Uptake Of Chylomicron Remnants Research Articles

Binding and uptake of chylomicron remnants by primary and THP-1 human monocyte-derived macrophages: determination of binding proteins

The binding and uptake of chylomicron remnants by human macrophages was studied in order to resolve paradoxical observations that have described the putative mechanisms by which postprandial lipoproteins induce foam cell formation. Chylomicron remnants bound to human monocyte-derived macrophages (HMMs) and to the transformed monocytic cell line THP-1 with high affinity (Kd of approx. 5.5 microg of chylomicron remnant protein/ml). Binding was found to be saturable for both cell types, and was strongly inhibited in the presence of unlabelled chylomicron remnants. Ligand blot studies with colloidal-gold-labelled chylomicron remnants identified two cell surface binding sites on both HMMs and THP-1 cells, with molecular masses of approx. 128 kDa and 43 kDa. The high-molecular-mass binding site was found to be the low-density lipoprotein (LDL) receptor, based on the strong inhibition of chylomicron remnant binding in the presence of unlabelled LDL, Fab2 antibody fragments to the LDL receptor or calcium chelators. Competition studies suggested that, in HMMs, the LDL receptor appeared to facilitate approximately half of the total chylomicron remnant uptake. In contrast, the LDL receptor was not significantly involved in macrophage uptake of chylomicron remnants by THP-1 cells. The identity of the 43 kDa binding site is presently unknown, but, importantly, expression was not inhibited as a consequence of sterol loading, which was induced by incubating HMMs and THP-1 cells with 25-hydroxycholesterol. In contrast, the expression of the LDL receptor was substantially attenuated following lipid loading. Collectively, our data suggest that, while the macrophage LDL receptor can bind chylomicron remnants and facilitate uptake in non-lipid-loaded HMMs, other sterol-insensitive sites are responsible for the unabated uptake of chylomicron remnants by macrophages. We propose that the 43 kDa macrophage chylomicron remnant binding protein may be a candidate for the sterol loading of macrophages.

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.

Role of sequestration in hepatic uptake of chylomicron remnants

Role of sequestration in hepatic uptake of chylomicron remnants

Relative roles of LDLr and LRP in the metabolism of chylomicron remnants in genetically manipulated mice

Remnant-like emulsions labeled with cholesteryl [13C]-oleate were prepared with lipid compositions similar to remnants derived from triacylglycerol-rich lipoproteins. When injected into the bloodstream of conscious mice, the remnant-like emulsions were metabolized in the liver leading to the appearance of 13CO2 in the breath. Previously, using this technique, we found that remnant metabolism was significantly impaired but not completely inhibited in mice lacking low density lipoprotein receptors (LDLr). We have now found in mice with non-functional low density lipoprotein receptor-related protein (LRP) that breath enrichment of 13CO2 was significantly decreased, indicating that the LRP also plays an important role in the metabolism of chylomicron remnants (CR). The enrichment of 13CO2 in the expired breath was negligible in mice lacking both LDLr and receptor-associated protein (−/−), essential for normal function of LRP. In mice pre-injected with gluthatione S-transferase–receptor-associated protein to block LRP binding, there was a marked inhibition of the appearance of 13CO2 in the expired breath of homozygous LDLr-deficient mice, supporting the role of LRP in vivo. Whether or not LDLr were present, in mouse and human fibroblast cells human apoE3 or E4 but not apoE2 were essential for binding of remnant-like emulsions, while lactoferrin and suramin completely inhibited binding. We conclude that in normal mice LDLr are important for the physiological metabolism of CR. When LDLr are absent the evidence supports a role for the LRP in the uptake of CR in liver cells and in fibroblasts, with binding characteristics for CR-associated apoE similar to LDLr. —Martins, I. J., E. Hone, C. Chi, U. Seydel. R. N. Martins, and T. G. Redgrave. Relative roles of LDLr and LRP in the metabolism of chylomicron remnants in genetically manipulated mice.

