Binding Of Chylomicron Remnants Research Articles

Protective effect of apolipoprotein E2 on coronary artery disease in African Americans is mediated through lipoprotein cholesterol

We studied the relationship of apolipoprotein E (apoE) isoforms and coronary artery disease (CAD) in 224 African Americans and 326 Caucasians undergoing diagnostic coronary angiography. The presence of CAD was defined as >50% stenosis in at least one artery. ApoE allele frequencies were 0.12, 0.62, and 0.26 for epsilon 2, epsilon 3, and epsilon 4, respectively, in African Americans and 0.08, 0.78, and 0.14 for epsilon 2, epsilon 3, and epsilon 4, respectively, in Caucasians. Among African Americans, CAD was present in 9 of 34 epsilon 2 carriers (26%), significantly smaller (P < 0.05) in proportion compared with 39 of 82 epsilon 3 carriers and 43 of 92 epsilon 4 carriers (48% and 47%, respectively), suggesting a protective effect of the epsilon 2 allele. No such difference was seen in Caucasians. In African Americans but not Caucasians, LDL cholesterol was lower in epsilon 2 carriers than in epsilon 3 and epsilon 4 carriers (106 vs. 127 and 134 mg/dl, respectively; P < 0.005). After adjusting for lipid levels, the association between apoE2 and CAD was no longer significant. Thus, the protective effect of apoE2 seen in African Americans could be explained by a favorable lipid profile in epsilon 2 carriers, whereas in Caucasians, the absence of such a protective effect could be attributable to the lack of effect of apoE2 on the lipid profile.

Endocytosis of hepatic lipase and lipoprotein lipase into rat liver hepatocytes in vivo is mediated by the low density lipoprotein receptor-related protein.

In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar ( approximately 10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP.

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.

Phagocytic degradation of chylomicron remnants by fibroblasts from subjects with homozygous familial hypercholesterolaemia.

1. Familial hypercholesterolaemia is a common genetic abnormality in man characterized by premature atherogenesis as a consequence of disturbed lipoprotein metabolism. Chylomicrons, which represent intestinally derived lipoproteins, are cleared poorly in familial hypercholesterolaemia which may explain the increased retention of chylomicron remnants in arterial fatty lesions. However, cellular uptake of chylomicron remnants in familial hypercholesterolaemia remains unclear. This study determined the quantitative significance of non-low-density lipoprotein receptor-mediated uptake in fibroblasts from subjects with homozygous familial hypercholesterolaemia. 2. The metabolism of chylomicron remnants was assessed in fibroblasts from subjects with homozygous familial hypercholesterolaemia who lack low-density lipoprotein receptors. Compared with fibroblasts from normolipidaemic subjects, binding and degradation of chylomicron remnants was reduced by about two-thirds. Nevertheless, degradation of chylomicron remnants in cells from subjects with familial hypercholesterolaemia persisted in the absence of functioning low-density lipoprotein receptors. 3. Binding of chylomicron remnants to fibroblasts from subjects with familial hypercholesterolaemia was saturable. Unlabelled chylomicron remnants competed efficiently for binding but unlabelled low-density lipoprotein or a monoclonal antibody specific to the human low-density lipoprotein receptor had little effect on binding or degradation. 4. Polyinosinic acid did not alter binding and degradation of chylomicron remnants by fibroblasts from subjects with familial hypercholesterolaemia, ruling out involvement of the scavenger receptor. Lactoferrin was found to inhibit binding and degradation of chylomicron remnants by fibroblasts from subjects with familial hypercholesterolaemia by approximately 50%, implicating involvement of the alpha 2-macroglobulin receptor. 5. Cytochalasin D blocked degradation of chylomicron remnants at 37 degrees C in fibroblasts from subjects with familial hypercholesterolaemia by more than 80% but had no effect on binding at 4 degrees C, consistent with a phagocytic uptake pathway. Collectively, the data suggest that chylomicron remnants bind to a cell-surface protein which initiates phagocytosis and that lactoferrin interferes with binding to this putative cell-surface protein. Phagocytic uptake of chylomicron remnants provides an additional mechanism whereby cells from subjects with familial hypercholesterolaemia can accumulate lipid.

