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.     

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.     

Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

Regulation of lipoprotein receptors on rat hepatomas in vivo

It has been shown previously that the rat hepatoma no. 7288C grown in vivo or in vitro expresses fewer receptors which recognize chylomicron remnants than does normal rat liver, and it was suggested that this may contribute to the deletion of dietary cholesterol-induced regulation of cholesterol synthesis in hepatomas (Barnard, G., Erickson, S. and Cooper, A. (1984) J. Clin. Invest. 74, 173-184). To investigate this further, Buffalo rats bearing hepatomas (HTC no. 7288C) were made hypercholesterolemic by feeding an atherogenic diet and hypocholesterolemic by ethinyl estradiol injections. Under all circumstances, tumor membranes had fewer receptors than liver membranes as measured by specific binding of [125I]chylomicron remnants. Ethinyl estradiol treatment increased the number of lipoprotein receptors 1.7-fold in liver membranes and 1.2-1.6-fold in tumor membranes, but hypercholesterolemia did not produce any significant changes in remnant binding to either liver or hepatoma membranes. Feeding an atherogenic diet induced a 2.4-fold increase in total cholesterol content in the liver, primarily as cholesterol ester; however, there was no change in total, free or ester cholesterol in the hepatomas. Acyl coenzyme A:cholesterol acyltransferase activity was low in this hepatoma line and neither treatment significantly affected its activity. One explanation for the lack of effect of the atherogenic diet on hepatoma cholesterol metabolism in addition to the decreased number of lipoprotein receptors might be the failure of access of lipoproteins to the tumor cell. To assess this, radioiodinated apo E-rich lipoproteins of various sizes were injected intravenously into rats with hepatomas. Their disappearance from the circulation was followed, and the uptake of each lipoprotein into a variety of tissues was determined. Chylomicron remnants were the most avidly removed particles. VLDLH, IDLH and HDLC were removed more slowly and less completely. None of the lipoproteins accumulated substantially in the tumors suggesting a limited access to the hepatoma tissue. Thus, in addition to the observed reduction in lipoprotein receptor number, limited lipoprotein access to the hepatoma tissue may be a significant factor in contributing to the apparent lack of feedback regulation of cholesterol synthesis by hepatoma tissue in vivo.     

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.     

Binding of rat chylomicrons and their remnants to the hepatic low-density-lipoprotein receptor and its role in remnant removal.

Binding and uptake of rat chylomicrons of different metabolic stages by the hepatic low-density-lipoprotein (LDL) receptor were studied. Pure chylomicrons, characterized by apolipoprotein B-48 devoid of contaminating B-100, were labelled in their cholesteryl esters. Lymph chylomicrons and serum chylomicrons, enriched in apolipoprotein E and the C-apolipoproteins, bound poorly to rat hepatic membranes. In contrast, chylomicron remnants, containing the apolipoproteins B-48 and E, bound with high affinity. Specific binding of remnants was virtually completely competed for by LDL free of apolipoprotein E. In addition, in ligand blots both remnants and LDL associated with the same protein with an Mr characteristic of the LDL receptor. Uptake of remnants during a single pass through isolated perfused rat livers was decreased to about 50% by an excess of LDL. It is concluded that rat chylomicron remnants are a ligand of the hepatic LDL receptor. The much higher affinity as compared with LDL is mediated by apolipoprotein E but not B-48, and is inhibited by the C-apolipoproteins. This explains why serum chylomicrons are not taken up by the liver, whereas remnants are rapidly removed from the circulation. Results from experiments in vivo suggest that the LDL receptor makes an important contribution to the hepatic uptake of remnants and may be the principal binding site of the liver responsible for remnant removal.     

