Triacylglycerol-rich lipoproteins derived from healthy donors fed different olive oils modulate cytokine secretion and cyclooxygenase-2 expression in macrophages: the potential role of oleanolic acid.

PURPOSE: Current evidence suggests that consumption of virgin olive oil (VOO) helps to protect against the development of atherosclerosis and that minor components such as oleanolic acid contribute to this effect. In this study, the effects of triacylglycerol-rich lipoproteins (TRLs) derived from olive oil on inflammatory processes in macrophages and how they are modulated by oleanolic acid was investigated. METHODS: TRLs isolated from healthy volunteers 2 and 4 h after a test meal containing VOO, pomace olive oil (POO) (the second pressing of olive oil, enriched in minor components) or POO enriched with oleanolic acid (OPOO) were incubated with macrophages derived from the human monocyte cell line, THP-1. RESULTS: All types of TRLs caused a decrease of about 50% in the secretion of monocyte chemoattractant protein-1 (MCP-1) by the cells. Interleukin (IL)-6 secretion was also significantly decreased by 2 and 4 h VOO TRLs and by 4 h OPOO TRLs. In contrast, increased IL-1ß secretion was observed with all 2 h TRL types, and increased tumour necrosis factor-α (TNF-α) production with 2 h VOO and POO, but not OPOO, TRLs. TRLs isolated after 4 h, however, had no significant effects on TNF-α secretion and increased IL-1ß secretion only when they were derived from VOO. Cyclooxygenase-2 (COX-2) mRNA expression was strongly down-regulated by all types of TRLs, but protein expression was significantly depressed only by 4 h OPOO TRLs. CONCLUSION: These findings demonstrate that TRLs derived from olive oil influence inflammatory processes in macrophages and suggest that oleanolic acid may have beneficial effects.

Influence of chylomicron remnants on human monocyte activation in vitro.

BACKGROUND AND AIMS: Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR), the lipoproteins which carry dietary fats in the blood, cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased, and clearance of CMR from blood may be delayed, however, whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here, the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model. METHODS AND RESULTS: Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment, and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast, exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1. CONCLUSION: Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis, and suggest that this may reflect direct interaction with circulating blood monocytes.

Introduction to the Biochemical Society Focused Meeting on Diet and Cardiovascular Health: chylomicron remnants and their emerging roles in vascular dysfunction in atherosclerosis.

Although it has been known for many years that dietary lipids influence the development of atherosclerosis, in the past this has been attributed to their effects on blood cholesterol levels. Recent work, however, has shown that CMRs (chylomicron remnants), the lipoproteins which carry dietary lipids in the blood, potentially have a direct role in initiating atherogenesis by influencing vascular function. The Diet and Cardiovascular Health: Chylomicron Remnants and Their Emerging Roles in Vascular Dysfunction in Atherosclerosis Meeting focused attention on studies which have shown that CMRs influence vascular function via interactions with cells of the artery wall, including endothelial cells and macrophages, and also highlighted the part played by CMRs in the development of premature atherosclerosis in conditions such as the metabolic syndrome, which are an increasing cause of heart disease in developed countries.

Comparison of the effects of dietary saturated, mono-unsaturated and polyunsaturated fatty acids on very-low-density lipoprotein secretion when delivered to hepatocytes in chylomicron remnant-like particles.

The effect of chylomicron remnant-like particles (CRLPs) enriched in saturated, mono-unsaturated or n-6 polyunsaturated fatty acids (derived from palm, olive or corn oil, respectively) on the secretion of VLDL (very-low-density lipoprotein) by rat hepatocytes in culture was investigated. CRLPs were incubated with cultured hepatocytes for 5 h. The medium was then removed and the secretion of cholesterol and triacylglycerol (TAG) into the whole medium during the following 16 h was determined. After exposure of the cells to olive oil as compared with corn and palm oil CRLPs, secretion of TAG into the medium was decreased. The TAG content of the cells was also lower in experiments with olive oil as compared with corn oil CRLPs. The levels of apoB48 (apolipoprotein B48) found in the medium remained unchanged after the exposure of the cells to the different types of remnants. These findings indicate that the type of fat in the diet directly affects VLDL lipid secretion on delivery to the liver in chylomicron remnants.

