol synthesis by chylomicron remnants, which is the major finding of these experiments, would be underestimated if the substrate pool was diluted. In order to investigate the effects of chylomicron remnants derived from fish oil on de novo lipid synthesis in J774 macrophages, the incorporation of [14C]acetate into cholesterol, non-esterified fatty acids and triacylglycerol was determined. In two previous studies the activity of 3hydroxy-3-methylglutaryl Co-enzyme A reductase (HMG CoA reductase), the rate-limiting enzyme in cholesterol synthesis, has been reported to be increased in J774 cells [42], but decreased in primary rabbit macrophages [43]. In our experiments, chylomicron remnants had no significant effect on the incorporation of [14C]acetate into cholesterol. Thus the influx of cholesterol from chylomicron remnants, at least at the concentrations used here, does not cause a decrease in de novo cholesterol synthesis in the macrophages, as occurs with LDL [44]. Incorporation of [14C]acetate into triacylglycerol was significantly reduced by the remnants (Fig. 1); however, the decrease of a similar magnitude (40 –50%) observed in incorporation into nonesterified fatty acids indicates that this change is due to depression of de novo fatty acid synthesis, which might be expected on delivery of relatively large amounts of triacylglycerol in chylomicron remnants to the cells. These results, therefore, do not conflict with our finding that triacylglycerol synthesis from [3H]oleate is increased by the particles. The enzyme DGAT catalyses the last and only committed step in triacylglycerol synthesis [30]. Levels of DGAT mRNA were low in the J774 cells (Table 6) and were not significantly affected by chylomicron remnants. It appears, therefore, that neither the increase in triacylglycerol synthesis caused by the particles, nor the differential effects of those enriched in n 3 or n 6 PUFA, can be attributed to up-regulation of transcription of this gene. It is possible,
however, that post-translational changes in the enzyme activity are involved, or that other factors influence the distribution of the substrate between triacylglycerol and phospholipid synthesis. Interestingly, the effects of oxidised chylomicron remnants on lipid synthesis, were generally similar to those of the native particles. The only difference observed was that prior oxidation of fish oil remnants abolished their effect in increasing phospholipid synthesis from glycerol. These findings are consistent with the similar effects of native and oxidised particles on the accumulation of triacylglycerol in the macrophages (Table 2), and indicate that stimulation of triacylglycerol synthesis is a major factor in foam cell induction by chylomicron remnants. Furthermore, it is clear from our results that the type of PUFA carried by chylomicron remnants is more important than their oxidation state in determining their effects on triacylglycerol metabolism in macrophages, with enrichment with n 3 PUFA as compared to n 6 PUFA leading to a marked reduction in synthesis and intracellular accumulation. Cholesteryl ester stores in macrophages undergo a continuous cycle of hydrolysis by nCEH, followed by reesterification by ACAT [29] in a process known as the cholesteryl ester cycle. In the present study, therefore, we investigated the effects of chylomicron remnants on cholesteryl ester metabolism by measuring the incorporation of [3H]oleate into cholesteryl ester, the activity of ACAT and nCEH, and the expression of mRNA for ACAT1, the form of ACAT expressed in macrophages [39]. ACAT activity and the levels of mRNA for ACAT1 in the cells were upregulated by corn, but not fish, oil remnants, but these changes were not reflected in an increase in the overall rate of cholesterol esterification (Tables 3, 5 and 6). This may be explained by the concomitant increase in nCEH activity observed in the presence of corn oil remnants, and is consistent with a previous report of reduced activity of ACAT in J774 macrophages exposed to n 3 as compared to n 6 PUFA in the free fatty acid form [45]. However, in contrast to our results, Yu and Mamo [43] found increased incorporation of [3H]oleate into cholesteryl ester in macrophages incubated with chylomicron remnants, while Ellsworth et al. [42] reported a decrease. The reason for these discrepancies is not clear, but the type of fat used to obtain the chylomicron remnants is not specified in either study, and our findings suggest that this may be important in the results obtained. Oxidation of the particles abolished the effects of corn oil remnants on ACAT activity and mRNA concentrations, but no other changes compared to native remnants were observed. It is interesting to note that oxidised LDL has also been found to have little effect on ACAT activity in murine macrophages, and this has been attributed to the retention of much of the sterol fraction in the lysosomal system as oxidised esters [46]. Taken together, therefore, our results indicate that chylomicron remnants enriched in n 6 PUFA increase the flux of cholesterol through the cholesteryl ester
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cycle in macrophages by up-regulating the activities of ACAT and nCEH at the transcriptional or post-transcriptionally level, respectively. These regulatory effects, however, are lost after oxidation of the particles, and are not exhibited at all by remnants enriched in n 3 PUFA. In summary, the studies presented here demonstrate that both native and oxidised chylomicron remnants induce lipid accumulation in J774 macrophages; that the major lipid accumulated is triacylglycerol, rather than cholesterol; and that increased synthesis of triacylglycerol from exogenous fatty acids is a major factor in the process. Importantly, however, they also demonstrate that the type of PUFA carried by the remnants profoundly affects their influence on lipid metabolism in the macrophages, with those enriched in n 6 PUFA causing a markedly higher level of triacylglycerol synthesis and accumulation compared to those enriched with n 3 PUFA. The oxidation state of the remnants, on the other hand, had relatively minor effects on the processes studied, and did not affect the amount of triacylglycerol accumulated in the cells. Since chylomicron remnants carry lipids from the diet, and their fatty acid composition reflects that of the fats consumed [24], these findings support the idea [7] that dietary fatty acids may have a role in the generation of foam cells. Thus, in addition to their well established effects in lowering blood triacylglycerol levels and depressing macrophage eicosanoid metabolism [22,47], dietary n 3, as compared to n 6, PUFA may also decrease the tendency of chylomicron remnants to promote foam cell formation.
Acknowledgements This work was supported by grants from the Wellcome Trust (Ref. no. 057282/Z/99) and Programma Ricerca Finalizzata 1999, Ministry of Health, Italy (Ref. ISS D70).
References [1] R. Ross, The pathogenesis of atherosclerosis: a perspective for the 1990s, Nature 362 (1993) 801 – 809. [2] D. Steinberg, Low density lipoprotein oxidation and its pathobiological significance, J. Biol. Chem. 272 (1997) 20963 – 20966. [3] M. Napolitano, R. Rivabene, M. Avella, K.M. Botham, E. Bravo, The internal redox balance of cells influences the metabolism of lipids of dietary origin by J774 macrophages: implications for foam cell formation, J. Vasc. Res. 38 (2001) 350 – 360. [4] K.V. Batt, K.M. Botham, B. Jackson, K.E. Suckling, Comparison of the effects of low density lipoprotein and chylomicron remnants on foam cell formation in the human monocytic cell line, THP-1, Atheroscler. Suppl. 2 (2001) 109. [5] K.C. Yu, J.C.L. Mamo, Chylomicron remnant-induced foam cell formation and cytotoxicity: a possible mechanism of cell death in atherosclerosis, Clin. Sci. (Lond.) 298 (2000) 183 – 192. [6] T.G. Redgrave, Formation and metabolism of chylomicrons, Int. Rev. Physiol. 28 (1983) 103 – 130. [7] J.C.L. Mamo, Atherosclerosis as a post-prandial disease, Endocrinol. Metab. 2 (1995) 229 – 244.