Effect of fish oil and coconut oil diet on the LDL receptor activity of rat liver plasma membranes

The influence of 4 weeks treatment with fish oil and coconut oil enriched diets on the chemical composition of rat liver plasma membranes and LDL and on the binding of LDL to liver membranes was investigated. Rats fed fish oil diet showed a total, LDL and HDL plasma cholesterol concentration lower than the values observed in rats fed coconut oil and to a lesser extent lower than those of rats fed standard laboratory diet. LDL of rats on fish oil diet had a relative percentage of cholesterol and phospholipid lower, while that of triacylglycerol was greater. Furthermore, fish oil feeding was associated with a greater concentration of n - 3 fatty acids and a lower arachidonic and linoleic acid content in LDL. Liver plasma membranes isolated from fish oil rats showed a higher percentage of n - 3 fatty acids, while only a trace amount of these fatty acids was found in control and coconut oil fed animals. In binding experiments performed with LDL and liver membranes from fish oil fed rats and control rats, binding affinity (Kd = 3.47 +/- 0.93 and 4.56 +/- 1.27, respectively) was significantly higher (P less than 0.05) as compared to that found using membranes and lipoprotein from coconut oil fed rats (Kd = 6.82 +/- 2.69). In cross-binding experiments performed with fish oil LDL and coconut oil liver plasma membranes or coconut oil LDL and fish oil liver plasma membranes, the LDL binding affinity was comparable and similar to that found in fish oil fed animals. No difference was found in the Bmax among all the groups of binding experiments. Our data seem to indicate that during fish oil diet the higher binding affinity of LDL to liver plasma membranes might be partly responsible of the hypocholesterolemic action of marine oil rich diet as compared to saturated diet. Furthermore, the modifications of binding affinity induced by changes of LDL and membrane source, suggest that lipoprotein and liver plasma membrane composition may be an important variable in binding studies.     

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.     

The fatty acid composition of chylomicron remnants influences their binding and internalization by isolated hepatocytes.

The binding and internalization of (125)I-labelled chylomicron remnants derived from palm, olive, corn, or fish oil (rich in saturated, monounsaturated, n-6, or n-3 polyunsaturated fatty acids, respectively) by hepatocytes from rats fed a low-fat diet or a diet supplemented with the corresponding fat for 21 days was investigated. In hepatocytes from rats fed the low-fat diet, the association of radioactivity with the cells at 4 degrees C (a measure of initial binding only) was similar with all types of remnants tested, but was more rapid at 37 degrees C (a measure of binding plus internalization) when fish oil, as compared to olive, corn or palm oil remnants, was used, and similar differences in the internalization of the particles were observed. In contrast, when hepatocytes from rats fed the fat-supplemented diets were used, the rate of association at 37 degrees C of remnants with cells from rats fed palm, corn or fish oil was similar, and higher than that found with cells from animals fed olive oil, and in this case these differences were mainly due to changes in the binding of the particles to the cells at 4 degrees C. Both excess low-density lipoprotein (LDL), which inhibits remnant uptake by the LDL receptor, and lactoferrin, which blocks the LDL receptor-related protein (LRP), were found to decrease the association of the remnants with cells from rats fed the low-fat and high-fat diets. However, in hepatocytes from animals given the low-fat diet, most of the differences between the various types of particle were retained in the presence of lactoferrin, but abolished in the presence of LDL. In contrast, in cells from rats fed the high-fat diets, the differences were reduced by both lactoferrin and LDL. These findings demonstrate that the hepatic uptake of chylomicron remnants is influenced both by the fatty acid composition of the particles, and by longer-term adaptive changes in liver tissue, and suggest that the former effects are mediated mainly by the LDL receptor, while the latter may involve both the LDL receptor and the LRP.     

A dot-blot assay for the low density lipoprotein receptor

We describe a new method for detecting the interaction of low density lipoprotein with its receptor using unmodified nitrocellulose as support for membrane protein. The method is specific and sensitive down to 3 micrograms of membrane protein. Unlabeled LDL, but not HDL, competes with 125I-labeled LDL for binding, and binding is abolished by pretreatment of the membranes with pronase and is dependent upon the presence of Ca2+. Furthermore, modification of arginine or lysine residues on LDL abolishes the lipoprotein interaction with the receptor protein supported on the nitrocellulose. When the membranes are solubilized with octyl glucoside, purification steps of the receptor can be directly followed with no interference of the detergent, therefore eliminating the need for its removal. The increased expression of LDL receptors on liver membranes from estradiol-treated rats was also demonstrated. We suggest, therefore, that this method can be used to detect the presence of LDL receptors on minute amounts of membrane protein.     

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.     