The induction of macrophage foam cell formation by chylomicron remnants.

The accumulation of foam cells in the artery wall causes fatty streaks, the first lesions in atherosclerosis. LDL (low-density lipoprotein) plays a major role in foam cell formation, although prior oxidation of the particles is required. Recent studies, however, have provided considerable evidence to indicate that CMRs (chylomicron remnants), which carry dietary lipids in the blood, induce foam cell formation without oxidation. We have shown that CMRs are taken up by macrophages and induce accumulation of both triacylglycerol and cholesterol, and that the rate of uptake and amount of lipid accumulated is influenced by the type of dietary fat in the particles. Furthermore, oxidation of CMRs, in striking contrast with LDL, inhibits, rather than enhances, their uptake and induction of lipid accumulation. In addition, the lipid accumulated after exposure of macrophages to CMRs is resistant to efflux, and this may be due to its sequestration in lysosomes. These findings demonstrate that CMRs induce pro-atherogenic changes in macrophages, and that their effects may be modulated by dietary factors including oxidized fats, lipophilic antioxidants and the type of fat present.

Dietary fats induce human monocyte activation in vitro.

In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is amplified, and migration of monocytes into the walls of the aorta and large arteries is increased, due partly to de novo expression or activation of monocyte adhesion molecules. Although there is increasing evidence that CMRs (chylomicron remnants) are strongly atherogenic, the outcomes of interactions between blood monocytes and circulating CMRs are not known. Here, we have studied the effects of CRLPs (CMR-like particles) on THP-1 human monocyte oxidative burst. The particles induced a significant increase in reactive oxygen species within 1 h, which persisted for 24 h. We suggest that monocyte-CMR interactions may be important in early atherosclerosis when many activated monocytes are found in susceptible areas of the artery wall.

Hypercholesterolaemia alters the responses of the plasma lipid profile and inflammatory markers to supplementation of the diet with n-3 polyunsaturated fatty acids from fish oil.

BACKGROUND: The influence of supplementing the diet with long-chain n-3 polyunsaturated fatty acids (PUFA) from fish oil on plasma lipids and lipid peroxides and the production of pro-inflammatory mediators in normolipidaemic and hypercholesterolaemic rats were studied. MATERIALS AND METHODS: Rats were divided into four groups and fed one of the following diets: a control diet (containing 4% corn oil); an n-3 PUFA diet [containing 4% eicospentaenoic (EPA) + docosahexaenoic (DHA)]; a hypercholesterolaemic diet (HCH); or a HCH + n-3 PUFA diet over a 4-week period. Plasma lipids, lipid peroxides, cytokines [tumour necrosis factor (TNF)alpha, interleukin (IL)-6, interferon (IFN)gamma] and mRNA for hepatic nuclear factor-4alpha (HNF4alpha) were determined. RESULTS: Plasma triglyceride (TG), but not cholesterol, levels were decreased by the n-3 PUFA as compared with the control diet (P < 0.001), but the addition of n-3 PUFA to the HCH diet decreased both the TG (P < 0.01) and cholesterol (P < 0.05) concentrations. Plasma lipid peroxides and expression HNF4alpha mRNA were increased by n-3 PUFA in the normolipidaemic (P < 0.05), but not in the hyperlipidaemic rats. Compared with the control diet group, plasma concentrations of TNFalpha and IL-6 were increased in the n-3 PUFA (P < 0.05) and HCH diet (P < 0.05, P < 0.01, respectively) groups, but not in animals given the HCH + n-3 PUFA diet, whereas IFNgamma levels were increased in hypercholesterolaemia (P < 0.05), but were unaffected by n-3 PUFA. CONCLUSION: These results demonstrate that the major effect of fish oil n-3 PUFA is to lower the TG levels in both normo- and hyperlipidaemia. Furthermore, in the hypercholesterolaemic state, fish oil n-3 PUFA induces additional beneficial changes in the immune and peroxidation responses.

Direct interaction of dietary lipids carried in chylomicron remnants with cells of the artery wall: implications for atherosclerosis development.

