Lack of Triglyceride-Lowering Properties of Fish Oil in Apolipoprotein E–Deficient Mice
Abstract—Fish oil is a potent triglyceride (TG)-lowering agent in humans. The goal of the present study was to assess the contribution of decreased triglyceride synthesis and of apoE in mediation of the triglyceride-lowering effect of fish oil. To this end, apoE-deficient mice and wild-type control mice were supplemented with either coconut oil, sunflower oil, or fish oil (20% wt/wt) for 2 weeks. Compared with coconut oil and sunflower oil, fish oil reduced the concentrations of cholesterol and triglycerides in the wild-type mice, whereas it had no effect on cholesterol concentration and it had a triglyceride–raising effect in apoE-deficient mice. The latter was due to increased triglyceride concentrations in the d<1.019 g/mL plasma density fraction. In apoE-deficient mice, but not in wild-type mice, the postprandial triglyceride area under the curve was higher after an intragastric load of fish oil than after a sunflower oil load. These data indicate an impairment of triglyceride metabolism in the fish oil–fed apoE-deficient mice. Compared with coconut oil and sunflower oil, fish oil lowered triglyceride production rates measured with the Triton method in both wild-type (P<0.0001) and apoE-deficient mice (P<0.0001). Similarly, in vitro lipoprotein lipase–mediated lipolysis of VLDL was lowered in the fish oil–fed wild-type and apoE-deficient mice, suggesting an alteration in VLDL lipolysis independent of the mice genotype. In conclusion, fish oil does not decrease triglyceride concentrations in apoE-deficient mice despite reducing triglyceride production rates, suggesting that decreased triglyceride synthesis is not sufficient to lower triglyceride concentrations in mice. ApoE appears to be necessary for fish oil to lower plasma triglyceride concentrations, indicating a critical role of apoE in this process.
- Received June 26, 2000.
- Accepted September 4, 2000.
Fish oil is an efficient triglyceride (TG)–lowering agent in both humans and animal models.1 2 Fish oil modulates the activity of several enzymes of both lipid and carbohydrate metabolisms. Its overall effect is to promote fatty acid oxidation and to decrease TG synthesis3 4 and therefore to lower VLDL production rate (PR) and plasma concentrations.5 6
Fish oil decreases the plasma concentration of VLDL and chylomicron remnants in patients with dysbetalipoproteinemia.7 The decreased hepatic TG synthesis usually induced by a fish oil diet certainly contributes to a reduction in the formation of TG-rich lipoproteins (TRLs) and their remnants. Alternatively, lower amounts of TRLs reduce the amount of lipoproteins that compete with remnants for receptor-mediated clearance, therefore promoting remnant uptake. The contribution of one of these pathways to the TG-lowering property of fish oil is not firmly established.
The apoE-deficient mice have a markedly elevated plasma cholesterol concentration due to the accumulation of VLDL plus IDL in their bloodstream.8 These particles are mainly cholesterol-rich remnants of chylomicron and VLDL. ApoE has a critical role in the clearance of lipoprotein remnants. It favors the anchoring of TRLs to cell surface heparan sulfate proteoglycan, which facilitates the interaction with lipoprotein lipase (LPL) and is an important ligand to lipoprotein cell receptors. Therefore, apoE deficiency in mice results in major defects in TG and VLDL clearance.
The goal of the present study was to assess the contribution of a decreased TG synthesis and the role of apoE in mediating the TRL-lowering properties of fish oil. To this end, we compared the effect of fish oil treatment in apoE-deficient and wild-type mice. Our hypothesis was that the absence of apoE prevents VLDL and chylomicron remnant uptake. This would allow us to measure the contribution of the synthetic pathway to the lipid-lowering properties of fish oil.
Studies were performed with apoE-deficient mice (Transgenic Alliance, IFFA CREDO) and wild-type mice (C57BL/6J). Before dietary studies, the animals were acclimated for 1 week in a temperature-controlled environment (20±1°C) and kept in the dark from 8 pm through 8 am in a room with low background noise. All experiments were performed with male mice (similar results were obtained with female mice).
