Postprandial phase time influences the uptake of TAG from postprandial TAG-rich lipoproteins by THP-1 macrophages.

Postprandial TAG-rich lipoproteins (TRL) can be taken up by macrophages, leading to the formation of foam cells, probably via receptor-mediated pathways. The present study was conducted to investigate whether the postprandial time point at which TRL are collected modulates this process. A meal containing refined olive oil was given to nine healthy young men and TRL were isolated from their serum at 2, 4 and 6 h postprandially. The lipid class and apoB compositions of TRL were determined by HPLC and SDS-PAGE, respectively. The accumulation of lipids in macrophages was determined after the incubation of THP-1 macrophages with TRL. The gene expression of candidate receptors was measured by real-time PCR. The highest concentrations of TAG, apoB48 and apoB100 in TRL were observed at 2 h after the consumption of the test meal. However, excessive intracellular TAG accumulation in THP-1 macrophages was observed in response to incubation with TRL isolated at 4 h, when their particle size (estimated as the TAG:apoB ratio) was intermediate. The abundance of mRNA transcripts in macrophages in response to incubation with TRL was down-regulated for LDL receptor (LDLR), slightly up-regulated for VLDL receptor and remained unaltered for LDLR-related protein, but no effect of the postprandial time point was observed. In contrast, the mRNA expression of scavenger receptors SRB1, SRA2 and CD36 was higher when cells were incubated with TRL isolated at 4 h after the consumption of the test meal. In conclusion, TRL led to excessive intracellular TAG accumulation in THP-1 macrophages, which was greater when cells were incubated with intermediate-sized postprandial TRL isolated at 4 h and was associated with a significant increase in the mRNA expression of scavenger receptors.

Suppression of VLDL secretion by cultured hepatocytes incubated with chylomicron remnants enriched in n-3 polyunsaturated fatty acids is regulated by hepatic nuclear factor-4alpha.

Dietary n-3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4alpha (HNF-4alpha) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n-3 PUFA) oil were incubated with cultured rat hepatocytes (24h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4alpha, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4alpha protein and mRNA levels were significantly decreased by CRLPs enriched in n-3 PUFA, but not SFA, and the expression of mRNA for HNF-4alpha target genes, including HNF-1alpha, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n-3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n-3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4alpha.

Differential influence of different dietary fatty acids on very low-density lipoprotein secretion when delivered to hepatocytes in chylomicron remnants.

The influence of dietary fats carried in chylomicron remnants on the hepatic secretion of very low-density lipoprotein (VLDL) was investigated using chylomicron remnant-like particles (CRLPs) and cultured rat hepatocytes as the experimental model. Chylomicron remnant-like particles containing triacylglycerol (TG) from palm, olive, or corn (enriched in saturated, monounsaturated, or n-6 polyunsaturated fatty acids) oil, respectively, were incubated with cultured hepatocytes for 5 hours. The medium was then removed and replaced with medium without CRLPs; and the secretion of TG, cholesterol, and apolipoprotein B48 during the following 16 hours was determined. Secretion of TG into the d less than 1.050-g/mL fraction containing VLDL was unaffected by olive CRLPs, but was significantly increased in cells exposed to palm or corn CRLPs in comparison with both olive CRLPs and control incubations without CRLPs. Secretion of apolipoprotein B48, however, was not changed by any of the CRLP types. Apolipoprotein B messenger RNA levels were decreased by olive and corn CRLPs, and 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA abundance was increased by palm CRLPs; but expression of other genes involved in the regulation of VLDL secretion was unaffected. These findings demonstrate that CRLPs enriched in saturated fatty acids or n-6 polyunsaturated fatty acids increase the secretion of TG in VLDL, possibly because of the secretion of larger particles, whereas those enriched in monounsaturated fatty acids have no effect. Thus, different dietary fats have differential effects on VLDL secretion directly when delivered to the liver in chylomicron remnants.

Differential modulation of hepatic very low-density lipoprotein secretion by triacylglycerol-rich lipoproteins derived from different oleic-acid rich dietary oils.