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 diurnal rhythms of cholesterol metabolism and plasma clearance of model chylomicrons: comparison of normal and genetically hypercholesterolemic rats (RICO)

Previous studies showed a slower clearance of cholesterol-labeled lymph chylomicrons in genetically hypercholesterolemic rats (RICO) compared with normocholesterolemic rats. In this study, we compared rates of lipolysis and remnant clearance in RICO versus control normocholesterolemic rats of the same strain (RAIF) or with control Wistar rats, by injecting chylomicron-like lipid emulsions labeled with ¹⁴C-triolein to trace lipolysis, and ³H-cholesteryl ester to trace remnant clearance. Our findings showed slower clearance of chylomicron remnants in RICO compared with control RAIF or with control Wistar rats. During the light period, the clearance of lipids from chylomicron-like lipid emulsions injected intravenously was significantly slower in RICO rats compared with normocholesterolemic control rats of the same strain, RAIF. Within the RICO group, clearance of emulsion triolein (TO) was faster during the dark period compared with the light period. In contrast, however, the clearance of the emulsion remnants traced by cholesteryl oleate (CO) was slower during the dark period. This behaviour was not found within the Wistar group, where the clearances of TO and CO were similar in the light and dark period. Hepatic clearance of chylomicron remnants is mediated primarily by the low density lipoprotein (LDL) receptor, the expression of which shows diurnal variation. In both Wistar and RICO rats, the expression of LDL receptors was highest during the dark period. The LDL receptors in hepatic microsomal membranes from RICO rats migrated faster on SDS polyacrylamide gel electrophoresis when compared with normal Wistar and the RAIF. However in hepatic plasma membranes the LDL receptors from RICO and Wistar rats appeared identical after immunoblotting. Furthermore the LDL receptors from RICO and Wistar rats responded similarly to treatment with neuraminidase. An alteration in post-translational processing of the LDL receptor could possibly account for the slower clearance of chylomicron remnants in the RICO.     

Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants

Vitamin A plays an important role in reducing infectious disease morbidity and mortality by enhancing immunity, an effect that is partly mediated by macrophages. Thus, knowing how these cells take up vitamin A is important. The results in the present study demonstrate that J774 macrophages efficiently take up chylomicron remnant retinyl esters and retinol-binding protein (retinol-RBP) bound retinol by specific and saturable mechanisms. The binding of (125)I-RBP to plasma membrane vesicles demonstrated that the macrophage receptor had a similar binding affinity, as was discovered previously for other cells. The B(max) for the macrophages was smaller than the values reported for placenta, bone marrow, and kidney, but larger than that reported for liver. The J774 cells also bound and took up [(3)H]retinol-RBP. Approximately 50 to 60% of the uptake may compete with excess unlabeled retinol-RBP and approximately 30 to 40% with excess transtyrethin. Following the uptake of [(3)H]retinol-RBP, an extensive esterification occurred: After 5 hours of incubation, 77.8 +/- 3.9% (SD; n = 3) of the cellular radioactivity was recovered as retinyl esters. The J774 cells also demonstrated saturable binding of chylomicron remnant [(3)H]retinyl esters, and a continuous uptake at 37 degrees C followed by an extensive hydrolysis of the retinyl esters. Binding could be inhibited by approximately 50% by excess unlabeled low density lipoprotein (LDL). In addition, lipoprotein lipase increased the binding of chylomicron remnant [(3)H]retinyl esters by approximately 30% and the uptake of chylomicron remnant [(3)H]retinyl ester by more than 300%. Furthermore, because sodium chlorate reduced binding with 40% and uptake with 55%, the results suggest that proteoglycans are involved in the uptake. Thus, the results suggest that both LDL receptor and LDL-related protein are involved in the uptake of chylomicron remnant [(3)H]retinyl ester in macrophages.     

Catabolism of HDL1 cholesteryl ester in the rat. Effect of ethinyl estradiol treatment.