Degradation of Chylomicron Remnants by Macrophages Occurs via Phagocytosis

Chylomicron remnants bound to rabbit alveolar macrophages with high-affinity (Kd = 3.3 +/- 0.71 microgram of protein/mL). The binding of chylomicron remnants was competitively inhibited in the presence of unlabeled remnants and to a lesser extent by unlabeled low-density lipoproteins. Pretreatment of cells with either trypsin or pronase inhibited degradation in a dose and time dependent manner, suggesting involvement of a cell surface protein. Chylomicron remnants were degraded by alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits, which are devoid of LDL receptor activity. Moreover, colchicine and monensin which are endocytotic and lysozomal inhibitors, respectively, did not have any effect on the degradation of chylomicron remnants by macrophages from normal rabbits. The absence of divalent cations was found to enhance chylomicron remnant degradation by macrophages. Activated alpha 2-macroglobulin and lactoferrin had no effect on chylomicron remnant degradation, indicating that the low-density lipoprotein receptor-related protein was not involved. In addition, the scavenger receptor inhibitors polyinosinic acid and fucoidan increased degradation of chylomicron remnant-ruling out uptake as a consequence of lipoprotein modification. Rather, the phagocytotic inhibitor cytochalasan D was found to significantly decrease chylomicron remnant degradation. Collectively, our data show that chylomicron remnants are metabolized by phagocytotic pathways initiated after binding to a cell surface protein which is distinct from the LDL receptor, LRP, or scavenger receptors.

Characterization of the chylomicron-remnant-recognition sites on parenchymal and Kupffer cells of rat liver. Selective inhibition of parenchymal cell recognition by lactoferrin.

Upon injection of chylomicrons into rats, chylomicron remnants are predominantly taken up by parenchymal cells, with a limited contribution (8.6% of the injected dose) by Kupffer cells. In vitro storage of partially processed chylomicron remnants for only 24 h leads, after in vivo injection, to an avid recognition by Kupffer cells (uptake up to 80% of the total liver-associated radioactivity). Lactoferrin greatly reduces the liver uptake of chylomicron remnants, which appears to be the consequence of a specific inhibition of the uptake by parenchymal cells. Kupffer-cell uptake is not influenced by lactoferrin. In vitro studies with isolated parenchymal and Kupffer cells show that both contain a specific recognition site for chylomicron remnants. The Kupffer-cell recognition site differs in several ways from the recognition site on parenchymal cells as follows. (a) The maximum level of binding is 3.7-fold higher/mg cell protein than with parenchymal cells. (b) Binding of chylomicron remnants is partially dependent on the presence of calcium, while binding to parenchymal cells is not. (c) beta-Migrating very-low-density lipoprotein is a less effective competitor for chylomicron-remnant binding to Kupffer cells compared to parenchymal cells. (d) Lactoferrin leaves Kupffer-cell binding uninfluenced, while it greatly reduces binding of chylomicron remnants to parenchymal cells. The properties of chylomicron-remnant recognition by parenchymal cells are consistent with apolipoprotein E being the determinant for recognition. It can be concluded that the chylomicron-remnant recognition site on Kupffer cells possesses properties which are distinct from the recognition site on parenchymal cells. It might be suggested that partially processed chylomicron remnants are specifically sensitive to a modification, which induces an avid interaction with the Kupffer cells. The recognition site for (modified) chylomicron remnants on Kupffer cells might function as a protection system against the occurrence of these potential atherogenic chylomicron-remnant particles in the blood.

Apoprotein-independent binding of chylomicron remnants to rat liver membranes

Rat lymph chylomicrons and chylomicron remnants were treated with trypsin or Pronase. The ability of the resulting apoprotein-free lipoproteins to be taken up by the isolated perfused rat liver, and to bind to isolated rat liver membranes, was examined. Compared with control lipoproteins, the apoprotein-free chylomicrons and remnants retained unaltered their capacity to be differentiated by the intact liver and by the isolated membranes. Further, control remnants and apoprotein-free remnants competed for binding to the isolated membranes. We conclude that apoproteins are not required for the hepatic differentiation between chylomicrons and remnants, and suggest that the lipoprotein phospholipids may play a direct role in this process.