263
[8] P. Benlian, P.L. De Gennes, L. Foubert, H. Zhang, S.E. Gagne, M. Hayden, Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene, N. Engl. J. Med. 335 (1996) 848 – 854. [9] P.H.E. Groot, W.A.H. van Stiphout, X.H. Krauss, H. Jansen, A. van Tol, E. van Ramshorst, S. Chin-On, A. Hofmann, S.R. Cresswell, L. Havekes, Postprandial lipoprotein metabolism in normolipidemic men with and without coronary heart disease, Arterioscler. Thromb. 11 (1991) 653 – 662. [10] S. Yla-Herttuala, O. Jaakkola, C. Enholm, M.J. Tikkanen, T. Solakivi, T. Sarkioja, T. Nikkari, Characterisation of two lipoproteins containing apolipoproteins B and E from lesion-free human aortic intima, J. Lipid Res. 29 (1988) 563 – 572. [11] S.D. Proctor, J.C.L. Mamo, Retention of fluorescent-labelled chylomicron remnants within the intima of the artery wall—evidence that plaque cholesterol may be derived from post-prandial lipoproteins, Eur. J. Clin. Investig. 28 (1998) 497 – 503. [12] D.J. Grieve, M. Avella, J. Elliott, K.M. Botham, Influence of chylomicron remnants on endothelial cell function in the isolated perfused rat aorta, Atherosclerosis 139 (1998) 273 – 281. [13] J.C.L. Mamo, J.R. Wheeler, Chylomicrons or their remnants penetrate rabbit thoracic aorta as efficiently as do smaller macromolecules, including low density lipoprotein, high density lipoprotein and albumin, Coron. Artery Dis. 5 (1994) 695 – 705. [14] S.H. Gianturco, S.A. Brown, D.P. Via, W.A. Bradley, The h-VLDL receptor pathway of murine P388D1 macrophages, J. Lipid Res. 27 (1986) 412 – 420. [15] T.P. Bersot, T.L. Innerarity, R.E. Pitas, R.C. Rall, K.H. Weisgraber, R.W. Mahley, Fat feeding in humans induces lipoproteins of density less than 1.006 that are rich in apolipoprotein (a) and that cause foam cell formation in macrophages, J. Clin. Invest. 77 (1986) 622 – 630. [16] C. Koo, M.E. Wernett-Hammond, Z. Garcia, M.J. Malloy, R. Uauy, C. East, D. Bilheimer, R.W. Mahley, T.L. Innerarity, Uptake of cholesterol-rich remnant lipoproteins by human monocyte-derived macrophages is mediated by low density lipoprotein receptors, J. Clin. Invest. 81 (1988) 1332 – 1340. [17] J.L. Ellsworth, F.B. Fong, F.B. Kraemer, A.D. Cooper, Differences in the processing of chylomicron remnants and h-VLDL by macrophages, J. Lipid Res. 31 (1990) 1399 – 1411. [18] J.C.L. Mamo, C.L. Ellsgood, K. Yu, S. Schumann, T.G. Redgrave, Chylomicron remnants metabolism by macrophages, Atherosclerosis 109 (1994) 107. [19] C.L. Elsegood, S. Pal, P.D. Roach, J.C.L. Mamo, Binding and uptake of chylomicron remnants by primary and THP-1 monocyte-derived macrophages: determination of binding proteins, Clin. Sci. (Lond.) 101 (2001) 111 – 119. [20] J.L. Ellsworth, F.B. Kraemer, A.D. Cooper, Transport of h-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway, J. Biol. Chem. 262 (1987) 2316 – 2325. [21] A.S. Truswell, Reducing the risk of coronary heart disease, Br. Med. J. 291 (1985) 34 – 37. [22] W.S. Harris, Dietary fish oils and blood lipids, Curr. Opin. Lipidol. 7 (1996) 3 – 7. [23] J.T. Thornberg, L.L. Rudel, How do polyunsaturated fatty acids lower lipids? Curr. Opin. Lipidol. 3 (1992) 17 – 21. [24] M.S. Lambert, K.M. Botham, P.A. Mayes, Modification of the composition of dietary oils and fats upon incorporation into chylomicron remnants, Br. J. Nutr. 76 (1996) 435 – 445. [25] M.S. Lambert, K.M. Botham, P.A. Mayes, Variations in composition of dietary fats affect hepatic uptake and metabolism of chylomicron remnants, Biochem. J. 310 (1995) 845 – 852. [26] M.S. Lambert, M.A. Avella, Y. Berhane, E. Shervill, K.M. Botham, The fatty acid composition of chylomicron remnants influences their binding and internalisation by isolated hepatocytes, Eur. J. Biochem. 268 (2001) 3983 – 3992. [27] X. Zheng, M. Avella, K.M. Botham, Comparison of the effects of
264
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
M. Napolitano et al. / Biochimica et Biophysica Acta 1631 (2003) 255–264 dietary n 3 and n 6 polyunsaturated fatty acids on very low density lipoprotein secretion when delivered to hepatocytes in chylomicron remnants, Biochem. J. 357 (2001) 481 – 487. S.D. Clarke, D.B. Jump, Dietary polyunsaturated fatty acid regulation of gene transcription, Annu. Rev. Nutr. 14 (1994) 83 – 98. M.S. Brown, J.L. Goldstein, Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis, Ann. Rev. Biochem. 52 (1983) 223 – 261. S. Cases, S.J. Smith, Y.W. Zheng, H.M. Myers, S.R. Lear, E. Sande, S. Novak, C. Collins, C.B. Welch, A.J. Lusis, S.K. Erickson, R.V. Farese, Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13018 – 13023. D.J. Grieve, M.A. Avella, J. Elliott, K.M. Botham, Influence of chylomicron remnants on endothelial cell function in the isolated perfused rat aorta, Atherosclerosis 139 (1998) 273 – 281. R.A. Davis, S.C. Engelhorn, S.H. Pangburn, D.B. Weinstein, D. Steinberg, Very low density lipoprotein synthesis and secretion by cultured rat hepatocytes, J. Biol. Chem. 254 (1979) 2010 – 2016. M.S. Brown, J.R. Faust, J.L. Goldstein, Induction of 3-Hydroxy-3methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase, J. Biol. Chem. 253 (1978) 1121 – 1128. M.J. Martinez, K.M. Botham, Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation – dephosphorylation, Biochim. Biophys. Acta 104 (1990) 90 – 98. K.E. Suckling, G.S. Boyd, C.G. Smellie, Properties of a solubilised and reconstituted preparation of acyl-CoA:cholesterol acyl transferase from rat liver, Biochim. Biophys. Acta 710 (1982) 154 – 163. M. Bradford, A sensitive method of quantification of microgram quantities of protein utilizing the principle of dye-binding, Anal. Biochem. 72 (1976) 248 – 252. K.M. Botham, E.N. Maldonado, Y. Chico, X. Zheng, M. Avella, B.
[38]
[39] [40] [41]
[42]
[43]
[44]
[45]
[46] [47]
Ochoa, The influence of chylomicron remnants on cholesteryl ester metabolism in cultured rat hepatocytes: comparison of the effects of particles enriched in n 3 or n 6 polyunsaturated fatty acids, Biochim. Biophys. Acta 1534 (2001) 96 – 109. H. Esterbauer, R.J. Shaur, H. Zollner, Chemistry and biochemistry of 4hydroxynonenal, malondialdehyde and related aldehydes, Free Radic. Biol. Med. 11 (1991) 81 – 128. C. Joyce, K. Skinner, R.A. Anderson, L.L. Rudel, Acyl-coenzyme A: cholesterol acyltransferase 2, Curr. Opin. Lipidol. 10 (1999) 89 – 95. M. Miller, Current perspectives on the management of hypertriglyceridemia, Am. Heart J. 140 (2000) 232 – 240. S.R. Bates, P.L. Murphy, Z.C. Feng, T. Kanazawa, G.S. Getz, Very low density lipoproteins promote triglyceride accumulation in macrophages, Arteriosclerosis 4 (1984) 103 – 114. J.L. Ellsworth, A.D. Cooper, F.B. Kraemer, Evidence that chylomicron remnants and h-VLDL are transported by the same receptor pathway in J774 murine macrophage-derived cells, J. Lipid Res. 27 (1986) 1062 – 1072. K.C. Yu, J.C.L. Mamo, Regulation of cholesterol synthesis and esterification in primary cultures of macrophages following uptake of chylomicron remnants, Biochem. Mol. Biol. Int. 41 (1997) 33 – 39. M. Ponec, J.A. Kempenaar, L. Havekes, J.G. Van der Schroeff, J.J. Emeis, B.J. Vermeer, Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte macrophages, Biochim. Biophys. Acta 666 (1981) 405 – 410. P.J. Davis, n 3 and n 6 Polyunsaturated fatty acids have different effects on acyl-CoA: cholesterol acyltransferase in J774 macrophages, Biochem. Cell. Biol. 70 (1992) 1313 – 1318. W. Jessup, L. Kritharides, Metabolism of oxidized LDL by macrophages, Curr. Opin. Lipidol. 11 (2000) 473 – 481. J.E. Kinsella, B. Lokesh, R.A. Stone, Dietary n 3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms, Am. J. Clin. Nutr. 52 (1991) 1 – 28.