Chylomicron-remnant-induced foam cell formation and cytotoxicity: a possible mechanism of cell death in atherosclerosis

The effects of chylomicron remnants on cytoplasmic lipid loading and cell viability were assessed in cultures of human monocyte-derived macrophages and rabbit arterial smooth muscle cells. At a cholesterol concentration of 150 microg/ml, chylomicron remnants induced substantial cytoplasmic lipid loading of macrophages, but not of smooth muscle cells, within 6 h of exposure. Chylomicron remnants were found to be cytotoxic to macrophages and smooth muscle cells, although the latter were generally more resistant. Chylomicron remnants contained no detectable oxysterols (>1 ng) and contained less non-esterified ('free') fatty acids than non-lipolysed nascent chylomicrons. Chylomicron-remnant-induced cytotoxicity appeared to be time- and dose-dependent. Macrophage and smooth muscle cell viability were inversely related to the production of superoxide free radicals and were significantly improved in the combined presence of superoxide dismutase and catalase. Collectively, our data suggest that, in macrophages, cell viability is compromised as a consequence of superoxide free radical production following uptake of chylomicron remnants. We would suggest that, in arterial smooth muscle cells, chylomicron-remnant-induced cell death also occurs as a consequence of superoxide free radical production. Our observations in the present study suggest that macrophage foam cells in atherosclerotic plaques might be derived from the cellular uptake of chylomicron remnants. Furthermore, arterial accumulation of chylomicron remnants might contribute to plaque destabilization as a consequence of cell death following superoxide free radical production by macrophages and smooth muscle cells.

Plasma clearance and liver uptake of chylomicron remnants generated by hepatic lipase lipolysis: evidence for a lactoferrin-sensitive and apolipoprotein E-independent pathway

Chylomicrons labeled with [3H]cholesterol and [14C]triglyceride fatty acids were lipolyzed by hepatic lipase (HL) in vitro and then injected intravenously into normal mice fed low- or high-fat diets, and into apolipoprotein (apo) E-deficient mice. In normal mice fed the high-fat diet and injected with non-lipolyzed chylomicrons, the plasma clearance and hepatic uptake of the resulting [3H]cholesterol-labeled remnants was markedly inhibited. In contrast, chylomicrons lipolyzed by HL were taken up equally rapidly by the livers of mice fed the low- and high-fat diets. The removal of non-lipolyzed chylomicrons lacking apoE from the plasma of apoE-deficient mice was inhibited, but not the removal of chylomicrons lipolyzed by HL. Pre-injection of lactoferrin into normal mice inhibited the plasma clearance of both non-lipolyzed chylomicrons and chylomicrons lipolyzed by HL. The removal of HL from the surface of the lipolyzed particles by proteolytic digestion did not affect their rapid uptake, indicating that the hepatic recognition of the lipoproteins was not mediated by HL. These observations support previous findings that phospholipolysis of chylomicrons by hepatic lipase generates remnant particles that are rapidly cleared from circulation by the liver. They also support the concept that chylomicron remnants can be taken up by the liver by an apolipoprotein E-independent mechanism. We hypothesize that this mechanism is modulated by the remnant phospholipids and that it may involve their interaction with a phospholipid-binding receptor on the surface of hepatocytes such as the class B scavenger receptor BI.—Crawford, S. E., and J. Borensztajn. Plasma clearance and liver uptake of chylomicron remnants generated by hepatic lipase lipolysis: evidence for a lactoferrin-sensitive and apolipoprotein E-independent pathway. J. Lipid Res. 1999. 40: 797–805.

Effects of hypercholesterolaemla on endothelial cell function and chylomicron remnant uptake by the rat aorta

Effects of hypercholesterolaemla on endothelial cell function and chylomicron remnant uptake by the rat aorta

Preparation and evaluation of an antibody to rat hepatic lipase: Effect on liver chylomicron remnant uptake

Preparation and evaluation of an antibody to rat hepatic lipase: Effect on liver chylomicron remnant uptake

Regulated turnover of a cell surface-associated pool of newly synthesized apolipoprotein E in HepG2 cells

