The apoprotein B-independent hepatic uptake of chylomicron remnants.

Rat lymph chylomicrons were treated with Pronase resulting in particles completely devoid of surface apoproteins. On re-incubation with serum, the Pronase-treated chylomicrons re-acquired, by transfer from other lipoproteins, all apoproteins except apoprotein B, which is water-insoluble and non-transferable. When two groups of rats were injected with [3H]cholesterol-labelled control or Pronase-treated chylomicrons, radioactivity was incorporated into the liver of both groups at similar rates. It is concluded that the remnants of the control and Pronase-treated chylomicrons formed in the vascular space were recognized and taken up by liver cells by a process that does not require apoprotein B.     

Uptake of phospholipid-depleted chylomicrons by the perfused rat liver.

1. Rat lymph chylomicrons were depleted of their surface phospholipids by treatment with pure phospholipase A2 from Crotalus adamanteus venom. 2. About 80% of the phospholipids could be removed from the chylomicrons without any apparent effect on their size, neutral lipid composition or qualitative profile of their tetramethylurea-soluble apoproteins. 3. Phospholipid-depleted chylomicrons were rapidly taken up whole by liver cells when perfused through isolated rat liver preparations. The rate of uptake was dependent on the extent of phospholipid depletion and reached a maximum (4-6.5-fold greater than control chylomicrons) when 80% of the phospholipids had been removed. 4. It is speculated that the hepatic uptake of phospholipid-depleted chylomicrons occurs by a mechanism to that of chylomicron-remnants uptake.     

The uptake of chylomicron remnants and very low density lipoprotein remnants by the perfused rat liver

The regulation of the hepatic uptake of chylomicron remnants and very-low-density lipoprotein (VLDL) remnants was studied in the rat using a nonrecirculating liver perfusion system. The hepatic removal of remnant lipoproteins was shown to be by receptor-mediated processes since the concentration-dependent uptake was saturable and reductive methylation of the particles reduced the uptake of each lipoprotein by two-thirds. Treatment of liver donor rats with 17 alpha-ethinyl estradiol resulted in a 2-fold increase in the hepatic uptake of VLDL remnants, while cholesterol feeding of liver donor rats caused complete suppression of the receptor-mediated uptake of VLDL remnants. Chylomicron remnant removal was unaffected by estradiol administration and only slightly diminished by cholesterol feeding. The results of competition studies also indicated that a specific chylomicron remnant receptor exists in the liver. Apoprotein E was shown to be required for the receptor-mediated uptake of both remnant lipoproteins. Chylomicron remnants which contained no apoprotein E and VLDL remnants which contained reductively methylated apoprotein E were removed by the liver to about one-third of the extent of native particles. Thus the hepatic uptake of remnant lipoproteins occurs by receptor-mediated processes and the specific removal of both particles is mediated by apoprotein E. In addition, the uptake of VLDL remnants is regulated by the same factors that control hepatic low-density lipoprotein removal, while chylomicron remnant removal is unaffected by these factors.     

Comparison of the metabolic effects of chylomicrons and their remnants on isolated hepatocytes

A comparison was made between the effects of chylomicrons and chylomicron remnants on metabolic processes of isolated hepatocytes. Since isolated triacylglycerol-rich lipoproteins are contaminated with nonesterified fatty acids, control incubations were conducted with an amount of fatty acid equivalent to the contaminating fatty acids present in the chylomicrons and the remnant preparations, respectively. Chylomicron remnants, produced in vitro by incubation of chylomicrons in postheparin rat plasma, caused marked inhibition of glycolysis, fatty acid synthesis, and cholesterol synthesis, along with marked stimulation of ketogenesis. These effects were traced to the release of nonesterified fatty acids from these remnant particles as a consequence of contamination with lipoprotein lipase, picked up by the particles during the incubation with rat plasma. Fatty acids inhibit glycolysis, cholesterol, and fatty acid synthesis, but enhance ketone body formation by isolated hepatocytes. Chylomicrons and remnants prepared in vivo by the injection of chylomicrons into functionally hepatectomized rats were not contaminated with lipoprotein lipase and did not inhibit glycolysis and cholesterol synthesis nor increase ketone body formation. These lipoprotein particles did, however, cause significant inhibition of fatty acid synthesis, with the chylomicrons being more effective on a protein basis than the remnants produced in vivo. The mechanism responsible for the inhibition of fatty acid synthesis by chylomicrons and remnants prepared in vivo remains to be resolved.     