Regulation of chylomicron remnant uptake in the human hepatoma cell-line Hep G2. Role of the low-density lipoprotein receptor

Uptake and degradation of chylomicron remnants by the human hepatoma cell line Hep G2 was studied. Mesenteric lymph was collected from rats and injected into hepatectomized rats to obtain chylomicron remnants. This remnant preparation was taken up and catabolized by Hep G2 cells. The uptake process was dependent on cell growth and was regulated by compactin (a HMG-CoA reductase inhibitor) which suppresses cholesterol synthesis and by mevalonolactone, which enhances cholesterol synthesis. A monoclonal anti LDL receptor antibody blocked binding of chylomicron remnants to Hep G2 cells to a degree, which was comparable to but generally lower than the suppression of low-density lipoprotein binding. The results thus indicate that in Hep G2 cells, chylomicron remnant uptake is regulated, similarly to low-density lipoprotein uptake and that a significant part of the remnant uptake is mediated through the LDL receptor.

Genetic Defects in Lipoprotein Metabolism

Certain proteins (called apolipoproteins B and E) on the surface of lipoprotein particles are responsible for mediating the binding of cholesterol-rich particles to specific lipoprotein receptors on the surface of cells and represent a major pathway controlling blood cholesterol levels. Three important disorders of lipoprotein metabolism, which provide insights into the molecular mechanisms responsible for the elevation of specific atherogenic lipoproteins, are the following: (1) Type III hyperlipoproteinemia results from specific mutations in apolipoprotein E that prevent the normal binding of chylomicron remnants and very-lowdensity lipoprotein remnants to lipoprotein receptors. Patients with this disorder who have elevated levels of these remnant lipoproteins develop atherosclerosis. (2) Familial defective apolipoprotein B-100 results from a single amino acid substitution in apolipoprotein B that prevents low-density lipoprotein from binding normally to the low-density lipoprotein receptor and elevates plasma cholesterol levels. (3) Familial hypercholesterolemia, which results in elevated levels of plasma low-density lipoprotein and premature atherosclerosis, is caused by a variety of mutations in the low-density lipoprotein receptor that interfere with the normal binding of lipoproteins to this receptor. These observations not only provide insights into the mechanisms responsible for normal lipoprotein metabolism, but also highlight the potential role of specific lipoproteins in atherogenesis. (<i>JAMA</i>. 1991;265:78-83)

Genetic defects in lipoprotein metabolism. Elevation of atherogenic lipoproteins caused by impaired catabolism

Certain proteins (called apolipoproteins B and E) on the surface of lipoprotein particles are responsible for mediating the binding of cholesterol-rich particles to specific lipoprotein receptors on the surface of cells and represent a major pathway controlling blood cholesterol levels. Three important disorders of lipoprotein metabolism, which provide insights into the molecular mechanisms responsible for the elevation of specific atherogenic lipoproteins, are the following: (1) Type III hyperlipoproteinemia results from specific mutations in apolipoprotein E that prevent the normal binding of chylomicron remnants and very-low-density lipoprotein remnants to lipoprotein receptors. Patients with this disorder who have elevated levels of these remnant lipoproteins develop atherosclerosis. (2) Familial defective apolipoprotein B-100 results from a single amino acid substitution in apolipoprotein B that prevents low-density lipoprotein from binding normally to the low-density lipoprotein receptor and elevates plasma cholesterol levels. (3) Familial hypercholesterolemia, which results in elevated levels of plasma low-density lipoprotein and premature atherosclerosis, is caused by a variety of mutations in the low-density lipoprotein receptor that interfere with the normal binding of lipoproteins to this receptor. These observations not only provide insights into the mechanisms responsible for normal lipoprotein metabolism, but also highlight the potential role of specific lipoproteins in atherogenesis.

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 lipoprotein receptors on a rat hepatoma cell line

The rat H-35 cultured hepatoma cell line expresses receptors for homologous lipoproteins. In previously reported experiments distinct receptors were identified for chylomicron remanants, HDL and LDL, by direct binding studies that yielded distinctive binding constants, cross competition assays, and by differential inhibitory effects of EDTA and suramin. In the present experiments, the regulation of expression of these receptors was assessed by growing cells either in the presence or absence of lipoproteins in the media and by growing cells to different densities (50–800 μg cell protein/dish). LDL binding to cells was increased by lipoprotein deprivation at all cell densities. LDL binding was inversely related to cell density when cells were grown in lipoprotein deficient serum (LPDS) but cell density did not affect LDL binding by cells grown in newborn calf serum (NBCS). By contrast HDL binding was not appreciably different whether cells were grown in NBCS or in LPDS. However, HDL binding was inversely related to cell density by cells grown either in LPDS or in NBCS. Binding of chylomicron remnants was increased by growth in LPDS at all densities, but altering growth density in either culture medium had little effect on the cellular binding of chylomicron remnants. The distinctive effects of these experimental perturbations on the binding of the 3 lipoprotein classes tend to confirm the presence of 3 separate receptor activities. The experiments also demonstrate that the responses at least of some of the receptors of the hepatoma cells in culture resemble those of hepatocytes in vivo and in vitro. Therefore, the H-35 hepatoma cell line could be a useful model in which to study receptor biosynthesis, structure, regulation and function.