Chylomicron remnant induction of lipid accumulation in J774 macrophages is associated with up-regulation of triacylglycerol synthesis which is not dependent on oxidation of the particles Mariarosaria Napolitano a, Michael Avella b, Kathleen M. Botham b, Elena Bravo a,* a
Laboratorio di Metabolismo e Biochimica Patologica, Istituto Superiore di Sanita´, Viale Regina Elena 299, 00161 Rome, Italy b Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Received 7 October 2002; received in revised form 25 February 2003; accepted 26 February 2003
Abstract The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n 6 PUFA) or fish oil (rich in n 3 PUFA) were prepared in vivo and oxidised by incubation with CuSO4. The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants ( < 2-fold). Triacylglycerol synthesis, as measured by the incorporation of [3H]oleate and [3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n 6 PUFA as compared with n 3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Chylomicron remnant; Triacylglycerol; Cholesterol esterification; Polyunsaturated fatty acid, Macrophage
1. Introduction One of the earliest events in the development of atherosclerotic lesions is the accumulation of lipid-laden macrophages in the artery wall. Monocyte-macrophages, which have invaded the arterial tissue, scavenge lipoproteins from the subendothelial space in an unregulated manner, becoming so engorged with lipid that they take on a foamy appearance, and are termed foam cells [1]. There is a great deal of evidence to implicate low density lipoprotein (LDL)
* Corresponding author. Tel.: +39-06-4990-2730; fax: +39-06-49387149. E-mail address: [email protected] (E. Bravo).
in foam cell formation, but it is also clear that native LDL is relatively benign in this respect, and that oxidative modification of the particles is required to induce extensive intracellular lipid accumulation [2]. Recent work in our laboratory and others, however, has established that chylomicron remnants, the lipoproteins which carry lipids from the diet from the intestine to the liver, are also able to induce macrophages to form foam cells [3– 5], although compared to the extensive literature on the effects of LDL, relatively little is known about the processes involved. After absorption by intestinal cells, fat and cholesterol from the diet are incorporated into large triacylglycerol-rich chylomicrons, which are secreted into lymph and enter the blood via the thoracic duct. These particles rapidly undergo lipolysis by lipoprotein lipase in extrahepatic capillary beds,
1388-1981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1388-1981(03)00042-8
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depleting their triacylglycerol content and leaving chylomicron remnants, which deliver the cholesterol and the remaining triacylglycerol to the liver [6]. These remnant lipoproteins are now believed to be strongly atherogenic for a number of reasons [7]. Hyperchylomicronaemia leads to premature atherosclerosis, although these patients have low LDL levels [8], men with coronary heart disease have raised post-prandial chylomicron remnant concentrations [9], and remnant-like particles containing apolipoprotein (apo) E have been isolated from human atherosclerotic plaque [10]. Moreover, it has been demonstrated that chylomicron remnants are able to penetrate the arterial wall as efficiently as LDL [7,11 –13]. Previous in vitro studies in our laboratory have shown that chylomicron remnants induce the murine macrophage cell line, J774, to form foam cells [3], and we have also observed this effect with chylomicron remnant-like particles and macrophages derived from the human monocyte cell line, THP-1 [4]. In addition, Yu and Mamo [5] have reported similar findings with human monocyte-derived macrophages. A striking difference between foam cell induction by LDL and chylomicron remnants highlighted in all these studies is that the remnant particles do not require prior oxidation to bring about their effects. Other workers have found that chylomicrons and a lipoprotein fraction from human blood enriched in chylomicron remnants cause accumulation of triacylglycerol, rather than cholesteryl ester, in mouse macrophages [14,15]. It is clear, therefore, that at least some of the cellular events which occur when foam cell formation is induced by chylomicron remnants are different from those triggered by oxidised LDL. It has been established that chylomicron remnants are taken up and degraded by macrophages in experiments with both primary cells and cell lines, including human monocyte-derived [16], mouse peritoneal and rabbit alveolar [17,18] macrophages, the human monocyte cell line, THP1 [19], and the murine cell lines, J774 and P388D1 [17,20]. Few studies, however, have investigated the effects of this uptake on macrophage lipid metabolism, and how these might lead to the massive lipid accumulation associated with foam cell formation. It has been known for many years that dietary polyunsaturated fats (PUFA) decrease the risk of the development of atherosclerosis [21, 22]. Both n 6 (abundant in plant oils) and n 3 PUFA (found in fish oils) are effective in this respect, although they bring about their effects by different mechanisms [23]. Since the function of chylomicron remnants is the transport in the blood of lipids of dietary origin, their fatty acid composition varies according to that of the fat consumed in the diet [24]. We have demonstrated that the type of PUFA (n 3 or n 6) predominating in chylomicron remnants alters their uptake and metabolism in the liver [25 – 27], and this contributes to the different ways in which these dietary fats retard the disease process. These findings, together with the fact that
dietary PUFA are known to play a role in the regulation of gene expression [28], suggest that the type of PUFA in chylomicron remnants may influence their effects on macrophage lipid metabolism. In the present work, we have investigated the influence of chylomicron remnants on lipid metabolism in J774 macrophages. Effects on lipid accumulation in the cells, the synthesis of triacylglycerol, cholesterol, cholesteryl ester and phospholipid from radiolabelled precursors, and the activity and/or expression of mRNA for enzymes regulating cholesteryl ester synthesis and hydrolysis and triacylglycerol synthesis were studied. Enzymes investigated were: acyl Co-enzyme A:cholesterol acyltransferase (ACAT), which regulated cholesterol esterification; lysosomal cholesteryl ester hydrolase (lCEH), which hydrolyses cholesteryl ester taken up from lipoproteins in the lysosomes; neutral cholesteryl ester hydrolase (nCEH), responsible for the hydrolysis of cytosolic cholesteryl ester stores [29]; and acyl Co-enzyme A:diacylglycerol acyltansferase (DGAT), which catalyses the rate-limiting step in triacylglycerol synthesis [30]. As our previous studies have shown that macrophages form foam cells on exposure to oxidised, as well as native, chylomicron remnant-like particles [4], both types of particle were tested. In addition, in order to investigate the possibility that the PUFA composition of the remnants is important in their effects on macrophage lipid metabolism, experiments were carried out using particles derived from both corn (rich in n 6 PUFA) and fish (rich in n 3 PUFA) oil.
