Very Low Density Lipoprotein Binding Research Articles

Shedding of syndecan-1 from human hepatocytes alters very low density lipoprotein clearance

We recently showed that the heparan sulfate proteoglycan syndecan-1 mediates hepatic clearance of triglyceride-rich lipoproteins in mice based on systemic deletion of syndecan-1 and hepatocyte-specific inactivation of sulfotransferases involved in heparan sulfate biosynthesis. Here, we show that syndecan-1 expressed on primary human hepatocytes and Hep3B human hepatoma cells can mediate binding and uptake of very low density lipoprotein (VLDL). Syndecan-1 also undergoes spontaneous shedding from primary human and murine hepatocytes and Hep3B cells. In human cells, phorbol myristic acid induces syndecan-1 shedding, resulting in accumulation of syndecan-1 ectodomains in the medium. Shedding occurs through a protein kinase C-dependent activation of ADAM17 (a disintegrin and metalloproteinase 17). Phorbol myristic acid stimulation significantly decreases DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate)-VLDL binding to cells, and shed syndecan-1 ectodomains bind to VLDL. Although mouse hepatocytes appear resistant to induced shedding in vitro, injection of lipopolysaccharide into mice results in loss of hepatic syndecan-1, accumulation of ectodomains in the plasma, impaired VLDL catabolism, and hypertriglyceridemia. These findings suggest that syndecan-1 mediates hepatic VLDL turnover in humans as well as in mice and that shedding might contribute to hypertriglyceridemia in patients with sepsis.

Synthesis of recombinant high density lipoprotein with apolipoprotein A-I and apolipoprotein A-V

It has been shown that apolipoprotein A-V (apoA-V) over-expression significantly lowers plasma triglyceride levels and decreases atherosclerotic lesion development. To assess the feasibility of recombinant high density lipoprotein (rHDL) reconstituted with apoA-V and apolipoprotein A-I (apoA-I) as a therapeutic agent for hyperlipidemic disorder and atherosclerosis, a series of rHDL were synthesized in vitro with various mass ratios of recombinant apoA-I and apoA-V. It is interesting to find that apoA-V of rHDL had no effect on lipoprotein lipase (LPL) activation in vitro and very low density lipoprotein (VLDL) clearance in HepG2 cells and in vivo. By contrast, LPL activation and VLDL clearance were inhibited by the addition of apoA-V to rHDL. Furthermore, the apoA-V of rHDL could not redistribute from rHDL to VLDL after incubation at 37°C for 30 min. These findings suggest that an increase of apoA-V in rHDL could not play a role in VLDL clearance in vitro and in vivo, which could, at least in part, attribute to the lost redistribution of apoA-V from rHDL to VLDL and LPL binding ability of apoA-V in rHDL. The therapeutic application of rHDL reconstituted with apoA-V and apoA-I might need the construction of rHDL from which apoA-V could freely redistribute to VLDL.

Mechanism for Endogenously Expressed ApoE Modulation of Adipocyte Very Low Density Lipoprotein Metabolism

Triglyceride-rich lipoproteins distribute energy in the form of fatty acids to peripheral tissues. We have previously shown that the absence of endogenous adipocyte apoE expression impairs adipocyte triglyceride acquisition from apoE-containing triglyceride-rich lipoproteins in vitro and in vivo. Studies were performed to evaluate the mechanism(s) for this impairment. We excluded a role for secreted apoE in accounting for the difference in very low density lipoprotein (VLDL)-induced adipocyte triglyceride accumulation using cross-incubation studies to show that secreted apoE did not enhance triglyceride synthesis in apoE knockout (EKO) adipocytes incubated with apoE-containing VLDL. Subsequent experiments established that both endocytic and lipase-mediated pathways for lipid acquisition from VLDL were impaired in EKO adipocytes. Binding and internalization of VLDL to EKO adipocytes were significantly lower due to decreased expression or redistribution of low density lipoprotein receptor family proteins. An important role for the VLDL receptor for contributing to differences in VLDL binding between wild-type and EKO adipocytes was identified. Lipoprotein lipase-dependent adipocyte lipogenesis was also significantly decreased in EKO adipocytes even though they secreted as much or more lipolytic activity. This decrease was related to impaired fatty acid internalization in EKO cells. Evaluation of potential mechanisms revealed reduced caveolin-1 and plasma membrane raft expression in EKO adipocytes. Increasing caveolin expression in EKO adipocytes increased fatty acid internalization. Our results establish a role for endogenous adipocyte apoE in VLDL-induced adipocyte lipogenesis by impacting both endocytic and lipoprotein lipase-mediated metabolic pathways. Reduced adipocyte apoE expression, for example that accompanying obesity, will suppress adipocyte acquisition of lipid from apoE-containing VLDL.