Minor components from dietary oils can modulate the atherogenic response of the TAG-rich lipoproteins (TRL) in which they are transported. In the present study we investigated the influence of TRL isolated from man after the intake of oleic acid-rich oils with different minor component compositions on VLDL secretion by rat primary hepatocytes. TRL were isolated from nine men after the intake of meals enriched with high-oleic sunflower oil (HOSO) or virgin olive oil (VOO) or VOO enriched with minor components (EVO). TRL were incubated with rat primary hepatocytes and the lipid accumulation was analysed in the cells and the secreted VLDL. The expression of genes for proteins related to hepatic lipid metabolism and VLDL production was also measured. Incubation of hepatocytes with TRL derived from HOSO as compared to VOO led to lower intracellular lipid accumulation and VLDL production despite higher mRNA expression for diacylglycerol-acyltransferase, microsomal TAG transfer protein, apoB and PPARalpha. When TRL derived from EVO were used there were no changes in VLDL secretion. These results suggest that incorporation of minor components from dietary high-oleic oils into TRL modulates the effect of these atherogenic particles on VLDL secretion.

Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes.

The influence of the oxidative state of chylomicron remnants (CMR) on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line, THP-1, during foam cell formation was investigated using chylomicron-remnant-like particles (CRLPs) at 3 different levels of oxidation. The oxidative state of CRLPs was varied by exposure to CuSO(4) (oxCRLPs) or incorporation of the antioxidant, probucol (pCRLPs) into the particles. oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs, and their rate of uptake was lower, while pCRLPs caused more lipid accumulation and were taken up faster. Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein (LDL) receptor related protein (80-90%), LDL receptor (-30-40%), CD36 (-40%) and phagocytosis (-35-40%) was blocked using lactoferrin, excess LDL, anti-CD36 and cytochalasin D, respectively, but blocking scavenger receptors-A or -B1 using poly inosinic acid or excess HDL had no effect. These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages. However, oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages, which occur mainly via the LRP with some contribution from the LDLr, while CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the particles. Thus, the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis.

Fatty acid composition of chylomicron remnant-like particles influences their uptake and induction of lipid accumulation in macrophages.

The influence of the fatty acid composition of chylomicron remnant-like particles (CRLPs) on their uptake and induction of lipid accumulation in macrophages was studied. CRLPs containing triacylglycerol enriched in saturated, monounsaturated, n-6 or n-3 polyunsaturated fatty acids derived from palm, olive, corn or fish oil, respectively, and macrophages derived from the human monocyte cell line THP-1 were used. Lipid accumulation (triacylglycerol and cholesterol) in the cells was measured after incubation with CRLPs for 5, 24 and 48 h, and uptake over 24 h was determined using CRLPs radiolabelled with [3H]triolein. Total lipid accumulation in the macrophages was significantly greater with palm CRLPs than with the other three types of particle. This was mainly due to increased triacylglycerol concentrations, whereas changes in cholesterol concentrations did not reach significance. There were no significant differences in lipid accumulation after incubation with olive, corn or fish CRLPs. Palm and olive CRLPs were taken up by the cells at a similar rate, which was considerably faster than that observed with corn and fish CRLPs. These findings demonstrate that CRLPs enriched in saturated or monounsaturated fatty acids are taken up more rapidly by macrophages than those enriched in n-6 or n-3 polyunsaturated fatty acids, and that the faster uptake rate results in greater lipid accumulation in the case of saturated fatty acid-rich particles, but not monounsaturated fatty acid-rich particles. Thus, dietary saturated fatty acids carried in chylomicron remnants may enhance their propensity to induce macrophage foam cell formation.

Uptake of triacylglycerol-rich lipoproteins of differing triacylglycerol molecular species and unsaponifiable content by liver cells.