2. Materials and methods 2.1. Animals and materials Male rats of the Wistar strain (300 – 350 g body weight) were used to obtain chylomicrons for chylomicron remnant preparation. The animals were fed a standard pellet diet and allowed food and water ad libitum. Climatic conditions (temperature 21– 22 jC, relative air humidity 50 + 5%) and the light cycle (12 h light, 12 h dark)) were kept constant. The J774.2 murine macrophage-like cell line was supplied by the American Type Culture Collection (Rockville, MD, USA). [9,10 3H(n)]oleate (9.2 Ci/mmol), [1(3)-3H]glycerol, [4-14C]cholesteryl oleate (60 mCi/mmol) and L-3 phosphatidylcholine 1-palmitoyl-2-(1-14C) linoleoyl (60 mCi/mmol) were obtained from NEN Life Science Products Inc. (Boston, MA, USA), and [2-14C]-acetic acid sodium salt (specific activity: 57 Ci/mmol), [1,2 3H(n)]cholesterol (35 – 50 Ci/mmol), [1-14C]oleoyl-CoA (56 mCi/mmol), and [1a,2a(n)-[3H]cholesteryl oleate (30 – 60 Ci/mmol) were purchased from Amersham Pharmacia Biotech Inc. (Milan, Italy). Fetal bovine serum (FBS), glutamine, antibiotics and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Hyclone (Europe Ltd). Fatty acid-
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free bovine serum albumin (BSA) and Menhaden fish oil were supplied by Sigma Chemical Company (St. Louis, MO, USA). 2.2. Preparation and oxidation of chylomicron remnants Chylomicrons were collected from rats after cannulation of the thoracic duct as described by Lambert et al. [24]. Briefly, animals were fed a single dose of 1 ml corn oil or Menhaden fish oil containing a-tocopherol (4 mg/ml) as an antioxidant by stomach tube, and approximately 1 h later the rats were anaesthetised and the thoracic duct was cannulated. The body wall was then sutured, the animals were placed in restraining cages and chyle was collected for 15 –18 h in the presence of ampicillin (0.1 mg/ml). Chylomicrons were isolated by ultracentrifugation (6 105 g min at 12 jC in a fixed angle rotor) after layering the chyle under 0.9% NaCl (d 1.006 g/ml), and harvested from the top fraction by tube slicing. Chylomicron remnants were prepared by incubation of the chylomicrons in vitro with rat post-heparin plasma [3]. After the incubation, chylomicron remnants were isolated by ultracentrifugation (6 107 g min at 12 jC), and further purified by a second ultracentrifugation (3.2 107 g min at 12 jC) after layering under 0.9% NaCl (d 1.006 g/ml). The top fraction was collected by tube slicing, and the remnants were dialysed overnight against culture medium prior to addition to the cell cultures. Chylomicron remnants were oxidised by incubation with CuSO4 (10 AM) for 18 h at 37 jC followed by dialysis according to Grieve et al. [31]. The oxidised remnants were further dialysed overnight against the culture medium before addition to the cell cultures. 2.3. Culture of J774 cells J774.2 cells were maintained in DMEM supplemented with penicillin (100 U/ml), streptomycin (100Ag/ml), glutamine (2 mM) and FBS (10%) at 37 jC in a humidified atmosphere of 95% air/5% CO2. For the experiments, cells (passage 6 –10) were seeded into 16-mm dishes at a density of 5 104 cells/ml and used on the fifth day of culture. Cell viability was determined by Trypan blue exclusion. 2.4. Experiments with radiolabelled substrates The amount of [3H]oleate incorporated into triacylglycerol, cholesteryl ester and phospholipid was determined as previously described [3]. Cells were incubated for 4 h at 37 jC in 5% CO2 in serum-free medium containing 0.326 mM oleic acid and 6 ACi/ml [3H]-oleate bound to fatty acid-free BSA (2%). After incubation, the cells were washed twice with phosphate-buffered saline (PBS), pH 7.4, and the lipids were extracted, separated by TLC and the radioactivity in the bands corresponding to cholesteryl ester, triacylglycerol
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and phospholipid was determined by liquid scintillation counting as before [3]. Incorporation of [3H]glycerol into triacylglycerol and phospholipid was determined using a modification of the method described by Davis et al. [32]. Cells were incubated for 4 h at 37 jC in 95% air/5% CO2 in serum-free medium containing [3H]glycerol (4 ACi/ml, 20 AM). After incubation, cells were washed twice with PBS. [14C]-phosphatidylcholine (900 dpm) was added to each tube as an internal standard and the lipids were extracted and separated by TLC [3]. The incorporation of [14C]-acetate into cholesterol, triacylglycerol and phospholipid was measured as described previously [33], with minor modifications. Cells were incubated in serum-free medium containing [14C]-sodium acetate (4 ACi/ml, 500 AM) at 37 jC in 95% air/5% CO2 for 4 h. After this time, the medium was removed and the cells were washed with PBS, and the lipids were extracted and separated by TLC as in Ref. [3]. [3H]cholesterol (1000 dpm/tube) was used as recovery marker. In all radiolabelling experiments, after extraction of cellular lipids, the cells were dissolved in 1 M NaOH for measurement of cellular protein. 2.5. Assay of enzyme activities nCEH and lCEH activity in cellular homogenates was determined by measuring the release of [14C]oleate from the cholesteryl[1-14C]oleate substrate according to Martinez and Botham [34]. Cells were homogenised in Tris –HCl buffer, pH 7.4, or in 10 mM acetic acid/Na acetate buffer pH 4.6, for nCEH or lCEH, respectively. ACAT activity was determined according to Suckling et al. [35] using [14C]oleoylCoA as the substrate (19.9 nmol/ tube, specific activity 3200 dpm/nmol). Lipids were extracted with chloroform/methanol (2:1 v/v), and [3H]cholesteryl oleate (1500 dpm/tube) was added to estimate recovery. Lipids were separated by TLC [3] and radioactivity in cholesteryl ester was determined by scintillation counting. 2.6. Analytical methods Triacylglycerol and total cholesterol concentrations in chylomicron remnants and cell samples were measured enzymatically using kits supplied by Boehringer Mannheim (Germany), and protein levels in cell samples were measured by the method of Bradford [36]. Cell viability was determined using by Trypan blue exclusion. The extent of oxidation of chylomicron remnants before and after incubation with CuSO4 was determined from the content of the aldehydic products of lipid peroxidation, 4hydroxy-2(E)-nonenal (4-HNE) + malondialdehyde (MDA), assayed using the Bioxytech LPO-586 assay (Oxis International Inc., Portland, OR, USA) according to the manufacturer’s instructions.
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Table 1 Characteristics of native and oxidised chylomicron remnants Lipid
Corn remnants
TG (Amol/ml) TC (Amol/ml) ratio TG/TC 4HNE F MDA (nmol/Amol TG)
Fish remnants
Native
Oxidised
Native
Oxidised
6.3 F 0.5(4) 0.55 F 0.07(4) 11.1 F 1.6(4) 4.7 F 1.7(4)
4.9 F 0.6(3) 0.48 F 0.04(3) 10.4 F 2.3(3) 18.9 F 7.3(3)*
5.4 F 0.1(4) 0.62 F 0.17(4) 9.9 F 1.6(4) 4.0 F 1.2(4)
5.4 F 0.3(4) 0.60 F 0.87(4) 10.7 F 2.1(4) 9.0 F 1.8(4)*
Chylomicron remnants derived from corn and fish oil were prepared and oxidised as described in Materials and methods, and their content of triacylglycerol, total cholesterol and 4HNE F malondialdehyde was determined. Data shown are the mean F S.E. and the number of separate experiments performed is given in parentheses. * P < 0.05 vs. corresponding native remnants.
Total RNA was extracted from the macrophages by homogenisation in guanidinium thiocyanate buffer (4 M) followed by layering over a CsCl (5.7 M) sodium acetate (25 mM) cushion and centrifugation at 31,000 rpm for 21 h. The relative abundance of mRNA transcripts for ACAT1, hormone sensitive lipase (HSL) and DGAT was then determined using reverse transcriptase-polymerase chain reaction (RTPCR). First strand cDNA synthesis was performed using Hotstar Taq reverse transcriptase (Qiagen, Crowley, West Sussex, UK) and amplification was carried out using the following primers for the appropriate mouse genes: ACAT1: sense, 5V-CCATTGATCTATTCCCTTGTCC-3V, antisense, 5V-GAGTCCTTGGGTAGTTGTCTCG-3V; DGAT: sense, 5V-GTGGTGATGCTGATCCTGAGT-3V, antisense, GAGTATGATGCCAGAGCAAAC-3V; under the following PCR conditions: initial denaturation/activation at 95 jC for 15 min followed by 30 (ACAT1) or 32 cycles (DGAT) consisting of denaturation at 94 jC for 30 s, annealing at 56 jC for 1 min and extension at 72 jC for 1 min, followed by final extension at 72 jC for 10 min. The products (ACAT1, 301 bp; DGAT, 329 bp) were analysed by electrophoresis using agarose gels (1.2% w/v) containing ethidium bromide (0.5Ag/ml). The bands were quantified using optical density volume analysis, and normalised with the values obtained for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples in the same assay system (in separate tubes). No effect on GAPDH mRNA expression was found with any of the treatments used. Preliminary experiments were carried out for each gene to ensure all measurements were made on the linear part of the curve.
Significance limits were determined using the paired Student’s t test.