Vascular-Directed Tissue Factor Pathway Inhibitor Overexpression Regulates Plasma Cholesterol and Reduces Atherosclerotic Plaque Development

Tissue factor pathway inhibitor (TFPI) is a potent regulator of the tissue factor pathway and is found in plasma in association with lipoproteins. To determine the role of TFPI in the development of atherosclerosis, we bred mice which overexpress TFPI into the apolipoprotein E-deficient (apoE(-/-)) background. On a high-fat diet, smooth muscle 22alpha (SM22alpha)-TFPI/apoE(-/-) mice were shown to have less aortic plaque burden compared to apoE(-/-) mice. Unexpectedly, SM22alpha-TFPI/apoE(-/-) had lower plasma cholesterol levels compared to apoE(-/-) mice. Furthermore, SM22alpha-TFPI mice fed a high-fat diet had lower cholesterol levels than did wild-type mice. Because TFPI is associated with lipoproteins and its carboxyl terminus (TFPIct) has been shown to be a ligand for the very-low-density lipoprotein (VLDL) receptor, we hypothesized that TFPI overexpression may regulate lipoprotein distribution. We quantified VLDL binding and uptake in vitro in mouse aortic smooth muscle cells from SM22alpha-TFPI and wild-type mice. Mouse aortic smooth muscle cells from SM22alpha-TFPI mice demonstrated higher VLDL binding and internalization compared to those from wild-type mice. Because SM22alpha-TFPI mice have increased circulating levels of TFPI antigen, we examined whether TFPIct may act to alter lipoprotein distribution. In vitro, TFPIct increased VLDL binding, uptake, and degradation in murine embryonic fibroblasts. Furthermore, this effect was blocked by heparinase treatment. In vivo, systemic administration of TFPIct reduced plasma cholesterol levels in apoE(-/-) mice. These studies suggest that overexpression of TFPI lowers plasma cholesterol through the interaction of its carboxyl terminus with lipoproteins and heparan sulfate proteoglycans.

Molecular Mechanism of Apolipoprotein E Binding to Lipoprotein Particles

The exchangeability of apolipoprotein (apo) E between lipoprotein particles such as very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) is critical for lipoprotein metabolism, but despite its importance, the kinetics and mechanism of apoE-lipoprotein interaction are not known. We have used surface plasmon resonance (SPR) to monitor in real time the reversible binding of apoE to human VLDL and HDL(3); biotinylated lipoproteins were immobilized on a streptavidin-coated SPR sensor chip, and solutions containing various human apoE molecules at different concentrations were passed across the surface. Analysis of the resultant sensorgrams indicated that the apoE3-lipoprotein interaction is a two-step process. After an initial interaction, the second slower step involves opening of the N-terminal helix bundle domain of the apoE molecule. Destabilization of this domain leads to more rapid interfacial rearrangement which is seen when the lipoprotein binding of apoE4 is compared to that of apoE3. The resultant differences in interfacial packing seem to underlie the differing abilities of apoE4 and apoE3 to bind to VLDL and HDL(3). The measured dissociation constants for apoE binding to these lipoprotein particles are in the micromolar range. C-Terminal truncations of apoE to remove the lipid binding region spanning residues 250-299 reduce the level of binding to both types of lipoprotein, but the effect is weaker with HDL(3); this suggests that protein-protein interactions are important for apoE binding to this lipoprotein while apoE-lipid interactions are more significant for VLDL binding. The two-step mechanism of lipoprotein binding exhibited by apoE is likely to apply to other members of the exchangeable apolipoprotein family.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