The fatty acid composition of dietary oils can modulate the incorporation of triacylglycerol-rich lipoproteins (TRL) into hepatocytes, thus affecting the atherogenicity of these particles. However, nothing is known about the effect of the unsaponifiable fraction of the oils. In the present study, we evaluated the influence of these components on the uptake of TRL by rat primary hepatocytes. TRL were isolated from human serum after the intake of meals enriched in high-oleic sunflower oil (HOSO), virgin olive oil (VOO) or VOO enriched in its own unsaponifiable fraction (EVO). HOSO and HOSO-TRL differed from VOO and EVO and their corresponding TRL in the composition of triacylglycerol molecular species and of the unsaponifiable fraction. Furthermore, the increase in the unsaponifiable fraction of VOO led to changes in the triacylglycerol molecular species in the EVO-TRL. On incubation with hepatocytes, HOSO-TRL were taken up at a faster rate than VOO-TRL or EVO-TRL. In addition, in comparison to VOO-TRL, HOSO-TRL increased the expression of mRNA for the LDL receptor-related protein receptor, which plays an important role in the internalisation of remnant lipoproteins. EVO-TRL also increased LDL receptor-related protein mRNA expression in comparison with VOO-TRL, but this change was not accompanied by a rise in the uptake rate, suggesting that the unsaponifiable fraction of VOO may inhibit LDL receptor-related protein expression or activity post-transcriptionally. In conclusion, TRL from dietary oils with differing triacylglycerol molecular species and unsaponifiable fraction content are taken up by liver cells at different rates, and this may be important in the atherogenicity of these particles.

Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron remnants.

The fate of cholesterol and triacylglycerol taken up and accumulated by macrophages after exposure to chylomicron remnants was investigated using macrophages derived from the human monocyte cell line THP-1 and chylomicron remnant-like particles containing human apolipoprotein (apo) E (CRLPs) as the experimental model. In THP-1 macrophages lipid loaded with CRLPs and incubated with various cholesterol acceptors for 24 h, the mass of cholesterol and cholesteryl ester found in the cells was not changed by HDL, HDL3 or lipid-free ApoA-I, although it was decreased by 38% by ApoA-I-phosphatidylcholine vesicles (ApoA-I-PC). After loading of the macrophages with [3H]cholesterol-labelled CRLPs, only about 5% of the label was effluxed in 24 h in the absence of cholesterol acceptors, and this increased to about 10% with ApoA-I or PC only, and to about 30% with apoA-I-PC. In similar experiments with [3H]triolein, only about 4% of the labelled triacylglycerol taken up by the cells was released into the medium in 24 h, and a large (>60%) and consistent proportion of the intracellular radioactivity remained associated with the triacylglycerol throughout this period. These results suggest that cholesterol and triacylglycerol derived from chylomicron remnants are not readily cleared from macrophages, and this is likely to contribute to the atherogenicity of the remnant lipoproteins.

Cholesterol esterification in human monocyte-derived macrophages is inhibited by protein kinase C with dual roles for mitogen activated protein kinases.

The possible role of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in the stimulation of cholesterol esterification by acetylated low density lipoprotein (acLDL) in human monocyte-derived macrophages (HMDM) was studied. Cholesterol esterification, as assessed by the rate of incorporation of [3H]-oleate into cholesteryl ester, was markedly higher in HMDM incubated with acLDL as compared to native LDL (nLDL). In the presence of the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM), however, the rate of incorporation was reduced by about 50% and 85% in incubations with nLDL and acLDL, respectively. Thus, the difference in the rate of cholesteryl esterification induced by the two types of lipoprotein was abolished by PMA, indicating that PKC activation inhibits the process, and this was confirmed by the finding that the PKC inhibitor calphostin C reversed the PMA-induced inhibition of cholesterol esterification. Incubation of HMDM with PMA was found to cause a considerable increase in the activation of p42/44 extracellular signal-regulated MAP kinases (ERK) and p38 MAP kinases, reaching a maximum at 30 min. In the presence of acLDL, the ERK inhibitor PD98059 decreased cholesterol esterification in HMDM by about 35%. In contrast, the p38 inhibitor SB203580 had no effect. However, when PMA was present in addition to SB203580, esterification was reduced to a level lower than that observed with PMA alone. These findings suggest that activation of ERK, but not p38, MAP kinases is involved in the induction of cholesterol esterification by acLDL in HMDM, while p38 MAP kinases may modulate the inhibitory effect of PKC, and thus provide evidence that MAP kinases play a role in the regulation of foam cell formation in human macrophages.

Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages.

The effects of low-density lipoprotein (LDL) and chylomicron remnants on lipid accumulation in human monocyte-derived macrophages (HMDMs) and in macrophages derived from the human monocyte cell line THP-1 were compared. The HMDMs or THP-1 macrophages were incubated with LDL, oxidized LDL (oxLDL), chylomicron remnant-like particles (CMR-LPs), or oxidized CMR-LPs (oxCMR-LPs), and the amount and type of lipid accumulated were determined. As expected, the lipid content of both cell types was increased markedly by oxLDL but not LDL, and this was due to a rise in cholesterol, cholesteryl ester (CE), and triacylglycerol (TG) levels. In contrast, both CMR-LPs and oxCMR-LPs caused a considerable increase in cellular lipid in HMDMs and THP-1 macrophages, but in this case there was a greater rise in the TG than in the cholesterol or CE content. Lipid accumulation in response to oxLDL, CMR-LPs, and oxCMR-LPs was prevented by the ACAT inhibitor CI976 in HMDMs but not in THP-1 macrophages, where TG levels remained markedly elevated. The rate of incorporation of [(3)H]oleate into CE and TG in THP-1 macrophages was increased by oxLDL, CMR-LPs, and oxCMR-LPs, but incorporation into TG was increased to a greater extent with CMR-LPs and oxCMR-LPs compared with oxLDL. These results demonstrate that both CMR-LPs and oxCMR-LPs cause lipid accumulation in human macrophages comparable to that seen with oxLDL and that oxidation of the remnant particles does not enhance this effect. They also demonstrate that a greater proportion of the lipid accumulated in response to CMR-LPs compared with oxLDL is TG rather than cholesterol or CE and that this is associated with a higher rate of TG synthesis. This study, therefore, provides further evidence to suggest that chylomicron remnants have a role in foam cell formation that is distinct from that of oxLDL.

Protection of chylomicron remnants from oxidation by incorporation of probucol into the particles enhances their uptake by human macrophages and increases lipid accumulation in the cells.

The effects of protection of chylomicron remnants from oxidation on their uptake and induction of lipid accumulation in macrophages were investigated using chylomicron remnant-like particles (CRLPs) containing the lipophilic antioxidant drug, probucol, and macrophages derived from the human monocyte cell line, THP-1. The total lipid content of THP-1 macrophages was markedly higher (x2.2) after 48 h of incubation of THP-1 macrophages with CRLPs containing probucol (pCRLPs) when compared to CRLPs without probucol, and this was because of increases in triacylglycerol (x2.3) and cholesterol (x1.8) levels, while cholesteryl ester concentrations were not significantly changed. Determination of the uptake of CRLPs and pCRLPs by the cells using particles labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3'3'-tetramethylindo-carbocyanine perchlorate showed that pCRLPs are taken up at a faster rate than CRLPs. The synthesis of triacylglycerol, as measured by the incorporation of [(3)H]oleate and [(3)H]glycerol, was also increased in macrophages incubated with pCRLPs as compared to CRLPs without probucol, but phospholipid and cholesteryl ester formation from [(3)H]oleate was unaffected. In addition, no differences between the effects of CRLPs and pCRLPs on the expression of mRNA for a range of genes believed to be involved in lipoprotein uptake, intracellular lipid metabolism and the efflux of cholesterol from macrophages was detected. These results suggest that antioxidants carried in chylomicron remnants enhance lipid accumulation in macrophages by increasing the rate of uptake of the particles and raising the intracellular synthesis of triacylglycerol, but not cholesteryl ester, and that these effects are brought about by changes at the post-transcriptional level. Antioxidants carried in chylomicron remnants therefore may promote the development of atherosclerosis, and this is likely to be particularly important in conditions where clearance of remnants from the circulation is delayed.

Incorporation of lycopene into chylomicron remnant-like particles enhances their induction of lipid accumulation in macrophages.