Newly synthesized apolipoprotein E (apoE) is incompletely secreted and partially degraded by hepatocytes. To evaluate current models concerning apoE's role in remnant lipoprotein clearance by the liver, we performed a detailed and quantitative pulse-chase analysis of apoE secretion, subcellular distribution, and proteolytic degradation by the human hepatoma cell line HepG2. Only about 35% of newly synthesized apoE were found to be secreted to the culture medium. As determined by a protease-protection assay, a substantial amount of newly synthesized apoE was transported to the cell surface, constituting more than half of the cellular apoE under steady-state conditions. A subpopulation representing almost 40% of newly secreted apoE was rapidly rebound to the cell surface, indicating a dynamic equilibrium between cell surface and secreted apoE. These pools of newly synthesized apoE were subject to proteolytic turnover that occurred in lysosomes, presumably after re-endocytosis. We found that the proteolytic turnover of cell surface and secreted apoE was regulated by the availability of apoE ligands, being almost completely suppressed by the presence of lipoprotein-containing human serum or isolated lipoproteins, namely LDL and HDL. The characteristics of regulated degradation of cell surface apoE shed new light on potential physiological functions of this pool of apoE. Our results provide evidence that hepatocytes are capable of forming a large pool of cell surface-associated apoE, thereby supporting the previously proposed secretion-recapture model for apoE.

Receptor and non-receptor mediated uptake of chylomicron remnants by the liver

The uptake of chylomicron remnants by rodent liver is mediated by proteins residing on the microvillous surface of hepatocytes and occurs in two steps. First, initial removal of the remnants from the blood occurs through binding to the low density lipoprotein (LDL) receptor via apo E and to hepatic lipase via polar lipids and proteins on the remnant surface. Second, chylomicron remnants are taken up into the cell mainly by the LDL receptor and follow the classical receptor-mediated pathway of endocytosis. The LDL receptor-related protein (LRP), which binds weakly to chylomicron remnants via apo E, does not appear to have a significant role in the initial removal process. The remnant particles can, however, be enriched with proteoglycan-bound apo E present on hepatocytic microvilli, which increases their affinity for LRP to the extent that they are subject to endocytosis by this receptor, particularly when the LDL receptor is deficient or down-regulated. Hepatic lipase can also mediate binding of remnants to LRP, for which it has high affinity. Lipolysis of remnant lipids by hepatic lipase may promote but is not required for interaction of remnants with the endocytic receptors. Proteoglycan-bound hepatic lipase may also mediate endocytosis of chylomicron remnants independent of apo E, so that hepatic catabolism of these particles is not completely dependent upon this apoprotein. Available data from experiments in vivo thus indicate redundancy of both steps of hepatic uptake of chylomicron remnants, consistent with the centrality of this process in nutrient delivery.

Multiple processes are involved in the uptake of chylomicron remnants by mouse peritoneal macrophages

The processes responsible for the uptake of chylomicron remnants by macrophages were investigated using freshly isolated cells from low density lipoprotein (LDL) receptor, very low density lipoprotein (VLDL) receptor and apolipoprotein E knockout mice. In peritoneal macrophages from normal mice, the metabolism of chylomicron remnants was inhibited 40% by anti-LDL receptor antibody and 60% by a high concentration of receptor-associated protein (RAP). Together they reduced the amount processed by 70%. Digestion of cell proteoglycans decreased remnant degradation by 20% while the addition of acetyl-LDL had no effect. When LDL receptors were absent, the absolute rates of metabolism were less than that of normal cells and were not inhibited by the anti-LDL receptor antibody; the rates, however, were reduced to less than half by RAP. These suggest that the LDL receptor-related protein (LRP) or another LDL receptor family member(s) contributes to chylomicron remnant uptake and becomes the major mechanism of uptake when LDL receptors are absent. In contrast, the VLDL receptor was not involved as its absence did not affect chylomicron remnant metabolism. Similarly, the absence of apoE production did not affect the amount of remnant uptake; however, the proportion that was sensitive to RAP was eliminated. The level of LRP expression was not altered in these cells whereas there was a decrease in LDL receptors. This suggests that the apoE content of chylomicron remnants is sufficient for its recognition by LDL receptors but additional apoE is required for its uptake by the LRP and that there is an up-regulation of a non-LDL receptor family mechanism in apoE deficiency. Together these studies suggest that even in the absence of LDL receptors or apoE secretion, chylomicron remnants could contribute to lipid accumulation in the artery wall during atherogenesis.

