Inhibitory effects of C apolipoproteins from rats and humans on the uptake of triglyceride-rich lipoproteins and their remnants by the perfused rat liver

Like rat C apolipoproteins, each of the C apolipoproteins from human blood plasma (C-I, C-II, C-III-1, and C-III-2) bound to small chylomicrons from mesenteric lymph of estradiol-treated rats and inhibited their uptake by the isolated perfused rat liver. This inhibitory effect of the C apolipoproteins was independent of apolipoprotein E, which is present only in trace amounts in these chylomicrons. Addition of rat apolipoprotein E to small chylomicrons from mesenteric lymph of normal rats did not displace C apolipoproteins and had no effect on the uptake of these particles by the perfused liver, indicating that an increased ratio of E apolipoproteins to C apolipoproteins on chylomicron particles, unaccompanied by depletion of the latter, may not promote recognition by the chylomicron remnant receptor. The hepatic uptake of remnants of rat hepatic very low density lipoproteins (VLDL) and small chylomicrons, which had been produced in functionally eviscerated rats, was also inhibited by addition of C apolipoproteins. These observations are consistent with the hypothesis that the addition of all of the C apolipoproteins to newly secreted chylomicrons and VLDL inhibits premature uptake of these particles by the liver and that depletion of all of these apolipoproteins from remnant particles facilitates their hepatic uptake. Remnants of chylomicrons and VLDL incubated with rat C apolipoproteins efficiently took up C-III apolipoproteins, but not apolipoprotein C-II (the activator protein for lipoprotein lipase). Preferential loss of apolipoprotein C-II during remnant formation may regulate the termination of triglyceride hydrolysis prior to complete removal of triglycerides from chylomicrons and VLDL.     

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.     

The kinetic parameters of the uptake of very-low-density lipoprotein remnant cholesteryl esters by perfused rat livers

The aim of this study was to determine the kinetic parameters of the hepatic uptake of VLDL remnant cholesteryl esters. Rat livers were perfused in situ with a broad range of remnant [3H]cholesteryl ester concentrations of known specific radioactivity. Following exactly 3 min of perfusion, hepatic lipids were extracted and labelled cholesteryl esters were separated by thin-layer chromatography and counted. The rate of cholesteryl ester uptake was a saturable process and the apparent kinetic parameters were determined from the Lineweaver-Burk plot of the data. Km and Vmax were calculated to be 72 microM and 35 nmol cholesteryl ester/min per g liver, respectively. For the purpose of comparison, we have expressed our kinetic parameters in terms of number of particles (Vmax = 0.022 nmol particles/min per g liver and Km = 45 nM) and compared our values with those obtained with chylomicron remnants by another group of investigators (Sherrill, B.C., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 1804-1807). We found that the maximal capacity for the removal of VLDL particles was similar to what was observed with rat chylomicron remnants. In contrast, the Km for the uptake process of VLDL remnant particles was approximately four times higher than that of rat chylomicron remnant particles. Our results are consistent with the hypothesis that hepatic removal of both chylomicron and VLDL remnants is mediated by the same receptor, but suggest that the affinity of VLDL remnants for the hepatic removal process is substantially lower, possibly due to structural differences between the two remnant particles.     

Contraceptive steroids increase hepatic uptake of chylomicron remnants in healthy young women

In an investigation of alterations in cholesterol metabolism during contraceptive steroid use, we studied plasma clearance of chylomicron remnants. Six healthy women were studied on and off contraceptive steroid therapy. Remnant clearance was measured from the disappearance of retinyl palmitate administered intravenously in plasma endogenously labeled with retinyl palmitate. We also measured cholesterol in HDL and its subfractions and postheparin lipoprotein lipase and hepatic triglyceride lipase activities. Plasma decay of retinyl palmitate was biexponential. The rapid component, reflecting chylomicron remnant removal, accounted for about 90% of the total clearance in all studies. During contraceptive steroid intake, both rapid and slow decay constants and the calculated plasma clearance rates were significantly increased (mean values: rapid decay constant, control 0.048 versus treated 0.101 min-1, P less than 0.05; slow decay constant, 0.004 versus 0.014 min-1, P less than 0.01; plasma clearance 74 versus 115 ml/min, P less than 0.025) indicating enhanced hepatic uptake of chylomicron remnants and probably an increased hepatic uptake of higher density lipoproteins (d greater than 1.006 g/ml). Total postheparin lipolytic activity and lipoprotein lipase activity were depressed in all six women (P less than 0.05) and hepatic triglyceride lipase activity was increased in four of five subjects. Contraceptive steroids also caused a decrease in the HDL2/HDL3 cholesterol ratio (P less than 0.05), implying impaired peripheral lipoprotein triglyceride hydrolysis and/or increased HDL2 clearance by hepatic triglyceride lipase. In conclusion, during intake of contraceptive steroids, the plasma clearance of chylomicron remnants and higher density lipoproteins was increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of chylomicron remnants by the liver: further evidence for the modulating role of phospholipids