Fifteen apoE-deficient and 15 wild-type mice of a similar age (±1 week) were used in the study. Before the dietary experiment, a blood sample was drawn for randomization of cholesterol concentrations. Animals were housed 2 or 3 per cage and given free access to a fat-free semipurified diet (UAR) supplemented (20% wt/wt) with fish oil (menhaden oil; Sigma-Aldrich Chimie SARL), coconut oil (Sigma-Aldrich Chimie SARL), or sunflower oil (BERTIN) for 2 weeks (5 mice per dietary group). Fish oil from menhaden contained 30% eicosapentaenoic acid and docosahexaenoic acid with a ratio of 1.2. At the end of the dietary intervention, mice were food deprived for 4 hours and exsanguinated via cardiac puncture while under anesthesia with diethylether. Blood samples were mixed with EDTA and kept at 4°C.
Lipoprotein Separation and Measurements
Plasma was separated through centrifugation (630g) for 20 minutes at 4°C. VLDL and IDL were separated through ultracentrifugation with a Beckman TL100 ultracentrifuge (Beckman Instruments France SA) from 150 μL plasma through 1 spin at d<1.019 g/mL, as described previously.9 Lipids were determined enzymatically with commercially available kits for TGs (Triglycerides GPO-PAP; Boehringer-Mannheim), cholesterol (Cholesterol C System; Boehringer-Mannheim), and phospholipids (Phospholipids PAP 150; BioMérieux). Lipid composition of the VLDL-IDL fraction was assessed in the d<1.019 g/mL lipoprotein fraction. The results of lipid composition are expressed in relative terms (percentage of total lipids).
Gel Filtration Chromatography
Gel filtration chromatography was performed with FPLC with a Superose 6 HR 10/30 column (Pharmacia LKB Biotechnology). The gel was allowed to equilibrate with PBS (10 mmol/L) containing 0.1 g/L EDTA and 0.1 g/L sodium azide; 200 μL plasma was eluted with the buffer at room temperature at a flow rate of 0.2 mL/min. Elution profiles were monitored at 280 nm and recorded with an analog-recorder chart tracing system (Pharmacia LKB Biotechnology). The effluents were collected in 0.24-mL fractions.
In Vivo Hepatic TG Production With Triton WR1339
Twelve apoE-deficient mice and 12 wild-type control mice were supplemented for 2 weeks with coconut oil, sunflower oil, or fish oil (20% wt/wt; 4 per dietary group). At the end of the dietary intervention, mice were food deprived for 4 hours. Each mouse was injected in the tail vein at 500 mg/kg body wt with a 150 g/L solution of Triton WR1339 (Sigma-Aldrich Chimie) in 9 g/L NaCl, as described elsewhere.10 Blood samples of 100 μL were drawn before the Triton WR1339 injection and 30, 60, 120, and 180 minutes later. Plasma TGs were measured in each sample. The TG PR was calculated as follows11 : PR=a×PV (in mg/h), where a is slope of the regression line of time (in hours) and TG concentrations (in mL) after Triton injection, and PV is plasma volume [body weight (in g)×0.033 (in mL)]
At 9 am, 15 chow-fed C57BL/6 wild-type and 15 apoE-deficient mice were intragastrically administered a bolus of 600 μL coconut oil, sunflower oil, or fish oil (5 mice per dietary group), and blood samples were taken 2 hours later. TRLs (d<1.019 g/mL) were isolated through ultracentrifugation from the pooled plasma. TRLs were incubated at 4 concentrations (range 0.06 to 0.35 mmol/L) during 5 minutes at 37°C in a 200-μL final volume solution that contained 0.1 mol/L Tris/HCl, pH 8.5, 12 g/L fatty acid–free BSA (Sigma-Aldrich Chimie), and 0.27 U of commercial bovine LPL (EC 188.8.131.52; Sigma Diagnostics). Reaction was stopped by the addition of 100 μL of ice-cold stop buffer (50 mmol/L KH2PO4, 1 mL/L Triton X-100, pH 6.9). A blank sample was prepared for each concentration. Nonesterified fatty acids (NEFAs) were quantified with use of the NEFA-C kit (WAKO Chemicals GmbH).