Accelerated lipoprotein uptake by transplantable hepatomas that express hepatic lipase

To test the hypothesis that hepatic lipase plays a key role in lipoprotein removal in vivo, a novel system was used. Hepatoma cells (HTC 7288c) were transfected with a cDNA encoding hepatic lipase in culture and grown as solid tumors in vivo. In culture, transfected cells degraded chylomicron remnants and low density lipoprotein (LDL) somewhat more efficiently than untransfected cells. Tumors from the transplanted cells produced hepatic lipase localized to the surface of tumors from transfected cells but not tumors from non-transfected cells, grown in the same rat. The tumors from transfected cells removed, per gm of tissue, 34% (P < 0.001) more 125I-labeled LDL than tumors from non-transfected cells in the same animal. The uptake of chylomicron remnants (by tumors from transfected cells) was also modestly enhanced (15 ± 6%, P < 0.005). There were no differences in the uptake of 125I-labeled albumin or 125I-labeled asialoglycoprotein. Compared to the liver, the untransfected tumors took up 12%, and the transfected tumors took up about 18% as much LDL per gram of tissue. The uptake of chylomicron remnants compared to liver was far lower. Both types of tumors had about twice as much LDL receptor related protein as the liver. Wild-type tumors had the highest level of LDL receptor, twice hepatic lipase-secreting tumors, and six times that of the liver.▪ Using the novel approach of transfecting transplantable tumor cells with hepatic lipase, the ability of hepatic lipase to facilitate the removal of apoB-containing lipoproteins was demonstrated. The liver still removes low density lipoprotein and especially chylomicron remnants more rapidly than the tumors, suggesting factors in addition to hepatic lipase and LDL receptor level play a major role in hepatic lipoprotein removal.—Donner, C., S. Choi, M. Komaromy, and A. D. Cooper. Accelerated lipoprotein uptake by transplantable hepatomas that express hepatic lipase.

The influence of chylomicron remnants on endothelial cell function in the isolated perfused rat aorta

A system for the perfusion of the isolated rat aorta which allowed the study of both the uptake of chylomicron remnants by the artery wall and their effects on endothelial function was developed. Perfusion for 2 h with 125I-labelled native or oxidised (by treatment with copper sulphate) chylomicron remnants showed that small amounts became associated with the artery wall (0.111±0.034 and 0.216±0.082 ng protein/mg tissue, respectively). Tests on endothelial function were carried out in vessel rings prepared after perfusion of the aortas in the presence or absence of chylomicron remnants for 2 h. After perfusion of the vessels with oxidised chylomicron remnants, the maximum response to phenylephrine (PE) was significantly increased (from 0.34±0.06 to 0.51±0.04 g/mg tissue; P<0.05), while the maximum % relaxation to carbachol (CCh) was significantly decreased (from 91.6±2.4 to 71.5±7.2; P<0.05) and the response to S-nitroso- N-acetylpenicillimine (SNAP) was unaffected. Perfusion with native chylomicron remnants showed a tendency to induce similar effects, although the changes observed did not reach statistical significance. As the lipoproteins were not present in the solution bathing the vessel rings during these tests, these effects can be attributed to perfusion of the aortas with chylomicron remnants, despite only small quantities being associated with the artery wall. The results suggest that oxidised chylomicron remnants influence vascular endothelial function by interfering with the l-arginine-nitric oxide (NO) pathway. The observed potentiation of contraction to PE may be due to inhibition of the basal release of NO or to the release of contractile factors. These findings support a role for dietary lipoproteins in the modulation of endothelial cell function which occurs in the pathogenesis of atherosclerosis.

Accumulation of chylomicron remnants in homozygous subjects with familial hypercholesterolaemia.

Post-prandial lipoprotein kinetics were investigated in subjects who lack functioning low-density lipoprotein (LDL) receptors [homozygous familial hypercholesterolaemia (FH)]. An oral fat load was given, and chylomicron plasma kinetics was determined by monitoring the clearance of triglyceride, retinyl palmitate and apolipoprotein B48, calculated as the area under the curve, for 7.5 h. In addition, the binding and uptake of chylomicron remnants by fibroblasts of FH and control subjects were assessed in vitro. Based on the plasma kinetics of chylomicron triglyceride, retinyl palmitate and apolipoprotein B48 after a lipid meal, chylomicron clearance was found to be substantially delayed compared with normolipidaemic control subjects. Consistent with involvement of the LDL receptor in chylomicron clearance, binding and uptake of chylomicron remnants by fibroblasts of FH subjects was found to be substantially less than in cells from control subjects. This study shows that, in addition to LDL, chylomicron metabolism is severely impaired in FH and that the LDL receptor is significantly involved in the clearance of post-prandial lipoproteins. Moreover, this study raises the possibility that in FH, and in other disorders in which LDL receptor expression is reduced, atherogenesis might be a post-prandial disease.

Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes

Chylomicron remnants transport cholesterol from the intestine, and are removed from the circulation principally by the liver. While hepatic receptors, including the low density lipoprotein (LDL) receptor account for endocytosis, heparan sulfate proteoglycans (HSPG) participate in the initial binding of remnants to liver cells. To explore the interactions between HSPG and endocytosis of remnants, in the present study the expression of HSPG was inhibited in HepG2 cells transfected by a synthetic antisense oligodeoxynucleotide SYN5. Immunofluorescent staining by a monoclonal anti-syndecan antibody showed significant reduction in the expression of syndecan in SYN5-treated cells compared with control cells. Remnant binding decreased by about 50–70% in SYN5-transfected cells. Monoclonal antibodies to either heparan sulphate or the LDL receptor decreased binding by about 60–65%. The glycosylation inhibitor β-nitrophenyl-xylopyranoside inhibited remnant uptake by 25%, whereas 4-nitrophenyl-β-d-galactopyranoside had no effect on remnant binding. Heparinase completely abolished binding at appropriate concentrations. Heparitinase was less effective than heparinase in inhibiting remnant binding. Suramin completely abolished the remnant binding. Poly-arginine, poly-lysine, and protamine all reduced remnant uptake by the cells, as did polybrene, a synthetic polycation, suggesting a role of cation–anion interactions in remnant binding. Brefeldin A, colchicine, and monensin caused the fluorescence associated with remnants to persist within the cells, confirming that blockers of tubulovesicular processes and Golgi function inhibit the intracellular transport and degradation of the remnants. Our results show that remnant binding to liver cells depends on the LDL receptor, on the expression of HSPG core proteins, and on the functionality of heparan sulfate in HSPG.—Zeng, B-J., B-C. Mortimer, I. J. Martins, U. Seydel, and T. G. Redgrave. Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes. J. Lipid Res. 1998. 39: 845–860.

Comparison of short- and long-term effects of different dietary fats on the hepatic uptake and metabolism of chylomicron remnants in rats.

The uptake and metabolism of [14C]oleate-labelled chylomicron remnants derived from olive oil, maize oil, palm oil, fish oil or butter fat was investigated using perfused livers from rats fed on the corresponding fat-supplemented diet (providing 40% of the dietary energy) or a low-fat diet for 21 d. The percentage of added [14C]oleate-labelled remnant removed from the perfusate was similar for livers from rats fed on the fat-supplemented diets irrespective of the type of fat fed, whereas livers from rats fed on the low-fat diet removed more labelled fish oil and butter fat remnants than olive, maize or palm oil remnants. Following hepatic uptake in the fat-supplemented groups, the oxidation of [14C]oleate-labelled remnant lipid from maize oil, fish oil, and butter fat remnants was greater than that of the lipids from olive and palm oil remnants, although only the oxidation of lipids from maize and palm oil remnants was increased by prior fat-supplementation of the diet. In addition, the livers from rats fed on the fish-oil-supplemented diet incorporated more [14C]oleate-labelled remnant lipid into phospholipid compared with the livers from rats fed on the other fat-supplemented diets or the low-fat diets. These investigations show that both prior fat feeding and the composition of the fat fed, as well as the fatty acid composition of the chylomicron remnant particles themselves, influence the uptake and metabolism of chylomicron remnants by the liver.

Regulation of hepatic chylomicron remnant uptake: effects of obesity and insulin resistance.

Regulation of hepatic chylomicron remnant uptake: effects of obesity and insulin resistance.

4.P.203 Mechanisms involved in the binding and uptake of chylomicron remnants by human macrophages

4.P.203 Mechanisms involved in the binding and uptake of chylomicron remnants by human macrophages

3.P.266 The role of the low-density-lipoprotein receptor in the uptake of chylomicron remnants by arterial smooth muscle cells

3.P.266 The role of the low-density-lipoprotein receptor in the uptake of chylomicron remnants by arterial smooth muscle cells

4.P.233 The uptake of chylomicron remnants and their effects on endothelial cell function in the perfused rat aorta

4.P.233 The uptake of chylomicron remnants and their effects on endothelial cell function in the perfused rat aorta