Nephrotic livers secrete normal VLDL that acquire structural and functional defects following interaction with HDL

Binding of very low-density lipoprotein (VLDL) isolated from serum of nephrotic rats VLDL to endothelial cells is defective. This defect is conferred on normal VLDL by prior incubation with high-density lipoprotein (HDL) from nephrotic, but not control rats. It is not known whether the defect is present in nascent VLDL (nVLDL) or is acquired after secretion. We test the hypothesis that VLDL is normal at the time of secretion from the liver and the defect in binding to endothelium is conferred following secretion through interaction with HDL. nVLDL was synthesized by and collected from isolated perfused livers from either control or nephrotic rats. nVLDL was labeled with 3H-oleate to measure binding and 35S methionine to evaluate apolipoprotein exchange and composition. To test whether HDL conferred a binding defect, nVLDL was incubated with HDL obtained either from control or nephrotic rats prior to measurement of binding. To distinguish the effects of proteinuria from reduced albumin concentration we additionally incubated nVLDL with HDL obtained from rats with hereditary analbuminemia. Both HDL and VLDL were reisolated by centrifugation prior to subsequent binding and lipolysis determination. Exchange of 35S-labeled apolipoprotein E (apoE) among the subsequent VLDL and HDL fractions was determined. To determine the effect of HDL on lipolysis, HDL-treated VLDL was exposed to lipoprotein lipase-coated 96-well plates and 3H-oleate release measured. To establish whether differences in apoE content could explain the differences in binding and lipolysis, apoE was restored to nephrotic VLDL and lipolysis and binding were subsequently measured. Binding of nephrotic nVLDL was greater than control nVLDL (0.58 +/- 0.13 vs. 0.75 +/- 0.07 ng protein bound/mg cell protein) (P= 0.04, N= 6). Lipolysis was similarly elevated (0.091 +/- 0.010 vs 0.064 +/- 0.002 nmol NEFA released/well/hour) (P < 0.05). Prior incubation with nephrotic HDL reduced binding of nVLDL obtained from either nephrotic or control livers (P= 0.02, N= 6). Treatment with nephrotic (vs. control) HDL reduced both binding (control nVLDL + control HDL, 0.64 +/- 0.02; control + nephrotic, 0.43 +/- 0.06; nephrotic + control, 0.69 +/- 0.05; and nephrotic + nephrotic, 0.62 +/- 0.04 mg VLDL protein/mg cell protein) and lipolysis (control nVLDL + control HDL, 0.053 +/- 0.004; control + nephrotic, 0.038 +/- 0.004; nephrotic + control, 0.069 +/- 0.004; and nephrotic + nephrotic, 0.062 +/- 0.004 nmol NEFA/well/hour) (P < 0.05 vs. nVLDL + control HDL) of nVLDL from either source. The apoE content of nVLDL coincubated with control HDL or analbuminemic HDL was increased compared nVLDL incubated with either no HDL or nephrotic HDL (P < 0.05). Similarly, the apoE/apoA-I ratio was reduced in HDL from nephrotic rats but not in HDL from controls (P < 0.05). Reintroduction of apoE to nephrotic VLDL resulted in increased binding. Unlike circulating VLDL, binding of nVLDL from isolated livers from nephrotic rats to endothelial cells is greater and its lipolysis is increased compared to control nVLDL. Decreased binding and lipolysis is conferred following incubation with HDL isolated from control, but not nephrotic rats and binding can be restored by reintroduction of apoE. Thus both defects are conferred on VLDL by exposure to HDL obtained from nephrotic animals, possibly a consequence of a failure of nephrotic HDL to enrich VLDL with apoE during clearance.