Eighteen wild-type and 18 apoE-deficient mice were supplemented for 2 weeks with either coconut oil, sunflower oil, or fish oil (20% wt/wt, 6 mice per dietary group). After a 4-hour fasting period, mice received an intravenous injection of heparin (100 U/kg body wt; Laboratoires Leo SA). Before the injection and 5 minutes after the injection, 100 μL of blood was collected retro-orbitally and stored on ice. Plasma TG concentrations were measured as described above.
Ten chow-fed mice of each genotype received an intragastric bolus of 600 μL of sunflower oil or fish oil (5 mice per dietary group). Approximately 50 μL of blood was collected retro-orbitally to measure plasma TG concentrations at different times (0, 1, 2, 3, 4, 5, 6, and 7 hours).
Two-way ANOVA (SPSS Release 7.5 for Windows; SPSS Institute Inc) was used to compare the effects of the various oils and genotypes on lipid and lipoprotein concentrations, lipoprotein lipid composition, and kinetic study parameters. Two-way ANOVA with diet (independent factor) and time (repeated measures) factors was used to assess the effect of susceptibility of TRLs after fat load. The Scheffé’s test was used for post hoc analysis.
Lipid and Lipoprotein Concentrations
Two-way ANOVA showed a statistically significant interaction between genotype and diet for all lipid and lipoprotein variables, suggesting that dietary responses differed in wild-type and transgenic mice (Table 1⇓). In wild-type mice, total cholesterol, TGs, and phospholipids were lower (P<0.05) in the fish oil group than in the coconut oil group. Similarly, VLDL-IDL cholesterol, VLDL-IDL TGs, VLDL-IDL phospholipids, LDL-HDL cholesterol, and LDL-HDL phospholipids were lower (P<0.05) in the fish oil group than in the coconut oil group. Sunflower oil supplementation was associated with lower concentrations (P<0.05) of total TGs, total phospholipids, VLDL-IDL TGs, VLDL-IDL phospholipids, and LDL-HDL phospholipids than coconut oil supplementation. Compared with wild-type animals, apoE-deficient mice had significantly higher concentrations (P<0.05) of total plasma cholesterol, phospholipids, and their respective VLDL-IDL lipoprotein fractions. In apoE-deficient mice, total phospholipids, LDL-HDL cholesterol, and LDL-HDL phospholipids were lower (P<0.05), and total TGs and VLDL-IDL TGs were higher (P<0.05) in the fish oil group than in the coconut oil group (Table 1⇓). Finally, total cholesterol, phospholipids, VLDL-IDL cholesterol, HDL-LDL cholesterol, and HDL-LDL phospholipid concentrations were lower (P<0.05) in the sunflower oil group than in the coconut oil group.
Gel Filtration Chromatography
Cholesterol (Figure 1⇓) and phospholipid concentrations in the HDL lipoprotein fractions were lower in wild-type mice supplemented with fish oil than in those fed with coconut oil. TG concentrations in the large lipoprotein fraction were also lower in wild-type mice supplemented with coconut oil than in those supplemented with fish oil (Figure 1⇓; please see http://atvb.ahajournals.org). In contrast, cholesterol, TG, and phospholipid concentrations in the large lipoprotein fraction were higher in the apoE-deficient mice in the fish oil group than in those in the coconut oil group. The apoE-deficient mice supplemented with sunflower oil had lower concentrations of cholesterol, TGs, and phospholipids in the large lipoprotein fraction and lower concentrations of cholesterol in the HDL fraction.
VLDL-Plus-IDL Lipid Composition
Two-way ANOVA showed a statistically significant interaction between genotype and diet for cholesterol (P<0.006), TG (P<0.0001), and phospholipid (P<0.025) composition, suggesting that dietary responses were different in wild-type and transgenic mice. In wild-type mice, the VLDL-IDL-cholesterol and VLDL-IDL-phospholipid compositions did not differ among dietary groups. TG concentration was slightly higher (P<0.05) in the sunflower oil group than in the coconut oil group (Figure 2⇓). Compared with the wild-type mice, VLDL-IDL fractions of apoE-deficient mice were richer in cholesterol (P<0.019) and phospholipids (P<0.042) and depleted in TGs (P<0.0001). In the apoE-deficient mice, the VLDL-IDL composition of the fish oil group had less (P<0.05) relative cholesterol and phospholipids and more (P<0.05) TGs than did that of the coconut oil or sunflower oil group. There was no difference in VLDL-IDL lipid composition between the coconut oil and sunflower oil diets.