3. Results 3.1. Characteristics of native and oxidised chylomicron remnants Our previous work has demonstrated that the fatty acid composition of chylomicron remnants prepared by the methods used in this study reflects that of the oil fed prior to collection of the chyle, and shows little variation from preparation to preparation [37]. Thus, in remnants derived from corn oil, the ratio of n 3:n 6 PUFA was 0.03, while that in particles derived from fish oil was 1.33, representing an increase of approximately 44-fold [37]. The lipid composition of four types of remnants tested (native and oxidised remnants derived from corn or fish oil) did not differ significantly in terms of their content of triacylglycerol, total cholesterol and triacylglycerol: total cholesterol ratio (Table 1). The content 4HNE + malondialdehyde, decomposition products of PUFA peroxidation, was determined in chylomicron remnants before and after incubation with CuSO4 (10 AM) for 18 h as a measure of the extent of oxidation of the particles [38]. Prior to incubation, the values obtained for corn and fish oil remnants were similar, and a significant increase was observed after incubation in both cases.
Table 2 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on triacylglycerol and total cholesterol levels in J774 macrophages Lipid
TG (nmol/mg protein) TC (nmol/mg protein)
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
65.0 F 3.6(4) 104.1 F 46.3(4)
410.6 F 46.3(4)** 175.6 F 43.7(4)
288.9 F 83.9(4)* 154.1 F 54.8(4)
57.6 F 2.7(3) 154.1 F 13.8(3)
101.9 F 9.5(3)* 127.5 F 47.0(3)
89.2 F 12.8(3)* 159.6 F 41.9(3)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 8 h, and the triacylglycerol and total cholesterol content of the cells was then determined. Data shown are the mean F S.E. and the number of separate experiments (with separate chylomicron remnant preparations) performed is given in parentheses. * P < 0.05 vs. corresponding control cells. ** P < 0.01 vs. corresponding control cells.
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Table 3 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the incorporation of [3H]oleate into cellullar lipids by J774 macrophages Lipid
TG CE PL
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
2.16 F 0.21(3) 0.206 F 0.092(3) 6.03 F 1.23(3)
8.78 F 1.50(3)* 0.158 F 0.053(3) 3.35 F 0.17(3)
5.61 F 2.1(2) 0.082 F 0.025(2) 2.66 F 0.44(2)
1.74 F 0.09(4) 0.167 F 0.007(4) 3.56 F 0.09(4)
2.40 F 0.31(4)* 0.176 F 0.020(4) 4.87 F 1.10(4)
2.60 F 0.45(4)* 0.205 F 0.014(4) 4.30 F 0.37(4)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 4 h in the presence of [3H]oleate (6 ACi/ml), and the incorporation of radioactivity into triacylglycerol, cholesteryl ester and phospholipid was determined. Data are expressed as nmol [3H]oleate/h/mg cell protein incorporated into each lipid and are the mean F S.E. (three or four experiments) or the mean F range (two experiments). The number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells.
3.2. The effect of chylomicron remnants on lipid accumulation in J774 macrophages J774 macrophages were incubated with or without native or oxidised chylomicron remnants derived from corn or fish oil for 4 h, and the triacylglycerol and total cholesterol content of the cells was then determined. The results are shown in Table 2. All four types of remnants tested caused significant increases in triacylglycerol levels in the cells. Remnants derived from corn oil, however, had a markedly greater effect than fish oil remnants, and this was the case with both native and oxidised particles (compared to control cells; corn oil remnants, native 6.3, oxidised 4.4; fish oil remnants, native 1.8, oxidised 1.6). The total cholesterol content of the macrophages was not significantly changed after incubation with native or oxidised fish oil remnants, and although the mean values obtained in experiments with both native and oxidised corn oil remnants were higher than the control values, because of the relatively large variation between individual experiments, these differences did not reach statistical significance. 3.3. Effect of chylomicron remnants on the incorporation of radiolabelled substrates into cellular lipids in J774 macrophages In order to investigate the effects of chylomicron remnants on lipid synthesis in J774 cells, their effects on the
incorporation of radiolabelled substrates into cellular lipids were determined. The influence of native or oxidised corn oil remnants on the incorporation of [3H]oleate into triacylglycerol, cholesteryl ester and phospholipid by the macrophages is shown in Table 3. The results showed that the native remnants caused a significant increase in incorporation into triacylglycerol over control cells incubated without remnants, and the mean incorporation was also increased by oxidised remnants. In the case of fish oil remnants, both native and oxidised particles caused a small but significant rise in incorporation into triacylglycerol (Table 3). The increases found with the native ( 1.35) and oxidised ( 1.49) fish oil remnants, however, was less marked than that seen with native ( 4.1) and oxidised ( 2.6) corn oil remnants (Table 3). In contrast to the results obtained with triacylglycerol, incorporation of [3H]oleate into cholesteryl ester and phospholipid was not significantly changed by any of the four types of remnants, although in experiments with native and oxidised corn oil particles there was a tendency towards a decrease in the amount of radiolabel found in phospholipid in comparison to control cells (Table 3). The incorporation of [3H]glycerol into triacylglycerol and phospholipid showed a generally similar pattern to that observed with [3H]oleate (Table 4). The amount of radiolabel incorporated into triacylglycerol was significantly increased in macrophages incubated with native and oxidised corn and fish oil remnants, as compared to those incubated in the absence of lipoproteins, with particles derived from corn oil (native 10.3, oxidised 14.5) having
Table 4 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the incorporation of [3H]glycerol into cellullar triacylglycerol and phospholipid by J774 macrophages Lipid
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
TG PL
0.13 F 0.03(3) 2.23 F 0.13(3)
1.34 F 0.27(3)** 0.77 F 0.23(3)**
1.93 F 0.54(2) 0.84 F 0.14(2)
0.13 F 0.03(4) 2.81 F 0.59(4)
0.57 F 0.12(4)* 3.90 F 0.95(4)*
0.40 F 0.11(4)* 2.47 F 0.70(4)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 4 h in the presence of [3H]glycerol (4 ACi/ml), and the incorporation of radioactivity into triacylglycerol and phospholipid was determined. Data are expressed as nmol [3H]glycerol/ h/mg cell protein incorporated into each lipid and are the mean F S.E. (three or four experiments) or mean F range (two experiments). The number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells. ** P < 0.01 vs. corresponding control cells.
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Fig. 1. Native ( – o – ) or oxidised ( – . – ) fish oil chylomicron remnants (5 – 20 Ag cholesterol/ml) were incubated with J774 macrophages for 4 h in the presence of [14C]acetate and the incorporation of radioactivity into (A) cholesterol, (B) triacylglycerol, and (C) nonesterified fatty acids was determined. Each point is the mean from four separate experiments (with separate chylomicron remnant preparations). Error bars show the S.E.