Lipoprotein binding to cultured human hepatoma cells.

Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man.

Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2.

The binding and metabolism of [3H]vitamin A-containing chylomicron (CM) remnants by the human hepatoma cell line HepG2 were studied. Mesenteric lymph chylomicrons were collected from [3H]retinol-fed rats and incubated with lipoprotein lipase to obtain CM remnants. At 4 degrees C, specific CM remnant binding was inhibited by an excess of unlabeled CM remnants. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 microgram triglyceride/ml). CM remnant uptake at 37 degrees C was greater than that of CM and at least 70 times more efficient than the pinocytosis of sucrose. CM remnant binding increased with the extent of lipolysis. Addition of human apolipoprotein E enhanced both CM remnant and CM binding. After internalization, HepG2 cells hydrolyzed CM remnant-[3H]retinyl esters, and radiolabeled metabolites accumulated. As a function of the concentration of [3H]retinoid initially bound to cells, retinol and retinyl esters accumulated as the major cell-associated metabolites. In contrast, retinol was the major metabolite in the medium only at low retinoid concentrations; other more polar metabolites accumulated at higher concentrations (greater than 110 pmol retinoid/mg cell protein). The accumulation in the medium of labeled metabolites derived from CM remnant-retinoid was reduced when cells were preincubated in unlabeled retinol-supplemented media. The specific activity of retinol in the medium indicated that CM remnant-vitamin A had mixed with the cellular store prior to its secretion as retinol. These results indicate that HepG2 cells internalize CM remnants in part by specific binding sites, and that the metabolism of CM remnant-retinoids by the HepG2 cell involves retinyl ester hydrolysis and the secretion of retinol and other more polar metabolites. These processes were regulated in part by the concentration of retinoid delivered by the CM remnant and by the initial retinoid content of the cell.

Binding of low-density lipoprotein and chylomicron remnants to the hepatic low-density lipoprotein receptor of dogs, rats and rabbits demonstrated by ligand blotting. Failure to detect a distinct chylomicron-remnant-binding protein by ligand blotting.

In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed.

Characterization of the estrogen-induced lipoprotein receptor of rat liver.

The ethinyl estradiol-induced lipoprotein receptor of rat liver was purified and characterized. Liver membranes were prepared from ethinyl estradiol-treated rats, solubilized, and subjected to DEAE chromatography. A fraction with a high specific activity for low density lipoprotein (LDL) binding was isolated and used to immunize mice. Hybridomas were prepared from their spleen cells, and a clone that secreted an IgG antibody, which cross-reacted with an ethinyl estradiol-induced protein of the same molecular weight as the bovine adrenal LDL receptor, was expanded. This antibody, designated P1B3, immunoprecipitated the induced lipoprotein receptor. P1B3 was used to purify the receptor, and a polyclonal antibody was raised against the pure protein. This antibody recognized a protein of similar molecular weight in rat liver, adult dog liver, and human skin fibroblasts, thus demonstrating that the induced rat lipoprotein receptor was related to the LDL receptor of other species. This receptor is present in normal rat liver, and its content is reduced by feeding an atherogenic diet, but not by feeding a diet containing 0.5% cholesterol. Moreover, cholestyramine supplementation of the diet did not induce the receptor on liver membranes. The polyclonal antibody could prevent the binding of LDL to liver membranes from control or ethinyl estradiol-treated rats. It decreased chylomicron remnant binding to membranes from ethinyl estradiol-treated membranes, but did not affect chylomicron remnant binding to liver membranes of untreated rats, a result compatible with the existence of a distinct receptor for these latter particles. The amount of LDL receptor-independent, specific remnant binding was the same in both control and ethinyl estradiol-treated rats. This is consistent with the concept that the remnant receptor is not regulated by this treatment. Based on the above, we conclude that the ethinyl estradiol-induced lipoprotein receptor of rat liver is biochemically and immunologically similar to the LDL receptor of other species. It is present on the liver of normal adult rats and could account for LDL as well as beta VLDL and HDLc removal. Although it may contribute to chylomicron remnant removal, there appears to be a second unrelated receptor or process which recognizes this lipoprotein.