The development of atherosclerotic lesions in the artery wall is a complex process involving the endothelium, lipid engorged macrophages (foam cells) and smooth muscle cells. In recent years it has become clear that chylomicron remnants, the lipoproteins which carry lipids of dietary origin in the blood, are strongly atherogenic, and there is increasing evidence to indicate that this is due to direct interaction of the remnant particles with cells of the artery wall. Chylomicron remnants have been demonstrated to inhibit endothelium dependent vasorelaxation and to activate signal transduction pathways associated with inflammation in cultured endothelial cells. They have also been shown to be taken up by smooth muscle cells and macrophages, and to cause the extensive lipid accumulation associated with foam cell formation, as well as influencing the expression of key genes regulating macrophage lipid uptake and metabolism. Furthermore, oxidative modification of the remnant particles is not required for many of these effects. Chylomicron remnants, therefore, have multiple direct effects on three major cell types of the arterial wall which are likely to promote the development of atherosclerotic lesions. These effects may be modulated by various lipids carried by the particles, including the type of fat (saturated or unsaturated or oxidised fat), micronutrients such as vitamins and carotenoids which have antioxidant properties, and orally administered lipophilic drugs. Delayed clearance of chylomicron remnants from the blood occurs in a number of dyslipidemias associated with premature atherosclerosis development, and the potentially atherogenic effects of the particles would clearly be enhanced in these circumstances. Thus, elucidation of the mechanisms involved will aid in the identification of new drug targets which may be particularly useful for these conditions.

Chylomicron-remnant-like particles modify production of vasoactive mediators by endothelial cells.

Endothelial-cell dysfunction is a critical initiating event in the pathogenesis of atherosclerosis. Although there is evidence to suggest that chylomicron remnants (CMRs), lipoproteins derived from the diet, influence endothelial-cell function to generate a pro-atherogenic phenotype, the mechanisms involved remain undefined. We have examined the effects of CMR-like particles (CMR-LPs) on human endothelial-cell function, focusing on the cyclo-oxygenase (COX) and nitric oxide synthase (NOS) pathways. CMR-LPs strongly enhanced the expression of the inducible cyclo-oxygenase COX-2 and increased prostacyclin synthesis in a biphasic manner. Studies with the COX-2-selective inhibitor NS-398 confirmed the COX-2 dependency of the later increase in prostanoid production. Pre-incubation with CMR-LPs reduced basal and thrombin-stimulated cGMP generation, whereas expression of endothelial NOS was not modified by remnant treatment.

The fatty acid composition of chylomicron remnants influences their propensity to oxidate.

BACKGROUND AND AIM: Although the replacement of saturated with unsaturated dietary fat has been advocated as a means of reducing the risk of cardiovascular disease, diets high in polyunsaturated fatty acids (PUFAs) may increase lipid peroxidation, thus contributing to the pathogenesis of atherosclerosis. As the susceptibility of individual fatty acids to oxidation directly depends on their degree of unsaturation, and the oxidative modification of lipoproteins may be an important determinant of atherogenesis, the aim of this study was to evaluate the susceptibility to auto-oxidation and copper-mediated oxidation of chylomicron remnants (CMRs) enriched in n-3 or n-6 PUFA. METHODS AND RESULTS: The remnants were prepared in vitro from chylomicrons obtained from rats given an oral dose of fish or corn oil, using rat plasma containing lipoprotein lipase. Their propensity to oxidate and the extent of the oxidation were estimated by measuring the formation of conjugated dienes and the detrimental products of lipid peroxidation. The results showed that: 1) the corn oil CMRs contained a relatively high proportion of n-6 PUFA (mainly linoleic acid), whereas the fish oil CMRs contained more n-3 PUFA, mainly eicosapentanoic and docosahexaenoic acids; 2) n-3-rich CMRs have a significantly lower propensity to oxidate than n-6-rich CMRs despite their 50% lower alpha-tocopherol content and 40% higher unsaturation index. CONCLUSION: Our data indicate that the precise allocation of n-3 PUFA within the lipid core of CMRs may play a pivotal role in lowering the susceptibility to oxidation of fish CMRs by overcoming the effects of unfavourable alpha-tocopherol concentration. Eating n-3 rather than n-6 PUFAs seems to make CMRs more resistant against free radical attack, which may contribute to attenuating their potential atherogenic properties.