Intracellular transport and degradation of chylomicron remnants in rat liver cells after in vivo endocytosis

The intracellular transport and degradation of in vivo endocytosed chylomicron remnants labelled with 125I in the protein moiety was studied in rat liver cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. Initially, the radioactivity was located in low-density endosomes and was sequentially transferred to light and dense lysosomes. Data from gel filtration of the light and dense lysosomal fractions showed radioactive material with a molecular weight of about 1000-2000, representing short peptide fragments or amino acids which remain attached to iodinated tyramine cellobiose. In addition, undegraded apoproteins accumulated in both types of lysosome. Our data suggest that endocytosed chylomicron remnant apoproteins are first located in low-density endosomes and are sequentially transferred to light and dense lysosomes. Furthermore, the degradation process starts in the light lysosomes.     

Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not alpha-macroglobulin.

Our recently found nonlipoprotein inhibitor of chylomicron remnant uptake, lactoferrin, has been investigated in vivo and in vitro. Lipoprotein lipase extracted triglycerides from chylomicrons, doubly labeled with [3H]retinol/[14C]oleate, in the presence of lactoferrin normally. The subsequent uptake of remnants into liver was retarded considerably. In the intact rat, chylomicron remnants (CRs), predominantly labeled in the apoB48 moiety by 125I, were excluded from the hepatic endosomal compartment in the presence of lactoferrin as shown in subcellular fractionation studies of rat livers. In tissue culture, internalization of [125I]chylomicron remnants was inhibited in the presence of 14 pM lactoferrin by 70%. Upon removal of lactoferrin, internalization was rapidly restored. Protease digestion eliminated the inhibitory effect completely. Modification of arginine residues with cyclohexanedione reversibly removed the inhibitory potency of lactoferrin. We located by molecular modeling an alpha-helical segment in lactoferrin on the exposed surface of the molecule containing the sequence Arg-X-X-Arg-Lys-X-Arg, which resembles the receptor recognition structure in apolipoprotein E (apoE). This firmly established ligand correspondence with apoE, the candidate ligand for CR recognition by the receptor. Finally, the postulated second function of low density lipoprotein receptor-related protein, uptake of alpha-2-macroglobulin (alpha 2M) was found to be distinct from lipoprotein binding, since lactoferrin inhibited CR but not alpha 2M internalization. In addition, CR uptake was not affected by alpha 2M. We conclude that if a bifunctional receptor were to operate, its diverse functions were exerted by independently operating substructures. The results of our in vivo and cell culture experiments are, however, entirely compatible with the existence of two receptors as well.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.     

Competition between chylomicrons and their remnants for plasma removal: a study with artificial emulsion models of chylomicrons

In previous studies, protein-free emulsions of defined lipid composition were shown capable of simulating either the metabolism of chylomicrons (chylomicron-like emulsion) or their remnants (remnant-like emulsion), depending on the content of free, unesterified cholesterol. To validate further the assumption that remnant-like and chylomicron-like emulsion have metabolic pathways in common with their natural counterparts, studies of competition for plasma removal were undertaken: the remnant-like emulsion labeled with [3H]triolein was injected sequentially twice in the carotid arteries of rats to compare the clearance of remnant-like emulsion of the second injection with the first (control). Prior to the second injection, a large bolus of the chylomicron-like emulsion or rat lymph chylomicron was injected, to check the hypothesis that remnant generated from chylomicron-like emulsion or natural chylomicrons could compete with and displace remnant-like emulsion particles from their tissue receptor sites. Experiments were also performed in rats treated with Triton WR-1339, to block the generation of remnants. Results showed that remnants derived from either natural chylomicrons or chylomicron-like emulsion both strongly competed with the remnant-like emulsion. In contrast, when transformation of remnants was prevented by Triton, the undegraded particles of chylomicron-like emulsion or natural chylomicron were unable to compete with or displace remnant-like emulsion from its sites of removal from the plasma. In agreement with plasma clearance data, the hepatic uptake of the remnant-like emulsion was inhibited by the surplus dose of natural chylomicrons. In contrast, the spleen uptake was unaffected by it.     