Rat lymph chylomicrons were treated with rat heparin-releasable hepatic lipase (HL) or with bovine milk lipoprotein lipase (LPL). The ability of the resulting particles to be taken up by the liver in vivo was assessed following their infusion into the portal vein of partially hepatectomized animals. The following observations were made: a) the rate of phospholipid depletion, relative to the rate of triglyceride hydrolysis, induced by HL was two- to threefold higher than that observed for LPL; b) the depletion of at least 57% of phospholipids from the surface of HL-treated chylomicrons caused no major alterations in the apoprotein profile of the particles; c) for the same extent of triglyceride hydrolysis, HL-treated chylomicrons were taken up by liver at a rate significantly higher (P less than 0.005) than LPL-treated particles; d) the liver uptake of HL-treated chylomicrons was competitively inhibited by endogenously generated chylomicron remnants, indicating that these two types of lipoproteins share the same process of recognition and uptake by liver cells. It is concluded that the in vivo changes in phospholipid content, or composition, on the surface of chylomicrons during their transformation into remnants, modulate the differentiation of these two particles by the hepatic remnant receptor.     

Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.     

Uptake and processing of remnants of chylomicrons and very low density lipoproteins by rat liver

In the rat, chylomicron remnants and very low density lipoprotein (VLDL) remnants are taken up into the liver by high affinity processes and appear to undergo degradation by lysosomes. The relationship of this catabolic process to the known pathways of uptake and degradation of low density lipoproteins (LDL) and the involvement of nonparenchymal cells are addressed in these studies. We have utilized both light and electron microscopic radioautography to determine whether the pathway of intracellular transport and catabolism resembles that established for LDL in hepatocytes. Radioiodinated plasma VLDL remnants and lymph chylomicron remnants were injected into femoral veins of rats and the livers were fixed by perfusion 3 to 30 minutes later. Quantitative light microscopic radioautography showed little or no accumulation of grains over Kupffer cells. Electromicroscopic radioautography confirmed these observations and, in addition, demonstrated that very few grains were associated with endothelial cells. The processing of the remnant particles closely resembled that of LDL. Following an initial association of grains with the parenchymal cell plasma membrane, frequently in regions in close proximity to clathrin-coated endocytic pits, the grains were found in endocytic vesicles just beneath the plasma membrane. By 15 minutes the grains were found over multivesicular bodies located in the Golgi-lysosome region of the cell. Thirty minutes after injection, radioautographic grains began to be associated with secondary lysosomes. These data indicate no significant role for nonparenchymal cells in the internalization and subsequent degradation of triglyceride-rich lipoproteins, and provide evidence that the processing of remnants as well as LDL follows the classical pathway of receptor-mediated endocytosis.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.     

Rapid initial removal of chylomicron remnants by the mouse liver does not require hepatically localized apolipoprotein E

Apolipoprotein E (apoE) is a ligand for the low density lipoprotein receptor (LDLR) and the low density lipoprotein receptor-related protein (LRP). The aim of the present study was to clarify the role of hepatically localized apoE in the rapid initial removal of chylomicron remnants by using the isolated perfused liver. Radiolabeled chylomicron remnants were perfused in a single nonrecirculating pass into the livers of C57BL/6J (wild-type) mice, apoE-knockout mice, and apoE/LDLR-knockout mice for a period of 20 min. Aliquots of the perfusate leaving the liver were collected at regular intervals and the rate of removal of radioactivity was determined. At a trace concentration of chylomicron remnants (0.05 microgram of protein per ml), wild-type mouse livers removed at a steady state of 50-55% of total chylomicron remnants perfused per pass; livers from apoE-knockout mice had the same capacity as wild-type mouse livers. When the concentration of remnants was increased to 12 microgram of protein per ml, a level at which it has been shown that LDL receptor and LRP are near saturation, the capacity of the wild-type mouse livers to remove chylomicron remnants was decreased to 10-25% per pass, confirming that the removal mechanisms were nearing saturation. However, instead of finding a greater reduction in the removal rates or impairment in chylomicron remnant removal, livers from apoE-knockout mice were just as efficient as those from wild-type mice in removing remnants. Livers of mice that lacked both apoE and the LDLR also had a similar rate of removal at relatively low remnant concentrations (0.05-0.5 microgram/ml), but had reduced capacity in removing remnants at a relatively high concentration (4-12 microgram/ml) of chylomicron remnants ( approximately 20% per pass). The rate of removal at these concentrations, however, was similar to that attributed to the LRP in previous studies. Chylomicron remnants, whose apolipoproteins were disrupted by trypsinization, were removed at a normal rate by wild-type mouse livers but there was almost no removal by apoE-knockout mouse livers. At higher concentrations, however, the removal of apolipoprotein-disrupted chylomicron remnants was decreased.Our present findings do not support the hypothesis that hepatically localized apoE is a critical factor in the rapid initial removal of chylomicron remnants by either of the major pathways but do suggest that hepatically localized apoE can be added to lipoproteins to accelerate their uptake, although this process may have a limited capacity to compensate for apoE deficiency on lipoproteins.     