Influence of the fatty acid composition of lipids in chylomicron remnants derived from fish or corn oil on the lipid profile of cultured rat hepatocytes.

The aim of this work was to characterise the lipid and fatty acid composition of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (PUFA) and to investigate their influence on the fatty acid profiles of the lipids of rat hepatocytes cultured in monolayers. Chylomicrons were prepared from the lymph collected from the thoracic duct of rats given an oral dose of fish or corn oil (high in n-3 and n-6 PUFA, respectively), and remnants were prepared in vitro from such chylomicrons using rat plasma containing lipoprotein lipase. The fatty acids predominating in the oils abounded also in their respective chylomicrons and remnants, especially in triacylglycerols. Chylomicrons as well as remnants contained small amounts of phospholipids and long-chain PUFA that were minor in, or absent from, the dietary oils, evidently provided by the intestinal epithelium. The incubation of hepatocytes for 6 h, with either n-3 or n-6 PUFA-rich remnants (0.25-0.75 mM triacylglycerol) resulted in a dose-dependent increase in the amount of triacylglycerols and phospholipids in the cells, which was not affected further by increasing the incubation time to 19 h. Whereas hepatocyte triacylglycerols mostly incorporated the PUFA predominating in each remnant type, the fatty acid profile of cell phospholipids was virtually unchanged. In addition, irrespective of whether they were enriched in n-3 or n-6 PUFA, remnants promoted a relative decrease in the amount of cholesteryl esters, a minor hepatocyte lipid class poor in PUFA. The results demonstrate that the hepatocyte fatty acid profile is modulated in a lipid-class specific way by the amount and type of dietary PUFA delivered to cells in chylomicron remnants.

Lipid synthesis in macrophages derived from the human cell line THP-1: modulation of the effects of native and oxidized chylomicron-remnant-like particles by oestrogen.

The effects of native and oxidized chylomicron remnants on the synthesis of cholesteryl ester and triacylglycerol in macrophages, and the way that this is influenced by exposure of the cells to oestrogen, was investigated using the human monocyte cell line THP-1 and chylomicron-remnant-like particles containing human apolipoprotein E (CRLPs). Synthesis of the lipids was measured by the incorporation of [(3)H]oleate into cholesteryl ester and triacylglycerol. CRLPs (5-40 microgram of cholesterol/ml) containing either trilinolein or triolein as the triacylglycerol component caused a dose-dependent decrease in cholesteryl ester formation, while triacylglycerol production was unchanged. After oxidation of the CRLPs, the level of thiobarbituric acid-reactive substances was increased by 6.3-fold and 2.2-fold in particles containing trilinolein and triolein respectively. Furthermore, CRLPs containing oxidized trilinolein lost their ability to down-regulate cholesterol esterification, while CRLPs containing oxidized triolein did not. Both types of oxidized CRLPs decreased triacylglycerol synthesis. Treatment of the macrophages with 17beta-oestradiol caused increases of approx. 94% and 34% in the synthesis of cholesteryl ester and triacylglycerol respectively in the absence of CRLPs. The differences between control and oestrogen-treated cells were abolished, however, when CRLPs (40 microgram of cholesterol/ml) were added to the incubations. In addition, in contrast with their lack of effect in control cells, CRLPs containing oxidized trilinolein decreased cholesterol esterification in oestrogen-treated cells by approx. 48%. These findings with CRLPs suggest that chylomicron remnants have significant effects on cholesteryl ester and triacylglycerol synthesis in macrophages, which may be modulated both by the oxidation state of the particles and by oestrogen.

Comparison of the effects of dietary n-3 and n-6 polyunsaturated fatty acids on very-low-density lipoprotein secretion when delivered to hepatocytes in chylomicron remnants.