Hepatic uptake of chylomicrons and triglyceride emulsions in rats fed diets of differing fat content

The hepatic removal of plasma chylomicrons was determined for rats fed the following diets: a) containing no triglyceride, b) regular chow diet with 4.5% of its mass as lipid and, c) a corn oil-supplemented chow with triglyceride accounting for 20% of the mass. The fractional hepatic uptake of either radiolabeled chylomicrons or a triglyceride emulsion was reciprocally related to the amount of lipid in the diet. The animals receiving only carbohydrate and protein calories had the most active hepatic uptake of particulate triglyceride and were observed to have a significant decrease in the plasma concentration of the C apolipoproteins. The addition of either C-I, C-II, or C-III apoproteins to the triglyceride emulsion prior to intravenous injection produced a significantly lower hepatic triglyceride recovery of emulsions containing apoC-III. When the plasma of animals fed a fat-free diet was supplemented with human C-III-1 apolipoprotein, the distribution into the liver of either enterally administered fatty acid or parenteral triglyceride was diminished. The triglyceride content in the liver of the rats fed fat-free or corn oil-supplemented diets was significantly greater than that of the control rats and composition was somewhat similar to that of lymph triglyceride. The studies indicate an important influence of dietary lipid on both the partition of plasma triglyceride into the liver and the steady state hepatic triglyceride content.     

Phospholipids as modulators of hepatic recognition of chylomicron remnants. Observations with emulsified lipoprotein lipids.

The lipids extracted from chylomicrons, chylomicron remnants generated in vivo and hepatic-lipase-treated chylomicrons were emulsified by sonication. These emulsified particles retained the capacity of the native lipoproteins to be differentiated by the liver in vivo, i.e. only the particles derived from remnant and hepatic-lipase-treated chylomicron lipids were efficiently taken up by the liver. To investigate the role of phospholipids in this differentiation process, the phospholipids of all three lipoprotein preparations were separated from the remaining lipids by silicic acid chromatography. The phospholipid-free lipid fraction of chylomicrons was then emulsified with the phospholipids derived from each of the three lipoprotein preparations. Only the particles emulsified with phospholipids derived from remnants and hepatic-lipase-treated chylomicrons were efficiently taken up by the liver in vivo. These results support the proposition that phospholipids modulate the hepatic differentiation between chylomicrons and remnants in vivo.     

Role of low density lipoprotein receptor-dependent and -independent sites in binding and uptake of chylomicron remnants in rat liver