Binding of chylomicron remnants and beta-very low density lipoproteins to hepatic and extrahepatic lipoprotein receptors. A process independent of apolipoprotein B48.

The ability of apolipoprotein (apo-) B48 to interact with lipoprotein receptors was investigated using three different types of lipoproteins. First, canine chylomicron remnants, which contained apo-B48 as their primary apoprotein constituent, were generated by the hydrolysis of chylomicrons with milk lipoprotein lipase. These apo-B48-containing chylomicron remnants are deficient in apo-E and reacted very poorly with apo-E receptors on adult dog liver membranes and the low density lipoprotein (apo-B,E) receptors on human fibroblasts. Addition of normal human apo-E3 restored the receptor binding activity of these lipoproteins. Second, beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs were subfractionated into distinct classes containing apo-E along with either apo-B48 or apo-B100. Both classes bound to the apo-B,E and apo-E receptors. Their binding was almost completely mediated by apo-E, as evidenced by the ability of the anti-apo-E to inhibit the receptor interaction. Third, beta-VLDL from type III hyperlipoproteinemic patients were subfractionated by immunoaffinity chromatography into lipoproteins containing apo-E plus either apo-B48 or apo-B100. Both subfractions bound poorly to apo-B,E and apo-E receptors due to the presence of defective apo-E2. However, the residual binding of the apo-B48-containing and apo-B100-containing human beta-VLDL was inhibited by the anti-apo-E. After lipase hydrolysis, apo-B100 became a more prominant determinant responsible for mediating receptor binding to the apo-B,E receptor. By contrast, lipase hydrolysis did not increase the binding activity of the apo-B48-containing beta-VLDL. These results indicate that apo-B48 does not play a direct role in mediating the interaction of lipoproteins with receptors on fibroblasts or liver membranes.

Lipoprotein metabolism by rat hepatomas. Studies on the etiology of defective dietary feedback inhibition of cholesterol synthesis.

In contrast to normal liver, it is known that in vivo hepatomas fail to decrease their rate of cholesterol biosynthesis in response to increased dietary cholesterol. From a consideration of the available data it has been hypothesized that the defect might lie in the delivery of cholesterol to the hepatoma cell. To study this further, lipoprotein interactions with rat hepatoma cells in tissue culture (HTC 7288C) and with the same cell line in vivo were investigated. HTC cells grown in a medium containing 10% calf serum exhibited saturable, specific, calcium-dependent binding of rat 125I-chylomicron remnants at 4 degrees C with half maximal saturation at 4.8 micrograms protein/ml and maximum binding of 96 ng protein/10(6) cells. At 4 degrees C, HTC cells also bound human 125I-low density lipoprotein (LDL) specifically, but bound it with a much lower affinity. These cells also exhibited specific binding for rat LDL and rat hypercholesterolemic very low density lipoprotein (VLDL). All these lipoproteins were degraded by HTC cells. Thus, it was concluded that hepatoma cells possess lipoprotein receptors that recognize and process LDL, VLDL, and chylomicron remnants. Overnight incubation of HTC cells in lipid-depleted medium containing 0.5 microM compactin increased binding of rat chylomicron remnants and of hypercholesterolemic VLDL approximately 1.7-fold without a significant change in binding affinity. LDL binding also increased, by approximately 3.5-fold. These changes were also observed when binding and internalization were measured at 37 degrees C. After HTC cells were incubated in lipid-depleted medium, the rate at which [14C]acetate was incorporated into [14C]cholesterol increased 2.5-fold. Inclusion of rat chylomicron remnants at 5-10 micrograms protein/ml prevented this increase in acetate incorporation or, if added after culture in lipid-depleted medium, reduced the increased levels back to control values. However, the rate of acetate incorporation into cholesterol by cells grown in complete medium was not decreased to levels below base line by rat chylomicron remnants. Inclusion of human LDL only partially prevented the rise or only partially reduced the increased levels back to control and did not reduce control levels below base line. Hypercholesterolemic VLDL, which contain more cholesterol per particle than chylomicron remnants, did reduce [14C]acetate incorporation to below control levels. Therefore, the intracellular mechanism for down regulation of cholesterol synthesis by lipoproteins is intact in these cells. Based on these results we hypothesized that a relative lack of lipoprotein receptors expressed by hepatomas in vivo in comparison with those expressed by normal liver would explain the apparent absence of feedback inhibition of cholesterol synthesis. Consistent with this hypothesis, the binding of chylomicron remnants to liver cell membranes was 3-5 times greater than to membranes from tumors grown in vivo subcutaneously or intramuscularly. Membranes from tumor cells grown in vitro bound remnants least well. It is proposed that the relative lack of receptors places the hepatoma at a disadvantage in competing with the liver for lipoproteins of dietary origin and may account for the lack of feedback regulation of cholesterol synthesis in hepatomas.