The present study was performed in control and ethinyl estradiol-treated rats in order to determine the mechanisms involved in the catabolism of HDL1 cholesteryl ester. Ligand blottings on liver membranes showed that purified HDL1, containing about 70% apolipoprotein E and 10% apolipoprotein AI, bind to the LDL receptor (130 kDa) and not to HB2 (100 kDa) or SR-BI (82 kDa), candidate HDL receptors. Immunoblots showed that the treatment increased the hepatic level of the LDL receptor five- to ten-fold, strongly decreased that of SRBI and did not change that of HB2. An in vivo kinetic study showed that the turnover of HDL1 cholesteryl ester is more rapid in treated than control rats. The liver participation (60%) in this clearance was not modified by the treatment. Therefore, it can be concluded that the catabolism of HDL1 cholesteryl ester, in control as in treated rats, is essentially ensured by the uptake of entire particles in the hepatocytes via LDL receptors.     

The very low density lipoprotein (VLDL) receptor – a peripheral lipoprotein receptor for remnant lipoproteins into fatty acid active tissues

The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.     

Isolation and characterization of the apolipoprotein E receptor from canine and human liver

Previous results have demonstrated that liver membranes possess two distinct lipoprotein receptors: a low density lipoprotein (LDL) receptor that binds lipoproteins containing either apolipoprotein (apo-) B or apo-E, and an apo-E-specific receptor that binds apo-E-containing lipoproteins, but not the apo-B-containing LDL. This study reports the isolation and purification of apo-B,E(LDL) and apo-E receptors from canine and human liver membranes. The receptors were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and were partially purified by DEAE-cellulose chromatography. The apo-B,E(LDL) receptor was isolated by affinity chromatography on LDL-Sepharose. The apo-E receptor, which did not bind to the LDL-Sepharose column, was then purified by using an HDLc (cholesterol-induced high density lipoprotein)-Sepharose affinity column and an immunoaffinity column. Characterization of the receptors revealed that the hepatic apo-B,E(LDL) receptor is similar to the extrahepatic LDL receptor with an apparent Mr = 130,000 on non-reducing sodium dodecyl sulfate-polyacrylamide gels. The apo-E receptor was found to be distinct from the apo-B,E(LDL) receptor, with an apparent Mr = 56,000. The purified apo-E receptor displayed Ca2+-dependent binding to apo-E-containing lipoproteins and did not bind to LDL or chemically modified apo-E HDLc. Antibodies raised against the apo-B,E(LDL) receptor cross-reacted with the apo-E receptor. However, an antibody prepared against the apo-E receptor did not react with the apo-B,E(LDL) receptor. The apo-E receptor also differed from the apo-B,E(LDL) receptor in amino acid composition, indicating that the apo-E receptor and the apo-B,E(LDL) receptor are two distinct proteins. Immunoblot characterization with anti-apo-E receptor immunoglobulin G indicated that the apo-E receptor is present in the hepatic membranes of man, dogs, rats, and mice and is localized to the rat liver parenchymal cells.     

Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and gp330, two members of the low density lipoprotein receptor gene family, share a multitude of cysteine-rich repeats. LRP has been shown to act as an endocytosis-mediating receptor for several ligands, including protease-antiprotease complexes and plasma lipoproteins. The former include alpha 2-macroglobulin-protease complexes and plasminogen activator inhibitor-activator complexes. The latter include chylomicron remnant-like particles designated beta-very low density lipoproteins (beta-VLDL) complexed with apoprotein E or lipoprotein lipase. The binding specificity of gp330 is unknown. In the current studies we show that gp330 from rat kidney membranes binds several of these ligands on nitrocellulose blots. We also show that both LRP and gp330 bind an additional ligand, bovine lactoferrin, which is known to inhibit the hepatic clearance of chylomicron remnants. Lactoferrin blocked the LRP-dependent stimulation of cholesteryl ester synthesis in cultured human fibroblasts elicited by apoprotein E-beta-VLDL or lipoprotein lipase-beta-VLDL complexes. Cross-competition experiments in fibroblasts showed that the multiple ligands recognize at least three distinct, but partially overlapping sites on the LRP molecule. Binding of all ligands to LRP and gp330 was inhibited by the 39-kDa protein, which co-purifies with the two receptors, suggesting that the 39-kDa protein is a universal regulator of ligand binding to both receptors. The correlation of the inhibitory effects of lactoferrin in vivo and in vitro support the notion that LRP functions as a chylomicron remnant receptor in liver. LRP and gp330 share a multiplicity of binding sites, and both may function as endocytosis-mediating receptors for a large number of ligands in different organs.     

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.