The effects of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (derived from fish or corn oil respectively) on the secretion of very-low-density lipoprotein (VLDL) lipid and apolipoprotein B (apoB) by rat hepatocytes in culture was investigated. Remnants were prepared in vivo from chylomicrons obtained from rats given an oral dose of fish or corn oil and incubated with cultured hepatocytes for up to 16 h. The medium was then removed and the secretion of cholesterol and triacylglycerol into the whole medium or the rho<1.050 g/ml fraction during the following 7-24 h was determined. After exposure of the cells to fish-oil as compared with corn-oil remnants, secretion of both cholesterol and triacylglycerol into the whole medium was decreased by 25-35%, and secretion into the rho<1.050 g/ml fraction was decreased by 20-25%. In addition, the levels of apoB48 found in the rho<1.050 g/ml fraction were significantly lower in cells treated with fish-oil rather than corn-oil remnants, although the levels of apoB100 remained unchanged. The expression of mRNA for apoB, as determined by reverse-transcriptase PCR, however, was not significantly changed after exposure of the cells to both types of remnants. These results demonstrate that the effects of dietary n-3 polyunsaturated fatty acids in depressing hepatic VLDL secretion occur directly when they are delivered to the liver from the intestine in chylomicron remnants, and that the secretion, but not the synthesis, of apoB is targeted.

Metabolism of chylomicron cholesterol is delayed by estrogen. An in vivo study in the rat.

In order to test the effects of estrogen on the clearance of cholesterol of dietary origin from the blood and its elimination from the body via the bile in an in vivo animal model, the fate of radioactivity from intravenously injected [3H]cholesterol-labeled chylomicrons was investigated in the rat. The labeled lipoproteins were administered intrajugularly to male rats previously given 17alpha ethinyl estradiol or the vehicle only, and the removal of the radioactivity from the blood and its uptake by the liver and secretion into bile was determined. Experiments were carried out in animals with or without prior drainage (20 hr) of the pool of bile acids in the enterohepatic circulation, to take account of the different demands of the liver for cholesterol in the two conditions. In rats without biliary drainage, estrogen treatment decreased the rate of removal of radioactivity from the blood by about 30% and the recovery of cholesterol in the liver by about 50% in the first 30 min after injection of the labeled chylomicrons. After biliary drainage, however, the recovery of label in the liver after 90 min was similar in estrogen-treated and control animals, although its secretion into bile was markedly reduced in the estrogen-treated group (total biliary secretion in 90 min was 26% of the value found in control rats). In addition, the apolipoprotein E (aopE) content of the serum total lipoproteins was markedly reduced by estrogen. These results provide direct evidence indicating that estrogen retards the elimination of dietary cholesterol from the body via the bile in the rat, and this is likely to be mainly due to a reduced level of apoE in chylomicrons. In view of this, we suggest that the hypothesis that estrogen increases the hepatic uptake of chylomicron cholesterol, and its excretion in the bile during contraceptive and hormone replacement therapy should be re-examined.

The internal redox balance of the cells influences the metabolism of lipids of dietary origin by J774 macrophages: implications for foam cell formation.

The effects of chylomicron remnants on lipid accumulation in J774 macrophages, and the incorporation of radioactivity from remnant lipids radiolabelled with [3H]oleate into cellular lipids was investigated. After 24 h of incubation with chylomicron remnants, there was considerable accumulation of lipid within the cells as assessed by staining with oil red O, indicating that the particles induce the formation of foam cells. Fatty acids released from the radiolabelled remnant lipids after uptake were found to be incorporated into cellular triacylglycerol (52%), phospholipid (37%) and cholesteryl ester (8%), but at higher remnant concentrations, the proportion used for triacylglycerol formation increased (up to 73%). When the macrophages were shifted into a pro-oxidising or pro-reducing state by incubation (24 h) with CuSO4 (2.5 microM) or N-acetylcysteine (5 mM), respectively, the incorporation of [3H]oleate from remnant lipid into cellular triacylglycerol and phospholipid was increased by 20-30% in the more oxidised as compared with the more reduced cells. These findings indicate that exposure of J774 macrophages to chylomicron remnants leads to the accumulation of lipid within the cells, and that this process is enhanced by pro-oxidising conditions. We conclude, therefore, that both lipids of dietary origin and the redox balance within macrophages may have a significant role in the induction of foam cell formation.