a considerably greater effect than those derived from fish oil (native 4.4, oxidised 3.1). In addition, when [3H]glycerol was used, effects on incorporation into phospholipid were seen, with native and oxidised corn remnants causing a decrease, while native fish oil remnants caused an increase. The effects of native and oxidised fish oil remnants on endogenous fatty acid, triacylglycerol and cholesterol synthesis in the macrophages were also investigated using [14C]acetate (Fig. 1). Cholesterol synthesis (Fig. 1A) was unaffected, but the formation of triacylglycerol (Fig. 1B) and fatty acid (Fig. 1C) was decreased by 40 –50%. There were no significant differences in the effects of native and oxidised particles. 3.4. Effect of chylomicron remnants on the activity of enzymes involved in cholesteryl ester synthesis and hydrolysis in J774 macrophages J774 macrophages were incubated in the presence or absence of native or oxidised chylomicron remnants derived from corn or fish oil, and the activities of ACAT, nCEH and lCEH were determined. The results are shown in Table 5. The activity of ACAT was increased by native, but not oxidised, corn oil remnants, while neither native nor oxidised fish oil remnants had any significant effect. nCEH
activity, on the other hand, was increased by both native and oxidised corn oil remnants, while the two types of fish oil remnants again had no effect. There was no change in the activity of lCEH after incubation of the cells with any of the four types of remnants under investigation. 3.5. Effect of chylomicron remnants on the expression of mRNA for enzymes involved in cholesteryl ester and triacylglycerol synthesis in J774 macrophages Recent studies have demonstrated that there are two forms of ACAT, designated ACAT1 and ACAT2. However, ACAT1 has been found in most tissues, while ACAT2 appears to be confined to the liver and intestine [39]. In the present study, therefore, the effects of chylomicron remnants on the expression of mRNA for ACAT1 in the J774 macrophages were measured. The results showed that the level of mRNA for this enzyme was increased by about 60% after incubation of the cells with native corn oil remnants. Oxidised corn oil remnants or native or oxidised fish oil remnants, in contrast, did not have any significant effect (Table 6). DGAT catalyses the terminal and sole committed step in triacylglycerol synthesis, and thus is important in the regulation of the process [30]. The amounts of DGAT
Table 5 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the activities of ACAT, nCEH and lCEH in J774 macrophages Enzyme
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
ACAT nCEH lCEH
4.32 F 1.25(4) 3.63 F 0.55(4) 36.7 F 14.7(3)
7.82 F 0.74(4)* 5.67 F 0.97(4)* 45.0 F 11.3(3)
3.21 F 0.90(4)yy 5.15 F 0.25(3)* 37.2 F 13.4(3)
5.20 F 0.72(5) 4.42 F 0.43(5) 39.5 F 10.3(4)
4.15 F 0.83(5) 4.94 F 0.58(5) 34.0 F 4.5(4)
4.83 F 0.94(5) 5.00 F 0.87(5) 42.5 F 12.1(4)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 8 h, and ACAT, nCEH and lCEH activity in the cells was then determined. Data are expressed as nmol product formed /h/mg cell protein and are the mean F S.E. The number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells. yy P < 0.01 vs. corresponding native remnants.
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Table 6 Effect of chylomicron remnants enriched in n 3 or n 6 PUFA on the expression of mRNA for ACAT1 and DGAT in J774 macrophages Gene
Corn remnants
Fish remnants
Control
Native
Oxidised
Control
Native
Oxidised
ACAT1 DGAT
0.65 F 0.22(3) 0.24 F 0.09(3)
1.03 F 0.33(3)* 0.36 F 0.13(3)
0.95 F 0.26(3) 0.30 F 0.06(3)
0.67 F 0.19(4) 0.38 F 0.17(3)
0.82 F 0.22(4) 0.26 F 0.9(3)
0.83 F 0.12(4) 0.25 F 0.06(3)
J774 macrophages were incubated with native or oxidised chylomicron remnants (10 Ag cholesterol/ml) derived from corn or fish oil for 8 h, and the relative abundance of mRNA transcripts for ACAT1 and DGAT in the cells was determined by RT-PCR. the bands were quantified by optical density analysis and the values were normalised using those obtained for GAPDH in the same system. Data shown are the mean F S.E. and the number of separate experiments performed (with separate chylomicron remnant preparations) is given in parentheses. * P < 0.05 vs. corresponding control cells.
mRNA found in the macrophages were relatively low, and no changes were detected after incubation in the presence of either native or oxidised corn or fish oil remnants.
4. Discussion Although elevated plasma LDL cholesterol levels are known to be a major factor in the development of atherosclerotic lesions containing foam cells engorged with cholesteryl esters [1,2], it is now recognised that plasma triacylglycerol levels are an independent risk factor for the disease [40]. Hypertriglyceridemia results when the clearance of chylomicron remnants from the blood is delayed post-prandially, and this has been found to lead to premature atherosclerosis development in the absence of raised LDL concentrations [8]. Previous work from our group has shown that the fatty acid composition of chylomicron remnants derived from corn oil (enriched in n 6 PUFA, mainly linoleic acid) or fish oil (enriched in n 3 PUFA, mainly eicosapentaenoic and docosahexaenoic acids) prepared by the methods used here reflects that of the parent oils, so that the ratio of n 3:n 6 PUFA was increased by 44-fold in fish as compared to corn oil remnants [37], although the overall distribution of lipids between triacylglycerol, total cholesterol and phospholipid was not changed. In the present study, oxidation of corn and fish oil remnants was found to cause a significant increase in their content of 4HNE + MDA, which is commonly used as an index of lipid peroxidation [38] (Table 1). As the triacylglycerol/total cholesterol ratio did not differ significantly in the four different types of remnants (native and oxidised corn, native and oxidised fish) (Table 1), the amount of triacylglycerol added to the cells was similar at each concentration of remnant cholesterol tested. Thus, as well as investigating the influence of chylomicron remnants on lipid metabolism in J774 macrophages, we have been able to observe the effects of changes in the ratio of n 3:n 6 PUFA, and in the oxidation state of the particles. Studies in our laboratory and others have demonstrated that chylomicron remnants cause lipid accumulation in macrophages [3– 5]. In general, triacylglycerol-rich lipoproteins, such as VLDL or h-VLDL, have been found to cause
the accumulation of triacylglycerol, rather than cholesterol [14,15,41], and preliminary experiments in our laboratory have demonstrated that chylomicron remnant-like particles have this effect in THP-1 macrophages [4] and unpublished results. This pattern was also observed in the present study, with cellular triacylglycerol levels being raised considerably after incubation of J774 macrophages with chylomicron remnants, while total cholesterol levels were not significantly changed (Table 2). It is well established that LDL must be chemically or oxidatively modified before it is able to induce macrophages to form foam cells [2]. Like other triacylglycerol-rich lipoproteins such as VLDL [46], however, chylomicron remnants have been found to cause foam cell formation without prior modification of the particles, and the present findings with J744 macrophages (Table 3) confirm this. In contrast, the type of PUFA in the particles has a clear effect on the amount of triacylglycerol accumulated, with remnants derived from corn oil causing a four- to sixfold increase over the levels found in control cells, while those derived from fish oil induced a rise of less than twofold (Table 3). In addition, the amount of total cholesterol in the cells tended to be increased in cells incubated with remnants derived from corn (increase observed in three out of four experiments), but not fish oil, although because of the relatively large variation between individual experiments this change did not reach significance. Thus, enrichment of chylomicron remnants with n 3 as compared to n 6 PUFA leads to decreased lipid accumulation in macrophages. Although it is known that macrophages take up and degrade chylomicron remnants [16 – 20], little information is available about the subsequent effects on intracellular lipid metabolism. In the present study, investigation of the effects of chylomicron remnants on lipid synthesis in the macrophages using the radiolabelled precursors [3H]oleate or [3H]glycerol showed that triacylglycerol synthesis was increased in the presence of the particles, and this is in agreement with a previous report [42]. Our results demonstrate further, however, that corn oil remnants cause a more marked rise than fish oil remnants (Tables 3 and 4). These results are consistent with our findings on triacylglycerol accumulation in the cells (Table 2), and suggest that remnants enriched with n 3 as compared to n 6 PUFA cause a lower level of accumulation because they stimulate