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.     

Obesity and post-prandial lipid metabolism. Feast or famine?

Both in Western countries and in third world countries there is an increasing incidence of obesity. Obesity per se or insulin resistance associated with obesity may increase cardiovascular risk factors including dyslipidemia, hypertension and Type 2 diabetes. Over the past decade the understanding has increased of specific mediators in the hypothalamus that are involved in regulating food intake and body weight. In obese humans fasting plasma lipids can be normal but postprandial lipid metabolism is abnormal with an accumulation of triglyceride-rich remnant lipoproteins. In viscerally obese men chylomicron remnant catabolism was markedly decreased when compared with lean individuals. The decreased clearance of chylomicron remnants in viscerally obese subjects may be explained by competition between chylomicron remnants and the increased hepatic production of VLDL for clearance by low density lipoprotein receptors. Increased food intake in rodent models of obesity was shown to be associated with a delay in the catabolism of remnant lipoprotein particles. Prevention of hyperphagia was found to correct the impairment in the metabolism of remnant lipoproteins. Under fasting and food restricted conditions the improvement of remnant metabolism was associated with an increased oxidation of remnant lipids as determined by a novel stable isotope breath test. Anti-obesity and lipid lowering drugs have been used for the treatment of obesity. Inhibitors of cholesterol synthesis inhibitors (statins) have been shown to be effective in treating dyslipidemia. Inhibition of cholesterol synthesis with Atorvastatin was shown to improve chylomicron metabolism by increasing chylomicron remnant catabolism in obese subjects as assessed by the newly developed stable isotope breath test.     

Comparison of the metabolism of chylomicrons and chylomicron remnants by the perfused liver

1. The hepatic metabolism of chylomicrons and chylomicron remnants was compared after adding approximately equal numbers of each lipoprotein particle to the perfusate of isolated livers. 2. At least 40% of the added remnants were metabolized by the liver compared with less than 3% for chylomicrons. 3. There was significantly more net removal of labelled remnants than of chylomicrons by the liver. 4. A greater proportion of labelled cholesterol than of labelled triacylglycerol fatty acids was transferred to the liver from each lipoprotein. 5. Cholesteryl esters of remnants were hydrolysed to triacylglycerol fatty lipoprotein. 5. Cholesteryl esters of remnants were hydrolysed to triacylglycerol fatty acids of remnants were oxidized to CO2 more extensively than those of chylomicrons. 6. There was greater oxidation of remnant glycerolipic [(1(-14)C]oleate than of glycerolipid [1(-14)C]palmitate. 7. A large fraction of the fatty acids of remnants, but not of chylomicrons, was transferred to phospholipids, which were released by the liver in a lipoprotein of relative density less than 1.006. 8. Label from remnants, but not from chylomicrons, was found in lipoproteins of relative density greater than 1.006, which were not released during perfusion but could be flushed out from the liver at the end of perfusion.     

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.     

Rates of removal and degradation of chylomicron remnants by isolated perfused rat liver

Chylomicron remnants are removed intact by isolated perfused rat livers and their lipid components are metabolized by the liver (Biochim. Biophys. Acta 488: 464, 1977). The present study provides quantitative information regarding these processes. When the lipoprotein concentration of the perfusate was constant, the removal of chylomicron remnants increases lineraly for 17 min. The rate of remnant removal was a hyperbolic function of the perfusate's remnant concentration. The removal rate had aV max of 28microgram cholesterol per g liver per min and an apparent Km of 64 microgram cholesterol per ml perfusate. Feeding the liver donors a diet containing 1% cholesterol or 4% cholesterol and 1% cholic acid failed to alter the hepatic removal rate. The cholesteryl ester removed from the remnants was hydrolyzed at a rate that was a small fraction of the removal rate (about 0.5% of removed cholesteryl ester per min). The rate of cholesteryl ester hydrolysis did not appear to approach saturation in the range studied. Studies of the lysosomal cholesteryl ester hydrolase suggested that this enzyme was not responsible for limiting the initial rate of hydrolysis, raising the possibility that the degradation rate is determined by the movement of the removed remnant to the site of hydrolysis.     

Regulation of apoprotein synthesis and secretion in the human hepatoma Hep G2. The effect of exogenous lipoprotein

The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

Effects of chylomicron remnants and beta-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglyceride- and cholesterol-rich) or with beta very low density lipoproteins (beta-VLDL) (cholesterol-rich). The medium was removed and the cells were incubated for and additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-3H]mevalonate. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with beta-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. In contrast, preincubation with beta-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with beta-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than an newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.     

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.