The fatty acid composition of chylomicron remnants influences their binding and internalization by isolated hepatocytes.

The binding and internalization of (125)I-labelled chylomicron remnants derived from palm, olive, corn, or fish oil (rich in saturated, monounsaturated, n-6, or n-3 polyunsaturated fatty acids, respectively) by hepatocytes from rats fed a low-fat diet or a diet supplemented with the corresponding fat for 21 days was investigated. In hepatocytes from rats fed the low-fat diet, the association of radioactivity with the cells at 4 degrees C (a measure of initial binding only) was similar with all types of remnants tested, but was more rapid at 37 degrees C (a measure of binding plus internalization) when fish oil, as compared to olive, corn or palm oil remnants, was used, and similar differences in the internalization of the particles were observed. In contrast, when hepatocytes from rats fed the fat-supplemented diets were used, the rate of association at 37 degrees C of remnants with cells from rats fed palm, corn or fish oil was similar, and higher than that found with cells from animals fed olive oil, and in this case these differences were mainly due to changes in the binding of the particles to the cells at 4 degrees C. Both excess low-density lipoprotein (LDL), which inhibits remnant uptake by the LDL receptor, and lactoferrin, which blocks the LDL receptor-related protein (LRP), were found to decrease the association of the remnants with cells from rats fed the low-fat and high-fat diets. However, in hepatocytes from animals given the low-fat diet, most of the differences between the various types of particle were retained in the presence of lactoferrin, but abolished in the presence of LDL. In contrast, in cells from rats fed the high-fat diets, the differences were reduced by both lactoferrin and LDL. These findings demonstrate that the hepatic uptake of chylomicron remnants is influenced both by the fatty acid composition of the particles, and by longer-term adaptive changes in liver tissue, and suggest that the former effects are mediated mainly by the LDL receptor, while the latter may involve both the LDL receptor and the LRP.

The differential hepatic uptake of chylomicron remnants of different fatty acid composition is not mediated by hepatic lipase.

The hypothesis that hepatic lipase mediates the differential hepatic uptake of chylomicron remnants of different fatty acid composition, demonstrated in previous work from our laboratory, was tested by investigating the effect of antibodies to the enzyme on the uptake of remnants enriched with saturated or n-3 polyunsaturated fatty acids by the perfused rat liver. After perfusion of rat livers with polyclonal antibodies to rat hepatic lipase raised in rabbits or with rabbit non-immune serum for 15 min, [3H]oleate-labelled chylomicron remnants, derived from chylomicrons of rats given a bolus of either palm (rich in saturated fatty acids) oil or fish (rich in n-3 polyunsaturated fatty acids) oil, were added. The disappearance of radioactivity from the perfusate during 120 min and its recovery in the liver at the end of the experiments were then measured. Although the rabbit anti-rat hepatic lipase antiserum was shown to inhibit hepatic lipase activity by up to 90%, and to bind extensively to hepatic sinusoidal surfaces when added to the perfusate, radioactivity from remnants of chylomicrons from rats given a bolus of fish oil as compared with palm oil disappeared from the perfusate and appeared in the liver more rapidly in the presence both the antiserum and the non-immune serum, and the differences between the uptake of the two types of remnants were similar. We conclude, therefore, that differential interaction with hepatic lipase is not responsible for the differences in the rate of removal of chylomicron remnants of different fatty acid composition from the blood.

Oxidation affects the regulation of hepatic lipid synthesis by chylomicron remnants.