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triacylglycerol synthesis to a lesser extent. The PUFA composition of remnants also had differential effects on phospholipid synthesis, with a decrease observed with corn oil particles, and an increase with those derived from fish oil (Table 4). This is consistent with the differences in the effects of corn and fish oil remnants on phospholipid synthesis in the liver observed in our earlier work [25]. These results suggest that the delivery of n 6 PUFA to the cells leads to the diversion of the fatty acid and glycerol substrates into triacylglycerol synthesis at the expense of phospholipid formation, while remnant-derived n 3 PUFA tends to be shared between the two pathways. One drawback of the radiolabelling experiments with [3H]oleate and [3H]glycerol is that the dilution of the intracellular substrate pools by fatty acids and glycerol derived from the remnants taken up by the cells may interfere with the results. The changes in triacylglycerol and phospholipid synthesis observed with the two different labels, however, were similar overall, suggesting that any such dilution effect did not influence the overall pattern of the findings. Furthermore, the up-regulation of triacylglycerol synthesis by chylomicron remnants, which is the major finding of these experiments, would be underestimated if the substrate pool was diluted. In order to investigate the effects of chylomicron remnants derived from fish oil on de novo lipid synthesis in J774 macrophages, the incorporation of [14C]acetate into cholesterol, non-esterified fatty acids and triacylglycerol was determined. In two previous studies the activity of 3hydroxy-3-methylglutaryl Co-enzyme A reductase (HMG CoA reductase), the rate-limiting enzyme in cholesterol synthesis, has been reported to be increased in J774 cells [42], but decreased in primary rabbit macrophages [43]. In our experiments, chylomicron remnants had no significant effect on the incorporation of [14C]acetate into cholesterol. Thus the influx of cholesterol from chylomicron remnants, at least at the concentrations used here, does not cause a decrease in de novo cholesterol synthesis in the macrophages, as occurs with LDL [44]. Incorporation of [14C]acetate into triacylglycerol was significantly reduced by the remnants (Fig. 1); however, the decrease of a similar magnitude (40 –50%) observed in incorporation into nonesterified fatty acids indicates that this change is due to depression of de novo fatty acid synthesis, which might be expected on delivery of relatively large amounts of triacylglycerol in chylomicron remnants to the cells. These results, therefore, do not conflict with our finding that triacylglycerol synthesis from [3H]oleate is increased by the particles. The enzyme DGAT catalyses the last and only committed step in triacylglycerol synthesis [30]. Levels of DGAT mRNA were low in the J774 cells (Table 6) and were not significantly affected by chylomicron remnants. It appears, therefore, that neither the increase in triacylglycerol synthesis caused by the particles, nor the differential effects of those enriched in n 3 or n 6 PUFA, can be attributed to up-regulation of transcription of this gene. It is possible,
however, that post-translational changes in the enzyme activity are involved, or that other factors influence the distribution of the substrate between triacylglycerol and phospholipid synthesis. Interestingly, the effects of oxidised chylomicron remnants on lipid synthesis, were generally similar to those of the native particles. The only difference observed was that prior oxidation of fish oil remnants abolished their effect in increasing phospholipid synthesis from glycerol. These findings are consistent with the similar effects of native and oxidised particles on the accumulation of triacylglycerol in the macrophages (Table 2), and indicate that stimulation of triacylglycerol synthesis is a major factor in foam cell induction by chylomicron remnants. Furthermore, it is clear from our results that the type of PUFA carried by chylomicron remnants is more important than their oxidation state in determining their effects on triacylglycerol metabolism in macrophages, with enrichment with n 3 PUFA as compared to n 6 PUFA leading to a marked reduction in synthesis and intracellular accumulation. Cholesteryl ester stores in macrophages undergo a continuous cycle of hydrolysis by nCEH, followed by reesterification by ACAT [29] in a process known as the cholesteryl ester cycle. In the present study, therefore, we investigated the effects of chylomicron remnants on cholesteryl ester metabolism by measuring the incorporation of [3H]oleate into cholesteryl ester, the activity of ACAT and nCEH, and the expression of mRNA for ACAT1, the form of ACAT expressed in macrophages [39]. ACAT activity and the levels of mRNA for ACAT1 in the cells were upregulated by corn, but not fish, oil remnants, but these changes were not reflected in an increase in the overall rate of cholesterol esterification (Tables 3, 5 and 6). This may be explained by the concomitant increase in nCEH activity observed in the presence of corn oil remnants, and is consistent with a previous report of reduced activity of ACAT in J774 macrophages exposed to n 3 as compared to n 6 PUFA in the free fatty acid form [45]. However, in contrast to our results, Yu and Mamo [43] found increased incorporation of [3H]oleate into cholesteryl ester in macrophages incubated with chylomicron remnants, while Ellsworth et al. [42] reported a decrease. The reason for these discrepancies is not clear, but the type of fat used to obtain the chylomicron remnants is not specified in either study, and our findings suggest that this may be important in the results obtained. Oxidation of the particles abolished the effects of corn oil remnants on ACAT activity and mRNA concentrations, but no other changes compared to native remnants were observed. It is interesting to note that oxidised LDL has also been found to have little effect on ACAT activity in murine macrophages, and this has been attributed to the retention of much of the sterol fraction in the lysosomal system as oxidised esters [46]. Taken together, therefore, our results indicate that chylomicron remnants enriched in n 6 PUFA increase the flux of cholesterol through the cholesteryl ester
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cycle in macrophages by up-regulating the activities of ACAT and nCEH at the transcriptional or post-transcriptionally level, respectively. These regulatory effects, however, are lost after oxidation of the particles, and are not exhibited at all by remnants enriched in n 3 PUFA. In summary, the studies presented here demonstrate that both native and oxidised chylomicron remnants induce lipid accumulation in J774 macrophages; that the major lipid accumulated is triacylglycerol, rather than cholesterol; and that increased synthesis of triacylglycerol from exogenous fatty acids is a major factor in the process. Importantly, however, they also demonstrate that the type of PUFA carried by the remnants profoundly affects their influence on lipid metabolism in the macrophages, with those enriched in n 6 PUFA causing a markedly higher level of triacylglycerol synthesis and accumulation compared to those enriched with n 3 PUFA. The oxidation state of the remnants, on the other hand, had relatively minor effects on the processes studied, and did not affect the amount of triacylglycerol accumulated in the cells. Since chylomicron remnants carry lipids from the diet, and their fatty acid composition reflects that of the fats consumed [24], these findings support the idea [7] that dietary fatty acids may have a role in the generation of foam cells. Thus, in addition to their well established effects in lowering blood triacylglycerol levels and depressing macrophage eicosanoid metabolism [22,47], dietary n 3, as compared to n 6, PUFA may also decrease the tendency of chylomicron remnants to promote foam cell formation.
Acknowledgements This work was supported by grants from the Wellcome Trust (Ref. no. 057282/Z/99) and Programma Ricerca Finalizzata 1999, Ministry of Health, Italy (Ref. ISS D70).
References [1] R. Ross, The pathogenesis of atherosclerosis: a perspective for the 1990s, Nature 362 (1993) 801 – 809. [2] D. Steinberg, Low density lipoprotein oxidation and its pathobiological significance, J. Biol. Chem. 272 (1997) 20963 – 20966. [3] M. Napolitano, R. Rivabene, M. Avella, K.M. Botham, E. Bravo, The internal redox balance of cells influences the metabolism of lipids of dietary origin by J774 macrophages: implications for foam cell formation, J. Vasc. Res. 38 (2001) 350 – 360. [4] K.V. Batt, K.M. Botham, B. Jackson, K.E. Suckling, Comparison of the effects of low density lipoprotein and chylomicron remnants on foam cell formation in the human monocytic cell line, THP-1, Atheroscler. Suppl. 2 (2001) 109. [5] K.C. Yu, J.C.L. Mamo, Chylomicron remnant-induced foam cell formation and cytotoxicity: a possible mechanism of cell death in atherosclerosis, Clin. Sci. (Lond.) 298 (2000) 183 – 192. [6] T.G. Redgrave, Formation and metabolism of chylomicrons, Int. Rev. Physiol. 28 (1983) 103 – 130. [7] J.C.L. Mamo, Atherosclerosis as a post-prandial disease, Endocrinol. Metab. 2 (1995) 229 – 244.