A Schistosoma japonicum very low-density lipoprotein-binding protein

Schistosomes acquire fatty acids from their hosts, although how these parasites bind human low-density lipoproteins (LDL) and like molecules that transport fatty acids is not understood. Because parasite surface-bound host LDL may provide the schistosome with cholesterol and other lipids, as well as aid immune avoidance, understanding this process may provide fundamental insights into lipid metabolism and host defense in schistosomes. To investigate molecular aspects of lipid acquisition by schistosomes, transcripts encoding a very (V)LDL-receptor ligand binding, cysteine-rich repeat-containing protein were isolated from Schistosoma japonicum cDNAs. The deduced amino acid sequence included 207 residues with an NH 2-terminal LDL ligand-binding Cys-rich motif and a COOH-terminal transmembrane (TM) domain. The ligand-binding domain was similar in sequence and structure to ligand-binding Cys-rich repeat domains from mammalian very low-density lipoprotein (VLDL) and LDL receptors, which are multi-domain proteins. This putative VLDL binding protein, designated S. japonicum very low-density lipoprotein binding protein (SVLBP), appeared to be membrane-associated, sensitive to reducing conditions, and included intra-molecular disulfide linkages. A three-dimensional (3D) model suggested that two of the three Cys residues form intra- and/or inter-molecular disulfide bridges that contribute to a patch of negative charge on the molecular surface, assumed to be associated with VLDL binding activity. SVLBP in membrane-associated and soluble fractions of adult schistosomes bound human plasma VLDL in vitro, and VLDL bound to recombinant SVLBP inhibited the binding of anti-recombinant SVLBP antibodies. Immunolocalization of SVLBP revealed prominent expression in the tegument and sub-tegument of adult male schistosomes. SVLBP may play a key role in lipid acquisition by S. japonicum.

The Low Density Lipoprotein Receptor-related Protein Complexes with Cell Surface Heparan Sulfate Proteoglycans to Regulate Proteoglycan-mediated Lipoprotein Catabolism

It has been proposed that clearance of cholesterol-enriched very low density lipoprotein (VLDL) particles occurs through a multistep process beginning with their initial binding to cell-surface heparan sulfate proteoglycans (HSPG), followed by their uptake into cells by a receptor-mediated process that utilizes members of the low density lipoprotein receptor (LDLR) family, including the low density lipoprotein receptor-related protein (LRP). We have further explored the relationship between HSPG binding of VLDL and its subsequent internalization by focusing on the LRP pathway using a cell line deficient in LDLR. In this study, we show that LRP and HSPG are part of a co-immunoprecipitable complex at the cell surface demonstrating a novel association for these two cell surface receptors. Cell surface binding assays show that this complex can be disrupted by an LRP-specific ligand binding antagonist, which in turn leads to increased VLDL binding and degradation. The increase in VLDL binding results from an increase in the availability of HSPG sites as treatment with heparinase or competitors of glycosaminoglycan chain addition eliminated the augmented binding. From these results we propose a model whereby LRP regulates the availability of VLDL binding sites at the cell surface by complexing with HSPG. Once HSPG dissociates from LRP, it is then able to bind and internalize VLDL independent of LRP endocytic activity. We conclude that HSPG and LRP together participate in VLDL clearance by means of a synergistic relationship.

Lipoprotein-stimulated mesangial cell proliferation and gene expression are regulated by lipoprotein lipase

Hyperlipidemia accelerates the progression of glomerular disease, and lipoproteins bind glomerular mesangial cells (MC) and induce proliferation and cytokine expression. In the vessel wall, the binding of lipoproteins to endothelial cells is markedly enhanced by lipoprotein lipase (LpL), synthesized by the underlying smooth muscle cells. While it is known that LpL is localized to the glomerulus, it is not known if and how it modulates the lipoprotein-mesangial interaction. Very low-density lipoprotein (VLDL) was isolated from rats and was used to treat cultured primary rat MCs. Binding studies were done with and without LpL and with/without pretreatment with heparanase, which degrades cell surface heparan sulfate proteoglycan (HSPG), known to modulate the LpL-lipoprotein interaction in blood vessels. VLDL/LpL was also used to assess MC proliferation and gene expression of the cytokine platelet-derived growth factor (PDGF). LpL enhanced VLDL binding to MCs by as much as 200-fold, and most of this effect was blocked by pretreatment with heparanase. LpL amplified VLDL-driven MC proliferation and increased VLDL-induced PDGF expression. Heparanase pretreatment of cells eliminated both of these amplifications. LpL alone increased MC proliferation and PDGF gene expression. As in the vessel wall, LpL enhances VLDL binding to MCs. MCs respond to LpL binding by proliferating and expressing cytokines such as PDGF. LpL may be a crucial paracrine mediator of the glomerular response to circulating lipoproteins, amplifying a response that includes cytokine elaboration, influx of circulating monocytes, and eventual sclerosis.