Lipid accumulation in macrophages exposed to chylomicron remnant-like particles containing the dietary antioxidant lycopene was investigated. After incubation with THP-1 macrophages (48h), chylomicron remnant-like particles containing lycopene (lycCRLPs) as compared to those without (CRLPs) caused significantly more lipid accumulation in the cells, and this was due to increases in both the triacylglycerol (+100%) and cholesterol (+62%) content. In addition, expression of mRNA for diacylglycerol acyltransferase (DGAT), a key enzyme in triacylglycerol synthesis, was significantly decreased by lycCRLPs, but not CRLPs. These findings suggest that lycopene from the diet may promote, rather than retard, lipid accumulation in macrophages during its transport in the blood in chylomicron remnants.

Chylomicron-remnant-like particles inhibit the basal nitric oxide pathway in porcine coronary artery and aortic endothelial cells.

The effects of chylomicron remnants on the activity of basally produced nitric oxide (NO) from porcine coronary artery rings and porcine aortic endothelial cells were studied by investigating the effects of chylomicron-remnant-like particles (CMR-LPs) containing porcine apolipoprotein E on the vessel tone of porcine coronary arteries and on cGMP release by aortic endothelial cells. CMR-LPs were oxidized by incubation with CuSO(4) (10 microM) for 18 h at 37 degrees C. N (omega)-nitro-L-arginine (L-NOARG) and oxidized CMR-LPs (oxCMR-LPs), but not native CMR-LPs, increased the vessel tone of static porcine coronary artery rings (increase in tone as a percentage of the tone induced by depolarizing Krebs-Henseleit solution: L-NOARG, 14.24 +/- 2.09; oxCMR-LPs, 4.98 +/- 0.88; and native CMR-LPs, 0.47 +/- 0.21). L-NOARG, endothelium removal and oxCMR-LPs also all significantly increased the maximum relaxation of the vessels to S -nitroso- N -acetyl-DL-penicillamine. In addition, oxCMR-LPs reduced the amounts of cGMP released by porcine aortic endothelial cells into the culture medium from 116 +/- 12.0 to 84.2 +/- 11.6 fmol/microg of cellular protein, mimicking the effects of L-NOARG. These results indicate that oxCMR-LPs, but not native CMR-LPs, inhibit the activity, production or release of NO from unstimulated porcine coronary and aortic endothelial cells. oxCMR-LPs mimicked the addition of L-NOARG and endothelium removal in these experimental systems, suggesting that the lipoproteins were interfering with the L-arginine/NO pathway. This study provides further evidence to support a role of chylomicron remnants in the development of atherosclerosis.

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.

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 CuSO(4). 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.

The effects of dietary n-3 polyunsaturated fatty acids delivered in chylomicron remnants on the transcription of genes regulating synthesis and secretion of very-low-density lipoprotein by the liver: modulation by cellular oxidative state.

The influence of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (PUFA) (derived from fish or corn oil, respectively) on the expression of mRNA for four genes involved in the regulation of the synthesis, assembly, and secretion of very-low-density lipoprotein (VLDL) in the liver was investigated in normal rat hepatocytes and after manipulation of the cellular oxidative state by incubation with N-acetyl cysteine (NAC) or CuSO(4). The four genes investigated were those encoding apolipoprotein B (apoB), the microsomal triacylglycerol transfer protein (MTP), and the enzymes acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2), which play a role in the regulation of triacylglycerol and cholesteryl ester synthesis, respectively. mRNA levels for apoB, MTP, and DGAT were unaffected by either fish or corn oil chylomicron remnants, but the amount of ACAT2 mRNA was significantly reduced after incubation of the hepatocytes with fish oil remnants as compared with corn oil remnants or without remnants. These findings indicate that the delivery of dietary n-3 PUFA to hepatocytes in chylomicron remnants downregulates the expression of mRNA for ACAT2, and this may play a role in their inhibition of VLDL secretion. However, when the cells were shifted into a pro-oxidizing or pro-reducing state by pretreatment with CuSO(4) (1 mM) or NAC (5 mM) for 24 hr, levels of mRNA for MTP were increased by about 2- or 4-fold, respectively, by fish oil remnants, whereas corn oil remnants had no significant effect. Fish oil remnants also caused a smaller increase in apoB mRNA in comparison with corn oil remnants in NAC-treated cells (+38%). These changes would be expected to lead to increased VLDL secretion rather than the decrease associated with dietary n-3 PUFA in normal conditions. These findings suggest that relatively minor changes in cellular redox levels can have a major influence on important liver functions such as VLDL synthesis and secretion.

