Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

ApoE is necessary and sufficient for the binding of large triglyceride-rich lipoproteins to the LDL receptor; apoB is unnecessary

Large triglyceride-rich very low density lipoproteins (VLDL) Sf 60-400 from hypertriglyceridemic (HTG) patients, but not VLDL from normal subjects, bind to the LDL receptor of human skin fibroblasts because they contain apolipoprotein E (apoE) of the correct conformation, accessible both to the LDL receptor and to specific proteolysis by alpha-thrombin. Trypsin treatment of HTG-VLDL Sf 60-400 causes extensive apoB hydrolysis (fragments less than 100,000 mol wt), total degradation of apoE, and thus complete loss of LDL receptor binding. The reincorporation of apoE (1 mol/mol VLDL) into trypsin-treated HTG-VLDL completely restored the ability of HTG-VLDL to interact with the LDL receptor, suggesting that apoE probably does not induce a conformational change in apoB which results in receptor recognition, nor is intact apoB necessary to maintain the appropriate conformation of apoE for LDL receptor binding. As a model of large triglyceride-rich VLDL Sf greater than 60, we fractionated Intralipid by the Lindgren method of cumulative flotation and prepared apoE-Intralipid complexes. Competitive binding studies demonstrated that apoE-Intralipid is at least as effective as LDL for uptake and degradation of 125I-labeled LDL. Control Intralipid complexes containing apoA-I instead of apoE do not compete with iodinated LDL. Since these TG-rich complexes contain no apoB, apoB is, therefore, not only not sufficient for receptor-mediated uptake of large particles, it is not necessary. ApoE of the correct conformation is not only necessary but is sufficient to mediate receptor binding of large triglyceride-rich particles to the LDL receptor.     

Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions

The atherogenicity theory for triglyceride-rich lipoproteins (TRLs; VLDL + intermediate density lipoprotein) generally cites the action of apolipoprotein C-III (apoC-III), a component of some TRLs, to retard their metabolism in plasma. We studied the kinetics of multiple TRL and LDL subfractions according to the content of apoC-III and apoE in 11 hypertriglyceridemic and normolipidemic persons. The liver secretes mainly two types of apoB lipoproteins: TRL with apoC-III and LDL without apoC-III. Approximately 45% of TRLs with apoC-III are secreted together with apoE. Contrary to expectation, TRLs with apoC-III but not apoE have fast catabolism, losing some or all of their apoC-III and becoming LDL. In contrast, apoE directs TRL flux toward rapid clearance, limiting LDL formation. Direct clearance of TRL with apoC-III is suppressed among particles also containing apoE. TRLs without apoC-III or apoE are a minor, slow-metabolizing precursor of LDL with little direct removal. Increased VLDL apoC-III levels are correlated with increased VLDL production rather than with slow particle turnover. Finally, hypertriglyceridemic subjects have significantly greater production of apoC-III-containing VLDL and global prolongation in residence time of all particle types. ApoE may be the key determinant of the metabolic fate of atherogenic apoC-III-containing TRLs in plasma, channeling them toward removal from the circulation and reducing the formation of LDLs, both those with apoC-III and the main type without apoC-III.     

The beta-VLDL receptor pathway of murine P388D1 macrophages

Very low density lipoproteins Sf 100-400 (VLDL1) from hypertriglyceridemic (HTG) subjects and chylomicrons cause receptor-mediated lipid engorgement in unstimulated macrophages in vitro via the beta-VLDL receptor pathway. We now report that the murine macrophage P388D1 cell line possesses the characteristics of the beta-VLDL receptor pathway observed previously in freshly isolated resident murine peritoneal macrophages or human monocyte-macrophages. HTG-VLDL1 isolated from the plasma of subjects with hypertriglyceridemia types 3, 4, and 5 interact with P388D1 macrophages in a high-affinity, curvilinear manner. beta-VLDL, HTG-VLDL1, chylomicrons, and thrombin-treated HTG-VLDL1 (which do not bind to the LDL receptor) compete efficiently and similarly for the uptake and degradation of HTG-VLDL1. LDL and acetyl LDL do not compete, indicating that uptake of HTG-VLDL1 is via neither the LDL receptor nor the acetyl LDL receptor. Binding of thrombin-treated HTG-VLDL1 to the beta-VLDL receptor indicates that the thrombin-accessible apoE, which is absolutely required for interaction of HTG-VLDL Sf greater than 60 with the LDL receptor, is not required for binding to the beta-VLDL receptor. The uptake and degradation of 125I-labeled HTG-VLDL1 is suppressed up to 80-90% by preincubation of the cells with sterols, acetyl LDL, or beta-VLDL, indicating that this process is not via the irrepressible chylomicron remnant (apoE) receptor. Chylomicrons, HTG-VLDL1, and thrombin-treated HTG-VLDL1-but not normal VLDL1, beta-VLDL, LDL, or acetyl LDL-produce massive triglyceride accumulation (10-20-fold mass increases in 4 hr) in P388D1 macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.     