Hypoalbuminemia and proteinuria contribute separately to reduced lipoprotein catabolism in the nephrotic syndrome

Hypertriglyceridemia is a result of reduced triglyceride (TG)-rich lipoprotein (TRL) catabolism and occurs in rats with nephrotic syndrome (NS) and in Nagase analbuminemic rats (NARs). While the heparin-releasable lipoprotein lipase (LpL) pool in NAR and in NS is similar, TG levels are significantly greater in NS, suggesting that factors other than reduced LpL alone act in NS but not in NARs. Furthermore, clearance of chylomicrons (CM) and very low-density lipoprotein (VLDL) is normal in vivo in NAR despite low LpL levels. We tested the hypotheses that impaired binding of VLDL and impaired VLDL-high density lipoprotein (HDL) interactions contribute to hyperlipidemia in NS. TG and apoB secretion was measured using Triton WR 1339. Clearance of CMs by perfused hearts from NS and NAR was determined. Binding of VLDL from control, NS and NAR to rat aortic endothelial cells (RAECs) was measured prior to and following incubation with HDL from NS, NARs, and control. ApoE, protein, and TG content was determined. TG levels were greatest in NS (516 +/- 95 mg/dL), intermediate in NAR (193 +/- 20), and least in control (97 +/- 16, P = 0.05), while in contrast, TG secretion was least in NS (178 +/- 33 mg/dL/hour) versus 212 +/- 17 in NAR and 294 +/- 15 in control (P < 0.001 vs. NS). Clearance of CMs by NS and NAR hearts was the same and significantly reduced versus control (P < 0.005). Binding of NS-VLDL to endothelial cells was reduced, while NAR-VLDL binding was increased versus control (P < 0.001). Incubation of NS-VLDL with control or NAR HDL increased VLDL binding compared with binding following incubation with NS HDL (P < 0.001). Increased TG levels in both NS and NAR are the result of decreased TRL clearance. TG levels are greater in NS because of the presence of a combined defect: (1) a decrease in endothelial-bound LpL that occurs as a consequence of reduced serum albumin concentration, and (2) a defect in VLDL binding to endothelial-bound LpL. This latter defect occurs only in the presence of proteinuria and is conferred by HDL.

Proteinuria and plasma compositional changes contribute to defective lipoprotein catabolism in the nephrotic syndrome by separate mechanisms

Triglyceride (TG)-rich lipoproteins are primarily increased in the nephrotic syndrome (NS) as a result of decreased catabolism. Lipoprotein lipase (LpL) is the rate limiting enzyme for lipolysis of TG. The biologically active endothelial bound LpL pool is reduced in NS providing one mechanism for decreased clearance of very low density lipoprotein (VLDL). LpL, however, is also reduced in the Nagase Analbuminemic Rat (NAR) to the same extent as in NS, suggesting that other factors contribute to decreased VLDL clearance. Hyperlipidemia worsens with the onset of proteinuria and is reduced when proteinuria abates. We established that while VLDL from NS rats bind poorly to bovine aortic endothelial cells (BAEC) in the presence of saturating LpL while, VLDL from NAR bind more avidly than control. We then established that rat aortic endothelial cells (RAEC) incubated with serum from NAR or from NS rats bind significantly less exogenous LpL. Thus decreased clearance of VLDL in NS results from: 1) reduced endothelial bound LpL; an effect of serum from animals with reduced oncotic pressure (π) that makes cells unable to bind LpL; and 2) an alteration in VLDL binding to endothelial bound LpL. The former has no relationship to proteinuria while the latter occurs as a consequence of proteinuria. These effects combine to suppress VLDL clearance.