In wild-type mice, the ingestion of a single bolus of sunflower oil was associated with a significant increase in serum TG concentrations that peaked 2 hours postprandially (Figure 3⇓). This peak was followed by a progressive decrease in TG concentrations to reach baseline values 4 hours after fat ingestion. Two-way ANOVA showed a statistically significant interaction between diet and time (P<0.013). In apoE-deficient mice, the increase in postprandial TGs after sunflower oil load was similar to that observed in the wild-type animals. Two-way ANOVA showed a statistically significant interaction between diet and time (P<0.006), suggesting a different response to diet in apoE-deficient mice. The TGs concentrations observed in fish oil–fed apoE-deficient mice remained higher than those observed in sunflower oil–fed animals at 3, 5, and 7 hours after the fat load (P<0.05).
Two-way ANOVA showed no evidence of a statistically significant interaction between diet and genotype, suggesting a similar effect of diet in both wild-type and apoE-deficient mice (Table 1⇑; please see http://atvb.ahajournals.org). In both groups, the TG PRs were low, intermediate, and high (P<0.0001) in animals fed fish oil, sunflower oil, and coconut oil, respectively.
In Vitro TRL Lipolytic Rate
In wild-type mice, in vitro LPL-mediated fatty acid release from TRLs was lower (ANOVA interaction of diet×TRLs, P<0.0001; main effect of diet, P<0.0001) in fish oil–fed animals than in those fed coconut oil or sunflower oil (Figure 4⇓). In apoE-deficient mice, fatty acid release was lower (ANOVA interaction of diet×TRLs, P<0.0001; main effect of diet, P<0.0001) in fish oil–fed mice than in sunflower oil– and coconut oil–fed mice.
In wild-type mice, the injection of heparin resulted in a reduction of total plasma TGs from baseline to an average of 0.2 g/L in the 3 dietary groups (Table 2⇓). Because baseline TG values were lower in fish oil–fed mice than in the other dietary groups, the absolute and relative differences were smaller (P<0.0001) in the fish oil group than in the coconut oil or sunflower oil groups. In apoE-deficient mice, the injection of heparin reduces plasma TGs from baseline to 0.21, 0.22, and 2.61 g/L in the coconut oil–, sunflower oil–, and fish oil–fed mice, respectively. The absolute TG difference between baseline and postheparin TG concentrations was greater (P<0.0001) and the relative difference was lower (P<0.0001) in fish oil–fed mice than in coconut oil– or sunflower oil–fed animals.
Earlier studies consistently demonstrated that a fish oil diet decreases TG synthesis and VLDL PRs. Thus, the goal of the present study was to assess the contribution of decreased TG synthesis and of apoE in the mediation of the TRL-lowering effect of fish oil. The results indicated that decreased TG synthesis is not sufficient to lower circulating TG concentrations and that apoE is necessary for fish oil to reduce TG concentrations in mice.
Fish oil supplementation in wild-type animals was associated with lower TG concentrations than was coconut oil and sunflower oil supplementation. In contrast, a fish oil diet in apoE-deficient mice resulted in a remarkable elevation of plasma TGs compared with the coconut oil and sunflower oil diets. Thus, fish oil increased, whereas sunflower oil decreased, VLDL TGs in the apoE-deficient mice, indicating major differences in the metabolic properties of these oils. In agreement with this hypothesis, several studies have shown that (n-6) and (n-3) fatty acids have different molecular impacts on the regulation of major enzymes of lipid metabolism.12 Moreover, electron spin resonance and fluorescence polarization studies have demonstrated that lipoproteins isolated from animals fed oils enriched in (n-6) and (n-3) fatty acids present different physicochemical properties.13 The lack of apoE in the transgenic mice appears to exacerbate all of these metabolic and chemical differences.