The role of the low density lipoprotein (LDL) receptor in the binding of chylomicron remnants to liver membranes and in their uptake by hepatocytes was assessed using a monospecific polyclonal antibody to the LDL receptor of the rat liver. The anti-LDL receptor antibody inhibited the binding and uptake of chylomicron remnants and LDL by the poorly differentiated rat hepatoma cell HTC 7288C as completely as did unlabeled lipoproteins. The antireceptor antibody, however, decreased binding of chylomicron remnants to liver membranes from normal rats by only about 10%. This was true for intact membranes and for solubilized reconstituted membranes and with both a crude membrane fraction as well as with purified sinusoidal membranes. Further, complete removal of the LDL receptor from solubilized membranes by immunoprecipitation with antireceptor antibody only decreased remnant binding to the reconstituted supernatant by 10% compared to solubilized, nonimmunoprecipitated membranes. Treatment of rats with ethinyl estradiol induced an increase in remnant binding by liver membranes. All of the increased binding could be inhibited by the antireceptor antibody. The LDL receptor-independent remnant binding site was not EDTA sensitive and was not affected by ethinyl estradiol treatment. LDL receptor-independent remnant binding was competed for by beta-VLDL = HDLc greater than rat LDL greater than human LDL (where VLDL is very low density lipoprotein, and HDL is high density lipoprotein). There was weak and incomplete competition by apoE-free HDL, probably due to removal of apoE from the remnant. The LDL receptor-independent remnant-binding site was also present in membranes prepared from isolated hepatocytes and had the same characteristics as the site on membranes prepared from whole liver. In contrast, when chylomicron remnants were incubated with a primary culture of rat hepatocytes, the anti-LDL receptor antibody prevented specific cell association by 84% and degradation of chylomicron remnants completely. Based on these studies, we conclude that although binding of chylomicron remnants to liver cell membranes is not dependent on the LDL receptor, their intact uptake by hepatocytes is.     

Regulation of the hepatic removal of chylomicron remnants and beta-very low density lipoproteins in the rat.

The contribution of the low density lipoprotein (LDL) receptor to the removal of chylomicron remnants was determined in vitro and in vivo by using interventions that up- or down-regulate the LDL receptor but not the LDL receptor-related protein (LRP). In vitro, chylomicron remnants and beta-very low density lipoprotein (VLDL) bind to the LDL receptor on endosomal membranes; their binding can be competed by LDL and beta-VLDL and the binding capacity is greatly augmented in membranes from estradiol-treated rats. Likewise, estradiol treatment almost doubled the removal of chylomicron remnants during a single pass through perfused rat livers. However, in vivo the removal of chylomicron remnants and beta-VLDL was very rapid even in untreated rats so that the effect of the stimulation by estradiol was barely detectable when trace amounts of lipoproteins were injected. Yet, when saturating doses of either lipoprotein were injected, the effect of estradiol treatment on the removal of chylomicron remnants and beta-VLDL was readily disclosed. In rats fed a diet containing lard, cholesterol, and bile acids, removal of chylomicron remnants or beta-VLDL was significantly retarded. Likewise, perfused livers from diet-fed rats removed only a mean of 16% of chylomicron remnants during a single passage as compared to 29% in livers from control animals. Also, when large doses of beta-VLDL had been infused into rats for 4 h, in subsequent perfusions of the livers the removal of chylomicron remnants was decreased to 11%. From these results it is concluded that the LDL receptor mediates the hepatic removal of a major fraction of chylomicron remnants and beta-VLDL.     

Inhibition of macrophage inflammatory cytokine secretion by chylomicron remnants is dependent on their uptake by the low density lipoprotein receptor

Secretion of pro-inflammatory chemokines and cytokines by macrophages is a contributory factor in the pathogenesis of atherosclerosis. In this study, the effects of chylomicron remnants (CMR), the lipoproteins which transport dietary fat in the blood, on the production of pro-inflammatory chemokine and cytokine secretion by macrophages was investigated using CMR-like particles (CRLPs) together with THP-1 macrophages or primary human macrophages (HMDM). Incubation of CRLPs or oxidized CRLPs (oxCRLPs) with HMDM or THP-1 macrophages for up to 24h led to a marked decrease in the secretion of the pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1) and the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β (-50-90%), but these effects were reduced or abolished when CRLPs protected from oxidation by incorporation of the antioxidant drug, probucol, (pCRLPs) were used. In macrophages transfected with siRNA targeted to the low density lipoprotein receptor (LDLr), neither CRLPs nor pCRLPs had any significant effect on chemokine/cytokine secretion, but in cells transfected with siRNA targeted to the LDLr-related protein 1 (LRP1) both types of particles inhibited secretion to a similar extent to that observed with CRLPs in mock transfected cells. These findings demonstrate that macrophage pro-inflammatory chemokine/cytokine secretion is down-regulated by CMR, and that these effects are positively related to the lipoprotein oxidative state. Furthermore, uptake via the LDLr is required for the down-regulation, while uptake via LRP1 does not bring about this effect. Thus, the receptor-mediated route of uptake of CMR plays a crucial role in modulating their effects on inflammatory processes in macrophages.     