The hypolipidemic action of bezafibrate therapy in hypertriglyceridemia is mediated by upregulation of lipoprotein lipase: no effects on VLDL substrate affinity to lipolysis or LDL receptor binding

Fibrates are regarded as drugs of choice in hypertriglyceridemia (HTG). Downregulation of apolipoprotein (apo) C-III gene expression and upregulation of lipoprotein lipase (LPL) gene expression have been suggested to explain the hypolipidemic action of fibrates. This study was designed to study the effects of bezafibrate therapy on very low density lipoprotein (VLDL) susceptibility to lipolysis, VLDL binding to the low density lipoprotein (LDL) receptor and postheparin LPL activities in patients with HTG. VLDL lipolysis was studied with heparan sulfate proteoglycan-bound LPL. Binding affinity of VLDL to the LDL receptor was determined in J774 cells with 125I-labeled control LDL. Eighteen HTG patients were randomized to receive, in a double-blind placebo-controlled cross-over fashion, 400 mg bezafibrate once daily for 6 weeks. In response to bezafibrate therapy, plasma triglyceride and apoC-III levels decreased by 69 and 42%, respectively. HTG VLDL was lipolyzed less efficiently compared to control VLDL, and lipolysis did not improve by bezafibrate therapy. VLDL binding affinity to the LDL receptor was comparable between the control group and HTG group, and did not change upon bezafibrate therapy. However, the post-heparin LPL activity in the HTG patients increased from 153 to 192 U/l ( P=0.025). A strong inverse relation was observed between the change in LPL activities and the change in triglyceride levels ( r=−0.62, P=0.006). In conclusion, the hypolipidemic action of bezafibrate therapy in HTG may be attributed to increased LPL activity, whereas VLDL susceptibility to lipolysis and LDL receptor binding are not affected.

Lipoprotein receptors in extraembryonic tissues of the chicken

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo.

Lipoprotein Receptors in Extraembryonic Tissues of the Chicken

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo.

Very-low-density lipoprotein binding to the apolipoprotein E receptor 2 is enhanced by lipoprotein lipase, and does not require apolipoprotein E

The apolipoprotein (apo)E receptor 2 (apoER2) is a recently cloned member of the low-density lipoprotein (LDL) receptor (LDLR) family, showing a high homology with both the LDLR and the very-low-density lipoprotein (VLDL) receptor (VLDLR). In the present study, the binding characteristics of the apoER2 with respect to apoE and lipoprotein lipase (LPL) were investigated. VLDL was isolated from both apoE-deficient mice and mice expressing the human APOE2 (Arg158 → Cys) and APOE3-Leiden isoforms on an Apoe-/-,Ldlr-/- double knock-out background. apoE-rich rabbit β-VLDL was used as a positive control for binding. Binding experiments performed with Chinese hamster ovary cells expressing the human apoER2 showed that the receptor was able to bind VLDL containing either of the apoE isoforms, as well as the apoE-deficient VLDL. Hence, in contrast with the VLDLR, the apoER2 is not strictly dependent on apoE for VLDL binding. Since LPL has been shown to enhance the binding of lipoproteins to several members of the LDLR family, including the LDLR-related protein, VLDL receptor, gp330 and the LDLR itself, VLDL binding experiments were performed in the presence of LPL. Addition of LPL resulted in a significant increase in apoER2 binding for all VLDL fractions used in this study. In conclusion, lipoprotein binding of VLDL to the apoER2 is enhanced in the presence of LPL, and is not restricted to apoE-containing lipoproteins.

Very-low-density lipoprotein binding to the apolipoprotein E receptor 2 is enhanced by lipoprotein lipase, and does not require apolipoprotein E.