Chylomicron-remnant-like particles inhibit receptor-mediated endothelium-dependent vasorelaxation in pig coronary arteries.

The influence of native and oxidized chylomicron-remnant-like particles (CMR-LPs) on endothelium-dependent relaxation in pig coronary arteries was studied. Artificial lipid particles of a size and lipid composition resembling chylomicron remnants and containing pig apolipoprotein E were used to investigate the effects of chylomicron remnants on the relaxation of isolated segments of pig coronary arteries in response to three endothelium dilators: 5-hydroxytryptamine (5-HT), bradykinin and the calcium ionophore A23187. CMR-LPs caused significant inhibition of the maximum relaxation response of the vessels to 5-HT, but not that to bradykinin or A23187 ( P <0.05). In contrast, CMR-LPs that had been oxidized by incubation with 10 microM CuSO(4) (oxidized CMR-LPs) were found to significantly reduce maximal relaxation to bradykinin by 13% ( P <0.05) and to reduce the sensitivity of the tissue to A23187 by 1.7-fold ( P <0.05). In experiments in which either the L-arginine/nitric oxide (NO) pathway or the endothelium-derived hyperpolarizing factor (EDHF) pathway was selectively inhibited, leaving the other intact, the inhibitory effect of oxidized CMR-LPs was observed only in vessels in which the -arginine/NO-mediated pathway was operative. Furthermore, the oxidized particles had no inhibitory effect on the relaxation of the vessel segments to the non-endothelium-dependent agonists S -nitro- N -acetylpenicillamine, 5'-( N -ethylcarboxamido)adenosine or pinacidil. These results demonstrate that CMR-LPs inhibit vascular relaxation in pig coronary arteries by an endothelium-dependent mechanism involving the L-arginine/NO pathway, but not the EDHF pathway, and provide evidence in support of a role for chylomicron remnants in the endothelial dysfunction associated with hypercholesterolaemia and atherogenesis.

The effects of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids on the transcription of genes regulating their uptake and metabolism by the liver: influence of cellular oxidative state.

The influence of chylomicron remnants enriched in n-6 or n-3 polyunsaturated fatty acids (PUFA) on the expression of mRNA for the low density lipoprotein receptor (LDLr), LDLr-related protein (LRP), and peroxisome proliferator activated receptor alpha (PPAR(alpha)) was investigated in normal hepatocytes and after manipulation of the cellular oxidative state by incubation with N-acetyl cysteine (NAC) or CuSO(4). In normal cells, mRNA levels for the LDLr were unaffected by incubation with chylomicron remnants, but those for the LRP and PPAR(alpha) were downregulated by remnants enriched in n-3 as compared to n-6 PUFA, suggesting that the transcription of these genes are influenced directly by the type of fatty acid delivered to the liver from the diet. Treatment with NAC or CuSO(4) was found to shift the hepatocytes into a pro-reducing or pro-oxidizing state, respectively. The abundance of mRNA for the LDLr, LRP, and PPAR(alpha) was increased after incubation with remnants enriched in n-3, but not n-6, PUFA in pro-reducing as compared to pro-oxidizing cells, and PPAR(alpha) mRNA levels were also decreased by remnants high in n-6 PUFA in the more reduced cells. These results indicate that the effects of fatty acids from the diet delivered to the liver in chylomicron remnants on the expression of hepatic genes regulating their uptake and metabolism are modulated by the redox state of the cells, and that the type of fatty acid carried by the particles also plays a part in determining the response observed.