Uptake of chylomicron remnants by the native LDL receptor in human monocyte-derived macrophages

Human chylomicron remnants were taken up by cultured human monocyte-derived macrophages. Competition studies using 125I-labeled and unlabeled lipoproteins demonstrated that the remnant particles were not taken up by the modified LDL (acetyl LDL) receptor in these cells, which also contain a receptor for native LDL. The data thus suggest that the apolipoprotein E- and B-containing remnant particles are mainly taken up through an extra-hepatic E and B receptor (the classical LDL receptor pathway) in macrophages as is the case in cultured human skin fibroblasts.     

Catabolism of chylomicron remnants in normolipidemic subjects in relation to the apoprotein E phenotype

The role of the various apolipoprotein E isoproteins in the removal of chylomicrons and their remnants from plasma was studied in 16 normolipidemic subjects with various apoE phenotypes: 5 homozygous for apoE-2, 6 heterozygous for apoE-2 (phenotype E3/2), and 5 without apoE-2 (phenotypes E3/3, E4/4, and E4/3). The subjects were given an oral fat load as cream (50 g/m2). Retinyl palmitate was added as a marker for chylomicrons and their remnants. Blood was sampled at regular time intervals for 8 hr. Remnant particles were isolated from the d less than 1.019 g/ml fraction by heparin-Sepharose chromatography (heparin-bound fraction) after removing the large chylomicrons by flotation at 7,8 X 10(5) g-min. All groups showed a rise in triglycerides in serum and in the chylomicron fraction between 3 and 6 hr to about twice the basal value, followed by a decrease to nearly fasting values. In the homozygous E-2 subjects, fasting lipids in the remnant fraction were increased. In all three groups the fat load did not induce a significant rise in the lipids of the remnant fraction. The homozygous E-2 group showed a strong continuing rise in the retinyl palmitate concentration in the chylomicron and remnant fractions up to 8 hr, whereas in the other groups its maximum was already reached at 5 hr at a much lower level. At 8 hr similar retinyl palmitate concentrations were found in both fractions in the heterozygous E-2 subjects compared to the non-E-2 subjects. These results indicate a delayed removal of chylomicrons and chylomicron remnants in normolipidemic homozygous E-2, but not in heterozygous E-2 subjects.     

Mechanisms of chylomicron uptake into lacteals

Right from birth, the lymphatics play a crucial role in dietary functions. A majority of the lipid absorbed from the newborn's lipid-rich diet enters the blood circulation through the lymphatic system, which transports triglyceride-loaded particles known as chylomicrons from the villi of the small intestine to the venous circulation near the heart. In light of the significance of this role, as well as the fact that lipid transport from the gut was one of the earliest discovered functions of the lymphatic vasculature, it is surprising that so little is known about how chylomicrons initially gain access to the lymphatic vessel. This review will focus on the current mechanisms thought to be important in this process and highlight important questions that need to be answered in the future.     

Hepatic vitamin A fat-storage cells and the metabolism of chylomicron cholesterol.

These experiments were designed to test the hypothesis that the vitamin A fat-storage cell removes chylomicron remnant cholesterol from hepatic portal venous blood; A modified Ficoll density gradient ultracentrifugation procedure was used to isolate from rat liver cellular fractions that were enriched in vitamin A. In rats fed a normal diet and in rats fed excess vitamin A isolated hepatocytes were fractionated 15 min after the intravenous injection of chylomicrons labelled in vivo with radioactive cholesterol. The results showed that cholesterol radioactivity was not concentrated in the vitamin A enriched cellular fractions, so it was concluded that the vitamin A fat-storage cell is not implicated in clearance of chylomicron remnants by the liver.