In the present study, isocaloric diets that contained 20% wt/wt fat were chosen to obtain rapid and clear lipid differences among diets. Comparable results were obtained with diets less enriched with fish oil, suggesting that the effect of fish oil was not related to the amount of oil provided in the diet (G. Asset et al, unpublished results). Thus, the findings of elevated TRL concentrations in fish oil–fed apoE-deficient mice, but not in wild-type animals with the same genetic background, suggest that apoE is necessary for fish oil to exert its TG-lowering effect. The apoE serves as an anchoring system to the cellular matrix for TRLs subsequently lipolysed by LPL.14 It also is a major ligand to cell receptors. Our findings suggest that apoE-mediated TRL clearance has an important role in the TG-lowering properties of fish oil.
Fish oil decreases hepatic TG PRs to the same extent in wild-type and apoE-deficient mice. These results are in agreement with previous studies that showed fish oil reduces TG synthesis in cultured hepatocytes15 and in isolated liver.16 The decrease in TG synthesis and VLDL production has been attributed to a reduced activity of the major enzymes of TG synthesis and to the stimulation of endogenous fatty acid oxidation, resulting in a lesser availability of fatty acids for TG formation.17 18 In this respect, apoE-deficient mice appear to react to fish oil supplementation as do wild-type mice and other animal models. Earlier studies by Kuipers et al19 showed that TG PRs are lower in apoE-deficient mice than in wild-type animals. Differences in experimental conditions such as the amount or type of fat in the diet may explain the lack of statistical significance between wild-type and apoE-deficient mice in the present study. Despite a clear decrease in TG synthesis rate, plasma TG concentrations were higher in fish oil–fed apoE-deficient mice than in the other dietary groups, suggesting that lowering TG PRs with fish oil is not sufficient to decrease TG concentrations in mice.
In apoE-deficient mice, but not in wild-type animals, fish oil treatment resulted in VLDL enriched in TGs, suggesting a defect in VLDL lipolysis. Theoretically, a decrease in LPL activity could explain VLDL TG enrichment. Previous studies, however, on the effect of fish oil on LPL mass and activity have been inconclusive;20 21 22 LPL activity was found to increase,20 remain unaffected,21 or decrease22 with fish oil treatment. Alternatively, we tested the hypothesis that TRLs from fish oil–fed apoE-deficient mice could be more resistant to LPL action than those from other oils. This hypothesis was supported by the finding that TRLs obtained after a single bolus of fish oil were less efficiently hydrolyzed than were TRLs obtained after a sunflower oil or coconut oil bolus. These results are consistent with earlier studies with fish oil emulsions23 or rat chylomicrons enriched with eicosapentaenoic acid and docosahexaenoic acid.24 However, the greater resistance to LPL-mediated lipolysis was observed with TRLs obtained from both wild-type and apoE-deficient mice, indicating an effect related to fish oil fatty acid composition but independent of the genotype.
From these results, the mechanism by which fish oil increases TG concentrations in apoE-deficient mice can be postulated. Fish oil decreased VLDL TG susceptibility to LPL in both wild-type and apoE-deficient mice. In apoE-deficient mice. this results in a major defect of lipoprotein clearance due to the absence of functional systems of TRL anchoring to endothelial cells and to the lack of remnant uptake by the liver. In wild-type mice, the presence of apoE facilitates the interaction between VLDL and endothelial cells to a point that overcomes the lower susceptibility to LPL-mediated lipolysis of fish oil–enriched TRLs, resulting in a normal processing of TRLs. The hypothesis that the impairment of TRL lipolysis by fish oil contributes to the defect in TG metabolism is supported by the observation that stimulation of lipolysis by heparin partially improves TG profile in fish oil–fed apoE-deficient mice. Alternatively, several studies have shown that fish oil treatment affects LDL receptor activity in rats25 26 and that the affinity of LDL toward cellular receptors is altered by fish oil feeding.27 28 Because the LDL-receptor pathway has an important role in the mediation of chylomicron remnant clearance in mice,29 a decrease in LDL receptor activity after fish oil feeding could very likely result in the accumulation of VLDL. Additional studies are necessary to evaluate this hypothesis.
In conclusion, the results of the present study indicate that decreased TG synthesis is not sufficient for fish oil to lower circulating TG concentrations in mice. Moreover, apoE is necessary for fish oil to lower TG concentrations, indicating a critical role of apoE in the plasma TG–lowering properties of fish oil.