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.     

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.     

巨噬细胞新型氧化低密度脂蛋白结合蛋白的研究

小鼠腹腔巨噬细胞（ＭＰＭ）膜上存在能结合氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的Ａ类清道夫受体（ＳＲ－Ａ）,但用配体印迹技术研究制备ＭＰＭ膜蛋白,发现还存在一种新型ｏｘ－ＬＤＬ膜结合蛋白,其分子量低于ＳＲ－Ａ,为９２ｋＤ它不结合乙酰化低密度脂蛋白ａｃ－ＬＤＬ）,与配体的结合也不受还原剂的影响,但唾液酸酶处理则明显减弱其与ｏｘ－ＬＤＬ的结合,未标记ｏｘ－ＬＤＬ能竞争性抑制（＾１２５Ｉ）ｏｘ－ＬＤＬ与９２ｋ     

Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL

Studies in humans and mice have shown that increased expression of apolipoprotein C-I (apoC-I) results in combined hyperlipidemia with a more pronounced effect on triglycerides (TGs) compared with total cholesterol (TC). The aim of this study was to elucidate the main reason for this effect using human apoC-I-expressing (APOC1) mice. Moderate plasma human apoC-I levels (i.e., 4-fold higher than human levels) caused a 12-fold increase in TG, along with a 2-fold increase in TC, mainly confined to VLDL. Cross-breeding of APOC1 mice on an apoE-deficient background resulted in a marked 55-fold increase in TG, confirming that the apoC-I-induced hyperlipidemia cannot merely be attributed to blockade of apoE-recognizing hepatic lipoprotein receptors. The plasma half-life of [3H]TG-VLDL-mimicking particles was 2-fold increased in APOC1 mice, suggesting that apoC-I reduces the lipolytic conversion of VLDL. Although total postheparin plasma LPL activity was not lower in APOC1 mice compared with controls, apoC-I was able to dose-dependently inhibit the LPL-mediated lipolysis of [3H]TG-VLDL-mimicking particles in vitro with a 60% efficiency compared with the main endogenous LPL inhibitor apoC-III. Finally, purified apoC-I impaired the clearance of [3H]TG-VLDL-mimicking particles independent of apoE-mediated hepatic uptake in lactoferrin-treated mice. Therefore, we conclude that apoC-I is a potent inhibitor of LPL-mediated TG-lipolysis.     

99mTechnetium-labeled low density lipoprotein: receptor recognition and intracellular sequestration of radiolabel

99MTechnetium-labeled low density lipoprotein (99MTc-labeled LDL) was developed to detect atherosclerosis by external imaging with the gamma scintillation camera (Lees, et al. J. Nucl. Med. 1985. 26: 1056-1062; Lees, et al. Arteriosclerosis. 1988. 8: 461-470). The present study examined high affinity LDL receptor recognition and intracellular sequestration of 99MTc-labeled LDL by fibroblasts. There were no significant differences between 99MTc-labeled LDL and 125I-labeled LDL in binding parameters or percent inhibition of accumulation, which indicated that 99MTc labeling did not alter receptor recognition of LDL. At 4 degrees C the Kd (+SE) for 99MTc-labeled LDL and 125I-labeled LDL, respectively, was 1.52 +/- 0.24 and 1.45 +/- 0.14 micrograms/ml; Bmax (+/- SE) was 5.45 +/- 0.48 and 4.89 +/- 0.25 ng/well, respectively. Binding was saturated at about 2 micrograms/ml. The complete linearity of 99MTc-labeled LDL accumulation from 0-6 h and the positive slope from 6-24 h indicated that radiolabel that entered cells as 99MTc-labeled LDL was sequestered; pulse-chase experiments, which measured residual cell-associated radioactivity out to 24 h, also showed that radiolabel was trapped. Because radiolabel sequestration was essentially complete, and because 99MTc-labeled LDL was recognized by the LDL receptor equally as well as 125I-labeled LDL, it should be useful not only for imaging atherosclerosis, but also for quantitatively determining sites of utilization and degradation of LDL.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.     