The apolipoprotein (apo)E receptor 2 (apoER2) is a recently cloned member of the low-density lipoprotein (LDL) receptor (LDLR) family, showing a high homology with both the LDLR and the very-low-density lipoprotein (VLDL) receptor (VLDLR). In the present study, the binding characteristics of the apoER2 with respect to apoE and lipoprotein lipase (LPL) were investigated. VLDL was isolated from both apoE-deficient mice and mice expressing the human APOE2 (Arg(158)-->Cys) and APOE3-Leiden isoforms on an Apoe(-/-),Ldlr(-/-) double knock-out background. apoE-rich rabbit beta-VLDL was used as a positive control for binding. Binding experiments performed with Chinese hamster ovary cells expressing the human apoER2 showed that the receptor was able to bind VLDL containing either of the apoE isoforms, as well as the apoE-deficient VLDL. Hence, in contrast with the VLDLR, the apoER2 is not strictly dependent on apoE for VLDL binding. Since LPL has been shown to enhance the binding of lipoproteins to several members of the LDLR family, including the LDLR-related protein, VLDL receptor, gp330 and the LDLR itself, VLDL binding experiments were performed in the presence of LPL. Addition of LPL resulted in a significant increase in apoER2 binding for all VLDL fractions used in this study. In conclusion, lipoprotein binding of VLDL to the apoER2 is enhanced in the presence of LPL, and is not restricted to apoE-containing lipoproteins.

Lipolytic remnants of human VLDL produced in vitro: effect of HDL levels in the lipolysis mixtures on the apoCs to apoE ratio and metabolic properties of VLDL core remnants

To determine the role of high-density lipoprotein (HDL) as an acceptor of lipolytic surface remnants of very low density lipoprotein (VLDL) in the metabolism of VLDL core remnants, we examined the effect of HDL levels in the VLDL lipolysis mixture on 1) the morphology and the apoCs to E ratio in VLDL core remnants and 2) the metabolic properties of VLDL core remnants in human hepatoma cell line HepG2 and human hepatocytes in the primary culture. Normolipidemic VLDL was lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL) in a lipolysis mixture containing a physiologic level of VLDL and albumin (30 mg VLDL-cholesterol (CH)/dl and 6% albumin) in the absence and presence of either a low HDL level (VLDL-CH:HDL-CH = 3:1) or a high HDL level (VLDL-CH:HDL-CH = 1:4). Lipolysis of VLDL in either the absence or presence of HDL resulted in the hydrolysis of >85% of VLDL-triglycerides (TG) and the conversion of VLDL into smaller and denser particles. In the absence of HDL, heterogeneous spherical particles with numerous surface vesicular materials were produced. In the presence of low or high HDL, spherical particles containing some or no detectable vesicular surface components were produced. The apoCs to apoE ratios, as determined by densitometric scanning of the SDS polyacrylamide gradient gel, were 2.89 in control VLDL and 2.27, 0.91, and 0.22 in VLDL core remnants produced in the absence and in the presence of low and high HDL levels, respectively. In vitro lipolysis of VLDL markedly increased binding to HepG2 cells at 4°C and internalization and degradation by human hepatocytes in primary culture at 37°C. However, the HDL-mediated decrease in the apoCs to apoE ratio had a minimal effect on binding, internalization, and degradation of VLDL core remnants by HepG2 cells and human hepatocytes in primary culture. In order to determine whether HepG2 bound VLDL and VLDL core remnants are deficient in apoCs, 125I-labeled VLDL and VLDL core remnants were added to HepG2 culture medium at 4°C. The bound particles were released by heparin, and the levels of 125I-labeled apoCs and apoE, relative to apoB, in the released particles were examined. When compared with those initially added to culture medium, the VLDL and VLDL core remnants released from HepG2 cells had a markedly increased (113%) level of apoE and a reduced (30–39%), but not absent, level of apoCs. We conclude that apoCs, as a minimum structural and/or functional component of VLDL and VLDL core remnants, may not have an inhibitory effect on the binding of VLDL or VLDL core remnants to hepatic apoE receptors. —Chung, B. H., and N. Dashti. Lipolytic remnants of human VLDL produced in vitro: effect of HDL levels in the lipolysis mixtures on the apoCs to apoE ratio and metabolic properties of VLDL core remnants.