This work was supported by grant Aliment 2000 96 G0182 from the Ministère de l’Enseignement Supérieur et de la Recherche and by an unrestricted grant from the Institut APPERT. Other major contributors were INSERM and the Institut Pasteur de Lille. We thank the Société BERTIN for kindly providing most of the oils for the study and Mrs Foster for editorial assistance.
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- ↵Harris WS. n-3 fatty acids and serum lipoproteins: animal studies. Am J Clin Nutr. 1997;65:1611S–1616S.
- ↵Brown AM, Castle J, Hebbachi AM, Gibbons GF. Administration of n-3 fatty acids in the diets of rats or directly to hepatocyte cultures results in different effects on hepatocellular apoB metabolism and secretion. Arterioscler Thromb Vasc Biol. 1999;19:106–114.
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- ↵Dallongeville J, Boulet L, Davignon J, Lussier-Cacan S. Fish oil supplementation reduces beta-very low density lipoprotein in type III dysbetalipoproteinemia. Arterioscler Thromb. 1991;11:864–871.
- ↵Brousseau T, Clavey V, Bard JM, Fruchart JC. Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles. Clin Chem. 1993;39:960–964.
- ↵Li X, Catalina F, Grundy SM, Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins. J Lipid Res. 1996;37:210–220.
- ↵Wassall SR, McCabe RC, Ehringer WD, Stillwell W. Effects of dietary fish oil on plasma high density lipoprotein: electron spin resonance and fluorescence polarization studies of lipid ordering and dynamics. J Biol Chem. 1992;267:8168–8174.
- ↵Mahley RW, Ji ZS. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J Lipid Res. 1999;40:1–16.
- ↵Brown AM, Baker PW, Gibbons GF. Changes in fatty acid metabolism in rat hepatocytes in response to dietary n-3 fatty acids are associated with changes in the intracellular metabolism and secretion of apolipoprotein B-48. J Lipid Res. 1997;38:469–481.
- ↵Moir AM, Park BS, Zammit VA. Quantification in vivo of the effects of different types of dietary fat on the loci of control involved in hepatic triacylglycerol secretion. Biochem J. 1995;308:537–542.
- ↵Harris WS, Lu G, Rambjor GS, Walen AI, Ontko JA, Cheng Q, Windsor SL. Influence of n-3 fatty acid supplementation on the endogenous activities of plasma lipases. Am J Clin Nutr. 1997;66:254–260.
- ↵Nozaki S, Garg A, Vega GL, Grundy SM. Postheparin lipolytic activity and plasma lipoprotein response to omega-3 polyunsaturated fatty acids in patients with primary hypertriglyceridemia. Am J Clin Nutr. 1991;53:638–642.
- ↵Montalto MB, Bensadoun A. Lipoprotein lipase synthesis and secretion: effects of concentration and type of fatty acids in adipocyte cell culture. J Lipid Res. 1993;34:397–407.
- ↵Oliveira FL, Rumsey SC, Schlotzer E, Hansen I, Carpentier YA, Deckelbaum RJ. Triglyceride hydrolysis of soy oil vs fish oil emulsions. J Parenteral Enteral Nutr. 1997;21:224–229.
- ↵Lindsey S, Pronczuk A, Hayes KC. Low density lipoprotein from humans supplemented with n-3 fatty acids depresses both LDL receptor activity and LDLr mRNA abundance in HepG2 cells. J Lipid Res. 1992;33:647–658.
- ↵Linga V, Leight MA, St, Parks JS. Dietary fish oil modification of cynomolgus monkey low density lipoproteins results in decreased binding and cholesteryl ester accumulation by cultured fibroblasts. J Lipid Res. 1993;34:769–778.
- ↵Ishibashi S, Perrey S, Chen Z, Osuga Ji, Shimada M, Ohashi K, Harada K, Yazaki Y, Yamada N. Role of the low density lipoprotein (LDL) receptor pathway in the metabolism of chylomicron remnants: a quantitative study in knockout mice lacking the LDL receptor, apolipoprotein E, or both. J Biol Chem. 1996;271:22422–22427.