The effects of native and oxidized chylomicron remnants on lipid synthesis in normal and oxidatively stressed liver cells were investigated using MET murine hepatocytes (MMH cells), a nontransformed mouse hepatocyte cell line that maintains a highly differentiated hepatic phenotype in culture. Lipid synthesis was determined by measuring the incorporation of [(3)H]oleate into cholesteryl ester, triacylglycerol, and phospholipid by the cells. The formation of cholesteryl ester and phospholipid was decreased by chylomicron remnants in a dose-dependent manner, while triacylglycerol synthesis was increased. Exposure of MMH cells to mild oxidative stress by incubation with CuSO(4) (2.5 microM) for 24 h led to significantly increased incorporation of [(3)H]oleate into triacylglycerol and phospholipid, but not cholesteryl ester, in the absence of chylomicron remnants. In the presence of the lipoproteins, however, similar effects to those found in untreated cells were observed. Oxidatively modified chylomicron remnants prepared by incubation with CuSO(4) (10 microM, 18 h, 37 degrees C) did not influence cholesteryl ester or phospholipid synthesis in MMH cells, but had a similar effect to that found with native remnants on triacylglycerol synthesis. These findings show that hepatic lipid metabolism is altered by exposure to mild oxidative stress and by lipids from the diet delivered to the liver in chylomicron remnants, and these effects may play a role in the development of atherosclerosis.

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.

The effect of chylomicron remnants derived from fish oil (rich in n-3 polyunsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids) on the formation and hydrolysis of cholesteryl esters in cultured rat hepatocytes was investigated. Hepatocytes were incubated with or without fish or corn oil chylomicron remnants (0.25-0.75 mM triacylglycerol), and the activity of acyl-CoA:cholesterol acyltranferase (ACAT) and cholesteryl ester hydrolases in the cytosol (cCEH) and endoplasmic reticulum (erCEH), and the expression of mRNA for ACAT1, ACAT2 and cCEH, and of enzyme protein for erCEH was determined. Addition of either type of remnants to hepatocyte cultures resulted in a decreased activity of erCEH, cCEH (after 6 and 19 h incubation), and of ACAT (after 6 h only). Hepatocyte levels of mRNA encoding ACAT1 and ACAT2 were not affected by either type of chylomicron remnants after 6 h of incubation, while ACAT2 mRNA levels were down-regulated by fish oil remnants as compared with corn oil remnants, and also with control cells in the long term (19 h). In contrast, cCEH mRNA levels were down-regulated by chylomicron remnants derived from corn oil but not fish oil. The expression of erCEH protein was induced in response to the inhibitory effect of both types of remnants on the activity of the enzyme, with corn oil remnants having a significantly greater effect. These findings demonstrate that dietary polyunsaturated fatty acids when delivered to hepatocytes in chylomicron remnants regulate the activity of the enzymes governing the intracellular cholesteryl ester balance, and suggest that dietary n-3 and n-6 polyunsaturated fatty acids or a metabolite thereof have differential effects on the expression of their genes at the mRNA and post-transcriptional levels.

The fatty acid composition of chylomicrons influences the rate of their lipolysis in vivo.

BACKGROUND AND AIMS: Previous work in our laboratory has shown that chylomicron triacylglycerol is lipolysed in vitro by lipoprotein lipase more rapidly when the particles are enriched with n-6 polyunsaturated as compared to saturated, monounsaturated or n-3 polyunsaturated fatty acids. It is possible, however, that this does not reflect the situation in vivo, where the active enzyme is bound to the vascular endothelium. The aim of the present study is to investigate the effect of the fatty acid composition of chylomicrons on their lipolysis in the rat in vivo. METHODS AND RESULTS: [3H]Oleate-labelled chylomicrons derived from palm, olive, corn or fish oil (enriched in saturated, monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids respectively) containing > 90% of the label in triacylglycerol were injected intravenously into functionally hepatectomised rats and blood samples were taken at time intervals up to 40 min. The radioactivity in serum triacylglycerol decreased significantly more rapidly when corn oil as compared to palm, olive or fish oil chylomicrons were used. Conversely, the radioactivity in serum free fatty acid derived from corn oil chylomicrons showed a faster increase than that derived from the other three types of particles. CONCLUSIONS: These results indicate that chylomicrons enriched with n-6 polyunsaturated fatty acids are converted to chylomicron remnants in vivo more rapidly than those enriched with saturated, monounsaturated or n-3 polyunsaturated fatty acids. This provides a partial explanation for the differential rate of removal from the blood of cholesterol carried in chylomicrons of different fatty acid composition demonstrated in previous work from this laboratory.