Rat high density lipoprotein subfraction (HDL3) uptake and catabolism by isolated rat liver parenchymal cells.

Rat liver parenchymal cell binding, uptake, and proteolytic degradation of rat 125I-labeled high density lipoprotein (HDL) subfraction, HDL3 (1.10 less than d less than 1.210 g/ml), in which apo-A-I is the major polypeptide, were investigated. Structural and metabolic integrity of the isolated cells was verified by trypan blue exclusion, low lactic dehydrogenase leakage, expected morphology, and gluconeogenesis from lactate and pyruvate. 125I-labeled HDL3 was incubated with 10 X 10(6) cells at 37 degrees and 4 degrees in albumin and Krebs-Henseleit bicarbonate buffer, pH 7.4. Binding and uptake were determined by radioactivity in washed cells. Proteolytic degradation was determined by trichloroacetic acid-soluble radioactivity in the incubation medium. At 37 degrees, maximum HDL3 binding (Bmax) and uptake occurred at 30 min with a Bmax of 31 ng/mg dry weight of cells. The apparent dissociation constant of the HDL3 receptor system (Kd) was 60 X 10(-8) M, based on Mr = 28,000 of apo-A-I, the predominant rat HDL3 protein. Proteolytic degradation showed a 15-min lag and then constant proteolysis. After 2 hours 5.8% of incubated 125I-labeled HDL3 was degraded. Sixty per cent of cell radioactivity at 37 degrees was trypsin-releasable. At 37 degrees, 125I-labeled HDL3 was incubated with cells in the presence of varying concentrations of native (cold) HDL3, very low density lipoproteins, and low density lipoproteins. Incubation with native HDL3 resulted in greatest inhibition of 125I-labeled HDL3 binding, uptake, and proteolytic degradation. When 125I-labeled HDL3 was preincubated with increasing amounts of HDL3 antiserum, binding and uptake by cells were decreased to complete inhibition. Cell binding, uptake, and proteolytic degradation of 125I-labeled HDL3 were markedly diminished at 4 degrees. Less than 1 mM chloroquine enhanced 125I-labeled HDL3 proteolysis but at 5 mM or greater, chloroquine inhibited proteolysis with 125I-labeled HDL3 accumulation in cells. L-[U-14C]Lysine-labeled HDL3 was bound, taken up, and degraded by cells as effectively as 125I-labeled HDL3. These data suggest that liver cell binding, uptake, and proteolytic degradation of rat HDL3 are actively performed and linked in the sequence:binding, then uptake, and finally proteolytic degradation. Furthermore, there may be a specific HDL3 (lipoprotein A) receptor of recognition site(s) on the plasma membrane. Finally, our data further support our previous reports of the important role of liver lysosomes in proteolytic degradation of HDL3.     

Identification of autoantibodies in human plasma recognizing an apoB-100 LDL receptor binding site peptide