Differential binding of triglyceride-rich lipoproteins to lipoprotein lipase

In comparison to very low density lipoprotein (VLDL), chylomicrons are cleared quickly from plasma. However, small changes in fasting plasma VLDL concentration substantially delay postprandial chylomicron triglyceride clearance. We hypothesized that differential binding to lipoprotein lipase (LPL), the first step in the lipolytic pathway, might explain these otherwise paradoxical relationships. Competition binding assays of different lipoproteins were performed in a solid phase assay with purified bovine LPL at 4°C. The results showed that chylomicrons, VLDL, and low density lipoprotein (LDL) were able to inhibit specific binding of 125I-labeled VLDL to the same extent (85.1% ± 13.1, 100% ± 6.8, 90.7% ± 23.2% inhibition, P = NS), but with markedly different efficiencies. The rank order of inhibition (Ki) was chylomicrons (0.27 ± 0.02 nm apoB) > VLDL (12.6 ± 3.11 nm apoB) > LDL (34.8 ± 11.1 nm apoB). By contrast, neither triglyceride (TG) liposomes, high density lipoprotein (HDL), nor LDL from patients with familial hypercholesterolemia were efficient at displacing the specific binding of 125I-labeled VLDL to LPL (30%, 39%, and no displacement, respectively). Importantly, smaller hydrolyzed chylomicrons had less affinity than the larger chylomicrons (Ki = 2.34 ± 0.85 nm vs. 0.27 ± 0.02 nm apoB respectively, P < 0.01). This was also true for hydrolyzed VLDL, although to a lesser extent. Chylomicrons from patients with LPL deficiency and VLDL from hypertriglyceridemic subjects were also studied.▪Taken together, our results indicate an inverse linear relationship between chylomicron size and Ki whereas none was present for VLDL. We hypothesize that the differences in binding affinity demonstrated in vitro when considered with the differences in particle number observed in vivo may largely explain the paradoxes we set out to study.—Xiang, S-Q., K. Cianflone, D. Kalant, and A. D. Sniderman. Differential binding of triglyceride-rich lipoproteins to lipoprotein lipase. J. Lipid Res. 1999. 40: 1655–1662.

Low density lipoprotein-receptor plays a major role in the binding of very low density lipoproteins and their remnants on HepG2 cells

The binding to HepG2 cells of very low density lipoproteins (VLDL) and their remnants (IDL) was alternatively, in the past, attributed to the low density lipoprotein receptor (LDLr) or to an apoE-specific receptor. In order to resolve this issue, we have compared the binding of those lipoproteins labelled with iodine-125 to normal and LDLr deficient HepG2 cells. Those deficient cells were obtained by a constitutive antisense strategy and their LDLr level is 14% the level of normal HepG2 cells. By saturation curve analysis, we show that VLDL and IDL bind to high and low affinity sites on cells. The low affinity binding was eliminated by conducting the assay in presence of a 200-fold excess of HDL3 respective to the concentrations of 125 I -labelled VLDL and IDL. For 125 I -VLDL high affinity binding to normal HepG2 cells, we found a dissociation constant ( K d) of 21.2±3.7 μg prot./ml (S.E., N=5) and a maximal binding capacity ( B max) of 0.0312±0.0063 μg prot./mg cell prot, while we have measured a K d of 5.3±0.8 and a B max of 0.0081±0.0014 with LDLr deficient cells. This indicates that LDLr is responsible for 74% of VLDL binding to HepG2 cells and that the non-LDLr high affinity receptor has a higher affinity for VLDL than LDLr. A 53% loss of 125 I -IDL binding capacity was measured with LDLr deficient cells compared with normal cells ( B max:0.028±0.005 versus 0.059±0.006), while no significant statistical difference was found between affinities. The study shows that the LDLr is almost the only contributor in VLDL binding, while it shares IDL binding capacity with another high affinity receptor. The physiological importance of LDLr is confirmed by an almost equivalent loss of IDL and VLDL degradation in LDLr deficient cells.