263
[8] P. Benlian, P.L. De Gennes, L. Foubert, H. Zhang, S.E. Gagne, M. Hayden, Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene, N. Engl. J. Med. 335 (1996) 848 – 854. [9] P.H.E. Groot, W.A.H. van Stiphout, X.H. Krauss, H. Jansen, A. van Tol, E. van Ramshorst, S. Chin-On, A. Hofmann, S.R. Cresswell, L. Havekes, Postprandial lipoprotein metabolism in normolipidemic men with and without coronary heart disease, Arterioscler. Thromb. 11 (1991) 653 – 662. [10] S. Yla-Herttuala, O. Jaakkola, C. Enholm, M.J. Tikkanen, T. Solakivi, T. Sarkioja, T. Nikkari, Characterisation of two lipoproteins containing apolipoproteins B and E from lesion-free human aortic intima, J. Lipid Res. 29 (1988) 563 – 572. [11] S.D. Proctor, J.C.L. Mamo, Retention of fluorescent-labelled chylomicron remnants within the intima of the artery wall—evidence that plaque cholesterol may be derived from post-prandial lipoproteins, Eur. J. Clin. Investig. 28 (1998) 497 – 503. [12] D.J. Grieve, M. Avella, J. Elliott, K.M. Botham, Influence of chylomicron remnants on endothelial cell function in the isolated perfused rat aorta, Atherosclerosis 139 (1998) 273 – 281. [13] J.C.L. Mamo, J.R. Wheeler, Chylomicrons or their remnants penetrate rabbit thoracic aorta as efficiently as do smaller macromolecules, including low density lipoprotein, high density lipoprotein and albumin, Coron. Artery Dis. 5 (1994) 695 – 705. [14] S.H. Gianturco, S.A. Brown, D.P. Via, W.A. Bradley, The h-VLDL receptor pathway of murine P388D1 macrophages, J. Lipid Res. 27 (1986) 412 – 420. [15] T.P. Bersot, T.L. Innerarity, R.E. Pitas, R.C. Rall, K.H. Weisgraber, R.W. Mahley, Fat feeding in humans induces lipoproteins of density less than 1.006 that are rich in apolipoprotein (a) and that cause foam cell formation in macrophages, J. Clin. Invest. 77 (1986) 622 – 630. [16] C. Koo, M.E. Wernett-Hammond, Z. Garcia, M.J. Malloy, R. Uauy, C. East, D. Bilheimer, R.W. Mahley, T.L. Innerarity, Uptake of cholesterol-rich remnant lipoproteins by human monocyte-derived macrophages is mediated by low density lipoprotein receptors, J. Clin. Invest. 81 (1988) 1332 – 1340. [17] J.L. Ellsworth, F.B. Fong, F.B. Kraemer, A.D. Cooper, Differences in the processing of chylomicron remnants and h-VLDL by macrophages, J. Lipid Res. 31 (1990) 1399 – 1411. [18] J.C.L. Mamo, C.L. Ellsgood, K. Yu, S. Schumann, T.G. Redgrave, Chylomicron remnants metabolism by macrophages, Atherosclerosis 109 (1994) 107. [19] C.L. Elsegood, S. Pal, P.D. Roach, J.C.L. Mamo, Binding and uptake of chylomicron remnants by primary and THP-1 monocyte-derived macrophages: determination of binding proteins, Clin. Sci. (Lond.) 101 (2001) 111 – 119. [20] J.L. Ellsworth, F.B. Kraemer, A.D. Cooper, Transport of h-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway, J. Biol. Chem. 262 (1987) 2316 – 2325. [21] A.S. Truswell, Reducing the risk of coronary heart disease, Br. Med. J. 291 (1985) 34 – 37. [22] W.S. Harris, Dietary fish oils and blood lipids, Curr. Opin. Lipidol. 7 (1996) 3 – 7. [23] J.T. Thornberg, L.L. Rudel, How do polyunsaturated fatty acids lower lipids? Curr. Opin. Lipidol. 3 (1992) 17 – 21. [24] M.S. Lambert, K.M. Botham, P.A. Mayes, Modification of the composition of dietary oils and fats upon incorporation into chylomicron remnants, Br. J. Nutr. 76 (1996) 435 – 445. [25] M.S. Lambert, K.M. Botham, P.A. Mayes, Variations in composition of dietary fats affect hepatic uptake and metabolism of chylomicron remnants, Biochem. J. 310 (1995) 845 – 852. [26] M.S. Lambert, M.A. Avella, Y. Berhane, E. Shervill, K.M. Botham, The fatty acid composition of chylomicron remnants influences their binding and internalisation by isolated hepatocytes, Eur. J. Biochem. 268 (2001) 3983 – 3992. [27] X. Zheng, M. Avella, K.M. Botham, Comparison of the effects of
264
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
M. Napolitano et al. / Biochimica et Biophysica Acta 1631 (2003) 255–264 dietary n 3 and n 6 polyunsaturated fatty acids on very low density lipoprotein secretion when delivered to hepatocytes in chylomicron remnants, Biochem. J. 357 (2001) 481 – 487. S.D. Clarke, D.B. Jump, Dietary polyunsaturated fatty acid regulation of gene transcription, Annu. Rev. Nutr. 14 (1994) 83 – 98. M.S. Brown, J.L. Goldstein, Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis, Ann. Rev. Biochem. 52 (1983) 223 – 261. S. Cases, S.J. Smith, Y.W. Zheng, H.M. Myers, S.R. Lear, E. Sande, S. Novak, C. Collins, C.B. Welch, A.J. Lusis, S.K. Erickson, R.V. Farese, Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13018 – 13023. D.J. Grieve, M.A. Avella, J. Elliott, K.M. Botham, Influence of chylomicron remnants on endothelial cell function in the isolated perfused rat aorta, Atherosclerosis 139 (1998) 273 – 281. R.A. Davis, S.C. Engelhorn, S.H. Pangburn, D.B. Weinstein, D. Steinberg, Very low density lipoprotein synthesis and secretion by cultured rat hepatocytes, J. Biol. Chem. 254 (1979) 2010 – 2016. M.S. Brown, J.R. Faust, J.L. Goldstein, Induction of 3-Hydroxy-3methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase, J. Biol. Chem. 253 (1978) 1121 – 1128. M.J. Martinez, K.M. Botham, Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation – dephosphorylation, Biochim. Biophys. Acta 104 (1990) 90 – 98. K.E. Suckling, G.S. Boyd, C.G. Smellie, Properties of a solubilised and reconstituted preparation of acyl-CoA:cholesterol acyl transferase from rat liver, Biochim. Biophys. Acta 710 (1982) 154 – 163. M. Bradford, A sensitive method of quantification of microgram quantities of protein utilizing the principle of dye-binding, Anal. Biochem. 72 (1976) 248 – 252. K.M. Botham, E.N. Maldonado, Y. Chico, X. Zheng, M. Avella, B.
[38]
[39] [40] [41]
[42]
[43]
[44]
[45]
[46] [47]
Ochoa, The influence of chylomicron remnants on cholesteryl ester metabolism in cultured rat hepatocytes: comparison of the effects of particles enriched in n 3 or n 6 polyunsaturated fatty acids, Biochim. Biophys. Acta 1534 (2001) 96 – 109. H. Esterbauer, R.J. Shaur, H. Zollner, Chemistry and biochemistry of 4hydroxynonenal, malondialdehyde and related aldehydes, Free Radic. Biol. Med. 11 (1991) 81 – 128. C. Joyce, K. Skinner, R.A. Anderson, L.L. Rudel, Acyl-coenzyme A: cholesterol acyltransferase 2, Curr. Opin. Lipidol. 10 (1999) 89 – 95. M. Miller, Current perspectives on the management of hypertriglyceridemia, Am. Heart J. 140 (2000) 232 – 240. S.R. Bates, P.L. Murphy, Z.C. Feng, T. Kanazawa, G.S. Getz, Very low density lipoproteins promote triglyceride accumulation in macrophages, Arteriosclerosis 4 (1984) 103 – 114. J.L. Ellsworth, A.D. Cooper, F.B. Kraemer, Evidence that chylomicron remnants and h-VLDL are transported by the same receptor pathway in J774 murine macrophage-derived cells, J. Lipid Res. 27 (1986) 1062 – 1072. K.C. Yu, J.C.L. Mamo, Regulation of cholesterol synthesis and esterification in primary cultures of macrophages following uptake of chylomicron remnants, Biochem. Mol. Biol. Int. 41 (1997) 33 – 39. M. Ponec, J.A. Kempenaar, L. Havekes, J.G. Van der Schroeff, J.J. Emeis, B.J. Vermeer, Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte macrophages, Biochim. Biophys. Acta 666 (1981) 405 – 410. P.J. Davis, n 3 and n 6 Polyunsaturated fatty acids have different effects on acyl-CoA: cholesterol acyltransferase in J774 macrophages, Biochem. Cell. Biol. 70 (1992) 1313 – 1318. W. Jessup, L. Kritharides, Metabolism of oxidized LDL by macrophages, Curr. Opin. Lipidol. 11 (2000) 473 – 481. J.E. Kinsella, B. Lokesh, R.A. Stone, Dietary n 3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms, Am. J. Clin. Nutr. 52 (1991) 1 – 28.