The aim of this study was to test the hypothesis that autoantibodies recognize amino acid sequences in the LDL receptor binding region of apolipoprotein B-100 (apoB-100). Autoantibodies against an unmodified or malondialdehyde (MDA)-modified LDL receptor binding site peptide were determined by ELISA in baseline plasma samples of 78 cases with coronary events and 149 matched controls recruited from the prospective Malm? Diet Cancer Study. IgG and IgM recognizing this peptide were detected in all subjects but did not differ between cases and controls. Inverse associations were observed between IgG against the native binding site and plasma oxidized LDL (r = -0.21, P < 0.005), but there were no significant associations with total or LDL cholesterol levels. In univariate analyses, inverse associations were found between baseline carotid intima-media thickness and IgG against the MDA-modified binding site (r = -0.14, P < 0.05), but this association was lost when controlling for other major cardiovascular risk factors. Specificity studies demonstrated that the binding of autoantibodies to these sequences could be inhibited by oxidized but not by native LDL. Autoantibodies recognizing the LDL receptor binding site in apoB-100 are frequently expressed. Their association with plasma oxidized LDL suggests that they have been generated in response to breakdown products of LDL oxidation, but their influence on cholesterol metabolism and the development of atherosclerosis appears limited.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

Abnormal clearance of postprandial Sf 100-400 plasma lipoproteins in insulin-dependent diabetes mellitus

Studies were carried out in three normolipidemic non-obese men with insulin-dependent diabetes mellitus (IDDM) and three normal men, to assess whether the clearance of postprandial Sf 100-400 lipoproteins is decreased in IDDM. Sf greater than 100 lipoproteins isolated from plasma 4.5 h after fat ingestion were labeled with 125I and injected into the same subject intravenously. ApoB radioactivity was measured over time in Sf greater than 400, Sf 100-400, and Sf 20-100 lipoproteins isolated from plasma and analyzed using a kinetic model that included both fast and slow delipidation cascades, where lipolysis and uptake of particles by the liver and other tissues were represented. Fractional catabolic rates of Sf 100-400 lipoproteins (min-1) were decreased in diabetic versus control subjects: fast = 0.170 +/- 0.126 versus 0.680 +/- 0.242 (mean +/- SD) (P less than 0.05, two-tailed) and slow = 0.011 +/- 0.006 versus 0.031 +/- 0.015 (P less than 0.05, one-tailed). Kinetic analysis showed that the data were consistent with decreased uptake by the tissues for the fast cascade (diabetic, 0.084 +/- 0.082, vs. control, 0.617 +/- 0.328, P less than 0.05, one-tailed). A similar trend was observed for the slow cascade. There were no significant differences between the two groups in the intraplasma lipolysis rates of Sf 100-400 particles. Analysis of the composition of the injected particles showed that they were total cholesterol (TC)- versus triglyceride (TG)-enriched (P less than 0.001, log-ratio analysis of composition) in IDDM subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-density lipoproteins modified by lipid transfer protein have altered biological activity

Low-density lipoproteins (LDL) were modified by incubation with very-low-density lipoproteins (VLDL) and lipid transfer protein(s) to yield LDL particles that were enriched in triacylglycerol, depleted in cholesteryl esters, and contained apolipoprotein C. The uptake and degradation of these 125I-labeled modified LDL particles by cultured skin fibroblasts was reduced by approx. 30% when compared with LDL that had not been exposed to lipid transfer protein. Incubation of fibroblasts for 24 h in the presence of modified LDL resulted in less inhibition of LDL receptor activity and sterol synthesis than did incubation with control LDL. Both the degradation of 125I-labeled modified LDL and the effect of unlabeled modified LDL on the regulation of LDL binding and sterol synthesis were progressively decreased as the extent of modification of the LDL was increased. Even when identical amounts of modified LDL or control LDL protein were degraded, less inhibition of LDL receptor activity and sterol synthesis was observed with modified LDL than with control LDL, suggesting that the effects of modified LDL on these regulatory events are related to both the reduced degradation of the modified lipoprotein particles and to the alteration in its chemical composition. Uptake and degradation of modified LDL by human monocyte-derived macrophages in culture was reduced in a manner similar to that observed in the cultured fibroblasts, and was considerably less than that observed with acetylated LDL. No differences were observed between modified LDL prepared by exposure to lipid transfer activity in the lipoprotein deficient fraction of serum or when partially purified lipid transfer was used. Modified LDL, with similar composition to that used in the experiments, has been observed in certain diabetic and non-diabetic hypertriglyceridemic states. Thus, it is possible that the cellular metabolism of LDL in vivo might be altered in the presence of hypertriglyceridemia.     

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.