DEFECTIVE CHYLOMICRON REMNANT CLEARANCE IN PATIENTS WITH FAMILIAL DYSBETAL IPOPROTEINEMIA

Familial dysbetalipoproteinemia results in part from a genetic defect in apolipoprotein E (apoE) which prevents normal binding of chylomicron remnants to a specific hepatic apoE receptor. We have developed a technique for measuring the consequent abnormality in chylomicron remnant clearance in these patients. Oral administration of retinol and Lipomul permits the labelling of nascent chylomicrons with retinyl palmitate (RP). RP is measured by high pressure liquid chromatography in plasma and in isolated lipoprotein fractions from 10-12 hourly blood samples. Clearance of chylomicron remnants (T½) is calculated from the rate of disappearance of RP from chylomicrons and very low density lipoproteins. The method was found to be safe and without side effects in 45 adults. Chylomicron remnant T½ was 1.5±0.5 hours in 7 people with normal fasting lipids. There was no change in low density lipoprotein RP; no RP was found in high density lipoprotein; no exchange of RP between lipoproteins was found. Five patients with familial dysbetalipoproteinemia were studied, including a teenage boy homozygous for the apoE 2 genetic variant (apoE 2/2) with severe type III hyperlipoproteinemia, generalized pruritic tuberoeruptive xanthoma and xanthoma striata palmaris. All patients failed to clear RP during the test, consistent with the failure of hepatic upake of chylomicron remnants carrying this apoE variant.

Characterization of chylomicron remnant binding to rat liver membranes.

The binding of chylomicron remnants to rat liver membranes was investigated using radioiodinated lipoproteins. The specific activity of binding increased in parallel with increased enrichment in plasma membrane markers. The yield of receptor activity, however, decreased with enrichment. Accordingly, a partially purified plasma membrane preparation was used for routine studies. Binding was saturable, with half maximal binding achieved at 4.6 micro g tetramethylurea-precipitable protein per ml. The rate of binding was time- and temperature-dependent. It could be inhibited only moderately by 10 mM EDTA. Chylomicron remnants appeared to bind to the membrane as a unit. The bound particle was richer in apoproteins of 20,000-50,000 molecular weight relative to low molecular weight apoproteins than the particles that were not bound. Lipoprotein particles containing only human apoB did not bind to liver membranes nor did they compete for the remnant binding site. Rat lipoproteins of d 1.019-1.063 g/ml did compete for remnant binding. When they were separated into apoB-rich (LDL) or apoE-rich (HDL(c)) fractions by block electrophoresis, the apoE-rich fraction was a more potent competitor. ApoE purified and reconstituted into dimyristoyl phosphatidylcholine vesicles was a potent competitor for the remnant binding site. Vesicles containing (125)I-labeled apoE bound to the membranes, and they could be displaced by unlabeled remnants. Dimyristoyl phosphatidylcholine vesicles themselves did not compete with either remnants or apoE-phospholipid vesicles. These results offer strong support for the hypothesis that the liver membrane chylomicron remnant receptor recognizes apoE with a high affinity, and this initiates the rapid removal of lipoproteins that contain this apoprotein.-Cooper, A. D., S. K. Erickson, R. Nutik, and M. A. Shrewsbury. Characterization of chylomicron remnant binding to rat liver membranes.